37? ajB/J Mo. 2 Vga

PHYSIOLOGICAL EFFECTS OF ASCARIS SUUM INTESTINAL MICROFLORA

ON 5-HYDROXYTRYPTAMINE LEVEL AND BINDING SITES IN THE

INTESTINAL EPITHELIAL CELLS

DISSERTATION

Presented to the Graduate Council of the

University of North Texas In Partial

Fulfillment of the Requirements

For the Degree of

DOCTOR OF PHILOSOPHY

By

Akbar Mohammadpour Shahkolahi B.S., M.A.

Denton, Texas

December, 1991

37? ajB/J Mo. 2 Vga

PHYSIOLOGICAL EFFECTS OF ASCARIS SUUM INTESTINAL MICROFLORA

ON 5-HYDROXYTRYPTAMINE LEVEL AND BINDING SITES IN THE

INTESTINAL EPITHELIAL CELLS

DISSERTATION

Presented to the Graduate Council of the

University of North Texas In Partial

Fulfillment of the Requirements

For the Degree of

DOCTOR OF PHILOSOPHY

By

Akbar Mohammadpour Shahkolahi B.S., M.A.

Denton, Texas

December, 1991

Shahkolahi, Akbar Mohammadpour, Physiological Effects of

Ascaris suum Intestinal Microflora on 5-Hydroxytrvptamine

Level and Binding Sites in the Intestinal Epithelial Cells.

Doctor of Philosophy (Biological Sciences), December, 1991,

114 pp., 6 Tables, 12 Figures, Bibliography, 112 Titles.

Serotonin (5-hydroxytryptamine, 5-HT) has been shown to

activate carbohydrate metabolism in adult female Ascaris

suum. Serotonin may be either absorbed directly from the

environment or synthesized de novo from the absorbed

L-tryptophan in adult female A. suum. The enzymes necessary

for the synthesis of 5-HT have been identified in both

intestine and muscle tissues. The serotonin absorbed from

the environment is obtained either from the host's

gastrointestinal contents or from the 5-HT producing bacteria

in the intestine of A. suum. Numerous 5-HT producing

bacteria were identified in the intestinal microflora. The

physiological contributions of 5-HT producing bacteria to the

5-HT level, turnover and binding sites in the intestinal

tissue of A. suum were investigated.

Using high performance liquid chromatography with

electrochemical detection, L-tryptophan and serotonin levels

of the intestinal tissue and its microflora were measured.

There was a significant decrease in the 5-HT level of

intestinal tissue as the 5-HT producing bacteria were

eliminated. The 5-HT turnover rates significantly increased

reaching the highest rate in intestine of worms incubated in

A. suum saline plus antibiotics for 48 hrs (301 ng 5-HT/mg

protein/hr). However, antibiotic treated intestinal tissue

showed an increase of more than 4 fold in its 5-HT turnover

rate which indicated that the elimination of 5-HT producing

bacteria stimulated the intestinal tissue to increase its

5-HT synthesis.

Serotonin receptor binding studies of the intestinal

tissue showed that elimination of intestinal microflora had

an effect on binding affinities and the apparent binding

densities. The [3H]-LSD binding sites increased in both

affinity and number in an anterior to posterior direction.

This directionality was altered when the intestinal

microflora were reduced.

Further characterization of 5-HT binding sites in

intestinal tissue indicated the presence of a specific

binding site in the intestinal membrane of A. suum.

L-tryptophan could possibly be transported across the

intestinal membrane independent of any 5-HT molecules

competing for the same site. However, L-tryptophan was found

to be capable of competing for [3H]-5-HT binding sites.

ACKNOWLEDGEMENTS

I wish to thank Dr. Manus J. Donahue for his technical

support and encouragement during the course of this project.

I wish to acknowledge the assistance provided by Dr. Andrew

S. Kester and to thank Dr. Judy Williams for her

contributions on the collaborated projects and her review of

the dissertation.

I extend my thanks to the other members of my committee,

Drs. Gerard A. 0'Donovan, Arthur J. Goven and Gerard R. Vela

for the instruction and guidance that each has given me.

I wish to thank my wife for her patience, continous

support and understanding during the completion of my

graduate education. I also wish to express my sincere

gratitude to my parents for the continous encouragement and

the support provided for my education. It is to them I

dedicate this volume.

TABLE OF CONTENTS

Page

AKNOWLEDGMENTS v

LIST OF TABLES viii

LIST OF FIGURES ix

LIST OF ABBREVIATIONS x

Chapter

I. INTRODUCTION

Historical Background on Helminths 1

Parasitic Nematodes 4

General Morphology and Life Cycle of Ascarids 9

Metabolism in Ascarids 14

Intestinal flora and the Metabolism of Tryptophan in

A. suum 19

Roles of Serotonin in A. suum 20

Possible Origin of Serotonin 23

Biosynthesis of Serotonin 27

Serotonin Uptake 30

Specific Aims 32

VI

II. MATERIALS AND METHODS

Experimental System 34

Serotonin Turnover Study 2 6

Serotonin Binding Sites in A. suum Intestinal

Tissue 39

Materials 42

III.RESULTS

Effects of Antibiotics on Intestinal Bacterial

Flora in A.suum 44

Tissue Perfusion Experiments 45

Binding Sites and Uptake of Serotonin 51

Competition Studies 53

IV. DISCUSSION

Discussion 55

Summary 61

APPENDIX A 63

APPENDIX B 70

BIBLIOGRAPHY 9 5

vn

LIST OF TABLES

Table Page

1. Bacterial Counts in Intestinal Tissue.of A. suum 64

2. Serotonin Levels and its turnover in whole A. suum

Intestinal Tissue '...65

3. Tryptophan and Serotonin levels in intestinal

segments of A. suum 66

4. Serotonin Turnover Rate in A. suum Intestinal

segments 67

5. Tryptophan Levels in A. suum Intestinal Tissue

and Microflora . 68

6. Serotonin Levels and Turnovers in A. suum Intestinal

Tissue and Microflora 69

Vlll

LIST OF FIGURES

Figure Page

1. HPLC Voltammogram From Intestinal Tissues of

A. suum 72

2. Dose-Response Cure 74

3. Dose-Response Curve 7 6

4. [3H] LSD Binding Intestinal Membranes 78

5. [3H] LSD Binding Intestinal Membranes 80

6. [3H] LSD Binding Intestinal Membranes 82

7. Inhibition of [3H]TRP Binding by 5-HT 84

8. Inhibition of [3H]TRP Binding by 5-HT 86

9. Inhibition of [3H]TRP Binding by 5-HT 88

10. Inhibition of [3H] Binding by TRP 90

11. Inhibition of [3H] Binding by TRP 92

12. Inhibition of [3H] Binding by TRP • 94

IX

LIST OF ABBREVIATIONS

AADC - aromatic amino acid decarboxylase

A. suum- Ascaris lumbricoides var. suum

ATP - adenosine triphosphate

BHI - brain heart infusion agar

cAMP - cyclic adenosine monophosphate

CFU - colony forming unit

CoA - coenzyme A, pantothenic acid

DAI - ' days after infection

DTP - guanosine triphosphate

GDP - guanosine diphosphate

HPLC-EC- high performance liquid chromatography with

electrochemical detector

LSD - lysergic acid diethylamide

MAO - monoamine oxidase

SBP - serotonin binding protein

TCA - tricarboxylic acid cycle, Kreb's cycle, oxidative

decarboxylation

TOH - tryptophan hydroxylase

TRP - tryptophan

5-HIAA - 5-hydroxyindoleacetic acid

5-HT - 5-hydroxytryptamin, serotonin

5-HTOL - 5-hydroxytryptophol

5-HTP - 5-hydroxytryptophan

XI

CHAPTER ONE

INTRODUCTION

Historical Background on Helminths

The helminths are trophoblastic metazoan organisms

commonly called "worms". All worms of medical importance

belong to one of two groups: the phylum Platyhelminthes or

"flatworms" and the phylum Nemathelminthes or "roundworms".

The former phylum, the flatworms, is divided into Trematodes

(flukes) and Cestode (tapeworms). The latter phylum includes

only the class Nematoda.

Nematodes are nonsegmented round worms and are the most

ubiquitous of all the multicellular animals. Most species of

nematodes are free living and inhabit soil and both fresh and

salt water, as well as other, more specialized habitats. The

majority of parasitic nematodes have developed a highly

specific biological dependence on a particular host and are

incapable of survival in any other host. A few have

succeeded in adapting to a variety of hosts. The following

is a brief history of nematodes (Levine, 1980) .

The earliest records of nematodes among the ancient

civilizations of the Mediterranean, Middle East, and Orient

were made some 2000 years ago. Indications of Ascaris (the

giant intestinal worm) and Dracunculus medinensis (the

guinea worm) are recorded in the Egyptian Ebers Papyrus, that

was probably recorded about 1550-1553 B.C. Human ascarids

were known to the ancient Greeks and Aristotle's Historia

animalium (384-322 B.C.) included two ascarids, the

tapeworm and the pinworm. In addition, nematodes are

mentioned in the Bible, and some interpret the passages of

Moses relating to Hebrew Laws of sanitation and hygiene as

emanating from his early learning from Egyptian physicians

about parasites. The great Arab scholar Avicenna (980-1037

C.E.) indicated human ascarids and Dracunculus and there is

references that the medieval falconers saw nematodes in their

birds (Albertus Magnus, 1200-1280).

In the mid-sixteenth century the invention of the

microscope made a major contribution to the advancement of

nematology. In 1656 Borellus discovered the first free-

living nematode. Studies by Tyson (1683) later described the

anatomy of nematodes and their eggs. From this point, the

interests in helminths grew among zoologists and

parasitologists and the first known information on helminths

was gathered by Rudolphi (1809-1819).

The discoveries of the past decades on nematodes in

plants and animals and the health and economical hazards that

they imposed on man shifted the focus of the studies and

opened a new phase of investigation in the nineteenth

century. The anatomical studies by Bojanus (1817-1821) and

life history and transmission studies by Owen (1835) led the

way to more important discoveries such as the alternation of

generations between free-living and parasitic phases and the

fact that invertebrates often act as intermediate hosts for

nemic parasites of higher vertebrates and humans. However,

it was Fedtschenko (1871) who finally solved the mystery of

the transmission of Dracunculus in drinking water

contaminated by the small aquatic crustacean Cyclops as the

intermediate host of this disease.

It was in 1878 that Manson discovered that mosquitoes

were the intermediate host and vectors of the human filirial

worm Wuchereria bancrofti. This finding led to the

discovery of mosquitoes as vectors for such diseases as

malaria and yellow fever. Through a series of

investigations, Stewart (1916) explained the migration of

Ascaris through the lungs.

Through these discoveries and continuous increase of

interest among scientists in recognizing the significance of

parasitic nematodes the history of nematodes entered .a new

era in the twentieth century. Today, the study of nematodes

has developed dramatically and researchers have contributed

much of their time to understanding the physiological and

ecological relationships between nematode and its host. One

of the strongest motives in the studying of nematodes has

been the demand to control diseases caused by the parasitic

forms in man, his domestic animals and his food crops. In

addition nematodes, due to their biologically simplistic

cellular organization, have also been used as beneficial

research models for application of modern experimental

technologies and methodologies.

Parasitic Nematodes

Nematodes are parasites of many plants, and many classes

of vertebrates and most invertebrates including annelids,

arthropods, and molluscs. They are found in soil, fresh

water, mountains and hot springs. The clinical aspects of

parasitic nematodes in man are outside the scope of this

discussion. However, basic review about different groups of

nematodes of medical importance, is necessary for

understanding their significance in the world. In addition

it is one of the predominant parasitic infections in Iran.

The parasitic nematodes of man, have a worldwide

distribution. They are organized into seven groups (Sun,

1988). The Rhabditida comprises free-living forms, some of

which are adapted to a parasitic existence (Strongyloides) .

Strongyloidiasis is a soil transmitted helminthic infection

that has a worldwide distribution, being particularly common

in tropical and subtropical regions.

The group Strongylida are better known as hookworms and

are characterized by infection stages which are free-living

larvae that infect through the skin (Ancylostoma, Nector)

and the mouth (Trichosstrongylus). Hookworm infection is

widely distributed in tropical and subtropical regions and

approximately one-fourth of the world's population have been

infected (Schad et al., 1984). This disease is second only

to ascariasis as the most common helminthic infection.

According to a report from the World Health Organization,

however, this parasitic infection has almost been eradicated

from Europe and the United States (1981).

The Oxyurida has one parasitic form generally recognized

in man: Entrobius vermicularis. The most prevalent

parasitic disease in the United States and Europe is

enterobiasis. The prevalence in the United States today is

approximately 30 to 40 million people (Moreno, 1971) .

The Spirurida is the largest group of parasitic nematodes

of man. It comprises many genera which require an

intermediate arthropod vector in their life cycles.

Dirofilariasis is worldwide in distribution among carnivores;

however, no human case of pulmonary dirofilariasis was

reported until 1961. Loiasis is another disease caused by

the eye worm of Africa, Loa loa. Loiasis is endemic only

to rain forest areas of central and West Africa.

Onchocerciasis is a filarial infection caused by Onchocerca

volvulus. It is estimated that from the 40 million people

who are infected, about two million have become blind (Connor

and Neafie, 1976). It is an ancient disease of tropical

Africa that has also become endemic to Central and South

America. According to the report from a World Health

Organization Expert Committee (WHO, 1984), 90.2 million

people have lymphatic filariasis. Lymphatic Filariasis

results from infection mainly by Wuchereia bancrofti ,

Burgia malayi or Brugia timori.

The Trichinelloidea has parasitic forms in the intestine

(Trichiuris, Trichinella), the respiratory passages

(Capillaria aerophila), the viscera (C. hepatica), the skin

(Anatrichosoma) and other sites. Trichuriasis (whipworm) is

worldwide in distribution, but it is most prevalent in

tropical and subtropical regions. The estimated worldwide

incidence is between 500-700 million (Pawlowski, 1984).

The Dioctophymatoidea has many species parasitic in

animals; only one, Dioctophyme renale has been reported

sporadically in man (Beaver et al., 1984)

The Ascaridida has many species of medical importance,

the adults of which inhabit mostly the gastrointestinal

tract. Development of certain groups of the Ascaridida

occur in soil (Ascaris, Toxocara), while other groups

7

require an intermediate host (Anisakis) (Chitwood, 1970) .

Ascariasis is cosmopolitan in distribution and the most

prevalent helminthic infection in the world: it is estimated

that one-fourth of the world population has this infection

(Pawlowski, 1978).

Ascaris lumbricoides is the human ascarid; it occurs in

the small intestine of man. World Health Organization (1971)

reported that over one billion people throughout the world

are infected by Ascaris lumbricoides. In industrialized

countries, such as Japan, the incidence has shown a dramatic

decrease, from 49 percent in 1949 to 0.7 percent in 1971, as

a result of repeated mass treatment (Pawlowski, 1978) . In

developing countries, however, the infection rate is still

high. In the Philippines, Indonesia and countries of

Southeast Asia, for instance, the rate of infection in rural

areas often exceeds 50 percent and occasionally reaches 100

percent (Beaver et al., 1984). The average infection rate

in most countries of Central and South America is

approximately 45 percent (Martin, 1972).

Ascariasis is probably the most inextricable health

hazard and economic burden to the livestock farming industry

as well. More than 80% of the swine in the United State are

infected with Ascaris suum (USDA reports, 1986) . On an

average statistical basis, an adult marketed hog carries 28

Ascaris suum in its intestinal tract and each worm weighs

3 g on an average (Martin et al., 1987). To maintain this

parasitic burden, the infected hog has to consume an extra

half a pound of food every day. The cost estimated, is at

least 233 million dollars annually to the swine industry from

Ascaris infection alone (Chaudhuri et al., 1987).

The strongest driving force in the study of nematodes has

been the need to control diseases caused by the parasitic

forms. Any steps taken to develop a suitable chemotherapy

towards these parasites should involve a detailed

investigation of the parasites biological development and

biochemistry. These animals have been used as model systems

for the study of many biochemical and physiological studies.

A unique feature of nematodes is that they show great

uniformity of structure, whether they are microscopic free-

living forms in soil or freshwater or parasitic worms of

plants or animals which can be up to 60 cm in length. All

nematodes also share the same basic life cycle comprising an

egg stage, four larval stages, and adult worms. One of the

most common nematodes that has been used as a model system to

study helminths is A. suum.

The subject of this research is the adult female A. suum

that lives in the upper intestine of the pig. Ascaridosis in

pigs creates an economic problem, mainly because it prevents

the weight gain in these animals. It also complicates the

immune system of the host and promote infection and alters

the feeding habit. It is estimated that 75 % of pigs in the

United States and Canada are infected by A. suum before they

have reached six months of age. It has been reported that

when pigs are infected with 20 or more worms at 8 weeks of

age failed to gain weight in direct proportion to the number

of worms (Chandler, 1950). The maximum number of worms found

in one pig was 109 and this pig did not gain any weight,

whereas uninfected pigs of the same age had gained an average

of 100 lbs. Obviously, the parasites are consuming Some of

the food. It has been indicated that the ascarids produce a

trypsin inhibiting substance which interferes with the host's

digestion of proteins (Krivoshta, 1939) . It has also been

shown that 20 adult worms consume 2.8 g of carbohydrate and

0.7 g of protein daily. Therefore, heavy infection, running

into the hundreds, cause the host to suffer in its diet.

General Morphology and Life Cycle of Ascarids

Ascarids are large fusiform nematodes, creamy-white in

color with an elongated cylindrical body which tapers at both

ends. The adult females measure 20-40 cm by 0.3-0.6 cm , and

the adult males 10-22 cm by 2-4 mm with a ventrally curved

tail and two copulatory spicules (Mozgovoi, 1968). The body

is covered with a thick, non-cellular structure called a

10

cuticle that is secreted by the underlying hypodermis. The

cuticle covers the outer layer of the body and also lines the

pharynx, excretory pore, rectum, cloaca, spicule pouches, and

vulva. The second layer is the hypodermis that may be

cellular or a syncytium. The hypodermis is composed of large

amounts of materials such as lipid and glycogen. Throughout

the hypodermis there are fibers that attach the cuticle to

the muscles. The use of the electron microscope by Bird

(1971) has provided a great deal of information about the

nematodes muscles. The longitudinal muscle layer lies

beneath the hypodermis, divided into four lengthwise bands by

four thickenings or ridges of the syncytium (Bird, 1971) .

The muscle cells are spindle shaped and obliquely striated.

They consist of three separate parts. The contractile

element which is long and in longitudinal form is attached

directly to the hypodermis and cuticle. The remainder is a

muscle cell body that contains the nucleus and lies free in

the pseudocoelom. Extending from the cell body and

contractile element are the processes "muscle arms". The arm

makes a synaptic connection at the surface of the dorsal or

ventral longitudinal nerves. The lack of circular muscles,

and the controlled synchrony of both dorsal or both ventral

nerve bands, permits bending of the muscle only in the

dorsoventral plane (Rosenbluth,1965).

The nervous system is composed of a nerve ring in the

11

esophageal region together with a large ventral, a smaller

dorsal and two or more lateral nerves running from this nerve

ring along the four hypodermal cords. The anterior end of

the Ascarids where the mouth is, has several nerves which

extend from the nerve ring and function as sense organs

(Levine, 1980).

The body cavity of Ascarids, like other nematodes, is a

pseudocoelom filled with fluid. This cavity forms the

hydrostatic skeleton of these animals giving them their

structure; in addition, the internal organs are covered with

this fluid. The pseudocoelomic site is where the nutrients,

waste products and oxygen from the environment pass through

(Harris et al., 1957). As a result, this fluid is a complex

solution containing a variety of proteins, fats,

carbohydrates, enzymes, nitrogenous compounds, inorganic ions

and hemoglobin. There is evidence of cells found in the

pseudocoelomic fluid called pseudocoelomocytes. It is

thought that they may be involved in phagocytosis and storage

of insoluble waste products (Chitwood and Chitwood, 1950) .

The alimentary system is composed of a mouth that opens

at the anterior tip of the worm, surrounded by lips, a buccal

cavity of varying shapes, a muscular and glandular pharynx,

which usually has a triradiate lumen, an intestine, rectum

and anus. The wall of the pharynx (esophagus) is composed of

muscle cells, several nerves and several types of glands.

12

The pharynx suck the food into the lumen of the pharynx and

pass it into the intestine against the hydrostatic pressure

on the wall of the pharynx and within the body contents.

The intestine is composed of a single layer of epithelial

cells that enclose the lumen. The cells often contain

numerous inclusion bodies of various types. These include

reserved food materials such as glycogen granules, fats and

fatty acid globules, and stored protein globules. Microvilli

cover the cell membrane that lines the lumen of the intestine

which increase the absorptive surface of the cell (Lee and

Atkinson, 1977). The intestinal tissue appear to be both

secretory and absorptive in function. The nutrients are

absorbed in the intestine in the form of sugars, amino acids

and fatty acids. A variety of bacteria, including

microaerophiles and anaerobes, can be isolated from the lumen

of the intestine.

The female reproductive system has one or two ovaries,

which open into an oviduct and uteri. The two uteri lead

into a vagina, which opens to the outside via the vulva.

Most males have one single testis which are tubular in shape,

a seminal vesicle and a vas deferens which open into a cloaca

(Chitwood and Chitwood 1950) .

The fluid in the body cavity helps in the distribution of

nutrients and oxygen in juveniles, and nematodes have no

special respiratory or circulatory systems. Regulation of

13

the body cavity fluid is probably a major role of the

excretory system, which consists of two canals that run in

the lateral epidermal ridges and unite near the anterior end

to form a single tube, opening ventrally through a pore

(Chitwood and Chitwood 1950).

Comparative studies of the life cycle of A. suum in swine

and A. lumbricoides in man have been reported by Douvres et

al. (1969). Mature females inhabiting the small intestine of

the host lay millions of tiny (45-75 X 35-50 um) eggs (Olson

et al., 1958) which then are fertilized and pass out of the

host with feces. In the soil, hatching of the eggs is

controlled by certain factors in order to develop to the

first-stage larva; moisture, oxygen tension and temperature

are important. The parasitic life cycle begins after

ingestion of fertilized eggs by a host, which hatch and

release larvae in the stomach and small intestine where they

undergo a migration around the body. Larvae penetrate the

intestinal mucosa and pass via the circulation to the lungs.

The larvae generally remain in the lungs where they develop

through the third stage. Eight Days After Infection (DAI),

third stage larvae begin tracheal migration by rupturing

pulmonary capillaries and entering the air sacs thence, up

the trachea to the area of epiglottis. Between eight and ten

DAI, larvae are coughed up, swallowed back through the

esophagus and stomach to the small intestine. In the small

14

intestine, the final molt to complete the development into

adult occurs. During this circulation, the individual worms

are increased from ym in length to cm in size. Only larvae

that have successfully complete the fourth ecdysis can

survive in the intestine and develop to maturity (Jaskoski,

1961; Nobel and Nobel, 1982). Thereafter, both male and

female adults copulate and female lay eggs that pass out with

the feces.

Metabolism in Ascarids

In general, Ascarids feed on the contents of the

alimentary canal of the host. Most uptake of nutrients of

Ascarids take place through the intestine. The intestine

functions in the digestion of particulate matter (bacteria,

erythrocytes, serum protein, etc.) and the absorption of

ingested low molecular weight nutrients. However, the

cuticle of Ascarids functions to some extend in the

absorption of nutrients and excretion of end products.

The metabolic activities of Ascarids are similar to those

of mammalian systems. Glycogen forms the main source of

carbohydrates as the principal energy reserve in these

animals. The amount of glycogen in the tissues of Ascaris

is about 20 percent of the dry weight of the animal and 70

percent of the dry weight of the muscle. The glycogen

15

molecule of Ascaris has been studied in detail and it has

been shown that there are some physiochemical differences in

comparison to mammalian glycogen.

A. suum has been considered as an obligate anaerobe due

to the lack of four enzymes of TCA cycle (Langer et al. ,

1980) and as a facultative anaerobe due to the presence of

cytochromes (Cheah, 1975). The tissues of adult A. suum

have been considered microaerophilic since some oxygen is

required for the parasite's survival (Saz, 1971). Oxygen

diffuses into the tissues as a result of the difference in

oxygen tensions between the surface and the internal regions

(Chance, et al., 1964). Diffusion of oxygen is induced by

muscular movements and aided by the hemolymph (perienteric

fluid) which bathes the pseudocoelomic surface of the

intestine, reproductive tract and muscle (Atkinson, 1976).

Generally, nematodes have different fatty acid

metabolisms depending on their living environment. The free

living, non-feeding, infective juveniles of several animal-

parasitic nematodes use up their stores of lipids as the

primary source of energy when they are starved (Nicholas,

1975; Von Brand, 1973). Adult Ascaris are unable to

utilize their stored lipids to any great extent.

Triglycerides are present in the ovaries of Ascaris to a

large extent and they are subsequently used by the

embryonating eggs. The embryonating eggs of A. suum carry

16

out S-oxidation (Ward et al., 1970).

During nonfeeding of the host the adult ascarids derive

most of their energy from stored glycogen (Fairbairn, 1970)

due to their inability for S-oxidation of fatty acids. In

A. suum carbohydrate is broken down by glycolysis, the

steps being similar to those in vertebrate tissue, with the

same relative rate of activity of the enzymes. However, it

is at the phosphoenolpyruvate (PEP) step that ascarids differ

from the vertebrates. The relative activities of

phosphorylase and hexokinase in muscle of Ascaris is

reversed as compared to mammalian muscle. The phosphorylase

functions at a higher rate in comparison to mammals and

hexokinase functions during times of limited activity

(Barret, 1973) .

The complete TCA cycle is present in many nematodes while

in adult ascarids it is absent or incomplete. However, the

TCA cycle and electron transport system are present in the

eggs, 1st, 2nd and 3rd stages of ascarids development (Von

Brand, 1973). Glycogen or glucose is broken down in the

muscle of Ascaris by glycolysis as far as

phosphoenolpyruvate (PEP). Since there is a deficiency of

pyruvate kinase (PK) and high activity of phosphoenolpyruvate

carboxykinase (PEPCK), PEP is carboxylated through the action

of PEPCK to form oxaloacetate. Oxaloacetate is reduced to

malate by cytoplasmic malate dehydrogenase. Malate is then

17

transported across the mitochondrial membrane where it

undergoes a dismutation reaction within the mitochondrium.

Malate may be reduced to fumarate via fumarase. Fumarate is

reduced to succinate by fumarate reductase and that is the

ATP producing step. Succinate may be converted to propionate

via fumarate reductase (Saz, 1971) . Propionate serves as a

precursor of branched-chain fatty acids (Saz, 1971). The

fatty acids are then excreted by the adult ascarid (Saz a-nd

Weil, 1960; 1962). Some of the malate may be converted to

pyruvate in the mitochondria. This pyruvate is not reduced

to lactate, instead is converted to acetate which is consumed

in fatty acids biosynthesis.

The pentose-phosphate pathway, which is an alternative

path way for carbohydrate oxidation in mammals (Lehninger,

1982) is also present in nematodes. Glucose 6-phosphate is

converted to ribose 5-phosphate with the reduction of 2 moles

of nicotinamide dinucleotide phosphate (NADP). In A. suum

the pathway is most active in the muscle tissue of males and

the reproductive tract of the females (Von Brand , 1973) .

The regulation of glycogen metabolism in the mammalian

skeletal muscle system has been established in great detail

in recent decades (Fischer and Krebs, 1955; Krebs and Beavo,

1979). The biochemistry of glycogen metabolism in A. suum

muscle suggests that it closely resembles that in the

mammalian skeletal system. The basis of this regulation lies

18

in the reversible covalent modification of enzymes by

phosphorylation and dephosphorylation initiated by the

accumulation of cAMP in muscle cells. The increase in cAMP

levels in mammalian skeletal muscle cells is under the

hormonal control of epinephrine (Sevilla and Fischer, 1969).

In invertebrates, the regulation of glycogen metabolism

is not yet completely understood. The presence of the

glycogenolytic enzyme, glycogen phosphorylase has been

demonstrated in A. suum muscle by Barrett and Beis (1973).

Recently, glycogen synthase from A. suum muscle tissue has

been purified to homogeneity (Hannigan et al.1985). The

enzymes glycogen phosphorylase and glycogen synthase have

been found to be interconvertible enzymes, like in mammalian

muscle, by Harris and Allen (1979) and Donahue and co-workers

(1981), respectively. The evidence in the literature

strongly suggests that cAMP and cAMP dependent protein kinase

mediated cascade regulating carbohydrate metabolism is

operative in A. suum muscle, analogous to the one existing

in mammals. This appears to be true for other host-parasite

systems too. Dendinger and Roberts (1977) showed with

Hymenolepis diminuta infected rat, that the presence of the

tapeworm depleted both host and parasite glycogen.

19

Intestinal Flora and the Metabolism of Tryptophan in A. suum

The bacterial flora of the proximal small intestine of

mammalian animals is predominantly Gram-positive in nature

generally consisting of Streptococci and Lactobacilli.

However, Enterobacteria and Bacteroides can occur fairly

frequently though in much lower numbers. In contrast, the

distal small intestine consists mostly of Gram-negative

bacteria including Enterobacteria, Proteus, Bacteroides and

Peptostreptococci. Escherichia coli was the most

frequently (58 isolates) isolated bacterial species from

about 133 isolates of the intestinal flora of A. suum (Hsu,

et al., 1986). All the anaerobic bacteria of the intestine

are either obligate or facultative anaerobes.

The intestinal flora convert aromatic L-amino acids to

metabolites (Gale, 1944). Several studies have shown the

formation of indole, indoleacetic acid, skatole (Lewis,

1962), and tryptamine from tryptophan (Yokoyama, et al.,

1974) when incubated with cultures of ruminal microorganisms.

L-5-hydroxytryptophan was degraded to 5-hydroxyindole and 5-

hydroxyskatole, but other analogues including D-tryptophan,

indolepropionate, indolebutyrate, indoleacrylate, tryptamine,

5-hydroxytryptamine, and tryptophol were not degraded

(Yokoyama, et al., 1974). The production of indole from

tryptophan is catalyzed by the enzyme tryptophanase with

20

liberation of ammonia and pyruvate. Studies on the

tryptophanase activity of various bacterial species have

shown that while E. coli has the highest levels of the

enzyme (Newton, et al., 1964), it is also present in other

species including Proteus species Cornybacterium species,

and Bacteroides (Chung, et al., 1975). Recent studies have

revealed that bacterial strains belonging to the

Micrococcaceae constitutively produce an aromatic L-amino

acid decarboxylase enzyme which has high activity towards

L-tryptophan, 5-hydroxy-L-tryptophan and L-phenylalanine

(Nakazawa, et al., 1977).

Previous work by Hsu et al., (1986) using conventional

fluorimetric methods showed that there are bacteria producing

5-HT in the intestine of A. suum. It was demonstrated that

all 20 bacterial isolates from Ascaris intestinal flora were

able to produce serotonin (Hsu et al., 1986). However, it

was not concluded whether or not the level of 5-HT produced

by these bacteria was a significant contributor to the tissue

levels of 5-HT.

Roles of Serotonin in A. suum

Serotonin, an ubiquitous indolealkylamine, was first

isolated by Rapport et al. (1949). This biogenic amine is

found throughout the animal kingdom and has been demonstrated

21

to elicit numerous physiological responses in different

animals. In mammals, 5-HT occurs in the central nervous

system (CNS), the pineal gland, the enterochromaffin cells of

the intestinal tract, the mast cells, and the platelets.

Serotonin is the precursor of melatonin and has both

neurotransmitter and hormonal functions in vertebrates

(Osborne, 1982) . It has been shown to be involved in

platelet aggregation and activation (Drummond, 1976) and in

carcinoid tumors (Ahlman, 1985) . Serotonin has been shown to

affect mood, food intake, sleep, cognition, aggressive

behavior, sexual activity perception of pain, and I

cardiovascular function (Barnes, 1988) .

In invertebrate species serotonin elicits numerous

physiological responses, which include stimulation of

motility in the parasitic flatworm Hymenoplepsis diminuta

(Mansour, 1979), activation of glycogenolysis in the

roundworm Ascaris suum (Donahue, 1981) and activation of

adenylate cyclase and cyclic AMP-dependent protein kinase

activities in Fasciola hepatica (Mansour, 1979) . Although

there are various physiological responses triggered by 5-HT

in parasitic helminths, perhaps the two most significant are

serotonin's participation in the regulation of motility and

carbohydrate metabolism. Serotonin stimulated the in vitro

rhythmic movement of Fasciola hepatica (Mansour, 1964),

Schistosoma mansoni (Barker et al., 1966; Hillman et al. ,

22

1974), Mescestoid.es corti (Hariri, 1974), Chlonorchis

sinensis, Taenia pisiformis (Hillman et al., 1974) and

Hymenolepsis diminuta (Mettrick et al., 1981). Reports

indicated that serotonin was able to stimulate glycolysis and

simultaneously decrease endogenous glycogen levels in the

cestode H. diminuta (Mettrick et al., 1981; Cyr et al.,

1982). Several studies by Ribero et al. (1983; 1987)

revealed that H. diminuta was capable of de novo synthesis

of 5-HT and that 5-HT stimulated increases in intracellular

cAMP were linked to membrane receptor binding and adenylate

cyclase activity. Serotonin has not been found to stimulate

motility in A. suum.

Serotonin has been reported to act as a hormone that

regulates carbohydrate metabolism in the muscle of A. suum

by producing a dose-dependent increase in cAMP levels and a

concomitant activation of glycogen phosphorylase and

subsequent inactivation of glycogen synthase (Donahue et

al., 1981). Abrahams et al. (1976) showed that in intact

F. hepatica the adenylate cyclase is activated by the

addition of serotonin to the incubation medium resulting in a

marked increase in the concentration of cAMP levels. Such an

effect may apply to other parasitic helminths too.

Schistosoma mansoni has been reported to contain adenylate

cyclase and that the enzyme is activated by serotonin

(Higashi et al., 1973) . Serotonin activates the cAMP-

23

dependent protein kinase and phosphofructokinase in addition

to activating adenylate cyclase in a wide variety of

parasitic helminths (Mansour, 1962; Higashi et al. , 1973;

Gentleman et al., 1976).

These data implicate serotonin as one of the most

effective indoleamine involved in increasing the

concentration of endogenous cAMP and thus acting as a major

metabolic regulator in parasitic helminths. In addition,

there are other potential regulators of metabolism which

include acetylcholine, garnma-amino butyric acid (GABA) ,

epinephrine, histamine and octopamine. These regulators

produced neither a significant change in activity of glycogen

synthethase or glycogen phosphorylase in A. suum. Serotonin

appears to be the only potential activator (Donahue et al.,

1981a). This is in accordance with the hypothesis that

glycogen metabolism in A. suum is regulated by a cAMP

initiated cascade of enzyme interconversions, which results

in the coordinated activation and inactivation of the

component enzymes in response to the signal triggered by

serotonin (Mansour, 1962).

Possible Origin of Serotonin

Despite its importance as a physiological regulator, the

origin of 5-HT in A. suum or any helminth had remained

24

uncertain for some time. Recently, in a series of reports,

the source of serotonin in these organisms has been

investigated. There appears to be three possible sources for

the serotonin found in A. suum. First, the host could be

secreting the 5-HT which is then absorbed by the parasite

(Mettrick et al., 1981). The enteroendocrine cells

(enterochromaffin cells) of the host's small intestine are

the presumed source of intralumenal 5-HT (Pentilla, 1966) .

It has been shown that the secretion of 5-HT from

enterochromaffin cells into the lumen is directly related to

intralumenal pressure (Toh, 1964; Bulbring et al., 1958) and

this release is probably regulated by either cholinergic,

adrenergic, and/or peptinergic neurons. Adrenergic

stimulation of enterochromaffin cells, releases 5-HT into the

circulation while cholinergic stimulation is responsible for

lumenal secretion during peristalsis,(Ahlman et al., 1976;

Gronstad et al., 1985).

Following a meal the intralumenal levels of 5-HT

increases by at least 3-fold over that observed during non-

feeding periods (Ferrara et al., 1987). In order to utilize

this host secreted 5-HT, the parasite must possess a

serotonin uptake and/or transport mechanism in the tissues

exposed to the 5-HT pool of the host. This mechanism of

serotonin uptake has been found in Schistosomes (Bennett and

Bueding, 1973). Mettrick et al., (1981) have reported the

25

presence of tegumental sites for the absorption of exogenous

(host) serotonin in H. diminuta. Schistosomes have been

reported to possess two types of serotonin uptake mechanisms

(Wood and Mansour, 1986). A passive component that

predominates at higher concentrations (5 to 100 yM) of 5-HT

is associated with the tegument and the main body of the

Schistosomes. A saturable component sensitive to ouabain as

well as imipramine (serotonin uptake inhibitor) is apparent

at low concentrations (0.05 to 5.0 yM) of 5-HT and is only

present in the main body.

Second, experiments have indicated that the cuticle of A.

suum is permeable to the aromatic amino acid L-tryptophan

(TRP), which the parasite can absorb from the lumen of the

host and use in the de novo synthesis of serotonin

(Chaudhuri et al., 1988). Intact liver flukes, F.

hepatica, were capable of synthesizing 5-HT from 5-

hydroxytryptophan (5-HTP) but not from TRP (Mansour et al.,

1970). Recently, the enzyme tryptophan hydroxylase (TOH)

which synthesizes 5-HTP from TRP was identified in helminths.

The decarboxylase in both adult and larval S. mansoni has

been characterized by Catto (1981). The enzyme is similar to

the mammalian aromatic L-amino decarboxylase (AADC). It has

a Km of 5.1 xl0~5 M, a Vmax of 1.1 nmol/30 min/mg protein and

is highly active (447 pmol of 5-HT formed/30 min/mg protein)

at pH 7.9 (Catto, 1981). The fundamental question of how

26

these parasitic helminths get 5-HT needs to be resolved.

Ribeiro and Webb's (1983) investigations also revealed that a

continous supply of TRP is necessary to maintain the

endogenous stores of 5-HT and 5-HTP in the parasitic cestode,

H. diminuta in its natural environment.

Third, the 5-HT could be synthesized by the bacterial

flora in the intestine of the parasite and transported across

the membrane. The presence of aromatic decarboxylase in

bacteria have been demonstrated in a number of different

microbial strains (Gale et al. , 1944; Guirard et al.,

1964). Bacterial strains belonging to the Micrococcaceae

showed especially high decarboxylase activity toward L-

tryptophan and 5-hydroxy-L-tryptophan (Nakazawa et al. ,

1977). It was suggested that 5-OH-tryptophan decarboxylase

is widely distributed among anaerobic bacteria. Hsu et al.

(1986) using conventional fluorimetric methods showed that

the intestinal tract had the highest concentration of

serotonin (108 ng/g) while the lowest concentration (12 ng/g)

was found in the reproductive tract. Using more sensitive

radioisotopic and chromatographic procedure Martin et al.

(1988) demonstrated the serotonin turnover in the intestine

of A. suum was 34.7 ng/mg protein/hr and concluded that the

level of serotonin in the intestine is higher than in other

tissues. These data do not discount the possibility of

serotonin uptake by the intestinal tissue from 5-HT producing

27

bacteria in the gut of the worms.

New analytical techniques, namely high performance liquid

chromatography (HPLC) with electro-chemical detection (EC)

(potentiostatic voltammetry) promises more refined approaches

to long standing areas of interest in the field of serotonin

metabolism. These methods allow rapid and simultaneous

analysis of 5-HT in a single sample of tissue without the

necessity of preparing derivatives or purification.

Deproteination is all that is required. The use of HPLC-EC

in this investigation should permit more conclusive studies

of regional 5-HT metabolism under different physiological

conditions.

Biosythesis of Serotonin

Serotonin is synthesized from the aromatic amino acid L-

tryptophan (TRP) in a two step reaction. Tryptophan, the

dietary amino acid is first converted to 5-HTP by TOH. The

5-HTP is then decarboxylated to 5-HT by AADC to

5-hydroxytryptamine (5-HT). The initial hydroxylation

involves two substrates, TRP and molecular oxygen, as well as

reduced pterine cofactor. Assay for TOH, the rate limiting

synthetic enzyme in mammals (Jequier et al., 1967),

indicated the enzyme's presence in A. suum. Like the

mammalian enzyme, TOH from Ascaris required

28

tetrahydropterine, was inactivated in the presence of excess

oxygen and could be reactived by Fe+2 and dithiothreitol

under anaerobic conditions (Chaudhuri et al., 1988).

Enzymatic activity was eight-fold higher in intestine than in

muscle tissue and the Km for the intestinal enzyme was 21.5

loM. The enzyme was partially inhibited by

parachlorophenylalanine (PCPA) an inhibitor of the mammalian

enzyme (Koe and Weissman 1966), and a Ki of 1.72 mM was

reported (Chaudhuri et al., 1988).

The second enzyme of the biosynthetic pathway, AADC, is a

pyridoxal-51-phosphate dependent enzyme which .converts 5-HTP

to 5-HT in serotonergic neurons, while in catecholaminergic

neurons it converts L-dopa to dopamine (Sourks, 1977). This

enzyme was also present in A. suum. The affinity of AADC

for 5-HTP is at least a hundred-fold greater than that for

TRP, and subsequently 5-HTP is preferentially decarboxylated

in serotonergic neurons tlchiayma et al., 1970). The

decarboxylase activity is present in brain extracts of rat in

far greater amounts than tryptophan hydroxylase (TOH)

(Ichiayma et al., 1968), for this reason the hydroxylation

of TRP rather than the decarboxylation of 5-HTP is presumed

to be the rate limiting step in 5-HT formation. In fact the

trace amount of 5-HTP present in brain tissues (Tappaz and

Pujol, 1980) indicates that it is decarboxylated almost as

rapidly as it is formed and also suggests that the

29

hydroxylation reaction is the rate limiting step in the 5-HT

biosynthesis.

Serotonin is catabolized by the enzyme monoamine oxidase

(MAO). Different isoenzymes of MAO has been in the mammalian

brain which differ in substrate and inhibitor specificity

(knoll and Magyark 1972). The enzyme is found in the outer

mitochondrial membranes of liver and brain cells in mammals,

(Tipton, 1967; Schraitman et al., 1967). There are reports

of presence of this enzyme in the tissues of Ascaris

lumbricoides and Ascaris galli (Mishra et al., 1978; 1985).

Mishra et al., (1985) demonstrated the presence of MAO in

the tissues of A. suum. When isolated intestine, muscle or

live parasites were perfused with TRP and 5-HT, a dose

dependent, saturable increase in the MAO products

5-hydroxyindoleacetic acid (5-HIAA) and secondarily

5-hydroxytryptophol (5-HTOL) were demonstrated (Chaudhuri et

al., 1988; Tyce, 1980). Inhibition of MAO by pargyline, in

vertebrates (Knoll and Magyark, 1972) demonstrated that a

decrease in the levels of 5-HIAA and 5-HTOL while

simultaneously increasing the level of 5-HT in muscle and

intestinal tissue of A. suum (Chaudhuri et al., 1988a;

Martin et al., 1988). Martin et al. (1988) quantitated

5-HT turnover in A. suum tissues and concluded that the

intestine was the primary tissue responsible for 5-HT

regulation since TOH activity was highest in intestinal

30

tissue.

These investigations suggest the presence of enzymes

necessary for metabolism of 5-HT in A. suum intestine and

muscle tissue. However, since the enzyme activity for the

synthesis of 5-HT is higher in the intestinal tissue it,

indicates that this tissue is the major site for absorption

or synthesis of 5-HT. The 5-HT in the parasite may originate

from either the host or the parasite itself. Since there are

also 5-HT producing bacteria found in the lumen of this

parasite it may be assumed that the bacterial flora present

in the intestine of the worm may be a source of this

important metabolic regulator.

Serotonin Uptake

One of the critical parameters in understanding the

biology of 5-HT is the uptake mechanism. Initial

investigations have reported the presence of a TRP/5-HT

transport system in A. suum intestinal tissue. Further

characterization of binding sites in A.suum muscle membrane

preparations have shown a 5-HT binding protein, possibly a

receptor (Chaudhuri et al., 1989).

Serotonin binding site was found in A. suum muscle and

intestine tissue, using [3H] lysergic acid diethylamide (LSD)

as ligand. It was concluded that the binding was three times

31

as abundant (per mg protein) in muscle than in intestine. It

has been shown that [3H] LSD binds with equal affinity to

5-HT^ and 5-HT2 receptors in the cell membranes prepared from

mammalian tissues (Peroutka, 1988). The Kd for LSD of the

receptor was 2.7 nM in intestine and 1.8 nM in muscle.

Binding of [3H] LSD in the presence of selective mammalian

5-HTj, 5-HT2 and 5-HT3 receptor agonists and antagonists

suggested that the binding site found in muscle was different

from receptor found in mammalian systems (Williams et al.,

1991). It was therefore proposed that the 5-HT receptor of

A. suum muscle be called 5-HTN. These data correlates well

with the hypothesis that 5-HT binding of a membrane receptor

is responsible for activation of glycogenolysis in the

muscle.

Investigations on serotonin transport in intestine and

muscle membrane preparations were performed using [3H] 5-HT

and cold imipramine, a specific 5-HT uptake inhibitor, in

platelets and vertebrate neurons (Langer et al., 1980;

Peroutka, 1988) or the 5-HT depletors reserpine and DL-p-

chloramphetamine (Pletcher et al., 1955; Brodie et al.,

1957). The results from these experiments indicated TRP and

5-HT may be absorbed by the same mechanism.

These initial investigations suggest that serotonin or

its amino acid precursors may be absorbed at the lumenal

surface of the intestine if they are present. During non-

32

feeding periods of the host, the worms are able of

synthesizing and/or storing 5-HT in the form of amorphous

patches (Martine et al., 1988).

Specific Aims

Initial investigations have demonstrated that A. suum

intestinal epithelial tissue is the major site of 5-HT

storage. Furthermore, 5-HT producing bacteria have been

found among the A. suum intestinal flora. The objective of

this investigation is to disclose the functional role of

these 5-HT producing bacteria in this tissue. The results

from this study will provide some understanding of the

regulatory factors involved in serotonin utilization in the

parasite A. suum. The specific aspects of this

investigation are as follows:

1.- Quantitative determination of serotonin level in the

whole and segmented intestinal tissues of A. suum in the

presence and absence of the microflora using high performance

liquid chromatography with electrochemical detection (HPLC-

EC) techniques.

2.- Quantitative determination of 5-HT levels produced by

the microflora in the intestine of A. suum over a period of

time using (HPLC-EC) techniques.

3.- Serotonin turnover studies of the whole and segmented

33

intestinal tissue in the presence and absence of intestinal

microflora.

4.- Serotonin binding studies of the intestine in the

presence and absence of intestinal microflora.

5.- Competition binding studies of Serotonin and

tryptophan in the presence and absence of intestinal

microflora.

CHAPTER TWO

MATERIALS AND METHODS

Experimental System

A. Animals:

Ascaris suum were collected at a local slaughterhouse and

transported to the laboratory in a salt solution (Harpur,

1963). Adult female parasites that were 2 5-3 0 cm in length

were used in these experiments. The worms were maintained in

a holding system (Donahue et al., 1981) that contained A.

suum saline (Harpur, 1963) modified to contain 5 mM MgCl2,

14 mM NH4C1, 111 mM NaCl, 10 mM N^HC03 , 24 mM KC1, 1 mM CaCl2

and 0.5 mM KH2P04, pH 7.0. The A. suum salt solution was

maintained at 37° C and saturated with 95 % N2 - 5 % C02.

B. Maintenance:

The worms were transferred to the laboratory and

maintained in the laboratory in two groups of antibiotic

treated and non-antibiotic treated worms for periods of 0,

24, 48, and 96 hrs. The antibiotic treated groups were kept

in A. suum saline solution containing the antibiotics

34

35

penicillin G (10,000 units/ml), streptomycin (1.5 mg/ml), and

tetracycline (0.5 mg/ml) (Bueding and Yale, 1951). The

antibiotic saline solutions were replaced every 24 hrs.

C. Tissue Preparation For Bacterial Culturing:

Representative worms (5-6) were randomly obtained from

the containers. The parasites were removed from the holding

container and dissected in prewarmed modified A. suum

saline. The intestines were isolated aseptically under both

aerobic and anaerobic environments (using an anaerobic

chamber). The intestine of each worm was cut into three

sections, anterior (the region anterior to the genital pore

of the female parasite), midgut (the region posterior to the

genital pore and 8-10 cm anterior to the anal opening) and

posterior (the hindmost 8-10 cm region). A sample from each

section was randomly selected and weighed for bacterial

culturing and the remaining samples were rapidly frozen in

liquid nitrogen. Each section was removed aseptically and

placed in a 5-ml sterile pre-weighed phosphate buffer saline

solution kept under an anaerobic environment. The weighed

tissue was transferred to a sterile glass homogenizer and

thoroughly homogenized (10 strokes). Bacteria were further

quantitated by triplicate plates and colony counts in brain

heart infusion (BHI) agar (Difco) and in anaerobic agar

(Difco). All plates were incubated at 37° C in anaerobic and

36

aerobic incubators and the colonies were counted 48 hrs

later. Serotonin levels of the samples were quantitated by

the HPLC-EC. All the frozen tissues were stored at -80° C

until used.

Serotonin Turnover Study

A. Tissue Perfusion and Extraction:

To determine the serotonin levels and 5-HT turnover rate,

the whole and/or the segments of isolated intestinal tissue

from adult female A.suum were perfused with 10 pM TRP or 10

yM TRP and 1 mM pargyline (PARG) for 3 min and the levels of

5-HT and TRP measured using HPLC-EC. The perfusion media,

maintained at 37°C, contained 95 % N2-5 % C02 saturated A.

suum saline. The tissue was homogenized and sonicated with

25 volumes of ice cold 1.0 M formic acid and acetone (15:85,

v/v) using a Brinkman polytron for 1 min (Kilts et

al.,1981). The homogenate was centrifuged at 1000 X g for

10 min at 4° C and the supernatant fluid extracted in a

separatory funnel with 3 volumes of heptane and chloroform

(8:1, v/v). The organic layer was discarded and the aqueous

layer was dried under nitrogen and lyophilized overnight.

The resultant material was then dissolved in 200-500 pi of

deionized water. The 5-HT and TRP were separated and

quantitated with HPLC-EC.

37

B. Whole Live Worm Perfusion:

To determine the serotonin levels and 5-HT turnover rate

of the intestinal tissue and intestinal microflora, live

adult female A. suum were perfused with 10 iaM TRP or 10 ioM

TRP and 1 mM pargyline (PARG) for 30 min and the levels of 5-

HT and TRP measured using HPLC-EC. The perfusion media,

maintained at 37°C, contained 95 % N2-5 % C02 saturated A.

suum saline. The worms were washed in A. suum saline three

times and cut open. The intestinal tissue was removed and

dissected. The intestinal contents were washed with 3 ml of

A. suum saline. The tissue and the intestinal contents were

divided into two halves. One half was dried and then

weighed; and the other half was processed for measuring the

5-HT and TRP. The intestinal contents containing microflora

were homogenized in a Potter Elvehjem homogenizer. The

intestinal contents were lyophilized overnight and the

resultant material was dissolved in 100 yl of water and

centrifuged at 10,000 rpm and the supernatant was used. The

intestinal- tissue was extracted as in part A. The 5-HT and

TRP were separated and quantitated with HPLC-EC.

C. Chromatography:

(i). Apparatus and Separation:

To measure the levels of serotonin in the isolated

intestinal tissues of A. suum and its production by bacteria

38

an Apple lie controlled Gilson HPLC system was used. The

system consisted of a solvent delivery pump (Model 302) in

conjunction with a manometric module (Model 802 B), a

controller (Model 502) and a gradient manager (Model 702),

all from Gilson International. The EC detection was

performed using a TL-5 glassy carbon electrode and an LC-4B

amperometric controller, both from Bioanalytical Systems

(West Lafayette, IN). The detector potential was maintained

at an oxidation potential of 0.75 V vs a Ag/AgCl reference

electrode. Chromatographic separations were performed using

a 25 cm x 4.6 mm internal diameter stainless steel column

packed with octadecylsilane (C18) on microparticulate (5 pm)

silica gel (Adsorbosphere ODS, HS reversed-phase, Alltech

Associates, Houston, TX). The mobile phase was a mixture of

0.1 M citrate, 0.075 M Na2HP04, 0.75 mM sodium octyl sulfate

and 14% methanol (v/v), pH 3.9. All separations were

performed isocratically at a flow rate of 1 ml/min,

KPSI=2.10, detector sensitivity was 5 nA/V (Bennett and

Snyder, 1976).

(ii). Standards:

Three sets of controls were run to minimize the error in

the quantitation of 5-HT by HPLC-EC voltammetry. One set

included a standard mixture of L-tryptophan (TRP) and

5-hydroxytryptamine (5-HT) containing 5.0 ng of each of the

39

metabolites. A second set of controls included the same

standard mixture, but was run through the extraction

procedures. The third set of controls included the standard

mixture and the tissues which were extracted together. All

three sets of controls were then assayed for 5-HT by HPLC-EC

and quantitated to evaluate the percentage loss which

occurred during the experimental procedure.

(iii). Statistics:

Data obtained were subjected to applicable statistical

procedures. Analysis were performed by using Anova test of

Statview Software (Macintosh) at appropriately selected

levels of significance (95 % confidence level).

Serotonin Binding Sites in A. suum Intestinal Tissue

A. Tissue Preparation for Binding Assay:

Tissue was prepared for the [3H] LSD binding assay using

the method of Peroutka and Snyder (1979) . Individual frozen

tissues were weighed and homogenized in 20 vols (v/g wet

weight) of iced buffer containing 40 mM Tris-Cl, pH 7.4, and

320 mM sucrose. The tissue was then homogenized in a

Brinkman polytron for 30 sec. The homogenate was then

centrifuged at 10,000 X g for 10 min. The supernatant was

removed and the pellet resuspended in an equivalent volume of

40

ice-cold distilled water and the centrifugation was repeated.

The pellet from the second centrifugation was rehomogenized

in an equivalent volume of buffer containing 40 mM Tris-Cl,

pH 7.4, and the centrifugation was repeated. The pellet

from this final centrifugation was resuspended in 5 vols 40

mM Tris-Cl, pH 7.4, and stored at -80° C prior to use. The

tissue was removed from the freezer and preincubated at 37° C

for 10 min in the storage buffer. The protein content in the

tissue was quantitated by the dye binding method of Bradford

(Bradford, 197 6), and the protein standard used was bovine

serum albumin.

B. Ligand-Receptor Binding Assay:

The binding of [3H]LSD to intestinal membranes of

intestine was assayed by the method of Bennett and Snyder

(1976) as modified by Peroutka and Snyder (1979) . Aliquots

of membrane suspensions from intestine were incubated with

1.0 nM to 5.0 nM of [3H]LSD (specific activity 62.9 Ci/mmol)

plus or minus a 1000-fold excess of mianserin as competitor

for 10 min at 37° C in a buffer containing 40 mM Tris-Cl, pH

7.4, 4.0 mM CaCl2, 5.7 mM ascorbic acid, and 10 viM pargyline-

C1 (total volume 300 ul). All samples were assayed in

triplicate. Membranes were collected by vacuum filtration

using Gelman GF/B filters with three rinses of ice cold 40 mM

Tris-Cl, pH 7.4 as described by Peroutka and Snyder (1979).

41

Radioactivity was determined in a LS 3801 Beckman Liquid

Scintillation Counter with 10 ml Universal cocktail (ICN

Radiochemicals). Specific binding was defined as that

binding that was displaced by a 1000-fold excess of non-

radioactive mianserin, as described by Peroutka and Snyder

(1979) . The non-specific binding ranged from 30 to 40% of

total binding for the intestinal membranes. Specific binding

was determined by subtracting cpm in the presence of

competitor (nonspecific binding) from cpm in the absence of

competitor (total binding). The equilibrium dissociation

constant (K^) and maximal number of binding sites (Bmax) were

estimated by Hofstee (1952) analysis.

C. Competition Binding Studies:

(i) [3H]5-HT binding and competition with unlabeled TRP:

Isolated intestinal tissues at different time intervals

(0 hr and 24 hrs plus and minus antibiotics) were perfused

with a fixed concentration (10-6 M, since the Kd = 1-2 yM) of

[3H] 5-HT (23.4 Ci. mmol _1) and various concentrations of

unlabeled TRP for 10 min at 37° C in A. suum saline. All

samples were assayed in triplicate. Individual samples were

washed three times in A. suum saline. Radioactivity was

determined in an LS 3801 Beckman Liquid Scintillation Counter

with 10 ml Universal cocktail (ICN Radiochemicals). The data

were plotted as a function of TRP concentration. The Cricket

42

Graphics Software Program (Great Valley Corporate Center) was

used for the graphical analysis of the binding data.

(ii) [3H]TRP binding and competition with unlabeled 5-HT:

Isolated intestinal tissues at different time intervals

(0 hr and 24 hrs plus and minus antibiotics) were perfused

with a fixed concentration (1CT7 M) of [3H] TRP (6 Ci. mmol

1) and various concentrations of unlabeled 5-HT for 10 min at

37° C in A. suum saline. All samples were assayed in

triplicates. Individual samples were washed three times in

A. suum saline. The data were plotted as a function of 5-

HT concentration. Radioactivity was determined in an LS 3801

Beckman Liquid Scintillation Counter with 10 ml Universal

cocktail (ICN Radiochemicals). The Cricket Graphics Software

Program was used for the graphical analysis of the binding

data.

Materials:

Sodium octylsulfate was obtained from Pflatz and Bauer

(Waterbury, CT); [2-3H]LSD (62.9 ci/mmol), [G-3H]5-HT (23.4

ci/mmol), [G-3H]TRP (6 ci/mmol) were obtained from Amersham

corporation (Arlington Heights, IL); mianserin hydrochloride,

was obtained from Research Biochemicals Inc., MA.

Unless otherwise specified, all reagents and supplies

43

were purchased from Sigma Chemical Company, St.Louis, MO or

Fisher Scientific Company, Pittsburgh, PA. All the chemicals

were of analytical grade and the HPLC solvents were of HPLC

grade. Protein concentrations were determined by the method

of Bradford (1976) using BioRad protein assay reagent.

CHAPTER THREE

RESULTS

Effects of Antibiotics on Intestinal Bacterial Flora in

Ascaris suum

To illustrate the direct effect of the antibiotics on the

intestinal bacterial flora, the worms were incubated for up

to 96 hrs in A. suum saline plus and minus antibiotics as

described in Methods. After 24 hrs of incubation in A. suum

saline plus and minus antibiotics, there was a significant

(95 %) decline in the number of bacteria in both anaerobic

and aerobic culturing. Since a large proportion of

intestinal bacteria are facultative and obligate anaerobes,

it is very possible that due to the physiological changes

that the worms go through from the time they are removed from

the porcine host and put in A. suum saline to time they are

used, a significant number of these bacteria have died off.

Table 1 demonstrates the bacterial populations of the

intestine when the worms were incubated in A. suum plus and

minus antibiotics for up to 96 hrs. The values were

expressed as Colony Forming Units (CFU)/g, wet weight of

intestinal tissue, and were the mean of three individual

44

45

experiments. The anterior section of the intestine was free

of bacteria when the worms were incubated in A. suum saline

with antibiotics, but the midgut and posterior sections

contained reduced number of bacteria after 24 hrs. The

anterior section stayed bacteria-free while the other two

sections showed the presence of bacteria after 48 hrs

incubation in A. suum saline plus and minus antibiotics.

The complete eradication of the bacterial flora took 96 hrs

for the whole intestine (Table 1). It was imperative that

the worms be placed in fresh antibiotic media each 24 hr

period or the bacteria were never eliminated. Since there

was a significant decrease in the intestinal bacterial

population after 24 hrs incubation in A. suum saline plus

and minus antibiotics the following investigation focused on

the physiological events that took place at this time period.

Tissue Perfusion Experiments

Serotonin is widely distributed throughout the

invertebrate phyla and functions as an important metabolic

regulator. Its occurrence in helminths is well established

but its origin is under investigation. Quantitative

measurement of 5-HT in the intestine was a preliminary step

of this investigation.

46

1. Quantitation of Serotonin and Tryptophan Levels:

A reverse-phase HPLC with an electrochemical detector was

used to separate and quantitate 5-HT and TRP from A. suum

intestinal tissue. An oxidation potential of 0.75 V was used

which was adjusted optimally for all metabolites. The

voltammograms (Fig. 1) were used to establish the

concentration of the metabolites in the samples. Isolated

intestinal tissue was perfused with different concentrations

of TRP and 5-HT (1 x 10"9 M to 1 x 10~4 M) as substrates in

order to determine whether endogenous levels of 5-HT could be

increased. Only perfusion with 1 x 10 ~4 M TRP caused an

increase in 5-HT levels. After 3 min perfusion, the 5-HT

levels increased to 135 +0.5 ng/g (Fig. 2 ).

When the intestine was perfused for 3 min with increasing

concentrations of TRP (1 x 10~4 M to 1 x 10~9 M) there was a

dose-dependent increase in 5-HT concentration up to a TRP

concentration of 1 x 10~4 M (Fig. 2). Above this

concentration of perfused TRP, the concentration of 5-HT

remained constant. When increasing concentrations of 5-HT

were perfused through isolated intestine for 3 min there was

a dose-dependent increase in 5-HT level (Fig. 3).

2. Serotonin Turnover in the Whole Intestinal Tissue:

Previous work (Martin et al., 1988) in our laboratory had

shown that all the metabolites of 5-HT were found in

47

intestinal tissue when perfused with TRP. It was concluded

that intestinal tissue contains the enzymes tryptophan

hydroxylase (TOH) and monoamine oxidase (MAO) needed for the

synthesis and degradation of 5-HT. The apparent 5-HT

turnover in intestine of A. suum was determined by

pharmacologically inhibiting MAO with pargyline. The

intestinal tissues were perfused for 3 min with 10 pM

tryptophan (TRP) alone or tryptophan with 1 mM pargyline

(PARG) to determine serotonin levels and turnover rates in

antibiotic treated and control tissues. Table 2 demonstrated

that there was 18-20 % more 5-HT when intestinal tissue

perfused with TRP plus PARG than tissue perfused with TRP

alone in the 0 hr group. This significant increase (p <

0.05) in 5-HT level following TRP plus PARG (160 +. 1.2 ng/g

tissue wet weight) relative to saline perfusion (91 ± 1.0

ng/g) demonstrated that MAO was indeed inhibited. This

increase in 5-HT levels was also observed following

incubation in the A. suum saline plus and minus antibiotics

at all time points. However, after 48 hrs of incubation in

A. suum saline plus and minus antibiotics the animals'

activity began to decline and by 96 hr the animals appeared

to be on the verge of death.

In TRP perfused, antibiotic treated animals, serotonin

levels were significantly decreased at 24 hr (p < 0.05).

However, serotonin levels were significantly increased

48

relative to non-antibiotic treated animals at 48 hr (p <

0.05) and at 96 hr (p < 0.05).

The TRP plus PARG data were used to calculate the 5-HT

turnover rate of the intestinal tissue of A. suum. Neckers1

(1982) method was used to calculate 5-HT turnover. Serotonin

turnover rate was increased both plus or minus antibiotics at

24 hrs and 48 hrs. Antibiotic treatment had no effect on 5-

HT turnover at the 24 hrs time point (p > 0.05). At both 48

hr and 96 hr time points 5-HT turnover was significantly

greater in the antibiotic treated animals (p < 0.05)

respectively.

3. Serotonin Turnover in the Intestinal Tissue Segments:

Since after 24 hrs of incubation in A. suum saline plus

and minus antibiotics showed a significant reduction in the

bacterial population, this time point was used for further

investigations to evaluate the 5-HT and TRP levels of

intestinal segments. Intestinal segments (anterior, midgut

and posterior) were perfused with TRP and TRP with PARG as

explained in the Methods. Results indicated that 5-HT and

TRP levels significantly decreased in all segments after 24

hrs incubation in A. suum saline plus and minus antibiotics

when only perfused in A. suum saline (Table 3) (P < 0.05) .

The 0 hr tissue showed 19-23 % increase of 5-HT levels when

perfused with TRP with PARG compared to TRP alone. The 5-HT

49

levels were significantly increased (80 - 140 %) after the

tissues had been incubated in A. suum saline plus and minus

antibiotics for 24 hrs. Anterior segments of all control and

experimental tissues showed significant differences in TRP or

5-HT levels when compared to their posterior intestinal

segments (p < 0.05).

The turnover study demonstrated that the there was a

significant (p < 0.001) increase in all segments of the

intestinal tissue after 24 hrs incubation in A. suum saline

plus or minus antibiotics (Table 4). The anterior segment of

the 24 hr antibiotic treated tissue showed the highest

turnover rate (145.9 ± 2.0 ng/mg protein/hr). Relative to

minus antibiotic animals, 5-HT turnover rates were higher in

the anterior segment and lower in the midgut of the

antibiotic treated animals (p < 0.05) No difference was

observed in the posterior segments.

4. Intestinal Microflora vs Tissue:

The following experiments with adult female A. suum

incubated in the A. suum saline plus 10 iaM TRP and/or TRP

with 1 mM PARG for 30 min. Since it takes approximately 30

min for materials to reach the posterior end of the worms

these experiments were designed to investigate whether the

available TRP could be synthesized into 5-HT by the

intestinal microflora. The intestinal microflora is mainly

50

the contents of the whole intestinal tract.

The intestinal tissue and the intestinal microflora of

the whole worms incubated in A. suum saline for 24 hrs plus

or minus antibiotics showed a 45 - 50 % drop in their TRP

levels when compared to the 0 hr groups (Table 5). There was

a 40 % increase in TRP level by the intestinal microflora and

100 % increase by the tissue when the whole worms were

incubated with TRP alone for 30 min and 100 to 400 % increase

respectively when incubated with TRP and PARG for 30 min.

A. suum incubation for 24 hrs, whether plus or minus

antibiotics, decreased the TRP levels of both the intestinal

tissue and microflora when perfused with TRP alone or TRP

with PARG (p = 0.005).

The intestinal microflora of the whole worms at 0 hr

showed 33 % higher 5-HT level than the intestinal tissue when

incubated in A. suum saline for 30 min (Table 6). The 5- HT

levels of both the intestinal tissue and the intestinal

microflora of the whole worms incubated in A. suum saline

for 24 hrs plus or minus antibiotics showed a decrease of

45 - 67 % when perfused with saline only (Table 6).

Following 24 hr incubation, 5-HT levels were higher in

intestinal tissue than in the microflora with both TRP and

TRP plus PARG (p = 0.0001). Antibiotic incubation resulted

in a significant (p < 0.05) decrease in 5-HT synthesis of the

microflora while it was increased in the tissue

51

(p < 0.005) .

In order to determine the 5-HT turnover in the intestinal

microflora and the intestinal tissue, the whole worms were

incubated with TRP alone and TRP with PARG for 30 min. At

the 0 hr time point the intestinal microflora had a three

fold higher turnover rate (10.2 +. 2.4 ng/mg protein/hr) than

the intestinal tissue (3.6 ± 0.4 ng/mg protein/hr). After 24

hrs incubation in A. suum saline plus or minus antibiotics

the intestinal microflora showed a decrease (6.6 + 2.9 and

6.9 + 2.0 ng/mg protein/hr respectively) in its turnover rate

and the intestinal tissue showed an increase in turnover rate

(12.1 + 0.7 and 15.5 + 1.5 ng/mg protein/hr) when compared to

the 0 hr control (Table 6).

Binding Sites and Uptake of Serotonin

To investigate the effects of bacterial flora on the

properties of serotonin binding sites in A. suum intestinal

tissue membranes, binding studies were performed using [3H]-

LSD after 24 hrs incubation in A. suum saline plus and minus

antibiotics. LSD has been shown to bind with equal affinity

to 5-HTj and 5-HT2 receptors in the membranes prepared from

mammalian nervous tissue (Peroutka, 1988) . A. suum

intestinal membranes were incubated with [3H]-LSD at

concentrations ranging from 1.0 nM to 5.0 nM. Figure 4

52

illustrates the specific binding of three sections (anterior,

midgut and posterior) of the 0 hr A. suum intestinal tissue.

Hofstee analysis of the specific binding data showed the

highest affinity (apparent Kd of 2.2 nM) to be in the

posterior intestinal section. Number of binding sites (Bmax)

was approximately 2.5 fold greater in the midgut and

posterior intestinal segment.

After 24 hrs incubation in A. suum saline minus

antibiotics the anterior and midgut intestinal segments

showed an increase in affinity (apparent Kd's of 2.0 and 1.0

nM respectively) when compared with the 0 hr time point.

There was a significant increase in the binding density of

the anterior segment (Bmax = 81 fmol/mg) while the midgut

showed a decrease in binding sites (Bmax = 64 fmol/mg)

(Fig. 5).

The apparent Kd's calculated from the binding studies done

with the anterior and posterior intestinal tissues from the

worms incubated in A. suum saline with antibiotics for 24

hrs indicated significantly lower affinity (p < 0.05) than

both control groups ( 0 and 24 hrs minus the antibiotics)

while the Bmax's were higher (Fig. 6) . However, the midgut

showed an increase in affinity (kd = 1.5 nM) and a decrease

in Bmax (61 fmol/mg) when compared to the anterior and

posterior segments. Although the [3H] LSD binding sites in

the anterior segments of 0 hr and 24 hrs without antibiotics

53

appeared to have higher affinities, the binding density

(Bmax) w a s greater in the anterior intestinal segment

incubated in antibiotics for 24 hrs. The affinity and number

of binding sites in the 0 hr intestinal tissue had anterior

to posterior direction. However, this directionality was not

present when the worms were incubated for 24 hrs in A. suum

saline plus and minus antibiotics.

Competition Studies

A membrane transport system that is specific for

serotonin has been described in a selective group of tissues

(Ross, 1982). A. suum intestinal perfusion studies showed

that both 5-HT and TRP are transported across the epithelial

membrane. In order to see whether 5-HT or TRP were binding

to the same binding sites in the intestinal tissue and

whether the reduction of bacterial population would have an

effect on this binding, a competition study was performed

using [3H]TRP and [3H]5-HT. Isolated intestinal tissues were

perfused with a fixed concentration of radioactive [3H]TRP

(0.1 \M) and increasing concentrations of unlabeled 5-HT.

The tissue was incubated for 10 min at 37° C and assayed for

radioactive [3H]TRP as described in Methods. The data were

plotted as a function of 5-HT concentration.

Even at 10~4 M, cold 5-HT did not significantly inhibit

54

binding of [3H]TRP (p > 0.05) to the 0 hr and 24 hr plus and

minus antibiotic intestinal tissues (Figs. 7, 8, 9). This

indicates that 5-HT is unable to compete for binding to

[3H]TRP binding sites. The intestinal tissues after 24 hrs

incubation in A. suum saline plus and minus antibiotics

showed a significant increase (25 - 50 %) in [3H]TRP binding

when there is no 5-HT competing (p = 0.005).

Noticeable differences in the percent inhibition of

[3H]5-HT binding was observed when intestinal tissues were

incubated with various concentrations of cold TRP. A

comparison of Figs. 10-12 reveals that in both the 0 hr

tissue and the 24 hr plus antibiotics tissue cold TRP was

able to compete for [3H]5-HT binding sites (50 % inhibition

at approximately 10-6 M TRP) . However, even at the highest

concentration (10~4 M) TRP was only able to compete for about

35 % of the [3H]5-HT binding sites in 24 hr minus antibiotics

tissue. Data from Figs. 7-12 suggest that a specific TRP

binding site and a binding site which interacts with both 5-

HT and TRP are present in A. suum intestine.

CHAPTER FOUR

DISCUSSION

Serotonin has been proposed as a putative hormone

activating glycogenolysis in Ascaris suum muscle tissue.

The origin of the serotonin which acts upon this tissue is

unclear. Is serotonin synthesized by A. suum intestinal

tissue or is it absorbed from the host or is it produced by

the intestinal bacterial flora? In the past, it has been

difficult to study the effects of 5-HT and its metabolites in

various tissues because the methods of measurement have been

difficult, unreliable, time consuming and tedious. The

method employed in this study was relatively fast, reliable

and extremely reproducible. Using an in situ perfusion

system and an electrochemical detector attached to the HPLC,

similar results for 5-HT to those observed using the more

conventional method of fluorescence were obtained. Hsu et

al. (1986) using a conventional spectrophotometer measured a

5-HT level of 107 ng/g wet weight in intestinal tissue from

adult female A. suum, as compared to HPLC values reported

here of 91 ng/g wet weight (Table 2). The HPLC-EC system,

however, allowed for the simultaneous measurement of several

5-HT metabolites. Mishra et al. (1981) using a

55

56

spectrofluorometer have reported 5-HT levels of Ascaris

lumbricoides of 2.1 yg/g for body wall and 1.9 yg/g for

intestine in adult females and 0.3 ug/g for body wall and 0.2

yg/g for intestine from Ascaridia galli. These levels are

higher than those observed here for A. suum. Since the

concentration of 5-HT found in the intestinal tissue was

higher than the muscle or the reproductive tract, it was

decided to investigate the role of intestinal flora as a

possible 5-HT source.

Previous investigations in this laboratory have

demonstrated the presence of intestinal bacterial flora and

measured the concentration of serotonin produced by them in

A. suum (Hsu et al., 1986) . It was suggested that the

bacterial isolates from the intestinal lumen of A. suum were

predominantly Gram-negative, of which many, e.g.,

Bacteroides spp., were able to produce a high concentration

of 5-HT. Moreover, the intestinal microflora were

predominantly facultative anaerobic organisms showing 5-OH-

tryptophan decarboxylase activity. The present study

provided a possible answer to one of the roles of intestinal

bacterial flora in these animals.

Previous reports in our laboratory have indicated that

TRP could be absorbed by the intestine of A. suum and

synthesized into 5-HT. It was also concluded that A. suum

intestinal tissue can absorb 5-HT directly from the

57

environment at a higher rate than the muscle tissue. The

complete elimination of A. suum 5-HT producing intestinal

bacteria resulted in a substantial (50%) drop in the

concentration of 5-HT (from 135 to 70 ng/g wet tissue) in the

intestinal tissue when perfused with TRP alone (Table 2).

The inhibition of monoamine oxidase by pargyline confirmed

the presence of this enzyme and indicated a significant

increase in the concentration of 5-HT when perfused with TRP

and PARG. The data were used to calculate the 5-HT turnover

in the intestine. The 5-HT turnover appeared highest in the

intestine of worms incubated in A. suum saline with

antibiotics for 48 hrs (301 ng 5-HT/mg protein/hr) as

compared to 5-HT turnover in intestine of worms incubated for

48 hrs in A. suum saline without antibiotics (195 ng 5-HT/mg

protein/hr).

When the 0 hr intestinal segments (anterior, midgut,

posterior) were incubated in TRP and then TRP with PARG there

was a slight increase (18 - 23 %) in 5-HT level in all

segments. The turnover rates of all three segments were not

statistically different from the turnover of the whole

intestine. After 24 hrs incubation in A. suum saline plus

and minus antibiotics the 5-HT turnover rate in all segments

showed over three fold increase. This significant increase

is possibly due to the elimination of a large number of 5-HT

producing intestinal microflora. Noticeably the anterior

58

segment of the intestine that was bacteria free after 24 hrs

incubation in A. suum saline with antibiotics showed the

highest 5-HT turnover (145.5 + 2.0 ng 5-HT/mg protein/hr).

There are several reports of the isolation of microbial

strains or microbial mixtures capable of carrying out the

decarboxylation of L-tryptophan. It has been shown that

several metabolites are formed from the degradation of L-

tryptophan by ruminal microorganisms (Yokoyama et al. ,

1974) . It has also been shown that all but 2 of the isolates

cultured from the intestinal microflora of A. suum secreted

detectable levels of serotonin ranging from 0.1 to 50 pg/lO9

cells (Hsu et al., 1986) . To study the 5-HT production of

the intestinal microflora the worms were incubated in 10 iaM

TRP and TRP with 1 mM PARG for 30 min. When the whole worms

were incubated in TRP, the TRP was absorbed by both

intestinal tissue and microflora (Table 5). After 24 hrs

incubation in A. suum saline plus and minus antibiotics the

TRP levels in both intestinal tissue and microflora

decreased. However, this reduction was statistically

significant for the intestinal microflora (p < 0.001).

The results summarized under Table 6 demonstrate that

intestinal microflora had higher 5-HT production than the

intestinal tissue when perfused with TRP alone. In addition

the 5-HT turnover rate in intestinal microflora at 0 hr is

over three fold higher than the intestinal tissue. The 5-HT

59

turnover rate significantly decreased in intestinal

microflora when the worms were incubated in A. suum saline

plus or minus antibiotics for 24 hrs. This significant

decrease in 5-HT turnover is possibly due to the elimination

of high number of intestinal bacterial flora. However, the

intestinal tissue showed an increase of more than 2 fold in

5-HT turnover rate which indicates that the elimination of 5-

HT producing bacteria in the intestinal microflora possibly

induced the intestinal tissue to increase 5-HT synthesis in

response to loss of bacterial 5-HT.

Evidence has been presented here demonstrates that

serotonin binding sites are present in the intestinal

membranes of Ascaris suum. These binding sites were highly

specific for [3H]-LSD. The [3H]-LSD binding sites in the

intestinal tissue of the 0 hr worms showed an increase in

binding affinity (Kd's =4.7, 3.6 and 2.2 nM respectively)

and number of binding sites (Bmax's = 33, 89.9 and 84

fmol/mg/protein) in an anterior to posterior direction (Fig.

4). After 24 hrs of incubation in A. suum saline plus

antibiotics, where more than 90 % of bacterial flora were

eliminated, the intestinal tissues showed a decrease in

binding affinity in the anterior and posterior regions (Kd's

of 5.8 and 9.9 nM) while the number of binding sites

increased (Bmax of 189 and 171 fmol/mg/protein) (Fig. 6) .

Collectively, these data indicate that the absence of

60

bacteria in the intestine of A. suum resulted in a

significant increase of 5-HT binding density with less

affinity.

A plasma membrane transport system that is specific for

serotonin has been described in a mammalian tissues (Ross,

1982). Initial investigations have indicated the presence of

an enteric TRP/5-HT transport system in A. suum and

pharmacological binding studies have demonstrated a 5-HT .

binding protein, possibly a receptor in muscle and intestinal

membrane preparations (Chaudhuri et al., 1989). Thus, to

determine whether TRP and 5-HT are competing with each other

for this transport system and/or if bacterial elimination

would have an effect on this system, a competition study was

performed. There was essentially no competition of 5-HT for

[3H]TRP binding sites among all three different tissue

samples (0 hr and 24 hrs plus or minus antibiotics). This

would indicate that the [3H]TRP binding site is specific for

TRP. However, it appears that TRP does compete for [3H] 5-HT

binding sites (Figs. 10 and 12). Incubation of intestinal

tissue with antibiotics for 24 hr appeared to facilitate the

ability of TRP to compete for [3H]5-HT binding sites.

The present study provides evidence that supports the

theory that the intestinal bacteria found in the parasitic

nematode Ascaris suum provide some of the serotonin

necessary for the regulation of glycogenolysis in this

61

parasite. Evidence is presented here that when there was a

significant reduction in the A. suum intestinal flora there

was also a significant reduction in the level of serotonin in

the intestinal tissue. The worms then need to reestablish

the normal levels of serotonin in the tissues. This may be

accomplished by absorbing serotonin from the host intestine

(Martin et al., 1988) or by the synthesis of serotonin by

the worms intestinal epithelial cells as measured by an

increase in the 5-HT turnover as seen here. The reduction in

bacterial number appeared to be one of the factors that

contributed to the changes in binding affinity and number of

binding sites. The data suggest that the bacteria play an

important role in the level of serotonin in the intestine of

this parasite.

Summary

In summary, from the data presented in this investigation

the following can be concluded:

1- Ascaris suum intestinal microflora are able to

synthesize 5-HT when perfused with TRP.

2-Elimination of intestinal microflora causes an increase

of 5-HT synthesis in intestinal tissue.

3-There is a directionality in [3H]-LSD binding to

intestinal tissue and the absence of intestinal microflora

62

alters this directionality.

4-There are two different binding sites for 5-HT, TRP and

in the presence of TRP, TRP would compete with 5-HT binding

site. In the absence of intestinal microflora the number of

binding sites for these ligands are significantly increased.

The data in this investigation provide support for the

theory that intestinal microflora is a source of serotonin

that functions as a hormone once absorbed by the A. suum

intestine.

63

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(0 <V i c > T5 LT) 2

66

TABLE 3 Tryptophan and. Serotonin Levels in Inteatinal

Segments of Ascstxs suum

Intestine Saline TRP * TRP+PARG*

^nt.ibiQ.

Ant. 48 • * ± 0.3 99 ± 0. 4 108 ± 1.7

TRP Mid. 43 ± 0.3 92 ± 4. 0 101 ±1.3

(ng/g) Pos . 40 ± 0.5 89 ± 0. 7 99 ± 0.6

Ant. 97 ± 0.9 136 ± 0. ,5 161 ± 0.2

5-HT Mid. 95 ± 0.9 136 ± 0. .8 159 ± 0.6

(ng/g) Pos. 86 ± 0.5 121 ± 0, .3 151 ± 0.3

24 n=_ -n.ir.U3 aim ibiot L.Z2. 24 n=_

Ant 25 ± 0.7 78 ± 0 .7 85 ± 4.0

TRP Mid. 24 ± i-o 68 ± 0 .9 71 ± 0.1

(ng/g) Pos. 21 ± 0.6 69 ± 2 .2 81 ± 0.4

Ant. 57 ±0.8 112 ± l .0 201 ± 0.7

5-HT Mid. 45 ± 0.3 95 ± 0 .3 193 ± 2.8

(ng/g) Pos. 48 ± 0.2 97 ± 1 .0 189 ± 2.7

hr „21US_ „anti Vnot.i Ant. 23 ± 1.2 83 ± 0.9 97 ± 1.0

TRP Mid. 20 ± 1.2 76 ± 2.6 84 ± 4.7

(ng/g) Pos. 19 ± 1.5 72 ± 1.7 77 ± 2.2

Ant. 31 ± 2.8 75 ± 3.5 175 ± I-7

5-HT Mid. 42 ± 2.3 88 ± 2.1 170 ± 1.6

(ng/g) Pos. 38 ± 2.0 65 ± 4.4 153 ± 1.3

Isolated intestinal segments were incubated with 10 (1M

tryptophan (TRP) ± 1 mM pargylin (PARG) for 3 min.

** The values were the mean of three experiments (± SD> and

standard deviation were calculated.

67

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70

APPENDIX B

FIGURES

71

Fig. l.HPLC Voltammogram from intestinal tissues of

Ascaris suum: The peaks of standard metabolites run on

HPLC-EC were: 1 = void volume; 2 = 5-HTP; 3 = 5-HIAA; 4 - 5 -

HTOL; 5 = TRP and 6 = 5-HT. Detector sensitivity (using a 1-

V recorder) was 5 nA/V. Chromatography procedure was as

described in Materials and Methods. When isolated intestinal

tissue was incubated with [3H]TRP for 10 min, the tissue

extracted, and the metabolites of 5-HT separated using HPLC,

the radioactivity was found to comigrate with TRP, 5-HTP and

5-HT.

72

FIGURE

rt| C

S W «

D a

5 7 9

MINUTES

73

Fig. 2.Dose-response curve: Isolated intestinal tissue

of A. suum was perfused by varying concentrations of TRP for

3 min. The intestine was frozen and extracted as described

in the Materials and Methods. The level of 5-HT was analysed

by HPLC-EC. Each point represents the mean + SD; n = 3 or

more.

CM

74

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00

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O \o

o o CM

(snnsTji q.3M £>/&u) 1 H - S

75

Fig. 3.Dose-response curve: Isolated intestinal tissue

of A. suum was perfused by varying concentrations of 5-HT

for 3 min. The intestine was frozen and extracted as

described in the Materials and Methods. The level of 5-HT

was analysed by HPLC-EC. Each point represents the mean +

SD; n = 3 or more.

CM

76

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CM o o

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( a n s s x j i 6 / 5 u ) x a A a i j h - S

77

Fig. 4. [3H]-LSD binding to intestinal membranes:

Isolated intestinal membranes of anterior ( I ), midgut ( O )

and posterior ( % ) segments from 0 hr samples were assayed for

[^H]-LSD binding (1.0 nM to 5.0 nM). Data shown are the mean of

three or more individual experiments and standard deviations

(± SD) were calculated. Hofstee analysis was used to estimate

K d (nM) and (fmol/mg) .

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m S E 6

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78

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I

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o CO

o v o

o o CM

( u T s ^ o a d B u i / x o u i j ) p u n o g Q S 1 - [ H E J

79

Fig. 5 [3H]-LSD binding to intestinal membranes:

Isolated intestinal membranes of anterior ( • ) , midgut ( O )

and posterior ( 0 ) segments from worms incubated in A. suum

saline minus antibiotics for 24 hrs were assayed for [3H]-LSD

binding (1.0 nM to 5.0 nM). Data shown are the mean of three or

more individual experiments and standard deviations (± SD) were

calculated. Hofstee analysis was used to estimate Kd (nM) and

Bmax (fmol/mg).

H H o 00 VP H II II II

* K m <u e e s 03 CQ 03

o ; O cn

<N iH on II ii II V T3 V * * K

• 0 •

80

LO

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r ^

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2

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N

o CM

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81

Fig. 6 [3H]-LSD binding to intestinal membranes:

Isolated intestinal membranes of anterior ( | ), midgut (O )

and posterior ( # ) segments from worms incubated in A. suum

saline plus antibiotics for 24 hrs were assayed for [3H]-LSD

binding (1.0 nM to 5.0 nM). Data shown are the mean of three or

more individual experiments and standard deviations (± SD) were

calculated. Hofstee analysis was used to estimate Kd and Bmax.

<T> CO H II * s ffl

U3 II * fl 6 CO

H r~ r-t II X rt g

82

VO

W

D 0 H Cn

S C

0 cn 1 r—i 93 CO

(u"ca.o<i Bm/xomj) putiog aSI-tHE]

83

Fig. 7.Inhibition of [3H]-TRP binding by 5-HT:

A. suum intestinal tissues from 0 hr control worms were

incubated with [3H]-TRP plus or minus varying concentrations

of unlabeled 5-HT; binding in the absence of competitor was

taken as 100 %. The binding in the presence of competitor

was expressed as a percentage of this maximum binding. Data

represent the mean percentage +. SD of three assays per

competitor concentration.

84

W (X D 0 H b

33 I

m

<w 0

c 0

•H 4J <d M -P G <0 0 c 0 u

0* 0 l-J 1

BuTpu-CH JO ^ u s s i a a

85

Pig. 8.Inhibition of [3H]-TRP binding by 5-HT:

A. suum intestinal tissues from worms incubated in A. suum

saline minus antibiotics for 24 hrs were incubated with [3H]-

TRP plus or minus varying concentrations of unlabeled 5-HT;

binding in the absence of competitor was taken as 100 %. The

binding in the presence of competitor was expressed as a

percentage of this maximum binding. Data represent the mean

percentage + SD of three assays per competitor concentration.

86

<N

00

W (X D CD H b

<—« 1 1 1 « 1 « 1 «•

KD

00

E-I X I

m

s CO

4-1 O

C o

•H +J <4 H 4J C <1) a c o u

O* 0

1

o ao

o vo

o o CM

S u T p u x g j o q . u a o i a < j

87

Fig. 9.Inhibition of [3H]-TRP binding by 5-HT:

A. suum intestinal tissues from worms incubated in A. suum

saline plus antibiotics for 24 hrs were incubated with [3H]~

TRP plus or minus varying concentrations of unlabeled 5-HT;

binding in the absence of competitor was taken as 100 %. The

binding in the presence of competitor was expressed as a

percentage of this maximum binding. Data represent the mean

percentage ± SD of three assays per competitor concentration.

88

G\

W X D 0 H fa

*—i 1 > 1 ' 1 « r

CM

V £

00

R S I

LD

4-1 0

a o •H -P (d n 4J a <D o c o u

Cn 0 Hi 1

o V 0

o *1»

o CM

B u T p u T S 5 0 q . u © 0 3 © < 3

89

Fig. 10.Inhibition of [3H]5-HT binding by TRP:

A. suum intestinal tissues from 0 hr worms were incubated

with [3H]5-HT plus or minus varying concentrations of

unlabeled TRP; binding in the absence of competitor was taken

as 100 %. The binding in the presence of competitor was

expressed as a percentage of this maximum binding. Data

represent the mean percentage + SD of three assays per

competitor concentration.

90

O t—i

w IX D O H h

CN

to

CO

CU cs

4-1 O

G 0

•H 4J rd u c 0) o e o u

CP 0 h4 1

fiUTpUTS 30 ^ U S O j a a

91

Fig. 11.Inhibition of [3H]5-HT binding by TRP:

A. suum intestinal tissues from worms incubated in A. suum

saline minus antibiotics for 24 hrs were incubated with

[3H]5-HT plus or minus varying concentrations of unlabeled

TRP; binding in the.absence of competitor was taken as 100 %.

The binding in the presence of competitor was. expressed as a

percentage of this maximum binding. Data represent the mean

percentage + SD of three assays per competitor concentration.

92

i H r—i

w &

D CD H fa

CM

10

CO

CU

H

4-1 0

£ 0

•H «P

•P C <1) O C 0 U

D* 0

1

fiu-rpuxa j o q . u a o ^ a a

93

Fig. 12.Inhibition of [3H]5-HT binding by TRP:

A. suurn intestinal tissues from worms incubated in A. suurn

saline plus antibiotics for 24 hrs were incubated with [3H]5-

HT plus or minus varying concentrations of unlabeled 5-HT;

binding in the absence of competitor was taken as 100 %. The

binding in the presence of competitor was expressed as a

percentage of this maximum binding. Data represent the mean

percentage + SD of three assays per competitor concentration.

94

CM tH

W cc o u H fa

&4 04 H

4-4 0

c 0 •H •P 03

-P C <D 0 G 0 U

0* 0 »-3 1

6uTpuxg fe^ox J© ^uaoaaa

BIBLIOGRAPHY

Abrahams, S.L., Northrup, J.K. and Mansour, T.E. Adenosine

cyclic 3',5'-monophosphate in the liver fluke, Fasciola

hepatica: I. Activation of adenylate cyclase by

5-hydroxytryptamine. Mol. Pharmacol. 12(1): 49-58.

(1976) .

Ahlman, H. Serotonin and the Carcinoid Syndrome, in Serotonin

and The Cardiovascular System, ed. by P.M. Vanhoutte. New

York, Raven Press. 199-212. (1985).

Atkinson, H.J., The Respiratory Physiology of Nematodes, in:

Organization £f Nematodes, ed. N.A. Croll Academic

Press, New York and London. (1976).

Barker, L.R., Bueding, E. and Timms, A.R. The possible role

of acetylcholine in Schistosoma mansoni. Br. J.

Pharmacol. Chemother. 26(3): 656-665. (1966).

Barnes, D.M. New Data Intensify the Agony Over Extasy. Sci.

239: 864-866. (1988).

95

96

Barrett, j. and Beis, I. Studies on glycolysis in the muscle

tissue of Ascaris lumbricoides (Nematoda). Comp.

Biochem. Phvsiol. 44 (3): 751-761. (1973).

Beaver, P.C., Jung R.C. and Cupp E.W. Clinical Parasitology.

Lea & Febiger, (1984).

Bennett, J.L. and Bueding, E. Uptake of 5-hydroxy-tryptamine

by Schistosoma mansoni. Mol. Pharmacol. £: 311-319.

(1973) .

Bennett, J.P.Jr. and Snyder S.H. Serotonin and lyseric acid

diethylamide binding in rat brain membranes: relationship

to postsynaptic serotonin receptors. Mol. Pharmacol. 12.:

373-389. (1976).

Bird, A.F. The Structure of Nematodes, Academic Press, London

and New York. (1971) .

Bradford, M. A rapid and sensitive method for the

quantitation of microgram quantitatives of protein

utilizing the principle of protein-dye binding. Anal.

Bioch. 72: 248-254. (1976).

Brodie, B.B., Tomick, E.G., Kuntzman, R. and Shore, P.A.

97

Mechanism of action of reserpine: Effect of reserpine on

capacity of tissue to bind serotonin. J. Pharm. and

Exper. Therap. 119:461-467. (1957).

Bueding, E., and Yale, H.W. Production of methyl-butyric acid

by bacteria-free Ascaris lumbricoides. <1. Biol. Chem.

193 : 411-423 . (1951) .

Bulbring, E. and Lin, R.C.Y. The effect of intralumenal

application of 5-hydroxytryptamine and 5-

hydroxytryptophan on peristalsis, The local production of

5-hydroxytryptamine and its release in relation to

intralumenal pressure and propulsive activity. J. Phvs.

(London). 140: 381-407. (1958).

Catto, B.A. Schistosoma mansoni: Decarboxylation of

5-hydroxytryptophan, L-dopa and L-histidine in adult and

larval schistosomes. Exot. Parasit. 51: 152-157 (1981).

Chandler, A.C. "Other intestinal nematodes", pp. 450-459. In:

A.C. Chandler (ed.) Introduction to parasitology. 8th ed.

New York, Jones Wiley & Sons Inc. (1950).

Chance, B., Schoener, B. and Schindler, F. The Intracellular

Oxidation-Reduction State, in: Oxvcren in the Animal

98

Organism. I.U.B. Symposium 31. eds. F. Dickens and E.

Neil Pergamon Press, Oxford. (1964).

Chaudhuri, J., Martin, R.E. and Donahue, M.J. What it costs

to feed worms. Successful Farming. Feb: 18-T. (1987) .

Chaudhuri, J., Martin, R.E. and Donahue, M.J. Evidence

for the absorption and synthesis of 5-hydroxytryptamine

in perfused muscle and intestinal tissue and whole worm

of adult Ascaris suum. Parasit. 96: 157-170. (1988).

Chaudhuri, J. and Donahue, M.J. Serotonin receptor in the

tissue of adult Ascaris suum. Mol. and Biochem. Parasit.

21: 191-198. (1989).

Chitwood, M. Nematodes of medical significance found in

market fish. Am. J- Trop. Med. Hvg. l£:599-602. (1970).

Chitwood B.G. and Chitwood M.B. An introduction to

Nematology, 2nd ed. Monumental Printing Co., Baltimore.

' (1950).

Chung, K., Anderson, G.M. and Fulk, G.E. Formation of

indoleacetic acid by intestinal anaerobes. J. Bacteriol.

124: 573. (1975).

99

Connor, D.H. and Neafie, R.C. Onchoceriasis. Path. &f Troo.

and Ext;raor. Pis. Washington, DC, Armed forces Institute

of pathology, pp. 3 60 (197 6) .

Cyr, D., Gruner, S. and Metrick, D.F. Hymenolepsis diminuta:

Uptake of 5-hydroxytryptamine (Serotonin), glucose, and

changes in worm glycogen levels. Can. J. Zoo. 61: 1469-

1474. (1982).

Dendinger, J.E. and Roberts, L.S. Glycogen synthase in the

rat tapeworm Hymenolepis diminuta - I. Enzyme activity

during development and crowding. Comt>. Biochem. Phvsiol.

5SB: 215-219 (1977) .

Douvres, F.W., Tromba F.G., and Malakatis, G.M. Morphogenesis

and Migration of Ascaris suum Larvae Developing to

fourth stage in swine. J. Parasit. 35: 689-712. (1969).

Donahue, M.J., Yacoub, N.J., Kaeini, M.R. and Harris, B.G.

Activity of enzymes regulating glycogen metabolism in

perfused muscle-cuticle sections of Ascaris suum

(Nematoda). J. Parasit. 67(3): 362-367. (1981).

Donahue, M.J., Yacoub, N.J., Michnoff, C.A., Masaracchia,

R.A. and Haris, B.G. Serotonin (5-hydroxytryptamine): A

100

possible regulator of glycogenolysis in perfused muscle

segments of Ascaris suum. Biochem. Biophvs. Res. Comm.

101: 112-117 (1981a).

Drummond, A.H. Interaction of Blood Platelets with Biogenic

Amines: Uptake, Stimulation and Receptor Binding, in

Platelets in Biology and Pathology, ed. by J.L. Gordon.

Amersterdam, Elesevier/North Holland. 201-239. (1976).

Fairbairn, D. Biochemical Adaptation and Loss of Genetic

Capacity in Helminth Parasites. Biol. Rev. 4£: 29-72.

(1970) .

Ferrara, A., Zinner, M.J. and Jaffe, B.M. Intraluminal

release of serotonin, substance P, and gastrin in the

canine small intestine. Dig. Pis. Sci. 32(3) : 289-294.

(1987) .

Fischer, E;H. and Krebs, E.G. Conversion of phosphorylase b

to phosphorylase a in muscle extracts. Biol. Client.

21£: 121-132 . (1955) .

Gale, E.F. and Epps, H.M.R. Studies on bacterial amino acid

decarboxylase. 3. Distribution and preparation of

codecarboxylase Biochem. J. 250-256. (1944).

101

Gentleman, S., Abraham, S.L. and Mansour, T.E. Adenosine

cyclic 35'-monophosphate in the liver fluke, Fasciola

hepatica: II. Activation of protein kinase by 5-

hydroxytryptamine. Mol. Pharmacol. 12(1) : 59-68.

(1976).

Gronstad, K.O., Dahlstrom, A., Florence, L., Zinner, M.J.,

Ahlman, H. and Jaffe, B.M. Regulatory mechanisms in the

endoluminal release of serotonin and substance p from the

female jejunum. Dig.Pis Sci. 32(4) : 393-400. (1985).

Guirard, B.M. and Snell, E.E. Nutritional requirements of

lactobacillus 30a for growth and histidine decarboxylase

production. J. Bact. 87: 370-376. (1964).

Hannigan, L.L., Donahue, M.J. and Masaracchia, R.A.

Comparative purification and characterization of

invertebrate muscle glycogen synthase from the porcine

parasite Ascaris suum. J. Biol. Chem. 260 (30): 16099-

16105. (1985).

Hariri, M. Quantitative measurements of endogenous levels of

acetylcholine and choline in tetrahidria of Mesocestoides

cori (Cestoda) by means of combined gas chromatography

Mass spectrometry. J. Parasit. 60 (2) : 227-230. (1974).

102

Harpur, R.P. Maintenance of Ascaris lumbricoides in vitro

III. Changes in the hydrostatic skeleton. Comp. Biochem.

Phvaiol. 11: 71-85.(1963).

Harris, J.E. and Crofton, H.D. Structure and function in the

Nematodes: Internal Pressure and Cuticular Structure in

Ascaris. J. Exp. Biol., 34.: 116-130 (1957).

Harris, B.G. and Allen, B.L. Purification and

characterization of glycogen, phosphorylase B from

Ascaris suum . Fed. Proc. 38: 656. (1979).

Higashi, G.I., Kreiner, P.W., Keirns, J.J. and Bitensky, M.W.

Adenosine 3',5'-cyclic monophosphate in Schistosoma

mansoni Life Sci. 13: 1211-1220. (1973).

Hillman, G.R., Olsen, N.J. and Senft, A.W. Effect of

methysergide and dihyroerogotamine on Schistosoma

mansoni. J[. Pharmacol. EXP . Ther. 188(3) : 529-532.

(1974) .

Hofstee, B.H. On the evaluation of the constants Vm,v and K„ uiciA m

in enzyme reactions. Sci. 116: 329-333. (1952).

Hsu, S-C., Johanson, K.R. and Donahue, M.J. The bacterial

103

flora of the intestine of Ascaris suum and 5-

hydroxytryptamine production. J. Parasit. 72 (4) : 545-549.

(1986) .

Ichiyama, A., Nakamura, S., Nishizuka, Y. and Hayaishi, 0.

Tryptophan-5-hydroxylase in mammalian brain. Adv.

Pharmacol. 6A: 5-17. (1968).

Ichiyama, A., Nakamura, S., Nishizuka, Y. and Hayaishi, O.

Enzyme studies on the biosynthesis of serotonin in

mammalian brain. jJ. Biol. Chem. 245: 1699-1709. (1970).

Jaskoski, B.J. As apparent swine Ascaris infection of man.

si. ML- Vet. Med. Assoc. 138: 504-520. (1961).

Jequier, E., Lovenberg, W. and Sojerdsma, A. Tryptophan

hydroxylase inhibition: The mechanism by which

p-chlorophenylalanine depletes rat brain serotonin.

Mol. Pharm. 2: 274-278. (1967).

Kilts, C.D., Breese, G.R. and Mailman, R.B. Simultaneous

quantification of dopamine, 5-hydroxytryptamine and four

metabolically related compounds by means of reversed-

phase high performed liquid chromatography with

electrochemical detection. J. Chromato. 225: 347-357.

104

(1981) .

Koe, B.K. and Weissman, A. p-chlorophenylalanine: A specific

depletor of brain serotonin. IJ. Pharmacol. EXP . Ther.

154: 499-516. (1966).

Knoll, J. and Magyar, K. Some puzzling pharmacological

effects of monoamine oxygenase inhibitors. Advanced

Biochemical Psvchopharmacoloav 5.: 393-408. (1972) .

Krebs, E.G. and Beavo, J.A. Phosphorylation-dephosphorylation

of enzymes. Annu. Rev. Biochem. 48: 923-959. (1979).

Krivoshta, E.E., "Epizootologiya i Profilaktika Pri

Askaridoze Svinei (Epizootology and Prophylaxis of

Ascaridosis of pigs)Trudv Rostovskoi Oblastnoi

Veterinarnoi Qpvtnoi Stantsii. 1:86-94. (1939).

Langer, S.Z., Moret, C., Raisman, R., Dubocovich, M.L. and

Briley, M. High-Affinity [3H] Imipramine Binding in Rat

Hypothalamus is Associated with the Uptake of Serotonin

but not Norepinephrine. Sci. (1980) .

Lee, D.L. and Atkinson H.J. Physiology of Nematodes. 2nd. ed.

Columbia University Press., New York. (1977).

105

Lehninger, A.L. Glycolysis: A Central Pathway of Glucose

Metabolism, in Principles of Biochemistry. New York,

Worth Publishers Inc. 397-430. (1982).

Lewis, T.R. and Emery, R.S. Intermediate products in the

catabolism of amino acids by rumen microorganisms.

Dairy Sci. 4£: 1363. (1962).

Levine, N.D. Nematode parasites of domestic animals and of

man. 2nd. ed. Burgess, Minneapolis. (1980) .

Mansour, T.E. Effect of serotonin on glycolysis in

homogenates f;rom the liver fluke, .Fasciola hepatica. J.

Pharmacol. Ex£. Ther. 135(1) : 94-101. (1962).

Mansour, T.E. The pharmacology and biochemistry of parasitic

helminths. Adv. Pharmacol. 2: 129-162. (1964).

Mansour, T.E. and Stone, D.B. Biochemical effects of lysergic

acid diethylamide on the liver fluke, Fasciola hepatica.

Biochem. Pharmacol. 19: 1137-1147. (1970).

Mansour, T.E. Chemotherapy of parasitic worms: New

Biochemical strategies. Sci. 205: 462-469. (1979).

Martin, L.K. Hookworm in Georgia. II. Survey of intestinal

106

helminth infection in members of rural households of

southeastern Georgia. Am- <!• Trop. Med. Hva. 21:930-943 .

(1972).

Martin, R.E., Foster, L.A., Kester, A.S. and Donahue, M.J.

Ascaris suum: Morophological characterization of

apparent cuticular pores by ionic permeability and

electron microscopy. Expt. Parasit. 63: 329-336. (1987)

Martin, R.E., Chaudhuri, J. and Donahue, M.J. Serotonin

(5-Hydroxytryptamine) turnover in adult female Ascaris

suum tissues. Comp. Pharm. and Toxicol. 92C: 307-310.

(1988).

Mettrick, D.F., Rahman, M.S. and Podesta, R.B. Effect of 5-

hydroxytryptamine on in. vitro glucose uptake and

glycogen reserves in Hymenolepsis diminuta. Mol.

Biochem. Parasit. 4: 217-223. (1981).

Mishra, S.K., Sen, R. and Ghatak, S. Monoamine oxidase and

metabolites of biogenic amines in human ascaris. Indian

J. Parasit. 2: 153-155. (1978).

Mishra, S.K., Agarwal, A., Sen, R. and Ghatak, S.

Characterization of mitochondrial monoamine oxidase of

107

Ascaridia galli . J. Helminth. 59: 101-107. (1985).

Moreno, E. Enterobiasis (Oxyuriasis, pinworm infection) in

Marcial-Rojas ed.: Pathology of Protozoal and Helminthic

Diseases. Baltimore,Williams & Wilkins pp. 760. (1971).

Mozgovoi, A.A., "Ascaris suum Goeze". and "Ascaridosis of

Pigs", In: A.K. Skrjabin (ed.) Ascaridata of Animals and

Man and the Disease Caused bv Them. Izdatel'stvo Akademii

Nauk, SSSR, Moskova. pp. 94-109 (1953). Translated from

Russian. Jeursalem, Israel Program for Scientific

Translations (1968).

Nakazawa, H., Sano, K.( Kumagai, H. and Yamada, H.

Distribution and Formation of Aromatic L-Amino Acid

Decarboxylase in Bacteria. Aaric. Biol. Chem. 41 (11):

2241-2247. (1977).

Neckers, L.M. Serotonin turnover and regulation. In Biology

of Serotonergic Transmission (Edited by Osborne N. N).

John Wiley & Son, New York. (1982).

Newton, W.A. and Snell, E.E. Catalytic properties of

tryptophanase, a multifunctional pyrodoxal phosphate

enzyme. Proc. Natl. Acad. Sci. 51: 382-386. (1964).

108

Nicholas, W.L. The Biology of Free-living Nematodes.

Clarendon Press, Oxford. (1975).

Nobel, E.R. and Nobel, G.A. Other Intestinal Nematodes , pp.

289-294. In: Lea & Febiger Philadelphia (ed.)

Parasitology. 5th ed. New York John Wiley & Sons Inc.

(1982).

Olsen, O.W. Hookworms, Uncinaria lucasi styles, 1901, in

fur seals, Callorhinus ursinus (Linn.), ori the Pribilof

Islands. Trans. |J. Am- Wildlife Conf. 21:152-175. (1958).

Osborne, N.N. Assay Distribution and Functions of Serotonin

in Nervous Tissues, in The Biolocrv of Serotonergic

Transmission. ed. by N.N. Osborne. New York, John Wiley

and Sons. Inc. 7-27. (1982).

Pawlowski, Z.S. Ascariasis. Clin. Gastroenterol 7: 157-178.

(1978).

Pawlowski, Z.S. Trichuriasis, in Warren KS, Mahmoud AAF

(eds): Trop. and Geo. Med. New York, McGraw-Hill, pp.380-

385 (1984).

Pentilla, A. Histochemical reactions of the enterochromaffin

109

cells and the 5-hydroxytryptamine content of the

mammalian duodenum. Acta Phvsiol. Scand. 69 (SUPPI. 281):

1-77. (1966).

Peroutka, S.J. and Snyder, S.H. Multiple serotonin receptors:

differential binding of [3H]-serotonin, [3H]-lysergic acid

diethylamide, [3H]-spiroperidol. Mol. Pharmacol. 16: 687-

699. (1979).

Peroutka, S.J. 5-Hydroxytryptamine receptor subtypes. Ann.

Rev. of Neuro. 11: 45-60. (1988).

Pletcher, A., Shore, P.A., and Brodie, B.B. Serotonin release

as a possible mechanism of reserpine Action. Sci. 122:

374-375. (1955).

Rapport, M.M. Serum Constrictor (Serotonin) V. The Presence

of Creatinine in the Complex. A Proposed Structure of the

Vasoconstrictor Principle. J. Biol. Chem. 180: 961-969.

(1949).

Ribeiro, P. and Webb, R.A. The synthesis of 5-

hydroxytryptamine from tryptophan and 5-hydroxytryptophan

in the cestode Hymenolepis diminuta. Int. Parasit. 1:

135-138. (1983).

110

Ribeiro, P. and Webb, R.A. The occurrence, synthesis and

metabolism of 5-hydroxytryptamine and 5-hydroxytryptophan

in the cestode Hymenolepis diminuta: A high performance

liquid chromatographic study. Comp. Biochem. Phvsiol.

79C : 159-164. (1984) .

Ribeiro, P. and Webb, R.A. Characterization of a serotonin

transporter and an adenylate cyclase-linked serotonin

receptor in the cestode Hymenolepsis dimiuta. Life Sci.

M : 755-768. (1987) .

Rosenbluth, J. "Ultrastructure of Somatic Muscle Cells in

Ascaris lumbricoides. II. Intermuscular Junctions,

Neuromuscular Junction; and Glycogen Stores", i. Cell

Biol.. 2£: 579-591. (1965).

Ross, S.B. In: Biology of serotonergic transmission.

(Osborne, N.N., ed) pp.159-195, John Wiley & Sons, New

York. (1982).

Saz, H.J. Comparative Biochemistry of Carbohydrates in

Nematodes and Cestodes, in Comp. Bioch.of Parasit. ed. by

H. Vanden Bossche. New York., Academic Press. 33-47.

(1971) .

Ill

Saz, H.J. and Weil, A. Pathway of Formation of a-

methylvalerate by Ascaris lumbricoides. >1. Biol. Chem.

237: 2053-2056. (1962).

Saz, H.J. and Weil, A. The Mechanism of Formation of

a-methylvalerate from Carbohydrate by Ascaris

lumbricoides Muscle. J. Biol. Chem. 235: 914-918. (1960).

Schad, G.A. and Banwell J.G. Hookworms, in Warren KS, Mahmoud

AAF (ed): Trop. Geo. Med. New York, McGraw-Hill, pp. 359

(1984) .

Schraitman, C., Erwin, V.G. and Greenawalt, J.W. The

submitochondrial localization of monoamine oxidase. «2.

Cell Biol. 22: 719-736. (1967).

Sevilla, C.L. and Fischer, E.H. The purification and

properties of rat muscle glycogen phosphorylase. Biochem.

8(5) : 2161-2171. (1969).

Sourkes, T.L. Enzymology of aromatic amino acid

decarboxylase, Structure and Function of Monoamine

Enzvmes (Eds. E. Usdin, N. Weiner and M.B.H. Youdin), pp.

477-495, Marcel Dekker, New York. (1977).

112

Sun, Tsieh. Introduction to helminths, Color atlas textbook

of diagnostic parasitology (Igaku-Shoin, Medical

Publishers, Inc.) 1140 Ave. of the Americas, New York,

N.Y. 10036. pp. 128 (1988).

Tappaz, M.L. and Pujol, J.F.M. Estimation of the rate of

tryptophan hydroxylation in vivo: A sensitive microassay

in discrete brain nuclei. J. Neurochem. 34: 933-940.

(1980) .

Tipton, K.F. The submitochondrial localization of monoamine

oxidase in rat liver and brain. Biochem. and Biophvs.

Acta. 135: 910-920. (1967).

Toh, C.C. Release of 5-Hydroxytryptamine (Serotonin) from the

Dog's Intestinal Tract. J. Phvs. (London). 126: 248-254.

(1964) .

Tyce, G.M. Biochemistry of serotonin. In serotonin and the

cardiovascular system, ed. by P.M. Vanhoutte. New York,

Raven Press. 1-13. (1980).

USDA report. Swine Nutrition AFS-3 6, pp. 1-7 (Ed. by Dept. of

Animal Sci., Purdue Univ., West Lafayette, IN, in

cooperation with the U.S. Department of Agriculture, Off.

113

of Govert. and Public Affairs, Washington D.C.) (1986).

Von, Brand, T. Biochemistry of Parasites (Academic Press, New

York. (1973).

Ward, K.H. and Fairbairn, D. Enzyme of b-Oxidation and their

Function During Development of Ascaris lumbricoides

eggs. Devel. Biol. 22.: 366-387. (1970).

Weinstein, P.A. In Stauber, L.A. ed. Host influence on

parasite physiology. Rutgers Univ. Press, New Brunswick,

N. J., 65-92. (1960).

Williams, J.A., Shahkolahi, A.M., Abbassi M. and Donahue M.J.

Identification of a Novel 5-HTN (Nematoda) receptor from

Ascaris suum muscle.(in press) . (1991) .

Wood, P.J. and Mansour, T.E. Schistosoma mansoni: Serotonin

uptake and its drug inhibition. Expt. Parasit. 62: 114-

119. (1986).

World Health Organization. Intestinal Protozoan and

Helminthic Infections. World Health Organization

Technical Reports Series 666. (1981).

World Health Organization: Lymphatic filariasis. Fourth

114

Report of the WHO Expert Committee on Filariasis.

Technical Report Series 702, Geneva (1984).

Yokoyama, M.T. and Carlson, J.R. Dissimilation of tryptophan

and related indolic compounds by ruminal microorganisms

in vitro. A P P I . Microbiol. 27: 540. (1974).