HCO3- secretion in mitochondria-rich cells is linked to an H+-ATPase.

Turtle bladder mitochondria-rich (MR) cells secrete H+ by an ATP-dependent process. MR cells also secrete HCO3- by an energy-requiring, Cl- -dependent process that may depend on a serosal H+-ATPase. To determine whether HCO3- is linked to an H+-ATPase, O2 consumption was assessed in MR and granular (G) cells exposed to H+ and HCO3- transport inhibitors alone and also with an H+-ATPase inhibitor. MR and G cells were separated by Ficoll density-gradient centrifugation and treated with ouabain and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) to maximally inhibit Na+ and luminal H+ transport. O2 consumption was measured before and after cells were additionally treated with 10 microM N-ethylmaleimide (NEM), an inhibitor of nonmitochondrial H+-ATPase. O2 consumption of MR cells fell after treatment with NEM (delta = 8.64 +/- 2.35 microliters O2.h-1.mg protein-1, P less than 0.025, n = 5). There was no significant difference in G cells similarly treated (delta = 1.61 +/- 0.62 microliters O2.h-1.mg protein-1, P greater than 0.05, n = 5). Because luminal H+ secretion is nearly abolished after treatment with SITS, the decline in O2 consumption of 44.4 +/- 7.11% after addition of NEM is probably due to inhibition of other non-mitochondrial H+-ATPases. In the intact bladder, HCO3- secretion was reduced by 35.1% after serosal application of NEM. Furthermore, in SITS-treated MR and G cells, ATP levels as measured by the luciferin-luciferase assay method were not appreciably different in the presence or absence of NEM.(ABSTRACT TRUNCATED AT 250 WORDS)

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.