2 DISTINCT MODES OF CA2-CELLS - CONCENTRATION, GLUCOSE DEPENDENCE ANDCA2+ ORIGIN( SIGNALING BY ACH IN RAT PANCREATIC BETA)

1. Calcium signalling by acetylcholine (ACh) in single rat pancreatic beta-cells was studied. The cytosolic free Ca2+ concentration ([Ca2+]( i)) was measured) by dual-wavelength fura-2 microfluorometry. 2. In th e presence of basal glucose (2.8 mM), 10(-6) to 10(-4)M ACh (high ACh) transiently increased [Ca2+](i). The [Ca2+](i) response to 10(-5) M A Ch was little altered under Ca2+-free conditions. Brief pulses of 10(- 5) M ACh evoked successive [Ca2+](i) responses, which were progressive ly inhibited by 0.2-0.5 mu M thapsigargin, a specific inhibitor of the endoplasmic reticulum (ER) Ca2+ pump. 3. Elevation of glucose to 8.3 mM, a concentration which stimulates insulin release, increased [Ca2+] (i) to an initial peak followed by a sustained, moderate elevation. Ad dition of 10(-8) to 10(-7) M ACh (low ACh) evoked a further increase i n [Ca2+](i). The [C2+](i) response to 10(-7) M ACh was completely inhi bited under Ca2+-free conditions by 1 mu M nitrendipine, a blocker of L-type Ca2+ channels, and by 100 mu M diazoxide, an opener of ATP-sens itive K+ channels. 4. In the presence of 8.3 mM glucose, [Ca2+], respo nses to 10(-5) nr ACh were reduced but not abolished by Ca2+-free cond itions, nitrendipine and diazoxide. Successive [Ca2+](i) transients in duced by 10(-5) M ACh pulses in the presence of nitrendipine were prog ressively inhibited by thapsigargin. 5. The results revealed two disti nct modes of Ca2+ signalling: low ACh increases [Ca2+](i) by stimulati ng Ca2+ influx through voltage-dependent L-type Ca2+ channels only in the p-cells in which glucose has already elevated [Ca2+](i), while hig h ACh increases [Ca2+](i) at basal as well as stimulatory glucose conc entrations by releasing Ca2+ from the ER. The former mechanism is like ly to relate to the potentiator action and the latter to the initiator action of ACh on insulin release. High ACh and elevated glucose provo ke both modes of Ca2+ signalling.