Turtle Bladder Research Articles

Effect of furosemide on ion transport in the turtle bladder: evidence for direct inhibition of active acid-base transport

The diuretic furosemide inhibits acid-base transport in the short-circuited turtle bladder. It inhibits luminal acidification when present in either mucosal or serosal bathing fluids, but decreases alkalinization only from the serosal side of the tissue. The inhibition of both acid-base transport processes is independent of ambient Cl −; and the disulfonic stilbene, SITS, an inhibitor of Cl −-HCO 3 − exchange, fails to prevent the furosemide-elicited inhibition of alkalinization. These results preclude an absolute requirement of a furosemide-sensitive Cl −-HCO 3 − exchange by these transport processes. The drug also interferes with the CO 2-induced stimulation of acidification and alkalinization. The inhibition of the residual acidification in acetazolamide-treated, acidotic bladders, however, suggests an action at sites other than cytosolic carbonic anhydrase. Although active Na + and Cl − reabsorption and tissue oxygen uptake are also decreased by furosemide, the rate of oxygen consumption uncoupled by 2,4-dinitrophenol is not diminished, indicating a primary inhibition of the various ion transport processes, not of metabolism. It is proposed that inhibition of transepithelial acid-base transport by furosemide in the turtle bladder includes inhibition of the acid-base pumps.

Carbon dioxide causes exocytosis of vesicles containing H+ pumps in isolated perfused proximal and collecting tubules.

In the turtle bladder it has recently been shown that CO2 stimulates H+ secretion, at least in part, by causing fusion of vesicles enriched in H+ pumps with the luminal plasma membrane. To test for the presence of this mechanism in the kidney we perfused collecting ducts and proximal straight tubules on the stage of an inverted epifluorescence microscope with fluorescein isothiocyanate dextran (70,000 mol wt) in CO2-free medium. After washout we noted punctate fluorescence in endocytic vesicles in some collecting ducts and in all proximal straight tubule cells. More cells took up fluorescent dextran in outer medullary than in cortical collecting ducts. Using the pH dependence of the excitation spectrum of fluorescein we found the pH of the vesicles to be acid (approximately pH 6). Addition of proton ionophores increased vesicular pH by 0.6 +/- 0.1 U, suggesting that the acidity of the vesicles was caused by H+ pumps. CO2 added to the medium (25 mmHg, pH 7.6 at 37 degrees C) reduced fluorescence intensity by 24 +/- 5% in cortical collecting ducts, 27 +/- 5% in medullary collecting ducts, and 25 +/- 5% in proximal straight tubules. Since this effect was prevented by the prior addition of colchicine to the bath, we believe that CO2 caused a decrease in cytoplasmic fluorescence by stimulating exocytotic fusion of the vesicles and thereby secretion of fluorescent dextran. This exocytotic fusion also occurred when tubules that were loaded with fluorescent dextran at a pCO2 of 37 mmHg were exposed isohydrically to a pCO2 of 114 mmHg; the mean decrease was 53 +/- 4%. We conclude that some cells in the collecting ducts and all cells in the proximal straight tubule incorporate fluorescent dextran into the apical cytoplasmic vesicles and acidify them with H+ pumps. CO2 causes fusion of these vesicles with the luminal membrane, but whether CO2 stimulates H+ secretion by increasing the number of functioning H+ pumps remains to be determined.

Inhibition of H+ secretion by lectins in turtle bladder: role of a cell type on acidification.

Because certain lectins have been shown to bind to the intercalated cell of the cortical collecting tubule, we investigated the effect of concanavalin A and wheat germ agglutinin on urinary acidification in isolated turtle bladders. After addition to the mucosal but not serosal fluid, concanavalin A and wheat germ agglutinin decreased H+ secretion in a dose-dependent manner, and these effects were specifically inhibited by the competitive antagonists of concanavalin A (alpha-methyl-D-mannoside) and of wheat germ agglutinin (N-acetylglucosamine). Concanavalin A decreased H+ secretion by decreasing both the proton motive force and the active conductance of protons. Although electroneutral HCO3 secretion was not inhibited by either lectin, Na transport was decreased by 18 and 25%, respectively, after concanavalin A and wheat germ agglutinin. Concanavalin A failed to inhibit O2 consumption by the granular cell fraction but significantly inhibited O2 consumption by the carbonic anhydrase rich cell fraction. Morphological studies utilizing peroxidase or fluorescein-labeled concanavalin A showed that concanavalin A stained one cell type and that this staining was specific since it could be blocked by the competitive antagonist alpha-methyl-D-mannoside. Studies utilizing double labeling with fluorescein concanavalin A and acridine orange suggested that both probes stain the same cell type. The data strongly suggest that concanavalin A interacts specifically with the cell responsible for H+ secretion.

Ammonia transport by the turtle bladder: relationship to H+ secretion.

The turtle bladder is apparently capable of transporting ammonia in the form of NH3 as well as NH+4. In the present study we examined the relationship of ammonia transport into the mucosal solution to H+ secretion and to chemical reactions in the unstirred layers. The relationship between ammonia transport and H+ secretion was examined before and after inhibition of H+ secretion by acetazolamide, SITS, and the putative inhibitor of the H+ pump dicyclohexylcarbodiimide. All these inhibitors caused a significant decrease in H+ secretion and led to a parallel decrease in the rate of ammonia transport with serosal pH at 6.4. Stimulation of H+ secretion by 1% CO2 increased both H+ secretion and ammonia transport at serosal pH 6.4. At serosal pH 6.4 the changes in H+ secretion were highly correlated with changes in ammonia transport. Ammonia transport at serosal pH 6.4 was electrogenic and inhibited by a low mucosal pH. In contrast, the diffusion of NH3 down an imposed concentration gradient was not correlated with changes in H+ secretion. Chemical reactions in the unstirred layers seem to influence ammonia transport in that increasing serosal nonvolatile buffer concentrations decreased the diffusion of NH3 down the concentration gradient from the serosal into the mucosal solution, probably by decreasing serosal NH3 concentration. Conversely, addition of uncouplers in the mucosal solution in an attempt to decrease the H+ concentration in the mucosal solution unstirred layer decreased the ammonia transport at serosal pH 6.4.

Plasma membrane proton-ATPase of a turtle bladder epithelial cell line.

Urinary acidification by the turtle bladder is mediated by a proton ATPase located in the apical membrane. The present study describes a proton ATPase in the plasma membrane of a cell line of turtle bladder epithelial cells. In the presence of ouabain to inhibit Na+,K+-ATPase and in the absence of Ca2+ to inhibit Ca2+-ATPase, we measured ATPase activity of the plasma membranes of the cultured cells. This ATPase was resistant to oligomycin but sensitive to dicyclohexylcarbodiimide, N-ethylmaleimide, and vanadate. In the presence of ATP, the ATPase was capable of acidification as assessed by quenching of acridine orange. Acidification could not be elicited by other nucleotides (GTP, UTP). Acidification was inhibited by dicyclohexylcarbodiimide, N-ethylmaleimide, and vanadate but was not affected by replacement of Na+ by K+. The acidification response was dependent on the presence of chloride, abolished in the presence of gluconate, and inhibited partially by nitrate. Experiments utilizing the voltage-sensitive dye 3,3'-dipropylthiodicarbocyanine iodide showed that the proton ATPase was electrogenic and capable of responding to a favorable electric gradient. In summary, the turtle bladder epithelial cell line has a plasma membrane proton ATPase which is similar to the proton ATPase of turtle bladder epithelium and thus should allow purification and characterization of this enzyme.

Effect of cyclic AMP on acidification in the isolated turtle bladder

Cyclic AMP (10 mM) has been demonstrated to inhibit hydrogen ion secretion in the isolated turtle bladder. These experiments were designed to study the effect of cyclic AMP on hydrogen ion secretion in the isolated turtle bladder using both the pH stat and the reverse short circuit current techniques. Sodium transport was measured as the short circuit current. Studies were carried out at 0% CO2 and 1% CO2 at pH 7.4. Cyclic AMP (10 mM) was added to either the serosal or mucosal solutions, and hydrogen ion secretion was measured from 0 to 120 min. In the presence or absence of carbon dioxide, cyclic AMP and dibutyryl cyclic AMP had no effect on hydrogen ion secretion in fasted turtles. The addition of theophylline (10 mM) to the serosal solution, with or without cyclic AMP had no effect on proton secretion. Sodium transport was unchanged from control following serosal or mucosal addition of 10 mM cyclic AMP in the presence or absence of carbon dioxide. In chronically bicarbonate-loaded turtles proton secretion was the same as control fasted turtles. In these animals, however, serosal administration of 10 mM cyclic AMP significantly stimulated bicarbonate secretion. Stimulation of bicarbonate secretion occurred in the presence of a 20 mM bicarbonate gradient. When there was no bicarbonate gradient, cyclic AMP was without effect; cyclic AMP had no effect on bicarbonate permeability when measured in the presence of acetazolamide. These results indicate that cyclic AMP has no effect on hydrogen ion secretion or sodium transport in the isolated turtle bladder when studied at two different rates of acidification (0 and 1% CO2). Cyclic AMP appears to stimulate active bicarbonate secretion.

An electrogenic proton-translocating adenosine triphosphatase from bovine kidney medulla.

Urinary acidification in the mammalian collecting tubule is similar to that in the turtle bladder, an epithelium whose H+ secretion is due to a luminal proton-translocating ATPase. We isolated a fraction from bovine renal medulla, which contains ATP-dependent proton transport. H+ transport was found to be electrogenic in that its rate was reduced by a membrane potential. H+ transport activity was inhibited by N-ethyl maleimide and dicyclohexyl carbodiimide, but not by oligomycin or vanadate; its activity did not depend on the presence of potassium, differentiating this ATPase from the mitochondrial F0-F1 ATPase and the gastric H+-K+ ATPase. H+ transport activity had a specific substrate requirement for ATP, distinguishing this pump from the lysosomal H+ ATPase, which uses guanosine or inosine triphosphate as well. The distribution of this H+ pump on linear sucrose density gradient was different from that of markers of lysosomes and basolateral membranes. These results show that the kidney medulla contains an H+ -translocating ATPase different from mitochondrial, gastric, and lysosomal proton pumps, but similar to the turtle bladder ATPase.

Ammonia transport by the turtle urinary bladder.

Ammonia transport across the turtle bladder was examined by adding NH4Cl to the serosal (S) or mucosal (M) solution. With appropriately fixed levels of pH and/or NH4Cl concentration the transepithelial flow of ammonia parallels the extracellular concentration of NH+4 while that of NH3 is kept constant and parallels the extracellular concentration of NH3 while that of NH+4 is kept constant. This suggests that NH+4 as well as NH3 traverses the bladder wall. The apparent S----M permeability to NH3 was 15-18 times greater than that to NH+4. At pH 6.4 in both S and M solutions, the net flow of ammonia was from S----M, but at pH 8.4 in the S and 6.4 in the M or vice versa ammonia transport was of the same magnitude in both directions. The relative permeability of the M membrane to NH+4 was less than that to Na and nearly the same as that to K. The relative permeability of the S membrane to NH+4 was greater than that to K. At S pH of 8.4 and M pH of 6.4, ammonia transport was a linear function of NH4Cl concentration. At S pH of 6.4 and M pH of 6.4, ammonia transport was a saturation function of NH4Cl concentration in that it was linear up to 5 mM and constant and maximal in excess of 7.5 mM. The net transport of methylamine directed from S to M was competitively inhibited by NH4Cl, suggesting that the two substances are transported through a common carrier system. At pH 6.4 in both S and M, the S addition of NH4Cl induced an increase in reverse short-circuit current, the magnitude of which approximated the chemically determined rate of ammonia transport. This means that ammonia transport at pH 6.4 is, at least in part, electrogenic due to the flow of ionic NH+4 through a transbladder conductive path. However, when the S pH was raised to 8.4 the increase in ammonia transport was not associated with an increase in current. The present study demonstrates that the turtle bladder is capable of transporting ammonia with different characteristics of NH+4 and NH3 transport.

Chloride-induced increment in short-circuiting current of the turtle bladder. Effects of in-vivo acid-base state

Evidence for the participation of conductive and non-conductive (exchange) transmembrane anion pathways in the luminal acidification, alkalinization, and chloride-reabsorptive functions of the turtle bladder is provided from the pattern of Cl −-induced changes in transepithelial electrical parameters of isolated urinary bladders from three groups of donor turtles: control or post-absorptive turtles (those killed 5 days after feeding); acidotic turtles (NH 4Cl-loaded); and alkalotic turtles (NaHCO 3-loaded). The predominance of each of the three aforementioned transport functions as well as the response to Cl −-addition is altered by the in-vivo electrolyte balance of the turtle. In post-absorptive bladders, which are poised for acidification and Cl − reabsorption, the mucosal and serosal addition of Cl − to Na +-free, (HCO 3 − + CO 2)-containing media increases the negative short-circuiting current ( I sc ). In acidotic bladders, which are poised for acidification but not Cl − reabsorption, mucosal Cl − addition has no effect on this I sc whereas serosal Cl − addition increases the negative I sc in a manner identical to that observed in the post-absorptive bladders. Alkalotic bladders do not possess an acidification function but instead are poised for Cl − reabsorption and cAMP-dependent electrogenic alkali secretion (positive I sc ). In these bladders, serosal Cl − addition is without effect while mucosal Cl − addition produces transient changes in this positive I sc . It is found that these results can be replicated by a model of the turtle bladder in which transmembrane Cl − and HCO 3 − conductive and exchange paths mediate transepithelial acidification, alkalinization and Cl − reabsorption.

Cellular response to acute respiratory acidosis in rat medullary collecting duct.

The collecting duct of the mammalian kidney is involved in urine acidification. Recent studies in the turtle bladder suggest that hydrogen ion secretion in response to elevated CO2 is regulated by insertion of hydrogen pumps into the luminal membrane of the mitochondria-rich cells. Because intercalated cells of the collecting duct are structurally similar to mitochondria-rich cells of the amphibian bladder, we studied the rat outer medullary collecting duct (OMCD) during respiratory acidosis to determine whether changes compatible with hydrogen ion secretion occur in the intercalated cells. Rats were studied during normal acid-base conditions and after 4-5 h of respiratory acidosis. After collection of physiologic data, the kidneys were fixed by in vivo perfusion and processed for electron microscopy. No changes were observed in the principal cells of the OMCD. Morphometric analysis revealed a significant increase in the surface density of the apical plasma membrane and a decrease in the number of tubulovesicular profiles in the apical region of the intercalated cells throughout the OMCD with respiratory acidosis. There were no changes in surface density of the basolateral membrane. These findings suggest that in response to respiratory acidosis there is transport of membrane from the tubulovesicular membrane compartment to the apical plasma membrane of the intercalated cells.

Chloride dependence of the HCO3 exit step in urinary acidification by the turtle bladder.

To characterize the efflux of HCO-3 across the basolateral membrane of the H+-secreting cells of the turtle bladder, we examined the effect of substitution of gluconate or methyl sulfate for Cl- on the rate of acidification (JH). JH was measured as the short-circuit current in bladders in which Na+ transport was abolished with 10(-4) M ouabain. In hemibladders bathed in normal Ringer solution (Cl- = 122 mM) JH was 44.9 microA. Substitution of the Cl- resulted in a marked reduction in JH (12.5 microA with gluconate and 7.5 microA with methyl sulfate). Addition of Cl- to the mucosal surface had no effect on JH. In contrast, serosal addition of Cl- restored JH to control. The apparent Km for Cl- in gluconate Ringer was 0.13 mM. Serosal furosemide (1 mM) inhibited JH by 55% in Cl- Ringer. We conclude that HCO-3 exit across the basolateral membrane of the H+-secreting cell occurs via a Cl-HCO3 exchanger that has a high affinity for chloride.

Aldosterone response in the turtle bladder is associated with an increase in ATP.

Urinary bladders from freshwater turtles, mounted as sacs, were stripped of their serosa and submucosa. This did not alter conductance. They were maintained in open circuit except for brief observation of short-circuit current (SCC) every 15 min. Potential difference (PD) averaged 68 +/- 14 mV and SCC 485 +/- 100 microA. Acetazolamide 10(-3) M increased SCC by 46 +/- 27 microA. Aldosterone 10(-7) M following acetazolamide resulted in a rise in SCC that began at about 75 min and reached a plateau between 3 and 5 h. SCC rose 127 +/- 15% compared with control bladder halves. ATP measured in perchloric acid extracts 5 h after addition of aldosterone increased by 33% (P less than 0.01) and (ATP)/(ADP) X (Pi) by 81% (P less than 0.01). These results support the view that the stimulatory effects of aldosterone on active sodium transport involve an increase in ATP and (ATP)/(ADP) X (Pi).

Regulation of proton transport in urinary epithelia.

In urinary epithelia, like the turtle bladder, protons are transported by a H+ translocating ATPase located in the luminal membrane. We have recently discovered that the H+ pump is stored in small vesicles that lie underneath the luminal membrane. CO2, a major regulator of H+ transport causes these vesicles to fuse with the membrane thereby inserting more H+ pumps. We have now isolated these vesicles from the turtle bladder and from beef kidney medulla. Based on inhibitor sensitivity and substrate specificity this proton translocating ATPase is different from the mitochondrial F0-F1 ATPase, yeast plasma membrane and the gastric H+,K+-ATPase. Solubilization and reconstitution of the enzyme into liposomes shows retention of transport activity and inhibitor sensitivity.

Characteristics of NH4+ and NH3 transport across the isolated turtle urinary bladder.

To evaluate the contribution of NH+4 movement to net ammonia flux across the turtle urinary bladder, studies were designed to determine the individual permeabilities of NH3 and NH+4. In these studies we examined the effect of varying the NH+4 concentration gradient while holding that for NH3 constant or the opposite maneuver on the unidirectional flux of ammonia. It was demonstrated that the changes in the unidirectional flux of ammonia was a linear function of the change in the concentration of the varied species. From the slope of these relationships we estimated the NH+4 permeability (4.9 X 10(-6) cm . s-1) and the NH3 permeability (2.6 X 10(-4) cm . s-1). In the absence of H+ transport the unidirectional fluxes of NH+4 were equal, and these fluxes changed in a predictable manner with the application of transepithelial electrical potentials. In the presence of H+ transport there was net NH+4 secretion due to an increase in the serosal-to-mucosal flux. Net secretion could be abolished by inhibiting H+ transport with acetazolamide. The addition of NH4Cl to the serosal solution resulted in a decrement in the rate of mucosal acidification. The subsequent addition of nonvolatile buffer to the serosal solution restored H+ transport and reduced the secretory NH+4 flux to the same extent as that produced by acetazolamide. We conclude that the turtle bladder has a significant passive permeability to NH+4. There also is apparent coupling between H+ transport and ammonia secretion. This coupling may result from titration of NH+4 in the serosal solution to NH3 by alkali generated behind the H+ pump and the diffusion of NH3 into the mucosal solution.

Role of membrane fusion in CO2 stimulation of proton secretion by turtle bladder.

The changes in cell structure produced during stimulation of proton secretion by CO2 in turtle bladder were examined using ultrastructural morphometric methods. One hour after CO2 addition, the area of the luminal membrane of the carbonic anhydrase-containing (CA) cell population was increased 2.5-fold and the volume percent of electron-lucent cytoplasmic vesicles in these CA cells was decreased by 61%. No changes were observed in the epithelial granular cells. These results suggest that during CO2 stimulation the vesicles fuse with the luminal membrane. CO2 stimulation of proton secretion is inhibited by the cytoskeleton-disrupting drugs colchicine and cytochalasin B and by 99% deuterium oxide as the Ringer solvent. Deuterium oxide also inhibits the decrease in cytoplasmic vesicles. Thus stimulation of proton secretion by turtle bladder CA cells depends to a large extent on vesicle fusion and the resultant increase in luminal surface area.

Stimulation of urinary acidification by insulin in the turtle bladder.

Addition of insulin to the substrate-containing serosal solution of freshly excised bladders or to that of bladders incubated overnight in substrate-enriched media increases the rate of H+ secretion to a greater extent in the latter (overnight group) than in the former. This effect can be blocked by pretreatment with anti-insulin antibody, suggesting that the stimulation of H+ secretion is a specific effect of insulin. The effect of insulin is concentration dependent with half-maximal stimulation of H+ secretion at 100 mU/ml and maximal stimulation at 250 mU/ml. Other characteristics of the insulin-induced stimulation of H+ secretion were its independence of any effect of Na transport and its absolute requirement for the presence of substrate (glucose or pyruvate) under aerobic conditions only. The proton-secreting action of insulin is associated with an increase in the proton-selective conductance in series with the proton pump while the estimated electromotive force of the proton pump remains constant. Finally, the insulin-induced and aldosterone-induced stimulations of proton secretion are mutually independent, as shown by the additivity of these events and by the fact that the effect of insulin was not blocked by pretreatment with cycloheximide. These data suggest that endogenous insulin modulates the rate of H+ secretion by the in vivo turtle bladder.

Effect of calcium ionophore A23187 on electrogenic acid-base transport in turtle bladder Inhibition of acidification and stimulation of alkalinization

Turtle bladders bathed on both surfaces with identical HCO − 3/CO 2-rich, Cl −-free Na + media and treated with ouabain and amiloride exhibit a transepithelial potential serosa electronegative to mucosa and a short-circuit current ( I sc ) which is a measure of the net luminal acidification rate. Addition of calcium ionophore A23187 (10 μM) to the mucosal side of the epithelium rapidly reverses the direction of the potential difference and I sc and decreases tissue resistance. The resulting positive I sc resembles that previously observed in response to isobutylmethylxanthine (IBMX) and cAMP analogs. Reversal of the I sc is enhanced in bladders from severely alkalotic turtles. In contrast, in severely acidotic turtles, ionophore A23187 decreases, but does not reverse, the I sc . The data suggest that, like IBMX and cAMP analogs, the Ca ionophore stimulates an electrogenic alkalinization mechanism, but, unlike the former agents inhibits the concurrent acidification process as well.

ATPase activity and ATP-dependent proton translocation in plasma membrane vesicles of turtle bladder epithelial cells

ATP-induced quenching of fluorescence of acridine orange (a pH probe) or Oxonol V (a potential difference probe) is evoked in turtle bladder membrane vesicles in suspending media of appropriate ionic composition and is insensitive to oligomycin, valinomycin, and ouabain. These effects are ascribed to a membrane-bound, ouabain-resistant ATPase which mediates an active electrogenic proton transport.

Active electrogenic mechanisms for alkali and acid transport in turtle bladders.

Immediately after mounting in the Ussing chamber between choline bicarbonate Ringer solutions devoid of exogenous Na and Cl, the serosal fluid is electronegative to the luminal fluid in bladders from postabsorptive and acidotic turtles; and electropositive in bladders from alkalotic turtles. In bladders from postprandial turtles, the electrical orientation, initially serosal positive, reverses to serosal negative. Serosal additions of 3-isobutyl-1-methylxanthine (IBMX) and adenosine 3',5'-cyclic monophosphate (cAMP) produce no changes in the negative short-circuiting current (Isc) of acidotic turtles but induce large positively-directed increases of Isc in bladders from other turtle groups. With IBMX and cAMP in the (HCO3 + CO2)-rich serosal fluid at pH 7.2 and with luminal pH maintained at 4.0-5.0, the rate at which titratable alkali enters the luminal fluid is electrochemically equal to the positive Isc; and this increased positive Isc is the same as that in the absence of transepithelial gradients. The effects of acetazolamide and 4-acetamido-4-isothiocyanostilbene-2,2'-disulfonic acid on positive and negative Isc are presented. It is concluded that isolated bladders from alkalotic, postprandial or postabsorptive turtles, but not those from acidotic turtles, possess an active electrogenic mechanism for a Na-independent Cl-independent secretion of bicarbonate. This transport process is accelerated by phosphodiesterase inhibitors (IBMX) and cAMP or its eight substituted derivatives.

Electrogenic proton transport in epithelial membranes.

Certain polar epithelial cells have strong transport capacities for protons and can be examined in vitro as part of an intact epithelial preparation. Recent studies in the isolated turtle bladder and other tight urinary epithelial indicate that the apical membranes of the carbonic anhydrase-containing cell population of these tissues contain an electrogenic proton pump which has the characteristics of a proton-translocating ATPase. The translocation of protons is tightly coupled to the energy of ATP hydrolysis. Since the pump translocates protons without coupling to the movement of other ions, it may be regarded as an "ideal" electrogenic pump. The apparent simplicity of the functional properties has led to extensive studies of the characteristics of this pump and of the cellular organization of the secondary acid-base flows in the turtle bladder. Over a rather wide range of electrochemical potential gradients, for protons (delta approximately microH) across the epithelium, the rate of H+ transport is nearly linear with delta approximately microH. The formalisms of equivalent circuit analysis and nonequilibrium thermodynamics have been useful in describing the behavior of the pump, but these approaches have obvious limitations. We have attempted to overcome some of these limitations by developing a more detailed set of assumptions about each of the transport step across the pump complex and to formulate a working model for proton transport in the turtle bladder than can account for several otherwise unexplained experimental results. The model suggests that the real pump is neither a simple electromotive force nor a constant current source. Depending on the conditions, it may behave as one or the other.