In vivo environmental temperature and the in vitro pattern of luminal acidification in turtle bladders. Evidence for HCO3 ion reabsorption.

In this study, it is shown how to transfer tared aliquots of (HCO3 + CO2)-containing luminal fluids directly into the mercury-sealed chamber of a modified Van Slyke apparatus and how to obtain direct as well as indirect manometric determinations of dissolved CO2 ([CO2]f) in each aliquot of such fluids. It is next shown that the pattern of in vitro luminal acidification in an isolated turtle bladder sac depends upon the prior in vivo ambient temperature to which the donor turtle had become adapted. Under in vivo conditions, the food intake, physical activity, and acid excretion of 32 degrees C-adapted turtles are greater than those of 21 degrees C or 26 degrees C-adapted turtles. Under in vitro conditions of incubating isolated bladder sacs (from 21, 26, and 32 degrees C turtles) in (HCO3 + CO2)-containing Ringer media at a single temperature (21 degrees C), the patterns of luminal acidification are as follows: (a) The rate of depletion of luminal [HCO3] is greatest in bladders from the 32 degrees C-adapted turtles. (b) Concomitant decreases in luminal [CO2]f, [HCO3], and pH (the 'CO2-decreasing patterns' of luminal acidification) develop in all bladders from 32 degrees C turtles, in half of those from 26 degrees C turtles, but in less than one-fifth of those from 21 degrees C-adapted turtles: and (c) a CO2-increasing pattern of luminal acidification is found in most of the bladders from 21 degrees C-adapted turtles. A postulated bicarbonate ion-reabsorbing pump is consistent with all of these patterns of luminal acidification.

Alkali secretion in the turtle bladder: up-regulation by the phospho-inositol cascade and inhibition by diphenylamine carboxylate (DPC).

The presently reported studies of factors involved in the regulation of acid-base excretory processes in isolated turtle urinary bladders have yielded the following data. (i) There exists in alkalotic and euhydric turtle bladders (but not in acidotic turtle bladders) a mechanism which drives a primary active electrogenic secretion of alkali; and the electrogenic output of this mechanism is up-regulated by exogenously added PDE inhibitors, such as theophylline or IBMX. (ii) VIP or cAMP up-regulates this alkali secretion in the presence of IBMX. (iii) Carbachol also initiates alkali secretion. But this action, independent of PDE activity, is probably associated with the phospho-inositol reaction cascade. (iv) Finally, low concentrations of mucosal DPC decreases the carbachol supported, but not the cAMP-supported alkali secretion.

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.

Vasoactive intestinal peptide stimulates alkali excretion in turtle urinary bladder.

The turtle urinary bladder possesses an active transport mechanism for the electrogenic secretion of alkali. This process is independent of exogenous Cl and Na, induced by cyclic AMP (cAMP), and potentiated in bladders from NaHCO3-loaded (alkalotic) turtles. In the present study, it is shown that the serosal addition of vasoactive intestinal peptide (VIP) induces rapidly developing parallel increases in alkali secretion and in the short-circuiting current carried by this secretion. The VIP-induced increment in alkali secretion is greater in the presence than in the absence of an exogenously added phosphodiesterase inhibitor. Additions of a cAMP analog subsequent to the VIP-induced alkali secretion fail to induce any further increase in alkalinization. These results provide evidence for the action of VIP as a hormonal up regulator of alkali excretion in the turtle urinary bladder.

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.

Bicarbonate and chloride transport in relation to the acidification or alkalinization of the urine.

Although the electrogenicity of the active reabsorption of sodium and bicarbonate (or secretion of protons) has been well-established in the short-circuited turtle bladder preparation, the nature of the active reabsorption of chloride and secretion of bicarbonate has been controversial. These processes have been ascribed to the separate actions of two discrete electrogenic pumps or to the single action of a metabolically driven electroneutral anion exchange mechanism. The present report deals with these transport processes per se; with the relations among serosal bicarbonate,glucose, and the reabsorption of Na and Cl; and finally with the application of the Heinz model for inversion of an active transport as a tentative single mechanism for the alkalinization and acidification of the urine.

Localization and characterization of transport-related elements in the plasma membrane of turtle bladder epithelial cells.

A mixed membrane preparation obtained from turtle bladder epithelial cells contains (Na+ + K+)-ATPase, adenylate cyclase and protein kinase, which interact with ouabain, norepinephrine and cyclic AMP, respectively. When such a preparation is obtained from bladders which had been preexposed to serosal fluids containing the tritiated form of 4,4'-diisothiocyano-2,2'-disulfonic stilbene, the subsequently isolated membrane proteins are enriched in tritium as well as in the afore-mentioned enzymes, none of which is inhibited. Free-flow electrophoresis separates the mixed membrane preparation into two distinguishable groups: one, construed as apical membranes, is enriched in norepinephrine-sensitive adenylate cyclase and cyclic AMP-sensitive protein kinase; the other, construed as basal-lateral membranes, is enriched in ouabain-sensitive ATPase and 4,4'-diisothiocyano-2,2'-disulfonic stilbene-binding proteins. The physiological counterparts of these enzymatically defined membrane markers are the mucosal sidedness of the transport effects of norepinephrine and cyclic AMP derivatives and the serosal sidedness of the transport effects of ouabain and disulfonic stilbenes in the intact turtle bladder. The discreteness and ion selectivity of each membrane-bound, transport-related element are discussed in relation to the corresponding characteristics of each transport process in vivo; the possibility of regulation of anion transport by adenylate cyclase-protein kinase system is also discussed.

Effects of pseudomonas toxin A, diphtheria toxin, and cholera toxin on electrical characteristics of turtle bladder.

Rapidly developing changes in the short-circuiting current (Isc), conductance (G), and potential (PD) of turtle bladders in Na-rich or Na-free media are seen after the mucosal addition, at 10 nM, of each of three toxins that contain ADP-ribosylation activity: Pseudomonas aeruginosa toxin A, diphtheria toxin, and cholera toxin. Toxin A irreversibility decreased the Isc, PD, and G of bladders in Na-rich media and the Isc and PD of bladders in Na-free media. Diphtheria or cholera toxin reversibly increased Isc and PD (not G), but only in Na-free media. The effects of toxin A in the turtle bladder, like those in other host cell systems, were eliminated by preexposure of this toxin to heat, specific antitoxin, or dithiothreitol and urea. Because exposure to this last condition increases the ADP-ribosylation activity of toxin A, it is suggested that the proenzyme is the required transport-inhibiting form of toxin A. The effects of all three toxins occurred rapidly, possibly before any of the possible intracellular ADP-ribosylation reactions are initiated. Whereas a recognition binding of toxin of toxin to receptors on the apical membrane completely accounts for the reversible effects of diphtheria or cholera toxin, this and additional toxin-membrane interactions (e.g., translocation) are needed to account for the irreversible effects of toxin A.

The effects of a disulphonic stilbene on chloride and bicarbonate transport in the turtle bladder.

1. In turtle bladders bathed by Na-free media containing Cl and HCO3, the mucosa is electropositive to the serosa. Serosally applied 4-acetamido-4'-isothiocyano-2,2'-disulphonic stilbene (SITS) reduces the p.d. and Isc by 100%, and reduces the rate of net Cl reabsorption in some bladders but not in others. 2. In the absence of exogenous HCO2 (Cl present), SITS reduces the p.d. and Isc by 100%, and reduces the rate of mucosal acidification by 80%; net Cl reabsorption is not detectable under these HCO3-free bathing conditions. 3. In the absence of exogenous Cl (HCO3, present), the mucosa is also electropositive to the serosa and serosally applied SITS reverses the orientation of the p.d. and Isc so that the mucosa becomes electronegative to the serosa. 4. This constitutes sufficient evidence for the active, electrogenic secretion of an anion, probably HCO3. These data are explained by an analogue of discrete, electrogenic pumps and paths for the reabsorption of Na, Cl, and HCO3, and the secretion of HCO3.

Amiloride-induced stimulation of HCO-3 reabsorption in turtle bladder.

Amiloride in the mucosal fluid (at concentrations of 5 . 10(-6) M to 10(-4) M) reversibly stimulates the HCO-3-dependent moiety of the short-circuiting current (Isc) in ouabain-treated turtle bladders bathed by Na-free Ringer solutions with or without Cl-. This effect is uniquely different from the known inhibitory effect of this agent on Na+ transport. Thus, any comprehensive hypothesis on the action of amiloride over a wide dosage-response range should take into account its effect on HCO-3 transport.

Effects of 4-acetamido-4'-isothiocyano-2,2-disulfonic stilbene on ion transport in turtle bladders.

The disulfonic stilbene (4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene) is found to be more potent than acetazolamide as an anion transport inhibitor in the turtle bladder, but less potent than acetazolamide as a carbonic anhydrase inhibitor. The anion-dependent (HCO-3,Cl-) moiety of the short-circuiting current is eliminated by 4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene, but only after its addition to the serosal bathing fluid. Whereas 4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene has no effect on Na+ transport across the bladder, it is more potent than ouabain as an inhibitor of microsomal (Na+ + K+)-ATPase of both turtle bladder and eel electric organ.

Effects of ouabain and amiloride on Na pathways in turtle bladders.

In a Na-rich bathing system, addition of amiloride to the mucosal fluid of turtle bladders produces decreased in the transepithelial potential difference (PD), short-circuiting current (I-sc), and conductance. Removal of amiloride results in complete reversal of these changes; and this reversibility is incomplete in amiloride-blocked bladders exposed to ouabain. In a Na-free bathing system, step increased in mucosal [Na] evoke rapid initial spikes in PD, Isc, and conductance, the magnitudes of which are independent of prior ouabain treatment. After these spikes, PD and Isc in the ouabain-treated bladder rapidly decay, while conductance remains unchanged and high. This unchanging conductance, plus the fact that ouabain inhibits half the microsomal (Na plus K). ATPase of this tissue within 1 min, suggests that ouabain inhibits Na pumping without changing tissue conductance. The delayed decrease in conductance (beginning 30 min after ouabain addition), a nonspecific and secondary effect of ouabain, is due to a concomitant collapse of the intercellular channels.