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N. S. Berrow, G. Milligan, and N. G. Morgan


Inhibition of insulin secretion from rat islets of Langerhans is known to involve at least one pertussis toxin-sensitive guanine-nucleotide binding (G) protein. We have used antisera raised against unique antigenic determinants of different members of the family of pertussis toxin-sensitive G proteins to identify these proteins in rat islets. Antiserum SG1, which recognizes both Gi1 and Gi2, reacted with an islet protein having an approximate M r of 40 000. Antiserum IlC (Gi1 specific) failed to recognize any islet proteins, suggesting that Gi2 is present in much greater amounts than Gi1. Indeed, Gi1 levels were below the detection limit of a sensitive immunogold/silver-staining method, indicating that it may be absent from the cells of rat islets.

Two different antisera were used to identify Go-like G proteins in rat islet homogenates. Both antisera reacted with a protein band which, under appropriate conditions, could be resolved to reveal two separate proteins of M r 39–40 000. Thus, at least two molecular forms of Go are present in rat islets.

Subcellular fractionation indicated that all three G proteins identified in this study (Gi2 and two forms of Go) are localized to islet membranes. No immunoreactivity could be detected in the cytosolic fraction.

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R. M. Haigh, C. T. Jones, and G. Milligan


Glucocorticoids are known to influence cardiovascular sensitivity to catecholamines but the molecular mechanisms are undefined. We recently showed that glucocorticoids control the coupling of adrenergic receptors to G protein. Alterations in the amount of G protein is one mechanism by which receptor-G protein coupling may be controlled. Therefore, we set out to measure the levels of G proteins in aorta from normal, adrenalectomized and dexamethasonetreated adrenalectomized rats. G proteins were measured in plasma membrane preparations by immunoblotting and horseradish peroxidase staining. After adrenalectomy there was a 53% (n = 5) decrease in the density of staining for Gi (ANOVA; P<0.05 compared to controls). Conversely, there was a 210% (n = 5) increase in the density of staining for Gs. The levels of Go and the β-subunit of G proteins were not changed by adrenalectomy. Dexamethasone-replacement treatment after adrenalectomy returned Gi and Gs close to control values. Go remained unaltered compared to controls but was 24% (n=3) less than the adrenalectomized values (ANOVA; P<0.05). The levels of β-subunit after dexamethasone replacement were significantly greater (ANOVA; P<0.05) than both the controls and adrenalectomized values. These results show that glucocorticoids can differentially regulate the amounts of G proteins in rat aorta as in other tissues. This may be an important mechanism by which steroids control receptor-G protein coupling and hence transmembrane signalling pathways in vascular smooth muscle.

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A. R. McLellan, S. Tawil, F. Lyall, G. Milligan, J. M. C. Connell, and C. J. Kenyon


Dexamethasone administration in vitro has been shown to increase adenylyl cyclase activity in vascular smooth muscle cells (VSMC) from renal arteries and in non-vascular cell lines. To investigate whether G proteins are involved in this response, cultured VSMC from mesenteric arteries of Sprague—Dawley rats were incubated in the presence and absence of 10 nm dexamethasone for 24 and 48 h. Basal and stimulated adenylyl cyclase activities were increased by approximately 50% after treatment with dexamethasone. The changes were neither specifically associated with ligands which stimulate adenylyl cyclase catalytic unit via Gs (isoproterenol and prostaglandin E1) nor with guanylylimidodiphosphate (0·1 nm), which inhibits the catalytic unit via Gi. This suggests that dexamethasone enhances adenylyl cyclase activity through changes at the level of the catalytic unit, rather than through the G proteins which modulate its activity. No differences were seen in immunoblotting studies of the levels of Giα2, G, Giα3 and β subunits. Similarly, dexamethasone had no effect on the expression of mRNA for Giα2 and G.

The results indicate that glucocorticoid-induced increases of adenylyl cyclase activity are due to changes at the level of the adenylyl cyclase catalytic unit rather than alteration of the levels or turnover of G, Giα2, Giα3 and β subunits in the membranes of VSMC.