Cholera toxin modulation of angiotensin II-stimulated inositol phosphate production in cultured vascular smooth muscle cells

Abstract

Activation of phospholipase C by angiotensin II in vascular smooth muscle has been postulated to be mediated by an unidentified GTP-binding protein (G-protein). Using a permeabilized preparation of myo-[3H]inositol-labelled cultured vascular smooth muscle cells, we examined the ability of a non-hydrolysable analogue of GTP, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), to stimulate inositol phosphate formation. GTP[S] (5 min exposure) stimulated inositol polyphosphate release by up to 3.8-fold in a dose-dependent manner, with an EC50 (concn. producing half-maximal stimulation) of approx. 50 microM. Inositol bisphosphate (IP2) and inositol trisphosphate (IP3) accumulations were also stimulated by NaF (5-20 mM). Furthermore, angiotensin II-induced inositol phosphate formation could be potentiated by a submaximal concentration of GTP[S] (10 microM), and this treatment appeared to interfere with the normal termination mechanism of the initial hormonal signal. The G-protein mediating angiotensin II-stimulated phospholipase C activation was insensitive to pertussis toxin at an exposure time and concentration which were sufficient to completely ADP-ribosylate all available substrate (100 ng/ml, 16 h). In contrast, a similar incubation with cholera toxin markedly inhibited angiotensin II-stimulated IP2 and IP3 release by 67 +/- 6% and 62 +/- 6% respectively. Cholera toxin appeared to inhibit angiotensin II stimulation of phospholipase C by a dual mechanism: it caused a 45% decrease in angiotensin II receptor number, and also inhibited G-protein transduction as assessed by GTP[S]-stimulated IP2 formation. This latter inhibition may be secondary to an increase in cyclic AMP, since it could be simulated by addition of dibutyryl cyclic AMP. Thus angiotensin II-stimulated inositol phosphate formation is cholera-toxin-sensitive, and is mediated by a pertussis-toxin-insensitive G-protein, which may be involved directly in termination of early signal generation.

G-Protein binding domains of the angiotensin II AT1A receptors mapped with synthetic peptides selected from the receptor sequence

“…It has been shown that Gα q proteins mediate AngII-induced activation of phospholipase C-β " [2,5], and that Gα "$ couples the AT "A R to Ca# + mobilization in the portal vein [3]. Although immunoblotting analysis showed that α subunits of G i and G s are expressed to a similar extent as G q in VSMC [31], we have shown that AngII-induced IP $ generation is not sensitive to pertussis toxin and that activation of adenylate cyclase is not induced by AngII in VSMC [19]. Furthermore, in the study by Shirai et al [15], a peptide corresponding to amino acids 306-320 was a potent activator of Gα i and Gα o , in contrast to the observation that a similar peptide had no effect on basal GTPase activity in our system.…”

“…Using membrane fractions, [$H]AngII binding was measured as described previously [19], with minor modifications. Briefly, the binding reaction was initiated by the addition of the membrane suspension (100-200 µg of protein) to the binding assay buffer containing the appropriate concentration of [$H]AngII.…”

Vascular smooth muscle cells from the Milan hypertensive rat exhibit decreased functional angiotensin II receptors.

“…To study the coupling of guanine nucleotide (GTP)-binding proteins to PLC, tritium-labeled VSM cells were permeabilized with saponin, 10 and phosphoinositol release determined in the absence and presence of a GTP analogue. In brief, [ 3 H]myoinositol-labeled VSM monolayers (35 mm) were washed with TBS.…”

“…11 Addition of the nonhydrolyzable guanosine-5'-O-(3-thiotriphosphate) (GTP-y-S) analogue to saponin-permeabilized VSM monolayers also resulted in inositol polyphosphate release. 10 These and synergism studies between GTP-y-S and Ang II demonstrated that the Ang H-induced PLC activation is coupled to a GTP-binding protein. 10 The occupied Ang II receptor also elicits activation of Na + -H + exchange in cultured VSM cells from SD rats.…”

“…10 These and synergism studies between GTP-y-S and Ang II demonstrated that the Ang H-induced PLC activation is coupled to a GTP-binding protein. 10 The occupied Ang II receptor also elicits activation of Na + -H + exchange in cultured VSM cells from SD rats. 12 -14 Cultured VSM cells from SHR, when compared with WKY cells, exhibited hyperresponsiveness to Ang II receptors as determined by stimulation of Na + -H + exchange and increased growth.…”