H+/ATP stoichiometry of proton pump of turtle urinary bladder

Urinary acidification in the turtle urinary bladder is due to a reversible proton-translocating ATPase. To estimate the H+/ATP stoichiometry of this pump, we measured the delta G'ATP in the epithelial cells and the maximum e.m.f. generated by the pump. The latter is the maximal transepithelial electrochemical gradient for protons placed across the epithelium that is needed to nullify the rate of transport and averaged 179 +/- 7 mV. The delta G'ATP averaged 50.1 kJ/mol. The H+/ATP stoichiometry of these bladders was 2.92 +/- 0.1. In other experiments, the bladders were poisoned by iodoacetate and cyanide and a variable transepithelial electrochemical gradient for protons was placed across them. It was noted that ATP synthesis occurred at a transepithelial electrochemical gradient for protons greater than 120 mV. The delta G'ATP in other bladders treated identically averaged 40.0 kJ/mol, giving a H+/ATP stoichiometry of 3.4 +/- 0.1. We conclude that the H+/ATP stoichiometry of the proton pump of turtle urinary bladder is approximately 3.     

Intracellular phosphate pools show compartmentalization of intracellular pH in turtle urinary bladder

The urinary bladder of the fresh water turtle is capable of acidification and Na transport, in vitro, and it has been extensively used as a model of distal nephron of the kidney. In the course of measuring intracellular pH of stripped turtle bladder mucosa with phosphorus nuclear magnetic resonance, we observed the consistent presence of two inorganic phosphorus resonances under aerobic conditions, indicating the existence of a pH gradient possibly between cytosol and mitochondrion. This pH gradient was collapsed by addition of N₂ and could be restored by reintroduction of oxygen. These observations demonstrate the existence of a spontaneous pH gradient between cytosol and mitochondria of turtle bladder epithelial cells.     

Metabolic pathways coupled to H+ transport in turtle urinary bladder

Summary Active H⁺ transport in the turtle urinary bladder is mediated by an ATPase. Although the source of ATP is usually mitochondrial oxidative phosphorylation, it is possible because of intracellular compartmentalization or cellular heterogeneity that one metabolic pathway exclusively provides ATP to the pump. To examine this we performed several types of experiments. In one, the coupling between the rate of transport and the rate of oxidation of¹⁴C-labeled substrates was studied. We found that there was coupling between H⁺ transport and glucose, butyrate, oleate, and -OH-butyrate oxidation. In another set of experiments we depleted turtle bladders of their endogenous substrates and tested the effect of a number of substrates on the rate of transport. We found that glucose, pyruvate, lactate, actetate, butyrate and -OH butyrate all stimulated H⁺ transport. In a third set of experiments we found no coupling between H⁺ transport and lactate production. Finally, we found that reduction of H⁺ transport by mucosal acidification resulted in an increase in epithelial cell ATP concentrations and a decrease in ADP levels.These results suggest that the H⁺ pump receives its ATP from carbohydrate and fatty acid oxidation. The changes in ATP and ADP levels provide an initial explanation for the coupling of H⁺ transport to the rate of cellular oxidative metabolism.     

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

The turtle urinary bladder acidifies the contents of its lumen by actively transporting protons. H+ secretion by the isolated bladder was measured simultaneously with the rate of 14CO2 evolution from [14C]glucose. The application of an adverse pH gradient resulted in a decline in the rate of H+ secretion (JH) and in the rate of glucose oxidation (JCO2). The changes in JH and JCO2 were linear functions of the pH difference across the membrane. Hence, JH and JCO2 were linearly related to each other. The slope, deltaJH/deltaJCO2 was found to be similar in half-bladders from the same animal but was seen to vary widely in a population of turtles. To investigate the effect of pH gradients on deltaJH/deltaJCO2, two experiments were performed in each of 14 hemibladders. In one, JH and JCO2 were altered by changing the luminal pH. In the other, they were altered by changing the ambient pCO2 while the luminal pH was kept constant. The average slope, deltaJH/deltaJCO2, in the presence of pH gradients was 14.45 eq-mol-1. In the absence of gradients in the same hemibladders it was 14.72, delta = 0.27 +/- 1.46. The results show that H+ transport is organized in such a way that leaks to protons in parallel to the pump are negligible. Analysis of the transport system by use of the Essig-Caplan linear irreversible thermodynamic formalism shows that the system is tightly coupled. The degree of coupling, q, given by that analysis was measured and found to be at or very near the maximum theoretical value.     

Relationship of transient electrical properties to active sodium transport by toad urinary bladder

Summary Application of voltage pulses of 10 mV for periods of 9 sec across toad urinary bladder elicits a rapid deflection in transepithelial current. Frequently, the current decays back towards its baseline value during the course of the polarizing pulse. This transient phenomenon can be induced, or its magnitude increased, by raising the mucosal or serosal Na⁺ concentration. The transient can be abolished by sufficiently hyperpolarizing the tissue (rendering serosa positive to mucosa), by inhibiting transcellular Na⁺ transport with amiloride or ouabain, and by increasing the serosal K⁺ concentration. Vasopressin increases net Na⁺ movement across toad bladder but does not elicit these transients. It is proposed as a working hypothesis for further study that the transient behavior characterized in this study reflects: (1) the partition of Na⁺ between the apical plasma membrane and contiguous fluid layers, (2) the partition of K⁺ between the basolateral plasma membrane and adjacent submucosal fluid layer, and (3) the negative feedback interaction between intracellular Na⁺ activity and Na⁺ permeability of the apical plasma membrane of the transporting cells.     

Adaptation to metabolic alkalosis by the turtle urinary bladder.

We studied the mechanism of adaptation to metabolic alkalosis by the turtle urinary bladder in vitro. Turtles were made alkalotic by administration of oral NaHCO3. Bladders removed from alkalotic turtles had an increased rate of HCO3- secretion in vitro as compared with that of control. H+ secretion, however, was not different, indicating that metabolic alkalosis selectively increases HCO3- secretion. Fluorescence microscopy was used to quantify the carbonic anhydrase cells. The total number of carbonic anhydrase cells was determined by mucosal staining of the bladder with 6-carboxyfluorescein diacetate. The number of HCO3(-)-secreting cells (beta cells) was quantified by mucosal staining with NBD-taurine and the number of H(+)-secreting cells (alpha cells) was calculated from the difference between the two. Metabolic alkalosis significantly increased the total number of 6-carboxyfluorescein positive cells and NBD-taurine-positive cells. The increase in the number of 6-carboxyfluorescein positive cells was totally accounted for by the increase in the NBD-taurine-positive cells without change in the number of alpha cells. If NBD-taurine accurately reflects the number of beta cells, these studies show that the adaptation to metabolic alkalosis is mediated, at least in part, by an increase in the number of HCO3(-)-secreting (beta) cells.     

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--HCO3- exchange, fails to prevent the furosemide-elicited inhibition of alkalinization. These results preclude an absolute requirement of a furosemide-sensitive Cl--HCO3- exchange by these transport processes. The drug also interferes with the CO2-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.     

Effects of luminal pH and luminal HCO3 on CI-HCO3 exchange across turtle urinary bladder

The Cl-HCO3 exchange rate across turtle urinary bladder was studied by measuring the net flux of 36Cl in paired tissues. Serosal bicarbonate concentration and pH were held constant (10 mM and 7.6, respectively) while luminal composition was altered. The rate of net chloride absorption was the same at a luminal pH of 5 or 7 and at luminal HCO-3 concentrations of 0 and 10 mM. These results indicate that the affinity of the exchanger for luminal Cl- is much higher than that for luminal HCO-3 or OH-. Vanadate, an inhibitor of a number of ATPases, had no significant effect on chloride absorption.     

The effect of pH on proton transport by bacteriorhodopsin

The pH-dependence of proton motion during the photocycle was investigated by measuring the photoelectric signals due to charge displacement inside bacteriorhodopsin molecules. Measurements were performed on purple membranes oriented in suspension and the kinetics of flash excited electric and light absorption signals was compared. It was found that in the pH range 4.5–8 the photocycle and the successive proton movements have identical kinetics, and do not depend on pH. In the pH range 8–10 both kinetics change, though differently; the charge motion decouples from the photocycle and the photocycle seems to split up into two parallel paths, the photoelectric signal becomes faster. However, the net proton transfer remains the same as at lower pH values. Above pH ≈ 10, the photocycle behaves differently and cannot be described by the parallel pathway model and the net proton displacement drops. The results are explained by the successive titration of two groups (probably tyrosine) participating in proton translocation.     

Proton transport and membrane shuttling in turtle bladder epithelium

Summary Proton secretion in the urinary bladder of the freshwater turtle is mediated by proton pumps located in the apical membrane of carbonic-anhydrase (CA)-rich cells. It has been proposed that the rate of proton transport is regulated by endocytotic and exocytotic fusion processes which alter the apical membrane area, and hence number of exposed pumps. Three techniques were used to study this process. Analyses of transepithelial impedance provided estimates of transport-associated changes in net membrane area, as well as other electrical parameters. Electron microscopy allowed visualization of the endocytotic vesicles thought to be involved in the process. Finally, uptake of a florescent fluid-phase markerprovided measurements of the rates of endocytosis. We report the following: (i) endocytotic and exocytotic processes occur primarily in the CA-rich cells; (ii) inhibition of proton transport resulting from 0.5mm acetazolamide (AZ) results in a decrease in the apical membrane area of approximately 0.47 cm²/cm² tissue; (iii) the apical membrane specific conductance of the CA-rich cells is approximately 220 S/F, and possibly represents a Cl^– conductance that may function in counter-ion flow; (iv) the decline in transport following AZ is not directly proportional to the decline in apical membrane area, suggesting that changes in pump kinetics are also involved in the regulation of transport; (v) the CA-rich cells exhibit a high rate of constitutive pinocytosis, and hence membrane shuttling, which appears to be independent of the rate of transport; (vi) AZ induces a transient increase in the rates of endocytosis and shuttling; and (vii) the transport-associated changes in apical membrane area may reflect an effect of AZ on a regulated endocytotic pathway which is distinct from the pinocytotic process.     

Thermodynamic analysis of active sodium transport and oxidative metabolism in toad urinary bladder.

Measurements of electrical current and oxygen consumption were carried out concurrently under voltage clamp conditions in 11 toad hemibladders. Inhibition of active transport with amiloride then permitted evaluation of the passive conductance and the rate of basal oxygen consumption Jbr, allowing the simultaneous determination of the rates of active sodium transport JaNa and suprabasal oxygen consumption Jsbr-JaNa and Jabr were linear functions of the electrical potential difference over a range of +/- 80 mV. This allowed the comprehensive application of a linear nonequilibrium thermodynamic formalism, leading to the evaluation of the affinity A (negative free energy) of the metabolic reaction driving transport, all phenomenological coefficients, and the degree of coupling q relating transport to metabolism. Values of A determined by two techniques were A1=56.0 +/- 5.8 and A2=58.2 +/- 6.5 kcal per mole. Values of q determined by two techniques agreed well and were less than 1, indicating incompleteness of coupling, and hence lack of fixed stoichiometry between Na transort and O2 consumption. The affinity and the electromotive force of sodium transport ENa are not closely correlated, reflecting the fact that ENa comprises both kinetic and energetic factors.     

Control of urea transport across toad urinary bladder by vasopressin: Effect of periodate oxidation of the mucosal cell surface

The Journal of Membrane Biology - Oxidation of toad urinary bladder epithelial cell membranes by periodate in the bathing medium altered vasopressin-stimulated transport of urea or of water and...     

Insulin-stimulated sodium transport in toad urinary bladder

Mammalian and teleost insulins increase active sodium transport by the toad urinary bladder at subnanomolar concentrations. This stimulation is evident within 15 min and persists for hours. Porcine proinsulin and a cross-linked derivative of bovine insulin are less effective than porcine insulin in stimulating the short-circuit current (SCC), indicating the specificity appropriate for activation of sodium transport through an insulin receptor. The initial stimulation by insulin of the SCC is not blocked by pretreatment with actinomycin D, puromycin, cycloheximide, or tunicamycin. However, in the presence of any one of these inhibitors the sustained increase in SCC is blocked and the rise is short-lived, lasting only 45 to 90 min. In amphotericin-treated bladders, the addition of insulin did not further stimulate SCC.     

Effect of sodium transport inhibition on active phosphate transport by toad bladder

Summary The relationship between active Na transport (estimated by the short-circuit (SCC)) and active inorganic phosphate (P_i) transport was studied in the toad bladder. When SCC was inhibited by amiloride, ouabaim, or removal of K from the serosal bathing solution, active P_i transport was totally inhibited. When Na was replaced isotonically by choline in either the mucosal bathing solution or both the mucosal and serosal bathing solutions, there was no measurable SCC or active P_i transport. These experiments are compatible with the hypothesis that active P_i transport occurs only in the presence of active Na transport.