220 T.C. COXTHE JOURNAL OF EXPERIMENTAL ZOOLOGY 279:220–227 (1997)

© 1997 WILEY-LISS, INC.

JEZ 837

Apical Regulation of Nonselective Cation Channelsby ATP in Larval Bullfrog Skin

THOMAS C. COX*Department of Physiology, Southern Illinois University, Carbondale,Illinois 62901

ABSTRACT The apical membrane of larval bullfrog skin contains a nonselective cation chan-nel that can be activated by apically applied amiloride and acetylcholine. In our search for otherligands that might activate this channel, ATP and other purinergics were tested. When ATP (10–1,000 µM) was added to the apical side of tadpole skin mounted in a modified Ussing chamber,there was a transient increase in short circuit current (Isc). The increase in Isc occurred witheither Na or K as the dominant cation in the apical solution. The response was larger in a cal-cium-free Ringer. ADP and AMP had similar but smaller effects than ATP. Adenosine and UTPwere without effect. The ATP response was blocked by W-7, atropine, curare, diltiazem, and suramin.These blockers also inhibit amiloride stimulation of Isc, suggesting that ATP activates a relatedtransport pathway. Studies with analogs of ATP suggest that the ATP binding site in tadpole skinhas characteristics in common with the P2x receptor found in other tissues. These results demon-strate that in addition to amiloride and acetylcholine, ATP stimulates cation transport atthe apical membrane of larval amphibian skin epithelia. J. Exp. Zool. 279:220–227, 1997.© 1997 Wiley-Liss, Inc.

*Correspondence to: Thomas C. Cox, Department of Physiology,Southern Illinois University, Carbondale, IL 62901. E-mail: tomcox@siu.edu

Received 7 March 1997; revision accepted 23 May 1997.

The amphibious adult frog maintains ion bal-ance by feeding and to some extent by absorptionacross the integument. In the adult frog skin, so-dium transport is well developed and largely regu-lated by hormones binding to receptors at thebasolateral membrane. In the obligately aquaticlarval frog, ion balance is achieved by feeding andabsorption across the gills. Ion transport acrossthe skin is negligible; nonselective cation chan-nels normally remain closed. In larval frog skin,amiloride and acetylcholine (ACh) applied on theapical side activate a relatively nonselective cat-ion channel (Cox, ’93, ’92; Cox and Alvarado, ’79;Hillyard and Van Driessche, ’89; Hillyard et al.,’82). This is in contrast to the skin of larval sala-manders, where short circuit current (Isc) is largerthan that observed in tadpole and is inhibited byamiloride (Cox, ’86).

Regulation of ion transport across epithelial tis-sue has been thought to be primarily accomplishedby processes initiated at the basolateral mem-brane. The discovery of apical receptors for ATP,UTP, and other compounds has lead to the studyof the apical membrane as a site of transport con-trol (Ecelbarger et al., ’94; Stutts et al., ’92). Re-cently, a new family of P2x receptors has beenidentified. These receptors are directly stimulatedby ATP and appear to function as nonselective cat-ion channels. They are structurally similar to the

amiloride-sensitive sodium channel, ENaC (Sur-prenant et al., ’95). Some forms have been identi-fied in the parotid and submandibular gland andthe nasal epithelium (Surprenant et al., ’95; Buellet al., ’96). Since ATP is known to stimulatetransepithelial ion transport in epithelia (Ecel-barger et al., ’94; Stutts et al., ’92), I tested theeffects of ATP on larval bullfrog skin. ATP acti-vates nonselective cation transport when appliedto the apical membrane. At the same concentra-tions, ATP has a greater stimulatory effect thandoes amiloride. The response reaches a peak andthen declines in a manner comparable to thatevoked by amiloride and ACh. The ATP effect islarger in divalent-free media, although it does notappear to induce the large pores seen in other celltypes (Dubyak and El-Moatassim, ’93; Surprenantet al., ’95, ’96). Initial pharmacological character-ization of the tadpole response suggests that tad-pole skin has a receptor similar to the P2xobserved in other systems (Dubyak and El-Moa-tassim, ’93; Zimmerman, ’94).

ATP ON FROG TADPOLE SKIN 221

METHODSRana catesbeiana tadpoles (stages XV–XVIII)

were collected locally or purchased from commer-cial suppliers. Tadpoles were anesthetized in 0.1%tricaine methane sulfonate buffered with 0.1% so-dium bicarbonate. The composite skin was dis-sected and mounted in modified Ussing chambers.Short circuit current (Isc) was measured, and so-lutions were changed as previously described (Cox,’92, ’93). The surface area of the chamber was 0.7cm2. Isc was sampled at 10 Hz using an IBM com-patible computer–based data acquisition system.

The KCl Ringer’s contained (in mM) 100 KCl,2.0 CaCl2, and 2.4 KHCO3, pH 7.4. NaCl Ringer’swas the same as above with 100 Na substitutedfor 100 K. NaCl Ringer’s was maintained in thebasolateral solution in all experiments. Calcium-free Ringer’s was made by deleting CaCl2 andadding 0.5 mM EGTA. N-methyl-D-glucamine(NMDG) Ringer’s was 100 mM NMDG, 3 mMHEPES, and 0.5 mM EGTA, pH 7.4. In someexperiments Ca-free Ringer’s was periodicallyflushed into the apical solution just prior to andduring drug addition. Because prolonged incuba-tion of the apical surface with Ca-free Ringer’sdestroys the integrity of epithelia (Contreras etal., ’92), KCl or NaCl Ringer’s (2 mM Ca) was usedfor drug washout. Amiloride, ATP, and other drugswere dissolved directly in Ringer’s solution orin a distilled water stock for later addition, withthe exception of W-7, which was dissolved in amethanol stock solution. The final methanol con-centration in the chamber never exceeded 0.5%.Methanol itself at these concentrations had no ef-fect on basal or amiloride-stimulated Isc. All so-lutions were flushed directly into the apicalchamber and removed from the top by vacuumwith no interruption in Isc (Cox, ’92, ’93).

Dose-response curves were fitted using a non-linear least squares algorithm (Microcal Origin)to the following Michaelis Menten type equa-tion which takes into account activation of theresponse:

Peak of Isc = (M * S/(Ka + S)). (1)

M is the maximum activation, Ka is the concen-tration at half maximal activation, and S is thedrug concentration. The peak of Isc was the maxi-mum value of Isc observed at each drug concen-tration. Since the response of skin to ATP variedfrom animal to animal, when comparisons weremade each skin was exposed to 1,000 µM ATP. All

of the other responses were reported as a percentof the 1,000 µM ATP response.

Values are expressed as mean ± SE (n). Differ-ences between means were established usingStudent’s t-test. Results were considered signifi-cant at the 95% confidence level. Amiloride was agift from Merck, Sharp & Dohme (West Point, PA).βγ-methylene-ATP and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7)were purchased from Research Biochemicals(Natick, MA). ATP, ADP, AMP, BzATP, UTP, ad-enosine, NMDG, atropine, curare, and suraminwere purchased from Sigma (St. Louis, MO).

RESULTSBasic response

In both NaCl and KCl Ringer’s, amiloride stimu-lates Isc abruptly. This is followed by a decay inIsc that probably represents block and desensiti-zation (Cox, ’92; Cox and Alvarado, ’79; Hillyardand Van Driessche, ’89; Hillyard et al., ’82). Inmost tadpole skins tested, ATP stimulated Isc ina manner similar to amiloride. Compare the firsttwo traces on the left panel of Figure 1. In thisexample, stimulation of Isc was about equal. Ingeneral, the ATP response was larger than thatto amiloride. In five paired experiments at highconcentrations (1,000 µM), the amiloride responsewas 54.4 ± 3.9% of the ATP response. At lowerconcentrations (100 µM), the amiloride responsewas 62.5 ± 4.1% of that with ATP. Direct com-parisons to these responses is complicated by thefact that amiloride at high concentrations appears

Fig. 1. Amiloride and ATP stimulation of Isc acrossfrog tadpole skin. Left panel: Stimulation by amiloride(100 µM) and ATP (100 µM) with 100 mM KCl, 2 mM CaRinger’s on the apical side. Amiloride or ATP was addedat the indicated arrow. Baseline current was near 0 µA.The second ATP response shows the occasionally observed(five skins) prolonged stimulation. The rapid dropoff dur-ing the decline in Isc indicates washout with drug-freeRinger’s. Right panel: ATP (100 µM) stimulation of Iscin NaCl or KCl Ringer’s (2 mM Ca).

222 T.C. COX

to stimulate and block Isc (Hillyard and VanDriessche, ’89), whereas ATP probably does nothave a blocking effect at the concentrations Itested (see below). A summary of the ATP responseat several concentrations is shown in Figure 3.The maximum projected ATP response was 106.5%of that observed at 1,000 µM ATP. The half-maxi-mal response occurred at 72 µM. In batches oftadpoles collected locally, occasionally (five skinsfrom two batches) a more prolonged ATP responsewas observed. This was characterized by anabrupt increase in Isc followed by a secondary risefor 3–5 seconds followed by a decline (Fig. 1, leftpanel, third trace).

In the presence of 2 mM calcium, the ATP (100µM) response was larger in KCl than in NaClRinger’s (Fig. 1, right panel). In four paired ex-periments, the average ATP response in NaCl was24.1 ± 4.3% of the response in KCl. When cal-cium was removed, the ATP response was in-creased (Fig. 2, top right panel). In four pairedexperiments (KCl Ringer’s), the ATP response inCa-free Ringer’s was 165 ± 25% of that observedwith 2 mM calcium. Previous studies have shownthat the amiloride response is also larger inCa-free Ringer’s (Cox, ’92; Hillyard and Van Dries-

sche, ’89). When calcium was removed, the differ-ence between ATP stimulation in Na and KRinger’s disappeared. In four paired experiments,the ratio of the maximum ATP response in K com-pared to Na was 1.09 ± 0.12. This was not differ-ent from 1.0. Similar to previous observations withamiloride (Hillyard and Van Driessche, ’89), ATPstimulates Isc in a comparable way whether Naor K is the dominant cation in the apical solu-tion. Experiments presented below were per-formed using either Na or K Ringer’s.

Analogs of ATPTo characterize further the apical membrane

purinergic response, adenosine, UTP, ADP, andAMP were tested on tadpole skin. As shown inFigure 2, bottom panel, ADP and AMP stimulatedIsc with similar time courses but decreased am-plitudes when compared to ATP. The responsesfor ADP and AMP are summarized in Figure 3.ADP appeared to have a similar effect to ATP butwith a lower affinity. AMP had a significantlylower maximum response than ATP or ADP (P <0.05). Adenosine and UTP had no effect at con-centrations up to 1,000 µM (data not shown).

βγ-methylene-ATP and BzATP have been usedto characterize purinergic receptors pharmacologi-cally (Dubyak and El-Moatassim, ’93; Surprenant

Fig. 2. Purinergic stimulation of Isc. Top left panel: Com-parison of stimulation by ATP and BzATP (50 µM) in Ca-freeNaCl Ringer’s. Top right panel: Effect of Ca removal on theATP response with KCl Ringer’s in the apical solution. Cen-ter panel: Concentration response of βγ-methylene-ATP.Numbers in parentheses are concentrations in micromolars.Bottom panel: Comparison of ATP, ADP, and AMP stimula-tion of Isc with 2 mM Ca KCl Ringer’s in the apical solution.

Fig. 3. Summary of concentration responses of ATP, ADP,AMP, and βγ-methylene-ATP in KCl, 2 mM Ca Ringer’s. Val-ues were normalized as a fraction of the response of eachskin to 1,000 µM ATP. Curves were fitted to equation 1. Val-ues at the extrapolated maximum concentration were 106.5,96.6, and 40.9% of the values at 1,000 µM ATP for ATP, ADP,and AMP, respectively. The concentrations for the half-maxi-mal responses were 72, 156, and 49 µM for ATP, ADP, andAMP, respectively. Values are means ± S.E. for three to sevenexperiments.

ATP ON FROG TADPOLE SKIN 223

et al., ’95, ’96). As shown in Figure 2, βγ-methy-lene-ATP was effective only at very high concentra-tions. At 50 and 100 µM, there was no detectableresponse. At 500 and 1,000 µM, the responses weresignificant. I did not test concentrations higher than1,000 µM. Data are summarized in Figure 3. Equa-tion 1 could not be fitted to the βγ-methylene-ATP data.

BzATP (50 µM) was tested in a Ca-free NaClRinger’s. As shown in Figure 2 (top left panel),the response was essentially identical to that ob-served with ATP. In five paired experiments, theBzATP response was 100.4 ± 9.8% of the ATP re-sponse. Similar results were obtained at 100 µM(data not shown).

Inhibitors of the ATP responseThe previous experiments have shown that ATP

opened channels that pass both Na and K. How-ever, ATP and BzATP are also known to open rela-tively large nonselective pores in some cell typesvia P2z receptors (Dubyak and El-Moatassim, ’93;Surprenant et al., ’95, ’96). This is particularlytrue in a divalent-free Ringer’s. To test for thispossibility in frog tadpole skin, K was replacedby NMDG (Ca-free Ringer’s). With a molecularweight of less than 200, NMDG is substantiallysmaller than other substances that permeate theATP or BzATP-induced pores (Dubyak and El-Moatassim, ’93). Therefore, if the pores areopened, NMDG replacement of K would not re-duce the initial transient in Isc. Examples of ex-periments of this type are presented in Figure 4.NMDG replacement of K completely eliminatedthe ATP/BzATP responses. This confirms that theIsc response is dependent on Na or K and thatthe initial purinergic response is not due to largepore formation.

Several drugs have been shown to inhibit stimu-lation of Isc by amiloride and acetylcholine (Cox,’92, ’93). These drugs were tested on the ATP re-sponse. As shown in Figure 5, atropine and W-7were quite effective at blocking the ATP response.Values for block by atropine and W-7 as well ascurare and diltiazem are summarized in Figure6. Suramin, a blocker of P2x receptors (Dubyakand El-Moatassim, ’93), was also shown to blockeffectively at 100 µM. Amiloride (100 µM)stimulation of Isc was also blocked by suramin.In five paired experiments in KCl, 2 mM CaRinger’s, suramin (100 µM) significantly re-duced the amiloride response to 49.1 ± 10.0%of control (P < 0.05).

In some cell types, zinc potentiates the stimu-

Fig. 4. Effect of replacement of K by NMDG on ATP (top)and BzATP (100 µM) (bottom) stimulation of Isc. After cal-cium removal (KCl Ringer’s), a test stimulation of ATP orBzATP was given. K was then replaced by NMDG, followedby another test pulse of ATP or BzATP.

Fig. 5. Examples of block of the ATP (100 µM) (top) re-sponse by atropine (100 µM) or W-7 (100 µM) (bottom) inthe apical solution. KCl, 2 mM Ca Ringer’s was used in theapical solution.

224 T.C. COX

lation of current by ATP (Brake et al., ’94; Li etal., ’93). To test for this effect in tadpole skin, ATPat 50, 100, and 1,000 µM was applied in the ab-sence and presence of 10 µM zinc. As shown inFigure 7, zinc blocked rather than potentiated theATP response at all ATP concentrations tested.

DISCUSSIONThe purpose of the present work was to par-

tially characterize the effect of ATP and relatedcompounds on ion transport across the larval bull-frog skin. ATP was shown to stimulate Na and Ktransport in a manner similar to amiloride andacetylcholine (Cox, ’92, ’93; Cox and Alvarado, ’79;Hillyard and Van Driessche, ’89; Hillyard et al.,

’82). Blockers of amiloride and acetylcholine stimu-lation also blocked the ATP effect. In addition,suramin, a well-known blocker of the P2x puri-nergic receptor, blocked both the ATP and theamiloride response in tadpole skin. This repre-sents the first report of purinergic activation ofcation transport in amphibian skin.

Nature of the ATP responseWhen ATP was perfused into the apical solu-

tion, Isc increased abruptly, reaching a peakwithin a few hundred milliseconds. Isc then de-clined towards zero over the next several seconds.This time course was similar to purinergic re-sponses seen in other cell types (Bean, ’92; Beanand Friel, ’90; Ecelbarger et al., ’94; Surprenantet al., ’95, ’96). In some skins (Fig. 1) there was asecondary transient rise in Isc after the initialabrupt increase. Whether this represents a varia-tion of the more typically observed response oractivation of second messenger systems or a sec-ond transport system cannot be determined at thistime. A secondary rise is Isc has also been ob-served after acetylcholine treatment in sometadpole skins (unpublished observations). Thisphenomenon will be studied in more detail whenit can be routinely obtained.

The direction of current flow in the ATP re-sponse is consistent with Na or K entry acrossthe apical membrane. This was confirmed by theexperiments where Na or K was replaced byNMDG in the apical solution. This eliminated theATP-stimulated current while maintaining a con-stant chloride gradient. ATP stimulation of chlo-ride current seen in other epithelia (Ecelbargeret al., ’94; Stutts et al., ’92) apparently does notoccur in larval frog skin under these conditions.

ATP stimulated current whether Na or K wasthe dominant cation in the apical solution. Thiswas particularly true in calcium-free Ringer’s (seebelow). With NaCl Ringer’s outside, this probablyrepresents a sodium influx into the cell down achemical and electrical gradient (Robinson andMills, ’87). With KCl Ringer’s outside, a chemicalgradient for K entry is presumed to be absent.Considering the substantial Isc response, theremust be a substantial electrical gradient for K en-try. This is also consistent with a preference forK over Na by the tadpole channel (Hillyard andVan Driessche, ’89). Current across the basolateralmembrane can be accounted for by Na flux throughthe pump (pump current) and K leak currentthrough basolateral K channels (Cox and Alvarado,’79; Hillyard and Van Driessche, ’92; Robinson and

Fig. 6. Summary of blocker effects on ATP (100 µM) stimu-lation of Isc. KCl, 2 mM Ca Ringer’s was used in all experi-ments. Blockers used were atropine (Atrop), W-7, curare (Cur),diltiazem (Dilt), and suramin (Sura) at 100 µM. Values aremean ± S.E. for at least four experiments each.

Fig. 7. Effect of 10 µM zinc on ATP stimulation of Isc. NaCl,2 mM Ca Ringer’s was used in all experiments. Values aremean ± S.E. for four experiments at each ATP concentration.

ATP ON FROG TADPOLE SKIN 225

Mills, ’87; Cox and Alvarado, ’83). Current flowthrough the shunt path cannot make a contribu-tion when NaCl Ringer’s is on both sides of theskin under short circuit conditions. When KClRinger’s is in the apical solution, the possibilitythat ATP opens up a cation selective shunt pathcannot be eliminated. However, the facts thatblocker inhibition and the time courses are verysimilar with either NaCl and KCl Ringer’s on theapical side argue against this possibility. Fluctua-tion analysis data also suggest that the currentfor Na and K is via a channel rather than througha paracellular pathway (Hillyard et al., ’82;Hillyard and Van Driessche, ’89).

Role of calciumAs mentioned above, ATP stimulates Isc with

either NaCl or KCl Ringer’s in the apical solu-tion. In the presence of 2 mM calcium, theresponse is two to three times larger in KCl com-pared to NaCl Ringer’s. A larger response in KClRinger’s has also been observed for amiloridestimulation (Hillyard and Van Driessche, ’89).When calcium was removed, there was a smallbut significant increase in Isc (Alvarado and Cox,’85; present study). This was generally larger withKCl Ringer’s in the apical solution. When ATP wasadded to either a calcium-free Na or K Ringer’s,the response was much greater. It has been sug-gested that calcium may block a negativelycharged binding site for amiloride or that calciummay block the channel itself (Alvarado and Cox,’85; Cox, ’92; Hillyard and Van Driessche, ’89).

In calcium-free EGTA Ringer’s, the concentra-tion of ATP–4 is greatly increased compared to 2mM calcium Ringer’s. ATP–4 has been shown toopen relatively large nonselective pores in othercell types (Dubyak and El-Moatassim, ’93; Sur-prenant et al., ’96). The fact that ATP had no ef-fect on Isc in NMDG Ringer’s in tadpole skinsuggests that nonselective pores are not opened,at least not during the time frame studied here.Using a calcium free Ringer’s also effectively elimi-nates calcium influx across the apical membrane.Since ATP is effective with apical calcium re-moved, calcium entry across the apical membraneis not likely to be the mechanism for the ATP re-sponse. Since the ATP stimulation of Isc is likelyto depolarize the basolateral membrane undershort circuit conditions, the possibility of calciumentry across the basolateral membrane throughvoltage-gated calcium channels has to be consid-ered. However, preliminary studies have shownthat loading or depleting cells of calcium does not

eliminate the ATP response (unpublished data)consistent with the idea that calcium may nothave a direct role as a cytosolic second messen-ger in the tadpole ATP response.

Relationship to the amiloride andacetylcholine responses

While the time course of the ATP response of-ten resembles that induced by amiloride, ATP andamiloride bear little resemblance to each other.The ring structure of adenosine looks a little likeamiloride, but adenosine alone was without effect.In addition, the number of negatively chargedphosphate groups was important to the response.ATP was much more effective than AMP. In con-trast, a positive charge appears to be importantfor the effect of amiloride and acetylcholine (Benoset al., ’95; Cox, ’92, ’93; Hillyard and Van Dries-sche, ’89). I favor the idea that ATP binds to adifferent site on the same receptor that bindsamiloride and acetylcholine. However, the possi-bility that different receptors are involved cannotbe eliminated at the present time.

Does ATP activate the same channels as doesamiloride? Several lines of evidence suggest thatit does. The time courses of amiloride and ATPstimulation are generally very similar. Currentsstimulated by ATP or amiloride are carried by ei-ther Na or K but not NMDG. The blockers W-7,curare, atropine, diltiazem, and suramin blockstimulation by both amiloride and ATP. Currentsstimulated by amiloride and ATP are larger in cal-cium-free Ringer’s. These observations suggestthat there is a common pathway of activation forthe two ligands. However, it is not known atpresent where the blockers exert their action.Suramin, curare, and atropine are thought to in-terfere with ligand binding (Cox, ’93; Dubyak andEl-Moatassim, ’93; Zimmerman, ’94). Since theyall block both the amiloride and ATP responsesin tadpole skin, it would appear that the ATP andamiloride binding sites are very near to each other.W-7 and calcium may directly block the channel.The above observations suggest that ATP andamiloride bind to nearby sites and stimulate acommon transport pathway. However, the inter-action between the two may be complex.

Relationship to purinergic receptorsStudies with ATP, adenosine, and related com-

pounds allow some comparisons of one receptortype with another. The lack of response of the tad-pole skin to adenosine places it in the category ofP2 purinergic receptors, of which there are at least

226 T.C. COX

five pharmacologically defined subtypes (Dubyakand El-Moatassim, ’93). Some of these can beeliminated from consideration using the classifi-cation scheme in Table 1 of Dubyak and El-Moatassim (’93). In P2t receptors, ATP is anantagonist. It clearly is not in tadpole skin. ForP2u receptors, UTP is the best agonist. For P2yreceptors, βγ-methylene-ATP has equal potencywith UTP, UTP is not effective in tadpole. We areleft with the P2x and P2z receptors. BzATP is ef-fective in tadpole skin, as it is on the P2z recep-tor. However, βγ-methylene-ATP and suramin arewithout effect on P2z receptors (Surprenant et al.,’96) and do work in tadpole skin. In some celltypes, zinc apparently increases the affinity of P2xreceptors for ATP (Brake et al., ’94; Li et al., ’93).In tadpole skin, zinc blocked rather than increasedthe ATP response, demonstrating that it does notshare this characteristic with those P2x receptors.P2x receptor agonists βγ-methylene-ATP, ATP, andADP and to a lesser extent AMP activate Isc inthe tadpole skin. Suramin also blocks both theP2x and the tadpole ATP response. P2x receptorsactivate cation-selective channels and show strongdesensitization similar to the ATP response in tad-pole skin (Bean, ’92; Bean and Friel, ’90). In sum-mary, the ATP receptor in the tadpole skin mostclosely resembles the P2x receptor (Dubyak andEl-Moatassim, ’93; Zimmerman, ’94).

There are complex interactions between P2xand acetylcholine receptors in PC12 cells andrat skeletal muscle (Dubyak and El-Moatassim,’93; Zimmerman, ’94). ATP has been shown topotentiate the postjunctional acetylcholine re-sponse (Zimmerman, ’94). Preliminary experi-ments in tadpole skin suggest that there maybe equally complex interactions among ATP,amiloride, and acetylcholine. This is currentlyunder study.

Function of the purinergic receptorSince ATP is not found in high concentrations

in extracellular spaces and is generally not veryplentiful in pond water, what function might apurinergic receptor in tadpole skin serve? One pos-sibility is that it is a relatively nonspecific recep-tor system that responds to several ligands notnormally found in the environment. This couldserve a sensory function and alert the tadpole tothe presence of unusual compounds in the pond.Additional support for a sensory role comes fromstudies of toads. An amiloride-sensitive process intoad skin has been shown to be involved in detec-tion of solution osmolarity (Von Seckendorff Hoff

and Hillyard, ’93). A role for ATP in this responsehas not been demonstrated.

Purinergic receptors have also been shown tobe involved in pain reception in other tissues(Bean, ’92). Alternative hypotheses might includethe possibility of a function in mucus secretion.ATP receptors on granules of the land slug trig-ger mucus release, probably after a sheer stress(Deyrup-Olsen et al., ’92). The tadpole skin has athick protective mucus layer. Another possibilityis that these receptors are involved in apoptosis,a process that removes the apical cell layer atmetamorphosis (Yoshizato, ’89). P2x purinergicreceptors are expressed in apoptotic thymocytes(Brake et al., ’94; Surprenant et al., ’95). It is alsointeresting to note that the P2x receptor moreclosely resembles the amiloride-blockable epithe-lial sodium channel, ENaC, than other channels(Benos et al., ’95; Canessa et al., ’93, ’94; Surpre-nant et al., ’95). Perhaps the tadpole channel isrelated to both ENaC and P2x. ENaC is relatedto the degenerin proteins of Caenorhabditiselegans (Canessa et al., ’93). Degenerins are in-volved in the sense of touch in the nematode.

ACKNOWLEDGMENTSThe excellent technical assistance of H. Dee

Gates is gratefully acknowledged. Support wasprovided by the American Heart Association, Illi-nois Affiliate.

LITERATURE CITEDAlvarado, R.H., and T.C. Cox (1985) Action of polyvalent cat-

ions on sodium transport across skin of larval and adultRana catesbeiana. J. Exp. Zool., 236:127–136.

Bean, B. (1992) Pharmacology and electrophysiology of ATP-activated ion channels. Trends Pharmacol. Sci., 13:87–90.

Bean, B.P., and D.D. Friel (1990) ATP-activated channels inexcitable cells. In: Ion Channels. T. Narahashi, ed. Plenum,New York, Vol. 2, pp. 169–203.

Benos, D.J., M.S. Awayda, I.I. Ismailov, and J.P. Johnson(1995) Structure and function of amiloride-sensitive Nachannels. J. Membr. Biol., 143:1–18.

Brake, A.J., M.J. Wagenbach, and D. Julius (1994) New struc-tural motif for ligand-gated ion channels defined by aniontropic ATP receptor. Nature, 371:519–523.

Buell, G., C. Lewis, R.A. North, and A. Surprenant (1996) Anantagonist insensitive P2x receptor expressed in epitheliaand brain. EMBO J, 15:55–62.

Canessa, C.M., J.-D. Horisberger, and B.C. Rossier (1993) Epi-thelial sodium channel related to proteins involved inneurodegeneration. Nature, 361:467–470.

Canessa, C.M., L. Schild, G. Buell, B. Thorens, I. Gautschi,J.D. Horisberger, and B.C. Rossier (1994) Amiloride sensi-tive epithelial Na channel is made of three homologous sub-units. Nature, 367:463–467.

Contreras, R.G., L. Gonzalez-Marsical, M.S. Balda, M.R.Garcia-Villegas, and M. Cereijido (1992) The role of calcium

ATP ON FROG TADPOLE SKIN 227

in the making of a transport epithelium. News Physiol. Sci.,7:105–108.

Cox, T.C. (1986) Ion transport across the skin of a larval sala-mander. Biochim. Biophys. Acta, 858:145–152.

Cox, T.C. (1992) Calcium channel blockers inhibit amiloridestimulated short-circuit current in frog tadpole skin. Am.J. Physiol., 263:R827–R833.

Cox, T.C. (1993) Low affinity mixed acetylcholine responsivereceptors at the apical membrane of frog tadpole skin. Am.J. Physiol., 264:C552–C558.

Cox, T.C., and R.H. Alvarado (1979) Electrical and transportcharacteristics of skin of larval Rana catesbeiana. Am. J.Physiol., 237:R74–R79.

Cox, T.C., and R.H. Alvarado (1983) Nystatin studies ofthe skin of larval Rana catesbeiana. Am. J. Physiol.,244:R58–R65.

Deyrup-Olsen, I., H. Louie, A.W. Martin, and D.L. Luchtel(1992) Triggering by ATP of product release by mucous gran-ules of the land slug Ariolimax columbianus. Am. J. Physiol.,262:C760–C765.

Dubyak, G., and C. El-Moatassim (1993) Signal transductionvia P2-purinergic receptors for extracellular ATP and othernucleotides. Am. J. Physiol., 265:C577–C606.

Ecelbarger, C.A., Y. Maeda, C.C. Gibson, and M.A. Knepper(1994) Extracellular ATP increases intracellular calcium inrat terminal collecting duct via a nucleotide receptor. Am.J. Physiol., 267:F998–F1006.

Hillyard, S.D., and W. Van Driessche (1989) Effect of amilorideon the poorly selective cation channel of larval bullfrog skin.Am. J. Physiol., 256:C168–C174.

Hillyard, S.D., and W. Van Driessche (1992) Verapamil blocks

basolateral K channels in the larval frog skin. Am. J.Physiol., 262:C1161–C1166.

Hillyard, S.D., W. Zeiske, and W. Van Driessche (1982) Poorlyselective cation channels in the skin of the larval frog (stageXIX). Pfluegers Arch., 394:287–293.

Li, C., R.W. Peoples, Z. Li, and F.F. Weight (1993) Zn+2 po-tentiates excitatory action of ATP on mammalian neurons.Proc. Natl. Acad. Sci. U.S.A., 90:8264–8267.

Robinson, D.H., and J.W. Mills (1987) Ouabain binding intadpole ventral skin I. Kinetics and effect on intracellularions. Am. J. Physiol., 253:R74–R79.

Stutts, M.J., T.C. Chinet, S.J. Mason, J.M. Fullton, L.L. Clarke,and R.C. Boucher (1992) Regulation of Cl– channels in nor-mal and cystic fibrosis airway epithelial cells by extracellu-lar ATP. Proc. Natl. Acad. Sci. U.S.A., 89:1621–1625.

Surprenant, A., G. Buell, and A. North (1995) P2x receptorsbring new structure to ligand-gated ion channels. TrendsNeurosci., 18:224–229.

Surprenant, A., F. Rassendren, E. Kawashima, R. North,and G. Buell (1996) The cytolytic P2z receptor for ex-tracellular ATP identified as a P2x receptor (P2x7). Sci-ence, 272:735–738.

Von Seckendorff Hoff, K., and S.D. Hillyard (1993) Toads tastesodium with their skin: Sensory function in a transportingepithelium. J. Exp. Biol., 183:347–351.

Yoshizato, K. (1989) Biochemistry and cell biology of amphib-ian metamorphosis with a special emphasis on the mecha-nism of removal of larval organs. Int. Rev. Cytol., 19:149–163.

Zimmerman, H. (1994) Signalling via ATP in the nervous sys-tem. Trends Neurosci., 17:420–426.