Evidence for the presence of G-proteins, adenylyl cyclase and phospholipase C activities in lymphatic smooth muscle cell membranes

  • Kelly J
  • Brazil D
  • Clyne C
 et al. 
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Abstract

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 G(s) 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 G(i) 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 β-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(ai2), G(aq), G(al1) and G(β1). 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) β, γ and δ 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 A1F4-, GTP[S], or purified G(βγ) subunits. Finally, no specific binding to membranes of the α1-adrenoceptor ligand [3H]prazosin or the α2-adrenoceptor ligand [3H]yohimbine was obtained. In conclusion, this study provides evidence for a G(s)-dependent stimulation of AC, and for the presence of G(i2) and G(q/l1), 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.

Author-supplied keywords

  • Adenylyl cyclase
  • G-proteins
  • Lymphatics
  • Phospholipase C
  • Plasma membranes
  • Smooth muscle cells

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Authors

  • James Kelly

  • Derek Brazil

  • Colin Clyne

  • Noel G. McHale

  • Peter Gierschik

  • Alan K. Keenan

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