Platelet‐derived growth factor and angiotensin II cause increases in cytosolic free calcium by different mechanisms in vascular smooth muscle cells

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Abstract
Platelet-derived growth factor (PDGF) and angiotensin II (All) are thought to mediate their biological effects in vascular smooth muscle cells (VSMCs) by causing alterations in cytosolic free calcium ([Ca2+]i). In this study we examine the pathways by which PDGF and All alter [Ca2 +]i in VSMCs. Addition of PDGF resulted in a rapid, transient, concentration-dependent increase in [Ca2+]i; this rise in [Ca2 +]i was blocked completely by preincubation of cells with ethylene glycol-bis (β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA) or CoCl2, by the voltage-sensitive Ca2 +-channel antagonists verapamil or nifedipine, by 12-O-tetradecanoylphorbol-13-acetate (TPA), or by pertussis toxin. All also caused an increase in [Ca2 +]i; however, All-stimulated alterations in [Ca2+]i displayed different kinetics compared with those caused by PDGF. Pretreatment of cells with 8-(diethylamine)-octyl-3,4,5-trimethyoxybenzoate hydrochloride (TMB-8), almost totally inhibited All-induced increases in [Ca2+]i. EGTA or CoCl2 only slightly diminished All-stimulated increases in [Ca2+]i. Nifedipine, verapamil, TPA, and pertussis toxin pretreatment were without effect on All-induced increases in [Ca2+]i. PDGF and All both stimulated increases in total inositol phosphate accumulation, although the one-half maximal concentration (ED50) for alterations in [Ca2+]i and phosphoinisitide hydrolysis differed by a factor of 10 for PDGF (3 × 10−10 M for Ca2+ vs. 2.5 × 10−9 M for phosphoinositide hydrolysis), but they were essentially identical for All (7.5 × 10−9 M for Ca2+ vs. 5.0 × 10−9 M for phosphoinositide hydrolysis). PDGF stimulated mitogenesis (as measured by [3H]-thymidine incorporation into DNA) in VSMCs with an ED50 similar to that for PDGF-induced alterations in phosphoinositide hydrolysis. PDGF-stimulated mitogenesis was blocked by pretreatment of cells with voltage-sensitive Ca2 + channel blockers, TPA, or pertussis toxin. These results suggest that PDGF and All cause alterations in [Ca2+]i in VSMCs by at least quantitatively distinct mechanisms. PDGF binding activates a pertussis-toxin-sensitive Ca2+ influx into cells via voltage-sensitive Ca2+ channels (blocked by EGTA, verapamil, and nifedipine), as well as stimulating phosphoinositide hydrolysis leading to release of Ca2+ from intracellular stores. All-induced alterations in [Ca2+]i are mainly the result of phosphoinositide hydrolysis and consequent entry of Ca2+ into the cytoplasm from intracellular stores. Our data also suggest that changes in [Ca2 +]i caused by PDGF are required for PDGF-stimulated mitogenesis.