Nucleotide metabolism by gastric glands and H⁺-K⁺-ATPase-enriched membranes

Abstract

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.

Syntaxin 3 is required for cAMP-induced acid secretion: streptolysin O-permeabilized gastric gland model

“…In addition, 1 mM of either AMP or ADP could substitute for 1 mM ATP in stimulation of AP accumulation by the SLO-permeabilized glands (data not shown). These data are very much like those obtained with ␣-toxinpermeabilized glands, in which it was shown that any adenine nucleotide source could provide the necessary energy intermediate (19). Even cAMP serves both as a second messenger to trigger PKA activation and a suitable source of ATP, through conversion to AMP by phosphodiesterase and subsequent resynthesis to ATP.…”

“…Direct stimulation of the protein kinase A (PKA) pathway in intact glands with DBcAMP and IBMX resulted in an 11.7-fold increase in AP ratio over resting glands (233.7 Ϯ 24.7 and 19.9 Ϯ 5.1, respectively). As with other permeabilized gland models (1,10,15,19,22), acid accumulation in SLO-treated glands requires substrates for oxidative phosphorylation to regenerate ATP required by the gastric proton pump. SLO-permeabilized glands incubated with ATP and cAMP but lacking pyruvate and succinate showed no significant increase in AP ratio (7.4 Ϯ 0.4) compared with those without the nucleotides (8.7 Ϯ 0.6).…”

“…It was then discovered that pore-forming bacterial toxins permeabilize gastric glands while allowing the transition of resting to a stimulated state (22). These toxins have been valuable in generating permeable cell systems that facilitate better understanding of the parietal cell activation process (19,21,26). Depending on the size of the plasma membrane pore, one can introduce a variety of molecules into the cell to facilitate or interfere with normal functional activity.…”

“…In previous work, we found ␣-toxin to be a superior permeabilization agent over digitonin and other pore-forming bacterial toxins. We determined (19) that stimulation was dependent on addition of cAMP, that addition of ATP led to synthesis of cAMP and therefore could trigger stimulation, and that addition of both of these nucleotides gave optimal results. As with other permeabilized preparations, ␣-toxin-treated glands required substrates of mitochondrial oxidative phosphorylation for regeneration of the ATP hydrolyzed for proton pumping (10).…”

“…Because of the small pore size produced (ϳ3-nm diameter) ␣-toxin allows the entry of only small molecules, such as nucleotides and phalloidin (8). This toxin was useful in determining the metabolic properties (19) and phosphoproteins associated with cell activation (26) in isolated gastric glands. Recent work has shown that treatment of glands with the saponin ester ␤-escin yields viable glands that allow entry of small polypeptides (1).…”