Effect of caveolin-1 scaffolding peptide and 17β-estradiol on intracellular Ca2+ kinetics evoked by angiotensin II in human vascular smooth muscle cells

Abstract

Caveolae are identifiable plasma membrane invaginations. The main structural proteins of caveolae are the caveolins. There are three caveolins expressed in mammals, designated Cav-1, Cav-2, and Cav-3. It has been postulated that Cav-1 acts as a scaffold protein for signaling proteins; these include ion channels, enzymes, and other ligand receptors like membrane-associated estrogen receptor (ER)α or ERβ. Caveolae-associated membrane proteins are involved in regulating some of the rapid estrogenic effects of 17β-estradiol. One important system related to the activity of ERα and caveolae is the renin-angiotensin system. Angiotensin II (ANG II) has numerous actions in vascular smooth muscle, including modulation of vasomotor tone, cell growth, apoptosis, phosphatidylinositol 3-kinase (PI3K)/Akt activation, and others. Many proteins associated with caveolae are in close relation with the scaffolding domain of Cav-1 (82–101 amino acid residues). It has been proposed that this peptide may acts as a kinase inhibitor. Therefore, to explore the ability of Cav-1 scaffolding peptide (CSP-1) to regulate ANG II function and analyze the relationship between ERα and ANG II type 1 and 2 (AT1 and AT2) receptors, we decided to study the effects of CSP-1 on ANG II-induced intracellular Ca2+ kinetics and the effect of 17β-estradiol on this modulation using human smooth muscle cells in culture, intracellular Ca2+ concentration measurements, immuno- and double-immunocytochemistry confocal analysis of receptor expression, immunoblot analysis, and immunocoprecipitation assays to demonstrate coexpression. We hypothesized that CSP-1 inhibits ANG II-mediated increases in intracellular Ca2+ concentrations by interfering with intracellular signaling including the PI3K/Akt pathway. We also hypothesize that AT2 receptors associate with Cav-1. Our results show that there is a close association of AT1, AT2, and ERα with Cav-1 in human arterial smooth muscle cells in culture. CSP-1 inhibits ANG II-induced intracellular signaling.