Involment Of Receptor Component Protein In The Transmembrane Signal Transduction Of Vascular Smooth Muscle Cells Proliferation Induced By Static Pressure

Objectives: This study was aimed to explore effect of CGRP-RCP on thetransmembrane signal transduction of vascular smooth muscle cells proliferationinduced by static pressure.Methods: After cultured with the serum-free medium for24h, A10VSMCwere subjected to different static pressure (0,80,100,120,140,160,180mmHg) fordifferent time (0,3,6,12,24h). Cell viability was detected by MTT assay; PCNAand RCP expression were determined by Western blot; RCP mRNA was detected byRT-PCR. After A10VSMC was respectively transfected with three RCP siRNAsequences (the final concentration of125nM), RCP mRNA was detected by RT-PCRin24h; RCP expression was determined by Western blot in48h. The expression ofRCP was knocked down by siRNA or induced by CGRP, cell viability was detectedby MTT assay and PCNA expression was determined by Western blot under thestatic pressure. After A10were dealt with CGRP (the final concentration of10nm)for30min and static pressure for5min,30min,1h,3h,6h, p-Akt was determined byWestern blot; the interaction between RCP and Caveolin-1or beween RCP and Gaswere detected by co-immunoprecipitation.Results: The cell viability and PCNA expression were increased with theelevation of static pressure and reached their peaks at120mmHg and a plateau aftersix hours. RCP mRNA and protein expression in A10VSMC were up-regulated withthe elevation of static pressure and reached their peaks at120mmHg and six hours.The sequence3is optimum for knocking RCP down after A10VSMC weretransfected with three RCP siRNA sequences. VSMC viability and PCNA expression were both significantly increased in the group of RCP siRNA under static pressurecompared to the group of scrambled siRNA(P<0.05). However, VSMC viability andPCNA expression were both significantly decreased in the group of CGRP understatic pressure compared to the group of no-CGRP(P<0.05). Caveolin-1expressionwas increased by CGRP pre-incubation but it was decreased by staticpressure(P<0.05); Disassociation beween Caveolin-1and RCP was occurred in staticpressure for5min, but they bound again under static pressure for30min compared tothe control. The level of p-Akt was significantly increased with the elevation of timeunder the static pressure in A10VSMC(P<0.05); The bind of RCP and Gas wassignificantly decreased by static pressure and CGRP.Conclusion: RCP participated in VSMC proliferation induced by staticpressure; Caveolin-1and Gas regulated PI3K/Akt signaling pathway induced bystatic pressure on regulation of RCP transmembrane signal transduction...