| β-adrenergic receptors (β - AR) are located on most of the blood vessels, where they mediate vasodilation effects of endogenous catecholamines and exert essential regulatory role of cardiovascular homeostasis. A large body of evidence recently revealed that β1-AR and β2-AR colocalized in vasculture, and more importantly, they mediated more physiological function other than vasodilation. However, the subtype specific effects of β-AR stimulation on regulating vascular smooth muscle cell (VSMC) function and the underlying mechanisms are largely unknown. Here, we examined whether pi-AR and P2-AR differ in regulating migration, apoptosis and proliferation of primary cultured rat aortic VSMC, and further explored possible mechanism.In the first part, the thoracic aortic vascular smooth cell primarily cultured from male Wistar rat were sustainly stimulated by selective β1-AR or β2-AR agonist. We found that stimulation of β1-AR significantly inhibited 5% FBS-mediated SMC migration to about 59%. The pi-AR inhibitory effect was totally reversed by pretreatment of cells with NO scavenger PTIO or NOS inhibitor L-NAME, suggesting that Pi-AR mediated inhibition of VSMC migration is through NO-dependent pathway, this conclusion was further supported by the ability of SANP, a NO donor, to inhibit SMC migration in a PTIO sensitive manner. Unlike β1-AR, β2-AR stimulation hadno effect on VSMC migration. However, inhibiting Gi with pertussis toxin, scavenging Gβγ with βARK-ct or blocking PI3K activation with LY294002 unmasked inhibitory effect of P2-AR stimulation. Phosphorylation of Akt, a substrate of PI3K, and ERKl/2 was markedly elevated by β2-AR stimulation. The inhibitory effect of P2-AR stimulation under the presence of pARK-ct overespression was also blocked by PTIO and L-NAME pretreatment.In the second part, specific β- AR subtypes were overexpressed by adeno virus delivery of recombined corresponding gene to VSMC. Hematoxylin-eosin staining, Hoechst33342 staining , flow cytometry(FCM) analysis as well as DNA agarose electrophoresis were performed to detect the effect on apoptosis by stimulation of endogenous and overexpressed β -AR subtype. Here we show that specific sustained stimulation of β1-AR and β2-AR, in either physiological or overexpressed state, promoted VSMC apoptosis in the essential medium containing 1% FBS, indicated by significant elevation of Hoechst33342 staining positive rate and decrease of viable cell percentage by FCM analysis. β2-AR stimulation exhibited much more significant proapoptotic effect than β1i-AR. The proapoptotic effect of both subtypes could be reversed by NO scavenger PTIO and NOS inhibitor L-NAME, while not by either PKA inhibitor or calcium channal blockade. In addition, Pi-AR and β2-AR stimulation downregulated the phosphorylation of ERKl/2, suggesting that MAPK signal pathway might mediat the proapoptotic effects of both P -AR stimulation.Finally, we investigated the effect on VSMC proliferation by stimulation of β1-AR and β2-AR by MTT assay and cell counting. Two subtype, especially β2-AR, exhibit significant inhibitory effect on VSMC proliferation,peak at the 3rd day after stimulation.Our results provide the first evidence that β1-AR and β2-AR stimulation exhibit opposite effects in regulating VSMC migration, with an inhibitory effect of Pi-AR and a concurrent inhibition and stimulation effect of β2-AR in rat VSMC. NO plays a critical role in both pi-AR and β2-AR mediated inhibition of VSMC migration, while Gi-Gβγ-PI3K-Akt pathway is involved in β2-AR stimulatory effect. Both P-AR subtype stimulation, especially β2-AR promote apoptosis in essential medium through NO signal pathway as well as inhibition of ERK1/2 phosphorylation. VSMC proliferation was inhibited by both subtype stimulation, especially β2-AR stimulation.In summary, β1-AR and β2-AR exhibit significant specificity and complexity on regulation of vascular smooth muscle cell migration, apoptosis as well as proliferation. β-AR system might be involved in the path...
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