No abstract

Remodeling of the apical membrane-cytoskeleton has been suggested to occur when gastric parietal cells are stimulated to secrete HCl. The present experiments assayed the relative amounts of F-actin and G-actin in gastric glands and parietal cells, as well as the changes in the state of actin on stimulation. Glands and cells were treated with a Nonidet P-40 extraction buffer for separation into detergent-soluble (supernatant) and detergent-insoluble (pellet) pools. Two actin assays were used to quantitate actin: the deoxyribonuclease I binding assay to measure G-actin and F-actin content in the two pools and a simple Western blot assay to quantitate the relative amounts of actin in the pools. Functional secretory responsiveness was assayed by aminopyrine accumulation. About 5% of the total parietal cell protein is actin, with about 90% of the actin present as F-actin. Stimulation of acid secretion resulted in no measurable change in the relative amounts of G-actin and cytoskeletal F-actin. Treatment of gastric glands with cytochalasin D inhibited acid secretion and resulted in a decrease in F-actin and an increase in G-actin. No inhibition of parietal cell secretion was observed when phalloidin was used to stabilize actin filaments. These data are consistent with the hypothesis that microfilamentous actin is essential for membrane recruitment underlying parietal cell secretion. Although the experiments do not eliminate the importance of rapid exchange between G- and F-actin for the secretory process, the parietal cell maintains actin in a highly polymerized state, and no measurable changes in the steady-state ratio of G-actin to F-actin are associated with stimulation to secrete acid.

It is generally believed that histamine-stimulated gastric acid secretion involves a transient elevation of intracellular Ca2+ and the adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) cascade through phosphorylation, whose actions ultimately effect the fusion of H(+)-K(+)-adenosinetriphosphatase (ATPase)-containing vesicles to the apical plasma membrane of parietal cells. To dissect the signaling events underlying gastric acid secretion, we have developed a permeabilized gastric gland model using Staphylococcus alpha-toxin. The advantage of this model is its ability to retain cytosolic components that are required for the secretory machinery. Here we show that acid secretion in alpha-toxin-permeabilized glands is a cAMP-dependent process, reaching a maximal stimulation at 100 microM cAMP. The cAMP-elicited acid secretion, as monitored by the accumulation of the weak base aminopyrine (AP), required functional mitochondria or exogenously supplied ATP. Maximal stimulation elicited by cAMP for AP uptake by permeabilized glands was 51-85% of intact glands. Moreover, secretory activity was potentiated by 0.1 mM ATP. The recruitment of H(+)-K(+)-ATPase-rich tubulovesicles into the apical plasma membrane was measured using biochemical and morphological assays, thus validating the cell activation processes in response to cAMP. From this permeabilized model, [gamma-32P]ATP was used to directly phosphorylate target proteins. A number of proteins whose phosphorylation-dephosphorylation is specifically modulated by cAMP were found. These studies establish the first permeabilized gland model in which the resting-to-secreting transition can be triggered and show that cAMP-mediated phosphorylation is correlated with secretory activity.

α-Toxin-permeabilized gastric glands represent a functional model in which acid secretion can be elicited by either adenosine 3′,5′-cyclic monophosphate (cAMP) or ATP, with proven morphological and functional transition between resting and secretory states [X. Yao, S. M. Karam, M. Ramilo, Q. Rong, A. Thibodeau, and J. G. Forte. Am. J. Physiol. 271 ( Cell Physiol. 40): C61–C73, 1996.] In this study we use α-toxin-permeabilized rabbit gastric glands to study energy metabolism and the interplay between nucleotides to support acid secretion, as indicated by the accumulation of aminopyrine (AP). When permeabilized glands were treated with a phosphodiesterase inhibitor, the secretory response to cAMP was inhibited, whereas the secretory response to ATP was potentiated. This implied that 1) ATP provided support not only as an energy source but also as substrate for adenylate cyclase, 2) activation of acid secretion by cAMP needed ATP, and 3) ATP and cAMP exchanged rapidly inside parietal cells. To address these issues, we tested the action of adenine nucleotides in the presence and absence of oxidizable substrates. All adenine nucleotides, including AMP, ADP, ATP, and cAMP, could individually enhance the glandular AP accumulation in the presence of substrates, whereas only a high concentration of ATP (5 mM) was able to support secretory activity in substrate-free buffer. Moreover, ATP could maintain 75–80% of maximal secretory activity in phosphate-free buffer; cAMP alone could not support secretion in phosphate-free buffer. In glands and in H+-K+-adenosinetriphosphatase-rich gastric microsomes, we showed the operation of adenylate kinase, creatine kinase, and ATP/ADP exchange activities. These enzymes, together with endogenous adenylate cyclase and phosphodiesterase, provide the recycling of nucleotides essential for the viability of α-toxin-permeabilized gastric glands and imply the importance of nucleotide recycling for energy metabolism in intact parietal cells.