INOSITOL TRISPHOSPHATE AND CYCLIC ADP RIBOSE AS LONG-RANGE MESSENGERSGENERATING LOCAL SUBCELLULAR CALCIUM SIGNALS

The process of messenger-mediated release of Ca2+ from intracellular s tores, which is of great importance in virtually all cell types includ ing neurons, can best be studied in cells lacking voltage-gated Ca2+ c hannels in the plasma membrane. In pancreatic acinar cells agonist-evo ked repetitive cytosolic Ca2+ spikes are due to release of Ca2+ via in ositoltrisphosphate (IP3) and ryanodine receptors and reuptake into th e stores via thapsigarin-sensitive Ca2+ pumps. At low acetylcholine (A Ch) or cholecystokinin concentrations the cytosolic Ca2+ spikes are mo stly confined to the secretory granule area of the polarized pancreati c acinar cells. Similar results can be obtained by intracellular infus ion of IP3 (or one of its non-metabolizable analogues) or cyclic ADP r ibose. This suggests that high affinity IP3 and ryanodine receptors ar e concentrated in the secretory granule area. We have generated an 'ar tificial synapse' on isolated acinar cells by having a cell-attached p atch pipette filled with ACh on the basal membrane. Initially, ACh is prevented from making contact with the receptors by the negative poten tial applied to the pipette. When the pipette polarity is switched to positive ACh can bind to its receptors. Using digital Ca2+ imaging it could be seen that the first cytosolic rise often occurred in the secr etory granule area, a considerable distance away from the site of the agonist-receptor interaction. This shows the long-range action of the messenger(s) IP3 and or cyclic ADP ribose generated by the ACh-recepto r interaction. The local Ca2+ spikes in the secretory granule area are sufficient for exocytotic secretory responses as seen in capacitance measurements. Ca2+ is used for many types of cellular signalling event s and localized subcellular Ca2+ spikes are necessary for compartmenta lization of the responses. There are specialized Ca2+ release sires no t only in the endoplasmic reticulum, hut also in the secretory granule area and in the inner nuclear membrane.