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 α₁, α₂, α₃, α₄, α₅, α₇, α₈, α_v, β₁, β₃, and β₄, and immunoblots of α₅, α_v, β₁, and β₃ 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 Ca ²⁺ 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.