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 muscle cells, guanine nucleotide and forskolin stimulated adenylyl cyclase (AC) activity in a concentration-dependent manner, indicative of the presence of the stimulatory G-protein Gs linked to AC. There was no significant enzyme inhibition by low concentrations of guanine nucleotide and no effect on basal or guanine nucleotide-stimulated activity following pertussis toxin treatment of cells, suggesting the absence of Gi linked to inhibition of AC. Furthermore, there was no effect of adrenaline, isoprenaline or clonidine on basal or forskolin-stimulated activities, nor was there any specific binding of the beta-adrenoceptor ligand [125I]cyanopindolol to membranes, suggesting that catecholamine receptors do not modulate AC activity in these membranes. Pertussis toxin-mediated ADP ribosylation of membrane proteins and Western immunoblotting analysis revealed the presence of G-protein subunits G alpha i2, G alpha q, and G beta 1. In experiments designed to identify a possible effector enzyme for these G-proteins, membranes were screened with a range of antibodies raised against phospholipase C (PLC) beta, gamma and delta isozymes. Though no evidence was obtained by Western blotting for any of these proteins, PLC activity was concentration-dependently stimulated by Ca2+, but not by AIF4-, GTP[S], or purified G beta gamma subunits. Finally, no specific binding to membranes of the alpha 1-adrenoceptor ligand [3H]prazosin or the alpha 2-adrenoceptor ligand [3H]yohimbine was obtained. In conclusion, this study provides evidence for a Gs-dependent stimulation of AC, and for the presence of Gi2 and Gq/11, which do not appear to regulate a PLC activity also identified in lymphatic smooth muscle cell membranes. Furthermore, neither AC nor PLC appear to be associated with catecholamine receptors.
Kelly
J
J
Department of Pharmacology, University College Dublin, Belfield, Ireland.
Brazil
D
D
Clyne
C
C
McHale
N G
NG
Gierschik
P
P
Keenan
A K
AK
D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000032:analysis*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D008208:Lymphatic System / Q000737:chemistry / Q000201:enzymology*; D008643:Mesentery / Q000276:immunology; D009130:Muscle, Smooth / Q000737:chemistry / Q000166:cytology / Q000201:enzymology*; D037342:Pertussis Toxin; D010738:Type C Phospholipases / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D010566:Virulence Factors, Bordetella; D005576:Colforsin; D006165:Guanylyl Imidodiphosphate; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases