| This study was divided into three sections.Section A:Expression of ERK and MKP-1 in vascular smooth muscle of spontaneously hypertensive rats with different agesObjective:To investigate the expression of extracellular signal-regulated kinases (ERKs) and mitogen-activated protein kinase phosphatase-1 (MKP-1) in thoracic aorta smooth muscles of spontaneously hypertensive rats (SHRs) and Wistar Kyoto rats (WKY) with different ages and the relationship between those and hypertension. Materials and Methods:1?The caudal arterial pressure was measured by tail-cuff,2?ERK activity in thoracic aorta smooth muscle was measured by immuno-precipitation;3?Protein expression of total ERK (t-ERK), phosphorylated-ERK (p-ERK) and mitogen-activated protein kinases phosphatase-1 (MKP-1) in thoracic aorta smooth muscle was detected by Western blot.4?MKP-1 mRNA in thoracic aorta smooth muscle was examined by RT-PCR.Result:1?The blood pressure of SHR was obviously higher than that of age-matched WKY (P?0.01), and elevated with age (P?0.05) and became stable after 14-week-old.2?There was no significant difference of t-ERK expression between SHR and age-matched WKY.3?The level of ERK activity, p-ERK and MKP-1 of SHR was higher than WKY in 5-week-old, and the level of ERK activity and p-ERK increased with age, while the expression of MKP-1 decreased with age (P?0.05)Conclusion:MKP-1 may play an important role in the development of hypertension in SHR, and the decrease of the expression of MKP-1 resulted in the activation of MAPK may induce vascular smooth muscle proliferation and hypertrophy.Section B:Effect of benazepril, losartan, carvedilol and hydralazine on pathway of ERK in thoracic aorta smooth muscles of SHRObjective:To investigate the effect of benazepril, losartan, carvedilol and hydralazine on pathway of ERKs in thoracic aorta smooth muscles of SHR.Methods:1?WKY rats were chosen as normal control group. Thirty-five 14-week-old SHR were randomly divided into 5 groups,7 rats each:benazepril group (10 mg-kg-1·d-1), losartan group (10 mg·kg-1·d-1), carvedilol group (30 mg·kg-1·d-1), hydralazine group (10 mg·kg-1·d-1) and model group.2?The measurement methods of ERK activity, t-ERK, p-ERK and MKP-1 in thoracic aorta smooth muscles are the same as those in part I. Results:1?Benazepril, losartan, carvedilol and hydralazine lowered the blood pressure after 10 weeks treatment (n=7, P<0.01)2. There was no significant difference of t-ERK expression in 6 groups rats (n=7, P>0.05)3?The level of ERK activity, p-ERK, MKP-1 and p-ERK/t-ERK in thoracic aorta smooth muscles in SHR benazepril, losartan and carvedilol groups were significantly lower than that in SHR hydralazine group and SHR model group (n=7, P<0.01), and similar to that in WKY group.4?There were no significant difference of ERK activity, p-ERK, MKP-1 and p-ERK/t-ERK between SHR hydralazine group and SHR model group (n=7, P>0.05)Conclusion:The level of ERK activity and MKP-1 expression are increased in thoracic aorta smooth muscles of SHR. Benazepril, losartan and carvedilol have the effect of suppression ERK activation and MKP-1 expression in aorta of SHR, while hydralazine could not. It suggested that benazepril, losartan and carvedilol may have altered other mechanisms involved in the reduction of ERK activation but not anti-hypertensive effect. Section C:Effects of Angiotensin II on ERK signaling pathway in cultured vascular smooth muscle cells from SHRObjective: To investigate the effects of angiotensin?(Ang?) on ERK signaling pathway incultured vascular smooth muscle cells (VSMCs) from SHR and WKY rats.Methods:1?VSMCs from SHR and WKY rats were treated with 1×10-7mmol/L Ang?for 24h in the absence or presence of 30 min of pre-treatment of valsartan (1×10-5mmol/L) or PD98059 (1×10-5mmol/L), selective inhibitor of ERKs-dependent pathways, when they were cultured in 20% calf serum medium. VSMCs of SHR and WKY cultured in serum-free medium were used as control groups.2?Among the different treatments, VSMCs from the SHR and WKY were devided into four groups:(1) control, (2) Ang?, (3) Ang?+valsartan, (4) Ang?+PD98059.3?The measurement methods of ERK activity, t-ERK, p-ERK, MKP-1 and MKP-1 mRNA in VSMCs are the same as those in part I.4?MKP-1 mRNA in VSMCs was examined by RT-PCR.Results:1?In VSMCs from WKY or SHR rats, ERK activity, p-ERK, MKP-1 and MKP-1 mRNA in Ang?group were higher than those in control group P?0.05).2?In both SHRs and WKYs, there were no significant differences in ERK activity, p-ERK, MKP-1 and MKP-1 mRNA among the control group, Ang?+valsartan group and Ang?+PD98059 group. ERK activity, p-ERK, MKP-1 and MKP-1 mRNA in SHRs were significantly higher than those in WKYs with same treatments (P<0.01).3?There was no significant difference in t-ERK among different groups and no difference in t-ERK between SHRs and WKYs (P>0.05).Conclusion:Ang?activates VSMCs ERK signaling pathways via Ang?type 1 (AT1) receptors. Ang II increased ERK activity and p-ERK, but not t-ERK, accompanied by an increase in MKP-1 mRNA expression and protein. Among the different treatments, ERK activity and p-ERK were higher in SHR than in WKY. Valsartan and PD98059 blocked Ang?-stimulated ERK activation. These results suggest that ERK signaling pathway plays an important role in the pathogenesis of hypertension. The effect of Ang?on SHR and WKY VSMCs ERK signaling pathway may be mediated by AT1 receptors, enhancing ERK activity and the amount of p-ERK, and then increasing MKP-1 mRNA and its expression. |
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