| Objective The aim of the study was to investigate the expression ofβ1-adrenergic receptor(β1-R) induced by norepinepherine(NE) and substance P(SP) individually in cultured neonatal rat cardiomyocytes,and explore effects of SP pretreatment on NE-inducedβ1-R expression.Methods(1)Culture and identification of cardiomyocytes.The model of primary cultured cardiomyocytes of Sprague-Dawley(SD) rats was established and observed.The neonatal rat cardiomyocytes were identified with morphology and immunocytochemical(IC) assay method under a reverse microscope.The cardiomyocytes obtained from 1-3 day old SD rats were cultured for 72 hours for experimental study.(2) Effects of NE on the expression ofβ1-R in cultured neonatal rat cardiomyocytes.Thirty holes of rat cardiomyocytes were randomly divided into 5 groups(6 holes in each group): control group and 10-9 mol·L-1,10-8 mol·L-1,10-7 mol·L-1,10-6 mol·L-1 NE groups.The cells were cultured with NE for 3 hours in NE groups.IC and flowcytometry(FCM) were used to detect and analyze the expression ofβ1-R in the cardiomyocytes.(3) Effects of SP on the expression ofβ1-R in cultured neonatal rat cardiomyocytes.Six holes of rat cardiomyocytes were randomly divided into 2 groups(3 holes in each group): normal control group and 10-7 mol·L-1 SP group.The cells were cultured with administration of SP for 3 hours in SP group.IC was used to detect the expression ofβ1-R in the cardiomyocytes. The other 15 holes of rat cardiomyocytes were divided into control group,10-9 mol·L-1,10-8 mol·L-1,10-7 mol·L-1,and 10-6 mol·L-1 SP groups.The cells were cultured with SP for 3 hours in SP groups.FCM was used to detect and analyze the expression ofβ1-R in the cardiomyocytes.(4) Effects of SP pretreatment on NE-inducedβ1-R expression in cultured rat cardiomyocytes.Twelve holes of the cardiomyocytes were randomly divided into 4 groups(3 holes in each group):control group,10-7 mol·L-1 NE group,10-6 mol·L-1 SP+10-7 mol·L-1 NE group and 10-6 mol·L-1 NK-1 receptor antagonist(S3144)+10-6 mol·L-1 SP+10-7 mol·L-1 NE group.FCM was used to detect and analyze the expression ofβ1-R in the cardiomyocytes.Results(1) The model of primary cultured cardiomyocytes of SD rats was successfully established.(2) The expression ofβ1-R in cultured neonatal rat cardiomyocytes in 10-9,10-8,10-7, 10-6 mol·L-1 NE groups were greater than that in control group.It was the highest in the expression of theβ1-R in 10-8 and 10-7 mol·L-1 NE groups observed with IC and FCM respectively.(3) The expression ofβ1-R in cultured neonatal rat cardiomyocytes in 10-7mol·L-1 SP group was lower than that in control group with IC(P＜0.05).Compared with control group,the expression ofβ1-R in 10-9,10-8,10-7,10-6mol·L-1 SP groups was decreased with FCM,but there was no significant difference in 10-9 mol·L-1 SP group.(4) Compared with control group,the expression ofβ1-R in NE group was significantly increased(P＜0.05) and it was also increased in S3144+SP+NE group,but there was no significant difference(P＞0.05).The expression ofβ1-R showed no significant difference between SP+NE group and control group(P＞0.05).Conclusions(1) NE could induce the up-regulation of the expression ofβ1-R in cultured neonatal rat cardiomyocytes.(2) SP could induce the down-regulation of the expression ofβ1-R in cultured neonatal rat cardiomyocytes.(3) SP pretreatment could inhibit NE-induced up-regulation ofβ1-R expression in cultured rat cardiomyocytes,which may be partly mediated by NK-1 receptor.
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