Localization and regulation of acid-base secretory currents from individual epithelial cells.

The turtle urinary bladder is composed of different epithelial cell types that are suspected to separately produce electrogenic acid and alkali excretion. We measured the electrical currents produced by individual cells, scanning a two-dimensional vibrating probe over the luminal surface of the bladder. Acidification (outward current) was produced by the type of epithelial cell rich in carbonic anhydrase (CA cells). The measured currents of these cells quantitatively accounted for the total epithelial acidification current. When alkali secretion was induced by adenosine 3',5'-cyclic monophosphate and acidification was inhibited (by luminal pH 4), we measured inward currents localized to a small number of epithelial cells in four bladders but found no localization in the other seven treated bladders. When alkali secretion was localized and induced without inhibiting acidification, we found both cells producing inward current and cells producing outward current, which demonstrated that the two transport functions can occur simultaneously. We conclude that net acid-base secretion can be determined by regulating the transport rates of separate cells.

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.

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.

Evidence for separate cellular origins of sodium and acid-base transport in the turtle bladder.

Transmembrane electrical parameters of the epithelial cells in short-circuited turtle bladders were measured to determine whether those cells participating in Na reabsorption also participate in electrogenic transepithelial acidification and alkalinization. Amiloride-induced increases in intracellular potential (Vsca), apical fractional resistance (FRa), and concomitant decreases in short-circuit current (Isc) denote the participation of the impaled cells in Na reabsorption. In bladders from postabsorptive turtles, amiloride increased Vsca by -45 mV, increased FRa by 37%, and decreased Isc from 36 to -10 microA/cm2. In bladders from NaHCO3-loaded turtles, amiloride increased Vsca by -21 mV, FRa by 21%, and decreased Isc from 22 to 0 microA/cm2. Neither the subsequent inhibition of the negative acidification current in postabsorptive bladders, nor stimulation of positive alkalinization current in bladders from NaHCO3-loaded turtles was associated with any transmembrane electrical change that could be attributed to changes in those transport processes. It is concluded that the electrogenic luminal acidification and alkalinization processes of the turtle bladder are not produced by, or electrically coupled to, those cells that are involved in Na reabsorption.

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.

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.

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.