J. exp. Biol. 163, 169-186 (1992) 169Printed in Great Britain © The Company of Biologists Limited 1992

THE IONIC BASIS OF THE HYPO-OSMOTICDEPOLARIZATION IN NEURONS FROM THE

OPISTHOBRANCH MOLLUSC ELYSIA CHLOROTICA

BY R. H. QUINN* AND S. K. PIERCE

Department of Zoology, University of Maryland, College Park, MD 20742, USA

Accepted 15 October 1991

Summary

The resting potential of identified cells (Parker cells) in the abdominal ganglionof Elysia chlorotica (Gould) depolarizes by about 30 mV in response to a 50%reduction in osmolality and returns to the original potential in 20 min. Cell volumerecovery requires approximately 2 h. Thus, recovery of the resting potential is notdependent on recovery of cell volume. The hypo-osmotic depolarization persistsfollowing inhibition of the electrogenic Na+/K+-ATPase with ouabain, and thelevels of extracellular K+ and C\~~ have little effect on the magnitude of thedepolarization, while decreasing extracellular Na+ concentration produces adepolarization of only 10 mV. This suggests that the hypo-osmotic depolarizationin Parker cells results mostly from increased relative permeability to Na+ .Following transfer from 920 to 460mosmolkg~1, Na+ , Cl~ and proline betaineleave the cells while intracellular K+ is conserved. Loss of intracellular Na+ andconservation of intracellular K+ are dependent on active transport by theNa+/K+-ATPase. Na+ and proline betaine leave the cells with a time course thatis much longer than that of the hypo-osmotic depolarization. Unlike the othersolutes, most of the reduction in intracellular Cl~ concentration occurs coinciden-tally with the hypo-osmotic depolarization. However, unlike the hypo-osmoticdepolarization, bulk loss of Cl~ does not require the reduction in osmolality, onlythe reduction in extracellular ion concentrations. There is no apparent relation-ship between membrane depolarization and the regulation of intracellularosmolytes in Elysia neurons following hypo-osmotic stress.

Introduction

In response to hypo-osmotic stress, membrane potential changes occur in somebut not all cell types. For example, a hypo-osmotic stress produces a membranedepolarization (sometimes preceded by a brief hyperpolarization) in Sabellapenicillus giant axon (Treherne and Pichon, 1978), Mytilus edulis cerebrovisceralconnectives (Willmer, 19786), Mya arenaria neurons (Beres and Pierce, 1981),

* Present address and address for correspondence: 360 Minor Hall, U.C. Berkeley, Berkeley,CA 94720, USA.

Key words: hypo-osmotic, membrane depolarization, neurons, Elysia chlorotica.

170 R. H . Q U I N N AND S. K. PIERCE

Limulus polyphemus cardiac ganglion follower cells (Prior and Pierce, 1981), ratbrain astrocytes (Kimelberg and O'Connor, 1988), Madin-Darby canine kidneycells (Volkl etal. 1988), Ehrlich ascites tumor cells (Lambert etal. 1989) andopossum kidney (OK) cell line (Ubl etal. 1989). Following depolarization, themembrane potential repolarizes back to, or slightly below, the original restingpotential (Prior and Pierce, 1981). In contrast, hypo-osmotic stress hyperpolarizesNecturus maculosus small intestine (Lau et al. 1984) and mouse hepatocytes(Howard and Wondergem, 1987). However, no membrane potential changeoccurs in Callinectes sapidus walking leg muscle (Lang and Gainer, 1969) orAmphiuma means tridactylum blood cells (Cala, 1980) in response to the stress.

The hypo-osmotically induced depolarization does not occur when ions aloneare reduced in the absence of an osmotic change (Carlson et al. 1978; Prior andPierce, 1981). Thus, the reduction in osmolality and not the reduction inextracellular ion concentrations causes the hypo-osmotic depolarization. Thepermeability changes that result in osmolyte loss and volume recovery followinghypo-osmotic stress also result from the reduction in osmolality rather than inextracellular ion concentrations (e.g. human red cells, Poznansky and Soloman,1972; bivalve ventricles, Pierce and Greenberg, 1973; Ehrlich ascites tumor cells,Hendil and Hoffman, 1974; flounder erythrocytes, Fugelli and Rohrs, 1980; sheepred cells, Dunham and Ellory 1981; flounder ventricles, Vislie, 1983). Thus, boththe hypo-osmotic depolarization and the permeability changes that controlosmolyte efflux and cell volume regulation result from the same stimulus.

However, a causal relationship between the membrane potential change andsolute efflux during hypo-osmotic stress has only been established in three celltypes. Hypo-osmotic stress activates a conductive efflux of Cl~ and K+ in humanlymphocytes (Grinstein etal. 1982), Chinese hamster ovary cells (Sarkadi etal.1984) and Ehrlich ascites tumor cells (Lambert et al. 1989). The hypo-osmoticallyactivated Cl~ permeability is greater than that for K+ , resulting in membranedepolarization (human lymphocytes, Grinstein et al. 1982; Chinese hamster ovarycells, Sarkadi etal. 1984). Similarly, the hypo-osmotic depolarization in Mytilusedulis connectives results from increased permeability to Cl~ (Willmer, 19786);however, a relationship to solute loss was not established.

The purpose of this study was to determine whether the hypo-osmoticdepolarization in neurons results from a volume-regulating efflux of ions. Theionic basis of the hypo-osmotic depolarization was examined to identify which ionsare involved, the levels of intracellular osmolytes were measured to determinewhich ones are responsible for cell volume regulation, and the time courses of thedepolarization and solute loss were compared to that of cell volume recovery totest for correlation between the events.

Ganglia from an opisthobranch mollusc, Elysia chlorotica, were chosen for thisstudy for several reasons. Elysia is an extremely euryhaline osmoconformer; bloodosmolarity parallels environmental osmolarity over a wide range (Pierce et al.1983). Thus, the individual cells from any tissue in this animal must be extremelycapable volume regulators. Also, the abdominal ganglion contains a pair of

Hypo-osmotic depolarization in mollusc neurons 111

identified somas, the Parker cells (Parker and Pierce, 1985), which are well suitedfor electrophysiological measurements. Since the cells are uniquely identifiable,the same cells could be utilized in every animal, thus reducing the variability thatmight be encountered by using random cells. The large size of these cells(approximately 100 pim in diameter) permits easy microelectrode penetration forintracellular recording, and impalement is usually not dislodged by cell swellingfollowing a change in osmolality from 920 to 460 mosmol kg"1. Parker cells arevery tolerant of hypo-osmotic stress and maintain excitability even after abruptdilution from 100 % to 25 % sea water. Also, the large size of these cells allows foroptical measurement of cell volume changes. Like most cells following hypo-osmotic stress, Parker cells swell and then partially recover their initial cellvolume. Finally, like other neurons, Parker cells transiently depolarize in responseto hypo-osmotic stress.

Materials and methodsMaintenance of animals

Elysia chlorotica were collected from a salt marsh near Menemsha on Martha'sVineyard Island, MA. The animals were maintained in Instant Ocean sea water at920 mosmol kg"' and 10°C on a 15 h dark, 9 h light cycle. Animals were allowed tocome to room temperature just prior to an experiment, and all experiments wererun at room temperature, 21.6±1.5°C (mean±standard deviation).

Solutions

Artificial seawater (ASW) solutions with differing ionic contents were mixedfrom stock solutions of individual, reagent grade salts using formulae adaptedfrom Costa and Pierce (1983) buffered to pH7.8 (Table 1). Concentrations ofindividual ionic species in the ASW were manipulated by substituting equimolaramounts of one salt for another as indicated. Quinidine and ouabain wereobtained from Sigma.

Membrane potential measurements

Circumesophageal nerve rings were dissected from animals that had beenacclimated to 920 mosmol kg"1 for at least 5 days. The ring of ganglia wastransferred to a bathing chamber containing 920 mosmol kg"1 ASW and secured tothe Sylgard bottom of the chamber by covering it with a nylon mesh held by glasspins. The chamber allowed for rapid superfusion of the tissue. During ionsubstitutions, the tissues were exposed to the ion-substituted ASW at 920 mos-mol kg"1 for 5min before perfusion with ion-substituted ASW at 460 mos-mol kg"1.

Membrane potentials were recorded intracellularly from one of an identifiedpair of pigmented somas, Parker cells, on the abdominal ganglion (Parker andPierce, 1985) by impalement with 3moll"1 KCl-filled glass micropipettes. Electri-cal connection to the KCl-filled pipettes was made by way of a Ag-AgCl pellet

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Hypo-osmotic depolarization in mollusc neurons 173

encased in a Lucite holder (WPI, Inc.). The reference electrode was a glass pipettefilled with 3 mol 1~' KC1 in 8 % agar and a chlorided silver wire sealed into the endof the pipette with epoxy. The electrodes were connected to a preamplifier (WPI,M-707) with outputs to an oscilloscope, a digital voltmeter and a chart recorder(Grass Instruments 79C). The amount of depolarization following osmoticreduction was calculated as the difference between the resting potential just priorto the reduction in osmolality and the peak of depolarization following osmoticdilution. Where action potentials occurred, the spikes were electronically filtered(half-amplitude frequency=0. lms) and the amount of depolarization was calcu-lated from the filtered trace. To estimate the effect of liquid junction potentials atthe reference electrode, each solution was tested for its effect with both electrodesin the bath, assuming that the liquid junction potential at the 3 mol I"1 KCl-filledmicroelectrode is negligible. The differences between the electrodes when goingfrom 920 mosmol kg"1 to 460 mosmol kg"1 in the following solutions were:1.0±0.4mV (normal ASW), 2.8±0.9.mV (Na+-free ASW), 3.6±0.6mV(Na+:Ca2+-free ASW), 2.0±2.0mV (Na+-free:low-Ca2+ ASW), 3.5±1.0mV[Na+-free (arginine) ASW], 3.2±0.4mV (low-Cl" ASW), 2.2±0.5mV (low-K+

ASW), 0.0mV (Ca2+-free ASW), microelectrode negative. These differences aretoo small and of the wrong polarity to account for the recorded membranepotential changes. In solutions using sucrose as a NaCl substitute, the differenceswere larger but again of the wrong polarity to account for the membrane potentialchanges: 7.8±1.4mV (Na+-free:low-Cr ASW) and 7.7±0.5mV (Na+:Ca2+-free:low-Cl" ASW), microelectrode negative.

Effect of ouabain on the hypo-osmotic depolarization

After impaling a Parker cell and establishing a stable resting potential in920 mosmol kg"1 ASW, the bath was perfused with 920 mosmol kg"1 ASW plus10"~4moll~' ouabain until a new stable potential was reached. To establishwhether the effect of ouabain was maximal at 10~4moll~1, the bath was thenperfused with ASW containing 10~3moll~1 ouabain in three trials. No additionaleffect was observed by increasing ouabain to 10~3moll~1; thus, the effect wasmaximal at 10~4moll~1. After reaching a stable potential in ouabain, theosmolality was reduced from 920 to 460mosmolkg~1.

Measuring the change in cell volume

Circumesophageal nerve rings were dissected from animals acclimated to920 mosmol kg" l for at least 5 days. The abdominal ganglia were separated fromthe nerve rings and transferred by pipette in a drop of 920 mosmol kg"1 ASW to amodified microscope slide that allowed exchange of the solution in a smallchamber under the coverslip. Each ganglion was oriented with a Parker cell facingup and the coverslip in contact with the surface of the ganglion. The slide was thenmounted on a compound microscope equipped with differential interferencecontrast (DIC) optics. The cell borders of a Parker cell were resolved usingoblique lighting, produced by setting the phase ring slightly off center, in

174 R. H . Q U I N N AND S. K. PIERCE

combination with DIC. The 920 mosmol kg~' ASW in the chamber was thenexchanged for 460mosmolkg~1 ASW or 920mosmolkg~' ASW. The solution inthe chamber was replaced with fresh solution every 5 min during the course of theexperiment. Video recordings were made using a camera (Dage MTI 67-ML)mounted on the microscope and connected to a video cassette recorder. Theserecordings were played back and the cross-sectional area of each Parker cell wasdetermined with an image analyser (Bioquant II, R&M Biometrics). The changein cell volume was expressed as a percentage of the initial cross-sectional area of asingle optical section of the cell.

Measurement of proline betaine

Circumesophageal nerve rings were dissected from animals acclimated to920mosmolkg~1. The nerve rings were then transferred by pipette directly toASW at 920 or 460mosmolkg~1 in groups of five. At the end of the incubationperiod (20, 60 or 120 min) each group of nerve rings was transferred to a filterpaper (Whatman no. 1). After brief blotting, each group was transferred to a smallpiece of cellophane, which served as a support for handling and weighing thegroup of nerve rings. The tissue was then frozen and freeze dried. The dried nerverings were then weighed to the nearest 10~7g on an ultramicro balance (Mettler,UM-7), homogenized in cold 40% ethanol, heated just to the boiling point for10min, and centrifuged at 20000g for 20 min (Pierce etal. 1984). The pellet wasdiscarded, and the supernatant was freeze dried. The residue was then dissolved in1 ml of double distilled water and filtered (0.2 mm). The sample was freeze driedagain and finally redissolved in 100ml of 0.040moll"1 NaH2PO3 buffer (pH4.0).

Proline betaine was measured by high performance liquid chromotography(HPLC) (ALTEX model 334) (Pierce et al. 1984) with a reverse-phase column(Microsorb-short one: C-18, 10cm ODS with 3mm diameter particles, RaininInstruments). Proline betaine was detected at 200nm (Gilson model HM) andchromatograms were analysed with a computer (Shimadzu C-RIA). The mobilephase was O.O4O1110IP1 NaH2PO4 (pH4.00). Standards were prepared usingproline betaine that had been purified from tissue extracts (Pierce etal. 1984).

Measurement of intracellular ions

The intracellular ion content of the ganglia was determined by measuring totaltissue ion content and then subtracting the ion content of the extracellular space(ECS) estimated using 14C-labelled polyethylene glycol (Mr 4000) as an ECSmarker. For each measurement, three circumesophageal nerve rings from animalsacclimated to 920 mosmol kg~l were pooled and incubated for either 20 or 90 minin ASW at 920 or 460 mosmol kg"1. The protocols for incubating the tissues indifferent experimental media, ECS determination and measurement of tissue Cl~are described in detail elsewhere (Quinn and Pierce, 1990). For Na+ and K+

determination, dried nerve rings were wet-ashed in concentrated HNO3 over-night. The acid was then dried under a stream of ultrapure N2, and the residue wasdissolved in 2 ml of diluting solution (6 % LiCl in 0.1 mol I"J HNO3). 1 ml of this

Hypo-osmotic depolarization in mollusc neurons 175

was taken for determination of ECS by liquid scintillation counting. An additional2 ml of diluting solution was added to the remainder, and [Na+] or [K+] wasmeasured by atomic absorption spectrophotometry (Perkin Elmer model 560).

Relationship between bulk loss of Cl~ and the hypo-osmotic depolarization

We tested whether Cl~ loss was dependent on the reduction in osmolality, as isthe hypo-osmotic depolarization, or was a response to the reduction in extracellu-lar ions alone. This was done by transferring ganglia from animals acclimated to920mosmolkg~1 to 920mosmolkg~1 ASW, 460mosmolkg~1 ASW or a solutionionically equivalent to 460mosmolkg~1 ASW with sucrose added to bring theosmolality up to 920 mosmol kg"' (iso-osmotic-hypoionic) for a period of 20min.Then intracellular Cl~ content was determined as previously described.

Active transport of Na+ and K+

Ouabain was used to test for a role of the Na+/K+-ATPase in the regulation ofthe intracellular content of K+ and Na+ following hypo-osmotic stress. Gangliafrom animals acclimated to 920mosmolkg~1 were transferred to 920 mosmol kg~ *or 460 mosmol kg"' ASW with or without ouabain (5xlO~4moll~1) for 20minbefore determination of intracellular K+ content and 90min before determinationof intracellular Na+ content.

Statistics

Analysis of variance and the Student-Newman-Keuls multiple-range compari-son (Sokal and Rohlf, 1969) were used to test for significant differences.Significance was accepted at P=s0.05.

ResultsCell volume regulation

Video images of Parker cells exposed to reduced osmolality initially show anincrease in cross-sectional area, indicating an increase in cell volume, followed bya decrease in cross-sectional area, indicating volume recovery (Fig. 1). Theaccumulated results from a number of such video recordings show that Parker cellsswell rapidly during the first 10 min following the reduction in osmolality, and thengradually recover towards the original volume over the next 90 min (Fig. 2).Volume recovery is significant by 60min.

The hypo-osmotic depolarization

In response to a reduction in osmolality from 920 to 460mosmolkg~1, Parkercells depolarize for 3 min, spike spontaneously, and then repolarize, taking20±3 min (N=9) to return to the original resting potential (Fig. 3). Thus, recoveryof the resting potential requires much less time than cell volume recovery. Thehypo-osmotic depolarization does not occur in a solution ionically equivalent to

176 R. H. QUINN AND S. K. PIERCE

Fig. 1. Photographs of a Parker cell taken from the video monitor of the imageanalyser. The photographs on the left show the cell at Omin (A), 20min (B) and120 min (C) following transfer from 920 to 460 mosmol kg"1 ASW. The photographs onthe right show the digitizer tracings of the cell at the same time points. The calculatedareas are A, 478 ,um2, B, 536 fim2, and C, 511 jim2. Scale bar, 100^m.

Hypo-osmotic depolarization in mollusc neurons

140-

_ 130-

177

15 30 45 60 75

Time (min)

90 105 120

Fig. 2. Change in cell volume of Parker cells after transfer from 920 to 920 mos-mol kg" or 460 mosmol kg"' « » ASW. The change in cell volume is expressed asa percentage of the initial cross-sectional area of an optical section of the cell. Errorbars show S.E.M. and N=6 (iso-osmotic) and 9 (hypo-osmotic).

-68 mV- --70mV

I920 to 460 mosmol kg

t14min 21 min

|50mV30 s

Fig. 3. Hypo-osmotic depolarization in a Parker cell. The upper trace is an intracellu-lar recording of the membrane potential. The lower trace is the same recording withthe spikes electronically filtered (see text). Dotted lines are at — 68mV.

460 mosmol kg"1 ASW while the osmolality is maintained at 920 mosmol kg"1

(sucrose) (Fig. 4). Furthermore, cells transferred from sucrose-substituted ASWat 920 mosmol kg"1 to ASW at 460 mosmol kg"L depolarize immediately(44±2mV, yV=3) (Fig. 4).

Manipulating the level of K+ or Cl~ in the ASW had no significant effect on thehypo-osmotic depolarization, while substituting Tris+ or arginine for Na+ signifi-cantly reduced the hypo-osmotic depolarization (Table 2). Although both Na+

substitutes significantly reduced the depolarization, Tris+ was more effective thanarginine. However, even in Na+-free Tris+, 10 mV of the depolarization was stillunaccounted for. The hypo-osmotic depolarization remaining in Na+-free solutionwas not affected by reducing or removing Ca2+ (Table 3).

178 R. H. QUINN AND S. K. PIERCE

100% ASW - ^ 50% ASW+sucrose920mosmolkg~' 920mosmolkg~1

-65 mV s -77mV

-80 mV'

40 mV

22min 50% ASW460 mosmol kg"'

Fig. 4. Unfiltered intracellular recording from a Parker cell showing the effect of iso-osmotic vs hypo-osmotic salinity reduction. In the upper trace, the ionic concentrationis reduced by 50 % while osmolality is kept iso-osmotic with 100 % ASW by addingsucrose. The lower trace is from the same cell at a later time. After 22min in 50%ASW+sucrose the osmolality is finally reduced by superfusion with 50% ASW.Dotted lines are at - 65mV (upper trace) and - 8 0 m V (lower trace).

Table 2. Hypo-osmotic depolarization in normal ASW vs Cl -, K+- or Na+-substituted ASW

Treatment(920 to 460 mosmol kg"1)

Normal ASWLow-CP ASWLow-K+ ASWNa+-free (arginine) ASWNa+-free (Tris+) ASW

See Table 1 for solution compositions.

Meandepolarization±s.E.M.

(mV)

31±136±333+321+310±l

N

10654

11

The hypo-osmotic depolarization remaining in the Na+-free solution wassignificantly increased by reducing Cl~ concentration, by replacing NaCl witheither sucrose or trismethanesulfonate (TrisMS) (Table 3). The Na+-free hypo-osmotic depolarization was also increased by quinidine (Table 3). Quinidine hadno effect on the membrane potential in iso-osmotic Na+-free solution.

Incubation of the Parker cells in 920 mosmol kg"1 ASW containing 10~4moll~J

(or 10~3moll~1) ouabain produced a small depolarization, 6±1 mV (N=6) aboveresting potential (Fig. 5). Subsequent osmotic reduction from 920 to 460

Hypo-osmotic depolarization in mollusc neurons 179

Table 3. Hypo-osmotic depolarization in Na+-free ASW: effect of Ca2+ -free, low-Cl~ and quinidine treatments

MeanTreatment depolarization ±S.E.M.(920 to 460 mosmol kg~') (mV) N

Na+-free (TrisHCl)Na+-free:low-Ca2+

Na+:Ca2+-freeNa+-free:low-Cr (sucrose)Na+-free:low-Cr (TrisMS)Na+-free (TrisHCl)+1 mmol I"1 quinidine

See Table 1 for solution compositions.

10±l12±411±229 ±423 ±225+2

113

14545

10 moll ouabain -

A

-65 mV

1 4 m i n 36 mVB hUomVt

10"3 moll"1 ouabain • ! 30 s

9 2 0 - • 460 mosmol kg"1

Fig. 5. Intracellular recording from a Parker cell showing the effect of ouabain on themembrane potential and the hypo-osmotic depolarization. A and B are simultaneousrecordings; B is filtered. The lower pair of traces is a continuation of the upper pair.Dotted lines for the upper pair of traces are at —71 mV, the resting potential of the cellbefore exposure to ouabain. Dotted lines for the lower pair of traces are at — 65 mV,the resting potential after treatment with 10~4moll~1 ouabain. Note that increasingthe concentration of ouabain to 10~3moll"' did not cause further depolarization,whereas reducing the osmolality did.

mosmol kg L produced an additional depolarization of 28±1 mV (N=6), which isnot significantly different from the hypo-osmotic depolarization observed withoutpre-treatment in ouabain (31±lmV, N=13).

180 R. H. QUINN AND S. K. PIERCE

Intracellular osmolytes

The main organic osmolyte in the ganglia, proline betaine, leaves the tissuethroughout the 2h period following hypo-osmotic stress (Fig. 6A), but notsignificantly so until after the 20min sampling period. After 2h, proline betainecontent is decreased from 784 /imolg"1 dry mass in iso-osmotic controls to4041umolg~

1 dry mass in hypo-osmotically exposed ganglia.Intracellular Na+ content declines in hypo-osmotically treated ganglia com-

pared with iso-osmotic controls, but not significantly until after the 20minsampling period (Fig. 6B). By the 90min sample, intracellular Na+ is reducedfrom 307 jumolg""1 dry mass in iso-osmotic controls to 250 jitmolg"1 dry mass inhypo-osmotically exposed ganglia. In ganglia exposed to ouabain (5x 10~4 mol I"1)for 90 min, intracellular Na+ content increases significantly in both iso-osmotic andhypo-osmotic media by 384 and 460^molg~1 dry mass, respectively, and there isno significant difference in intracellular Na+ concentration between iso-osmoti-cally and hypo-osmotically treated ganglia (Fig. 6C). Thus, the loss of Na+ thatfollows hypo-osmotic stress is inhibited by ouabain.

There is no significant difference in intracellular K+ content between hypo-osmotically treated ganglia and iso-osmotic controls during the 90 min test period.Thus, unlike the other osmolytes, intracellular K+ is conserved following hypo-osmotic stress. Ouabain (5xlO~4molP ] for 20min) causes a significant decreasein intracellular K+ concentration in both iso-osmotic and hypo-osmotic media by114 and 172^molg~1 dry mass, respectively; and intracellular K+ concentration issignificantly lower in hypo-osmotically treated ganglia in the presence of ouabain(Fig. 6D). Thus, the ability of the cells to conserve K+ following hypo-osmoticstress is inhibited by ouabain.

Intracellular CP content is reduced significantly by 20 min of hypo-osmoticstress. The majority of intracellular C P loss occurs during the first 20 min (Quinnand Pierce, 1990). The intracellular CP content is also reduced significantly from330 jumolg"1 dry mass in iso-osmotic controls to 199 jttmol g~'dry mass in iso-osmotic-hypoionically treated ganglia (Fig. 6E). Further, there is no significantdifference in intracellular CP content between hypo-osmotic and iso-osmotic-hypoionically treated ganglia (Fig. 6E). Thus, the reduction in osmolality is notrequired for the loss of CP from the cells, only the accompanying decrease in[cr]o.

Discussion

It has been suggested that membrane potential changes during osmotic stressmight accompany cell volume recovery (Gilles, 1987). Such a theory must presumeeither that the time course of osmolyte loss is contolled by the membrane potentialchanges or that osmolyte loss by electrogenic pathways follows the full time courseof volume regulation. A portion of the solute lost during hypo-osmotic stressconsists of organic osmolytes, such as free amino acids or quaternary ammoniumcompounds, which have no net charge. If these osmolytes were to leave the cells

Hypo-osmotic depolarization in mollusc neurons 181

2 7

ito 3

1000-

8

182 R. H. QUINN AND S. K. PIERCE

by electrically neutral pathways, at least a portion of cell volume regulation wouldoccur in the absence of membrane potential changes. The loss of these organicosmolytes often occurs more slowly than the loss of inorganic ions and accounts formost of the cell's capacity to recover volume (Pierce, 1982). Thus, even ifinorganic ions leave the cells by conductive pathways, volume recovery may occurover a time course that is very different from that of the hypo-osmotic depolar-ization. Indeed, the depolarization in Parker cells occurs before the peak in cellvolume (which is probably limited by ion loss), and recovery of the resting po-tential occurs before volume recovery, which is produced by proline betaine loss.

The results of the ion substitution experiments indicate that Na+ permeabilityaccounts for most of the hypo-osmotic depolarization in Parker cells. This couldoccur in two ways, either by an increased relative permeability to Na+ or byinhibition of the Na+/K+-ATPase in the presence of an already existing andrelatively large Na+ leak. Since the depolarization was not altered by ouabain, theresults suggest that increased relative permeability to Na+ is the case. Theactivation of a permeability pathway favoring an inward movement of Na+ is of noosmotic benefit to a cell that needs to lose solute in order to regulate volume.However, the influx of Na+ by this pathway may be very small and rapidlyopposed by the Na+/K+-ATPase. Indeed intracellular Na+ content does notincrease during hypo-osmotic exposure.

The gradient for Na+ is strongly inward and the observed reduction inintracellular Na+ concentration following hypo-osmotic stress does not occur inthe absence of active transport. Similarly, the reduction in intracellular Na+

concentration in Mercierella enigmatica axons following salinity reduction isouabain-sensitive (Benson and Treherne, 1978), but in the mammalian kidneycortex (Gilles et al. 1983) and Limulus polyphemus heart (Warren, 1982) it is not.In most cell types, aniso-osmotic volume recovery is not inhibited by ouabain,indicating that the Na+/K+-ATPase is not required for volume recovery (Roriveand Gilles, 1979). The present data indicate that the Na+/K+-ATPase cancontribute a part of the total osmolyte loss.

There are at least two explanations for the hypo-osmotic depolarization thatremains in the absence of Na+. First, the differences between our results obtainedwith arginine and Tris+ suggest that Tris+ may not be completely impermeant,thus causing some depolarization. The permeability of Na+ channels to organiccations varies in different tissues and even Tris+ can be highly permeant(Edwards, 1982). Second, it is possible that other ions contribute to thedepolarization. Our results rule out Ca2+ in that regard, since reducing orremoving Ca2+ in Na+-free ASW does not affect the level of depolarization.However, the increased hypo-osmotic depolarization that occurs when Cl~concentration is reduced in Na+-free ASW indicates that an increased relativepermeability to Cl~ may account for the remaining depolarization. This Cl~component of the hypo-osmotic depolarization could also represent an osmoticallyactivated pathway for Cl~ loss from the cells.

Most of the Cl~ loss from Elysia cells occurs during the first 20 min after transfer

Hypo-osmotic depolarization in mollusc neurons 183

to hypo-osmotic ASW. Thus, unlike that for the other osmolytes, the time courseof Cl~ loss correlates with that of the hypo-osmotic depolarization (Quinn andPierce, 1990). However, an equal loss of CP occurs from a reduction of ions alonein iso-osmotic media (iso-osmotic-hypoionic), a condition in which a hyperpolar-ization rather than a depolarization occurs. This result indicates that an osmoti-cally activated pathway is not required for a substantial loss of CP from the cellsand that bulk loss of CP and the hypo-osmotic depolarization are unrelated.

Since the hypo-osmotic stress causes cellular swelling, it is possible that astretch-activated Na+ channel is responsible for the depolarization. In strikingsimilarity to our results from Parker cells, a stretch-activated channel isolated fromopossum kidney cells conducts an inward cation current, but in the absence ofextracellular Na+ carries an outward CP current (Ubl et al. 1988). The activationof such a channel would result in membrane depolarization in the presence orabsence of Na+, given an outward gradient for CP . A channel with similarproperties may be responsible for the hypo-osmotic depolarization in Parker cells.Such a channel may be utilized in neuronal functions such as the electromechanicaltransduction described in axons and stretch receptors (Sachs, 1986).

Volume-activated K+ conductances in Ehrlich ascites tumor cells, humanperipheral blood lymphocytes and pancreatic /3-cells are inhibited by quinine(Hoffmann etal. 1984; Sarkadi et al. 1984; Marcstrom et al. 1990). Thus, theincrease in the CP component induced by quinidine could result from inhibition ofa volume-activated K+ conductance, possibly a means for K+ to leave the cells andserve as a counterion for loss of C\~. However, intracellular K+ content isunchanged in Elysia ganglia following hypo-osmotic stress. Thus, K+ does notcontribute to cell volume regulation. This result contrasts with those from anumber of other cell types in which hypo-osmotic stress activates efflux of K+

along with CP , either by separate conductances or by electrically neutralcotransport or exchange mechanisms (Eveloff and Warnoch, 1987; Hoffmann andSimonsen, 1989). Intracellular K+ concentration is also reduced in axons from theeuryhaline osmoconformers Mytilus (Willmer, 1978a) and Mercierella (Bensonand Treherne, 1978) following salinity reduction, but not in proportion to thereduction in extracellular K+ concentration. Treherne (1980) concluded that thesemolluscan neurons require a certain amount of intracellular K+ for maintenance oftheir excitability characteristics at low salinity. Failure to reduce [K+]j inproportion to [K+]o increases the K

+ ratio across the membrane at low salinities,resulting in a hyperpolarized resting potential. The increased ratio compensatesfor the reduction in action potential overshoot produced by the reduction inextracellular Na+ concentration, thus maintaining the amplitude and rate of rise ofthe action potential (Treherne, 1980). A hyperpolarized resting potential alsooccurs in Parker cells after acclimation to low salinity (Parker and Pierce, 1985).Thus, Elysia neurons may conserve K+ in order to maintain excitability duringexposure to very dilute sea water.

In Elysia ganglia, the effect of ouabain on intracellular K+ concentration inhypo-osmotic vs iso-osmotic media indicates that activity of the Na+/K+-ATPase

184 R. H. QUINN AND S. K. PIERCE

is required for the maintenance of intracellular K+ concentration during low-salinity stress. Since extracellular K+ concentration was reduced by half whenElysia ganglia were transferred from 920 to 460mosmolkg~1, the completeconservation of intracellular K+ content would require either a reduced K+ leak orincreased active transport. A 76% increase in Na+ pump sites, measured bybinding of [3H]ouabain, occurs in Mytilus axons after acclimation to 25% seawater from 100% sea water, indicating that active transport by the Na+ /K+-ATPase increases at low salinity (Willmer, 1978c). A similar response may accountfor the conservation of intracellular K+ in Elysia ganglia during low-salinity stress.

In conclusion, we have observed a depolarization in neurons which occurs onlyin response to a reduction in osmolality and not to an iso-osmotic reduction inions. The electrophysiological evidence indicates that this depolarization isprimarily the result of an increased relative permeability to Na+ and is, therefore,not the result of bulk loss of intracellular ions. The measurements of intracellularsolutes support that conclusion, since the only osmolyte that is reduced coincidentwith the depolarization, Cl~, is equally reduced by an iso-osmotic reduction inions, a manipulation that produces hyperpolarization rather than depolarization.Thus, the hypo-osmotic depolarization in neurons has no apparent relationship tocell volume regulation. Possibly, the hypo-osmotic depolarization results fromsome other neuronal property, such as transduction of mechanical stimuli(osmotically induced stretch) into electrical signals.

This work was supported in part by NSF grant no. DCB-8710067.

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