H + transport in urinary epithelia.

H+ transport

in urinary

epithelia

AL-AWQATI, QAIS. H+ transport in urinary epithelia. Am. J. Physiol. 235(2): F’77-FM, 1978 or Am. J. Physiol.: Renal Fluid Electrolyte Physiol. 4(2): F77-F88, 1978. - This review of urinary acidification is primarily based on studies in isolated epithelia such as the turtle bladder. Despite the lack of unambiguous proof, the wealth of indirect evidence suggests that the cause of bicarbonate absorption is H+ secretion into the lumen. The mechanisms that regulate H+ transport are discussed. The electrochemical gradient for protons across the membrane is found to be the most fundamental regulator not only of passive movement but also of active transport. CO* and aldosterone stimulate H+ transport, the latter by a mechanism apparently separate from the effect of this hormone on sodium transport. Although carbonic anhydrase activity is important for optimal function of the H+ pump, the results with carbonic anhydrase inhibitors need to be interpreted with caution. The evidence for Na:H exchange is reviewed and found to be not very persuasive. The metabolic pathways that fuel H+ transport are found to be all the major energyyielding reactions in the cell, but particular prominence is given to the new discovery of the role of the pentose shunt in energizing transport. Finally, I discuss the important role of H+ transport in energy transduction in subcellular organelles. electrochemical urinary acidification; hydrase activity; membrane transport

THIS EDITORIAL REVIEW FOCUSES ON aspects of urinary acidification that have been studied in the turtle bladder, a tissue which is functionally analogous to the mammalian collecting tubule. The advantages of using this bladder preparation include the ability to control the. electrochemical gradient across the membrane, thereby making it possible for the various regulatory influences to be studied, and to measure both active and passive fluxes simultaneously, thereby allowing study of the various active components of transport. The ability to distinguish among the various active and passive fluxes has led to a rigorous description of net acid excretion in this tissue, which in turn has served as a heuristic model for H+ transport in the kidney.

Distinction Between H+ Secretion and HCO, Absorption Arguments over whether H+ secretion or HCO, absorption is the cause of urinary acidification are as old as the field itself. The major problem that has prevented a satisfactory resolution of this question is the presence of COZ. In a completely COP-free system the removal of HCO, should abolish acidification if it were due to HCO, reabsorption. However, only if all metabolic CO, can be removed by inhibition of oxidative reactions will the system be CO, free, but this inhibition will undoubtedly suppress transport, hence the dilemma. Further, mea0363-6127/78/0000-0000$01.25

Copyright

0 1978 the American

gradient;

aldosterone;

carbonic

an-

surement of the various components of the CO, system have proved to be difficult. Despite all this, many ingenious experiments have been performed both in the kidney and in the turtle bladder, the results of which provide a wealth of evidence in favor of H+ secretion. Excellent reviews of these experiments have been presented by Rector (72) and Steinmetz (87). In the present paper, I will touch on some recent issues of major interest. Evidence of HC03 absorption. Addition of “maximal” doses of carbonic anhydrase inhibitors decreases HCO, absorption (50, 73, 95). The remaining rate is higher than can be accounted for by intracellular hydration of CO, by an uncatalyzed reaction (54, 72). This led Maren to conclude that HCO, ion as such must be transported to account for the residual rate (55). However, Rector (72) has suggested that carbonic acid formed in the lumen from the combination of H+ and HCO, could diffise into the cell to act as a proton donor to the pump. This ingenious explanation could completely account for the discrepancy between the uncatalyzed rate and the rate of H+ secretion, but unfortunately carbonic acid diffusion cannot be measured to test this hypothesis.’ e---

l I attempt here to calculate the permeability coefficient of carbonic acid that will be needed to account for Rector’s suggestion. Afi er carbonic anhydrase inhibition the rate of HCO, absorption is approximately 2 nmo1/cm2 s -l (51) and the luminal pH is -6.3. Using

Physiological

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Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 15, 2018. Copyright © 1978 American Physiological Society. All rights reserved.

Q. AL-AWQATI

F78 However, .recent developments have obviated the necessity for this. Studies on the isolated perfused proximal straight tubule have shown that acetazolamide completely inhibits HCO, absorption in doses of low5 M (47, 56). Maren’s hypothesis assumes that the dose of acetazolamide which was given results in complete inhibition of carbonic anhydrase, and that in the absence of carbonic anhydrase OH-- cannot leave the cell from the peritubular side. The first assumption now appears to be incorrect. The complete inhibition of transport on in vitro addition of the drug raises the possibility that the in vivo results in the rat may have been confounded by some pharmacological problem of drug delivery. Furthermore, recent studies by Gutknecht et al. (28) on CO, diffusion in lipid bilayers have shown that 1 mM acetazolamide does not completely *abolish the effect of carbonic anhydrase in this well-defined system. Moreover, Itada and Forster (37) have demonstrated that the catalytic activity of intracellular carbonic anhydrase in red cells is quite different from that in free solution, raising the possibility that drug-enzyme interactions are also different. The assumption that H+ secretion is completely dependent on carbonic anhydrase activity may also be incorrect. It assumes that in the absence of this enzyme OH- ions formed by the proton pump cannot leave the cell unaided; however, the electrochemical gradient in the tubule favors anion exit from the peritubular side, and there is no evidence which states that such movement cannot take place. Finally, the observation that acetazolamide can completely inhibit acid secretion in renal tubule (56) and turtle bladder (80) need not be construed as evidence for the complete dependence of H+ transport on carbonic anhydrase activity, since it appears that this drug inhibits the transport of OH- (or HCO,- ) across the peritubular membrane (22). CO, equilibria in the kidney and turtle bladder. The addition of protons or removal of HCO, from the urine should result in a Pco2 increase with the former and a Pco2 decrease with the latter (Fig. 1). Therefore, for membranes not freely permeable to COz, a change in PP.__ a pK of 3.57 and Pcop of 40 mmHg the carbonic acid concentration will be -25 PM. In order to account for most of the flux the permeability coefficient will have to be J = P (AC) 2 nmo1/cm2 s-l = P (25 nmol/cm3> P = 0.08 cm/s This is an overestimate since the flux is given per square centimeter of the area of a cylinder. The proximal tubular brush border increases the surface area by about 40-fold. Thus, the permeability coefficient in the membrane will be (0.08/40) (2 x 10v3 cm/s). Unfortunately it is not possible to directly measure the permeability coefficient of carbonic acid with present techniques. However, a reasonable estimate can be obtained by comparing it to that of formic acid in egg lecithin membranes at room temperature, which is 2 x 10s4 cm/s (100). Carbonic acid is more hydrophilic than formic acid because of the extra oxygen, hence its permeability coefficient would be lower. This discrepancy would suggest that carbonic acid diffusion cannot account for H+ secretion. However, it should be pointed out that the permeability of membranes to small hydrophilic molecules can vary by one or two orders of magnitude depending on the structure of the membrane (19).

BLOOO

CELL

LUMEN

+ + HC03 tl H2C03

‘02

FIG.

1. A model for reabsorption

It+

Hz0

of HCO, by H+ secretion.

Pco2 should signify one or the other process. Schwartz et al. (79) found an increase in Pco2 when they placed a CO, electrode in the cavity of the turtle bladder, while previous studies by Schilb and Brodsky (76) demonstrated a reduction in Pcoz. However, to measure Pco2 in the latter study, fluid from the bladder was removed in a syringe containing large amounts of mineral oil. Since the partition coefficient of CO, between saline and mineral oil is of the order of 1.8 in favor of the oil, it is not surprising that they did not find a high Pco2 in their fluid. Further, the pH of the luminal medium in their experiments was initially 6.5 and declined to 4.8. Since it is now well established that the rate of acid secretion is inhibited by a low luminal pH (6, 88) (Fig. 2), the amount of acid secreted into the luminal fluid in their experiments would be very small, and consequently the expected change in Pco2 would be trivial even if the collection of the sample were adequate. A second approach to the distinction between H+ and -HCO, transport involves assessment of the disequilibrium pH. If H+ transport proceeds continuously in an HC03-containing solution, the steady-state H&O3 concentration will be higher than the equilibrium condition if the dehydration of H&O, is slower than its rate of formation.2 Rector et al. (73) and Vieira and Malnic (95) measured the in situ pH and found that it was not different from the equilibrium pH; however, addition of acetazolamide reduced the luminal pH. This disequilibrium pH is strong evidence against HCO, reabsorption and favors H+ secretion. The absence of an initial disequilibrium pH, and its appearance afier acetazolamide, suggested that there was luminal carbonic anhydrase which was acting to facilitate the dehydration of carbonic acid. Recent studies support this view, since isolated brush border membranes from renal proximal tubules appear to contain carbonic anhydrase (99). 2 The rate of uncatalyzed

carbonic acid dehydration is given by J = k *&CO, where k is the dehydration constant (= 90 s-l ) (15) and the carbonic acid concentration at pH 6.3 and pK 3.57 is 27 PM. The amount formed in a tubule segment 1 mm long with a radius of 15 pm is 1.7 x 10V3 nmol/s. The flux of H+ is 4 nmo1/cm2 8-l (51), which should equal the rate of formation of carbonic acid. For a tubule segment 1 mm long and 15 pm radius (surface area = 9.4 x lOa cm2) the rate of formation of carbonic acid will be 3.7 nmol/s. Since the rate of formation is the same as the rate of secretion it is apparent that in the steady state the carbonic acid concentration will be higher than the equilibrium level, leading to a lower pH than the equilibrium PH.


EDITORIAL

F79

REVIEW 30

25

O---O e--e

W APH

A 20 :: e I5 3 2

IO

.

5

.

I

A* (mV) ApH

(units)

24 .4

.S

I.4

1.9

2.4

2.9

Effect of electrochemical gradient on net H+ transport in turtle bladder. In 8 experiments net rate of H+ transport in ouabaintreated bladders was measured by pH-stat method or short-circuit current method. In each bladder the luminal pH was decreased in several steps from 7.4 down to 4.6. Serosal pH was 7.4 at all times. In same bladders the potential across the bladder was clamped at varying values with mucosal side positive to serosa. In these experiments both sides of the membrane were bathed by solutions at pH 7.4. Average resistance of those bladders was 5,000 0 cm*. (Drawn from data in Ref. 3.) FIG.

2.

An alternative cause of the disequilibrium pH is a high intraluminal PcolL . Recent measurements have shown that the transepithelial CO, permeability is so high that a APco, is unlikely to develop in the proximal tubule (97). This possibility was supported by the finding of Du Bose et al. (13) that the Pcoz in luminal fluid and cortical blood is the same. However, Sohtell and Karlmark (85) found a 16-mmHg difference in Pco~. Clearly this is an area in which new methods for the measurement of Pco, in the tubule will be very useful. A third approach to this problem involves the relation of the distribution of CO, to the transported species. If one removes HCO, from the medium, urinary acidification does not stop. However, there is sufficient production of metabolic CO, to allow for formation of HCO, in the luminal fluid which could then be transported. Of course, the concentration of HCO, will be very low, in the micromolar range; thus, the putative HCO, pump must have a high affinity. (However, that is not a serious problem since H+ pumps find no difficulty in transporting protons from nanomolar solutions.) In the absence of added HCO,, CO, is the only source of HCO,, and enough CO, should enter the luminal solution to account for sufficient HCO, formation. Schwartz et al. (79) measured the CO, production by the turtle bladder in an important experiment. They found that the bladder produced 2.40 pmol/h. Further, they were able to measure the CO, production from the mucosal and serosal solutions simultaneously. They found that 0.8 pmol/h escaped from the mucosal side while 1.6 left from the serosal side. The rate of transport was about 1 pmol/h. If all urinary acidification occurred by HCO, reabsorption, then 1 pmol/h of HCO, (formed from CO, hydration in the luminal medium) would have to be transported to the serosal medium. There, in the steady state, it would dehydrate and the CO, would escape. The amount of CO, that should have gone into the lumen would have to be 0.8 + 1.0 = 1.8 pmol/h.

Similarly the amount of CO, going into the serosal side would be 1.6 - 1.0 = 0.6 pmol/h. Accordingly, the permeability of the luminal membrane to CO, should be 1.8/0.6, or 3 times that of the serosal side. It seems unlikely that one cell membrane can have 3 times the permeability of CO, of the other, since CO2 is a very permeant and lipid-soluble gas. Further, on addition of acetazolamide (causing a decline of H+ transport to near zero levels) the CO, flux into both solutions became nearly equal. Unless acetazolamide alters the permeability of cell membranes to CO,, an unlikely event, the intrinsic permeability to CO, of both cell membranes is the same. The experiments of Schwartz et al. (79) thus provide strong evidence against HCO, absorption in the turtle bladder. H+ or OH- transport. 2 It is often stated that no distinction can be made between proton secretion and OH- absorption. But if one were able to change the pH on both sides of the pump, one could then make the distinction: the conductance of the pump should increase as the pH declines if the pump were a proton pump, while the opposite should happen if it were a hydroxyl pump. This distinction has been made for the gramicidin channel in lipid bilayers (34). Regulation of H+ Transport OH-

or HCO,-

exit from the antiluminal

border.

Active H+ translocation across the luminal border results in accumulation of OH- in the cell. Since the cell is electrically negative to the blood, there is a sufficient driving force to move anions out into the blood. The presence of a favorable driving force is a necessary condition for passive exit, but in order to produce large fluxes the permeability of the membrane would have to be high. It is highly improbable that passive diffusion of OH- or HCO,- occurs through the lipid membrane phase because of its low dielectric constant. Therefore, some special mechanism, probably a protein, must exist to allow rapid exit of these anions. Such proteins have been described in red cells and appear to facilitate anion transport. This function is inhibited by a number of sulfonic acid derivatives of stilbene such as 4-acetamido4’.isothiocyano-2,2’-disulfonic stilbene (SITS) (74). In both kidney and turtle bladder, application of SITS to the. antiluminal border irreversibly inhibits net acid secretion (10, 16, 91). Recently Cohen et al. (10) demonstrated that the mechanism of this inhibition of H+ transport in the turtle bladder was due to inhibition of OH- (or HCO,-) exit, since intracellular pH measured by 5,5-dimethyl-2,4=oxazolidinedione (DMO) distribution was increased. Recent studies by Cohen et al. (9) have also demonstrated that raising the HCO,- concentration in the serosal medium reduces the rate of H+ transport. This observation suggests that HCO,- exit from the serosal membrane is a passive process whose direction depends on the magnitude of the driving force. As the HCOsconcentration outside the cell becomes higher the electrochemical gradient for HCO,- (or OH-) becomes less favorable to its exit. This would presumably lead to cellular accumulation of OH- or HCOa- and consequent inhibition of H+ secretion.


F80 Relation of H+ transport to transepithelial electrochemical gradients. Transepithelial electrochemical gradients are the fundamental driving forces for passive ion movements. In an epithelium such as the renal tubule or turtle bladder, secretion of hydrogen by the pump results in lowering the pH of the urine. The low pH will act as a driving force for passive H+ movement from the lumen to the blood, and the net transport rate in the presence of an acid urine will be the sum of the active movement into the lumen minus the passive flow out of the lumen. If the membrane is very permeable to protons, the passive flow will be so large that a small pH difference can effectively nullify net H+ secretion. The larger the proton permeability the smaller is the pH difference that can develop across the membrane. The luminal pH, however, is not determined only by the amount of protons added minus those that leak out. The contribution of proton-consuming reactions will become large if the load of buffer perfusing the tubule is significant. In the first part of the proximal tubule the HCOs concentration is high and will end up consuming large amounts of protons. The “disequilibrium pH” resulting can be dissipated by luminal carbonic anhydrase (73, 95). In the proximal tubule all these factors will tend to maintain the luminal pH near 7.4. Although no measurement of proton permeability is available, it is probable that the proximal tubule has a higher permeability than the collecting duct. The proton permeability of the proximal tubule is not very high since the luminal pH can drop to about 5.9 (51). In the turtle bladder the passive proton permeability is negligible (61, 88). It is not widely appreciated that the active transport rate is sensitive to the gradient. In the turtle bladder, acidifying the urinary surface results in an inhibition of net transport (88) (Fig. 2). S,ince the permeability of the bladder to protons is very low, it follows that net transport is a reasonable approximation of the active transport rate. Direct measurement of the activity of the pump showed that the pump itself is sensitive to concentration gradients (6) (Fig. 3). This sensitivity to adverse gradients leads to an interesting property of the system, in that transport would lead to urinary acidification which in turn would shut off transport. This (‘negative-feedback” loop has some implications for the regulation of the amount of protons transported. For instance, the proximal tubule, being a leaky epithelium, cannot maintain large gradients, and, therefore, the rate of transport will always be higher; while in the collecting duct the tightness of the epithelium leads to large pH gradients on the order of 2-3 units lower than blood pH, which would tend to decrease the rate of H+ transport. Based on the change of pH of a droplet of known HCO,- composition, Malnic and Giebisch and their coworkers (23, 24, 52) have observed that the decline in pH follows first-order kinetics. They have suggested that the distal tubule pump is sensitive to a pH gradient, while in the proximal tubule decline in acidification is due to a leak in parallel to a gradient-insensitive pump. There is at the moment no information that can distinguish between these two transport systems. Cer-

Q. AL-AWQATI

Time Mucoso I PH 1 6.55

1 4.78

1 5.62

(hours

1 7.1 I 1

1 693

I

3. Effect of transepithelial pH gradients on active transport and glucose oxidation. Rate of transport was measured as shortcircuit current. Rate of 14C0, production from uniformly labeled glucose wan measured by an ionization chamber method (6). Acidification of mucosal side (at constant serosal pH) is seen to inhibit both transport (J,, lower panel) and glucose oxidation (J&,, , upper panel). Removal of ambient CO2 (at CO, free) also caused a simultaneous inhibition of transport and metabolism. These 2 kinds of maneuvers lead one to conclude that there is coupling between transport and cellular metabolism. (Reprinted, with permission, from Ref. 6.) FIG.

tainly the finding of a single-rate constant is no evidence one way or another, as Giebisch and Malnic (23) recognize. A priori there is no reason to suspect that the H+ pump in the proximal tubule will not be affected by a decrease in luminal pH. All H+ pumps (indeed all ion pumps) when tested for this phenomenon exhibit a relation to the transepithelial electrochemical gradient. If the H+ pump of the proximal tubule is insensitive to the gradient, a special mechanism will have to be invoked. In order to examine the relation of active transport to electrochemical gradients it would be necessary to obtain independent estimates of passive proton permeability and active transport rates, ashasbeen done in the turtle bladder (6). A further complication. lies in the effect of pH on passive pathways. It is well known that the selectivity of certain channels to ions is influenced to a significant degree by pH (59). Depending on the apparent pK of the channel, a decrease in pH may increase or decrease cation (i.e., proton) permeability. The relation between gradient and flux, therefore, might not be linear. Indeed, it had been shown in the proximal tubule that pH has an important effect on Na+ and Cl- permeability (11, 20). Role of carbon dioxide and carbonic anhydrase. It is well known that the rate of H+ transport is enhanced by CO, both in the kidney and in the turtle bladder. This dependence is not absolute, since H+ transport can proceed in the complete absence of CO2 produced by inhibition of oxidative metabolism (82). Addition of exogenous CO, increases the rate of transport up to about a Pcoz of 40 mmHg in the turtle bladder (78). In the kidney higher Pco2 values cause larger increases in acid excretion. The cause of the effect of CO, is not well understood. It is ascribed usually to disposal of OH“behind” the pump by converting it to HCOs in a carbonic anhydrase-sensitive step. Why this should have an effect is not clear, but it could increase the local concentration of protons next to the pump so that a


EDITORIAL

F81

REVIEW

gradient is set up across the luminal membrane which could stimulate transport. The rate of H+ secretion is linearly related to the pH difference across the membrane, so that at a pH difference of 3 units the pump is completely inhibited (3, 88) (Fig. 2). Substitution of 5% CO, by CO&ee air reduced H+ transport by about 75%. If the effect of this removal was due solely to accumulation of OH-, then the cell pH must have gone up by =2 pH units. (Each pH unit should give a change of about 33%.) If at that moment one decreases the luminal pH by 1 unit the H+ rate should be abolished. When this was done, it was found that the ApH necessary to nullify the H+ rate was the same regardless of the ambient PCO, (3) (Fig. 4). Therefore, one cannot invoke a substantial change in the cell H+ or OH- concentration as a cause of the changes in transport. Understanding the role of carbonic anhydrase in facilitating H+ transport is clearly dependent on understanding the mechanism of effect of CO,. A number of added problems relate to the effect of membrane-bound as opposed to cytoplasmic enzyme. The recent demonstration that both brush border and basolateral membranes contain enzyme has aroused questions regarding the function of membrane-bound carbonic anhydrase. Is it a carrier for HCO,- or OH- or does it serve as a catalyst for CO, hydration in the unstirred layers next to the membranes? Friimter et al. (22) have recently shown the HCOs permeability of the peritubular border measured electrically is decreased by acetazolamide. However, it is not possible from these studies to state unequivocally which ion conductance is being decreased, OH- or HC03-. The effect of carbonic anhydrase is frequently investigated with use of inhibitors by assuming that these agents have no effect other than that on carbonic

1.0

2.0

3.0

ApH FIG. 4. Effect of ambient CO, on relation of H+ transport to the transepithelial pH difference. Rate of transport measured as shortcircuit current in response to increasing pH gradient (lumen more acid than serosa) was tested in presence of different CO, concentrations in gassing mixture. Rate of transport was normalized to rate at 1% CO, to allow placement of unpaired results on the same graph. It is seen that CO, affects slope of relation of H+ transport on the applied gradient. Maximal pH difference needed to nullify I-I+ transport was approximately the same in all experiments. (Reprinted from Ref. 3. )

anhydrase. It is now well established that the most frequently used inhibitor, acetazolamide, not only inhibits some transport systems such as chloride (63) and OH (HCO,) exit from the cell (22), but also inhibits certain enzymes that may be important for H+ transport. For instance, Norby and co-workers (64) recently demonstrated that acetazolamide inhibits glucose-6phosphate dehydrogenase, the rate-limiting enzyme in the hexose monophosphate pathway. These authors speculate that this agent might decrease H+ transport by inhibiting the flux through this pathway, which they had previously shown to be important for H+ transport, In the kidney, in vivo results clearly demonstrate enhancement of urinary acid excretion by CO,. Indeed, this is the mechanism for the so-called renal compensation of respiratory acid-base changes. Micropuncture experiments have also shown enhancement of transport by increasing peritubular Pco2 (12). No change in transport rate was seen on decreasing PcoB at constant pH. This may be due to the difficulty of significantly reducing the cell Pco*, since the perfusing volume is small with respect to the cell volume. Recent studies in the whole animal have shown that changing Pcoz changes the plasma HCO, concentration in the same direction regardless of the initial acid-base status of the animal (49). Aldosterone. It remains difficult to ascribe a direct effect of aldosterone on renal H+ transport. This is due in large part to the well-known effect of this hormone on Na+ transport, which would be expected to lead to an increase in the potential difference across the collecting tubule (lumen negative) with consequent enhancement of H+ secretion. Sebastian et al. (36, 83) have concluded that this hormone increases the rate of H+ transport, probably by a direct effect. In adrenalectomized animals and in patients with hypoaldosteronism, they demonstrated that H+ secretion is reduced at any level of urinary pH but that the ability to generate a minimally low urinary pH is unimpaired. It appears now that aldosterone does have a direct effect on H+ transport independent of its effect on Na transport, since Ludens and Fanestil (48) and AlAwqati et al. (4) have demonstrated that aldosterone can stimulate H+ transport in the toad and turtle bladders. The effect is independent of that on Na+ transport, appears earlier than the effect on Na+ transport, occurs in the absence of electrochemical gradients, and even when Na+ transport is completely inhibited by ouabain (1, 4). The mechanism of action of aldosterone on H+ transport has not been examined with the detail with which its effect on Na+ transport has been studied. Based on an equivalent circuit analysis (see below, Theoretical Methods in Analysis of H+ Transport), the conductance f or protons in the active transport pathway was found to be increased. However, the force of the H+ pump, given as the maximum electrochemical gradient needed to nullify active transport, was unchanged (Fig. 5, center panel ). The physical significance of the increase in active conductance is not yet apparent. Goodman et al. (25) have recently suggested that aldosterone might stimulate Na+ transport by altering


F82

Q. AL-AWQATI

brone (20 h)

Mucosal

7.4

6.4

5.4 Mucosal

pIi

4.4 pH

i 0.02

0.04

Suprabasal nmol.

0.06

0.06

J&

min -Qmg

dw-f

FIG. 5. Effect of aldosterone on coupling between H+ transport and glucose oxidation. One member of a pair of hemibladders from same animal was exposed to aldosterone. Rate of H+ transport and 14C02 production fkom uniformly labeled glucose was examined by applying adverse pH gradients across the membrane (lumen more acid than serosa). Results demonstrate linearity of transport (J,) and glucose oxidation (J &) in applied pH gradient and in each other. Both control and aldosterone-treated bladders exhibit this linearity. Aldosterone is seen to increase slope of transport on applied gradient. Slope of transport on glucose oxidation is also seen to be increased. In other experiments in which exposure to aldosterone was of a shorter duration the effect of hormone on relation between JH and Jz$ was found to be unaltered. Slope of JH on ApH was increased in both short-term and long-term exposures. (Reprinted, with permission, from Ref. 5.)

the fluidity of the luminal membrane. Traditionally the hormone is thought to have either a “transport” effect (as described above) or a metabolic effect. The latter could be either an increase of the driving force of metabolism, i.e., the AG of ATP hydrolysis, or an increase in the stoichiometry between transport and metabolism so that more protons are pumped per mole of ATP hydrolyzed. Recently I found that the effect of this hormone on the coupling ratio between transport and 14C02 produced from uniformly labeled glucose was unchanged. However, if the hormone was allowed to

exert its effect for 20 h, there was a change in this gearing ratio so that more protons are transported per mole of glucose oxidized (Fig. 5, tower panel). Using the thermodynamic analysis mentioned below, I calculated that the driving force of glucose oxidation was decreased by aldosterone in the first few hours of its effect, while 20 h later it was increased (1). The driving force for glucose oxidation is related to the logarithm of the ratio of the reactants to the products and, therefore, is some function of the ratio of ATP to ADP x Pi. A decrease in this ratio could occur if aldosterone stimulated H+ transport primarily by an effect on the pump, thereby leading to increased utilization of ATP. Aldosterone has a multiplicity of effects not only on several transport processes but also within each transport process. Its effect on sodium transport is also thought to involve both conductance effects (29, 41, 84) and metabolic effects (14, 46, 75). Whether these phenomena are related to a single fundamental process is yet to be determined. Lifschitz et al. (45) have shown that the inhibitor of protein synthesis, actinomycin D, blocks the effect of aldosterone on renal sodium absorption but not on H+ secretion or potassium excretion. An intriguing result is the recent finding by Mueller and Steinmetz (60) that spironolactone blocks the effect of aldosterone on sodium transport but has no effect on H+ transport in turtle bladder. This competitive antagonist of aldosterone has agonistic properties on H+ secretion, since it stimulates H+ tiansport in steroid-depleted turtle bladders. It is tempting to speculate that the aldosterone effect on H+ transport is mediated by a different receptorfrom that which activates the sodium transport system. In a careful examination of the effects of inhibitors of protein synthesis on the stimulation of H+ secretion by toad bladders, Fanestil et al. (18) found the astonishing result that cycloheximide stimulates H+ secretion and this effect is not additive with that of aldosterone, suggesting that both agents may act by inhibiting protein synthesis. It is possible, therefore, that aldosterone stimulates H+ transport by a mechanism of derepression. In this context it is intriguing to note that one of the components of the proton-translocating ATPase of mitochondria is a small-molecular-weight protein that is an inhibitor of the pump (69). When this cytoplasmic protein is removed transport is stimulated. The H+ pump of turtle bladder may have a local inhibitor as well, the removal of which might “de-repress” the transport apparatus, leading to stimulation of acidifi_ cation. Relation of H+ transport to cellular metabolism. A number of approaches can be used to define the specific metabolic pathways that are linked to transport. In one, a variety of specific inhibitors can be used to block certain metabolic reactions and to evaluate their effect on transport activity. In another, the rate of oxidation of specifically-labeled substrates is measured and the coupling between the rate of transport and the rate of oxidation can be examined. In a third approach, substrates can be added to epithelia depleted from endogenous energy stores with the expectation that if their metabolism leads to the formation of the relevant high-


EDITORIAL

REVIEW

energy intermediates one will see an increase in the rate of transport. All three of these methods have been applied to H+ transport in the turtle bladder. Schwartz and Steinmetz (82) found that inhibition of oxidative phosphorylation by anaerobiosis or cyanide led to an inhibition of H+ transport; however, some residual rate remained, suggesting that H+ transport can be supported by anaerobic pathways. Addition of 2deoxy-n-glucose (2-DG) (an agent that blocks the flux of glucose through its metabolic pathways) resulted in a substantial inhibition of H+ transport. The combination of 2-deoxyglucose and deoxygenation virtually abolished H+ transport. Addition of pyruvate after 2-DG stimulated transport, although not to the initial levels. Addition of glucose after 2-DG restored transport to initial rates. During inhibition of transport by cyanide or deoxygenation, attempts to stimulate H+ transport by CO, resulted in only small increases, indicating that metabolic inhibitors result in a decrease in the supply of energy sources to the degree that they become rate limiting for transport. Turtle bladders, unlike the kidney, have a high glycogen content (42), which might explain why exogenous substrates do not, as a rule, exert any effect on the rate of transport. However, incubation of the bladder for 18 h can result in significant depletion of glycogen (42) and probably other endogenous stores, allowing the investigation of the stimulator-y effects of a variety of substrates. Addition of glucose to the serosal side uniformly results in an increase in the rate of transport (2, 3). In paired hemibladders, glucose results in a larger stimulation than pyruvate, even though the simultaneously measured rates of sodium transport are enhanced equally (4). This result suggests that pathways other than oxidative phosphorylation contribute a significant amount to the support of H+ transport. The addition of lactate and acetate also stimulate H+ transport, but Krebs cycle intermediates have no effect, possibly because of difficulty in gaining access to the compartment. Short-chain fatty acids such as butyrate do not stimulate transport (2) even though they are avidly oxidized by the tissue (1). Furthermore, addition of P-OH butyrate resulted in an increase in H+ transport (2), but the addition of two glucogenic amino acids, glutamate and alanine, to depleted bladders caused no increase in the rate of H+ transport. These results show that all of the major energy-yielding reactions of the cell participate in the support of H+ transport in the turtle bladder. Of great interest has been the recent demonstration by Norby and Schwartz (65) of coupling of H+ transport to [lJ4C]glucose oxidation but much less so to [S14C]glucose oxidation, which provides compelling evidence for a participation of the pentose phosphate pathway of glucose metabolism in H+ transport. These investigators also demonstrated that acetazolamide inhibited glucose-g-phosphate dehydrogenase, the ratelimiting enzyme in this pathway (64). The specific activity of this enzyme is quite high in the turtle bladder (64). Using the specific-yield method of Katz and Wood (38), Norby and Schwartz (65) calculated that stimulation of H+ transport led not only to an increase

F83 in the pentose shunt flux but also to a decrease in the glycolytic flux. Therefore, although there is abundant evidence for a contribution of oxidative phosphorylation to the support of transport, it seems that a significant (although it is not clear how large) proportion of the energy available is obtained from the pentose cycle. What do the above experiments tell us about the nature of the H+ pump? The H+ pump is probably either an ATPase or a redox pump, but unfortunately the studies mentioned above are unable to distinguish between the two possibilities. Although it would seem that a role for oxidative phosphorylation and, hence, ATP production would tend to support an ATPase pump, this is by no means conclusive proof. Nor does the participation of the pentose shunt prove the redox pump (by supplying reducing equivalents in the form of NADPH). It appears now that the cytoplasmic redox potential and the free energy of ATP hydrolysis are probably in equilibrium, and recent work by Wilson et al. (98) has shown that in a number of systems changes in the redox potential lead to directional changes in the AG of ATP hydrolysis. Na:H exchange -active transport, passive diffusion, or merely facilitated confusion? Renal acid excretion exhibits some reciprocal relation with Na or K excretion, originating the idea that there may be some ion exchange mechanism which is responsible for H+ transport. In the turtle bladder there is unequivocal evidence that there is no exchange between H+ transport and the transport of other ions. In the kidney, the evidence for direct coupling between Na+ and H+ transport is ambiguous, because Na+ transport results in an electric potential difference with the lumen being negative, which can act as a driving force to facilitate H+ secretion. In the turtle bladder, H+ transport continues unaf= fected if Na+, K+, or Cl- are removed from the medium (89), and inhibition of sodium transport by ouabain has no effect on H+ transport. Since Na+ transport is tightly coupled to metabolism, inhibiting Na+ transport will lead to a decrease in metabolic CO2 production (6, 81). If the ambient Pco2 is very low this decrease in sodiumdependent CO, production will tend to decrease H+ transport. However, if the Pco, of the medium is kept at a reasonably high level there will be no effect. Recent studies (44) have demonstrated that amiloride decreases H+ transport by about 20%. The simplest interpretation of this phenomenon is that amiloride in the turtle bladder, as elsewhere (30, 77), hyperpolarizes the luminal membrane potential, rendering the electrical gradient from cell to lumen more adverse for H+ transport. The effect of amiloride demonstrates that both Na and H+ are transported from the same cell. In the renal tubule the evidence for Na:H exchange has not been compelling. One major new finding that has given strength to this view is the presence in brush border vesicles of a neutral Na:H exchange process (61). Many investigators see this process as the proximate cause of H+ secretion. In this view, the mechanism of urinary acidification in the proximal tubule is considered passive although it relies on the potential energy of the sodium gradient across the brush border created by active sodium transport. The driving force of this


F84 exchange is not only the sodium concentration gradient but also the H+ concentration gradient. When these two gradients are equal in magnitude but different in sign no net transport should result. The sodium concentration in the lumen is about 150 meq/liter while in the proximal tubular cell it is -50 meq/liter, and the cell pH is ~7.3 (90). On purely energetic grounds one would expect no net H+ transport when the luminal pH declines to 6.8, but the luminal pH in the proximal tubule can go down to levels as low as 5.9 (12). Clearly this exchange cannot be the mechanism of H+ secretion, because at this luminal pH one would expect H+ absorption and sodium secretion. In fact, for this exchanger to continue to secrete H+ at a pH of 5.9 the cell sodium concentration (or rather activity) will have to be less than 5 meq/liter, an unlikely event. Alternatively, the presence of Na:H exchange may be an artifact -of the condition under which it was examined. The stoichiometry between Na+ and H+ in this exchanger may be variable, as has been found for the proton-amino acid co-transport system in bacterial membrane vesicles (71). Either the cell potential, cell pH, the presence of ATP, or other unidentified factors may. convert this exchanger into a “pure” H+ pump. It is worth pointing out that not everyone finds coupling between Na+ and H+ transport in micropuncture experiments. For example, Malnic and de Mello Aires (50) and Green and Giebisch (26, 27) concluded that there is no coupling between Na+ transport and H+ secretion. Recent studies by Ullrich et al. (91) demonstrate that addition of ouabain completely inhibits sodium reabsorption but has no effect on H+ secretion. However, they had previously shown (93) that decreasing luminal sodium concentration decreases H+ secretion. Burg and his co-workers (8, 56) have consistently shown inhibition of net bicarbonate absorption on removal of sodium from the medium or when sodium transport has been inhibited by ouabain or absence of potassium. The studies in isolated perfused proximal tubule appear to provide compelling evidence for cou-pling between sodium absorption and H+ secretion. However, the major problem with these studies is that net HCO, absorption is being measured in a segment that has been previously shown to be leaky to HC09 (5, 96) Electrophysiological studies are supportive of the lack of Na:H exchange. Inhibition of H+ transport by acetazolamide results in an increase in the luminal negativgenerates an active ity, suggesting that H+ transport tran ,sport potential (7, 21, 22). Although complete inhibition of sodium transport does not result in the appearante of a lumen-positive potential (7, 21) this segment is so leaky to electrolytes that it may not be possible to interpret these potentials very readily. Integration of the various regulatory influences. It appears that the respon se to electrochemica .1gradient is the most fundamental property of the H+ pump. This follows from observations that the pump responds to it under all variety of situations, with or without CO*, with or without metabolic stimulation and inhibition, in the presence or absence of aldosterone, and even when OH- exit is blocked (3, 4, 10). Aldosterone also

Q. AL-AWQATI

seems to affect the system in starved and in fed bladders in the absence or presence of ambient CO, (4). Interestingly enough, metabolic inhibition (82) or substrate depletion (4) severely limits the ability of turtle bladders to respond to CO*. It is evident, however, th .at these effects are additive under many experimental conditions. In the whole animal this problem also appears to occur. For instance, changing an animal’s Pcoz always results in a simultaneous change in renal HCO, absorption regardless of the initial condition. In metabolic acidosis, hypercapnia causes an increase and hypocapnia causes a decrease in plasma HC03 (49). Similarly, in metabolic alkalosis these changes in PcoB result in changes in HCOs. Molecular Mechanisms in Proton Transport

In oxidative and photosynthetic phosphorylation, electron transport through sites on the mitochondrial and chloroplast membranes leads to a vectorial transport of protons, with consequent development of a potential and a pH difference across the membrane. In parallel with this system is a reversible proton .-transloeating ATPase. The electrochemical gradient for protons formed by the respiratory chain is used to drive protons through the ATPase into the mitochondria, causing the synthesis of ATP. Uncouplers of oxidative phosphorylations are now known to be proton carriers (35) which collapse the electrochemical gradient by allowing protons to enter the mitochondria passively. Protons are then pumped out of the mitochondria by the ATPase, leading to hydrolysis of ATP. The collapse of the electrochemical gradient will le ad to an enhancement of electron transport, since the grad ient was oriented in a way that was adverse to electron (proton) flow. This simultaneous enhancement of respiration and inhibition of phosphorylation (actually stimulation of ATP hydrolysis) leads to the observed uncoupling of oxidation and phosphorylation. This view of energy conversion, the chemiosmotic theory, first proposed by Mitchell (58), has now acquired immense strength from reconstitution experiments (70). Individual components of the system have been isolated and incorporated into artificial membranes and found to perform their various transport functions and ATP synthesis or hydrolysis. The recent review by Hinkle and McCarty (33) is highly recommended for a very lucid exposition. The proton translocating ATPase in mitochondria and bacteria is now purified and its various components isolated. It appears to contain a proton “channel” that is embedded in the membrane. This proteolipid has apparently important carboxyl groups for proton transport, since addition of dicyclohexyl carbodiimide (DCCD) which covalently binds to carboxyl groups decreases the permeability of the channel to protons (67, 70). The catalytic subunit has a number of proteins that hydrolyze (or synthesize) ATP plus others that confer sensitivity to various inhibitors (70). The isolation of a photosensitive protein from Halobacterium halobium has caused immense excitement in the field. Bacteriorhodopsin, when energized by light, emits protons vectorially, causing the development of


EDITORIAL

F85

REVIEW

an electrochemical gradient. Numerous studies on its mechanism of action by Stoeckenius’s laboratory (66) and others have demonstrated with remarkable elegance that the chromophore is rhodopsin. The structure of the protein has now been identified by Henderson and Unwin (31, 32) to be an alpha helix. Theoretical studies by Nagle and Morowitz (62) have given rise to a possible mechanism of proton translocation in a protein, where the fundamental structural element is seen to be a continuous chain of hydrogen bonds. From studies in ice, such chains are known to have high proton conductantes and can function as “proton wires.” Theoretical Methods of H+ Transport

-

Jn

L”

PMF

Jn

L,

Jn

L,

1::. JM

h

PMF

PMF

J”

L,

PMF

PMF

Jn

L,

PW

in Analysis

One simplified equivalent circuit that has been frequently used to model transepithelial sodium transport (94) has also proved to be useful for H+ transport in the turtle bladder (3). The circuit has two limbs in parallel, one for active transport, made of a battery and a conductance in series, while the other represents “passive” transport and is composed of a “shunt” conductance. The parallel conductance models flow in intercellular pathways as well as through cells that do not participate in active transport and through damaged cells. It had been demonstrated experimentally that this passive proton conductance of the turtle bladder is negligible (6, 88). This made the evaluation of the elements of the active limb experimentally simple, since in the absence of any passive leaks the net rate of H+ transport will be equal to the rate of active transport even in the presence of electrochemical gradients (6). The force of the battery (the proton motive force of the H+ pump or the PMF) will be given by the maximum gradient needed to nullify the rate of the pump, while the conductance in the active pathway is given by the slope of active transport on the applied electrochemical gradient (Fig. 2). In ascribing physical significance to the two circuit elements of active transport it is customary to equate the PMF with the energy-requiring step in the transport process and to give passive properties to the series conductance. Such passive properties might include the delivery of protons to the pump, exit of OH- or HCO,from the pump or through the serosal barrier of the cell, movement of protons through the pump itself, and the number of pumping sites. There is, however, no a priori reason why any of the functions described should not be metabolically dependent. In analyzing the H+ transport system with this equivalent circuit we found that CO, stimulates H+ transport by a mechanism that involves the series conductance alone and that acetazolamide inhibits H+ transport by inhibiting this series conductance without changing the PMF. These results are compatible with the effects of these agents on the supply and disposal of protons or OH-, and in a way validate the method used. Interestingly, maneuvers that changed cellular metabolism, such as the ones depicted in Fig. 6, also changed the series conductance. Their effect on the PMF was much smaller. One can speculate on the physical significance of this by claiming that metabolically induced ‘changes in the configuration of

6. Effect of various maneuvers on relation of H+ transport (J, ) to applied pH gradient. In each set of experiments rate of H+ transport in absence of electrochemical gradients (J, ), slope of J, on applied pH gradient (L,), and maximal pH difference needed to nullify transport (PMF) was evaluated before and after an experimental maneuver. Values of these 3 parameters in experimental period was normalized to those in control period. Transport was stimulated by increasing CO, and by addition of glucose to substratedepleted bladders. It was inhibited by acetazolamide, deoxygenation, addition of 10 mM %deoxy-n-glucose, and overnight depletion. (Reprinted from Ref. 3.) FIG.

the proton channels could lead to an effect that can be expressed as a change in conductance. We were, however, rather surprised to find that these metabolic maneuvers had little or no effect on the PMF despite large changes in the rate of H+ transport (Fig. 6). Another approach that has been used is the irreversible thermodynamic approach first given by Kedem (39) and later formalized in detail for the case of transepithelial ion transport by Essig and Caplan (17). This formalism relies on the observation that active transport across membranes is a flow of matter that is coupled to a metabolic reaction. Accordingly, the rate of H+ transport (JH) will be coupled to its conjugate driving force gradient across the mem(clt-c the electrochemical brat;) as well as to the free energy of hydrolysis of the driving metabolic reaction (the affinity or A). Similarly the rate of metabolism (J,> is driven by A as well as by AiiH J H=LH*AfiH+brA Jr = bH*AbH

+ L,A

Each flow is coupled to its conjugate driving force by a “straight” coefficient (L and I+) and to the other driving force by a %ross-coefficient” (Lr and l&H)* The cross-coefficients are assumed to be equal-by Onsager symmetry. Beauwens and Al-Awqati, using the rate of uniformly labeled glucose oxidation as J,, found that JH and Jr were highly linear functions of Ab,+ (Figs. 5 and 6), allowing the use of this formalism. A number of interesting conclusions follow from this formalism. In one the PMF (taken from the equivalent circuit analysis) will be seen to equal


Q. AL-AWQATI

F86 PMF = (Afi.),,

= o = Fb

A H

This indicates that the PMF is not a fundamental thermodynamic parameter in that it is a composite function including both energetic (A) and conductance factors (L,). The series conductance in the equivalent circuits will be given by I&. The lack of response of the PMF to a variety of maneuvers that are thought to change the affinity may be due to the observation that h itself is metabolically dependent. Therefore, the changes in & and A tend to cancel each other, leaving the PMF unchanged. An important contribution of irreversible thermodynamics is in its clarification of the issues of coupling, stoichiometry, and efficiency. One important concept introduced by Kedem and Caplan (40) is a uantitative analysis of the coupling between two flows. The observation that changes in H+ transport are accompanied by changes in the rate of glucose oxidation is proof that the two- flows are coupled. However, the tightness of coupling cannot be discerned from these observations alone. The degree of coupling, q, was introduced as a parameter that quantifies the tightness of coupling. This is given by q2 = -=&HA2

I+&

@JH/aJh + o @JH/~JAAG = o

The degree of coupling varies between 1 and 0 with the former being the maximum and 0 being for uncoupled reactions. The degree of coupling can be measured by the ratio of the slope of JH on Jr obtained in the absence of a gradient to that obtained by changing the electrochemical gradient. We found that for the H+ transport system the degree of coupling is not significantly different from 1. The implications of this result

are several. First it indicates that the flows of JH and Jr are so tightly coupled that if H+ are forced to go through the pump by a reversed gradient ATP may be synthesized (if ATP hydrolysis is the coupled reaction). Second, it appears that the eficiency of energy conversion by the pump is solely determined by q (40). If q is 1, then the maximum yield of electroosmotic energy can be obtained from the free energy of hydrolysis of the driving reaction, which would be given by AG = (PMF) z where AG is the free energy of the reaction, PMF is the maximum electrochemical gradient that can be generated by the pump, and z is the stoichiometry of the pump in moles of H+ per mole of ATP hydrolyzed. The affinity of the driving reaction is the free energy available for the coupled reaction. It may not bear any relation to the AG calculated from cell homogenates; however, it is likely that it will be in equilibrium with the cytoplasmic AG. In recent studies on the mechanism of action of aldosterone it appeared that this hormone stimulated transport by increasing L (4). In the first few hours of its effect preliminary results showed that the finity declined, while 20 h later it appeared that there was an increase in the affinity (1). The early effect is indicative of the fact that initially, at least, the increase in transport by conductance factors happens faster than the stimulation of the metabolic machinery. Later there is stimulation of metabolism so that the affinity increases. Recently Edelman (14) has shown that citrate synthase activity is increased by aldosterone. This may lead to an increase in the ATP/ADP* Pi ratio which will be expressed as an increase in the affinity. This work was supported in part by Grant HL-21849 from the

National

Institutes

of Health.

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2. AL-AWQATI, Q. Metabolic pathways linked to H+ transport in turtle bladder (Abstract). Kidney Intern. 12: 547,1977. 3. AL-AWQATI, Q., A. MUELLER, AND P. R. STEINMETZ. Transport of H+ against electrochemical gradients in turtle urinary bladder. Am. J. Physiol. 233: F502-F508, 1977 or Am. 3. Physiol.: Renal Fluid EZkctroZyte PhysioZ. 2: F502-F508, 1977. 4. AL-AWQATI, Q., L. H. NORBY, A. MUELLER, AND P. R. STEINMETZ. Characteristics of stimulation of H+ transport by aldosterone in turtle urinary bladder. J. CZin. Invest. 58: 351-358, 1976.

5. BANK, N., AND H. S. AYNEDJIAN. A microperfusion study of bicarbonate accumulation in the proximal tubule of the rat kidney. J. Chin. Invest. 46: 95402, 1967. 6. BEAUWENS, R., AND Q. AL-AWQATI. Active H+ transport in the turtle urinary bladder. Coupling of transport to glucose oxidation. J. Gen. Physiol. 68: 421-439,1976. 7. BERRY, C. A., AND F. C. RECTOR, JR. Electrogenic H+/HCO, transport in the rabbit proximal convoluted tubule (PCT) (Abstract). Proc. Intern. Congr. NephroZ., 7th, MontreaZ, 1978. In press. 8. BURG, M., AND N. GREEN. Bicarbonate transport by isolated perfused rabbit proximal convoluted tubules. Am. J. Physiol. 233: F307-F314,1977 or Am. J. Physiol.: RenaZ FZuid Electrolyte Physiol. 2: F307-F314, 1977. 9. COHEN, L. H., A. MUELLER, AND P. R. STEINMETZ. Inhibition of the H+ current by sejr~sal HCO,- in turtle bladder (Abstract). Kidnev Intern. 12: 554.1977.

L. H., A. MUELLER, AND P. R. STEINMETZ. Inhibition of the HCO, exit step in urinary acidification by a disulfonic stilbene. J. CZin. Inuest. 61: 981-986, 1978. DE MELLO, G. B., A. G. LOPES, AND G. MALNIC. Conductances, diffusion and streaming potentials in the rat proximal tubule. J. Physiol. tondon 260: 553-569,1976. DE MELLO AIRES, M., AND G. MALNI~. Peritubular pH and PCO, in renal tubular acidification. Am. J. Physiol. 228: 17661774, 1975. Du BOSE, T. D., N. W. CARTER, AND J. P. KOKKO. Determination of in situ pCOz in the rat nephron (Abstract). Kidney Intern. 12: 555, 1977. EDELMAN, I. S. Candidate mediators in the action of aldosterone on Na transport. In: Membrane Transport Processes, edited by J. F. Hoffman. New York: Raven Press, 1978, vol. I, p. l25140. EDSALL, J. T., AND J. WYMAN. Biophysical Chemistry. New York: Academic, vol. I, 1958. EHRENSPECK, G., AND W. A. BRODSKY. Effects of 4-acetamido4’0isothiocyano-2,2’-disulfonic stilbene on ion transport in turtle bladders. B&him. Biophys. Acta 419: 555-558, 1976. ESSIG, A., AND S. R. CAPLAN. Energetics of active transport processes. Biophys. J. 8: 1434-1457, 1968. FANESTIL, D. D., M. E. BAKER, D. A. VAUGHN, AND J. H. LUDENS. Urinary acidification. A novel unifying model explaining stimulation by both aldosterone (aldo) and inhibitors of RNA and protein synthesis (IRNAPS) (Abstract). Kidney Intern. 10: 96A, 1976. FINKELSTEIN, A. Water and nonelectrolyte permeability of lipid bilaver membranes. J. Gen. Phvsiol. 68: 127-135.1976.

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Qais Al-Awqati Departments of Medicine and Physiology, Columbia University College of Physicians and Surgeons, New York City 10032


Transport of H+ against electrochemical gradients in turtle urinary bladder.

Solute transport across isolated epithelia.

Acid-base transport systems in gastrointestinal epithelia.

Isotonic water transport in secretory epithelia.

Active H+ transport in the turtle urinary bladder. Coupling of transport to glucose oxidation.

Membrane fluidity and transport properties in epithelia.

Transport across epithelia: some basic principles.

Transport across epithelia. A kinetic evaluation.

Second-messenger regulation of sodium transport in mammalian airway epithelia.

Characteristics of stimulation of H+ transport by aldosterone in turtle urinary bladder.

Eicosanoids: their function in renal epithelia ion transport.

Paracellular calcium transport across renal and intestinal epithelia.

Relationship between the rate of H+ transport and pathways of glucose metabolism by turtle urinary bladder.

Physiological role for vanadate-inhibitable active H+ transport: a new model for distal urinary acidification.

Sodium-dependent and -independent nucleobase transport in cultured LLC-PK1 epithelia.

Ion transport in cultured epithelia from human sweat glands: comparison of normal and cystic fibrosis tissues.

Modulation of cytoskeletal organization and cytosolic granule distribution by verapamil in amphibian urinary epithelia.

Amide transport channels across toad urinary bladder.

A new model for regulation of sodium transport in high resistance epithelia.

On the influence of a leaky tight junction on water and solute transport in epithelia.

Polarized sorting in epithelia.

Actions of hydrogen sulfide on sodium transport processes across native distal lung epithelia (Xenopus laevis).

Basolateral Mg2+ extrusion via CNNM4 mediates transcellular Mg2+ transport across epithelia: a mouse model.

Effects of 5-hydroxytryptamine and 5-hydroxytryptamine receptor agonists on ion transport across mammalian airway epithelia.