In rat pituitary GH4C1 cells, activation of transfected dopamine D2 receptors (long, D2L, or short, D2S, form) and endogenously expressed somatostatin and muscarinic M4 receptors induced inhibition of cAMP synthesis and of Bay K 8644-induced calcium entry via pertussis toxin-sensitive G proteins. To analyze the role of alpha 0 and alpha i2 in relaying of these signals, alpha 0 or alpha i2 antisense constructs were separately and stably transfected into GH4C1 cells. Reverse transcription-polymerase chain reaction and Western blot analyses indicated specific ablation of alpha 0 or alpha i2 in the antisense transfectant clones. Elimination of alpha 0 selectively abolished receptor-mediated inhibition of calcium entry. Notably, the action of dopamine D2L receptor was partially (about 30%) retained. By contrast, depletion of alpha i2 selectively impaired receptor-mediated inhibition of cAMP accumulation. Inhibition of basal cAMP synthesis by any of the four receptors studied was blocked in alpha i2-depleted clones. Additionally, dopamine D2L, somatostatin and muscarinic M4 receptor-mediated inhibition of vasoactive intestinal peptide-stimulated cAMP formation was also abolished. Remarkably, somatostatin even potentiated (by 30%) the action of vasoactive intestinal peptide in alpha i2-antisense clones. In contrast, the action of dopamine D2S receptor on stimulated cAMP synthesis remained largely unaltered. The results demonstrate that alpha 0 specifically triggers receptor-induced closure of calcium channels, whereas alpha i2 specifically mediates inhibition of adenylyl cyclase in GH4C1 cells. Furthermore, the data suggest that G1 protein specificity in receptor coupling to inhibition of adenylyl cyclase depends critically on the activity state of the enzyme. Moreover, the results indicate an essential difference in coupling of dopamine D2L and D2S receptors to G proteins.