The Ca2+-selective fluorescent probe quin 2 was used to measure changes in the concentration of free cytosolic [Ca2+] in isolated rabbit gastric glands. Both carbachol and cholecystokinin octapeptide (CCK-8) were found to increase transiently intracellular Ca2+ concentration, [( Ca2+]i) with maximal increases from approximately 0.15 to 0.5 microM occurring within 4-6 s following secretagogue addition. Increases in [Ca2+]i were dose dependent and inhibited by appropriate antagonists. Prestimulation with either carbachol or CCK-8 effectively prevented increases in [Ca2+]i in response to the other agonist. Acute removal of extracellular Ca2+ slightly reduced the increase in [Ca2+]i that occurred following secretagogue addition but had no effect on pepsinogen secretion. Severe Ca2+ depletion resulted in potent inhibition of the quin 2 signal and suppressed basal pepsinogen release and reduced, but did not totally block, pepsinogen release in response to carbachol and CCK-8. Gastrin stimulation also elevated [Ca2+]i in glands, but this agonist was only 40-50% as effective as CCK-8. The cAMP-dependent agonists histamine and forskolin increased [Ca2+]i to approximately the same degree as gastrin. There was a definite lag in the rise in [Ca2+]i following simulation with histamine and forskolin compared with carbachol and CCK-8, which suggests that additional biochemical events occur between agonist-receptor binding and the rise in [Ca2+]i observed with the cAMP-dependent agonists. Both cAMP-dependent and -independent agonists induced an increase in autofluorescence that was slower than the rise in [Ca2+]i but equally affected by extracellular Ca2+ depletion.

The in vitro release of pepsinogen was studied using a preparation of isolated gastric glands from rabbits. The pepsinogen content of the glands was estimated to be about 700 U/mg dry wt. Spontaneous release of pepsinogen was found to be less than 1% of the total per hour and relatively constant for at least 2 h. Pepsinogen release was stimulated in a dose-dependent manner by both carbachol and isoproterenol. The cholinergic and beta-adrenergic stimulation was selectively inhibited by atropine and propranolol, respectively. Removal of external calcium inhibited the responses to both isoproterenol (partially) and carbachol (completely). Several agents, including histamine, prostaglandin (E2), and synthetic secretin, were found not to stimulate pepsinogen release. However, a crude secretin preparation (Boots) was found to produce significant stimulation. Dibutyryl cAMP increased pepsinogen release in a dose-dependent manner. Isoproterenol was found to increase the cAMP content of gastric glands and to stimulate adenylyl cyclase activity in homogenates. The beta-adrenergic stimulation of adenylyl cyclase was found to be selective for a population of gastric cells that was relatively depleted of parietal cells and distinct from the histamine-stimulated adenylyl cyclase activity. The results indicate that pepsinogen secretion by the gastric chief cell is regulated, in part, by separate cholinergic and beta-adrenergic mechanisms and that both calcium and cAMP play a role in this regulation.

Isolated gastric glands were used to study the mechanism of acid secretory inhibition by thiocyanate (SCN). It was found that SCN does not act as a competitive antagonist of histamine nor does SCN prevent the increase in cellular cAMP associated with histamine stimulation. SCN modifies but does not prevent the expansion of parietal cell canaliculi, indicating that this characteristic morphological transition does not require the actual formation of hydrochloric acid. Low doses (less than 5 mM) of SCN were found to inhibit aminopyrine accumulation, an index of acid formation, but do not inhibit either resting or stimulated respiration. Higher doses (greater than 10 mM) of SCN produce significant inhibition of stimulated but not resting respiration. These results indicate that SCN has two actions, i.e., inhibition of acid formation that requires low doses and inhibition of oxidative metabolism that requires higher doses. Incubation of glands in high-K+ (108 mM) medium leads to formation of an acid gradient in the absence of other secretagogues. The gradient was found to be transient, and its formation does not require oxidative metabolism, indicating that continued proton pumping is not required. Low doses of SCN were found to be more effective in dissipating the high-K+-induced gradient than a similar gradient induced by histamine stimulation. These results support the hypothesis that SCN inhibits acid secretion by increasing the rate of proton-gradient dissipation rather than interfering with a proton-pump mechanism.

Cellular responses induced in human neutrophils by the synthetic diacylglycerol, 1-oleoyl-2-acetyl-glycerol (OAG), paralleled those induced by phorbol myristate acetate (PMA). Like PMA, OAG caused the preferential release of enzymes from specific granules and promoted superoxide (O2-) generation. The efficacy of OAG was similar to that for PMA, but its potency was lower by four orders of magnitude. First derivative kinetic analysis showed that rates of O2- generation elicited by PMA decayed exponentially in a first order manner; the half life was found to be 21 +/- 6 min. Results obtained in studies carried out with high OAG concentrations were similar except that after 40 min, the rate of decay increased and became complex order. This difference was attributed to the greater susceptibility of OAG to metabolic alteration, and was reflected in the NADPH oxidase activity of granule rich membrane fractions (GRF) of cells stimulated for 90 min with PMA or OAG. It was found that the O2- generating activity of the PMA treated GRF was significantly greater than that for the OAG treated fraction. Current evidence indicates that cellular responses arise from direct activation of protein kinase C by PMA-OAG. The stability of this complex and the bypassing of normal regulatory constraints may account for the relative longevity of the PMA-OAG O2- respiratory burst.