Cholera toxin modulation of angiotensin II-stimulated inositol phosphate production in cultured vascular smooth muscle cells.
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.
Socorro
L
L
Department of Medicine, Brigham and Women's Hospital, Boston, MA.
Alexander
R W
RW
Griendling
K K
KK
D000246:Adenosine Diphosphate Ribose / Q000032:analysis; D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000032:analysis; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology*; D003864:Depression, Chemical; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011945:Receptors, Angiotensin / Q000378:metabolism; D012969:Sodium Fluoride / Q000494:pharmacology; D013873:Thionucleotides / Q000378:metabolism
D011945:Receptors, Angiotensin; D013873:Thionucleotides; D000804:Angiotensin II; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D003994:Bucladesine; D015544:Inositol 1,4,5-Trisphosphate; D006160:Guanosine Triphosphate; D012969:Sodium Fluoride; D002772:Cholera Toxin; D002118:Calcium