Isolated cells from rat portal vein smooth muscle in short-term primary culture were studied using patch-clamp technique (whole-cell configuration). In order to study a calcium-activated chloride current, the potassium currents were blocked by intracellular cesium diffusion. Without EGTA in the pipette solution, depolarizing voltage pulses from a holding potential of -70 mV to positive potentials activated an early inward and a late outward current. The latter persisted as a long-lasting inward tail current when the membrane was repolarized to -70 mV. The outward current measured at the end of the pulse and the tail current were blocked by extracellular cobalt, after replacement of external calcium with barium, after removal of external calcium, and when the calcium concentration of the pipette solution was less than 0.5 microM, suggesting that they were calcium-dependent. The tail current decay was voltage sensitive, becoming faster with hyperpolarization. The reversal potential of the calcium-activated current was near the equilibrium potential for chloride ions, and was shifted as predicted by the Nernst equation when the extracellular or intracellular chloride concentration was changed. The calcium-activated current was blocked by adding micromolar concentrations of niflumic acid or millimolar concentrations of DIDS. This effect of compounds known to interfere with chloride channels together with the data on the equilibrium potential for chloride ions indicated above suggested the existence of a calcium-activated chloride current in vascular smooth muscle cells.

Previous results have shown that in rat portal vein myocytes the ␤␥ 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 PI3K␣ and PI3K␥ and no other class I PI3K isoforms. In the intracellular presence of an anti-p110␥ antibody infused by the patch clamp pipette, both angiotensin II-and G␤␥-mediated stimulation of Ca 2؉ channel current were inhibited, whereas intracellular application of an anti-p110␣ antibody had no effect. The anti-PI3K␥ antibody also inhibited the angiotensin IIand G␤␥-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-PI3K␥ antibody, whereas the anti-PI3K␣ antibody had no effect. The specificity of the anti-PI3K␥ antibody used in functional experiments was ascertained by showing that this antibody did not recognize recombinant PI3K␣ in Western blot experiments. Moreover, anti-PI3K␥ antibody inhibited the stimulatory effect of intracellularly infused recombinant PI3K␥ on Ca 2؉ channel current without altering the effect of recombinant PI3K␣. Our results show that, although both PI3K␥ and PI3K␣ 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 PI3K␥ isoform.

Abstract-Heterodimeric class I phosphoinositide 3-kinase (PI3K) has been shown to be involved in the stimulation of voltage-gated Ca 2ϩ channels by various mediators. In this study, we bring evidences that vascular L-type Ca 2ϩ channels can be modulated by both tyrosine kinase-regulated class Ia and G protein-regulated class Ib PI3Ks. Purified recombinant PI3Ks increased the peak Ca 2ϩ channel current density when applied intracellularly. Furthermore, PI3K␣-, ␤-, and ␦-mediated stimulations of Ca 2ϩ channel currents were increased by preactivation by a phosphotyrosyl peptide, whereas PI3K␥-and ␤-mediated effects were increased by G␤␥. In freshly isolated and cultured vascular myocytes, angiotensin II and G␤␥ stimulated L-type Ca 2ϩ channel current. In contrast, platelet-derived growth factor (PDGF)-BB and the phosphotyrosyl peptide did not stimulate Ca 2ϩ channel current in freshly isolated cells despite the presence of endogenous PDGF receptors and PI3K␣ and PI3K␥. Interestingly, when endogenous PI3K␤ expression arose in cultured myocytes, both PDGF and phosphotyrosyl peptide stimulated Ca 2ϩ channels through PI3K␤, as revealed by the inhibitory effect of an anti-PI3K␤ antibody. These results suggest that endogenous PI3K␤ but not PI3K␣ is specifically involved in PDGF receptor-

Abstract-Modulation 6 -8 This mechanism seems to underlie the increased vascular spontaneous tone observed in hypertensive rats. 9 However, how PI3K is able to regulate Ca 2ϩ channels activity remains to be elucidated. 10 Class I PI3Ks are enzymes that selectively phosphorylate the 3Ј-OH position of the PI(4,5)P 2 inositol ring in vivo to generate PI(3,4,5)P 3 , further metabolized by inositol lipid phosphatases to PI(3,4)P 2 . PI(3,4)P 2 and PI(3,4,5)P 3 are absent in resting cells, increase on class I PI3K activation during cellular stimulation, and interact with pleckstrin homology (PH) domains of cellular proteins to transduce signal. Class I PI3Ks have been subclassified according to their structure and mode of activation by cell surface receptors. Class IA PI3Ks are heterodimers composed of a catalytic subunit (the ubiquitous p110␣ or more tissue-restricted p110␤, or p110␦) tightly complexed to a regulatory adapter subunit (p85␣, p85␤, p55, or their splice variants). These regulatory subunits dock the holoenzyme to the membrane through interactions with specific phosphotyrosyl-containing sequences within receptor tyrosine kinases or other membrane-associated proteins. Class IB PI3K is composed of the p110␥ catalytic subunit associated with a p101 regulatory protein. PI3K␥ is specifically stimulated by G␤␥ dimers liberated on G protein-coupled receptor (GPCR) activation. The p110␥ catalytic subunit contains all the structural elements necessary for G␤␥-induced stimulation. The p101 noncatalytic regulatory subunit, which is able to bind lipid substrates, increase p110␥ activity. 11 PI3K␤ is synergistically activated by GPCRs and receptor tyrosine kinases. Like for other class IA enzymes, phosphotyrosyl peptide interaction with the p85 regulatory subunit leads to activation of PI3K␤. However, p110␤ catalytic subunits can be directly activated by membrane-bound ␤␥ dimers. 12 Original