Integrin ligands mobilize Ca2+ from ryanodine receptor-gated stores and lysosome-related acidic organelles in pulmonary arterial smooth muscle cells.
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.
Umesh
Anita
A
Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA.
Thompson
Michael A
MA
Chini
Eduardo N
EN
Yip
Kay-Pong
KP
Sham
James S K
JS
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002478:Cells, Cultured; D005453:Fluorescence; D016023:Integrins / Q000378:metabolism*; D008024:Ligands; D008247:Lysosomes / Q000378:metabolism*; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D015388:Organelles / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology; D011485:Protein Binding; D011651:Pulmonary Artery / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*
D016023:Integrins; D008024:Ligands; D009842:Oligopeptides; D010446:Peptide Fragments; D012333:RNA, Messenger; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D002118:Calcium