Abstract
Extracellular matrix (ECM) protein receptors, or integrins, participate in vascular remodeling and the systemic myogenic response. Synthetic ligands and ECM fragments regulate the vascular smooth muscle cell contractile state by altering intracellular Ca2+ levels ([Ca2+]i). Information on the Ca2+ effect of integrins in vascular smooth muscle cells is limited, but nonexistent in pulmonary arterial smooth muscle cells (PASMCs). We therefore characterized integrin expression in endothelium-denuded pulmonary arteries, and explored [Ca2+]i mobilization pathways induced by soluble ligands in rat PASMCs. Reverse transcriptase-PCR showed mRNA expression of integrins alpha1, alpha2, alpha3, alpha4, alpha5, alpha7, alpha8, alpha(v), beta1, beta3, and beta4, and immunoblots of alpha5, alpha(v), beta1, and beta3 confirmed protein expression. Exposure of PASMCs to integrin-binding peptides (0.5 mM) containing the arginine-glycine-aspartate (RGD) motif elicited [Ca2+]i responses with an order of potency of GRGDNP > GRGDSP > GRGDTP = cyclo-RGD. Pharmacological analysis revealed that the GRGDSP-induced Ca2+ response was unrelated to Ca2+ influx and the inositol triphosphate receptor-gated Ca2+ store, but partially blocked by ryanodine or inhibition of lysosome-related acidic organelles with bafilomycin A1. Simultaneous inhibition of both pathways was necessary to abolish the response. GRGDSP treatment increased cyclic ADP-ribose, the endogenous activator of ryanodine receptors, by 70%. GRGDSP also rapidly reduced Lysotracker Red accumulation, confirming direct modulation of acidic organelles. These data are the first demonstration of integrin-mediated Ca2+ regulation in PASMCs. The presence of an array of integrins, and activation of ryanodine-sensitive Ca2+ stores and lysosome-like organelles by GRGDSP suggest important roles for integrin-dependent Ca2+ signaling in regulating PASMC function.
Highlights
Integrins participate in controlling systemic vascular tone by invoking changes in smooth muscle [Ca2ϩ]i, resulting in relaxation or contraction
We characterize intracellular Ca2ϩ signaling by integrins in rat pulmonary arterial smooth muscle cells (PASMCs)
We show the presence of multiple integrins in PASMCs, and the initiation of intracellular Ca2ϩ
Summary
Isolation and Culture of PASMCs—PASMCs were enzymatically isolated and transiently cultured as previously described [17]. Arteries were incubated in ice-cold HBSS (30 min), and in reduced Ca2ϩ (20 M) HBSS (20 min, room temperature), upon which they were digested in reduced Ca2ϩ HBSS containing collagenase (Type I, 1750 units/ml), papain (9.5 units/ml), bovine serum albumin (2 mg/ml), and dithiothreitol (1 mM), at 37 °C for 18 min. RNA Isolation and RT-PCR—Intralobar pulmonary arteries and aorta were removed, cleaned of connective tissue, denuded of endothelium as above, frozen in liquid nitrogen, and kept at Ϫ80 °C until use. Total protein from cultured PASMCs were isolated by scraping cells with a rubber policeman in the ice-cold Tris-HCl buffer and processed as described for the smooth muscle tissue. After washing in PBST, membranes were incubated with horseradish peroxidase-coupled donkey anti-rabbit or sheep anti-mouse secondary antibodies (Amersham Biosciences) diluted in 1% bovine serum albumin/ PBST (1 h, room temperature), again washed extensively, and detected using enhanced chemiluminescence (Amersham Biosciences). Images were acquired with a Zeiss LSM-510 inverted confocal microscope (Carl Zeiss Inc., Germany) using
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