Male Sprague-Dawley rats were injected ip with 1 microgram pertussis toxin (PTX)/100 g BW. The rats were killed 24, 48, and 72 h after injection, and their pancreases were removed. At each time point, insulin secretion by isolated islets was measured under basal and glucose-stimulated conditions and in the absence and presence of norepinephrine. cAMP levels were measured under basal and forskolin-stimulated conditions in the absence and presence of norepinephrine. PTX-induced ADP ribosylation of Gi/Go proteins in vivo was monitored by ADP ribosylation in vitro using PTX and 32P-labeled NAD and also by Western blotting. At 24 h, 1 microM norepinephrine inhibited glucose-stimulated insulin secretion by 92% in the control islets, but by only 53% in the PTX-treated islets; at 48 h, norepinephrine still inhibited secretion (by 40%) in the PTX-treated islets; at 72 h, the inhibitory effect of norepinephrine was abolished. Therefore, contrary to recent suggestions, all of the effect of norepinephrine to inhibit insulin release is PTX sensitive. The effects of PTX on the ability of norepinephrine to lower cAMP levels were similar to those observed for the inhibition of insulin release. PTX partially blocked the effect of norepinephrine to lower cAMP levels at 24 and 48 h, and the block was complete after 72 h. The extent of the in vivo ADP ribosylation of the Gi/Go proteins, monitored at each time point by in vitro [32P]ADP-ribosylation and Western blotting, demonstrated a profound ADP ribosylation at 48 and 72 h. As detected by Western blotting, the rates of ADP ribosylation by PTX and the onset of decreased expression varied among the different G-proteins. G alpha o was virtually eliminated after only 24 h of PTX treatment. G alpha i2 was markedly affected by 48 h; G alpha i3 was little affected until 72 h.
Inhibition of insulin secretion by galanin is pertussis toxin (PTX) sensitive, suggesting the activation of one or more heterotrimeric (alpha, beta, gamma) G-proteins (Gi/Go). Multiple effectors, including the K+ATP and L-type Ca2+ channels, adenylyl cyclase, and an as yet unidentified system at a site close to exocytosis, are modulated by galanin. Therefore, it is necessary to delineate the particular G-proteins activated by the galanin receptor as a first step to understanding its net cellular response. During specific conditions, cholera toxin (CTX) can ADP-ribosylate the alpha i/alpha o-subunits of the PTX-sensitive substrates but only during receptor/G-protein interaction. Therefore, we used CTX-catalyzed ADP ribosylation to identify galanin receptor-associated G-protein alpha-subunits in RINm5F cells. Galanin enhanced the ADP ribosylation of membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in two bands at 39,000 and 42,000 M(r). This labeling was blocked in membranes prepared from PTX-treated cells, enhanced by Mg2+, and showed a biphasic dependence on exogenous guanine nucleotides. Identification of the CTX ADP-ribosylated G-proteins by immunoprecipitation with selective antisera indicate activation by the galanin receptor of alpha i1 and alpha i3, which have the same mobility on SDS-PAGE (42,000 M(r)), and alpha i2 (39,000 M(r)). These studies provide evidence for the activation of multiple G-proteins by receptors for galanin in RINm5F cells.
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