G-PROTEIN-DEPENDENT INHIBITION OF L-TYPE CA2-CELLS( CURRENTS BY ACETYLCHOLINE IN MOUSE PANCREATIC B)

1. The effect of acetylcholine (ACh) on voltage-dependent Ca2+ current s in mouse pancreatic B-cells was studied using the whole-cell configu ration of the patch-clamp technique. 2. ACh (0.25-250 mu M) reversibly and dose-dependently inhibited the Ca2+ current elicited by depolariz ations from -80 mV to +10 mV. Maximal inhibition was observed at conce ntrations > 25 mu M where it amounted to similar to 35%. The effect wa s voltage independent and prevented by atropine (10 mu M) suggesting t hat it wax mediated by muscarinic receptors. 3. The inhibitory action of ACh on the Ca2+ current was abolished when the cytoplasmic solution contained GDP beta S (2 mM) and became irreversible when the non-hydr olysable GTP analogue GTP gamma S (10 mu M) was included in the pipett e. This indicates the participation of G proteins in the inhibitory ef fect of ACh but pretreatment of the cells with either pertussis or cho lera toxin failed to prevent the effect of ACh on the Ca2+ current. 4. ACh remained equally effective as an inhibitor of the whole-cell Ca2 current in the presence of the L-type Ca2+ channel agonist (-)-Bay K 8644 and after partial inhibition of the current by nifedipine. Additi on of omega-agatoxin IVA, omega-conotoxin GVIA or omega-conotoxin MVII C neither affected the peak Ca2+ current amplitude nor the extent of i nhibition produced by ACh. These pharmacological properties indicate t hat ACh acts by inhibiting L-type Ca2+ channels. 5. The inhibitory act ion of ACh on the B-cell Ca2+ current was not secondary to elevation o f [Ca2+](i) and ACh remained equally effective as an inhibitor when Ba 2+ was used as the charge carrier, when [Ca2+](i) was buffered to low concentrations using EGTA and under experimental conditions preventing the mobilization of Ca2+ from intracellular stores. 6. These results suggest that ACh reduces the whole-cell Ca2+ current in the B-cell thr ough a G protein-regulated, voltage- and Ca2+-independent inhibition o f L-type Ca2+ channels.