Potentiation of amylase secretion by the combination of isoproterenol and substance P was examined in perfused rat parotid acinar cells. Combined additions of substance P and isoproterenol evoked biphasic changes in amylase secretion, an initial large peak and the following sustained plateau: the magnitudes of the both responses were higher than the sum of the responses induced by each agonist alone. Isoproterenol also increased the maximum response and the apparent affinity (EC50) for substance P to evoke the initial peak response; the EC50 values were about 20 and 0.8 nM, respectively, in the absence and the presence of isoproterenol. On the other hand, 1 nM substance P was sufficient for evoking the maximum potentiation of the sustained plateau response. Substance P did not change the EC50 for isoproterenol. The effect of isoproterenol was mimicked with dibutyryl cyclic AMP and agonists that increase parotid cyclic AMP. Omission of Ca2+ or addition of 5 mM nickel chloride almost completely abolished the potentiation of the sustained plateau, but little decreased that of the initial peak. Depletion of Ca2+ in InsP3-sensitive intracellular stores with thapsigargin, on the other hand, decreased the initial peak response, but not the sustained plateau, to substance P. The potentiation was also observed between isoproterenol and Ca2+ ionophores. Switching to the solutions containing higher concentrations of Ca2+ during the continuous stimulation with isoproterenol or IBMX evoked a large, but transient, response of amylase secretion. Time course of changes in amylase secretion induced by isoproterenol and substance P in combination was very similar to that of substance P, but not of isoproterenol. Isoproterenol did not enhance the effect of substance P on [Ca2+]i. These results show that the potentiation is mainly, if not totally, caused by cyclic AMP-induced enhancement of the potency and the efficacy in the pathway regulated by Ca2+.

Isolated parotid acinar cells were perifused in small columns by embedding them in Bio-Gel P-2 beads as an inert supporting matrix, and the effect of carbamylcholine, substance P, and isoproterenol on the rate of amylase release was examined by measuring amylase activity in the effluent. Amylase release by continuous stimulation with carbamylcholine and substance P was biphasic. They caused a rapid and large increase in the rate of amylase release that reached maximum 30 to 60s after the onset of stimulation, followed by a rapid decline to a lower sustained level that was maintained as long as the agonists were present. The rapid decline in the rate of amylase release was due to rapid development of refractoriness. Repeated 1 min pulse stimulation with these secretagogues showed that recovery from refractoriness was also rapid in onset, and 1 min of washout was sufficient to cause significant recovery from refractoriness for both carbamylcholine and substance P. Recovery, however, was not complete after 10 min of washout. Amylase release by continuous stimulation with isoproterenol, on the other hand, developed more slowly with the peak rate being attained at about 6 min after the onset of stimulation. Refractoriness was not observed in the effect of isoproterenol. The maximum effect in the rate of amylase release attained by carbamylcholine or substance P was higher than that by isoproterenol. These results suggest that the apparent small effect of carbamylcholine and substance P on amylase release reported earlier by using batch systems is probably due to the rapid development of refractoriness to these secretagogues, but not to isoproterenol.

A column perfusion system was applied to rat parotid acinar cells to clarify the roles of Ca2+ and protein kinase C (PKC) in the mechanisms of carbachol (CCh)‐induced amylase secretion. CCh evoked a biphasic response of amylase secretion with an initial rapid and large peak that reached maximum at about 10 s followed by a sustained plateau. The time profile and the dose‐response relationship paralleled with those of cytosolic free Ca2+ concentration ([Ca2+]i). The CCh‐induced sustained response of amylase secretion maintained by Ca2+ influx into cells was ATP dependent, while the initial peak response regulated by Ca2+ released from InsP3‐sensitive stores was relatively ATP independent. Restoration of extracellular Ca2+ during continuous stimulation with CCh in Ca2+‐free medium evoked a second rapid and large peak of amylase secretion. Phorbol 12,13‐dibutyrate (PDBu) potentiated the CCh‐induced amylase secretion in both the initial peak and the sustained plateau without enhancing CCh‐induced [Ca2+]i changes. PKC inhibitors such as Ro 31–8220 inhibited the potentiating effect of PDBu but only slightly reduced amylase secretion induced by CCh alone. These results suggest that a CCh‐induced rise in [Ca2+]i triggers the final fusion and/or exocytosis of amylase secretion. CCh also has some ability to promote ATP‐dependent priming of secretory granules that, together with Ca2+ influxed into cells, contributes to the CCh‐induced sustained plateau of amylase secretion. PDBu‐induced activation of PKC promotes the priming of secretory granules, thereby enhancing the efficacy for Ca2+ to trigger fusion/exocytosis.

Rat parotid acinar cells were perfused in small quartz columns to examine the role of cAMP and Ca(2+) in the priming and fusion/exocytosis of amylase secretion. Carbachol (CCh) evoked a biphasic response of amylase secretion with an initial rapidly occurring large peak and a subsequent sustained plateau. Isoproterenol produced slowly increasing amylase secretion that reached the plateau greater than that induced by CCh. Combined stimulation with isoproterenol and CCh greatly potentiated amylase secretion. The rise and decay of amylase secretion induced by the combined stimulation was similar to those induced by CCh but not by isoproterenol, suggesting that the potentiation is caused by isoproterenol-induced modification of the CCh effect. Concentration-dependent responses of CCh-induced amylase secretion with and without isoproterenol showed that isoproterenol greatly enhances both the sensitivity and maximum effect of CCh. Similar potentiation was observed when the Ca(2+) effect was directly examined in cells permeabilized to Ca(2+) with ionomycin instead of CCh. In a Ca(2+)-free medium, CCh evoked an initial peak but did not produce a sustained plateau. Isoproterenol did not enhance the effect of CCh on [Ca(2+)](i). 2,4-Dintrophenol and carbonyl cyanide m-chlorophenyl hydrazone did not decrease the CCh-induced initial peak of amylase secretion but markedly decreased the sustained responses induced by isoproterenol and CCh. These results suggest that CCh, via Ca(2+), has two distinct effects on amylase secretion: triggering of fusion/exocytosis and the priming of secretory granules. Isoproterenol, via cyclic AMP, also has two distinct effects: direct stimulation of priming and enhancement of the sensitivity to the Ca(2+)effects. Thus, isoproterenol stimulates amylase secretion by increasing the primed pools of secretory granules, whereas CCh increases the flux of secretory granules into/from the primed pools, which is greatly enhanced by isoproterenol.

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