There is now convincing evidence that in addition to the vacuolar-type H(+)-ATPase, a gastric-type H+/K(+)-ATPase participates in acidification by the distal nephron. To determine whether a similar pump exists in the turtle bladder, we examined the dependence of acid secretion on mucosal K+, and the effects of supposedly specific inhibitors of the gastric H+/K(+)-ATPase, omeprazole and SCH 28080. In CO2-stimulated bladders both drugs produced dose-dependent inhibition of electrogenic H+ secretion measured as the reverse short-circuit current (RSCC). At the highest concentrations tested, H+ secretion decreased 45 +/- 16% with mucosal and 20 +/- 7% with serosal omeprazole (P < 0.01). SCH 28080 at 400 microM produced essentially complete inhibition of H+ secretion with either mucosal or serosal application. When H+ secretion was purposefully inhibited by DIDS or an adverse mucosal pH gradient, SCH 28080 had no effect on RSCC. Removing mucosal K+ (measured K+ < 50 microM), with or without mucosal barium, had no effect on RSCC. The inhibition of RSCC by omeprazole was reversed by mercaptoethanol. Finally, HCO3 secretion, as measured by either RSCC or pH-stat titration, increased significantly in response to 400 microM SCH 28080. The results demonstrate that these compounds inhibit acid secretion by the turtle bladder but stimulate the secretion of base. In view of the total independence of acid secretion on potassium, it is unlikely that any of the bladder's acid secretion is mediated by an H+/K(+)-ATPase. The most reasonable interpretation of the data is that omeprazole and SCH 28080, previously thought to be specific inhibitors of the H+/K(+)-ATPase, also inhibit the vacuolar H(+)-ATPase of the turtle bladder. The results also indicate that HCO3 secretion by the bladder employ a different mechanism of H+ transport than is used for acid secretion; there is no simple reversal of polarity in the acid- versus base-secreting cells.

The effects of phorbol myristate acetate (PMA) on acid secretion by the turtle urinary bladder were examined to evaluate the importance of protein phosphorylation in modulating the distal acidification system. In HCO3-free PO4 buffer 0.2 mM mucosal PMA inhibited reverse short-circuit current (RSCC) by 42%. The inhibition of RSCC was dose dependent, and RSCC approached zero at high concentrations of PMA. PMA also inhibited acid secretion measured titrimetrically but had no effect on RSCC from bladders in which proton secretion was selectively inhibited by an adverse pH gradient or serosal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The inhibition by PMA was duplicated by 1-oleoyl-2-acetyl-rac-glycerol, but not by an inactive phorbol ester. The PMA inhibition was much more potent with mucosal compared with serosal application. The PMA inhibition caused a significant 0.15 pH unit reduction of carbonic anhydrase (CA) cell cytoplasmic pH and a change in the CA cell morphology. Unlike the inhibition of proton transport induced by acetazolamide, the PMA inhibition was neither reversed nor prevented by sodium azide. We conclude that PMA decreases basal acid secretion and blocks the stimulatory effects of CO2 by an azide-insensitive mechanism distinct from that of acetazolamide. In view of recent findings that PMA stimulates HCO3 secretion by the turtle bladder, these results suggest that kinase C-mediated protein phosphorylation may be a central event in the transition from the secretion of acid to the secretion of base.

Annals of the New York Academy of SciencesVolume 574, Issue 1 p. 489-490 Phorbol Myristate Acetate Controls the Mode of Add/Base Transport in the Turtle Urinary Bladder MARK GRABER, MARK GRABER VAMC Northport, New York 11768Search for more papers by this authorPHILIP DEVINE, PHILIP DEVINE VAMC Northport, New York 11768Search for more papers by this authorDENISE COACHMAN, DENISE COACHMAN VAMC Northport, New York 11768Search for more papers by this authorTROY DIXON, TROY DIXON SUNY at Stony Brook Stony Brook, New York 11794Search for more papers by this author MARK GRABER, MARK GRABER VAMC Northport, New York 11768Search for more papers by this authorPHILIP DEVINE, PHILIP DEVINE VAMC Northport, New York 11768Search for more papers by this authorDENISE COACHMAN, DENISE COACHMAN VAMC Northport, New York 11768Search for more papers by this authorTROY DIXON, TROY DIXON SUNY at Stony Brook Stony Brook, New York 11794Search for more papers by this author First published: December 1989 https://doi.org/10.1111/j.1749-6632.1989.tb25189.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume574, Issue1Bicarbonate, Chloride, and Proton Transport SystemsDecember 1989Pages 489-490 RelatedInformation

Acetazolamide (ACZL) inhibits luminal acidification by the turtle urinary bladder, a process thought to be mediated by the drug's ability to inhibit carbonic anhydrase (CA) and thus elevate cell pH. To test the hypothesis that these transport changes are actually mediated by changes of cell pH, we measured this parameter in single, identifiable mucosal cells using 4-methylumbelliferone and fluorescence microscopy. In control bladders 5 X 10(-4) M ACZL inhibited proton transport by 80 +/- 6%, and alkalinized cell pH, especially in a subpopulation of CA cells. A much larger cell alkalinization was induced by serosal HCO3- but proton transport fell only 30 +/- 7%. When cell pH was clamped at approximately 7.0 using 50 mM dimethyloxazolidinedione, or when cell pH was acidified using 7.5 mM propionate, transport rates still declined by 74 +/- 2, and 100 +/- 12%, respectively, in response to ACZL. In propionate-acidified bladders, 1 mM sodium azide blocked the inhibition of transport seen with 5 X 10(-4) M ACZL and reversed the inhibition with 10(-5) M ACZL. The apical endocytosis rate was increased by ACZL in normal and propionate-acidified bladders, but was not stimulated by alkalinizing the cell with NH4Cl. We conclude that ACZL can induce cellular alkalinization in this tissue, but that this pH change is not required for the inhibition of transport, or the ACZL-associated stimulation of endocytosis. The drug's ability to inhibit acidification appears to be the result of an azide-sensitive mechanism that has yet to be defined.

Mucosal cells of the toad and turtle urinary bladder have cell membranes that exclude charged fluorescent dyes. We describe a simple and effective method of transiently permeabilizing the apical membranes of mucosal cells in situ by applying mild mechanical stress to the bladder's mucosal face. The technique produces clusters of individually distinguishable granular cells, which are loaded with and retain membrane-impermeant fluoroprobes and macromolecules. Carbonic anhydrase-rich cells are resistant to the permeabilization. The apical membranes regain functional integrity after permeabilization, as indicated by a return of transepithelial electrical resistance, an inability to load cells except immediately after the stress, and the observation that loaded cells behave identically to normal cells in regulating cell pH. With the use of this technique, BCECF and fura-2 were loaded into granular cells and used successfully to follow cell pH and cell calcium. In granular cells loaded with these dyes or Lucifer Yellow, there was no detectable spread of dye into adjacent cells. This lack of dye coupling was confirmed by use of conventional iontophoresis of dye into normal granular cells. Electrical coupling was also undetectable between granular cells separated by distances less than 30 micron. We conclude that none of the mature cells of the bladder surface are directly coupled.