Characteristics of the isolated apical plasmalemma and intracellular tubulovesicles of the gastric acid secreting cells: demonstration of secretagogue-induced membrane mobilization

Abstract

Separation of the gradient-purified gastric microsome into two membrane subfractions of distinct enzymatic and phospholipid composition has been achieved by mild SDS (0.033% w/v) treatment followed by sucrose gradient centrifugation of the pig and rabbit gastric microsomes. While the high-density membranes had all of the (H+,K+)-ATPase and K+-pNPPase activities and revealed a single major 100-kDa band on SDS-PAGE, the low-density membranes contained all of the 5'-nucleotidase and nearly all of the Mg2+-ATPase. In the present study, the low-density subfraction has been characterized to be derived from the apical membranes and the high-density one from the intracellular tubulovesicular membranes of the parietal cells. Such characterization was based primarily on sole dependency of the apical plasma membranes on the endogenous activator for (H+,K+)-ATPase activity, differential sensitivity of the activator (AF)-dependent and -independent (H+,K+)-ATPase on micromolar vanadate and Ca2+, specific vitamin B12 binding ability of the apical plasmalemma, phospholipid and protein profiles of the two membrane subfractions, and other parameters. The AF, mentioned previously, has recently been implicated as a cytosolic regulator of the gastric (H+,K+)-ATPase [Bandopadhyay et al. (1987) J. Biol. Chem. 262, 5664-5670]. Two different forms (i.e., AF-dependent and -independent forms) of the (H+,K+)-ATPase are suggested to be present in the tubulovesicles on the basis of differential vanadate sensitivity while the AF-dependent form alone is present in the apical membranes. The data have been discussed in terms of stimulation-induced membrane transformation characteristic of the H+-secreting epithelia including the acid-secreting cells of the stomach.

Mechanisms through which ammonia regulates cortical collecting duct net proton secretion

“…Elevated PCO 2 appears to stimulate CCD H ϩ -K ϩ -ATPase through vesicular insertion into CCD intercalated cell apical plasma membranes (52). Recycling of H ϩ -K ϩ -ATPase between cytosolic vesicles and plasma membranes is a major mechanism of its regulation in both the stomach (23,34,41) and the kidney (46) and appears to involve a tyrosine-based signal in the cytoplasmic tail of the H ϩ -K ϩ -ATPase ␤-subunit (8, 46) and involvement of SNARE proteins (Ref. 32 and present study).…”

The parietal cell gastric H, K-ATPase also functions as the Na, K-ATPase and Ca-ATPase in altered states

“…Thus, the phosphatidyl choline content of APM and TV was 67 and 33 µ moles/mg protein respectively with corresponding phosphatidyl choline/phosphatidyl ethanolamine ratios being 1.38 and 0.87 for APM and TV. Also, the phosphatidyl inositol and phosphatidyl serine content of APM were 24 and 8 µ moles/mg protein, respectively, about twice as much as that of TV 17 . It may be noted that the APM and TV have different buoyant densities of 1.06 and 1.115 respectively, with a nearly equal phospholipid to cholesterol molar ratio of 0.64.…”

“…It may be noted that the APM and TV have different buoyant densities of 1.06 and 1.115 respectively, with a nearly equal phospholipid to cholesterol molar ratio of 0.64. The identity of APM was based on exclusive 5´-nucleotidase activity, unique vitamin B12 binding ability and characteristic quantitative differences in phospholipid make up from that of TV 17 .…”

“…Under this condition, the apical membrane-located H, K-ATPase system would be acting as a provisional device for pumping out calcium prior to the onset of acid secretion 17, 18 .…”

“…Following our current evidence, the critical interplay among the HAF, H, K-ATPase and Ca in parietal cells is depicted in this diagram. While the pump molecules integral to the tubulovesicle (TV) are stimulated appreciably by the HAF, those associated with the apical plasma membrane (APM) are absolutely dependent on the HAF for their function, revealing the essential nature of the HAF in gastric proton-pump function 17 . For the ATPase assay 9 the desired amount of HAF (as indicated by the prior dose response study) was first pre-incubated with 5 µg of APM for 10 minutes at 37°C in 2 mM Pipes buffer (pH 7.4).…”