MOLECULAR AND FUNCTIONAL-STUDIES OF INHIBITORY G-PROTEIN IN RINM5F CELLS

Inhibitory G proteins (G(i)) play an important role in cell proliferat ion. In order to characterize G(i) proteins in RINm5F (RIN) cells, we first established RIN cells in cell culture. Immunoblot analysis was p erformed on extracted G proteins using Western blot techniques and a G (i)-specific antibody. We identified three prominent bands consistent with three distinct inhibitory cu subunits of membrane-bound G protein (G(i)) in RIN cells. In contrast, we identified only one prominent di stinct inhibitory alpha subunit of G protein in an equal quantity of m embrane-protein in our control (normal rat pancreas). In several cell types, G(i) is known to mediate the inhibitory action of somatostatin on intracellular cyclic AMP (cAMP) accumulation. Therefore, we studied the action of the long-acting analogue of somatostatin, octreotide (S MS), on basal and 3-isobutyl-1-methylxanthine-stimulated cAMP accumula tion in RIN cells. SMS did not inhibit cAMP accumulation or tritiated thymidine incorporation into DNA (TTID) in RIN cells. However, when tr eatment with SMS is supplemented with the nonhydrolyzable analogue of guanine nucleotide, Gpp(NH)p (Gpp), which is known to dissociate G pro teins into its constitutive subunits, then SMS + Gpp induced an inhibi tory action and significantly reduced cAMP accumulation and TTID. Thes e data are consistent with the concept of qualitatively and functional ly altered inhibitory G protein expression in the insulin-producing, i slet cell (RINm5F) rat insulinoma tumor cell line. Further study of hu man tumors will lead to new insights into the clinical implications of G protein-mediated signal transduction in insulinoma. (C) 1994 Academ ic Press, Inc.