Volume density, distribution, and ultrastructure of secretory and basolateral membranes and mitochondria predict parietal cell secretory (dys)function

Abstract

Acid secretion in gastric parietal cells requires highly coordinated membrane transport and vesicle trafficking. Histologically, consensus defines acid secretion as the ratio of the volume density (Vd) of canalicular and apical membranes (CAMs) to tubulovesicular (TV) membranes, a value which varies widely under normal conditions. Examination of numerous achlorhydric mice made it clear that this paradigm is discrepant when used to assess most mice with genetic mutations affecting acid secretion. Vd of organelles in parietal cells of 6 genetically engineered mouse strains was obtained to identify a stable histological phenotype of acid secretion. We confirmed that CAM to TV ratio fairly represented secretory activity in untreated and secretion-inhibited wild-type (WT) mice and in NHE2-/- mice as well, though the response was significantly attenuated in the latter. However, high CAM to TV ratios wrongly posed as active acid secretion in AE2-/-, GHKAalpha-/-, and NHE4-/- mice. Achlorhydric genotypes also had a significantly higher Vd of basolateral membrane than WT mice, and reduced Vd of mitochondria and canaliculi. The Vd of mitochondria, and ratio of the Vd of basolateral membranes/Vd of mitochondria were preferred predictors of the level of acid secretion. Alterations in acid secretion, then, cause significant changes not only in the Vd of secretory membranes but also in mitochondria and basolateral membranes.

Figure 1

WT parietal cells (left and right). Right panel shows tubulovesicles similar to those boxed in (a). (b) Parietal cells from omeprazole treated mice: WT (left) and NHE2−/− (right). Insets show tubulovesicles from areas similar to those boxed in WT and NHE2−/− omeprazole-treated mice, respectively. Microvilli were significantly longer and the Vd of mitochondria was reduced in NHE2−/− parietal cells compared to WT. Tubulovesicles in NHE2−/− parietal cells were mostly vesicular, and often contained electron-dense cores. N: nucleus; arrowheads: mitochondria; arrows: canaliculi, boxes surround TV.

Figure 2

Vd of secretory membranes with and without inhibition of acid secretion with omeprazole (omep) or vagotomy (vagot) is shown for WT and NHE2−/− parietal cells. Black bars are WT, and grey bars are NHE2−/−. N: sample size. Asterisks denote significances of at least P = .05.

Figure 3

Volume density (Vd) of various membrane and organellar components of the parietal cells of WT and 6 achlorhydric genotypes are shown. Significant differences from WT (P < .05): white bars; marginal significances (P < .1): grey bars; and WT, to which all comparisons were made, black. The number of animals used in each comparison is given in the upper left graph, and names of the genotypes are below the bottom graphs in each column. Total secretory membranes = TV + CAM + BLM, being all membranes involved in balanced transport activities directly involved in acid secretion.

Figure 4

(a) AE2−/− parietal cells showed a reduction in TV, canalicular and mitochondrial Vd, and, seen at higher magnification, cytoplasm contained occasional polysomes but generally reduced numbers of organelles (compared to WT and NHE2−/− (see Figure 1)). Microvilli were mostly confined to the apical membrane, incipient canaliculi were equivocally identified, and electron-dense material often filled the gastric gland lumen (G). (b) The GHKAα−/− parietal cells showed short microvilli, round vesicles instead of TV, cytoplasmic glycogen, frequent canalicular dilatation, and occasionally, large mitochondria with whorled cristi. (c) and (d). The two genotypes that seemed to have the most organelle-depleted cytoplasm were the NHE4−/− (c) and the NHE2−/− AE2−/− double KO (d). Parietal cells in both of these genotypes were very sparsely inter-populated with organelles, having significant reductions in Vd of both membranes and mitochondria. (e) NHE2−/− GHKAα−/− parietal cells were quite similar to the GHKAα−/− single knockout. The latter two were the only genotypes which showed cytoplasmic glycogen or whorled cristi in mitochondria, and short stubby microvilli. For all panels: arrow heads: mitochondria; white arrowheads: mitochondria with whorled cristi; arrows: canaliculi, N: nucleus; Z (white) zymogen granule.

Figure 5

WT (a) basolateral membrane shows mostly parallel or slightly undulating infoldings. AE2−/− (b) and NHE4−/− (c) showed the significantly increased Vd of basolateral membranes infoldings typical of achlorhydric mice. In addition, loops and interdigitations were broadened in width. Arrowheads: mitochondria; N: nucleus, interdigitations: long arrows; bar: 1 micron for all images.

Figure 6

Lamellar and dense granular bodies were uncommon in WT parietal cell cytoplasm (see Figure 1). Lamellar inclusions were increased in NHE2−/−, AE2−/− and NHE2−/− AE2−/− mice. Lamellar inclusions decreased in the NHE2−/− parietal cells when secretion was inhibited. In GHKAα−/− mice, inclusions were almost always dense and granular. L, lamellar inclusions, G dense granular inclusions; bar: 1 micron for all.