MECHANISM OF ACTION OF EXTRACELLULAR CALCIUM ON ISOPRENALINE-EVOKED AMYLASE SECRETION FROM ISOLATED RAT PAROTID-GLANDS

1. The effects of extracellular Ca2+ deprivation on amylase secretion, 45Ca efflux and cyclic adenosine 3'',5''-monophosphate (cyclic AMP) metabolism were investigated in incubated parotid glands of young rats. 2. Reducing the extracellular Ca2+ concentration from 2.5 .times. 10-3 M to 10-6 M had no effect on amylase secretion but did increase the rate of 45Ca efflux from unstimulated glands. 3. Isoprenaline (10-5 M) increased amylase secretion and the rate of 45Ca efflux in media containing 2.5 .times. 10-3 M-Ca2+, and also increased the parotid cyclic AMP content. 4. When glands were pre-incubated for either 10, 20, or 30 min in media containing either 10-3 or 10-6 M-Ca2+ there was no effect on the peak rate of amylase secretion stimulated by isoprenaline but the response took longer to develop. The effects of the same experimental manoeuvre on the net change in 45Ca efflux stimulated by isoprenaline were consistent with the idea that the .beta.-adrenergic agonist mobilizes Ca2+ from a number of intracellular pools. 5. When glands were pre-incubated for 100 min in Ca2+-depleted buffers and then exposed to isoprenaline a triphasic effect on amylase secretion was observed. The secretory response was depressed when the extracellular Ca2+ concentration was lowered from 2.5 .times. 10-3 M to 10-3 M; remained unaffected when the extracellular Ca2+ was further reduced to 10-5 M; and was virtually abolished when the extracellular Ca2+ concentration was decreased to 10-6 M. 6. When glands were pre-incubated for 100 min in media containing Ca2+ at concentrations above 10-5 M and then stimulated with isoprenaline parallel changes in cyclic AMP content and amylase secretion were observed. At lower Ca2+ concentrations there was no further reduction in cyclic AMP content. 7. The lipophilic cyclic AMP analogue N6,O2-dibutyryl adenosine 3'',5''-monophosphate (dibutyryl cyclic AMP) mimicked the effects of isoprenaline on amylase secretion and 45Ca efflux. However, the amylase secretory response took longer to develop and the 45Ca efflux response was of shorter duration. These resposnes were reduced after pre-incubation of glands for 100 min in a medium containing 10-6 M-Ca2+. 8. Ca2+ at concentrations up to 10-7 M enhanced parotid adenylate cyclase activity but higher concentrations were inhibitory. The same pattern of Ca2+ sensitivity was seen for basal and also isoprenaline and F-stimulated activities. 9. The results show that isoprenaline can mobilize Ca2+ from a number of intracellular pools which may exchange at different rates with the extracellular medium. This Ca2+ may be required at a minumum of two sites in the amylase secretory pathway. At the catalytic subunit of the parotid adenylate cyclase where it may modulate the generation of endogenous cyclic AMP, and at another site which is closer to the release mechanism where it may also modulate the effects of cyclic AMP on amylase release.