function of the epithelial channel cells of the …function of channel cells of land slugs 303 roach...

J. exp. Biol. 121, 301-314 (1986) 3 0 1Printed in Great Britain © The Company o/Biobgists Limited 1986

FUNCTION OF THE EPITHELIAL CHANNEL CELLSOF THE BODY WALL OF A TERRESTRIAL SLUG,

ARIOUMAX COLUMBIANUS

BY A. W. MARTIN AND I. DEYRUP-OLSEN

Department of Zoology, University of Washington, Seattle, USA

Accepted 3 October 1985

SUMMARY

The body wall of the land slug Ariolimax columbianus Gould (Arionidae) secretesfluid in response to mechanical stimulation. This fluid is a product of specializedchannel cells, with addition of mucus. Channel cell function can be explainedin terms of ultrafiltration of blood components into the central channel, andmodification of this ultrafiltrate, prior to extrusion from the body surface, by cellulartransport of univalent ions but without change in osmotic pressure. Sodium andchloride ions are conserved, and potassium and bicarbonate ions are transferred outinto the channel cell product. Agents known to affect epithelial transports and ionprocessing in a variety of animals - ouabain, amiloride, furosemide, SITS andacetazolamide — depress the function of the slug channel cells.

INTRODUCTION

Secretions of the epithelium of the body surface are of critical importance insurvival of terrestrial gastropods. In slugs, as compared with the shell-bearing snails,the skin is particularly vulnerable to excess gain or loss of water, and to mechanical orchemical damage by physical objects in the environment, by predators, and byparasites. Controlled and often voluminous secretions containing water, ions, mucusglycoproteins, lectins and a variety of other constituents are elicited by mechanicaland chemical stimulation of the slug skin. These secretions can flood the skin surface,removing or neutralizing toxic materials, lubricating and moistening it, and perhapsserving to correct hyperhydration. The secretions are formed by epithelial cellsincluding exceptionally large and complex structures, the mucus cells (Wondrak,1968) and channel cells (Luchtel, Deyrup-Olsen & Martin, 1984). The latter havebeen described in a variety of terrestrial pulmonates, under names includingalbumen cells and protein cells (Zill, 1924; Roth, 1929; Campion, 1961). Aremarkable property of channel cells is their ability to allow passage from thehaemocoel to the body surface of fluid containing such large molecules and molecularaggregates as haemoglobin (Mr68000), slug haemocyanin (Afr9xl06) and carbonparticles (Simkiss & Wilbur, 1977; Deyrup-Olsen & Martin, 1982; Luchtel et al.1984). Mucus and channel cells are readily activated by mechanical stimulation.

Key words: channel cells, Ariolimax columbianus, epithelial transport, slug.

302 A. W. MARTIN AND I. DEYRUP-OLSEN

Even a light touch on the skin provokes local secretion by these cells. Repeated,gentle stroking of the skin, for example with a glass rod, results in a suddenoutpouring of a large volume of secretion over the surface of the mantle and back ofthe foot. This secretion has two clearly distinguishable components - mucus, oftenpresent in the form of membrane-bound vesicles, and fluid identified, because it cancontain exogenous material of high molecular weight, as derived at least in part as aproduct of the channel cells. A filtration process appears to be involved, sincephysiologically inert materials such as high molecular weight dextrans and carbonparticles traverse the body wall at concentrations related to their particle size(Deyrup-Olsen & Martin, 1982; Luchtel et al. 1984). Burton (1965), observingslime formation elicited by stimulation of the foot of Helix pomatia, suggested thatthis fluid was formed partly by cell secretion and partly by filtration from the blood.

We have addressed three specific questions in the present study. (1) What fractionof the fluid secretion of the body wall has passed through the channel cells? (2) Towhat extent is the filtered product modified, with respect to ionic composition,before it is lost from the body? (3) What cellular processes are involved in modifyingthis fluid output?

METHODS

Experiments were carried out with Ariolimax columbianus, large (20-50 g) slugsnative to the Pacific North-west of the United States and Canada. The animals werecollected on Tatoosh Island, off the north-west coast of the United States, andmaintained in the laboratory at 10°C. They were housed in plastic containers, in amoist atmosphere, with continuous access to food (lettuce; yams or potatoes;occasionally also commercial trout food or cat food). The slugs thrived under theseconditions, growing and eventually laying eggs. Prior to experiments, they wereplaced at room temperature (approx. 19°C) for 24-48 h.

Two preparations were used: intact slugs and an in vitro preparation of the bodywall. Although skin secretions in slugs can be elicited by mechanical, chemical andelectrical stimuli, we used chiefly mechanical stimulation of the body surface, inorder to limit the variables in the experimental protocols. In typical experiments withintact animals, a slug was placed on an inclined glass plate and the skin of the mantleand back of the foot was gently stroked with a pipe-cleaner (cotton-covered wire), at arate of about 2 strokes along the body per second. Fluid secretions, formed duringthe procedure, dripped into a tared Petri dish below the glass plate, and could thenbe measured and analysed for various constituents. The total amount of material(fluid and formed mucus) secreted was determined by the change in the slug's weightresulting from stimulation. The procedure for the in vitro experiments, using a sac ofslug skin, has been described in detail elsewhere (Deyrup-Olsen & Martin, 1982).Briefly, the posterior body wall, freed of viscera, formed a sac termed the posteriorchamber (PC) which was tied to a glass manometer tube filled with slug Ringersolution and arranged to maintain a constant hydrostatic pressure of about 11 Torrwithin the sac. The Ringer solution was prepared according to the formula given by

Function of channel cells of land slugs 303

Roach (1963); it was modified, as a result of our analyses of Ariolimax blood, toincrease the osmotic pressure to loOmosmoll"1 and the concentration of calciumions to Smmoll"1. Various agents, known to affect transport processes and per-meability to univalent ions in other systems, were administered by injection intothe PC through a fine polyethylene tube threaded through the manometer tube.Mechanical stimulation was applied by stroking the skin lightly with a glass rod, andthe resulting fluid secretion was collected and analysed as in the in vivo experiments.Measurements could also be made of the amount of mucous secretion which collectedon the rod. Supplementing the experiments with mechanical stimulation, some testswere made in which fluid secretion was elicited by injection into the PC of argininevasotocin (AVT, l-2nmol; Sawyer, Deyrup-Olsen & Martin, 1983); alternatively,secretion was inhibited by administration of norepinephrine (1 /imol).

Histological preparations (Bouin's fixative; paraffin; haematoxylin and eosinstaining) were made of the body wall, both with untreated PC preparations and withPCs that had been relaxed and distended by successive treatments (at 5- to 15-minintervals) with atropine sulphate, hexamethonium bromide, 5-hydroxytryptamineand acetylcholine (1 /tfnol of each agent injected into the PC).

In order to determine what fraction of the fluid, emerging through the body wall inresponse to stimulation, was filtered directly from blood or Ringer solution, thefollowing procedure was used. A solution of beef haemoglobin (in methaemoglobinform, twice recrystallized) in Ringer solution was dialysed extensively (10°C) againstslug Ringer solution. The resulting haemoglobin solution (ZOmgmF1) was injectedinto intact slugs or PC preparations. In a typical experiment, after a 30-min to 2-hperiod allowed for distribution of the haemoglobin in body fluids, mechanicalstimulation was applied. Fluid containing haemoglobin emerged through the bodywall, and blood or PC contents were sampled. The concentrations of haemoglobin inthese fluids were compared (Beckman B spectrophotometer, 540 nm). In a fewexperiments, India ink (Pelikan), which had been dialysed extensively against slugRinger solution, was injected into the PC.

The fluid collected during mechanical stimulation, centrifuged to provide amucus-free supernatant solution, was analysed for the following components: Na+

and K+ ions (Orion ion meter and ion specific electrodes); Cl~ ions (amperometric,Cotlove chloridometer); inorganic phosphate ions (method of Fiske & SubbaRow);HCO3~ ions (titration to pH4 of protein-free filtrates prepared with ethanol, givingthe sum of phosphate and HCC>3~ ions, with subtraction of phosphate to giveHCO3-); osmotic pressure (Wescor vapour pressure osmometer). Preliminary testsindicated that Ca2+ and Mg2"1" contributed little to the overall osmotic pressure,hence the changes in these ions were not assessed in the present study.

The potential roles of several known membrane processes in channel cell functionwere investigated by varying the ionic composition of the Ringer solution byreduction of Na+ or Cl~ ions (in vitro PC preparations only), and by administrationof pharmacological agents known to affect membrane functions of vertebrateepithelia. These agents included the following, obtained from Sigma ChemicalCompany, Aldrich Chemical Company, and Merck Sharp & Dohme: ouabain,


amiloride, furosemide, 4-acetamido-4'-isothiocyanate-stilbene-2,2'-disulphonic acid(SITS) and 9-anthracenecarboxylic acid (9-AC) (mechanisms of action reviewedbriefly in Reuss et al. 1984; Warnock et al. 1984). They were dissolved in Ringersolution and generally given at concentrations of 10~4 or 10~3moll~1 in PC exper-iments, or in doses of about 1 ^molg"1 body weight in intact animals. Where effectsof various agents were compared, the significance of the results was determined byuse of the <-test.

RESULTS

When a glass rod or pipe-cleaner was passed repeatedly over the surface of the skinof a PC or an intact slug, local muscle contractions and a slight outpouring of mucousfluid, quickly adherent to the stroking object, occurred. After about 30-90 s, amarked change took place in the response: free fluid, carrying a rich complement ofmucus vesicles giving the fluid a milky white appearance, emerged on the surface ofthe skin and dripped off the lower end of the body. The rapid flow continued forabout 1-2 min, then slowed abruptly and generally ceased within 3 min, even thoughthe stroking stimulation was continued. The patterns of response in intact slugs andPC preparations were similar. In intact animals, the response did not recur on theday of the experiment, but it could be elicited predictably on days subsequent to theday of initial stimulation. In the case of the PC, after stimulation had stopped and thesystem had been left undisturbed for 15 min or more, the response could sometimesbe elicited again.

The volumes of fluid traversing the body wall were large, averaging 21 -6 ± 12-8 %(N = 30) of the blood volume in intact slugs; blood volume was estimated as 26% ofbody weight (Martin & Deyrup-Olsen, 1982). In the case of the PC, the fluid outputaveraged 0-47 ±0-12 (N = 25)gg~l of PC tissue. Mucus, confined in vesicles orformed as a sticky mass on the glass rod or pipe-cleaner, was released consistently. Itwas quantified in an approximate way in terms of its wet weight, and, in the case ofthe PC preparation, averaged 0-063 ± 0-041 (N = 6) gg"1 PC, or about 12% of thetotal output. Formed mucus represented 60-4 ± 8-2% of the total stimulated outputof the intact animal. We found that the fluid outputs of the PC preparations weresignificantly less in the period 0—15 min after the PC was set up than in the sub-sequent 30min to 2h; in this latter period the response was stabilized at about0-5 gg"1 of PC tissue. Therefore, tests of conditions affecting fluid output werecarried out consistently in the time span 30—90 min after preparation of the PCsystem.

When haemoglobin was present in blood or Ringer solution, effective mechanicalstimulation of the body caused outpouring of fluid. This fluid contained haemo-globin at levels varying widely from experiment to experiment, yet in each test theconcentration in the fluid was essentially identical with the concentration in bodycontents. Thus, the ratios of haemoglobin in skin fluid vs Ringer solution (PCpreparation) or blood (intact slug) were respectively 1-02 ±0-02 (N=6) and0-97 ± 0-06 (N= 5). These results confirmed our earlier finding that haemoglobin


passes freely through the body wall in response to stimulation by AVT (Luchtel et al.1984). Furthermore, the results indicated that the processes of bulk flow of fluid andhaemoglobin movement through the body wall depended on the same route, since,were the processes separate, consistent identity of concentrations of haemoglobin inblood and secreted fluid would require fortuitously equal operation of the separateprocesses. We had shown previously (Luchtel et al. 1984) that the channel cells ofthe skin are the routes of passage of carbon particles. We concluded, therefore, thatchannel cells are also the site of fluid flow through the body wall. This structuredoes not take up and store haemoglobin, as indicated by the following experiment.The PC was filled with haemoglobin solution, allowed to stand undisturbed for30min, then drained, rinsed and filled with haemoglobin-free solution. Next, fluidoutput was elicited by mechanical stimulation. This fluid contained little or nohaemoglobin.

In independent tests, it was possible to exclude the mucus cells as routes ofhaemoglobin and bulk fluid output. This production of fluid was suppressed bytreatment of the PC with norepinephrine (1 /imolPC"1), but sticky mucus was stilldischarged in response to mechanical stimulation. If carbon particles were present inthe PC in these conditions, they were not released into the mucus mass.

The large magnitudes of the flow of material through the stimulated body wallwere not surprising in view of the histology of this organ (Fig. 1). In the case ofthe PC fully expanded following treatment with neurotropic agents, it may be seenthat about half the body wall is made up of epithelial channel and mucus cells.These discharge on the body surface, and have underlying muscle organized inapproximately alternating layers of cells oriented transversely and longitudinallywith respect to the main axis of the body. Tissue from normal, unrelaxed PCs had astrikingly different appearance, with the mucus and channel cells, as well as themuscle layers, contorted into complex configurations effectively masking the basiccellular organization of the body wall. Observations on six different areas of thedorsal surface of the foot showed that mucus and channel cells were present inapproximately equal numbers. Mucus cells generally appeared larger than channelcells, and we estimated that the latter comprise 20 % of the total volume of these giantcells of the body wall. On this basis, it is possible to make preliminary calculations ofthe rate of fluid flow through channel cells. If these have a total weight, in a 5-g PC,of 20 % of the combined mucus cell + channel cell mass (estimated as half the PCweight, or 2-5 g), the channel cells would represent about 0-5g. They would pass,according to the average figure given above for fluid output by a 5-g PC, 2-35 g in5 min. This flow through the cells, in volume per minute, is about equal to thecombined volume of the channel cells. In a similar manner, the output of the mucuscells can be estimated. The sticky mucus formed represented about 15% of thevolume of these large vesicular cells.

The concentrations of ions in the stimulated output of PC preparations differedlittle from the corresponding values in the Ringer solution filling the sacs. The mostcommon difference observed was a modest elevation of K concentration of the fluidoutput, up to about twice the level in the sac. In contrast, analysis of the fluid given


PMli I

Fig. 1. (A) Segment of cross-section of entire body wall of Ariolimax PC, relaxed bytreatment with neurotropic agents. The very large channel cells (locations marked withvertical lines) and mucus cells connect apically with the small microvillous cells of theepithelium, facing the outer (o) surface of the skin. Layers of muscle cells, and connectivetissue cells, lie deeper and surround the inside (i) of the body cavity. After fixation withBouin's fluid, channel cells appear fairly homogeneous and are stained strongly witheosin, appearing grey in the photograph. Mucus cells are stained little or not at all withhaematoxylin/eosin, and have a light, vesicular appearance in the photograph. Thebracket indicates the area shown enlarged in Fig. IB. (B) Details of epithelial cells:e, layer of microvillous cells; ch, channel cell; m, mucus cell. Scale bars, 200 fan.

off by the body wall of intact slugs (Fig. 2) demonstrated that the channel cellproduct in vivo, even though produced passively by filtration, was not merely anunmodified ultrafiltrate of blood. Compared with blood, the channel cell product washigher in K+ and HCC>3~ ions, and lower in Na+ and Cl~ ions. Blood and channelcell fluid were equivalent in osmotic pressure. The results showed that, as fluidtraversed the body wall, there was a net exchange of Na+ ions for K+ ions, and Cl~ions were retained, presumably in exchange for HCO3~ ions. The standarddeviations, hence the variances, of the concentrations of Na+ and K+ ions in the fluidreleased by the body wall were considerably greater than the corresponding variancesin blood ion concentrations (Fig. 2). The higher variances of fluid output wererelated to the wide variations in flow rates (Fig. 3). At low rates of fluid formation,the Na concentrations were relatively lower, and the K concentrations higher, than athigh flow rates.

Analogous net ion exchanges by epithelial membranes are well known in the case ofvertebrate epithelia, and we tested whether conditions which interfere with iontransports in vertebrates might reveal mechanisms of operation of the slug skin informing fluid. For the most part, we used the in vitro system for these studies.


Manipulation of Na+ ions proved to have profound effects on channel cell function(Fig. 4). If choline replaced 56% or 100% of the Na+ ions in Ringer solution, theresponse to mechanical stimulation was deeply depressed. This was also true whenamiloride (known to block Na transport) or ouabain (which inhibits Na/K-ATPase)were present in the medium. Together, the results indicated that functioning of thechannel cell — including, apparently, the patency of the route through which fluidtraverses the cell to the exterior - depends on Na transport as well as on the continuedmaintenance by the Na/K pump of the normal gradient of these ions across cellmembranes.

The differences in composition of blood and the fluid produced by the body wallsuggested further that Cl~ and HCC>3~ ions play parts in formation of this fluid. Asshown in Fig. 5, SITS, a powerful pharmacological agent known to block CI/HCO3exchange in vertebrate epithelia, inhibited fluid output on mechanical stimulation ofthe body. Furosemide, which inhibits coupled Na/Cl (or Na + K/Cl) transport, and9-AC, a fairly specific blocker of membrane Cl channels, had similar effects.Acetazolamide (AZ) was tested, since this agent is an effective inhibitor of carbonicanhydrase in a wide variety of animal phyla. At relatively high concentrations (10~2,10~3molP') AZ reduced markedly the response to mechanical stimulation,presumably by diminishing the availability of cellular HCO3. The results suggestedthat Cl-depleted Ringer solution, like Na-depleted Ringer solution, would fail tosupport a mechanical response. This prediction proved to be incorrect, however,since fluid output was not significantly depressed in experiments in which 86 % of theCl~ in Ringer solution had been replaced with nitrate or gluconate ions (Fig. 5).

80

I 40

I

T

It

T1

i

Na

(7,18)K

(10,21)

Cl

(8,19)

HCO3

(6,9)

200

osm

(6,10)

100

o

I

Fig. 2. Comparison of ion concentrations and osmotic pressures of blood (circlesrepresent means ± standard deviations, S.D.) and fluid emerging through the body wall(crosses represent means ± S.D.) of intact slugs in response to 5-min mechanicalstimulation of the body surface. Numbers in parentheses represent numbers of tests: onblood, first number; body fluid output, second number. Differences between blood andbody fluid concentrations are significant for Na ( P < 0 0 1 ) , K and Cl (P<0-005) andHCO3 (P< 0-05). Osmotic pressures (P,™) do not differ.


A Sodium

75 £

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Z 25

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i

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i

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o

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.-I;-;-."-:-:

O o.o—

10-5 10 1-5 2-0 2-5

75

L 50

0-5 10

Flow rate (

1-5 20

in"1 35g~')

2-5

Fig. 3. (A) Sodium concentration as a function of flow rate of fluid released by the bodywall of intact slugs during 5-min stimulation of the body surface (mantle and back offoot). Flow rates are normalized to the mean body weight in this series of experiments,35 g. The filled circle and shaded area with central line represent blood Na concentration(72 ± 8 mmol 1~ ,N=7). The line, derived by linear regression, gives the relationship ofNa concentration (CN.) to flow rate (f): CN, = 9-4f + 25-8. (B) Potassium concentration(CK) as a function of flow rate (f). The filled circle and shaded area with central linerepresent blood K concentration (4± lmmolP 1 , iV=7). The relationship derived bylinear regression is CK = — 7-6f + 52-6.

Moreover, in such Cl-depleted Ringer solution, furosemide totally failed to blockfluid output.

The agents amiloride, ouabain, furosemide and SITS blocked (partially or com-pletely) the response of the PC to AVT as well as to mechanical stimulation. Some ofthe results with the in vitro preparation were checked in experiments on intact slugs,although we did not attempt to impose on the slug such drastic conditions as partialreplacement of blood Na+ or Cl~ ions. The effects of injections of amiloride,furosemide and SITS (1/imolg"1) are summarized in Fig. 6. Fluid output wasdepressed, as was the animal's general activity, by each of these agents. In exper-iments with both intact slugs and PC preparations, it was found that conditionswhich blocked channel cell function did not interfere with the release of mucus(mucus cell product) by the body wall.


DISCUSSION

The epithelium of the body wall of Ariolimax columbianus is made up chiefly ofthree cell types - the microvillous epithelial cells and the giant mucus and channelcells that extend deeply into the body wall. These are connected at their apicalmargins by junctional complexes of distinctive structure (Luchtel et al. 1984). In ourultrastructural studies of the skin through which carbon particles were passingrapidly, we found no evidence of paracellular passage of these particles, nor werethey ever seen in the microvillous or mucus cells. Rather, carbon particles wereclearly and consistently observed in the peripheral cisternae and the large centralreservoirs of the channel cells. The route for passage of particles from the haemocoelto the outside of the body wall involves a selective barrier, as shown by the relativelylow concentrations of carbon particles, large dextran and haemocyanin molecules influid traversing the body wall as compared with blood (Deyrup-Olsen & Martin,1982; Luchtel et al. 1984). Haemoglobin, on the other hand, is filtered freely.Electron micrographs of sacs filled with carbon particles indicated that the barrier islocated at the basement membrane enveloping the basal aspects of channel, as well as

3-0 -

2-0

3O

1 1-0

c

Control CholineA B

AmilA B

Ou

Fig. 4. Effects of conditions altering Na availability or transport on response of PC to5-min mechanical stimulation. Data on fluid output normalized to 5g PC; circles andbars give means ±S.D. Control (TV = 25); choline = choline Ringer solution with 56%(A, AT = 4) or 100% (B, N = 4 ) of Na replaced with choline; Amil A = 1 0 " 3 m o i r 'amiloride in normal Ringer solution (N = 11); Amil B = 10~3moll~' amiloride in Cl-depleted Ringer solution (N = 4); Ou = 10~3 mol I"1 ouabain, (N=6). Responses in allexperimental conditions differ significantly from control responses ( P < 0-001).


30

2-0

23C

10

S

C F(25,10) (5,5)

I I IS AZ AC-9

(5,10) (10,5) (7,4)

Fig. 5. Effects of conditions altering Cl availability or transport on response of PC to5-min mechanical stimulation. Data on fluid output normalized to Sg PC. Plain barsrepresent means ± s.D. values in tests with normal Ringer solution. Shaded bars givemeans ± S . D . values in Cl-depleted Ringer solution. Paired numbers in parenthesesrepresent numbers of experiments in normal Ringer, Cl-depleted Ringer solution.C = control (no additive); F = furosemide lO^mol l" 1 ; S = SITS 10~3 moll"1; AZ =acetazolamide 10~z and 10~3 mol P 1 (results for the two dose levels did not differ); AC-910~3moll~'. Results with epithelial agents in normal Ringer solution were comparedwith the corresponding control, and differed significantly from it (P<0-001). In Cl-depleted Ringer, F did not depress the response, whereas the S, AZ and AC-9 responseswere significantly below the control level (P< 0-001).

other epithelial, cells. It is interesting that we have found no evidence for a para-cellular route from the inside of the body to the epithelial surface, since paracellularmovements of inulin, peroxidase and lanthanum ions in the opposite direction — fromthe exterior into or through the skin - have been observed in the limacid slugsAgriolimax reticulatus and Umax maxtmus by Ryder & Bowen (1977) and Prior &Uglem (1984). The pathway whereby large particles pass from the blood and emergeon the body surface offers a low resistance route for movement of fluid as well. Ourevidence indicates that the transport processes which modify the composition of thisfluid - conserving sodium and chloride ions, but increasing the concentrations ofpotassium and bicarbonate ions — are sited in the channel cells rather than, for


example, in the microvillous epithelial cells. Thus, agents that block epithelial iontransports (e.g. amiloride, SITS) do not merely alter the composition of the bodywall fluid, but instead partially or completely block the process of output itself.As yet, there is no indication as to whether the blockage of filtration resultsfrom physical closure of the channel cell cisternae, from alterations of membranepotentials, or from other cell changes.

Comparison of the effects of 9-AC and SITS with those of furosemide lead to theidea that Cl may play a distinctive role in channel cell function. All three agentsinhibited the fluid output in normal Ringer solution, indicating that cell Cl per-meability and transport play a part in the response. The failure of furosemide (butnot of the other agents) to affect the response in Cl-depleted Ringer solution might beexplained as follows: channel cells (or cells controlling these) at rest have relativelyhigh intracellular Cl activities maintained by a furosemide-sensitive Cl transport.Activation of these cells would result from the opening of Cl channels and celldepolarization by diffusion of Cl~ ions down their electrochemical gradient.According to this hypothesis, in normal Ringer solution furosemide would block Cltransport, allowing gradual dissipation of the Cl gradient and hence loss of excit-ability. But in Cl-depleted Ringer solution, a transmembrane gradient for Cl couldexist, even in the presence of furosemide, until the intracellular Cl initially presenthad diffused from the cell. Therefore, furosemide would fail to block the response ofthe PC filled with Cl-depleted Ringer solution. This interpretation can be testedcritically only by direct measurements of membrane potential and conductancechanges, and we have not done this. Nevertheless, the results are consistent with thehypothesis that the channel cells are activated, directly or indirectly, by membranedepolarization resulting from Cl movement.

40

•a

8

3o

•p'3C

20

I O

Ic Amil

IF

Fig. 6. Fluid output in response to mechanical stimulation of the body wall of intactslugs without (C) or with injections of 1 /rniol g~' of amiloride (Amil), furosemide (F) orSITS (S). Control values (JV=31) differed significantly from: Amil (N=4), P<0-02;F (N=4),P<0-05; S (AT = 4), P<0-01.


Outside

Ultrafiltration

, HCO3

Fig. 7. Channel cell features observed or inferred from electron micrographs are listed onthe left side of the diagram. Processes inferred from experiments with mechanicalstimulation of the skin are listed on the right side of the diagram. (1) Epithelial cell withmicrovillous apical surface; (2) channel cell; (3) route of egress from central channel tobody surface; (4) mitochondrion; (5) central channel; (6) opening of cisternae to exteriorand interior of channel cell, as inferred from positions of injected carbon particles withinthe system; (7) basement membrane; (8) nucleus.

Fig. 7 is a diagram summarizing our current concept of channel cell function,based on data from electron micrographs (Luchtel et al. 1984) and the presentresults. Burton (1965), in a study of Helix pomatia, noted that the skin secretion('slime') formed early in an animal's reaction to stimulation was relatively rich in K+

ions, but that the concentration of these fell, and that of Na+ ions increased, asstimulation proceeded. He suggested that the secretion was formed in part byfiltration from the blood and in part by cell activity. Burton's observations andconclusions are in full accord with our findings, in which we locate both processes ina single cell type, the channel cell.

The copious flow of fluid through the slug's skin during repetitive mechanicalstimulation is similar to the outpouring of fluid elicited by chemical stimuli, such asdirect application to the skin of sodium chloride crystals or hypertonic solutions.Thus, although we have used primarily mechanical stimulation, in order to simplifyour experimental protocols, the response we have studied is probably a commonreaction to a variety of noxious stimuli. Presumably the channel cells normallyfunction also to provide the fluid medium in which mucus vesicles discharge theircontents in the development of the mucous secretion, or slime, of varying


consistency formed by slugs under different environmental conditions and inresponse to varying stresses. The partial substitution of K+ for Na+ ions, and ofHCC>3~ for Cl~ ions, in blood filtrate traversing the channel cells would appear to behighly adaptive, in view of the fact that slugs, like most other terrestrial pulmonates,are herbivores. Potassium ions are relatively abundant in their diets, whereas Na+

and C\~ are not. Because of this relative K+ excess, it is also possible that the channelcells fuction as an extra-renal route for excretion of K+ ions (Martin & Deyrup-Olsen, 1985).

The responses to mechanical stimulation were quite similar in intact animals andin in vitro PC preparations. The time courses of the responses were closely parallel,as were the directions of change in composition of fluids traversing the body wall. Inboth systems, channel cell and mucus cell functions were clearly separable, as shown,for example, by the fact that fluid output could be depressed by epithelial blockingagents without an effect on mucus output. On the other hand, there were markedquantitative differences in both the volume of fluid produced (proportionally greaterin vitro) and the changes in ion concentrations (greater in the intact animals). Thevolumes of fluid traversing the channel cells - and the contrasts between the in vivoand in vitro systems — must depend on at least three factors: (1) the patency of thechannel cell's route to the outside; (2) the volume available for filtration — withdifferences in the limited blood volume of the intact animal, and the unlimitedsupply available to the PC; (3) the hydrostatic pressure within the haemocoel (intactslug) or PC (Luchtel et al. 1984). In the intact animal, the pressure was controlled bycontraction or relaxation of the body musculature, whereas in the in vitro preparationit was determined by the conditions imposed by the experimenter. Thus the totalresponse could differ between the two systems because of differences in patterns oftrans-wall pressure. In part, also, it is probable that the normal slug, with cerebralganglia and neurohormonal mechanisms intact, has a wider repertory of processescontrolling the function of the body wall than does the preparation.

Activation of the channel cells by mechanical stimulation of the body surface mustdepend on a sequence of processes: excitation of mechanoreceptors of the body wall;conduction in sensory nerve fibres of impulses to a synapse, resulting ultimately inexcitation of the channel cell. Numerous neurones and synapses may intervene.Previously we have shown that channel cells of the in vitro preparation can beactivated by injections of AVT as well as by extracts of slug head ganglia containingan endogenous peptide resembling AVT (Sawyer et al. 1984). Both acetylcholineand 5-hydroxytryptamine also stimulate secretion by the body wall (Deyrup-Olsen &Martin, 1982), whereas norepinephrine and atropine block the fluid output (A. W.Martin & I. Deyrup-Olsen, unpublished observations). Fluid production is deeplydepressed in slugs suffering from dehydration (Deyrup-Olsen & Martin, 1984).These and other observations that we have made in the course of our studies areevidence that the slug can exert neurohormonal control over its channel cells.Indeed, such control must be crucial for survival of these animals, since skinsecretions are both important in protection of the skin surface, and potential routesfor life threatening losses of body fluids. The analysis of the neuroendocrine control


of the channel cell is proving to be complicated and difficult, and will be dealt withelsewhere. Finally, it is of interest to note that, while differing greatly in detail, thechannel cell of the slug skin and the vertebrate nephron show some intriguingparallels in structure and function. In each, a primary fluid is formed byultrafiltration through an epithelial basement membrane. The composition of thisultrafiltrate is modified by membrane transports and exchanges of Na+, K+, Cl~ andHCC>3~ ions. In both kidney and skin, these processes show sensitivities topharmacological agents such as ouabain and the loop diuretics, amiloride andfurosemide. These parallels represent a striking illustration of the fundamentalsimilarities of molluscan and vertebrate physiology at the level of cell function.

We are grateful to Dr R. T. Paine for collecting the slugs used in this study, and toP. M. Brunner for preparation of the histological sections.

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function of the epithelial channel cells of the …function of channel cells of land slugs 303 roach...

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