Inhibition of Voltage-Dependent Ca2+Currents and Activation of Pertussis Toxin-Sensitive G-Proteins via Muscarinic Receptors in GH3Cells

Abstract

In the rat pituitary cell line GH₃, carbachol inhibits PRL secretion in a pertussis toxin-sensitive manner. For elucidation of the underlying mechanisms, we studied the effect of carbachol on voltage-dependent Ca²⁺ currents. Under voltage-clamp conditions, carbachol inhibited whole-cell Ca²⁺ currents by about 25%. This inhibitory action of carbachol was not observed in cells treated with pertussis toxin, indicating the involvement of a pertussis toxin-sensitive G-protein. In membranes of GH₃ cells, carbachol stimulated a pertussis toxin-sensitive highaffinity GTPase. In immunoblot experiments with peptide antisera, we identified two forms of the G_i α-subunit (41 and 40 kDa) and two forms of the G_o α-subunit (40 and 39 kDa). The 40-kDa G_i α-subunit was recognized by an antibody specific for the G_i2 α-subunit, and the 39-kDa G_o α-subunit was detected by an antibody specific for the G_o2 α-subunit. Incubation of membranes with the photoreactive GTP analog [α-³²P]GTP azidoanilide resulted in photolabeling of 40- and 39-kDa pertussis toxin substrates comigrating with G-protein α-subunits of the corresponding molecular masses. Carbachol dose-dependently stimulated incorporation of the photoreactive GTP analog into the 39-kDa pertussis toxin substrate and, to a lesser extent, into 40-kDa pertussis toxin substrates. The data indicate that muscarinic receptors of GH₃ cells couple preferentially to G_o, which is likely to be involved in the inhibition of secretion, possibly by conferring an inhibitory effect to voltage-dependent Ca²⁺ channels.