The effects of manganese, cobalt and calcium on amylase secretion and calcium homeostasis in rat pancreas

1. Mn 2+ evoked an atropine‐resistant secretion of amylase from the isolated pancreas of the young rat. The lowest effective concentration of Mn 2+ was 10 −3 m . The response to 10 −2 m ‐Mn 2+ was biphasic, an initial peak being followed by a slow sustained rise in amylase output. The maximal effect of 10 −2 m ‐Mn 2+ was to double the basal rate of amylase secretion after 70 min incubation. 2. Co 2+ (10 −2 m ) also stimulated amylase secretion. The maximal rate, about three times the basal value, was attained after 20 min incubation. Atropine partially inhibited this effect. 3. Ca 2+ (10 −2 m ) evoked an atropine‐resistant amylase secretion similar in both magnitude and time course to the sustained phase observed with 10 −2 m ‐Mn 2+ . 4. Mn 2+ (10 −4 ‐10 −2 m ) also increased the rate of 45 Ca efflux from the gland. Maximal efflux rates were attained after 30 min incubation and thereafter declined to basal values. A small increase was also observed with 10 −2 m ‐Co 2+ , but not with 10 −2 m ‐Ca 2+ . The effect of Co 2+ was almost completely abolished by atropine. 5. Reducing the extracellular Ca 2+ concentration from 2·5 × 10 −3 to 10 −5 m did not reduce amylase secretion in response to 10 −2 m ‐Mn 2+ , but secretion was abolished in a Ca 2+ ‐free medium containing EGTA. The increase in 45 Ca efflux rate evoked by Mn 2+ was inversely related to the extracellular Ca 2+ concentration. 6. Mn 2+ (10 −2 m ) increased the concentration of cyclic 3′,5′‐guanosine monophosphate (cyclic GMP) within the pancreas. Also, Mn 2+ accumulated within the cellular pool of the gland. The time course of both these effects was similar to the time course of 45 Ca efflux. 7. Mn 2+ displaced Ca 2+ bound to isolated pancreatic microsomal membranes. The cation‐binding sites on these membranes probably have a higher affinity for Mn 2+ than Ca 2+ . 8. We conclude that Mn 2+ stimulates enzyme secretion by displacing membrane‐bound Ca 2+ , the resulting increase in cytosolic Ca 2+ concentration activating the secretory mechanism. 9. Mn 2+ partially inhibited amylase secretion stimulated by optimal doses of either acetylcholine (ACh) or caerulein. Maximal inhibition (about 60%) occurred with 10 −3 m ‐Mn 2+ (i.e. the lowest concentration required to stimulate secretion in the absence of secretagogues). Decreasing the extracellular Ca 2+ concentration reduced the inhibitory effect of Mn 2+ . 10. When glands were exposed to ACh and Mn 2+ simultaneously, the time required for inhibitory effects to develop was inversely related to the dose of ACh and the concentration of Mn 2+ . 11. Mn 2+ did not alter the acceleration of 45 Ca efflux evoked by ACh or by caerulein in a medium containing 2·5 × 10 −3 m ‐Ca 2+ . However, under conditions of Ca 2+ deprivation ACh‐stimulated 45 Ca efflux was greatly enhanced. 12. Mn 2+ reduced the total amount of Ca 2+ accumulated into the cellular pool of the pancreas after 60 min incubation, but had no effect on the initial, rapid phase of Ca 2+ uptake. 13. The effects of Mn 2+ on the relationship between ACh dose, amylase release and the extracellular Ca 2+ concentration suggest that the inhibitory actions of Mn 2+ cannot be explained by a simple, competitive interaction with the stimulant or with extracellular Ca 2+ . However, the time course of inhibition is consistent with a requirement for Mn 2+ to accumulate within the acinar cells. 14. Mn 2+ partially inhibited amylase secretion stimulated by hyperosmolarity and also increased the 45 Ca efflux rate under these conditions. 15. Our results are not consistent with Mn 2+ exerting its inhibitory effect on secretagogue‐stimulated enzyme secretion solely by blocking Ca 2+ influx from the extracellular space. We conclude that inhibition probably depends on the ability of Mn 2+ to displace Ca 2+ from binding sites involved in secretion, presumably coupled with a reduced ability of Mn 2+ to replace Ca 2+ in the secretory process.