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.     

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/CO₂-rich, Cl^?-free Na⁺ media and treated with ouabain and amiloride exhibit a transepithelial potential serosa electronegative to mucosa and a short-circuit current ($\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$) 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 $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ and decreases tissue resistance. The resulting positive $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ resembles that previously observed in response to isobutylmethylxanthine (IBMX) and cAMP analogs. Reversal of the $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ is enhanced in bladders from severely alkalotic turtles. In contrast, in severely acidotic turtles, ionophore A23187 decreases, but does not reverse, the $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$. 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.     

Barium inhibition of sodium ion transport in toad bladder

The effect of Ba²⁺ on Na⁺ transport and electrical characteristics of toad bladder was determined from change produced in short circuit current (I_sc, epithelial, apical and basal-lateral potentials (ψ_t, ψ_a, ψ_b), epithelial and membrane resistances (R_t, R_a, R_b) and shunt resistance (R_s). Mucosal Ba²⁺ had no effect. Serosal Ba²⁺ reduced I_sc, ψ_t, ψ_a, and ψ_b, but had no effect on R_t, R_a, R_b and R_s. Minimal effective Ba²⁺ concentration was 5 · 10^?5 M. The phenomenon was reversed by Ba²⁺ removal, but not by 86 mM serosal K⁺. Ba²⁺ inhibition of I_sc did not impair the response to vasopressin which was quantitatively the same as controls. ψ_a with Ba²⁺ equalled ψ_b. After Ba²⁺ inhibition, ouabain produced no further decrease in ψ_t and I_sc. Ba²⁺ exposure after ouabain did not decrease ψ_t and I_sc. The results suggest that Ba²⁺ inhibits the basal-lateral electrogenic Na⁺ pump.     

Control of active proton transport in turtle urinary bladder by cell pH

The rate of active H+ secretion (JH) across the luminal cell membrane of the turtle bladder decreases linearly with the chemical (delta pH) or electrical potential gradient (delta psi) against which secretion occurs. To examine the control of JH from the cell side of the pump, acid-base changes were imposed on the cellular compartment by increasing serosal[HCO3-] at constant PCO2 or by varying PCO2 at constant [HCO3-]. When serosal [HCO3-] was increased from 0 to 60 mM, cell [H+] decreased, as estimated by the 5,5-dimethyloxazoladine-2,4- dione method. JH was a saturable function of cell [H+], with an apparent Km of 25 nM. When PCO2 was varied between 1 and 20% at various serosal Km of 25 nM. When PCO2 was varied between 1 and 20% at various serosal [HCO3-], the PCO2 required to reach a maximal JH increased with [HCO3-] so that JH was a function of cell [H+] rather than of cell [HCO3-] or CO2. The proton pump was controlled asymmetrically with respect to the pH component of the electrochemical potential for protons, microH. On the cell side of the pump, a delta pH of < 1 U was required to vary JH between maximal and zero values, whereas on the luminal side a delta pH of 3 U was required. Cell [H+] regulates JH by determining the availability of H+ to the pump in a relationship resembling Michaelis-Menten kinetics. Increasing luminal [H+] generates an energy barrier at a luminal pH near 4.4 that equals the free energy (per H+ translocated) of the metabolic driving reaction.     

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 HCO3-/CO2-rich, Cl--free Na+ media and treated with ouabain and amiloride exhibit a transepithelial potential serosa electronegative to mucosa and a short-circuit current (Isc) which is a measure of the net luminal acidification rate. Addition of calcium ionophore A23187 (10 microM) to the mucosal side of the epithelium rapidly reverses the direction of the potential difference and Isc and decreases tissue resistance. The resulting positive Isc resembles that previously observed in response to isobutylmethylxanthine (IBMX) and cAMP analogs. Reversal of the Isc is enhanced in bladders from severely alkalotic turtles. In contrast, in severely acidotic turtles, ionophore A23187 decreases, but does not reverse, the Isc. 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.     

Vanadate inhibition of ATP-dependent H+ transport in membrane vesicles from turtle bladder epithelial cells

The ATP-dependent proton transport into vesicles of a mixed membrane fraction obtained from turtle bladder epithelial cells consists of at least two kinetically defined moieties: one, which is maximally inhibited by 25% with nanomolar levels of vanadate, but not inhibited at all with equimolar levels of N-ethylmaleimide, and another, which is maximally inhibited by 70% with micromolar levels of N-ethylmaleimide and by 25% with equimolar levels of vanadate. In contrast to the transport function, the associated enzymatic function (the ouabain-resistant ATPase activity) in these membranes, not inhibited by nanomolar levels of vanadate or N-ethylmaleimide, is maximally inhibited by 40% with micromolar levels of vanadate and by 13% with equimolar levels of N-ethylmaleimide. Independent of these kinetic differences between the enzyme and the transport functions, membranes containing the N-ethylmaleimide-sensitive proton transport function are electrophoretically separable from those containing the vanadate-sensitive transport function. For example, the kinetically defined, vanadate-sensitive proton transport function is recovered exclusively and kinetically identified in one of four electrophoretic membrane fractions, EF-II; while the N-ethylmaleimide-sensitive function is recovered in EF-III as well as in EF-II. Membranes of EF-IV, maximally enriched in ouabain-resistant ATPase activity, possess no proton transport function at all, even in the absence of N-ethylmaleimide or vanadate. Additional data under in vivo as well as under in vitro conditions are required to prove that the vanadate-sensitive proton transport in these vesicles is an in vitro manifestation of the mechanism responsible for generating the vanadate-sensitive luminal acidification process under in vivo conditions in the intact turtle bladder.     

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 (NH4Cl-loaded); and alkalotic turtles (NaHCO3-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, (HCO3- + CO2)-containing media increases the negative short-circuiting current (Isc). In acidotic bladders, which are poised for acidification but not Cl- reabsorption, mucosal Cl- addition has no effect on this Isc whereas serosal Cl- addition increases the negative Isc 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 Isc). In these bladders, serosal Cl- addition is without effect while mucosal Cl- addition produces transient changes in this positive Isc. It is found that these results can be replicated by a model of the turtle bladder in which transmembrane Cl- and HCO3- conductive and exchange paths mediate transepithelial acidification, alkalinization and Cl- reabsorption.     

Effect of 3-isobutyl-1-methylxanthine on HCO3− transport in turtle bladder: Evidence for electrogenic HCO3− secretion

Ouabain-treated turtle bladders bathed on both surfaces by identical HCO₃^?/CO₂-containing, Cl^?-free Na⁺ media exhibit a short-circuit current (I_sc) and transepithelial potential (p.d.) serosa electronegative to mucosa. Addition of 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cyclic nucleotide phosphodiesterase, rapidly reverses the direction of the I_sc and p.d.. The IBMX-induced reversal of I_sc and p.d. is (1) dependent on the presence of HCO₃^? (and CO₂) in the serosal bathing fluid, (2) independent of Na⁺ and other ions in the bathing medium, (3) decreased by inhibitors of carbonic anhydrase or oxidative metabolism, (4) increased by the serosal addition of cyclic AMP or the disulfonic stilbene, SITS. The results constitute evidence that the reversed I_sc elicited by IBMX represents electrogenic secretion of HCO₃^?.     

Effects of valinomycin on vanadate-sensitive and vanadate-resistant H+ transport in vesicles from turtle bladder epithelium: evidence for a K+/H+ exchanger

The vanadate-sensitive component of the ATP-dependent H+ gradient formed in isolated vesicles from a urinary epithelium was abolished by valinomycin omission. This suggests that vanadate-sensitive H+ transport has an absolute requirement for intravesicular K+ and that the transport may be due to a K+/H+ exchanger. Sensitivity to the inhibitor SCH28080 supports this conclusion. On the other hand, valinomycin affects the initial velocity of vanadate-resistant transport without altering its maximum gradient. This is consistent with the development of a membrane potential consequent to electrogenic uniport H+ transport.     

The effect of catecholamines on ion transport in the toad bladder

Noradrenalin (8 · 10⁻⁶ M) and adrenalin (6 · 10⁻⁶ and 6 · 10⁻⁷ M) were found to cause marked stimulation of short-circuit current (S.C.C.) in isolated toad bladder, but isoprenalin (8 · 10⁻⁷ M) was found to be without effect. The percentage rise in S.C.C. due to noradrenalin was found to be inversely proportional to the initial S.C.C. or total conductance of the bladder. Again in the case of noradrenalin the rise in S.C.C. was almost completely abolished by α-adrenergic blockade but not by β-blockade. This rise in S.C.C. was found not to be significantly different from the rise in net Na⁺ flux. Bidirectional Cl⁻ fluxes were estimated using ⁸²Br as a companion radionuclide to ³⁶Cl. No significant net Cl⁻ flux was apparent, either before or after addition of any of the three catecholamines tested. However, in some cases the unidirectional Cl⁻ fluxes rose markedly following addition of noradrenalin or of adrenalin and this change was not reflected in a change in total conductance. This anomaly was noted to occur in bladders whose initial conductance was of the order of 0.5 kΩ⁻¹ · cm⁻² or greater. The evidence presented suggests that two actions of catecholamines on ion transport in toad bladder are (a) to increase Na⁺ transport via stimulation of α-adrenergic sites and (b) at the concentrations tested to cause an increase in passive Cl permeability in bladders whose initial conductance is high.     

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.     

Cooperativity in the inhibition of P-glycoprotein-mediated daunorubicin transport: evidence for half-of-the-sites reactivity

P-Glycoprotein (Pgp) is an important transport enzyme composed of two homologous domains and transports a wide range of structurally diverse xenobiotics from the cell. Recent studies have indicated that allosteric interactions occur between the nucleotide binding domains and between the substrate binding domains of the two halves, but the extent of this interaction as well as the means by which the enzyme can transport such a wide variety of substrates has not been elucidated. Herein, the Pgp-mediated transport of a marker substrate, daunorubicin (DNR), out of viable cells was examined in the presence of a variety of other known substrates of Pgp. For most of the typical Pgp substrates examined, the relationship between inhibition of DNR efflux and competing substrate concentration was sigmoidal and therefore not a simple mutually exclusive competitive inhibition of transport. The Hill coefficient ranged from about 3 to 5 for the inhibition of transport of DNR. This negative cooperativity in combination with recent evidence, including several examples of noncompetitive inhibition between the homologous halves of Pgp, indicates a "half-of-the-sites" reactivity. Our data support the mechanistic proposal that substrate binding at one putative transport binding site precludes activity at another unequal site; many of the substrates examined exert a negative allosteric effect on the other transport site (and vice versa). A half-of-the-sites reactivity model would account for many of these observations and may be critical to the efficiency of Pgp substrate transport of a broad spectrum of compounds.     

Chloride-bicarbonate exchange in the urinary bladder of the turtle. Independence from sodium ion

The rates of Cl^? absorption and HCO^?₃ secretion were not different in turtle urinary bladders bathed in Na⁺-containing and solutions.These results in turtle bladder are inconsistent with Na⁺-anion cotransport but can be accounted for by a Cl^?/HCO^?₃ exchange system.