734
OTHER EPITHELIA
[47] Turtle Colon: Keeping Track of Transporters Apical and Basolateral Membranes
[47]
in the
B y DAVID C. DAWSON and DEAN CHANG
T h e Study of Parts Our nearly 20-year romance with the turtle colon began, as many things do in science, by accident. In 1973 one of us (D.C.D.) had just arrived at the University of Iowa as an Assistant Professor of Physiology and Biophysics having completed a 2-year postdoctoral stint in the laboratory of Peter F. Curran at Yale University. In Peter Curran's lab we studied active Na + absorption using the colon of an amphibian, the toad, Bufo marinus. 1 We chose the toad colon because it appeared as though it might be a good model system in which we could study the subcellular "parts" involved in active Na + absorption. We were looking, in a sense, for a better frog skin. The initial attempt to continue studies of the toad colon at Iowa was thwarted by the Iowa winter: our first shipment of toads from Colombia, South America arrived frozen solid! At this point it seemed important to study these parts, regardless of their origin, so we called Phillip Steinmetz, a long-time student of turtles who had recently arrived as Iowa's new Chief of Nephrology. He assured us that turtles possessed a colon and within a few days we had determined that the turtle colon transported Na + better than its amphibian counterpart and that, as an additional bonus, there was much more of it. Furthermore, sharing the animals made good ecological sense so it was resolved to continue the pursuit of active Na + absorption using the turtle colon. Seventeen years later it appeared that the turtle colon was a good choice for a model for Na + absorbing epithelium. It is a prodigious Na -+transport machine with rates of absorption far exceeding those in the frog skin or toad bladder. The epithelium is a single layer of cells, lacking the crypts and folds characteristic of mammalian colon. The isolated tissue is quite hardy and can be maintained in vitro for several days and subjected to a variety of experimental maneuvers (such as apical permeabilization) while maintaining its functional properties. In addition salt absorption is subject to both stimulatory (aldosterone) and inhibitory (cholinergic) control. In this chapter we will describe our attempts to identify and characterize the
D. C. Dawsonand P. F. Curran, J. Membr. Biol. 28, 295 (1976). METHODS IN ENZYMOLOGY, VOL. 192
Copyright © 1990 by Academic Press, Inc. All rights ofrqmxluetion in any form rc~rved.
[47]
TURTLE COLON
735
Blood
K÷
Na"~K"
FIG. 1. Right: Electron micrograph showing the columnar cells which compose the epithelial cell layer of the turtle colon. Le~: The Koefoed-Johnsen-Ussing model for active Na + absorption. Sodium ion is presumed to enter the cell across the terminal membrane via amiloride-sensitive Na + channels and exit the cell across the basolateral membrane via the Na+,K+-ATPase.
individual transporters which comprise the molecular basis for the ion transport properties of the turtle colon. One of the vexing problems associated with epithelial cells is their tendency to lose their highly differentiated properties when they are isolated from one another. In order to obtain information about individual apical and basolateral transporters it has been necessary, therefore, to design experimental strategies which permit some degree of functional isolation of the transporters of interest while preserving the structural integrity of the cell layer. In several instances the turtle colon has proved to be uniquely suited to this type of experimental approach. Transepithelial Transport
Cellular and Noncellular Paths Figure 1 shows an electron micrograph of the colonic epithelial cell layer juxtaposed with the model used by Koefoed-Johnsen and Ussing2 to describe active Na + absorption by frog skin. The long columnar cells of the turtle colon form a single, flat cell layer and, as might be expected, ions can traverse the cell layer by way of cellular and paracellular paths. In our initial description of the properties of the colon3 we used 3H-labeled mannitol as a marker for the paracellular path and concluded that solute diffusion between the cells behaved much as it would in free solution. This 2 V. Koefoed-Johnsen and H. H. Ussing, Acta Physiol. Scand. 42, 298 (1958). 3 D. C. Dawson, J. Membr. Biol. 37, 213 (1977).
736
OTHER EPITHELIA
[471
and later studies4 were all consistent with the notion that the paracellular path behaved as a watery leak with no special selectivity properties. With identical solutions bathing both sides of the tissue the transepithelial electrical potential difference would constitute a driving force for net transepithelial movement of Na ÷, K +, and CI- via this extracellular leak path. The transepithelial potential difference across isolated colonic sheets, stripped of musculature, varies from about 30 to 130 mV, lumen negative, and the transepithelial resistance is in the range of 500 to 200 f~ • cm 2, placing this epithelium in the class of "moderately tight" epithelia. Our earliest estimates of cellular and paracellular conductances, 0.6 and 1.4 mSc/cm 2, respectively, were probably biased by the tissue damage which is inevitable in conventional Ussing chambers. More recent experiments suggest that values of 1.48 for the cellular conductance and 0.65 for the shunt conductance are more typical. Active Transport o f N a + and K +
Electrogenic Na + absorption is the dominant transport process in the turtle colon, comprising greater than 90% of the short-circuit current (I,~) which is virtually abolished by mucosal amiloride. Sodium ion absorption is also abolished by serosal ouabain. Absorption is stimulated by aldosterone 5 but is inhibited by muscarinic cholinergic agonists~ as well by adenosine and histamine. 7 The basis for the small residual 1,~ observed in the presence of mucosal amiloride is, at present, unknown. Potassium ion is both actively absorbed and actively secreted by turtle colon 4 but at rates which are of the order of 100-fold less than that of Na ÷. In some tissues the net flow was absorptive under short-circuit conditions, but little is known about this process except that it was blocked by mucosal (but not serosal) ouabain and orthovandate (M + S). The secretory transport, when present, was blocked by amiloride under short-circuit conditions, a result which was taken to reflect an effect of the reduced turnover of the basolateral Na ÷, K+-ATPase and hyperpolarization of the apical membrane potential. Potassium ion secretion was enhanced by serosal barium and blocked by mucosal barium, suggesting that the net rate of secretion depended on the ratio of the apical to basolateral K + conductances. As expected for a conductive transport process the cellular secretory rate was highly dependent on transmural potential. 8 The normal, lumennegative open cicuit potential enhanced K + secretion so that net trans4 D. R. Halm and D. C. Dawson, Am. J. Physiol. 246, C315 (1984). 5 D. R. Halm and D. C. Dawson, Pfluegers Arch. 403, 236 (1985). 6 C. J. Venglarik and D. C. Dawson, Am. J. Physiol. 251, C563 (1986). C. J. Vengladk. Cholinergic Regulation of Salt Absorption by Turtle Colon: Dual Control of Sodium and Chloride Transport. Ph.D. Thesis, University of Michigan, 1988. s D. R. Halm and D. C. Dawson, Am. J. Physiol. 247, C26 (1984).
[47]
TURTLE COLON
737
mural K + flow in vivo would be expected to be secretory. The relatively small rate of K + secretion (compared to Na + absorption) may indicate that not all of the Na+-absorbing cells contain the apical K + channels necessary for the exit o f K + toward the lumen, or simply that apical K + conductance is quite small in all of the cells. A Cellular CI- Leak?
The initial description3 of the transport of NaC1 by the colon produced no evidence for any net movement of C1- under short-circuit conditions, but in some experiments there was the suggestion of a cellular component of the transmural flux of ~sC1-. More recent experiments of Vengiarik and Dawson9 suggested, in fact, that a relatively small proportion of turtles (-5%) exhibited a substantial, transceUular C1- leak pathway which had several interesting properties. The appearance of the CI- leak was accompanied by a dramatic increase in tissue conductance which could not be attributed to the paracellular path. The conductive leak was highly C1selective as judged by C1- currents (induced by imposing transmural C1gradients) and transmural ~sc1- fluxes. The C1- leak path was inactivated by carbachol (as was the net active transport of Na +) but was specifically inhibited by experimental maneuvers which were expected to elevate cellular cyclic AMP (phosphodiesterase inhibitors, forskolin, cAMP derivafives). These maneuvers did not affect Na + absorption. Net C1- transport occurred only in the presence of an applied driving force, suggesting that the presence of this highly anion-selecfive leak pathway could facilitate NaC1 absorption under open circuit conditions. The salt absorptive process could be envisioned as being driven by active Na + absorption (the engine of salt absorption) but also dependent on the conductance of the anion-selective leak path. In addition, the inhibitory regulation of salt absorption by cholinergic agonists was seen as having a dual mechanism: inhibition of active Na + absorption and attenuation of the C1- leak path. Although these were intriguing thoughts, further study of this transmural path has been frustrated by the fact that it appears in such a small percentage of the turtles examined. Apical Ion Channels As expected from the large component of electrogenic Na + absorption the dominant components of the apical membrane of the turtle colon appears to be amilofide-sensitive, Na+-selective channels. We examined these initially by measuring initial rates of ZZNa+uptake from the mucosal 9 C. J. Venglarik and D. C. Dawson, Fed. Proc., Fed. Am. Soc. Exp. Biol. 46, 496 (1987).
738
OTHER EPITHELIA
[47]
bath 1° and later using blocker-induced Na + channel noise?' Thompson and Dawson ~° showed that a 30-sec tracer uptake from the mucosal path provided an estimate of the rate coefficient for Z2Na+ entry across the apical membrane of the transporting cells. At low mucosal Na + concentrations the entry rate coefficient was highly correlated with the amiloride-sensitive, transmural conductance as expected if the apical membrane was the major resistance barrier in the transporting cells. Other studies provided evidence that these apical channels were highly permeable to Li+ but that the presence ofLi + did not markedly affect Na + entry, as if the two ions did not interact in the conduction process/2 This result would be consistent with the notion that the apical amiloride-sensitive channels can accommodate either Na + or Li+ but contain only one ion at any instant. Kirk and Dawson 13 obtained evidence that the apical Na + conductance can be modulated in response to changes in intracellular Na + concentration. Experimental maneuvers which would be expected to lead to an increase in cytosolic Na + concentration produced an inhibition of the Na + conductance of the apical membrane. In ouabain-treated tissues this inhibition could be reversed by simply depleting cellular Na +. Wilkinson and Dawson H applied the techniques of Van Driessche and Lindemann ~4to study blocker-induced fluctuations in apical Na + currents. Using the weak Na + channel blocker, CDPC, 15 they observed blocker-induced Lorentzian components in the power density spectra which were indicative of reversible blockade of open Na + channels. Under control conditions single-channel Na + currents averaged 0.43 pA and the density of Na + channels was 250 × 106 channels/cm 2. Carbachol, which inhibited Na + absorption, caused a decrease in the number of open Na + channels but did not alter the single-channel current. The result was consistent with the notion that inhibitory cholinergic control of Na + absorption was associated with decrease in the conductance of both the apical and the basolateral membranes such that the fractional resistance was not greatly altered. Noise analysis also produced evidence for apical K + channels. 16 In some instances it was possible to detect a "spontaneous" Lorentzian component in the power density spectrum with a comer frequency of about 15 Hz. This Lorentzian appeared to be unrelated to Na + absorption. It was abolished by mucosal barium, however, and the plateau values were aug-
,0 S. M. Thompson and D. C. Dawson, J. Membr. Biol. 42, 357 (1978). H D. J. Wilkinson and D. C. Dawson, FASEBJ. in press (1989). ,2 S. M. Thompson and D. C. Dawson, J. Gen. Physiol. 72, 269 (1978). 23 K. L. Kirk and D. C. Dawson, Pfluegers Arch. 403, 82 (1985). 14W. Van Driessche and B. Lindemann, Nature (London) 282, 519 (1979). 15 S. I. Helman and L. M. Baxendale, 95, 647 (1990). 26D. J. Wilkinson and D. C. Dawson, Am. J. Physiol. 259, 668 (1990).
[47]
TURTLE COLON
739
mented by applying a serosa-positive transmural potential. This behavior suggested that these fluctuations emanated from the apical channels which mediated the potential-sensitive K + secretion documented by Halm and Dawson. s Basolateral Transporters
Permeabilized Cell Layers In most studies of isolated sheets of turtle colon the tissue is stripped of circular and longitudinal musculature, but a layer of connective tissue and a muscularis mucosa remains on the serosal side. The thickness of the layer is roughly 5 to 10 times that of the single layer of mucosal cells so that the shortest route to the basolateral membrane is through the apical membrane? 7,~s For this reason we have explored a number of approaches to chemically modifying the apical membrane so that it no longer constitutes a significant barrier to transcellular ion flow. The object of any such strategy is to reach a compromise between two goals: that of eliminating the apical membrane as a permeability barrier and that of restricting or controlling the changes in intracellular composition which occur as a result of the permeabilization process. To this end we used the pore-forming, polyene antibiotic, amphotedcin B, to permeabilize the apical membrane to monovalent cations. The principal advantage of amphoteriein as a permeabilizing tool is its relative selectivity. The resulting pores are relatively nonselective for monovalent cations so that the membrane becomes freely permeable to Na + and K +. The pores appear to be impermeable to divalent cations, however, and six-carbon sugars. Monovalent anions are only about one-seventh as permeant as cations but the C1- permeability is sufficient to increase salt entry into the cells. One consequence of this is that cells exposed to amphotericin will gain salt if bathed by mucosal NaCI or KCI, a fact exploited by Germann et aL ~9 to induce swelling in turtle colon cells. In our earliest experiments we investigated a number of different anion replacements in order to find one which would attenuate cell swelling but would also preserve cellular transport. We settled on benzene sulfonate and used this in a number of experiments although we later learned that this anion may, itself, promote some degree of cell swelling. ~9 It is interesting that the effects of amphotericin appear to be completely reversible even though permeabilization can induce substantial changes in cell volume and cellular ionic composition. 17D. C. Dawson, Curr. Top. Membr. Transp. 211,41 (1987). ~8D. C. Dawson, D. J. Willdnson, and N. W. Riehards, Curr. Top. Membr. Transp. 37, 191 (1990). 19W. J. Germann, S. A. Ernst, and D. C. Dawson, J. Gen. Physiol. 88, 253 (1986).
740
OTHER EPITHELIA
[47]
Na+/K + P u m p and Potassium Conductance
Our first experiments with amphotericin-permeabilized colon were designed to determine if the basolateral membranes contained the two elements basic to the Koefoed-Johnsen-Ussing model, a basolateral K + conductance and an Na+/K + exchange pump. 2° A K + conductance was identified by imposing a transmural K + gradient and noting that the resulting K + current was blocked by serosal barium. Using tissues treated with serosal barium (but not ouabain) it was then possible to show that in the absence oftransmural gradients a current appeared which exhibited an obligatory dependence on mucosal Na + and serosal K + and was inhibited by ouabain. Transmural flux determinations using these permeabilized tissues confirmed that the current was due to opposing net Na + and K + fluxes in the ratio 3Na+: 2K +. Halm and Dawson 2~ arrived at the same estimate of the pump stoichiometry by studying the kinetics of activation by mucosal (cellular) Na + and serosal K +. Kirk and Dawson 22 investigated the properties of the basolateral K + conductance and found that under the conditions of these experiments (benzene sulfonate as the anion) it was possible to observe basolateral currents carried by K ÷, Rb +, or Th +, all of which were blocked by serosal barium. Furthermore, 42K+ fluxes revealed coupling between the flows of tracer and abundant species which was indicative of single filing. Subsequent experiments by Germann et al. 23 revealed that the basolateral K + conductance (gK) observed in tissues bathed by benzene sulfonate ringers was more complex than we had initially guessed. Germann et al. 23 showed that it was possible to identify two distinct components to the basolateral K + conductance in permeabilized cells. One did not discriminate between K + and Rb +, was blocked by serosal barium (but not by quinidine or lidocaine), was inactivated by cholinergic agonists, and was present under conditions which prevented the cells from swelling. Because normal active transport was supported equally well by K + or Rb + and was inactivated by cholinergic agonists6 this conductance was dubbed the "resting" K + conductance. If the cells were swollen by the entry of salt or urea, a second conductance was activated which could be distinguished by a marked preference for K + over Rb +, sensitivity to quinidine and lidocaine, and marked single-filing behavior. In addition this conductance could be activated by swelling in cell layers which had previously been exposed to carbachol to inactivate the resting gK. Germann et a/.~9 pro2o K. L. Kirk, D. R. Halm, and D. C. Dawson, Nature (London) 287, 237 (1980). 2mD. R. Halm and D. C. Dawson, J. Gen. Physiol. 82, 315 (1983). 22 K. L. Kirk and D. C. Dawson, J. Gen. Physiol. 82, 297 (1983). 23 W. J. Germann, M. E. Lowy, S. A. Ernst, and D. C. Dawson, J. Gen. Physiol. 88, 237 (1986).
[47]
TURTLE COLON
741
posed that this second conductance was due to a second population of channels which was not active under resting conditions but was activated by cell swelling. In retrospect, it seems clear that the earlier results of Kirk and Dawson~ represented the properties of a mixed channel population. The Rb + currents are likely to have been due to the resting conductance whereas the strong single-filing probably reflected a component of the swelling-induced gK. Dawson, Van Driessche, and Helman 24 used noise analysis to investigate the possible induction of basolateral K + channels by cell swelling. They showed that under conditions of cell swelling it was possible to detect a lidocaine-induced, Lorentzian component in the power density spectrum of the basolateral K + current. Under nonswelling conditions this lidocaineinduced Lorentzian was absent. This result was consistent with the notion that cell swelling activated a population of lidocaine-blockable channels in the basolateral membrane which was not active under normal osmotic conditions. Analysis of the blocker-induced noise provided an estimate of 20 pS for the single-channel conductance. Richards and Dawson25 used single-channel recording techniques to investigate K + channels in isolated colonic cells. In cells bathed in KC1-Ringer's solution they identified a 20-pS channel which was blocked by lidocaine and quinidine and appears to be the molecular basis for the swelling activated gK. Venglarik and Dawson~ investigated the possible role of the resting gk in the inhibitory cholinergic control of Na + absorption. They showed that cholinergic agonists, either exogenous or released from submucosal nerves, inactivated both active Na + absorption and basolateral gK. The cholinergic response of gK could be duplicated by exposing the tissue to a calcium ionophore, ionomycin, suggesting that perturbing intracellular calcium activity was sufficient to trigger the chain of events which led to the inactivation of basolateral gK" Richards and Dawson, 26,27 however, had recorded in isolated cells several types of channels which were activated by cytoplasmic calcium, suggesting that calcium might be involved in both activation and inactivation of components of the basolateral gK. CI- Channels
Venglarik et al. 2s identified a basolateral CI- conductance in amphotericin-permeabilized colonic cells. This conductance had several properties 24D. C. Dawson, W. VanDriessch¢, and S. I. Helman, Am. £ Physiol. 254, C165 (1988). 25N. W. Richards and D. C. Dawson, Am. J. Physiol. 251, C85 (1986). 26N. W. Richards and D. C. Dawson, Biophys. J. 51, 344a (1987). 27N. W. Richards and D. C. Dawson, FASEB £ 3, A1149 (1989). 2s C. J. Venglafik, J. L. Keller, and D. C. Dawson, Fed. Proc., Fed. Am. Soc. Exp. Biol. 45, 2082 (1986).
742
OTHER EPITHELIA
[47]
which suggested that it is related to the transceUular C1- leak which is present in a small percentage of colons studied. The conductance, which could be identified by ion substitution, was inactivated by muscarinic cholinergic agonists, as was the resting basolateral K + conductance. Basolateral go-, however, was specifically inhibited by experimental maneuvers which are expected to raise the cellular levels of cyclic AMP. Exposure to forskolin, phosphodiesterase inhibitors [3-isobutyl-l-methylxanthine (IBMX), for example] or cAMP derivatives (8 cAMP) all produced inactivation of get- but had little or no effect on gK- As opposed to the transcellular CI- leak this basolateral conductance was present in virtually all cell layers examined. At present, however, the physiological role of this conductance and the significance of the possible regulation by cAMP have yet to be determined.
Role of lntracellular Cae+: Digitonin-Permeabilized Cells Questions concerning a possible role for cytoplasmic calcium in the control of basolateral gK led Chang and Dawson29 to develop a preparation of the isolated turtle colon which was apically permeabilized by the detergent, digitonin. Exposure of the apical membranes to digitonin (20/tM) led to the release of the cytoplasmic enzyme, lactate dehydrogenase (LDH), from the ceils and rendered the apical membrane highly permeable to monovalent and divalent cations as well as to organic buffers for pH (PIPES) and calcium (EGTA). Somewhat surprisingly the basolateral membrane retained its functional integrity despite this chemical onslaught. Chang and Dawson 29 speculated that the long columnar shapes of the epithelial cells may have contributed to this result. In an attempt to at least partially mimic the intracellular milieu Chang and Dawson employed a mucosal solution which consisted primarily of potassium aspartate along with an EGTA (5 m M ) buffer system designed so that the free calcium concentration could be readily controlled in the range of 10-9 to 10--6 M. 3° The pH was buffered to 6.6 to exploit the optimal Ca2+-buffering range of EGTA. In the presence of about 10-9 M mucosal calcium digitonin permeabilization did not lead to a transcellular K + current. Raising the free calcium to 10-7 Mr, however, led to the prompt development of a K + current which could be rapidly inactivated by adding additional EDTA to reduce the free calcium. This type of result led to the conclusion that the basolateral membranes contained a K + conductance which could be activated by increases in cytosolic calcium. Similar experiments conducted using K+-free solutions in the presence of a C1- gradient 29 D. Chang and D. C. Dawson, J. Gen. Physiol. 82, 281 (1988). ~oD. Chang, P. S. Hsieh and D. C. Dawson, Comput. Biol. Med. 18(5), 351 (1988).
[47]
TURTLE COLON
743
were used to identify a calcium-activated basolateral C1- conductance. Both the K + and C1- conductances exhibited spontaneous inactivation in the continued presence of calcium. The nature of this inactivation is not understood at present. The activation of basolateral conductances in digitonin-permeabilized cells was also sensitive to cytoplasmic pH. 3~ Raising the cytosolic pH from 6.6 to 7.4 shifted the calcium activation dose response to the left so that there was greater activation at any given concentration of free calcium. These results suggested that the calcium activity and intracellular pH could interact to determine the degree of activation of basolateral conductances, and that the mechanism of this interaction is a proton modification of the calcium activation site. Regulation of basolateral conductance by pH is particularly interesting in fight of the demonstration of a basolateral Na+/H + exchanger32 which could couple intracellular pH to transmembrahe Na + gradients. The observation of a basolateral K + conductance which could be activated by calcium in digitonin-permeabilized cells raised the question of the relation of such conductances to the previously defined "resting" and "swelling activated" basolateral gK. Interestingly, the calcium-activated K + conductance did not match up perfectly with either of these. Calcium-activated K+ currents could be carried by Rb +, but were blocked by quinidine. The Ca2+-activated gK was blocked by barium, but only from the cytoplasmic side, whereas both of the K + currents identified in amphotericintreated cells were blocked by serosally applied barium. In an effort to identify the channels that were the basis for the calcium-activated conductance Richards and Dawson27 exploited the observation that in digitonintreated cells Ca2+-activated gx was blocked by n-phenyl anthranilic acid (DPC). Somewhat surprisingly this effect turned out to be relatively specific. The compound did not block the 20-pS swelling activated channel nor did it block a "big K +'' channel recorded from isolated colonic cells. The compound did, however, block a very flickery, inwardly rectifying channel which could be recorded from isolated colonic cells. The channel was activated by cytoplasmic calcium, and could play some role in the resting K + conductance of the basolateral membranes. Na+/H + Exchange and Na + Channels Kirk and Dawson33showed that it was possible to measure transcellular Na + currents and fluxes using colons which had been treated with ouabain 3t D. Chan~ D. C. Dawson, FASEB J. 2, A1284 (1988). 32 M. A. Post and D. C. Dawson, FASEB J. 4, A549 (1990). 33 K. L. Kirk and D. C. Dawson, J. Gen. Physiol. 82, 497 (1983).
744
OTHER EPITHELIA
[47]
to abolish active transport and exposed to a steep, serosa to mucosa Na + gradient (I 12 m M : 2 mM). In this condition a mucosal amiloride-sensitive, cellular component of the S to M Na + current and transmural 22Na+ flux was evident. Analysis oftranscellular Na + flow in the absence of active transport suggested that the cells normally responsible for active Na + absorption contained, in the basolateral membranes, a cation exchanger which could carry out Na+:Na + or Na+:Li + exchange and a cation channel conductive to Na + and Li+. Kirk and Dawson 33 proposed that basolateral Na+/Li + exchange was the basis for the ouabain-sensitive active Li+ absorption described by Sarracino and Dawson. ~ The free energy for Li + transport was thought to reside in the basolateral Na + gradient maintained by the Na+,K+-ATPase which, as shown by Halm and Dawson, 21 was not activated by cytoplasmic Li +. In more recent experiments Post and Dawson 32 explored the properties of basolateral Na + transport elements using amphotericin-permeabilized cell layers.They showed that the basolateral membrane contains two sodium-selective, amiloride-inhibitable transport activities which may or may not reside in the same membrane protein. One is an Na+-selective conductance and the other is an Na+/H + exchanger. Interestingly, the activity of both of these transporters was modulated by cell volume. Exchange flow and conductive flow were largest in shrunken cells and were markedly attenuated by cell swelling. The presence of both a swelling activated K + conductance and shrinkage-activated Na+/H + exchange (and Na + conductance) may suggest that these elements represent the two poles of a push-pull system for volume regulation. A Model for Salt Absorption Figure 2 shows a working model which incorporates most of the transport "parts" which have been identified in the turtle colon epithelial cells. The basic elements of the Kocfoed-Johnsen-Ussing model remain the foundation for the ability of the cell to carry out active, transccllular transport of Na +. In our current model we envision the resting basolateral K + conductance as serving to recycle much of the K + which enters the cell via the Na+,K+-ATPasc, although a relatively small fraction leaks out across the apical membrane to produce K + secretion. A negative feedback, "self-regulation" loop is shown to suggest that increases in cytosolic Na + concentration can act, via some as yet unidentified mechanism, to shut down the apical Na + conductance. Possible pathways for inhibitory cholincrgic regulation are shown in keeping with the evidence that the agonistinduced decline in active absorption is accompanied by decreases in both S. M. Sarracino and D. C. Dawson,J. Membr. Biol. 46, 295 (1979).
[47]
TURTLE COLON
LumenL.~,
745
) Blood
Na+ I ( "÷
--C
Cl-
--]- -
Fio. 2. Current working hypothesisfor the disposition of cellular transporters in the Na+-transportingcellsof the turtle colon. See text for details. basolateral gx and apical gNa+. The hypothetical model cell is indicated as possessing C1- channels in the apical and basolateral membranes, although the apical channel is shown as being tentative because the transcellular conductance is observed so infrequently. If present, the series arrangement of C1- channels would provide a conductive, transcellular C1- leak path which could augment NaC1 absorption. In keeping with the observations of Venglarik et at2S the basolateral CI- conductance is also shown as being attenuated as a result of muscarinic agonist binding to membrane receptoil.
Depending on the prevailing osmotic conditions at least three additional transport elements might be present in the basolateral membranes. Cell swelling is associated with the activation of a specific basolateral conductance which is blocked by quinidine or lidocaine. This same condition inactivates two Na+-selective elements, an Na+/H + exchanger and an Na+-selective conductance, whereas the latter are fully activated in shrunken cells. It is possible that these transport elements play a role in the maintenance of cellular homeostasis as well as the regulation oftranscellular transport. These mechanisms could play a role in the regulation of cell volume but it might also be that cell volume is one component of the intracellular coupling network which permits "crosstalk" between the apical and basolateral membranes. Acknowledgments
The research described herein was supported by the National Institute for Diabetes, Digestive and Kidney Disease, The Cystic Fibrosis Foundation, and the AmericanHeart Association of Iowa and Michigan. Steve Thompson, Dave Luneman, Kevin Kirk, Dan Halm, Jane Nelson,BillGermann,JeffKeller,ChuckSole,Dan Le G-ault,CharlesVenglarik, Neff Richards,Marc Post,Dan Wilkinson,Melinda Lowy, and Nancy Kushman allplayedan important rolein the studiesdescribedherein.
OTHER EPITHELIA
[47] Turtle Colon: Keeping Track of Transporters Apical and Basolateral Membranes
[47]
in the
B y DAVID C. DAWSON and DEAN CHANG
T h e Study of Parts Our nearly 20-year romance with the turtle colon began, as many things do in science, by accident. In 1973 one of us (D.C.D.) had just arrived at the University of Iowa as an Assistant Professor of Physiology and Biophysics having completed a 2-year postdoctoral stint in the laboratory of Peter F. Curran at Yale University. In Peter Curran's lab we studied active Na + absorption using the colon of an amphibian, the toad, Bufo marinus. 1 We chose the toad colon because it appeared as though it might be a good model system in which we could study the subcellular "parts" involved in active Na + absorption. We were looking, in a sense, for a better frog skin. The initial attempt to continue studies of the toad colon at Iowa was thwarted by the Iowa winter: our first shipment of toads from Colombia, South America arrived frozen solid! At this point it seemed important to study these parts, regardless of their origin, so we called Phillip Steinmetz, a long-time student of turtles who had recently arrived as Iowa's new Chief of Nephrology. He assured us that turtles possessed a colon and within a few days we had determined that the turtle colon transported Na + better than its amphibian counterpart and that, as an additional bonus, there was much more of it. Furthermore, sharing the animals made good ecological sense so it was resolved to continue the pursuit of active Na + absorption using the turtle colon. Seventeen years later it appeared that the turtle colon was a good choice for a model for Na + absorbing epithelium. It is a prodigious Na -+transport machine with rates of absorption far exceeding those in the frog skin or toad bladder. The epithelium is a single layer of cells, lacking the crypts and folds characteristic of mammalian colon. The isolated tissue is quite hardy and can be maintained in vitro for several days and subjected to a variety of experimental maneuvers (such as apical permeabilization) while maintaining its functional properties. In addition salt absorption is subject to both stimulatory (aldosterone) and inhibitory (cholinergic) control. In this chapter we will describe our attempts to identify and characterize the
D. C. Dawsonand P. F. Curran, J. Membr. Biol. 28, 295 (1976). METHODS IN ENZYMOLOGY, VOL. 192
Copyright © 1990 by Academic Press, Inc. All rights ofrqmxluetion in any form rc~rved.
[47]
TURTLE COLON
735
Blood
K÷
Na"~K"
FIG. 1. Right: Electron micrograph showing the columnar cells which compose the epithelial cell layer of the turtle colon. Le~: The Koefoed-Johnsen-Ussing model for active Na + absorption. Sodium ion is presumed to enter the cell across the terminal membrane via amiloride-sensitive Na + channels and exit the cell across the basolateral membrane via the Na+,K+-ATPase.
individual transporters which comprise the molecular basis for the ion transport properties of the turtle colon. One of the vexing problems associated with epithelial cells is their tendency to lose their highly differentiated properties when they are isolated from one another. In order to obtain information about individual apical and basolateral transporters it has been necessary, therefore, to design experimental strategies which permit some degree of functional isolation of the transporters of interest while preserving the structural integrity of the cell layer. In several instances the turtle colon has proved to be uniquely suited to this type of experimental approach. Transepithelial Transport
Cellular and Noncellular Paths Figure 1 shows an electron micrograph of the colonic epithelial cell layer juxtaposed with the model used by Koefoed-Johnsen and Ussing2 to describe active Na + absorption by frog skin. The long columnar cells of the turtle colon form a single, flat cell layer and, as might be expected, ions can traverse the cell layer by way of cellular and paracellular paths. In our initial description of the properties of the colon3 we used 3H-labeled mannitol as a marker for the paracellular path and concluded that solute diffusion between the cells behaved much as it would in free solution. This 2 V. Koefoed-Johnsen and H. H. Ussing, Acta Physiol. Scand. 42, 298 (1958). 3 D. C. Dawson, J. Membr. Biol. 37, 213 (1977).
736
OTHER EPITHELIA
[471
and later studies4 were all consistent with the notion that the paracellular path behaved as a watery leak with no special selectivity properties. With identical solutions bathing both sides of the tissue the transepithelial electrical potential difference would constitute a driving force for net transepithelial movement of Na ÷, K +, and CI- via this extracellular leak path. The transepithelial potential difference across isolated colonic sheets, stripped of musculature, varies from about 30 to 130 mV, lumen negative, and the transepithelial resistance is in the range of 500 to 200 f~ • cm 2, placing this epithelium in the class of "moderately tight" epithelia. Our earliest estimates of cellular and paracellular conductances, 0.6 and 1.4 mSc/cm 2, respectively, were probably biased by the tissue damage which is inevitable in conventional Ussing chambers. More recent experiments suggest that values of 1.48 for the cellular conductance and 0.65 for the shunt conductance are more typical. Active Transport o f N a + and K +
Electrogenic Na + absorption is the dominant transport process in the turtle colon, comprising greater than 90% of the short-circuit current (I,~) which is virtually abolished by mucosal amiloride. Sodium ion absorption is also abolished by serosal ouabain. Absorption is stimulated by aldosterone 5 but is inhibited by muscarinic cholinergic agonists~ as well by adenosine and histamine. 7 The basis for the small residual 1,~ observed in the presence of mucosal amiloride is, at present, unknown. Potassium ion is both actively absorbed and actively secreted by turtle colon 4 but at rates which are of the order of 100-fold less than that of Na ÷. In some tissues the net flow was absorptive under short-circuit conditions, but little is known about this process except that it was blocked by mucosal (but not serosal) ouabain and orthovandate (M + S). The secretory transport, when present, was blocked by amiloride under short-circuit conditions, a result which was taken to reflect an effect of the reduced turnover of the basolateral Na ÷, K+-ATPase and hyperpolarization of the apical membrane potential. Potassium ion secretion was enhanced by serosal barium and blocked by mucosal barium, suggesting that the net rate of secretion depended on the ratio of the apical to basolateral K + conductances. As expected for a conductive transport process the cellular secretory rate was highly dependent on transmural potential. 8 The normal, lumennegative open cicuit potential enhanced K + secretion so that net trans4 D. R. Halm and D. C. Dawson, Am. J. Physiol. 246, C315 (1984). 5 D. R. Halm and D. C. Dawson, Pfluegers Arch. 403, 236 (1985). 6 C. J. Venglarik and D. C. Dawson, Am. J. Physiol. 251, C563 (1986). C. J. Vengladk. Cholinergic Regulation of Salt Absorption by Turtle Colon: Dual Control of Sodium and Chloride Transport. Ph.D. Thesis, University of Michigan, 1988. s D. R. Halm and D. C. Dawson, Am. J. Physiol. 247, C26 (1984).
[47]
TURTLE COLON
737
mural K + flow in vivo would be expected to be secretory. The relatively small rate of K + secretion (compared to Na + absorption) may indicate that not all of the Na+-absorbing cells contain the apical K + channels necessary for the exit o f K + toward the lumen, or simply that apical K + conductance is quite small in all of the cells. A Cellular CI- Leak?
The initial description3 of the transport of NaC1 by the colon produced no evidence for any net movement of C1- under short-circuit conditions, but in some experiments there was the suggestion of a cellular component of the transmural flux of ~sC1-. More recent experiments of Vengiarik and Dawson9 suggested, in fact, that a relatively small proportion of turtles (-5%) exhibited a substantial, transceUular C1- leak pathway which had several interesting properties. The appearance of the CI- leak was accompanied by a dramatic increase in tissue conductance which could not be attributed to the paracellular path. The conductive leak was highly C1selective as judged by C1- currents (induced by imposing transmural C1gradients) and transmural ~sc1- fluxes. The C1- leak path was inactivated by carbachol (as was the net active transport of Na +) but was specifically inhibited by experimental maneuvers which were expected to elevate cellular cyclic AMP (phosphodiesterase inhibitors, forskolin, cAMP derivafives). These maneuvers did not affect Na + absorption. Net C1- transport occurred only in the presence of an applied driving force, suggesting that the presence of this highly anion-selecfive leak pathway could facilitate NaC1 absorption under open circuit conditions. The salt absorptive process could be envisioned as being driven by active Na + absorption (the engine of salt absorption) but also dependent on the conductance of the anion-selective leak path. In addition, the inhibitory regulation of salt absorption by cholinergic agonists was seen as having a dual mechanism: inhibition of active Na + absorption and attenuation of the C1- leak path. Although these were intriguing thoughts, further study of this transmural path has been frustrated by the fact that it appears in such a small percentage of the turtles examined. Apical Ion Channels As expected from the large component of electrogenic Na + absorption the dominant components of the apical membrane of the turtle colon appears to be amilofide-sensitive, Na+-selective channels. We examined these initially by measuring initial rates of ZZNa+uptake from the mucosal 9 C. J. Venglarik and D. C. Dawson, Fed. Proc., Fed. Am. Soc. Exp. Biol. 46, 496 (1987).
738
OTHER EPITHELIA
[47]
bath 1° and later using blocker-induced Na + channel noise?' Thompson and Dawson ~° showed that a 30-sec tracer uptake from the mucosal path provided an estimate of the rate coefficient for Z2Na+ entry across the apical membrane of the transporting cells. At low mucosal Na + concentrations the entry rate coefficient was highly correlated with the amiloride-sensitive, transmural conductance as expected if the apical membrane was the major resistance barrier in the transporting cells. Other studies provided evidence that these apical channels were highly permeable to Li+ but that the presence ofLi + did not markedly affect Na + entry, as if the two ions did not interact in the conduction process/2 This result would be consistent with the notion that the apical amiloride-sensitive channels can accommodate either Na + or Li+ but contain only one ion at any instant. Kirk and Dawson 13 obtained evidence that the apical Na + conductance can be modulated in response to changes in intracellular Na + concentration. Experimental maneuvers which would be expected to lead to an increase in cytosolic Na + concentration produced an inhibition of the Na + conductance of the apical membrane. In ouabain-treated tissues this inhibition could be reversed by simply depleting cellular Na +. Wilkinson and Dawson H applied the techniques of Van Driessche and Lindemann ~4to study blocker-induced fluctuations in apical Na + currents. Using the weak Na + channel blocker, CDPC, 15 they observed blocker-induced Lorentzian components in the power density spectra which were indicative of reversible blockade of open Na + channels. Under control conditions single-channel Na + currents averaged 0.43 pA and the density of Na + channels was 250 × 106 channels/cm 2. Carbachol, which inhibited Na + absorption, caused a decrease in the number of open Na + channels but did not alter the single-channel current. The result was consistent with the notion that inhibitory cholinergic control of Na + absorption was associated with decrease in the conductance of both the apical and the basolateral membranes such that the fractional resistance was not greatly altered. Noise analysis also produced evidence for apical K + channels. 16 In some instances it was possible to detect a "spontaneous" Lorentzian component in the power density spectrum with a comer frequency of about 15 Hz. This Lorentzian appeared to be unrelated to Na + absorption. It was abolished by mucosal barium, however, and the plateau values were aug-
,0 S. M. Thompson and D. C. Dawson, J. Membr. Biol. 42, 357 (1978). H D. J. Wilkinson and D. C. Dawson, FASEBJ. in press (1989). ,2 S. M. Thompson and D. C. Dawson, J. Gen. Physiol. 72, 269 (1978). 23 K. L. Kirk and D. C. Dawson, Pfluegers Arch. 403, 82 (1985). 14W. Van Driessche and B. Lindemann, Nature (London) 282, 519 (1979). 15 S. I. Helman and L. M. Baxendale, 95, 647 (1990). 26D. J. Wilkinson and D. C. Dawson, Am. J. Physiol. 259, 668 (1990).
[47]
TURTLE COLON
739
mented by applying a serosa-positive transmural potential. This behavior suggested that these fluctuations emanated from the apical channels which mediated the potential-sensitive K + secretion documented by Halm and Dawson. s Basolateral Transporters
Permeabilized Cell Layers In most studies of isolated sheets of turtle colon the tissue is stripped of circular and longitudinal musculature, but a layer of connective tissue and a muscularis mucosa remains on the serosal side. The thickness of the layer is roughly 5 to 10 times that of the single layer of mucosal cells so that the shortest route to the basolateral membrane is through the apical membrane? 7,~s For this reason we have explored a number of approaches to chemically modifying the apical membrane so that it no longer constitutes a significant barrier to transcellular ion flow. The object of any such strategy is to reach a compromise between two goals: that of eliminating the apical membrane as a permeability barrier and that of restricting or controlling the changes in intracellular composition which occur as a result of the permeabilization process. To this end we used the pore-forming, polyene antibiotic, amphotedcin B, to permeabilize the apical membrane to monovalent cations. The principal advantage of amphoteriein as a permeabilizing tool is its relative selectivity. The resulting pores are relatively nonselective for monovalent cations so that the membrane becomes freely permeable to Na + and K +. The pores appear to be impermeable to divalent cations, however, and six-carbon sugars. Monovalent anions are only about one-seventh as permeant as cations but the C1- permeability is sufficient to increase salt entry into the cells. One consequence of this is that cells exposed to amphotericin will gain salt if bathed by mucosal NaCI or KCI, a fact exploited by Germann et aL ~9 to induce swelling in turtle colon cells. In our earliest experiments we investigated a number of different anion replacements in order to find one which would attenuate cell swelling but would also preserve cellular transport. We settled on benzene sulfonate and used this in a number of experiments although we later learned that this anion may, itself, promote some degree of cell swelling. ~9 It is interesting that the effects of amphotericin appear to be completely reversible even though permeabilization can induce substantial changes in cell volume and cellular ionic composition. 17D. C. Dawson, Curr. Top. Membr. Transp. 211,41 (1987). ~8D. C. Dawson, D. J. Willdnson, and N. W. Riehards, Curr. Top. Membr. Transp. 37, 191 (1990). 19W. J. Germann, S. A. Ernst, and D. C. Dawson, J. Gen. Physiol. 88, 253 (1986).
740
OTHER EPITHELIA
[47]
Na+/K + P u m p and Potassium Conductance
Our first experiments with amphotericin-permeabilized colon were designed to determine if the basolateral membranes contained the two elements basic to the Koefoed-Johnsen-Ussing model, a basolateral K + conductance and an Na+/K + exchange pump. 2° A K + conductance was identified by imposing a transmural K + gradient and noting that the resulting K + current was blocked by serosal barium. Using tissues treated with serosal barium (but not ouabain) it was then possible to show that in the absence oftransmural gradients a current appeared which exhibited an obligatory dependence on mucosal Na + and serosal K + and was inhibited by ouabain. Transmural flux determinations using these permeabilized tissues confirmed that the current was due to opposing net Na + and K + fluxes in the ratio 3Na+: 2K +. Halm and Dawson 2~ arrived at the same estimate of the pump stoichiometry by studying the kinetics of activation by mucosal (cellular) Na + and serosal K +. Kirk and Dawson 22 investigated the properties of the basolateral K + conductance and found that under the conditions of these experiments (benzene sulfonate as the anion) it was possible to observe basolateral currents carried by K ÷, Rb +, or Th +, all of which were blocked by serosal barium. Furthermore, 42K+ fluxes revealed coupling between the flows of tracer and abundant species which was indicative of single filing. Subsequent experiments by Germann et al. 23 revealed that the basolateral K + conductance (gK) observed in tissues bathed by benzene sulfonate ringers was more complex than we had initially guessed. Germann et al. 23 showed that it was possible to identify two distinct components to the basolateral K + conductance in permeabilized cells. One did not discriminate between K + and Rb +, was blocked by serosal barium (but not by quinidine or lidocaine), was inactivated by cholinergic agonists, and was present under conditions which prevented the cells from swelling. Because normal active transport was supported equally well by K + or Rb + and was inactivated by cholinergic agonists6 this conductance was dubbed the "resting" K + conductance. If the cells were swollen by the entry of salt or urea, a second conductance was activated which could be distinguished by a marked preference for K + over Rb +, sensitivity to quinidine and lidocaine, and marked single-filing behavior. In addition this conductance could be activated by swelling in cell layers which had previously been exposed to carbachol to inactivate the resting gK. Germann et a/.~9 pro2o K. L. Kirk, D. R. Halm, and D. C. Dawson, Nature (London) 287, 237 (1980). 2mD. R. Halm and D. C. Dawson, J. Gen. Physiol. 82, 315 (1983). 22 K. L. Kirk and D. C. Dawson, J. Gen. Physiol. 82, 297 (1983). 23 W. J. Germann, M. E. Lowy, S. A. Ernst, and D. C. Dawson, J. Gen. Physiol. 88, 237 (1986).
[47]
TURTLE COLON
741
posed that this second conductance was due to a second population of channels which was not active under resting conditions but was activated by cell swelling. In retrospect, it seems clear that the earlier results of Kirk and Dawson~ represented the properties of a mixed channel population. The Rb + currents are likely to have been due to the resting conductance whereas the strong single-filing probably reflected a component of the swelling-induced gK. Dawson, Van Driessche, and Helman 24 used noise analysis to investigate the possible induction of basolateral K + channels by cell swelling. They showed that under conditions of cell swelling it was possible to detect a lidocaine-induced, Lorentzian component in the power density spectrum of the basolateral K + current. Under nonswelling conditions this lidocaineinduced Lorentzian was absent. This result was consistent with the notion that cell swelling activated a population of lidocaine-blockable channels in the basolateral membrane which was not active under normal osmotic conditions. Analysis of the blocker-induced noise provided an estimate of 20 pS for the single-channel conductance. Richards and Dawson25 used single-channel recording techniques to investigate K + channels in isolated colonic cells. In cells bathed in KC1-Ringer's solution they identified a 20-pS channel which was blocked by lidocaine and quinidine and appears to be the molecular basis for the swelling activated gK. Venglarik and Dawson~ investigated the possible role of the resting gk in the inhibitory cholinergic control of Na + absorption. They showed that cholinergic agonists, either exogenous or released from submucosal nerves, inactivated both active Na + absorption and basolateral gK. The cholinergic response of gK could be duplicated by exposing the tissue to a calcium ionophore, ionomycin, suggesting that perturbing intracellular calcium activity was sufficient to trigger the chain of events which led to the inactivation of basolateral gK" Richards and Dawson, 26,27 however, had recorded in isolated cells several types of channels which were activated by cytoplasmic calcium, suggesting that calcium might be involved in both activation and inactivation of components of the basolateral gK. CI- Channels
Venglarik et al. 2s identified a basolateral CI- conductance in amphotericin-permeabilized colonic cells. This conductance had several properties 24D. C. Dawson, W. VanDriessch¢, and S. I. Helman, Am. £ Physiol. 254, C165 (1988). 25N. W. Richards and D. C. Dawson, Am. J. Physiol. 251, C85 (1986). 26N. W. Richards and D. C. Dawson, Biophys. J. 51, 344a (1987). 27N. W. Richards and D. C. Dawson, FASEB £ 3, A1149 (1989). 2s C. J. Venglafik, J. L. Keller, and D. C. Dawson, Fed. Proc., Fed. Am. Soc. Exp. Biol. 45, 2082 (1986).
742
OTHER EPITHELIA
[47]
which suggested that it is related to the transceUular C1- leak which is present in a small percentage of colons studied. The conductance, which could be identified by ion substitution, was inactivated by muscarinic cholinergic agonists, as was the resting basolateral K + conductance. Basolateral go-, however, was specifically inhibited by experimental maneuvers which are expected to raise the cellular levels of cyclic AMP. Exposure to forskolin, phosphodiesterase inhibitors [3-isobutyl-l-methylxanthine (IBMX), for example] or cAMP derivatives (8 cAMP) all produced inactivation of get- but had little or no effect on gK- As opposed to the transcellular CI- leak this basolateral conductance was present in virtually all cell layers examined. At present, however, the physiological role of this conductance and the significance of the possible regulation by cAMP have yet to be determined.
Role of lntracellular Cae+: Digitonin-Permeabilized Cells Questions concerning a possible role for cytoplasmic calcium in the control of basolateral gK led Chang and Dawson29 to develop a preparation of the isolated turtle colon which was apically permeabilized by the detergent, digitonin. Exposure of the apical membranes to digitonin (20/tM) led to the release of the cytoplasmic enzyme, lactate dehydrogenase (LDH), from the ceils and rendered the apical membrane highly permeable to monovalent and divalent cations as well as to organic buffers for pH (PIPES) and calcium (EGTA). Somewhat surprisingly the basolateral membrane retained its functional integrity despite this chemical onslaught. Chang and Dawson 29 speculated that the long columnar shapes of the epithelial cells may have contributed to this result. In an attempt to at least partially mimic the intracellular milieu Chang and Dawson employed a mucosal solution which consisted primarily of potassium aspartate along with an EGTA (5 m M ) buffer system designed so that the free calcium concentration could be readily controlled in the range of 10-9 to 10--6 M. 3° The pH was buffered to 6.6 to exploit the optimal Ca2+-buffering range of EGTA. In the presence of about 10-9 M mucosal calcium digitonin permeabilization did not lead to a transcellular K + current. Raising the free calcium to 10-7 Mr, however, led to the prompt development of a K + current which could be rapidly inactivated by adding additional EDTA to reduce the free calcium. This type of result led to the conclusion that the basolateral membranes contained a K + conductance which could be activated by increases in cytosolic calcium. Similar experiments conducted using K+-free solutions in the presence of a C1- gradient 29 D. Chang and D. C. Dawson, J. Gen. Physiol. 82, 281 (1988). ~oD. Chang, P. S. Hsieh and D. C. Dawson, Comput. Biol. Med. 18(5), 351 (1988).
[47]
TURTLE COLON
743
were used to identify a calcium-activated basolateral C1- conductance. Both the K + and C1- conductances exhibited spontaneous inactivation in the continued presence of calcium. The nature of this inactivation is not understood at present. The activation of basolateral conductances in digitonin-permeabilized cells was also sensitive to cytoplasmic pH. 3~ Raising the cytosolic pH from 6.6 to 7.4 shifted the calcium activation dose response to the left so that there was greater activation at any given concentration of free calcium. These results suggested that the calcium activity and intracellular pH could interact to determine the degree of activation of basolateral conductances, and that the mechanism of this interaction is a proton modification of the calcium activation site. Regulation of basolateral conductance by pH is particularly interesting in fight of the demonstration of a basolateral Na+/H + exchanger32 which could couple intracellular pH to transmembrahe Na + gradients. The observation of a basolateral K + conductance which could be activated by calcium in digitonin-permeabilized cells raised the question of the relation of such conductances to the previously defined "resting" and "swelling activated" basolateral gK. Interestingly, the calcium-activated K + conductance did not match up perfectly with either of these. Calcium-activated K+ currents could be carried by Rb +, but were blocked by quinidine. The Ca2+-activated gK was blocked by barium, but only from the cytoplasmic side, whereas both of the K + currents identified in amphotericintreated cells were blocked by serosally applied barium. In an effort to identify the channels that were the basis for the calcium-activated conductance Richards and Dawson27 exploited the observation that in digitonintreated cells Ca2+-activated gx was blocked by n-phenyl anthranilic acid (DPC). Somewhat surprisingly this effect turned out to be relatively specific. The compound did not block the 20-pS swelling activated channel nor did it block a "big K +'' channel recorded from isolated colonic cells. The compound did, however, block a very flickery, inwardly rectifying channel which could be recorded from isolated colonic cells. The channel was activated by cytoplasmic calcium, and could play some role in the resting K + conductance of the basolateral membranes. Na+/H + Exchange and Na + Channels Kirk and Dawson33showed that it was possible to measure transcellular Na + currents and fluxes using colons which had been treated with ouabain 3t D. Chan~ D. C. Dawson, FASEB J. 2, A1284 (1988). 32 M. A. Post and D. C. Dawson, FASEB J. 4, A549 (1990). 33 K. L. Kirk and D. C. Dawson, J. Gen. Physiol. 82, 497 (1983).
744
OTHER EPITHELIA
[47]
to abolish active transport and exposed to a steep, serosa to mucosa Na + gradient (I 12 m M : 2 mM). In this condition a mucosal amiloride-sensitive, cellular component of the S to M Na + current and transmural 22Na+ flux was evident. Analysis oftranscellular Na + flow in the absence of active transport suggested that the cells normally responsible for active Na + absorption contained, in the basolateral membranes, a cation exchanger which could carry out Na+:Na + or Na+:Li + exchange and a cation channel conductive to Na + and Li+. Kirk and Dawson 33 proposed that basolateral Na+/Li + exchange was the basis for the ouabain-sensitive active Li+ absorption described by Sarracino and Dawson. ~ The free energy for Li + transport was thought to reside in the basolateral Na + gradient maintained by the Na+,K+-ATPase which, as shown by Halm and Dawson, 21 was not activated by cytoplasmic Li +. In more recent experiments Post and Dawson 32 explored the properties of basolateral Na + transport elements using amphotericin-permeabilized cell layers.They showed that the basolateral membrane contains two sodium-selective, amiloride-inhibitable transport activities which may or may not reside in the same membrane protein. One is an Na+-selective conductance and the other is an Na+/H + exchanger. Interestingly, the activity of both of these transporters was modulated by cell volume. Exchange flow and conductive flow were largest in shrunken cells and were markedly attenuated by cell swelling. The presence of both a swelling activated K + conductance and shrinkage-activated Na+/H + exchange (and Na + conductance) may suggest that these elements represent the two poles of a push-pull system for volume regulation. A Model for Salt Absorption Figure 2 shows a working model which incorporates most of the transport "parts" which have been identified in the turtle colon epithelial cells. The basic elements of the Kocfoed-Johnsen-Ussing model remain the foundation for the ability of the cell to carry out active, transccllular transport of Na +. In our current model we envision the resting basolateral K + conductance as serving to recycle much of the K + which enters the cell via the Na+,K+-ATPasc, although a relatively small fraction leaks out across the apical membrane to produce K + secretion. A negative feedback, "self-regulation" loop is shown to suggest that increases in cytosolic Na + concentration can act, via some as yet unidentified mechanism, to shut down the apical Na + conductance. Possible pathways for inhibitory cholincrgic regulation are shown in keeping with the evidence that the agonistinduced decline in active absorption is accompanied by decreases in both S. M. Sarracino and D. C. Dawson,J. Membr. Biol. 46, 295 (1979).
[47]
TURTLE COLON
LumenL.~,
745
) Blood
Na+ I ( "÷
--C
Cl-
--]- -
Fio. 2. Current working hypothesisfor the disposition of cellular transporters in the Na+-transportingcellsof the turtle colon. See text for details. basolateral gx and apical gNa+. The hypothetical model cell is indicated as possessing C1- channels in the apical and basolateral membranes, although the apical channel is shown as being tentative because the transcellular conductance is observed so infrequently. If present, the series arrangement of C1- channels would provide a conductive, transcellular C1- leak path which could augment NaC1 absorption. In keeping with the observations of Venglarik et at2S the basolateral CI- conductance is also shown as being attenuated as a result of muscarinic agonist binding to membrane receptoil.
Depending on the prevailing osmotic conditions at least three additional transport elements might be present in the basolateral membranes. Cell swelling is associated with the activation of a specific basolateral conductance which is blocked by quinidine or lidocaine. This same condition inactivates two Na+-selective elements, an Na+/H + exchanger and an Na+-selective conductance, whereas the latter are fully activated in shrunken cells. It is possible that these transport elements play a role in the maintenance of cellular homeostasis as well as the regulation oftranscellular transport. These mechanisms could play a role in the regulation of cell volume but it might also be that cell volume is one component of the intracellular coupling network which permits "crosstalk" between the apical and basolateral membranes. Acknowledgments
The research described herein was supported by the National Institute for Diabetes, Digestive and Kidney Disease, The Cystic Fibrosis Foundation, and the AmericanHeart Association of Iowa and Michigan. Steve Thompson, Dave Luneman, Kevin Kirk, Dan Halm, Jane Nelson,BillGermann,JeffKeller,ChuckSole,Dan Le G-ault,CharlesVenglarik, Neff Richards,Marc Post,Dan Wilkinson,Melinda Lowy, and Nancy Kushman allplayedan important rolein the studiesdescribedherein.
