EVIDENCE FOR THE PRESENCE OF G-PROTEINS, ADENYLYL-CYCLASE AND PHOSPHOLIPASE-C ACTIVITIES IN LYMPHATIC SMOOTH-MUSCLE CELL-MEMBRANES

In plasma membranes derived from bovine mesenteric lymphatic smooth mu scle cells, guanine nucleotide and forskolin stimulated adenylyl cycla se (AC) activity in a concentration-dependent manner, indicative of th e presence of the stimulatory G-protein G(s) linked to AC. There was n o significant enzyme inhibition by low concentrations of guanine nucle otide and no effect on basal or guanine nucleotide-stimulated activity following pertussis toxin treatment of cells, suggesting the absence of G(1) linked to inhibition of AC. Furthermore, there was no effect o f adrenaline, isoprenaline or clonidine on basal or forskolin-stimulat ed activities, nor was there any specific binding of the beta-adrenoce ptor ligand [I-125]cyanopindolol to membranes, suggesting that cate-ch olamine receptors do not modulate AC activity in these membranes. Pert ussis toxin-mediated ADP ribosylation of membrane proteins and Western immunoblotting analysis revealed the presence of G-protein subunits G (alpha l2), G(alpha q), G(alpha 11) and G(beta 1). In experiments desi gned to identify a possible effector enzyme for these G-proteins, memb ranes were screened with a range of antibodies raised against phosphol ipase C (PLC) beta, gamma and delta isozymes. Though no evidence was o btained by Western blotting for any of these proteins, PLC activity wa s concentration-dependently stimulated by Ca2+, but not by AlF4-, GTP[ S], or purified G(beta gamma) subunits. Finally, no specific binding t o membranes of the alpha(1)-adrenoceptor ligand [H-3]prazosin or the a lpha(2)-adrenoceptor ligand [H-3]yohimbine was obtained. In conclusion , this study provides evidence for a G(s)-dependent stimulation of AC, and for the presence of G(2) and G(q11), which do not appear to regul ate a PLC activity also identified in lymphatic smooth muscle cell mem branes. Furthermore, neither AC nor PLC appear to be associated with c atecholamine receptors. Copyright(C) 1996 Elsevier Science Inc.