BAFILOMYCIN A1 AT NANOMOLAR CONCENTRATIONS SATURABLY INHIBITS A PORTION OF TURTLE BLADDER ACIDIFICATION CURRENT

SUMMARY An earlier report indicated that acid secretion in turtle urinary bladder is driven by an unusual vacuolar H+-ATPase and that the ATPase accounts for essentially all acid secreted. These results, however, are difficult to reconcile with the acid transporters currently ascribed to the renal collecting duct. Here, we re-examine the effect of bafilomycin A1, an inhibitor of vacuolar (V-type) H+-ATPases, on acid secretion by intact isolated bladders from Pseudemys scriptaturtles. Serosal-side bafilomycin had no effect on the transepithelial acidification current (AC). In the mucosal solution, bafilomycin inhibited the AC, with inhibition developing over the range 0.1-10 nmol l-1, with a sigmoidal dose—response curve, and an IC50 of 0.47 nmol l-1. At saturation, approximately 70 % of H+ secretion was inhibited. The remaining 30 % could be abolished by 30 μmol l-1 Sch-28080, which is a level that in other systems is known to inhibit H+/K+-ATPase transport activity specifically and essentially completely. When the order of addition was reversed (Sch-28080 first), there was no change in the magnitude of the effect produced by either inhibitor, and the two together again eliminated the AC. The data indicate that baseline acid secretion in intact bladders is due (i) in part to a highly bafilomycin-sensitive process, with sensitivity typical of vacuolar H+ ATPases; and (ii) in part to a more bafilomycin-resistant process that is sensitive to Sch-28080.

Effects of A-23187 and verapamil on the active transport enzymes in turtle bladder epithelial cells

These experiments were designed to examine the effects of A-23187 (5 x 10(-4) M) and verapamil (100 microM) on membrane transport, 45Ca fluxes, and adenosine-triphosphatase (ATPase) activities in turtle bladder. In the intact membrane, the calcium ionophore decreased proton secretion and sodium transport [short-circuit current (SCC)] to approximately the same degree (by approximately 55% at 30 min). During the same period of time, verapamil decreased SCC (by approximately 58%), but proton secretion was unaffected. The turtle bladder membrane is composed predominantly of two cell types: 1) the mitochondrial-rich cells (MR cells) thought to be involved in proton (and bicarbonate) secretion containing significant H(+)-ATPase and Ca(2+)-ATPase and 2) the granular cells (G cells), postulated important in sodium reabsorption, having abundant Na(+)-K(+)-ATPase. That Na(+)-K(+)-ATPase activity was unchanged by either a calcium ionophore or a calcium channel blocker suggests that the decrease in SCC noted in the intact membrane is not directly mediated by changes in the sodium "pump." The decrease of H(+)-ATPase in MR cells, which resulted after the A-23187, suggests that it probably exerts a direct action on the proton pump, which decreases hydrogen ion secretion. The increase in ATP-dependent 45Ca transport seen after the ionophore (or the decrease in ATP-independent 45Ca transport after verapamil) most likely reflects increased (or decreased) Ca2+ availability within the cytosol, and the high (or low) cell calcium could decrease the SCC. These results thus suggest that cytosolic Ca2+ reciprocally sets, by different mechanisms, the rate of proton secretion in MR cells and the sodium reabsorption in G cells.

Sch-28080 inhibits bafilomycin-sensitive H+ secretion in turtle bladder independently of luminal [K+]

To explore the possible contribution of an H-K-adenosine-triphosphatase (H-K-ATPase) to H+ secretion (JH) in the isolated turtle bladder, we measured electrogenic JH (JeH) as short-circuit current and total JH (JTH) by pH stat titration in the presence of ouabain at different ambient K+ concentration ([K+]) and during luminal addition of a known gastric H-K-ATPase inhibitor, Schering (Sch)-28080. JH was not reduced by decreasing ambient [K+] to undetectable or very low levels (< 0.05 mM by atomic absorption) and luminal BaCl2 addition to further reduce local [K+] at the apical membrane. These K(+)-removal studies indicate that H+ transport is not coupled to countertransport of K+. JTH did not exceed JeH at any point: in K(+)-free solutions JTH was 0.73 +/- 0.05, and JeH was 0.95 +/- 0.08 mumol/h; in standard (3.5 mM) K+ solutions JTH was 0.72 +/- 0.05 and JeH 0.98 +/- 0.06 mumol/h; in high (118 mM) K+ solutions JTH was 0.65 +/- 0.07 and JeH 0.94 +/- 0.08 mumol/h. Sch-28080 caused a rapid inhibition of JH, with similar half-maximal inhibitory concentrations (IC50) in K(+)-free, standard [K+], and high [K+] solutions. Bafilomycin inhibited JeH and JTH with an IC50 of approximately 100 nM. The observed non-potassium-competitive inhibition of JH by Sch-28080 and the bafilomycin sensitivity distinguish the H-ATPase of the turtle bladder from the gastric H-K-ATPase. The rapidity of the inhibition by Sch-28080 suggests that it acts at an accessible luminal site of the ATPase.

Carbonic anhydrase in turtle bladder mitochondrial-rich luminal and subluminal cells

Bladders from March-April turtles were processed for carbonic anhydrase (CA) cytochemically using the method of D.A. Riley, S. Ellis, and J. Bain (Neuroscience 13: 189, 1984). CA-positive cells comprised 11.1 +/- 0.7% of mucosal epithelial cells. Microplicated (MP) cells comprised 47.2 +/- 1.8% of CA-positive cells and displayed at least two distinct staining patterns: the first was characterized by reaction product that filled the luminal one-third, including the terminal web and microplicae. These cells possessed extensive microplicae, a morphological feature of ongoing H+ secretion. The second was characterized by reaction product distributed throughout cells, excluding the terminal web and microplicae, with greatest intensity in the luminal one-third below the terminal web. These cells possessed flattened microplicae, a morphological feature of diminished H+ secretion. Microvillated (MV) cells comprised 6.0 +/- 1.0% of CA-reactive cells. The basal layer was occupied by 46.8 +/- 1.7% of CA-positive cells, which were termed subluminal (SL) cells. SL cells were mitochondrial rich and did not contact the lumen. Extracellular CA staining was common between the lateral margins of contiguous mitochondrial-rich or non-mitochondrial-rich cells.

Carbonic anhydrase and proton secretion in turtle bladder mitochondrial-rich cells

Bladders from actively feeding turtles were processed for carbonic anhydrase (CA) cytochemically. CA-positive cells were identified as microplicated (MP) cells, microvillated (MV) cells, and subluminal (SL) cells. After acute enhancement of H+ secretion with 5% CO2, MP cells displayed extensive microplicae and a reduced density of apical subplasmalemmal vesicles, and they were CA reactive throughout a large part of the cytoplasm including the microplicae. After acute inhibition of H+ secretion with a pH 4.5 mucosal bath, CA staining was excluded from the microplicae and apical subplasmalemmal region of most MP cells, whereas microplicae varied from extensive to reduced, and subapical vesicle density remained elevated. MV cells were characterized by basolateral staining with sparing of the MV and apical subplasmalemmal region in all settings except 1) after 5% CO2 and 2) when MV cells were found in areas in which MP cells were stained to the lumen. These results indicate that CA is active at the site of H+ secretion in MP cells and is correlated with the acute acid-base status of the bladder.