Role of calcium in exocrine pancreas secretion. VII. Effect of sodium on enzyme secretion and calcium metabolism in rabbit pancreatic fragments and acinar cells.

1. The role of sodium in the pancreatic stimulus-secretion coupling has been studied. 2. Reduction of the extracellular sodium concentration or addition of ouabain to the medium inhibits the stimulation of enzyme secretion by carbachol. 3. Incubation in low sodium medium or in the presence of ouabain increases the exchangeable calcium content, but not the total calcium content of acinar cells. 4. Depending on preincubation time and the substrate replacing sodium in the low sodium medium, the carbachol-induced Ca45(2)+ efflux may be blocked, but it is not blocked by addition of ouabain to a normal Na+ medium. 5. Stimulation of enzyme secretion by the calcium ionophore A23187 in the presence of external calcium is inhibited in low sodium as well as in ouabain containing media. 6. These findings suggest that reduction of the Na+ gradient across the plasma membrane blocks the coupling between intracellular calcium release and exocytosis, while under certain conditions it also blocks the coupling between hormone-receptor interactions and intracellular calcium release.

Role of the tryptophan residue in the interaction of pancreozymin with its receptor.

1. Analogues of the C-terminal octapeptide and tetrapeptide of pancreozymin with a modified tryptophan residue have been tested on the rat pancreas adenylate cyclase activity, on the enzyme and fluid secretion of the rat pancreas in vivo and on the amylase release from rabbit pancreatic fragments. 2. Fluorination of the tryptophan residue in position 5 or 6 does not influence the effect of the peptides on any of the measured parameters. 3. Methylation of the nitrogen atom in the indolyl ring, which eliminates hydrogen bond formation, markedly reduces the affinity of the peptides for the adenylate cyclase activity and for the amylase release in rabbit pancreatic fragments. The effects on fluid and enzyme secretion in the rat pancreas in vivo are reduced nearly as much. 4. Tetrafluorination of the tryptophan residue, which reduces its charge donor capacity, causes a still larger reduction in activity and affinity of the octapeptide. 5. The tetrafluorinated tetrapeptide stimulates the adenylate cyclase activity and the enzyme and fluid secretion in vivo more than the unmodified tetrapeptide, which may be due to its increased hydrophobicity. 6. Replacement of the nitrogen atom in the indolyl ring of tryptophan by a sulfur or an oxygen atom, which also reduces the charge donor capacity, leads again to a large reduction in the affinity and activity of both the octapeptide and the tetrapeptide. 7. These findings suggest that the charge donor capacity of the tryptophan residue is of primary importance for the biologic activity of pancreozymin, while hydrogen bond formation and hydrophobicity are of secondary importance.

Rat pancreas adenylate cyclase: VII. Effect of extracellular calcium on pancreozymin-induced cyclic AMP formation.

1. The effect of stimulation of adenylate cyclase by pancreozymin-C-octapeptide on the cyclic AMP level of rat pancreatic fragments has been investigated. 2. In normal Krebs-Ringer bicarbonate medium pancreozymin-C-octapeptide causes a slight increase in pancreatic cyclic AMP level; this increase can be considerably enhanced by incubation in a calcium-free incubation medium. 3. The dose-response curve for pancreozymin-C-octapeptide in calcium-free medium is shifted to lower peptide concentrations, compared to the curve in normal Krebs-Ringer bicarbonate medium. 4. The maximal stimulatory effect of pancreozymin-C-octapeptide is obtained at a 1-methyl-3-isobutylxanthine concentration of 10 mM. 5. It suffices to lower the Ca2+-concentration of the medium from 2.5 to 1.5 mM to get the maximal increase in cyclic AMP content under influence of pancreozymin-C-octapeptide. 6. It is concluded that extracellular calcium antagonizes the stimulation of adenylate cyclase by pancreozymin-C-octapeptide. This suggests that a low cytoplasmic Ca2+-concentration is required for the maximal response of acinar cell adenylate cyclase to pancreozymin.

Role of calcium in exocrine pancreatic secretion. IV. Calcium movements in isolated acinar cells of rabbit pancreas.

1. A study has been made of the calcium movements in isolated acinar cells of rabbit pancreas in relation to the process of enzyme secretion. 2. After 90 min, the 45Ca2+ uptake level of the acinar cells reaches a steady state level which depends on the extracellular calcium concentration: it increases with increasing calcium concentration. 3. Carbachol, in addition to stimulating enzyme secretion, causes a decrease in the 45Ca2+ content of pre-loaded acinar cells. This decrease is virtually independent of the extracellular calcium concentration. It is followed by an uptake of 45Ca2+ which only in a medium with 2.5 mM Ca2+ leads to a significantly higher 45Ca2+ level than the steady state level. 4. Carbachol, added to acinar cells not preloaded with 45Ca2+, increases the 45Ca2+ uptake in medium with 2.5 mM and in that with 0.1 mM Ca2+. 5. The amount of enzyme released by carbachol depends on the extracellular calcium concentration: it is larger in media with higher calcium concentration. 6. It is concluded that the increase in cytoplasmic calcium concentration which takes place immediately upon addition of a stimulus and which is necessary for the stimulation of the enzyme secretion, is caused by a release of calcium from an intracellular pool and not by an influx of calcium from the extracellular medium. 7. The results suggest that there are at least three different calcium pools in the pancreatic acinar cells.