Characterization and Regdation of a Moult Cycle-Dependent Inwardly-RectiSing Chlorîde Current in the Epidermal Cells of the Beetle Tenehrlo ntoliror

Ian John Watson

Submitted in partial fulfilment of the requirements for the degree of

Master of Science

Faculty of Graduate Studies The University of Western Onîario

London, Ontario July, 1997

O Ian John Watson 1997

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ABSTMCT

The insect epidermis is involved in fluid transport at the moult, a process

involving the transport of ions and water. The whole-ceIl patch-clamp method was used

to detect an inwardly rectiQing current in epidermal cells from newly-moulted beetle

iarvae. This current is CI--selective and is activated by membrane hyperpolarization, the

presence of CAMP in the patch pipette. and by exposinç the cells to hyposmotic saline.

Evidence that the inwardly rectifying current is a CI- current is based on the observations

that: the inwardly rectieing current did not decrease in amplitude nor did its reversa1

potential shift when the bath Na+ was removed; the inwardly rectifying current remained

afier pipene K' was replaced with CS' and tetraethylammoniumL and bath K* was

repiaced with ~ a " ; the inward current was suppressed by several stiibene derivatives

and diphenylamine-2-carboxylate, known anion channel blockers; and reversa1 potentials

in cells recorded in the presence or absence of pipette CAMP were similar to those

predicted by the Nernst equation for Cl-.

This inwardly rectiwing current reaches its peak at the moult and it is greatly

reduced in mid-instar epidennal cells. Epidermal cells increase in volume at the moult

and the CI- current may allow them to reguiate their volume. I propose that the Cl-

current is a component of a second messenger-dependent mechanisrn driving fluid

secretion by the epidermis at the moult.

iii

ACKNOWLEDGEMENTS

1 would like to thank Dr. Stanley Caveney for the opportunity to work in his

laboratory. His enthusiasm, devotion to his work and his passion for biology were

inspiring. 1 especially would like to thank hirn for the many hours he spent editing my

thesis.

1 would like to acknowledge the friendship of everyoiie who spent time in the lab

during my stay at U.W.O. including, Heather, Jenny, Erika, Dennis, Chantel, Miyo, Kim,

Rod, Tabita and Xiujan.

1 would especially like to thank Tabita Malutan, Oana Marcu and Eck Harris for

their friendship and support. Through Our many hours of discussion 1 leamed a lot about

life, science, patience and the pursuit of happiness. They liffed my spirits when I most

needed help.

1 thank al1 of those around the department who helped me directly or indirectly

with my work, especially, Ian Craig, Mary Martin, Jane Sexsmith and Melina Buragina.

1 thank the members of my advisory cornmittee, Dr. G. Kelly and Dr. S. Sims for

their helpful suggestions. 1 also would like to thank the rnembers of my exarnining

board, Dr. G. Kelly, Dr. J. Steele, Dr. J. Dixon and Dr. L. Milligan for their creative

comments and thoughtful questions.

Finally, 1 would like to thank my wife for her suppon and love. Without her 1

never would have had the confidence to start a Master's degree or have the perseverance

to cornpiete it.

TABLE OF CONTENTS

. . ......... ........... CERTIFICATE OF EXAMINATION .... ,.. I I

ABSTRACT ....................... ,.. ............................................................................ iii ACKNOWLEDGE LMENTS ........ ... ...... ..., iv TABLE OF CONTENTS ..................................................................................... v

. * .............................................................................................. LIST OF FIGURES vil

................................................................... .................. LIST OF TABLES .... ix .............................................. LIST OF ABBREVIATIONS .. x

1 . INTRODUCTION ........................................................................................ 1

.......................................... i? . MATERMLS AND iMETEIODS ........... ... 8

n . i

11.2

III . III . I

111.2 III . 3 III.4 In . 5 III . 6

III . 7

III . 8 ILI . 9 III . 10 III . 1 1

Isolation of Cuticle-Attached Cells .............................................. 8 II . 1.1 Beetle Culture and Staging .................................................... 8 II . 1.2 Dissection ........................................................................ 8 II . 2.3 Chernicals .............................................................................. 8

U . 1 .3.1 Saline Recipes ........................................................... - 9 ...................................................... U . 1.3.2Enzyme Solutions 9

II . 1.4 Enzyme Treatment ................. ... ........................................ 9 II . 1 -5 Scraping ................................................................................. 9 II . 1.6 Recording Charnber and Superfusion .............................. 1 1 Whole-ce11 Recording ........................................................................ 12 11.2.1 Correction for Liquid Junction Potentials ......... .. ..... .. .... 14 IL 2.2 Calculating Membrane Potentials ........................................ 14 IL2.3 Cell Capacitance .................................................................... 15

RESULTS ....................................................................................... 16

Activation of an Inwardly Rectifying Current in Epidemal Cells ................................................................................. 16 The Inwardly Rectifjing Current is a CI' Current ................. .. ....... 16 The Inwardly RectiQing Cwent is Not a K' Cunent ....................... 25 The Inwardly Rectifying Current is Not a ~ a + Current ..................... 75 Pipette CAMP Reduces the Current Activation Time ....................... -30 Effects of Bath Osrnolarity on the Cl- Current in the Presence and Absence of Pipette CAMP ........................................................... 30 CAMP in the Pipette Shifts Sensitivity to Bath Saline Osrnolarity ........................................................................................ 35 Ion Selectivity of CAMP Activated Current ....................................... 40 Pharmacological Inhibition of the Inwardly RectiQing Current ...... -40 The Inwardly Recti fying Current is Developmentaliy Regulated ..... 43 The CAMP-Activated Current Could Not be Switched on by Serotonin or Leukokinin 1 .......................................................... 50

III . 12 The CAMP-Activated Current Could Not be Switched off with a Proton Pump Blocker .............................................................. 50

. N DISCUSSION ................. ....... ........................................................................ 51

Cf is the Major Contibutor to the Inwardly RectiQing Current in Epidermal Cells ............................................................................. 51 Regulation of the Inwardly RectiQing Current ................................. 51

................... ......... Cornparison with CI' Currents in Other Cells ... 51 CAMP Alters the Rate of Activation But Not the Amplitude of the

................................................................... .................. Current ... 53 The Possible Role of CAMP and Hormones in the Activation

............................................................................... of the Cl- Curent 54 Functional Significance of the Cl- Channel in the Insect

........................................................................................... Epidermis 55 .................. Possible Models for the Cl- Current in Insect Epidermis 56

IV.7.1 Moulting Fluid Resorption ......................................... 5 6 IV.7.2 Volume Reduction ................................................................ 6 0 IV.7.3 Moulting Fluid Secretion ............... .... ............................. 60

V . CONCLUSIONS .......... ... ............................................................................. 67

REFERENCES .......................................................................................... 68

CURRICULUM VITA ............................................................................................ 73

LIST OF FIGURES

Figure 1.

Figure 7.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

Figure I 1.

Figure 12.

Figure 13.

Figure 14.

Figure 1 5

Trnebrzo moliror epidermal cells are capable of regulating their ..... volume in the response to a hyposmotic stress .................... ... 6

Activation of the inwardly rectifying current .................................... 18

Hyposmotic treamient increases the amplitude of the rectiQing current in cells that displayed a srna11 initial current on

. ..... hyperpolanzation .. ................................................................. 10

33 .................................... Selectivity of the inwardly rectiwing curent -- The observed reversal potentials fit the Nernst prediction for a Cl- current ........................................................................................... 24

Removal of K' from the bath saline and pipette does not alter the magnitude of the current strength or change reversa1 potentials ............................................................................................ 27

Na'-fiee bath saline does not change the reversa1 potential or ..................... ................................................... current strength .... 29

The presence of 200 p.M CAMP or 1 mM cGMP in the pipette shortened the time necessary for the current to reach K maximum amplitude ......................................................................................... 32

The amplitude of the inwardly rectiSing current is increased ............ by CAMP, membrane hyperpolarization and osmotic stress ..34

CAMP alters cell sensitivity to changes in bath osmolarity. .............. 37

Pipette CAMP increases the amplitude of the initial current recorded from the cells used to compile Figure 10.. ......................... 39

Cl- is the dominant ion in the current seen in the presence ............................................................................................. of CAMP 42

DPC largely abolishes the inwardly rectiSing current ...................... 45

Several Cl- chamel blockers suppress the inwardl y rectifying ................................................................................................ current 47

The inwardly rectiQing current is developmentaily re ylated.. ....... -49

Figure 16 . Model proposing how a CI- current could be involved in rnoulting fluid resorption ................ ... .......................................................... 58

Figure 17 . Volume reduction model .................................................................. 63

Figure 1 8 . Model proposing how a Cl' current could be involved in the secretion of rnoulting fluid .................. .. ......................................... 64

Table 1 .

Table 2.

Table 3

LIST OF TABLES

. . ......... ..................-........................*....... Solution compositions. ... 10

Ionic selectivity of the activated current ............................................ 25

Ionic selectivity of the CAMP-activated current ................................ JO

LIST OF ABBREVIATIONS

"C CAMP cGMf cm DIDS DNDS DPC EGTA

n PA PF PIPES R SITS T TEACI TBS t-Test

Degrees Celsius Adenosine 3 ',S.-monophosphate Guanosine 3''s'-monophosphate Centimeter 4'4'-diisothiocyanostilbene-1,2'-disulphonic acid 4,4?-dinitrostilbene-2,2'-disulphonic acid di phenylarnine-2-carboxylate Ethylene glycol-bis@-aminoethyl ether) N,N,N7,N'-tetracetic acid Reversa1 potential predicted by the Nernst equation for chloride Observed reversal potential Faraday's constant Gigaohm Grams Hertz Current-voltage relationship Degrees Kelvin Megaohms Micrometer Micromoiar Millimeter Millimolar Millivolt Milliosmols per liter Sample size Picoamp PicoFarads Piperazine-N,N'-bis-2-ethanesulfonic acid Gas constant 4-acetamido-4'-isothiocyanostil bene-2,T-disul phonic acid Temperature (degrees Celsius) Tetraethylammonium chloride Tenebrio Bath Solution Student's t-Test Time required to reach '/, maximum current amplitude 5- hydroxytryptamine

1 INTRODUCTION

The arthropod integument consists of an extracellular matrix, the cuticle, formed

by an underlying transporting epithelium, the epidermis. The properties of the cuticle

have been recognized as key contnbutors to the adaptive success of the phylum

Arthropoda, which includes the Insecta (Gullan and Cranston, 1994). Due to the

cuticle's rigidity and its limited ability to expand, insects are forced to shed their old

cuticle and produce a larger one in order to increase in size. As an insect is dependent on

its cuticle for structural integrity and protection against dehydration, a new cuticle must

fom before the old one is shed. Before an insect sheds its old cuticle it is enzymatically

degraded to allow the cuticle to split dunng ecdysis and to extract valuable nitrogen from

proteins and chitin that rnakes up the bulk of the old cuticle. Cuticle degradation begins

at apolysis, a process in which epidermal cells retract from the inner surface of the old

endocuticle and fil1 the formed space, known as the exuvial space, with a mixture of the

inactive foms of enzymes including proteinases, peptidases, chitinases, P-N-

acetylgl ucosaminidases, phophatases, esterases and phenoloxidases. This solution is

referred to as moulting gel (Passoneau and Williams, 1953). Beneath the moulting gel a

new cuticle is laid down. Activation of the moulting gel coincides with the transport into

the exuvial space of additional fluid and the conversion of the gel into a moulting fluid

(Katzenellenbogen and Kafatos, 197 1 ). According to Jungreis ( 1974), fluid is transported

into the ecdysial space by the active transport of KHCOl and other ions by the epiderrnis.

Jungreis (1 979) claimed that, in Hyaiophora cercopia and in Manducu sextu, the high

osmotic potential of the moulting fluid relative to the hemolymph then draws water into

the exuvial space. The moulting fluid

absorbed before the old cuticte is shed.

and the digested components of the cuticle are

The mechanism by which the moulting fluid is absorbed is presently not fully

understood (Reynolds and Samuels, 1996). Wigglesworth, based on his work with

Rhodnius ~rolrrus, supported the concept that moulting fluid was resorbed through the

new cuticle by the underlying epidermis ( Wigglesworth, 1 933: Wigglesworth, 1 965).

Observations made by othen working with H. cecropio pupae supported Wigglesworth's

claim (Lensky et cil., 1970; Passoneau and Williams, 1953). To explain how moulting

fluid could pass through the new cuticle, Wiggleswonh commented that the new cuticle

is not waterproofed at the time of resorption and therefore could allow its passage

(Wigglesworth, 1965). Locke (1966) observed the existence of 3 nm pores in the new

outer epicuticle of Cafpodes erhliu~ during the pharate stage of the moulting cycle.

These pores could act as macromolecular sieves allowing non-proteins to pass to the

epidermal cells below (Reynolds and Samuels, 1996). Once across the new cuticle, the

moulting fluid must then pass across the epidermal tissue in order to enter the

hemolyrnph. Jungeeis ( 1 979) was unable to conclusively show the existence of an ion

current that could carry the moulting fluid back across the epidermis. Observations by

Locke (1969), Delachambre (1 971 ), and Maucharnp and Hubert ( 1984) also support the

idea of retrieval of moulting fluid by pinocytosis.

An alternative method to epidermal-based resorption is anal ancilor oral imbibing

of the moulting fluid. Watcher (1930) working on Bombyx morr and Zacharuk (1973;

1976) on elaterid beetle larvae, suggested that the moulting fluid was swallowed.

Comell and Pan (1983) observed that the moulting fluid in Monduca sexta was imbibed

through the mouth and anus. Therefore, the epidermis may function mainly as a

secretoiy epithelium. Research is still required to solve the problem of the fate of the

moulting fluid.

Volume regulation is an impomnt function in man! cells. If a ce11 is exposed to

a hyposmotic solution, rapid transmembrane rnovement of water into the ceIl occurs until

the equilibrïum is restored. This rnay lead to the dilution of cytoplasm, the distortion of

organelles and the stretching of the plasma membrane. If the volume increase is too

great the ce11 may lyse. Typically, the complete or partial restoration of ce11 volume is

accomplished by the efflux of intracellular ~smotically active inorganic solutes (CI-, K+,

Na+, and HCOZ') andior organic molecules (amino acids, polyols and methylamines)

(Deaton and Pierce, 1994). This process would lower intracellular osmotic pressure

resulting in the net movement of water out of the cell. Osmolyte mechanisms involve

various channels (Kt and Cl-), cotransport systems (K'/c 1-, ~ a * / ~ l - , N~'/K+/C 1-).

exchangers ( ~ a + / r , and Cl-MC03-), and pumps (K' or Cl') (Chamberlin and

Strange, 1989). How these mechanisms are activated is poorly understood. Tension-

regulated ion channels, changes in cytoskeletal structure, dilution of cytoplasm and

changes in density and distribution of plasma membrane components have al1 been

suggested as possible mechanisms that could activate the reylatory volume decrease

mechanism (Strange et al., 1 996).

CI' channels have been shown to aid in ce11 volume regulation in many venebrate

cells (Valverde et al., 1992; Diaz et al., 1993; Kunzelrnann et al., 1992: Kelly et al.,

1994; Strange et of., 1996). By using hyposmotic stress and an electrical stimulus, cells

ln vitro are induced to activate a mechanism that appears to regulate ce11 volume.

Whereas most cells are unlikely to encounter the large osmotic stresses imposed

experirnentally in vrtro except under pathological conditions in situ, ce11 sweiling is a

normal physiological activity in certain cells. For example, in rat Leydig cells swelling

occurs pnor to the release of testosterone (Podesta et ui., 1991). Simiiarly, in insect

epidennis ceils at the moult, the production of structural materials for the new cuticle,

the possible manufacture of digestive enzymes, the claimed absorption of breakdown

products of the old cuticle or moulting fluid could lead to the increased volume. The

epidermal cells of Tenebrio moiilor double in volume during the moult cycle and then

retum to a smaller size shortly after the shedding of the old cuticle. Previously. 1 have

demonstrated that the epidermal cells of this insect are capable of regulating their volume

when exposed to a hyposmotic environment and that the mechanism involved is sensitive

to anion channel blockers (Figure 1 ) (Watson, 1995).

The beetle epidermal cells are involved in fluid transport at the moult, a process

involving the transport of ions and water. In this study 1 report that an inwardly

rectifiing current discovered by Dennis Churchill in these epidermal cells (Churchill,

1993) results from the movement of CI* across the plasma membrane. 1 show that this CI-

current is sensitive to osmotic perturbations and various inhibitors of Cf channel activity.

1 demonstrate that this current is only present during the moult cycle and that CAMP

and/or cGMP play a role in its activation. Cl- movement has been implicated in fluid

secretion in a variety of insect ce11 types (Phillips and Hanrahan, 1984; Cooper and

Jungreis, 1985; Prince and Bemdge, 1973; O'Donnell et al., 1996). Consequently 1

Figure 1. Tenebrio molifor epidermal cells are capable of regulating their volume in response to a hyposmotic stress. Cuticle-attached isolated cells were perfused with hyposmotic saline. They recovered rapidly on removal of the hyposmotic saline but did not show significant volume regulation during the stress. The cells swelled to a greater extent when treated subsequently with the anion channel blocker diphenylamine-2- carboxylate (DPC) under hyposmotic conditions, suggesting that there is a regulatory volume mechanisrn that prevents the cells from excessive swelling ( from Watson, 1 995).

methods: The average size of the cells (n=4) during the 12 minutes of the experiment were used as the reference size for the calculation of percent change for the rest of the measurements. Images were captured every 2 minutes using an Intel 486-DX2 66 Mhz based computer mnning the Northern Exposure irnaginy sohare comected to an inverted Zeiss phase-contrast microscope (3OOX rnagnification) and was used to determine the cross-sectional area of the cells.]

200 uM DPC 335 moçmoül

-5 l

Minutes

speculate on the possible role(s) this CI' current plays in fluid transport across the

epidermis during the moult cycle. The chloride channel may help: (1) resorb moulting

fiuid pnor to ecdysis; (2) reduce the epidemal cells to their pre-moult size afier the

moult cycle is complete; (3) move fluid into the exuvial space to activate the moulting

gel.

11 MATERIALS AND METHODS

11.1 Isolation of Cuticle-Attached Cells

II.1.i Beetle Culture and Staging

The larvae of Tenebrio molzror (flour beetle) was raised on a flour-bran-yeast

mixture at 77 OC in a II hour lightldark cycle.

Last instar larvae (weight, 0.09 to O. 1 1 g) were collected immediately afier

ecdysis (tertned newly-moulted larvae) or 6 days after ecdysis (mid-instar larvae).

II. 1.2 Dissection

Dissection was similar to that previously described by Caveney and

Blennerhassett ( 1980). Immediately before dissection, the mealworm was anaesthetized

by submersion in 70% ethanol for four minutes. The insect was then pinned to wax

molded to firmly hold the rnealworm. The ventral stemites from segment II to VI1 were

removed and placed into a 35 mm Petri dish containing Tenebno bath saline (TBS. 41 5

mosmol/l). Fat and muscle stubs were rernoved and the segments then transferred to

fresh TBS in a 35 mm Petri dish and maintained at 28 OC.

U.1.3 Chernicals

4,4'-Diisothiocyanostilbene-2,27-disulphnic acid (DIDS), 4,4'-dinitrostilbene-

2,2'-disulphonic acid (DNDS), 4-acetamido-4'-isothiocyanostilbene-2,3'-disu~phonic

acid (SITS), diphenylamine-2-carboxylate (DPC), piperazine-N,N7-bis-2-ethanesulfonic

acid (PIPES), dimethylsulfoxide (DMSO) and ethylene glycol-bis(b-arninoethyl ether

N,N,N7N'-teuaacetic acid (EGTA), leukokinin 1, serotonin (5-HT, 5-hydroxytryptamine),

adenosine 3',5'-cyclic monophosphate (CAMP), 3',5'-cyclic monophosphate (cGMP).

bromo and dibutml CAMP and bafilomycin Al were from Signa (St. Louis. Missouri).

Al1 salts were analytical grade from BDH (Toronto, Ontario).

II. 1.3.1 Saline Recipes

Solution osmolarity was detemined by fieezing point depression (posmette.

Precision Syst., Natick, MA, USA). Pipette and bath saline pH were adjusted using KOH

and NaOH, respectively. Compositions of salines are show in Table 1 .

LI. 1.3.2 Enzyme Solutions

Enzyme stock solutions were made up in TBS and stored at -20 O C . Enzyme

solutions were made by diluting 1% stock of pronase E (type XXV) (Sigma, St. Louis,

Missouri) in TBS to a concentration of O. 1%.

II.1.4 Enzyme Treatment

One to five houn after dissection, the segments were placed for 90 seconds in a

35 mm Petri dish containing a 0.1% pronase TBS solution. The pronase removed the

basal lamina but did not strip the epidermis from the cuticle. The segments were then

placed into fresh TBS and maintained at 28 OC.

11.1.5 Scraping

Single cells were isolated by clamping the segments into a recording charnber

(Churchill, 1993) and scraping the segments with a g las pipette with its tip broken to a

diarneter of two to seven Fm (Churchill, 1993). The scraping procedure was camed out

on an inverted phase contrast microscope (mode1 IM-35, Car1 Zeiss, New York, NY)

fitted with phase optics (40X Neofluar objective, NA 0.75) at 400X mapification. A

broken pipette was inserted onto a rnicromanipulator and then its tip was lowered

through the epidermal layer down to the cuticle. The microscope stage was moved in

such a way that the pipette fonned a series of furrows one to two cells wide. A second

set of furrotvs was made at 45 degrees to the first set foming islands of one or two cells.

The segment was transfened to a 35 mm Petri dish and lefi for one how to allow the

damaged cells to die. The scraped segments were then placed back into the recording

chamber and vacuum cieaned with a broken glass pipette (10 Fm diameter). Removing

debris left from the dead cells prevented plugging of the whole-ce11 recording pipene.

Care was taken not to subject the healthy cells to suction currents that could damage their

membranes. The isolated cells were typically two to three cells apart, thereby

elirninating coupling via gap junctions.

11.1.6 Recording Cbamber and Superfusion

A cuticle segment containing isolated cells was clamped into a 0.5 ml Plexiglas

recording chamber (Churchill, 1993) and was constantly perfused at a rate of 1

ml/minute. Perfusion was achieved by gravity feed from 60 ml syringes. Each 60 ml

reservoir had its own Tefion needle-valve leading to a cornmon distributor (rnodified

plastic 1 ml pipette tip). At the beginning of the experiment, each reservoir was

individually tumed on to assure that the Tygon (Canlab, Toronto) tubing lead from the

needle-valve to the distributor was hl1 of saline. This assured instant flow fiorn each

reservoir when it was used dunng the experiment. The distributor was connected to a

22.5 gauge needle, which in tum was connected to PE 50 tubing (Canlab, Toronto). The

PE 50 tube kept the flow rate to 7 mlhinute. To minimize dead space the PE 50 tube

was only 25 cm long. The PE 50 tube rested on the bottom of the bath and was held

under the saline by surface adhesion forces. A vacuum was used to aspirate the bath.

Tygon tubing was slipped over the out channel of the bath and was connected to PE 50

via 12 and 22.5 gauge needles glued end to end. The PE 50 was then attached to Tygon

tubing that lead «, the building vacuum systern, which siowed the removal rate of saline

to match the fil1 rate. Al1 parts of the perfusion system were cleaned daily with distilled

water and dried with pressured air.

ïI.2 Whole-ce11 Recording

Al1 expenments were carried out on single ceils attached to the cuticle. The room

temperature was kept to 26 to 32 OC by a ceramic heater. The cuticle segments were

studied using whole-ce11 voltage-clamp technique (Hamill et ai., 198 1 ) within three hours

of being prepared.

Patch-pipettes were prepared on the &y of use. A two stage vertical pipette

puller (mode! 750, Kopf, Tujunga, California) was used to form 1 to 2 pm tip diameter

pipettes from 131.12 mm OD/D thin-walled borosilicate glass capillary tubing without

filament (TW 150-4, World Precision Instruments (WPI), Sarasota, Florida). Pipettes

were tipfilled by suction and then backfilled through a 0.2 mm low protein-binding

syringe filter with a fine quartz metal-free pipette-filling needle (MF28G-5, (WPI)).

Once filled, the pipette tip was coated with beeswax (Fisher Scientific, Toronto) to

reduce stray capacitance across the pipette wall. The pipettes typically showed a

resistance of 1 to 10 M n . The pipette was attached to a hydraulic micromanipuiator

(mode1 MO-207 attached to a MN-2/3 3-D micromanipulator, Narishige, Japan ). The

head stage was connected to a List LU EPC-7 patch-clamp voltage-clamp amplifier

(Medical Systems, New York). The ion-sensing element of the pipette was a Teflon

coated silver wire (AGT1010, 0.25 mm diameter, WPI) with the exposed tip chlonded

with 5% Javex for two hours before use.

Current signals were monitored by a digital storage oscilloscope (model 2720,

Tehonix, Washington) and recorded with a four-charnel ink chart recorder (model RS

3400, Gould, Cleveland, Ohio) and stored on an IBM compatible Intel 486-100 MHz

cornputer using a 125 lcHz TL- 1 DMA A/D interface (Axon Instruments).

Single ce11 studies used Axon Instniments' Pclamp stimulation, acquisition and

analysis sofhvare (version 6.02). Data was filtered at 1000 Hz using an eight pole low-

p a s Bessel filter (Senes 900, Frequency Devices, Haverhill, Massachusetts).

Back pressure ( 10 cm H,O) was applied to the pipette using a U-tube before it was

lowered into the bath to prevent debris blockage of the pipette tip. Before the pipette was

lowered into the bath saline, an electrical protocoi of O mV holding voltage and 25 msec

1 mV voltage pulses at 2 Hz was passed to the pipette. The amplifier was set to gain of

10 mVIpA. A gigaohm-seal was fomed between the pipette and cell membrane by

placing the pipette ont0 the membrane of a single ce11 and applying gentle mouth suction

(approximately 2 cm of HzO). Once a gigaohrn-seal (25GR) was formed, the stimulation

protocol was changed to a holding voltage of -30 mV while supplying 50 rnsec - 10 m V

voltage pulses at 1 Hz. Stray capacitance was cancelled using the capacitance

compensation control of the patch-clamp amplifier. Mouth suction was used to rupture

the membrane.

U.2.1 Correction for Liquid Junction Potentials

The liquid junction potentials between the pipette and bath solutions were detected as the

shift in the voltage measured when the composition of the saline in the bath was altered

relative to that in the pipette. AI1 data reported were corrected as needed. The rneasured

liquid junction potentials for the following pipette solutions were (pipette solutions

negative with reference to bath) 5 mV (33 m M CI-), 5 mV (80 rnM CI-) and I rnV ( 1 16

mM CI-) when the salines were changed fiom 415 mosmolll saline to 335 mosmol/l

saline. The measured liquid junction potential for the 26 rnM CI- bath (1 16 m M pipette

saline) experiments was 2 mV (electrode soiutions negative with reference to bath) when

the saline was changed from 415 mosmoVl ( 1 19 mM Cl-) to 335 mosmoVl (26 mM CI-).

Liquid junction potentials were reduced by placing the ground wire in a Petri dish

containing TBS, with a 3 M KCI 1% agar bridge connecting the Petri dish to the

recording chamber. This maintained a constant junction potential at the ground. Cl'

contamination of the bath saline was considered minimal since the bath was perf'used at a

rate of 2 ml/min.

II.2.2 Calculating Membrane Potentials

The Nernst equation was used to calculate reversal potentials based on the various

interna1 and external Cl- concentrations (Nernst, f 888).

&-=RTE In [C17J[C17,

R is the gas constant, T is 30 1 O K and F is the Faraday constant. Reversai potential (ERcv)

refers to the point where the current reversed sign. Since the background ("leakagee")

current was negligibie and had the same reversal potential as the inwardly rectihing

current, 1 regarded the point where the current crossed zero on the x-axis of a current-

voltage (IV) curve to be an appropriate measure of the reversal potentiai. This is

equivalent to the Nernst potential provided one ion is responsible for the current (Hille,

1992).

ïI.2.3 Ce11 Capacitance

Cell capacitance values were obtained by determining the area under the filter

compensated capacitance spike using Clampex version 6.02 (Axon Instruments).

III. RESULTS

m.1 Activation of an Inwardly Rectifying Current in Epidermal Cells

The currents associated with isolated cuticle-attached cells were measured in the

whole-ceIl patch-clamp configuration under conditions of equal intemal and external

osmolarities (4 15 mosmol/l). The cells were stimulated within one minute of gigaseal

formation and were heid at a holding potential of -30 mV and stepped from -100 to -60

mV for 2.25 seconds in 10 rnV increments separated by one second intervals. In

approximately 60% of the cells @=?O), a large inwardly rectifjing current spontaneously

activated (Figure 2). In the remaining cells, the current was of smaller amplitude but

could be fully activated by exposure to a hyposmotic saline (335 mosmol/l) (Figure 3).

Once activated, the current could not be suppressed by the reintroduction of 415

mosmol/l saline (data not shown). The maximum current recorded at -100 mV in both

groups of cells was not significantly different (t-Test p<O.OS).

m.2 The inwardly Rectifying Current is a CI- Current

Reversa1 potentials were detennined €rom the current-voltage (1-V) relationship

for induced currents within three to eight minutes after perfusion with hyposmotic saline

(335 mosmolA) (Figure 4). In normal bath and pipette salines, the reversai potential was

close to that predicted for chloride (Table 2). To test whether CI- was the dominant

component of the current, gluconate' was used to substitute for CI- in the pipene and bath

CI- solutions. The resulting reversal potentials (ERmr) were compared to those predicted

by the Nernst equation for Cl- (&). Table 2 and Figure 5 show the results of these

experiments.

Figure 2. Activation of the inwardly rectifiing current. Whole-ce11 current traces are shown in response to voltage jumps (-100 to +60 mV in 10 mV increments) applied From a holding potential of -30 mV. Zero current level is indicated by the dashed line between the traces. (A) Current measured 30 seconds after breaking into the cell under isosmotic conditions (4 15 mosmoVl bath saline and 415 mosmoVl pipette). (B) The inwardly rectifying current 4 minutes afier breaking into the ce11 under isosmotic conditions. (C) The current-voltage relationships for the initial current (m) and the current at peak activation (*). Ce11 capacitance was 6.2 pF.

Figure 3. Hyposrnotic treatment increases the amplitude of the rectiming current in cells that displayed a small initial current on hyperpolanzation. The current was induced by - 100 mV pulses in 4 15 mosrnolA saline. Adding hyposmotic saline at the time indicated by the vertical line increased the strength of the current in the cell. Time O refers to the fint pulse after the membrane was ruptured.

O 30 60 90 120 150 180 210 240 270 300 330 360

Seconds

Figure 4. Selectivity of the inwardly rectifying current. The reversa1 potential of the activated current could be altered by the substitution of bath or pipette Cl- with gluconate-. The symbols are the average values of 16 cells exposed to various intemal and extemai chloride concentrations with bars representing standard deviations. Currents showed inward rectification passing through the zero current axis close to the reversal potentials of -1, - 10, -33 and +38 mV predicted by the Nernst equation for Cl' (see next figure) for these four sets of solutions.

200 -

-400 - - - a- - 33 mM CI Pipettefi 19

mM CI Bath Saline - e- - 80 mM Pipette/ll9 mM

-600 - Cl Bath Saline - * - I l 6 mM CI Pipettell lS

mM Cl Bath Saline - 116 rnM CI Pipette126 -800 - mM CI Bath Saline

-1000 -

Figure 5. The observed reversa1 potentials fit the Nernst prediction for a Cl- current. The reversal potentials (ER=,-) show in Figure 4 and Iisted in Table 2 are plotted against the predicted reversai potentials (&,-). The lest squares best fit of the data (solid line) has a slope of 0.82. The dashed line, dope of 1 , is the predicted relationship for a CI- current. Values shown as means t SD, where larger than the symbol size.

50 - I

# #

40 - # - m .- c, 30 - C QI C1 O e 9

Q f! 0,

Best Fit Line - - - Predicted Reversal -30 - Potential

4 0 -

Obsented Reversal Potential

Table 2. Ionic Selectivity of the Activated Cu rrent - -- Pipette CI- mM - . Bath CI' mM Expected Ecl- - - Observed ER, 33 119 -3 3 -34 + 7 80 119 - 10 -11 2 4 116 119 - 1 -5 + 3 116 26 +38 +28 +, 5

Values are means f standard deviation (n=4). Reversai potentials (ERev) were measured at peak current amplitude.

[IL3 The Inwardly Rectifying Current is Not a K' Current

Even though the results above are consistent with the current being produced by

Cl', I wanted to rule out the possibility that the current was produced by Kt. To do this,

whole-ce11 currents were recorded under conditions where K' currents were blocked with

~ a " replacing K' in the extracellular solution and Cs' and TEA' replacing K' in the

electrode. Hyperpolanzation elicited an inwardly rectifjing current similar to that seen

in the absence of K' charnel blockers (Figure 6). Removal of K' fiom the bath and

pipette solutions did not change the reversa1 potential (E& = -1 and ERev = 4 f 7? n=5)

nor did it affect current amplihide (Figure 6).

III.4 The Inwardly Rectifying Current is Not a ~ a + Current

Since the inwardly rectibing cunent could also be due to ~ a ' entw, extracellular

~ a * was replaced with choline and hyperpolarizing stimulations were used to evoke the

inwardly recti@ng current. This manipulation did not change the reversa1 potentiai (Eci-

= -1 and E R . = -6 t 2) nor did it decrease the strength of the current strength when

compared to that in the presence of ~ a ' in the extracellular saline (n=5) (Figure 7).

Figure 6. Removal of K' from the bath saline and pipette does not alter the magnitude of the current strength or change reversal potentials. Whole-ce11 currents were induced in 335 mosmolA saline with pipette K* replaced with CsCl and TEACI. Five minutes afier the current was at maximum level (m), the bath saline was replaced with 335 mosmoVl K' free saline (K' replaced with ~ a ' + ) (+).The magnitude of the current is unaffected by the removal of K' and the reversal potential remained close to that predicted for Cl- (expected Ecl- = -1, observed ERrr = 4 f 7, n=4). Measurements for the 1-V graph followed the same protocol used in figure 2 and were performed five minutes after pefising with K+ free saline. Perfusion rate was 2 ml/minute and the bath volume was < 0.25 ml.

Figure 7. Na' fiee bath saline does not change the reversal potential or current strength. Once a maximum current strength had been obtained with 335 mosmol/I saline containing ~a+(*) , the saline was changed to a ~ a * free saline (m) (Na' replaced with choline+). The magnitude of the current is unaffected by the removal of ~ a * and the reversa1 potential remained close to that predicted for CI- (expected Ecl- = -1, observed ERev = -6 f 2, n=5). Measurements for the 1-V graph followed the same protocol used in Figure 1 and were performed five minutes afier p e f i i n g with the ~ a ' free saline. Perfusion rate was 2 murninute and the bath volume was 4 0.25 ml. Each point represents four cells. Bars represent standard deviations.

II1.5 Pipette cAMP Reduces the Current Activation Time

Newly moulted epidermal cells were incubated in 415 mosmol/l saline and then

subjected to the whoie-ce11 patch-clamp with either 200 p M CAMP or 1 mM cGMP in the

pipette solution (Figure 8). The average time taken to reach 1/2 rnâuimurn current

amplitude (t,,) was significantly reduced by adding cAMP to the pipette solution (49 + 20 seconds, n=7) (t-Test ~ ~ 0 . 0 5 ) compared to the tirne in the absence of CAMP (1 17 +_ 69

seconds, n=7). Cells patch-clarnped with pipettes containing 1 mM cGMP showed a

significant reduction in tlE (4 1 f 2 1, n=3) (t-Test pc0.05) compared to control ceils and

was sirnilar to that seen in cells exposed to CAMP via the pipette.

EI.6 Effects of Bath Osmolarity on the CI- Current in the Presence and Absence of Pipette cAMP

Cells were bathed for 10 minutes in hyperosmotic saline of 455 mosmoV! or

greater. When the cells were patched with pipettes containing 200 p M CAMP, the CI-

current seen on hyperpolarization was small and did not increase until the osmotic

strength of the bath saline was lowered to 415 mosmol/l (Figure 9 A). When the

hyperosmotic saline was reapplied the current retumed to its initial level (Figure 9 A).

When the cells were preincubated in a saline of slightly lower osmotic strength (445

mosmoUl), the current increased in a m p h d e without the application of hyposrnotic

saline (Figure 9 B). In the absence of CAMP in the pipette the current remained small in

similarly treated cells (Figure 9 C). The current increased on application of a less

hyperosrnotic (4 15 mosmoM) saline followed by the application of a stimulus protocol of

-100 to +60 mV (Figure 9 C). As seen when CAMP was present in the pipette solutions

Figure 8. The presence of 200 pM CAMP or 1 mM cGMP in the pipette shortened the tirne necessaiy for the current to reach '/1 maximum amplitude. Cells were incubated in 415 mosmoVl saline and then whole-ce11 patch-clarnped with CAMP or cGMP in the pipette saline. Currents were recorded with -100 mV pulses of 2.25 seconds. The scale bar applies to al1 three traces.

no nucleotide

Figure 9. The amplitude of the inwardly rectieing current is increased by CAMP, membrane hyperpolarization and osmotic stress. (A) Isolated cuticle-attached single cells incubated in 455 mosmol/l saline for 10 minutes were exposed to 4 15 mosmoVl saline and then retumed to 455 mosmolfl saline after maximum current was achieved at a membrane potential of -100 mV. (B,C) Current time courses for single cells recorded with (B) CAMP in the patch pipette and (C) without CAMP in the pipette. Both cells were preincubated in 445 mosmol/l saline for 10 minutes. Application of 41 5 mosmoln saline in (C) did not induce the current seen in (A). However, application of the standard stimulus protocol (black bar) was subsequently able to induce the current. Scale bar refers to al1 traces.

(Figure 9 A). the current strength decreased to its original size after the reapplication of

the hyperosmotic saline. The maximum current seen when the cells were exposed to 4 15

mosmo1A saline (Figures 9 A and C) was 841 + 104 pA with no pipette CAMP and 1050 f

180 pA with 200 pM pipette C A M P and was not statistically different &Test ~ 0 . 0 5 )

Incubation of newly moulted epidermal cells in 4 15 mosmol/l saline containinç

500 pM of 8-bromoadenosine 3'5 ' cyclic monophosphate ( ~ 6 ) or N",2'-0-

dibutyryladenosine 3'5'-cyclic monophosphate (n=4) unexpectedly did not decrease

cwrent activation time

EI.7 CAMP in the Pipette Shifts Sensitivity to Bath Saline Osmolarity

To determine in more detail how pipette CAMP (200 pM) affects the CI' current,

the current was measured over a broad range of bath osmolarity in the presence and

absence of CAMP in the pipette. Figure 10 shows how the current is affected by

osmolarity in these two conditions. The maximum currents were not statistically

different (t-Test ~0.05) for 415 and 335 mosmoVl saline, although the minimum

currents were statistically different The presence of 200 ph4 CAMP in the pipette

appears to raise the set point at which the current is activated by low osmolanty by

approximately 20 mosrnol/l. (n=3- 1 1 cells).

The current initially recorded at three minutes after patching ont0 a ce11 (n=3-11

cells) (t-Test ~0.05) in the presence 200 pM pipette CAMP showed a significant

increase of approximately 200 pA (Figure 1 1 ). However, the points within each

treatment group (with or without CAMP) are not significantly (t-Test ~ 0 . 0 5 ) different.

Two of the four cells recorded with CAMP in the pipette at a bath osmolality of 455

Figure 10. CAMP alten ce11 sensitivity to changes in bath osrnolariry. This is seen as a change in the amplitude of the peak current recorded on exposure to a given hyperosmotic saline. Cells were preincubated in vanous bath solutions for 10 minutes and then subjected to whole-ce11 patch-clamp with (e) CAMP (200 pM) or without (m) CAMP in the pipette solution (415 mosrnol/l) and stimulated with -100 mV of 2.25 seconds. The vertical dashed line represents the nominally isosmotic condition. Graphed are the maximum steady state currents recorded from these cells. Each point is the mean f standard deviation, n= 3 to 12 cells. Missing bars are obscured by the symbols.

330 350 370 390 41 0 430 450 470 490 51 0

Bath Osmolarity (mosmol/l)

Figure 1 1 . Pipette CAMP increases the amplitude of the initial current recorded from the cells used to compile Figure 10. Cells were preincubated in various bath solutions for 10 minutes with (*) CAMP (200 PM) or without (W) CAMP in the pipette. Each point represents the initial cunent at a -100 mV membrane potential after performing a step protocol stimulation experiment shown in Figure 2. Each point represents 3 to 12 cells with bars represent standard deviations. Missing bars are obscured by the symbols.

O 330 350 370 390 410 430 450 470 490 510

Bath Osmolarity (mosmolll)

mosmol/l (749 t 1 PA), and the 10 cells recorded with 465 and 495 mosmolil bath saline

did not show an increase in their current. This was simiiar for some of the cells without

CAMP in the pipette. Three of five ceils bathed in 435 mosmol/l saline showed no

change in current (80 + 20 PA), six of eight cells in 445 mosmol!l bath saline showed no

change (60 f 6 PA) and none of the three recorded at 455 rnosmol/l showed change. This

suggests that these are the osmolarities at which the current cannot be activated.

LU.8 Ion Selectivity of CAMP-Activated Curren t

To ver@ that CAMP activates a Cl' current, reversa1 potentials were detemined

from the current-voltage (1-V) relationship one to four minutes after perfusion with

isosmotic saline (415 mosmol/I) (Figure 12). The cells were held at -30 mV and stepped

from - 100 to +60 mV using the protocol descnbed earlier. Pipette CI- concentration was

lowered by substituting Cl- with gluconate-. The recorded revenals potential were

compared to those predicted by the Nernst equation.

Table 3. Ionie Selectivity of the CAMP Activated Current Pipette . - CI- - - mM - - - Bath - Cl- mM Expected - Ea- Observed ER, 33 149 -33 -37 1: 8 116 2 49 -6 -7 I 2 - - - - - -- - - - - - - - - - - . - -. . . - - - - - - -

Values are means + standard deviation (n=4). ~eve&al~otent ials (ERer) were measured at peak current amplitude.

m.9 Pharmacological In bibition of the Inwardly Rectiwiog Current

The effects of anion channel blocken on the osmotically and CAMP-activated

current were determined (Figure 13 and 14). Perfusion with hyposmotic saline

containing 250 pM 4,4 '-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), 500 pM

Figure 12. Cl- is the dominant ion in the current seen in the presence of CAMP. The 1-V graph shows cells in the whole-ce11 configuration with 1 16 m M Cl- and 33 rnM Cl* in the pipette (* and respectively) and 149 mM Cl' in the bath saline. The reversal potentials closely followed those predicted by the Nernst equation (see Table 3). Symbols represent the average of four cells and the bars represent standard deviations. Al1 salines were at 4 15 mosmol/l.

4,4'-dinitrostilbene-7,2'-disulphonic acid (DNDS), 1 m M 4-acetamido-4'-

isothiocyanostilbene-2,2'-disulphonic acid (SITS) or 200 p M diphenylamine-2-

carboxylate (DPC) resuited in the suppression of the inward current. Inward currents

were evoked by a -100 mV 2.25 seconds msec voltage pulses applied from a holding

potential of -30 mV. DIDS reduced the current within 3 minutes by 50 + 6 O h ( n 4 ) but

was not reversible. DNDS reduced the current within 6 minutes by 28 + 12 % (n=rl) and

was reversible. SITS reduced the current within 4 minutes by 15 + 1 ?6 and was

reversible (n=3). DPC reduced the current within 3 minutes by 31 + 1 1 YO (n=4) (Figure

13 & 14) and by 28 + 11 % (n=4) with 200 pM CAMP in the pipettes. Both treatrnents

with DPC were reversible .

m.10 The Inwardly Rectifying Curreat is Developmentally Regulated

A comparison was made of current magnitude in cells isolated from newly

moulted and intermoult larval epidermis. Whole-ce11 currents were evoked in mid-instar

epidermal cells bathed in hyposmotic saline with a holding potential of -30 mV and

stepped from -100 to +60 mV increments o f 2.25 seconds with one second intervals. At a

membrane potential of -100 mV the resulting current was 35% of that observed in newly

moulted cells (n=4) (Figure 15 A). When 200 CAMP was placed in the pipette, the

resulting current at a membrane potential of -1 00 mV in mid-instar cells was only 10% of

that observed in newly moulted cells (n=3) (Figure 15 B).

Figure 13. DPC largely abolishes the inwardly rectiSing current. The current was measured at -100 mV membrane potential. A 335 mosrnol/l saline containing 200 p M DPC (335 + DPC) was applied at 30 and 330 seconds. The first treatment reduced the current to -300 PA. Washing with 335 mosmolll saline did not fully restore the original current. Reapplying the 335 mosmolA plus DPC saline reduced the current more than seen dunng the first exposure. Washing with 415 mosmolfl saline increased current amplitude but not to the level seen initially Salines were changed at rimes when the current was stable.

335 + DPV 335 + DPC

O XI Q 80 12û 150 180 210 240 270 300 330 330 420 450 (80 510 540 570 600 630

Seconds

Figure 14. Several CI- channel bloc kers suppress the inwardly recti Qing curent. Peak currents were fiat obtained at a membrane potential of a - 100 mV in 335 mosmoVl saline and then the cells were perfused with 335 mosmolA saline containing 250 jM 44'- diisothiocyanostiIbene-2,T-disulphonic acid (DIDS), 500 pM 4,4'-dinitrostilbene-3,2'- disulphonic acid (DNDS), 1 rnM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS) and 200 pM DPC. The last bar shows the resuits obtained fiom using I mM CM in the pipette and 200 p M DPC in the bath saline. Bars represent standard deviations.

DNDS SlTS DPC CAMP + 200 pM DPC

Figure 15. The inwardly rectifiing current is developmentally regulated. The current in epidermal cells from 6 day (mid-instar) larvae (*) is weak compared to that in cells from newly moulted larvae (m), both (A) in the absence of CAMP and (B) with 200 pM CAMP in the pipette. (A) The single whole-ce11 current in mid-instar cells bathed in hyposmotic saline (335 mosmolA) reduced by DPC (.).(B) Whole-ce11 currents under isosmotic (4 15 mosmolA bath and pipette saline) conditions with 200 pM CAMP in the pipette. Each point represents four cells and bars represent standard deviations

IlI.11 The CAMP-Activated Current Couid Not be Switched on by Serotooin or Leukokinin I

Newly moulted epidermal cells were incubated with known insect hormones such

as 5-KT and leukokinin to see whether they were able to induce current profiles similar

to those seen with 100 pM pipette CAMP. 5-KT in insect salivary glands (Prince and

Bemdge, 1973) and Malpighian tubules (O'Donnell et a/., 1996) works through a CAMP-

second messenger cascade. After obtaining a whole ce11 configuration with isosmotic

pipette (no CAMP) and bath saline, the cells were perfused with (n=5 per treatment)

either 1 mM 5-EIT or 100 pM Ieukokinin I for 10 minutes at

26 O C . Neither treatment sigmficantly increased the current strength.

iII.12 The CAMP-Activated Current Could Not be Switched Off with a Proton Pump Blocker

A H' V-ATPase found in the apical membrane of the Drosophile rndanog~srer

Malpighian tubule (Maddrell and ODomell, 1992) has been shown to be the primary

active ion purnp in the apical membrane in many insect epithelia (Harvey and Wieczorek.

1997). The H' V-ATPase blocker, bafilomycin Al, was used to see if tuming off this

pump affected CI' currents. Newly moulted epidermal cells clamped in the whole-cell

configuration with 200 pii4 CAMP in the pipette were incubated at 26 OC for 10 minutes

in 2 pM bafilomycin A once the current reached a maximum steady state. No change in

the current strength was seen.

W. DISCUSSION

IV.1 CI- is the Major Contributor to the Inwardly Rectifying Current in Epidermal Cells

This study demonstrates that an inwardly rectifying Cl'-selective current in beetle

epidermal cells may be activated by hyperpolanzation, intracellular CAMP and by

osmotic stress. Evidence that the inwardly rectiQing current is a CI- current is based on

the following observations: (1) the inwardly rectifying current did not decrease in

amplitude nor did its reversal potential shift when the bath ~a was removed; (7) the

inwardly rectiQing current remained after pipette R was replaced with CS+ and TEA'

and bath K+ was replaced with ~ a ' + , a treatment that blocks many types of K* channels

(Standen, 1988; Kelly et al., 1994); (3) the inward current was suppressed by the anion

chamel blockers DDS, DNDS, SITS (Kelly et ai., 1994; Meng and Weinman, 1996) and

DPC (with and without pipette C A M P ) (Zhang el al., 1994) which have been shown to

suppress CI- currents in other ce11 types; (4) reversa1 potentials in cells recorded in the

presence or absence of pipette CAMP were similar to those predicted by the Nernst

equation for Cl-. Least-squares analysis of the ERer suggests the recorded current is

predominately the result of Cl- flux.

W.2 Regulatioo of the Inwardly Rectifying Current

About 60% of the cells immersed in bath saline isosmotic to the pipette saline

exhibited an inwardly rectifying current when stimulated by applying a voltage. In other

ce11 types this has been attributed to an osmotic imbalance that may occur upon

esrablishing the whole-ce11 recording configuration under apparently isosmotic

condit~ons (Worrell et al.. 1989; Kelly et uL, 1994). The CI- current in the beetle

epidermis is clearly sensitive to bath osmolarity because current decreased as saline

hyperosmolarity increased. It is possible the channel involved is a stretch-activated

mechanosensitive receptor (Oliet and Bourque, 1993) that, once tumed on, remains

active until the membrane retums back to its normal size. Extreme hyperosmotic saline

may shrink the ce11 suficiently to negate any mechanical stretch caused by the patch-

clamp pipette or hydrostatic pressure resulting from the pipette saline.

Canine red blood cells are reported to possess a volume-sensing mechanism that

is activated by changes in macromolecular crowding (Parker et ai., 1995). The attractive

interactions between the small electrolytes and the volume sensing proteins reduces the

thermodynarnic activity of the proteins, thereby reducing macromolecular crowding

without changing ce11 volume. The ce11 perceives the reduced crowding as swelling and

therefore activates volume reduction mechanisms. It is possible that the ions used in the

pipette saline are electncally different to those natively found in the beetle cytoplasm and

alter the interaction between the cytoplasmic proteins.

W.3 Cornparison with Cl- Currents in Other Cells

Osrnotically induced CI- currents have been described in a number of ce11 types

(for generai reviews see; (Lewis and Donaldson, 1990; Hoffmann and Simonsen, 1989)).

it has been shown that hyposmotic perturbations result in an increase in Cl- permeability

and regulatory volume decrease (Lewis et al., 1993; Ross et al., 1994; Zhang et al., 1994:

McCann et al., 1989; Kelly et al., 1994). Notably, in al1 of these examples the measured

Cl- exhibited outward rectification. Staley (1994), however, reported an inwardly

rectifjing chlonde current in the CA1 and dentate gyms regions of adult rat hippocampal

slice preparations and Noulin et a/. (1996) recently reported both a swelling and CAMP-

activated inwardly rectifjing CI- current in rat Leydig cells. This current was shown to

be sensitive to SITS. A cloned channel called C1C2 has been show to be activated by a

hyperpolarizing stimulus (Thiemann et ai., 1 992). Hyperpolarization-activated chloride

charnels in Apfysia neurons (Chesnoy-Marchais, 1994) and rat osteoblastic cells

(Chesnoy-Marchais and Fritsch, 1994) have been shown to exist.

Unlike the hyposmotically-induced CI- cunent in most other cells, the current

found in the beetle epidemal cells did not switch off when the cells were retumed to

saline isosmotic to the pipette saline. This, however, could be due to experimental

problems caused by the pipette or pipette saline, as outlined above.

W.4 CAMP Alters the Rate of Activation But Not the Amplitude of the Current

There was no significant difference in the size of the inwardly rectifjing current

recorded in isolated epidemal cells with and without CM in the patch pipette..

However, the time to reach maximum current strength was reduced by CAMP in the

pipette. This suggests that CAMP does not stimulate a separate population of CI-

charnels. A plausible explmation is that the addition of CAMP to the pipette corrects for

the loss of endogenous CAMP dialyzed out of the cells once the whole-cell patch-clamp

configuration was established. The slow increase in current strength in the absence of

pipette CAMP could be due to the time required to manufacture CAMP andior to a

mechanism îhat opens the channel being down regulated at low CAMP concentrations.

These possible explanations are supported by the observation that the current strength

was greater in experirnents containing pipette CAMP over most of the range of

osmolarities tested. Whatever the function of CAMP, it is evidently involved in the

activation of the Cl- channels.

This shifl in current sensitivity to bath saline osmolanty was also reported in the

CAMP- and swelling-activated outwardly rectiSinç chloride channel found in rat

hepatocytes (Meng and Weinman, 1996). These authon suggested that by reylating

intracellular CAMP concentration the ce11 is able to control the point at which the

swelling activated CI- chamel is stimulated. Such a mechanism in which extemal sigals

influence the ability of cells to regulate their volume via secondary messengers could

also be present in insects.

N.5 The Possible Role of c M P and Hormones in the Activation of the CI- Curreat

O'Domeii et al. ( 1996) found that anion and cation transport are under separate

hormonal and second messenger control. In their model, cGMP and CAMP are secondary

rnessengers that stimulate an apical V-ATPase to pump protons into the luminal space of

the Malpighian tubule. The resuiting concentration gradient drives K' into the luminal

side via a K'M+ antiporter. CI-, which is under the control of the intemal ~ a "

concentration and leukokinin, flows down its electro-chemical gradient and enters the

lumen via an anion channel. Addition of bafilomycin Al did not affect cunent strength.

as clamping the membrane potential bypassed the need for an active V-ATPase / K'M'

systern to produce the lumen-positive/intracellular-negative environment needed to move

Cf across the apical membrane out of the cell.

To further study the mechanism proposed by O 'Do~e i l er UL (1996), 1 tried to

elicit the CI' current by treating the cells with the hormones serotonin (5-

hydroxytryptamine) or leukokinin 1. Serotonin has been show to be an important insect

hormone that stimulates fluid secretion across salivary epithelia (Prince and Bemdge,

1973). O'Domeii et al. (1996) have show that leukokinin 1 increased CI- permeability

in Drosophila malpighian tubules. Both of these processes are thought to involve CAMP

acting on Cl- channels. The lack of reaction of epidermal cells to these hormones could

be due to the fact they do not control CI- current activity in this tissue. Altematively, the

membrane receptors involved were damaged by the pronase treatrnent used to strip the

basal lamina fiom the epidermal cells.

IV.6 Functional Significance of the CI- Channel in the losect Epidermis

Volume regulation is important for ce11 survival. Cells encounter fluctuations in

ambient osmotic conditions, although probably not as great as those used to demonstrate

the existence of volume regulating mechanisms in vitro. While the inwardly rectifying

Cl- current in the insect epidennis may play a role in volume regulation, its primary

physiological significance may lie elsewhere. Mid-instar epidermal cells in Tenebrio

molifor do not display the large inwardly rectiQing current detected in newly moulted

epidermal cells, implying that the CI' charnel is not a constitutive mechanism protecting

against environmentally or experimentally imposed osmotic stress. Rather its role in

volume regulation may be under hormonal control. Epidermal cells are known to double

in volume during the moult cycle, and then return to their original size by a mechanism

not presently understood.

W.7 Possible Models for the CI- Current in Insect Epidermis

Ln general terms, the CI' current may be pan of a mechanism used by the

epidemal cells to: ( 1 ) Resorb moulting fluid prior to ecdysis; (7) Reduce their size after

moulting; (3) Move water into rhe exuvial space to activate the rnoulting gel.

N.7.1 Moulting Fluid Resorption

The location on the ce11 surface of the Cl' chamel in the beetle epidermis should

influence its function. 1 attempted to determine on which face (apical/basolateral) of the

cell the charnels were located by using the cell-attached configuration. Unfominately

these expenments were unsuccesshl. Work done by others on different insect cells may

explain the function of the CI- current. Cooper and Jungreis (1985) using an Ussing

chamber to record Cl- currents in M a n d m sexra epidermis found that there was a net

chlonde movement from the exuvial space towards the hemolymph side of the

integurnentary epithelium. The possible role of this chloride current in terms of the

resorption of moulting fluid is difficult to reconcile with Cornell and Pan's (1983)

observations that M a n d m imbibes (via the mouth and anus) the moulting fluid and does

not resorb it through the integument. Similarly, C. Yarerna (work in progress) has found

that another caterpillar also imbibes its rnouiting fluid and that linle nuid is absorbed

across the epidennis. Cooper and Jungreis ( 1985) therefore suggested that CI' resorption

during moulting fluid resorption rnay be a cellular response to changing hormonal

conditions and may not have any thing to do with fluid movement. The chloride current

may reduce intemal ce11 chionde concentrations that may inhibit enzymes (Cooper and

Jungreis, 1985).

Figure 16. Mode1 proposing how a Cl' current could be involved in moulting fluid resorption. Hormone (X) stimulates a CAMP andor cGMP pathway to activate a mechanism that draws digested components of the cuticle and inorganic ions from the exuvial space into the cell. The increased intracellular solute level causes the osmotically obligated movernent of water tnto the cell. The resulting ceIl swelling and/or the hormonal stimulation activates a volume regulatory mechanism involving Cl' efnux throught charnels on the basolateral surface of the cell. Fluid moves out of the ce11 and into the hemolymph, thereby reducirig the cell volume. In this way the components of moulting fluid could be transported across the epidermis from the exuvial space to the hemolymph.

Exuvial Space

K+ CI-

Fluid K' CI-

Hemolymph

Phillips et aL( 1988) proposed that an electrogenic Cl' pump exists on the luminal side

and a chloride channel on the basolateral side in the hindgut epithelium of the desert

locust (Schzstocercu gregurra). The pump and channel are apparent1 y both stimulated by

CAMP. Locusts excrete a strongly hyperosmotic secondary unne by re-absorption of a

hyposmotic fluid through solute-dnven osmosis in the hindgut. Under hormonal

stimulation the apical CI- pump actively rnoves CI- into the cell with K' following down

its electrochemical gradient. The increased solute level leads to osmotically obligated

movement of water into the cell. CI- then flows down its concentration gradient into the

hemolymph across the basolateral surface of the epithelium along with other anions and

cations. This again forces water to flow down its concentration gradient but this time

into the hemolymph. Assurning a mechanism of this type is also found in the epithelial

cells underlying the cuticle of the entire animal, and Comell and Pan's observation does

not apply for Tenebrio mulitor, this would suggest a resorptive role for the CI' channel

that ai& in moulting fluid absorption. However, unlike pnmary urine in locust, moulting

fluid of A4anducu (Jungreis, 1978) contains little Cl- in cornparison to cations and other

anions such as HC03-. If the Cl- channel characterized in Tenebrio molitor does in fact

play an absorptive role it is possible that it rnay be a non-selective anion channel and

experimentai salines simply highlighted its ability to carry Cl-. The findings Phillips et

al. (1988) and Cooper and Jungreis (1985) would place the Cl* channel on the basal side

of the Tenebrio rnolttor epidennal cell.

IV.7.2 Volume Reduction

Assuming Corne11 and Pan ( 1983) are correct, then it is possible the Cl- channel is

pan of a mechanism by which the epithelial cells retum to their original size before the

moulting process began. The enzymes and proteins manufactured by the epidermal cells

to digest the old cuticle and construct the new cuticle could act as osmolytes. This would

result in the movement of water into the cells causing them to swell. Funhermore

Tomlin er al., (1992) have show that the Tenebrio rnolitor epidermal cells are efficient

at taking up glutamate. When they do, the epidermal cells become swollen. If glutamate

and other amino acids are byproducts of cuticle digestion and are taken up by the cell,

then the cells may swell. The ability for the Cl- channel to be stimulated by CAMP would

allow the cell to reduce its solute load thereby decreasing its size even in hyperosmotic

conditions. As the glutamate is processed and diffuses from the ce11 CI- and other ions

re-enter the ceil to balance its solute load. Again this would place the Cl- channel on the

basal side of the cell.

iV.7.3 Moulting Fluid Secretion

The most likely role for the Cl- current is in fluid secretion. The Cl- channels

could be situated on the apical side of the epithelial cells where they could aid in moving

fluid into the exuvial space during moulting gel activation (Katzenellenbogen and

Kafatos, 1971). The discovery of the apically located V-ATPase pump and the K'W

antiport in the Malpighian tubules of Rhodnius and Drosophzla (O'Donnell el al., 1996)

and the sensilla of Manduca sexta and Anfhernea pernyi (Klein and Zimmermann, 199 1 )

supports this conclusion. The V-ATPase pump along with the K'/H+ antiport has been

Figure 17. Volume reduction model. Insect epidermal cells are known to increase in volume during the moult cycle. The e-es and proteins manufactured by the epidermal ce11 to digest the old cuticle and constmct the new cuticle could act as osmolytes drawing water into the cell. Furthemore the absorption of cuticle digestion byproducts (amino acids and sugars) could act as osmolytes causing the cells to swell. The activation of the Cl- charnel on the basolateral plasma membrane would allow the ce11 to reduce its solute load thereby decreasing its size once moulting ras completed. The rnovement of CI- would be accompanied by other ions. This mechanism could be under the control of an unknown hormone (X) that through CAMP andor cGMP may determine when this volume regulatory mechanism is activated or deactivated.

Exuvial Space

Hemolymph

Exuvial Space

Hemolymph

Figure 18. Mode1 proposing how a Cl- current could be involved in the secretion of moulting fluid. The epidemis activates the moulting gel located in the exuvial space by secreting a Kt nch Buid into this space. Hormone (X) may activate pumps and charnels via cyclic-nucleotide pathways resulting in the transport of inorganic ions into the exuvial space. This resulting concentration gradient would move fluid from the hemolymph across the epidemis into the exuvial space.

Exuvial Space

Hemolymph

s h o m to be responsible for moving cations into the tubule lumen. Cl* moves passively

through its channel down an electrochemical gradient and water then follows. A similar

chloride movement occurs in the Malpighian tubules of the tsetse fly ( (;/ossrnu rnorsrruns

morsifans) (Isaacson and Nicolson, 1996), in which CM has been shown to cause the

tubule cells to swell. Fluid excretion into the lumen then follows.

This mode1 is supponed by the observation that the Cl- current is strongest in

epidennal cells when the membrane is hyperpolarized. Asswninp there is a cation pump

on the apical side of the cell, movement of cations from the ce11 would cause it to

become hyperpolarized which in tum would activate the chlonde channel. The presence

of a cation purnp may also explain why no cation current was detected. Experimentally,

the cells were hyperpolarized by an applied voltage possibly explaining why the current

was active under isosmotic conditions. Once tumed on it remained active. The

hormonal message that causes the cells to swell may also be responsible for the

production and/or insertion of the CI- channel protein in the plasma membrane andior

activation. It is also possible the simple act of swelling is somehow responsible for

activating secretion. If this were the case, the hyperosmotic saline would reduce the size

of the cell, hence turning off the secretory rnechanism. CAMP could be the messenger

that activates the channels or it could keep the secretory process going when the

hemolymph is hyperosmotic to the moulting fluid. Jungreis (1978) showed that in

M a n d m the osmotic pressure of the hemolymph was highest at the begtming of the

moult cycle and became isosmotic to the exuvial space later in the moult cycle. In this

report it was shown that CAMP did activate the cunent in hyperosmotic conditions.

Therefore, the innacellular concentration of CAMP would decrease as the moulting cycle

proceeded because it would no longer be needed to activate the current. Delachambre et

uf. (1979) have show that CAMP levels in the epidermis, peak at the time of the moult

fluid production.

The models assume that the movement of cations from the ceIl is balanced

electrically by Cl' effiux. This does not eliminate the possibility that cation channels do

not exist, but under the conditions used in this work Cl- was the major current recorded in

these cells. It is likely that rn siru the natural current is actually the net result of a

combination of various ions with Cl- contributing to the bulk of the current. Further work

is required to identiQ the components of the current. The administration of various K-

and ~ a - channel blockers could help with this problern. Furthemore, the specificity of

the anion channel to chlonde should be f ~ h e r investigated. It is possible this channel is

also responsible for the movement of other anions (phosphate, organic anions, OH' and

HC03') under normal physiological conditions and that the experimental conditions used

here artificially emphasized its selectivity for Cl-.

V. CONCLUSIONS

1 have shown previously (Watson, 1995) that the epidermal ceils of newiy-

moulted beetle larvae are capable of volume regdation afier being induced to swell. The

mechanism involved may be primarily for moving moulting fluid into/out from the

exuvial spaces rather than a defensive mechanism against hyposmotic stress. An

inwardly rectiQing current is activated in these cells during osmotic stress, exposure to

CAMP and by electncal stimulation. This current is the result of the efflux of anions

(mainly CI') from these cells. Since the anion charnels generating this current are under

the control of intracellular CAMP and/or cGMP, this inwardly recti@ing current is

presurnably stimulated under normal conditions by a yet-to-be îdentified honnone(s) or

neuropeptide(s).

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