Phosphoinositide 3-kinase gamma mediates angiotensin II-induced stimulation of L-type calcium channels in vascular myocytes.
Previous results have shown that in rat portal vein myocytes the betagamma dimer of the G(13) protein transduces the angiotensin II-induced stimulation of calcium channels and increase in intracellular Ca(2+) concentration through activation of phosphoinositide 3-kinase (PI3K). In the present work we determined which class I PI3K isoforms were involved in this regulation. Western blot analysis indicated that rat portal vein myocytes expressed only PI3Kalpha and PI3Kgamma and no other class I PI3K isoforms. In the intracellular presence of an anti-p110gamma antibody infused by the patch clamp pipette, both angiotensin II- and Gbetagamma-mediated stimulation of Ca(2+) channel current were inhibited, whereas intracellular application of an anti-p110alpha antibody had no effect. The anti-PI3Kgamma antibody also inhibited the angiotensin II- and Gbetagamma-induced production of phosphatidylinositol 3,4,5-trisphosphate. In Indo-1 loaded cells, the angiotensin II-induced increase in [Ca(2+)](i) was inhibited by intracellular application of the anti-PI3Kgamma antibody, whereas the anti-PI3Kalpha antibody had no effect. The specificity of the anti-PI3Kgamma antibody used in functional experiments was ascertained by showing that this antibody did not recognize recombinant PI3Kalpha in Western blot experiments. Moreover, anti-PI3Kgamma antibody inhibited the stimulatory effect of intracellularly infused recombinant PI3Kgamma on Ca(2+) channel current without altering the effect of recombinant PI3Kalpha. Our results show that, although both PI3Kgamma and PI3Kalpha are expressed in vascular myocytes, the angiotensin II-induced stimulation of vascular L-type calcium channel and increase of [Ca(2+)](i) involves only the PI3Kgamma isoform.
Quignard
J F
JF
Laboratoire de Signalisation et Interactions Cellulaires, CNRS UMR 5017, Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux, France.
Mironneau
J
J
Carricaburu
V
V
Fournier
B
B
Babich
A
A
Nurnberg
B
B
Mironneau
C
C
Macrez
N
N
D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001464:Barium / Q000494:pharmacology; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000502:physiology*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D058544:Class Ib Phosphatidylinositol 3-Kinase; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008861:Microsomes / Q000201:enzymology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D018408:Patch-Clamp Techniques; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011169:Portal Vein / Q000502:physiology; D021122:Protein Subunits; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D000906:Antibodies; D020746:Calcium Channels, L-Type; D007527:Isoenzymes; D021122:Protein Subunits; D011994:Recombinant Proteins; D000804:Angiotensin II; D001464:Barium; D015240:Phorbol 12,13-Dibutyrate; D019869:Phosphatidylinositol 3-Kinases; D058544:Class Ib Phosphatidylinositol 3-Kinase; C546765:Pik3cg protein, rat; D002118:Calcium