Sodium-coupled chloride transport by epithelial tissues FRIZZELL, RAYMOND A., MICHAEL FIELD, AND STANLEY C. SCHULTZ. Sodium-coupled chZoride transport by epithelial tissues. Am. J. Physiol. 236(l): Fl-F8, 1979 or Am. J. Physiol.: Renal Fluid Electrolyte Physiol. 5(l): Fl-FS, 1979. -There is compelling evidence that active Cl absorption by a variety of epithelia, widely distributed throughout the animal kingdom, is the result of an electrically neutral Na-coupled transport process at the luminal membrane and that the energy for transcellular Cl movement is derived from the Na gradient across that barrier. These cotransport processes are found predominantly in “leaky” or “moderately leaky” epithelia and permit these tissues to absorb Na and Cl with high degrees of efficacy. In addition, there is a growing body of evidence that cyclic AMP and Ca-induced electrogenic Cl secretion by a wide variety of epithelia may involve electrically neutral, Na-coupled Cl entry across the contraluminal membrane and that the energy for these secretory processes is derived from the Na-gradient across that barrier. A model for electrogenie Cl secretion that accounts for the available data is presented. Cl absorption; Na gradient tion; model for electrogenic

; co-transport Cl secretion

ERA OF THE STUDY of ion transport by epithelia, in vitro, was ushered in by the brilliant studies of Ussing and his collaborators (65) on frog skin and, later, those of Leaf (39) and his collaborators on toad urinary bladder. For some years thereafter Na occupied center stage in this unfolding drama, with Cl relegated to the unglamorous role of a passive partner. Recently, however, there has been an upsurge in interest in transepithelial Cl transport, sparked by the recognition that active Cl transport is a far more widespread phenomenon than was previously appreciated and that it may be the primary ionic movement responsible for a number of secretory processes stimulated by elevations in intracellular levels of cyclic 3 ’ ,5’adenosine monophosphate (CAMP) and/or calcium. In vitro studies have fairly well established three modes of transepithelial Cl transport. The first is a strictly passive mode driven by transepithelial differences in concentration and electrical potential as exemplified by frog skin (65) and toad urinary bladder (39). The second involves an electrically silent Cl-HCO, exchange found, for example, in turtle urinary bladder (40), in some species of mammalian colon (2’7, 35), and, perhaps, in pancreatic ducts (35, 62). Finally, in recent years it has become apparent that Cl absorption by a variety of epithelia is coupled in an electrically neutral fashion to Na transport and there is strongly suggestive

THE MODERN

0363-6127/79/0000-0000$01.25

Copyright

0 1979 the American

Physiological

processes; electrogenic

Cl secre-

evidence that a number of Cl secretory processes1 also involve a Na-dependent movement across one of the limiting cell membranes. Currently there is no compelling evidence for “primary active” transport of Cl directly linked to a source of metabolic energy in animal epithelia. This Editorial Review will focus on Na-coupled transepithelial Cl transport. We will first summarize the rather compelling evidence for Na-coupled Cl absorption and then turn our attention to the more speculative issue of Cl secretion. Electrically

Neutral

NaCl Absorption

The existence of a mechanism capable of mediating electrically neutral (one-for-one) NaCl absorption was first strongly suggested by the findings of Diamond (17, 181, Wheeler (68), and Dietschy (19) that Na and Cl are both actively absorbed by fish and rabbit gallbladder at near equal rates, and that a) these tissues are characterized by negligibly small transepithelial electrical potential differences (PD), b) replacement of Na with nonabsorbed cations abolishes active Cl absorption with l Absorption hollow organ direction. Society

is defined and secretion

as a net movement from refers to a net movement

the lumen of a in the opposite

Fl

Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (084.054.057.240) on January 18, 2019.

F2

FRoIZZELL,

FIELD,

AND

SCHULTZ

no change in the PD, and c) replacement of Cl with cell even if the efficiency of energy transfer is relatively nonabsorbed anions abolishes active Na absorption with low. no change in PD. These findings differ strikingly from Martin and Diamond (42) found that approximately those in other epithelia (65) where transepithelial Cl 25 mol of NaCl were absorbed actively by rabbit galltransport is passively (electrically) coupled to Na ab- bladder for every mole of O2 consumed above the basal level and, a&r comparing their data with those resorption. These studies, however, provided no insight into the possible site(s) of interaction between Na and ported for other epithelia, concluded: “Thus, the gallCl. Subsequent studies of the unidirectional influxes of bladder, in which both Na and Cl transport are active, Na and Cl from the mucosal solution into rabbit gall- pumps approximately twice as many ions activeZy per bladder epithelial cells by Frizzell et al. (26) and Cre- extra oxygen consumed as do epithelia which transport only Na actively.” This conclusion, drawn well before maschi and Henin (14) disclosed the presence of one-forone coupling between the movements of Na and Cl the- notion that &tive Cl transport by gallbladder may remains a most across the mucosal membrane. Electrophysiologic stud- be energized by the Na gradient, ies employing intracellular microelectrodes ’ (26, 32) ex- articulate expressi .on of the energy transducing function cluded the possibility that this interaction could be the of Na-coupled transport process?The teleologic significance of coupled NaCl transport will be considered result of electrical coupling and provided compelling evidence for the mediated entry of NaCl into the cell in below. A model of the NaCl co-transport process in gallbladthe form of a neutral complex.* Nellans et al. (44) had der is illustrated in Fig. 1. Although it seems quite described a similar process in the brush border of rabbit ileum somewhat earlier and, in both ileum and gall- certain that the coupled entry step brings about the bladder, this coupled influx process appears to be in- uphill movement of Cl into the cell, energized by the hibited by procedures that lead to an elevation of downhill flow of Na, the mechanisms responsible for Na and Cl exit from the cell are unclear. As is the case for intracellular CAMP (26,44). activity, In addition to providing a reasonable explanation for all epithelia studied to date, Na-K-ATPase the earlier findings of Diamond (17, 18) and others (19, ubiqu tously associated w ith the Na-K active exchange 68), the localization of the coupled mechanism to the PWP has been identified in the basolateral m .embranes epithelial cells (43, 63), and indirect mucosal membrane raised the attractive possibility that a of gallbladder evidence has been presented which implicates this the energy necessary for the active transcellular transpump mechanism in transcellular Na transport (47). port of Cl might be derived from coupling to the electroHowever, this evidence is circumstantial and, at the chemical potential difference for Na across that barrier, analogous to Na-coupled sugar and amino acid transport by small intestine and renal proximal tubule (54, TABLE 1. Intracellular chloride activities 55). The findings that the intracellular concentration of in some epithelia Cl exceeds that expected for a passive distribution (14, 26) and declines toward the equilibrium value when the Na-Ringer Na-Free Ringer tissue is bathed in a Na-free medium (26) lent encouragement to this possibility; but, given the uncertainties -42 that becloud the measurement and interpretation of Rabbit gallbladder -49 35 2.4 19 1 20 Necturus proximal -52 25 2.3 -61 10 1.4 59 intracellular concentrations, these observations cannot renal tubule be considered conclusive. Amphiuma small -33 1.4 Recently, Dtiey et al. (20) have determined intracelintestine lular Cl activities in rabbit gallbladder in the presence Bullfrog small 71 2.3 3 intestine and absence of extracellular Na using Cl-selective (liquid ion exchanger) microelectrodes; their results are + is the electrical potential difference across the luminal memgiven in Table 1. In the presence of Na, the thermodybran? with respect to the luminal solution, hcl is the intracellular namic activity of cell Cl is 2-3 times that predicted by Cl activity, and R is the ratio of the measured activity to that predicted for a passive distribution of an anion. the Nernst equation for a passively distributed anion, whereas in the absence of Na there is excellent agreement between the predicted and the observed activities. Furthermore, as discussed by Duffey et al. (20), al- MUCOSAL SEROSAL though the intracellular Na activity is not known, it is SOLUTION CELL SOLUTION likely to be well below the estimated intracellular Na concentrations of 66-80 mM (14, 26). Accordingly, the electrochemical potential difference for Na across the mucosal membrane is almost certainly far more than that required to energize uphill Cl movement into the ‘LThe cell interior is 50-60 mV negative with respect to the mucosal solution and is not significantly affected by replacement of Cl with nontransported anions (e.g., sulfate, isethionate). Therefore, electrical coupling between Na and Cl entry is precluded.

FIG.

1.

Model for NaCl co-transport

by rabbit gallbladder

(26).

Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (084.054.057.240) on January 18, 2019.

EDITORIAL

F3

REVIEW

present time, a direct relation between transcellular Na TABLE 2. Epithelial tissues that appear to possess coupled NaCl absorptive mechanisms transport and K uptake across the ba solateral membranes has not been demonstra ted for gallbladder or, Mammals for that matter, for any other Na-transporting epitheHuman ileum (64) lium (46, 52). Furthermore, although it seems certain Rabbit ileum (44) that Cl exit from the cell is a downhill process directed Rabbit gallbladder (26) along a favorable electrochemical potential gradient, it Rat colon (5) is not clear wheth .er the Cl conductan .ce of the basolatBovine rumen (13) era1 membrane is sufficiently high to permit a strictly Amphibia diffusional outflow. The available data suggest that this Necturus gallbladder (31) barrier is relativelv impermeable to Cl and raise the Necturus proximal renal tubule (59) possibility that theWexit bf this anion is nonconductive, Bullfrog small intestine (48) Frog skin (66, 67) i.e., coupled to the co-transport of a cation or the countertransport of another anion ( 1% 20). Clearly, Fish additional studies aimed at defining the active and Flounder intestine (24) passive properties of the basolateral membrane are Sculpin intestine (34) necessary. Marine eel intestine (58) Flounder (seawater acclimated) urinary bladder (49) Rabbit gallbladder provided an ideal preparation for Trout (fresh water) urinary bladder (38) the demopstration and characterization of NaCl cotransport and certainly remains the tissue of choice for Molluscs the further study of this process inasmuch as the Aplysia intestine (30) epithelium is comprised of a single layer of what apArthropods pears to be, histologically, one cell type (61, and NaCl Prawn intestine (1) (or more generally, Na anion)” co-transport is the only transcellular transport process present. The results cited above, therefore, are not complicated by the presFinally, it is of some interest to note that with the ence of heterogeneous cell types or multiple ion transexception of a few reported instances (e.g., 49, 66, 67) port processes, and the interpretation of these results in NaCl co-transport processes appear to be largely reterms of the model illustrated in Fig. 1 seems eminently reasonable. The conclusive demonstration of NaCl co- stricted to that class of epithelia referred to as “leaky” or “moderately leaky” because of the presence of relatransport becomes somewhat more difficult when tistively low-resistance paracellular shunt pathways (53). sues are characterized by heterogeneous cell populaIn general, these epithelia are characterized by relations and several mechanisms for transcellular ion tively high rates of Na and Cl absorption accompanied transport that may reside in different cell types [e.g., by the isotonic equivalent of water. The biologic “advanrabbit ileum, which in addition to NaCl co-transport tage” conferred on these epithelia by the possession of possesses mechanisms for Na-coupled nonelectrolyte transport and for transcellular Na transport that is NaCl co-transport processes can be appreciated from the following considerations. apparently uncoupled to the movement of other solutes In “tight” epithelia such as frog skin and toad urinary (55)]. Nonetheless, there is now a considerable body of bladder, Na is actively absorbed and Cl is “dragged -evidence for the presence of NaCl co-transport processes along,” passively driven by the transepithelial PD esin epithelia from a wide variety of species ranging down tablished by the movement of Na. Therefore, one might the phylogenetic scale from man through arthropods. loosely refer to this phenomenon as NaCl “co-transport” Some examples are listed in Table 2. This listing should in which the coupling is electrical. Obviously, the not be considered exhaustive, but is meant to illustrate energy invested in the mechanism responsible for active the widespread distribution of these processes throughNa absorption also indirectly energizes Cl absorption. out the animal kingdom. In most instances the evidence However, electrical coupling is not a very efficacious for co-transport is that both Na and Cl are actively way to ensure rapid rates-of NaCl absorption by leaky absorbed, that replacement of Na with nonabsorbed epithelia in which low-resistance shunt pathways precations reduces or abolishes Cl absorption, and that of significant transepithelial replacement of Cl with nonabsorbed anions reduces the clude the development PDs. Accordingly, as pointed out by Frizzell et al. (26), rate of active Na absorption to the same degree. In tt in spite of the fact that total tissue conductance [of several instances, evidence for co-transport has been is very large, it is nonetheless supplemented by measurements of intracellular Cl ac- rabbit gallbladder] grossly insufficient to permit pure electrical coupling of tivities (a$‘) which have uniformly indicated active passive Cl transport to active Na transport or passive accumulation by the cells; these studies are summarized Na transport to active Cl transport at the observed rates in Table 1. even if the entire tissue conductance passively driven ion .” Furthermore,

3 Rabbit gallbladder also absorbs HCO, and some other anions in what appears to be an electrically silent manner. Thus, the cotransport system appears to have a high specificity for Na but is less demanding with respect to the co-transported anion (70).

is assigned

to the

it can be readily shown that the data of Diamond and Martin (42) on the energetics of NaCl transport by rabbit gallbladder and those of Frizzell et al. (26) on the electrical properties of that tissue absolutely preclude the possibility of electri-

Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (084.054.057.240) on January 18, 2019.

F4

FRIZZELL,

tally coupled Cl absorption on purely energetic grounds? In short, the biological function of many leaky epithelia is to absorb Na and Cl at relatively rapid rates against minimal transepithelial electrochemical potential differences. In these systems the efficiency of energy conversion (i.e., the ratio of osmotic work performed to the associated investment of metabolic energy) may be vanishingly small. However, a far more important biological parameter, in the case of many epithelia, is the rate of transport per unit of energy invested, or the efficacy of the transport process (22), rather than the steepness of the gradients that can be generated or sustained. The evolution of mediated, neutral NaCl cotransport processes in leaky epithelia assures the attainment of a high level of efficacy which could not possibly be achieved by electrical coupling between the flows of these ions. “Nonneutral”

Na-Coupled

MUCOSAL SOLUTION

FIELD,

AND

SCHULTZ

SEROSAL SOLUTION

A

Cl Absorption

Although generally ignored because of its small magnitude (~1 mV), there is usually an electrical potential difference across rabbit gallbladder oriented such that the serosal solution is electrically negative with respect to the mucosal solution (26, 41). An ingenious explanation for this finding was suggested by Machen and Diamond (41), namely, that the PD is the result of a NaCl diffusion potential between the lateral intercellular spaces and the mucosal solution across cation-selective tight junctions. In accordance with the “standing gradient” hypothesis advanced earlier, they argued that Na and Cl are accumulated in the lateral spaces where they achieve a concentration somewhat greater than that in the mucosal solution. This concentration difference provides the driving force for the backdiffusion of NaCl into the mucosal solution. Since the junctions are more permeable to Na than to Cl, this recycling process generates a PD oriented in the observed direction. This notion is illustrated in Fig. 2A. Recent studies (24) of Na and Cl transport by the isolated intestinal mucosa of the winter flounder, Pseudopleuronectes americanus, strongly suggest a similar model, but in this instance the transepithelial PD is as much as 5 mV, serosa negative; although this value is small by comparison with tight epithelia it is by no means negligible. The central findings consistent with the model illustrated in Fig. 2A are the following. a) As shown in Table 3, under short-circuit conditions both Na and Cl are actively absorbed but the rate of Cl absorption exceeds that of Na; the short-circuit current J From measurements of the Cl conductance of rabbit gallbladder, Frizzell et al. (26) have estimated that the transepithelial electrical potential difference (&,s> would have to be 34 mV, serosa positive, in order to drive net Cl absorption at the observed rate of 14 peq/cm* per h. Since the conductance of the paracellular shunt pathway accounts for at least 95% of the entire tissue conductance, the “electromotive force” of the Na pump (ENa) would have to be (34/0.05) = 680 mV in order to generate this ems. The energy that can be obtained from the hydrolysis of 1 mol of ATP is probably in the range of 400-600 mV (i.e., 8,000-12,000 callmol). Therefore, even if 1 Na were actively pumped per ATP there would be scarcely enough energy to generate the required E,,; if 3 Na were pumped per ATP hydrolyzed, the required E,, is energetically impossible.

FIG. 2. A : model proposed by Machen and Diamond (41) to account for small serosa-negative PD across rabbit gallbladder. B: model proposed by Field et al. (24) to account for the fact that under short-circuit conditions, the rate of active Cl absorption by flounder small intestine exceeds that of active Na absorption. Clearly, when the model illustrated in A is short circuited, a current must flow across the intercellular space comprised of cations (Na) flowing from serosal to mucosal solution and/or anions (Cl) flowing in opposite direction. If the permeability (transference number) of the tight junctions for Na is greater than that for Cl, most of the current passing through the junctions will be due to recycling of Na. TABLE

flounder

Control

3. Na and Cl transport small intestine

13.6

11.6

2.0

Na-free Cl-free

13.8

13.9

-O.l*

by short-circuited

8.4

3.0

5.4

-3.4

-4.0

6.3*

6.2*

O.l*

-0.2”

0

o.o*

*

-0.2”

Data from Field et al. (24). J&-., is the unidirectional flux of i from the mucosal solution to the serosal solution; J& is the unidirectional flux in the opposite direction; Jiet = Jk* - Jk,. All fluxes and the short-circuit current (I,,) are in peq/cm2 per h. $m is the transepithelial electrical potential difference with respect to the mucosal solution in mV. * Differ from control byP < 0.05.

(Zsc>is in excellent agreement with the algebraic sum of the Na and Cl currents. However, in the absence of Na, active Cl absorption ceases and the Zsc disappears; and in the absence of Cl, active Na absorption ceases and the Zsc again disappears. b) Frizzell et al. (28) have recently demonstrated that the unidirectional influx of Na into the transporting cells of winter flounder is abolished when Cl in the mucosal solution is replaced with sulfate, and that the unidirectional influx of Cl is abolished when Na is replaced with choline. The absolute decreases observed in these experiments were similar in magnitude and large enough to account for the net flux changes shown

Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (084.054.057.240) on January 18, 2019.

EDITORIAL

F5

REVIEW

in Table 3, suggesting that there is a one-for-one neutral coupled NaCl entry mechanism at the mucosal membrane and that this mechanism provides most, if not all, of the Cl transported across the epithelium. Frizzell et al. (28) also demonstrated that furosemide, when added to the mucosal solution, inhibits the coupled entry of NaCl, thereby reducing the transepithelial net Cl flux and PD. c) M. E. Dtiey, R. A. Frizzell, and S. G. Schultz (unpublished o b servations) have measured intracellular Cl activities in flounder intestinal cells using Clselective microelectrodes. They found that a$l is approximately 3 times that predicted by the Nernst equation for a passively distributed monovalent anion; however, in the absence of Na, acC1does not differ from the value predicted for a passive distribution. The data obtained from studies on flounder small intestine are in strict qualitative agreement with those described above for rabbit gallbladder, the only exception being that JEA1 exceeds Ji$ under short-circuit conditions. However, as pointed out by Field et al. (24) this observation can be readily accommodated by the model illustrated in Fig. 2B, since under short-circuit conditions (i.e., in the presence of an external current), the requirement for electroneutral flow across the epithelium per se is relieved. Ion transport in flounder small intestine, therefore, appears to be an instance in which the principal mechanism responsible for active Na and Cl transport is a tightly coupled, neutral NaCl entry mechanism but in which, at the same time, there is a significant serosanegative PD. It is tempting to speculate that such a mechanism may be responsible for active Cl absorption by the ascending thick limb of the loop of Henle (9, 10, 50) and studies explicitly designed to examine this possibility are clearly indicated. Electrogenic

Chloride

Secretion

The first clear-cut example of electrogenic Cl secretion was provided in 1955 by Hogben (33) who demonstrated that the transepithelial electrical potential difference and short-circuit current across frog gastric mucosa, in vitro, can be accounted for by active Cl transport from the serosal (“nutrient”) solution to the mucosal (“secretory”) solution. For a while thereafter, this appeared to be a peculiarity of gastric mucosa, but during the past lo-15 years other compelling examples of electrogenic Cl secretion have been reported and a pattern of common characteristics appears to be emerging. Among these “shared” properties are the following. Electrogenic Cl secretion is dependent on the presence of Na in the solution bathing the basolateral or contraluminal membrane of the secretory cells and is inhibited by the presence of ouabain in that solution. Cl secretion is rapidly elicited or enhanced by procedures that result in an elevation of intracellular levels of CAMP (see, for example, Table 4). In many instances, physiological “primary messengers” such as hormones and/or neurotransmitters have been identified. In other instances, CAMP remains a second messenger in search of a first.

Cl secretion may be elicited by Ca ionophores (e.g., A23187) which, in the presence of extracellular Ca, bring about an increase in cytoplasmic free Ca activity (Table 4). In some instances, procedures that result in an increase in cell CAMP also increase the rate of Ca exchange by the tissue, suggesting that they lead to an increase in cytoplasmic Ca activity by bringing about the release of sequestered Ca from intracellular structures. In several cases, Cl secretion is inhibited by the presence of furosemide in the solution bathing the contraluminal surface of the secretory cells. Table 5 lists examples of tissues that possess electrogenie Cl secretory processes and indicates the extent to which they conform to the pattern described above. These observations suggest the model for cAMPstimulated electrogenic Cl secretion illustrated in Fig. 3. According to this model: a) At the basolateral membrane, a neutral NaCl entry mechanism mediates the movement of Cl into the cell against an electrochemical potential difference by coupling its flow to the flow of Na down its electrochemical potential difference. This coupled uptake is inhibited by furosemide, as is the case for the coupled NaCl absorptive process in flounder small intestine (24, 28) . TABLE 4. Effects of CAMP and A23187 on sodium and chloride fluxes across rabbit colon \a J m-+s

J::,,,

J,‘,al

Pm

Control + CAMP

3.1 4.2

1.5 2.3

1.6 1.9

5.5 5.0

4.3 6.4*

1.2 -1.4

1.6 3.8*

4.5 6.3*

Control + A23187

3.4 3.4

1.5 1.6

1.9 1.8

6.3 6.1

4.9 7.5*

1.4 -1.4”

2.2 4.6*

4.6 6.5*

J“l

+\

J“'

\ -111

I SC

1lCl

Gl

the unidirectional flux of i from the mucosal JL+s designates solution to the serosal solution; JL+,,, is the unidirectional flux in the opposite direction; and Ji,, = Jb-, - Jj,-,, . All values are in peq/cm’ per h with the exception of G, (the tissue conductance) which is expressed in mmho/cm T Data on the effects of CAMP are from Ref. 27 and those dealing with the effects of A23187 are from Ref. 25. Standard errors have been omitted for clarity. * Differ from control by P < 0.05. TABLE 5. Possible examples of Na-coupled electrogenic Cl secretion

Tissue

Rabbit ileum Rabbit colon Frog stomach Dogfish rectal gland Frog cornea Killifish operculum Canine tracheai epithelium

Sodium Dependent

Inh&~~,b”

Stimu%tIP

+ (45) +m + (51)

+(*) +w +(15,

+ (57)

+ (57)

+ (57)

+(ll, 71, 72) + (16)

+tll) + (16)

+ (73) +(16)

+ La

+ca

+(2)

* M. Field, unpublished lished observations.

observations. $ F. Al-Bazzaz,

51)

+ (23) + (27) +(51)

Stimulated by Ca-Ionophore

Inhibited . bZ3t$Z-

+(7) + (25)

+(*) +(-u

+ (57)

+(12)

* R. A. Frizzell, personal communication.

+ (74) +(16) +c#3

unpub-

Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (084.054.057.240) on January 18, 2019.

FRIZZELL,

F6 MUCOSAL SOLUTION

CELL

SEROSAL SOLUTION

rosemide

FIG. 3. A working model for electrogenic CAMP. See text for discussion.

Cl secretion induced by

b) Na carried into the cell by means of the coupled mechanism is extruded by the ouabain-sensitive Na-K exchange pump which has been identified in the basolateral membranes of a wide variety of epithelial cells (43, 60, 61, 63) [’inc 1u d ing secretory cells (22, 37)]. Therefofe, Na simply recycles across the basolateral membrane and the Na gradient across that membrane is maintained by the energy-dependent pump mechanism. c) Active Cl secretion is the result of passive Cl exit from the cell across the luminal or mucosal membranes down a favorable electrochemical potential difference. d) An increase in cell CAMP and/or cytosolic free Ca could stimulate Cl secretion either by activating a latent, coupled NaCl entry mechanism at the basolatera1 membrane or by increasing the permeability of the apical membrane to Cl, and, thereby, ease its diffusional outflow from the cell. The latter possibility seems more attractive for the following reasons. First, as shown in Table 4, Cl secretion induced in rabbit colon with either CAMP or A23187 is accompanied by a large increase in tissue conductance (Gt ). Since the bidirectional Na fluxes and the unidirectional flux of Cl from mucosa to serosa are not affected, it seems reasonable to conclude tha .t the conductance of the pa .racel lular shunt pathway is no t significant] .y affected and that most, if not all, of the increase originates in the transcellular pathway (27). Second, the electrophysiologic studies of Klyce and Wong (37) demonstrate, quite clearly, that active Cl secretion by rabbit cornea, induced by epinephrine acting via CAMP, is associated with a marked decrease in the resistance of the outer barrier (“tear side”) which would correspond to the apical or luminal membrane of “hollow” organs. This decrease in resistance was not observed in Cl-free mediums, suggesting that it represents a rather specific increase in the Cl permeabi .lity of that barrier. Berridge et al. (4) have demonstrated a calcium-dependent decrease in the apical resistance of insect salivary gland cells in response to serotonin and CAMP, and similar findings have been reported for pancreatic exocrine cells in response to secretagogues and Ca-ionophores (cf. Ref. 29) Accordingly, at present there does not seem to be any cogent reason to speculate that increa .ses in cell CAMP and/or cytosol .ic free Ca activ ,ate a latent NaCl cotransport process. Instead, it seems far more reasonable to postulate that this mechanism 1s active in the absence of secretagogues and serves to “prime” the cells

FIELD,

AND SCHULTZ

for a secretory stimulus that “triggers” an increase in the Cl permeability of the apical membrane. An increase in cytosolic Ca could conceivably bring about an increase in the Cl permeability of the apical membrane simply by interacting with membrane components that bear fixed negative charges. Although the model illustrated in Fig. 3 can accommodate all of the available data on CAMP-induced active Cl secretory processes by a variety of epithelia, there are a number of ttmissing links” that await direct experimental confrontation. Thus: a) Although Cl secretion is Na dependent, there is no direct evidence for Na-coupled Cl transport across the basolateral membranes. The importance of distinguishing between Na dependence and Na coupling has been stressed and illustrated previously (56). b) There are no direct measurements of Cl activities in single secretory cells under “resting” and “secreting” conditions, and it may be very difficult to obtain such data in tissues characterized by a heterogeneous cell population. The data reported by Klyce and Wong (37) suggest that the activity of Cl in rabbit cornea is greater than that in the “aqueous” and “tear” sides but the interpretation of these experimental results is complicated by the complex multicellular structure of this tissue.5 c) Needless to say, further studies are needed in order to establish, unequivocally, the role of intracellular CAMP and/or Ca in the activation of the secretory process. In short, this model is presented as a working hypothesis in the hope that it will serve to stimulate future investigations whose results will critically test its basic tenets; nothing more can be asked of any model. Some Speculations on “Active” Transepithelial Chloride Transport

It should be apparent from this brief review that what is often referred to as “active” Cl absorption or secretion by a variety of epithelia, widely distributed throughout the animal kingdom, is in fact the result of NaCl cotransport across one of the limiting membranes where the energy for Cl transport against an electrochemical potential difference is derived from the Na gradient across that barrier. This Na gradient, in turn, is the result of active extrusion of Na from the cell mediated by a well-defined enzymatic reaction. Accordingly, Cl transport is not directly linked to a source of metabolic energy and should be referred to as “secondary active transport.” The only other well-defined mode of transepithelial Cl transport in which Cl is propelled against an electrochemical potential difference appears to involve a onefor-one exchange of Cl for HCO, (cf. Ref. 40). Although this mechanism requires further study, it is not unreasonable to suggest, on the basis of available data, that the energy for uphill Cl transport may be derived from coupling to the countertransport of HCO:{ out of the cell 5 Preliminary studies by M. E. Duffey, Schultz on the Cl-secreting rectal gland accznthias (see Table 5), indicate that the 6-8 times that expected for an equilibrium

R. A. Frizzell, and S. G. of the dogfish, Sgualus intracellular Cl activity is distribution.

Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (084.054.057.240) on January 18, 2019.

EDITORIAL

F7

REVIEW

down its electrochemical potential gradient? Finally, although a Na-activated plasma membrane ATPase -has bee6 well characterized in a number of epithelia, there is at present no compelling evidence for a- Cl-activated ATPase in any of the Cl-transporting epithelia. Earlier findings of an anion-sensitive ATPase in plasma membrane preparations appear to be due to contamination with fragments of mitochondrial membranes (8). It may not be too presumptuous, therefore, to speculate that there are no Cl transport mechanisms in animal epithelia that are energized by direct coupling V

to the flow of a metabolic reaction (i.e., “primary active transport”). Certainly, every effort should be made to rigorously establish or exclude the participation of Cl in co-transport or countertransport processes in those tissues which are capable of t&w@rting Cl against an electrochemical potential difference. These studies were supported by research grants from the National Institute of Arthritis, Metabolism, and Digestive Diseases (AM-16275, AM-18199, and AM-21345). R. A. Frizzell is the recipient of a Research Career Development Award from the National Institute of Arthritis, Metabolism, and Digestive Diseases (AM-00173).

REFERENCES 1. AHEARN, G. A., L. A. MAGINNISS, Y. K. SONG, AND A. TORNQUIST. Intestinal water and ion transport in fresh water malacostracan prawns (crustacea). In: Water Relations in Membrane Transport in Plants and Animals, edited by A. M. Jungreis, T. K. Hodges, A. Kleinzeller, and S. G. Schultz. New York: Academic, 1977, p. 129-142. 2. AL-BAZZAZ, F., AND Q. AL-AWQATI. Interaction between Na and Cl transport in canine tracheal mucosa. J. AppZ. Physiol. In press. 3. ARMSTRONG, W. McD., W. WOJTKOWSKI, AND W. R. BIXENMAN. A new solid-state microelectrode for measuring intracellular chloride activities. Biochim. Biophys. Actu 465: 165-170, 1977. 4. BERRIDGE, M. J., B. D. LINDLEY, AND W. T. PRINCE. Membrane permeability changes during stimulation of isolated salivary glands of CuZZiphoru by 5-hydroxytryptamine. J. PhysioZ. London 244: 549-567, 1975. 5. BINDER, H. J., AND C. L. RAWLINS. Electrolyte transport across isolated large intestinal mucosa. Am. J. Physiol. 225: 1232-1239, 1973. 6. BLOM, H., AND H. F. HELANDER. Quantitative electronmicroscopical studies on in vitro incubated rabbit gallbladder epithelium. J. Membr. BioZ. 37: 45-61, 1977. 7. BOLTON, J. E., AND M. FIELD. Ca ionophore-stimulated ion secretion in rabbit ileal mucosa: relation to actions of cyclic 3’,5’-AMP and carbamylcholine. J. Membr. BioZ. 35: 159-173, 1977. 8. BONTING, S. L., J. M. M. VAN AMELSVOORT, AND J. J. H. H. M. DE PONT. Is anion-sensitive ATPase a plasma membrane located transport system? In: Gastric Ion Transport, edited by K. J. Obrink and G. Flemstrom. Uppsala: Acta Physiol. Stand., Special Suppl., 1978. 9. BURG, M. B., AND N. GREEN. Function of the thick ascending limb of Henle’s loop. Am. J. Physiol. 224: 659-668, 1973. 10. BURG, M. B., L. STONER, J. CARDINAL, AND N. GREEN. Furosemide effect on isolated perfused tubules. Am. J. Physiol. 225: 119124, 1973. 11. CANDIA, 0. A. Ouabain and sodium effects on chloride fluxes across the isolated bullfrog cornea. Am. J. PhysioZ. 223: 10536 Short-circuited in vitro preparations of rabbit descending colon bathed by a normal Ringer solution containing 20 mM HCOs and equilibrated with a mixture of 95% O,-5% CO, “actively” absorb Cl. This process is .electrically silent and almost certainly involves a one-for-one exchange of Cl for HCO, across the apical membrane (27). The electrical potential difference across the apical membrane under short-circuit conditions is 46 mV, cell interior negative with respect to the mucosal solution. Therefore, if HCO, is at electrochemical equilibrium across this membrane, the intracellular HCO, concentration predicted by the Nernst equation should be approximately 3.6 mM. Assuming that the intracellular Pco2 is equal to that in the bathing medium (40 mmHg), the intracellular pH would be 6.6 in the presence of 3.6 mM HCO,. This pH is considerably lower than that estimated for most cells. Even if the cell pH were approximately 7.0, as estimated for a variety of nonepithelial cells, the intracellular HCO, concentration would be 9.5 mM, or 2.6 times the predicted equilibrium value. Therefore, tight coupling between HCO, efflux across the apical membrane and Cl influx (“countertransport” or “antiport”) could raise cell Cl to levels well above its equilibrium value, and, in a manner analogous to NaCl co-transport, energize transcellular Cl transport.

1057, 1972. 12. CANDIA, 0. A., R. MONTOREANO, AND S. M. PODOS. Effect of the ionophore A23187 on chloride transport across isolated frog cornea. Am. J. Physiol. 233: F94-FlOl, 1977 or Am. J. Physiol: Renal FZuid Electrolyte Physiol. 2: F94-F101, 1977. 13. CHIEN, W., AND C. E. STEVENS. Coupled active transport of Na and Cl across forestomach epithelium. Am. J. Physiol. 223: 9971003, 1972. 14. CREMASCHI, D., AND S. HENIN. Na and Cl transepithelial routes in rabbit gallbladder. Tracer analysis of the transports. Pfluegers Arch. 361: 33-41, 1975. 15. DAVENPORT, H. W. Effect of ouabain on acid secretion and electrolyte content of frog gastric mucosa. Proc. Sot. Exp. BioZ. Med. 110: 613-615, 1962. 16. DEGNAN, K. J., K. J. KARNAKY, AND J. A. ZADUNAISKY. Active chloride transport in the in vitro opercular skin of a teleost (Fundulus heteroclitus), a gill-like epithelium rich in chloride cells. J. Physiol. London 271: 155-191, 1977. 17. DIAMOND, J. M. Transport of salt and water in rabbit and guinea pig gallbladder. J. Gen. Physiol. 48: 1-14, 1964. 18. DIAMOND, J. M. The mechanism of solute transport by the gallbladder. J. Physiol. London 161: 474-502, 1962. 19. DIETSCHY, J. M. Water and solute movement across the wall of the everted rabbit gallbladder. Gastroenterology 47: 395-408, 1964. 20. DUFFEY, M. E., K. TURNHEIM, R. A. FRIZZELL, AND S. G. SCHULTZ. Intracellular chloride activities in rabbit gallbladder: direct evidence for the role of the sodium-gradient in energizing “uphill” chloride transport. J. Membr. BioZ. 42: 229-245, 1978. 21. ERNST, S. A. Transport adenosine triphosphatase cytochemistry. II. Cytochemical localization of ouabain-sensitive, potassiumdependent activity in the secretory epithelium of the avian salt gland. J. Histochem. Cytochem. 20: 23-38, 1972. 22. ESSIG, A., AND S. R. CAPLAN. Energetics of active transport processes. J. Gen. PhysioZ. 8: 1434-1457, 1968. 23. FIELD, M. Ion transport in rabbit ileal mucosa. II. Effects of cyclic 3’,5’-AMP. Am. J. Physiol. 221: 992-997, 1971. 24. FIELD, M., K. J. KARNAKY, P. L. SMITH, J. E. BOLT~N, and W. B. KINTER. Ion transport across the isolated intestinal mucosa of the winter flounder, PseudopZeuronectes americanus. I. Functional and structural properties of cellular and paracellular pathways for Na and Cl. J. Membr. BioZ. In press. 25. FRIZZELL, R. A. Active chloride secretion by rabbit colon: calcium-dependent stimulation by ionophore A23187. J. Membr. Biol. 35: 175-187, 1977. 26. FRIZ~ELL, R. A., M. DUGAS, and S. G. SCHULTZ. Sodium chloride transport by rabbit gallbladder: direct evidence for a coupled NaCl influx processes. J. Gen. Physiol. 65: 769-795, 1975. 27. FRIZZELL, R. A., M. J. KOCH, AND S. G. SCHULTZ. Ion transport by rabbit colon. I. Active and passive components. J. Membr. BioZ. 27: 297-316, 1976. 28. FRIZZELL, R. A., P. L. SMITH, E. VOSBURGH, AND M. FIELD. Coupled sodium-chloride influx across brush border of flounder intestine. J. Membr. BioZ. In press. 29. GARDINER, J. D. Regulation of pancreatic exocrine function in vitro: initial steps in the actions of secretagogues. Ann. Rev. Physiol. In press. 30. GERENCSER, G. A., S. K. HONG, AND G. MALVIN. Metabolic dependence of active chloride transport in isolated aplysia intestine. Federation Proc. 35: 464, 1976.

Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (084.054.057.240) on January 18, 2019.

FS

FRIZZELL,

31. GRAF, J., AND G. GIEBISCH. Intracellular sodium activity and sodium transport in Necturus gallbladder epithelium. J. Membr. BioZ. In press. 32. HENIN, S., AND D. CREMASCHI. Intracellular ion route in rabbit gallbladder. Electric properties of the epithelial cells. PfZuegers Arch. 355: 125-139, 1975. 33. HOGBEN, C. A. M. Active transport of chloride by isolated frog gastric mucosal potential. Am. J. PhysioZ. 180: 641-649, 1955. 34. HOUSE, C. R., AND K. GREEN. Ion and water transport in isolated intestine of the marine teleost, Cottus scorpius. J. Exp. BioZ. 42: 177-189, 1965. 35. HUBEL, K. A. The ins and outs of bicarbonate in the alimentary tract. Gustroenterology 56: 647-651, 1968. 36. KARNAKY, K. J., JR., L. B. KINTER, W. B. KINTER, AND C. E. STIRLING. Teleost chloride cell. II. Autoradiographic localization of gill Na,K-ATPase in killifish FunduZus heteroczitus adapted to low and high salinity environments. J. CeZZ BioZ. 70: 157-177, 1976. 37. KLYCE, S. D., AND R. K. S. WONG. Site and mode of adrenaline action on chloride transport across the rabbit cornea1 epithelium. J. PhysioZ. London 266: 777-799, 1977. 38. LAHLOU, B., AND B. FOSSAT. Mechanisme du transport de l’eau et du se1 a travers la vessie urinaire d’un Poisson teleosteen en eau deuce, la truite arc-en-ciel. C. R. Acad. Sci. 273: 2108, 1971. 39. LEAF, A. Transepithelial transport and its hormonal control in toad bladder. Ergeb. Physiol. BioZ. Chem. Exp. Pharmakol. 56: 216-263, 1965. 40. LESLIE, B. R., J. H. SCHWARTZ, AND P. R. STEINMETZ. Coupling between Cl absorption and HCO, secretion in turtle urinary bladder. Am. J. PhysioZ. 25: 610-617, 1973. 41. MACHEN, T. E., AND J. M. DIAMOND. An estimate of the salt concentration in the lateral intercellular spaces of rabbit gallbladder during maximal fluid transport. J. Membr. BioZ. 1: 194213, 1969. 42. MARTIN, D. W., AND J. M. DIAMOND. Energetics of coupled active transport of sodium and chloride. J. Gen. Physiol. 50: 295315, 1966. 43 MILLS, J. W., AND D. R. DIBONA. Distribution of Na pump sites in the frog gallbladder. Nature 271: 273-275, 1978. 44 NELLANS, H. N., R. A. FRIZZELL, AND S. G. SCHULTZ. Coupled sodium-chloride influx across the brush border of rabbit ileum. Am. J. Physiol. 225: 467-475, 1973. 45 NELLANS, H. N., R. A. FRIZZELL, AND S. G. SCHULTZ. Brush border processes and transepithelial Na and Cl transport by rabbit ileum. Am. J. PhysioZ. 226: 1131-1141, 1974. 46 NELLANS, H. N., AND S. G. SCHULTZ. Relations among transepithelial sodium transport, potassium exchange and cell volume in rabbit ileum. J. Gen. Physiol. 68: 441-463, 1976. 47 OS, C. H., VAN, AND J. F. G. SLEEGERS. Correlation between (Na-K)-activated ATPase activities and the rate of isotonic fluid transport of gallbladder epithelium. Biochim. Biophys. Acta 241: 89-96, 1971. 48. QUAY, J. F., AND W. M. ARMSTRONG. Sodium and chloride transport by isolated bullfrog small intestine. Am. J. PhysioZ. 217: 694-702, 1969. 49. RENFRO, J. L. Interdependence of active Na and Cl transport by the isolated urinary bladder of the teleost, Pseudopleuronectes americanus. J. Exp. ZooZ. 199: 383-390, 1977. 50. MOCHA, A. S., AND J. P. KOKKO. Sodium, chloride and water transport in the medullary thick ascending limb of Henle. Evidence for active chloride transport. J. CZin. Invest. 52: 612623, 1973. 51. SACHS, G., J. G. SPENNEY, AND M. LEWIN. H+ transport: regulation and mechanism in gastric mucosa and membrane vesicles. PhysioZ. Reu. 58: 106-173, 1978. 52. SCHULTZ, S. G. Is a coupled Na-K exchange pump involved in active transepithelial Na transport? A status report. In: Membrane Transport Processes, edited by J. F. Hoffman. New York: Raven Press, 1978, vol. 1, p. 213-227.

53. 54.

55. 56.

57.

58.

59. 60. 61.

62.

63.

64.

65. 66.

67.

68. 69.

70.

71. 72. 73.

74.

FIELD,

AND

SCHULTZ

SCHULTZ, S. G. The role of paracellular pathways in isotonic fluid transport. YaZe J. BioZ. Med. 50: 99-113, 1977. SCHULTZ, S. G. Sodium-coupled solute transport by small intestine: a status report. Am. J. PhysioZ. 233: E249-E254, 1977 or Am. J. Physiol.: EndocrinoZ. Metab. Gastrointest. Physiol. 2: E249-E254, 1977. SCHULTZ, S. G., AND P. F. CURRAN. Coupled transport of sodium and organic solutes. Physiol. Rev. 50: 637-718, 1970. SCHULTZ, S. G., R. A. FRIZZELL, AND H. N. NELLANS. Ion transport by mammalian small intestine. Ann. Rev. Physiol. 36: 51-91, 1974. SILVA, P., J. STOW, M. FIELD, L. FINE, J. N. FORREST, AND F. H. EPSTEIN. Mechanism of active chloride secretion by shark rectal gland: role of Na-K-ATPase in chloride transport. Am. J. PhysioZ. 233: F298-F306, 1977 or Am. J. PhysioZ.: RenaZ Fluid Electrolyte PhysioZ. 2: F298-F306, 1977. SKADHAUGE, E. Coupling of transmural flows of NaCl and water in the intestine of the eel (AnguiZZa anguilla). J. Exp. BioZ. 60: 535-546, 1974. SPRING, K. A., AND G. KIMURA. Chloride reabsorption by renal proximal tubules ofNecturus. J. Membr. BioZ. 38: 233-254, 1978. STIRLING, C. E. Radioautographic localization of sodium pump sites in rabbit intestine. J. CeZZ BioZ. 53: 704-714, 1972. STIRLING, C. E . High-resolution autoradiography of “H-ouabain binding in salt transporting epithelia. J. Microsc. 106: 145-157, 1976. SWANSON, C. H., AND A. K. SOLOMON. A micropuncture investigation of the whole tissue mechanism of electrolyte secretion by the in vitro rabbit pancreas. J. Gen. PhysioZ. 62: 407-429, 1973. TORMEY, J. M. Anatomical methods for studying transport across epithelia. In: Water ReZations in Membrane Transport in Plants and Animals, edited by A. M. Jungreis, T. K. Hodges, A. Kleinzeller, and S. G. Schultz. New York: Academic, 1977, p. 233-248. TURNBERG, L. A., F. A. BIEBERDORF, S. G. MORAWSKI, AND J. S. FORDTRAN. Interrelationships of chloride, bicarbonate, sodium and hydrogen transport in the human ileum. J. CZin. Invest. 49: 557-567, 1970. USSING, H. H. The AZkaZi Metal Ions in Biology. Berlin: Springer-Verlag, 1960. WATLINGTON, C. O., AND F. JESSEE, JR. Net Cl flux in shortcircuit skin of Rana pipiens: ouabain sensitivity and Na+K dependence. Biochim. Biophys. Acta 382: 204-212, 1975. WATLINGTON, C. O., S. D. JESSEE, AND G. BALDWIN. Ouabain, acetazolamide, and Cl- flux in isolated frog skin: evidence for two distinct active Cl- transport mechanisms. Am. J. PhysioZ. 232: F550-F558, 1977 or Am. J. Physiol.: Renal Fluid Electrolyte Physiol. 1: F550-F558, 1977. WHEELER, H. 0. Transport of electrolytes and water across wall of rabbit gallbladder. Am. J. Physiol. 205: 427-438, 1963. WHITE, J. F. Activity of chloride in absorptive cells of Amphiuma small intestine. Am. J. PhysioZ. 232: E553-E559, 1977 or Am. J. PhysioZ.: EndocrinoZ. Metab. Gastrointest. Physiol. 1: E553-E559, 1977. WHITLOCK, R. T., AND H. 0. WHEELER. Anion transport by isolated rabbit gallbladders. Am. J. Physiol. 213: 1199-1204, 1967. ZADUNAISKY, J. A. Active transport of chloride in frog cornea. Am. J. PhysioZ. 211: 506-512, 1966. ZADUNAISKY, J. A. Sodium activation of chloride transport in the frog cornea. Biochim. Biophys. Acta 282: 255-257, 1972. ZADUNAISKY, J. A., M. A. LANDE, M. CHALFIE, AND A. H. NEUFELD. Ion pumps in the cornea and their stimulation by epinephrine and cyclic AMP. Exp. Eye Res. 15: 577-584, 1973. ZADUNAISKY, J. A., AND B. SPINOWITZ. Drugs affecting the transport and permeability of the cornea1 epithelium. In: Drugs and OcuZar Tissues, edited by S. Dikstein. Basel: Karger, 1977, p. 57-78.

Raymond A. Frizzell, Michael Field, and Stanley G. Schultz Department of Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; Department of Medicine, University of Chicago, Chicago, Illinois 60637; and Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672 Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (084.054.057.240) on January 18, 2019.