Integrin Ligands Mobilize Ca2+ from Ryanodine Receptor-gated Stores and Lysosome-related Acidic Organelles in Pulmonary Arterial Smooth Muscle Cells

Abstract

store, but partially blocked by ryanodine or inhibition of lysosomerelated 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 Ca 2؉ regulation in PASMCs. The presence of an array of integrins, and activation of ryanodine-sensitive Ca 2؉ stores and lysosome-like organelles by GRGDSP suggest important roles for integrin-dependent Ca 2؉ signaling in regulating PASMC function. Extracellular matrix (ECM)2 protein receptors, or integrins, comprise a superfamily of structurally related heterodimeric transmembrane receptors that mediate cell-cell and cell-ECM interactions. Integrins physically bridge the ECM and cytoskeleton, and act as transducers of "outside-in" and "inside-out" signaling (1). Extracellular integrin ligation changes [Ca 2ϩ ] i in a variety of cell types, including platelets, neutrophils, monocytes, lymphocytes, fibroblasts, endothelial cells, osteoclasts, neurons, glomerulosa cells, epithelial and smooth muscle cells (2-5). Integrin ligation also activates other intracellular signaling molecules, including H ϩ and a plethora of protein kinases such as the focal adhesion kinase, Rac, and extracellular signal regulated kinases (1, 6). As a result, a myriad of cellular functions such as differentiation, proliferation, migration, apoptosis, and mechanosensing for shear and tension are affected (6, 7).Integrins participate in controlling systemic vascular tone by invoking changes in smooth muscle [Ca 2ϩ ] i , resulting in relaxation or contraction. Synthetic ligands carrying the prototypic integrin-binding tripeptide motif, arginine-glycine-aspartate (RGD), decrease [Ca 2ϩ ] i causing vasodilation in rat cremaster muscle arterioles (8, 9). On the other hand, RGD-containing peptides constrict rat renal afferent arterioles through increased [Ca 2ϩ ] i (10, 11). Other integrin binding sequences also exist, such as leucine-aspartate-valine (LDV), which elevates [Ca 2ϩ ] i and causes vasoconstriction of rat cremaster muscle arterioles (12). Integrins ␣ 5 ␤ 1 and ␣ v ␤ 3 are, furthermore, directly involved in the vascular myogenic behavior, where blockade of either integrin inhibits myogenic constriction of skeletal muscle arterioles (13).Apart from regulating tone, integrins play an important role in vascular pathogenesis. This is evidenced by the remodeling and deposition of ECM components seen in various vascular diseases such as atherosclerosis, hypertension, and restenosis (5). In the pulmonary vasculature, remodeling occurs in chronic obstructive pulmonary disease, asthma, scleroderma, and pulmonary hypertension (14). Interestingly, pulmonary vascular remodeling has been linked to integrin activation in rat models recapitulating pulmonary hypertension. Previo...

CD38 Mediates Angiotensin II–Induced Intracellular Ca2+ Release in Rat Pulmonary Arterial Smooth Muscle Cells

“…Ca 21 signaling in pulmonary arterial smooth muscle cells (PASMCs) is important in many different physiological processes, including contraction, proliferation, migration, and gene transcription. CD38 is a master Ca 21 regulator that catalyzes the formation of the endogenous Ca 21 -mobilizing messengers cyclic adenosine diphosphate ribose and nicotinic acid adenosine dinucleotide phosphate (NAADP) for the activation of ryanodine receptors of sarcoplasmic reticulum and NAADP-sensitive Ca 21 release channels in endolysosomes, respectively.…”

“…CD38 is a master Ca 21 regulator that catalyzes the formation of the endogenous Ca 21 -mobilizing messengers cyclic adenosine diphosphate ribose and nicotinic acid adenosine dinucleotide phosphate (NAADP) for the activation of ryanodine receptors of sarcoplasmic reticulum and NAADP-sensitive Ca 21 release channels in endolysosomes, respectively. However, its physiological roles in PASMCs have not been examined systematically.…”

“…release from endolysosomes (4). Recent studies have identified the two-pore channels (TPC1 and TPC2) as the NAADP-activated Ca 21 release channels (5,6). It has been suggested that NAADP binds directly to TPCs or to an accessory protein of the TPC complex to activate endolysosomal Ca 21 release (7).…”

“…In general, these vasoconstrictory agonists of G-protein-coupled receptors activate phospholipase C (PLC) to synthesize inositol trisphosphate (IP 3 ) and diacyglycerol from phosphatidylinositol bisphosphate. IP 3 activates IP 3 receptors to release Ca 21 from SR in VSMCs. Evidence from other studies suggests that Ang II and ET-1 can also activate NADPH oxidases (NOXs) to increase the production of reactive oxygen species (ROS) in VSMCs (17).…”

Integrin α4 is involved in the regulation of glioma-induced motility of bone marrow mesenchymal stem cells

“…3.0 was used for reverse transcription (Takara, Dalian, China). For routine PCR, the primer sequences of were: integrin α4, 5'-ATCTAGTTTTTA CACACAGGATTT-3' (forward) and, 5'-TGTCAATGTCGC CAAGATT-3' (reverse) (23); GAPDH, 5'-CCGTATCGGACG CCTGGTTA-3' (forward) and, 5'-TCTCGCTCCTGGAAG ATGGTG-3' (reverse). The length of the amplified fragment was 533 bp for integrin α4 and 207 bp for GAPDH.…”