G-protein Specificity in Signaling Pathways That Mobilize Calcium in Insulin-Secreting β-TC3 Cells

Fuel- and receptor-induced signal transduction pathways were investigated in β-TC3 cells, an insulin-secreting cell line. An increase of glucose concentration from 0 to 15 mM and stimulation with 0.5 mM carbachol resulted in up to a twofold increase in insulin secretion by β-TC3 cells, and their simultaneous addition increased insulin release eightfold. In single fura 2–loaded cells, a potentiating effect of carbachol was also observed on glucose-induced intracellular Ca²⁺ mobilization. Immunoblotting with antibodies raised to the COOH-terminal of G-protein α-subunits showed that Gα_i, Gα_o, and Gα_q are present in β-TC3 cells in commensurable quantities. The novel technique of microinjection of anti-Gα antibodies into single β-cell was used to probe the functional role of these G-proteins. Microinjection of anti-Gα_i antibodies into β-TC3 cells had no effect on glucose- and carbachol-induced Ca²⁺ mobilization. However, anti-Gα_q completely inhibited the Ca²⁺-mobilizing effect of carbachol, but not of glucose, within 5 min. Microinjection of anti-Gα_o antibodies had no effect on carbachol-induced Ca²⁺ mobilization. Microinjection of anti-Gα_i and anti-Gα_q antibodies had a minimal effect on glucose-induced Ca²⁺ mobilization (<8% of cells nonresponding), but microinjection of anti-Gα_o increased the proportion of nonresponding cells to 37%. The results suggest that, in β-TC3 cells, distinct signal transduction pathways with specific G-protein involvement may interact with secretagogue-induced Ca²⁺ mobilization and, ultimately, with insulin secretion.