Abstract
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.
Highlights
Ʈ To whom correspondence should be addressed: Laboratoire de Signalisation et Interactions Cellulaires, CNRS UMR 5017, Universite Bordeaux 2, 146 rue Leo Saignat, 33076 Bordeaux, France
We have shown that rat portal vein myocytes express both phosphoinositide 3-kinase (PI3K)␣ and -␥, but only PI3K␥ is involved in the regulation of L-type calcium channels by Angiotensin II (AII)
The inactive isoform of PDBu (4␣ PDBu) did not stimulate the Ba2ϩ current in the same batches of cells that were stimulated by PDBu. These results suggest that the stimulatory effects of AII and G␥ on L-type Ca2ϩ channels are mediated via activation of the PI3K␥, which appears to be the upstream effector of protein kinase C (PKC)
Summary
Ʈ To whom correspondence should be addressed: Laboratoire de Signalisation et Interactions Cellulaires, CNRS UMR 5017, Universite Bordeaux 2, 146 rue Leo Saignat, 33076 Bordeaux, France. In A7r5 smooth muscle cells, AII stimulates calcium channels via a tyrosine kinase and a undetermined phosphoinositide 3-kinase (PI3K) [2]. G protein-coupled receptors may stimulate a p85-dependent PI3K as described in human vascular smooth muscle cells [17], and PI3K has been shown to be activated by G␥ [15, 18]. The purpose of this study was to investigate which types of class I PI3K isoforms could be activated by AII to stimulate voltage-gated calcium channel activity. The same antibodies were used in patch clamp experiments to identify the isoforms involved in the transduction pathway coupling AII receptors to stimulation of Ca2ϩ channels. We have shown that rat portal vein myocytes express both PI3K␣ and -␥, but only PI3K␥ is involved in the regulation of L-type calcium channels by AII
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