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Initiation of acid secretion in the gastric mucosa is accompanied by a morphological transformation in which the acid pump, the H+/K(+)-ATPase, translocates from a cytoplasmic vesicular location to the secretory surface lining the canaliculi. Associated with the morphological changes, activation of K+ and Cl- pathways are necessary to supply K+ to the extracytoplasmic face of the pump. Although the pump in the secretory membrane is known to secrete acid, it is not known whether activation of the KCl pathway occurs in the tubulovesicular membrane prior to the formation of the canaliculus, or when the pump is in the secretory membrane. The cellular site of activation of acid secretion in the rabbit gastric parietal cell was investigated using the covalent binding of [3H]omeprazole as a probe of acid secretion in rabbit gastric glands that were undergoing stimulation in vitro. This compound depends on an acidic environment for activation and covalent binding to the H+/K(+)-ATPase. Electron microscopic autoradiography showed that activation of the enzyme occurred only when it was present in the canalicular membrane and not when it was present in the cytoplasmic tubulovesicular membrane. Hence there is likely to be a physical separation of K+ and/or Cl- pathways from the ATPase in the resting cell, and stimulation of acid secretion is dependent on colocalization of these pathways in the canalicular membrane.

Isolated rabbit gastric glands were used to study the nature of the muscarinic cholinergic responses of parietal cells. Carbachol (CCh, 100 microM) stimulation of acid secretion, as measured by the accumulation of aminopyrine, was inhibited by the M1 antagonist, pirenzepine, with an IC50 of 13 microM; by the M2 antagonist, 11,2-(diethylamino)methyl-1 piperidinyl acetyl-5,11-dihydro-6H-pyrido 2,3-b 1,4 benzodiazepin-6-one (AF-DX 116), with an IC50 of 110 microM; and by the M1/M3 antagonist, diphenyl-acetoxy-4-methylpiperidinemethiodide (4-DAMP), with an IC50 of 35 nM. The three antagonists displayed equivalent IC50 values for the inhibition of carbachol-stimulated production of 14CO2 from radiolabeled glucose, which is a measure of the turnover of the H,K-ATPase, the final step of acid secretion. Intracellular calcium levels were measured in gastric glands loaded with FURA 2. Carbachol was shown to both release calcium from an intracellular pool and to promote calcium entry across the plasma membrane. The calcium entry was inhibitable by 20 microM La3+. The relative potency of the three muscarinic antagonists for inhibition of calcium entry was essentially the same as for inhibition of acid secretion or pump related glucose oxidation. Image analysis of the glands showed the effects of carbachol, and of the antagonists, on intracellular calcium were occurring largely in the parietal cell. The rise in cell calcium due to release of calcium from intracellular stores was inhibited by 4-DAMP with an IC50 of 1.7 nM, suggesting that the release pathway was regulated by a low affinity M3 muscarinic receptor or state; Ca entry and acid secretion are regulated by a high affinity M3 muscarinic receptor or state, inhibited by higher 4-DAMP concentrations (greater than 30 nM), suggesting that it is the steady-state elevation of Ca that is related to parietal cell function rather than the [Ca]i transient. Displacement of 3H N-methyl scopolamine (NMS) binding to purified parietal cells by CCh showed the presence of two affinities for CCh, but only a single affinity for 4-DAMP and lower affinity for pirenzepine and AFDX 116, providing further evidence for the parietal cell location of the [Ca]i response. Elevation of steady-state [Ca]i levels with either ionomycin or arachidonic acid did not replicate M3 stimulation of acid secretion or glucose oxidation, hence elevation of [Ca]i is necessary but not sufficient for acid secretion.

1. Models are presented for (a) HK ATPase acting in the presence of K and Cl conductances; (b) a pH regulatory system where Na/H exchange is regulated directly by second messenger and the anion exchanger is activated secondarily to the rise in cell pH; (c) vesicle fusion and K and Cl conductances activation in the gastric parietal cell. 2. It is suggested that H transport involves protonation and deprotonation of histidine groups as well as the motion of these groups relative to the membrane barrier. 3. The HK ATPase would have a voltage generating and voltage sensitive step in the forward direction. 4. Given net electroneutrality the K transport reaction would also be charge translocating and voltage sensitive.

Digitonin was used to render isolated gastric glands permeable. This procedure was found to release cellular lactic dehydrogenase without disrupting the parietal cell's ability to generate proton gradients. Optimal conditions for permeabilizing the glands were found to depend on the ratio of digitonin to gland concentration. Stimulation of the glands with histamine, forskolin, or 8-bromo-cAMP prior to digitonin treatment resulted in a marked enhancement of the subsequent ATP-dependent acid formation. This enhancement was not found with the cholinergic agonist carbachol. These results indicate that preservation of the active secreting state does not require the continued presence of soluble factors. Characterization of the ATP-dependent acid formation in prestimulated permeable glands showed a dependence on exogenous substrate and inhibition by the mitochondrial inhibitors oligomycin and atractyloside. Moreover, it was found that ADP could replace ATP in promoting acid formation. These results are interpreted to show that mitochondrial oxidative phosphorylation can serve as an in situ ATP-recycling system to provide a local supply of ATP for proton transport. The overall study demonstrates that the digitonin-permeabilized gastric gland preparation is a valuable model system for studying mechanisms of gastric proton transport.

A fluorescence-quench method using acridine orange as the probe was employed to monitor acid formation in situ by detergent-permeabilized gastric glands. In KCl medium, the addition of ATP to the permeabilized glands resulted in a rapid decrease in fluorescence and addition of valinomycin resulted in a second phase of fluorescence quench. The fluorescence was restored by addition of the H+-K+-ATPase inhibitor, Sch 28080. An ATP-dependent fluorescence quench was observed also in K2SO4 or K+-isethionate medium; however, valinomycin was ineffective in the Cl-free media. The ATP-dependent quench could be reversed or prevented by the electrogenic protonophore, tetrachlorosalicylanilide (TCS), in KCl medium but not in Cl-free media. The results with TCS are interpreted as demonstrating a large Cl- conductance in the secretory membrane, whereas the results with valinomycin indicate that resting membranes lack a K+ conductance. The data suggest that a complex KCl pathway that may demonstrate a Cl- conductance is used to activate acid secretion.