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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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