G-protein specificity in signaling pathways that mobilize calcium in insulin-secreting beta-TC3 cells.
Fuel- and receptor-induced signal transduction pathways were investigated in beta-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 beta-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 alpha-subunits showed that G alpha i, G alpha o, and G alpha q are present in beta-TC3 cells in commensurable quantities. The novel technique of microinjection of anti-G alpha antibodies into single beta-cell was used to probe the functional role of these G-proteins. Microinjection of anti-G alpha i antibodies into beta-TC3 cells had no effect on glucose- and carbachol-induced Ca2+ mobilization. However, anti-G alpha q completely inhibited the Ca(2+)-mobilizing effect of carbachol, but not of glucose, within 5 min. Microinjection of anti-G alpha o antibodies had no effect on carbachol-induced Ca2+ mobilization. Microinjection of anti-G alpha i and anti-G alpha q antibodies had a minimal effect on glucose-induced Ca2+ mobilization (< 8% of cells nonresponding), but microinjection of anti-G alpha o increased the proportion of nonresponding cells to 37%. The results suggest that, in beta-TC3 cells, distinct signal transduction pathways with specific G-protein involvement may interact with secretagogue-induced Ca2+ mobilization and, ultimately, with insulin secretion.
Baffy
G
G
Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia 19104-6089.
Yang
L
L
Wolf
B A
BA
Williamson
J R
JR
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005947:Glucose / Q000502:physiology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D015398:Signal Transduction
D000906:Antibodies; D007328:Insulin; D002217:Carbachol; D019204:GTP-Binding Proteins; D005947:Glucose; D002118:Calcium