The Activity And Regulatory Mechanism Of TRPP2/TRPV4 Channel Complex In Mesenteric Arteries Endothelial Cells From Salt-sensitive Hypertension Rats

Background and purposeWe have previously found that TRPP2 and TRPV4 could assemble a functional channel complex in HUVECs and act as a mechanosensitive sensor influencing NO production via mediating flow-induced Ca²⁺ entry into cells.The aim of this study was to determine whether TRPP2/TRPV4 channel complex exists in mesenteric artery endothelial cells and to investigate the role of it in salt-sensitive hypertension.MethodsCoimmunoprecipitation and double immunostaining were used to explore the physical interaction of TRPP2 and TRPV4 in MAECs.Inside-out patch-clamp was performed to study electrophysiological properties of TRPP2/TRPV4 channel complex.SS and SR rats were fed with normal salt diet?0.3% NaCl?and high salt diet?8% NaCl?,respectively.Systolic blood pressure?SBP?was measured in conscious rats by tail-cuff method at the 0 and 21 day.TRPP2/TRPV4 channel activity was recorded from inside-out patch-clamp configuration in the split-open mesenteric arteries from the rats under different experimental conditions.The effect of aldosterone on the TRPP2/TRPV4 channel activity in MAECs was studied by patch-clamp.DAF-FM and Fluo-3 were used to detect the effect of aldosterone on [NO]i and [Ca²⁺]i in MAECs.The inside-out patch-clamp was used to detect the effect of NO on TRPP2/TRPV4 channel activity in MAECs.ResultsCoimmunoprecipitation,double immunostaining and patch-clamp demonstrated that TRPP2 and TRPV4 could assemble a 21.7-pS single channel conductance complex in MAECs.High salt intake resulted in an increase in blood pressure in SS rats.The basal blood pressure in SS rats was higher than that in SR rats?132 ± 5 vs.118 ± 5,p < 0.01?.There was not significant increase in blood pressure on 0 and 21 days after high salt diet in SR rats.The data obtained from open-split mesenteric arteries patch-clamp showed that the activity of TRPP2/TRPV4 was reduced in SS rats fed high salt diet?NPo: 0.34 ± 0.04 vs.0.16 ± 0.01,p < 0.01?;by contrast,the activity of TRPP2/TRPV4 channel in open-split mesenteric arteries were slight changed in SR rats fed high salt diet?NPo: 0.36 ± 0.03 vs.0.35 ± 0.03?.Application of 100 nmol/L aldosterone in the bath for 10 min,the NPo of TRPP2/TRPV4 channel complex did not decrease compared with control group;however,treatment of aldosterone to MAECs for 24 h,the NPo of TRPP2/TRPV4 decreased significantly.The results obtained from the fluorescence microscope demonstrated that NO and Ca²⁺ level in MAECs reduced after aldosterone treatment,and the MR-antagonist?eplererone?attenuated the negative effect of aldosterone on NO and Ca²⁺ level.The NO donor?SNP?increased the NPo of TRPP2/TRPV4 channel in MAECs?NPo: 0.89 ± 0.05 vs.0.37 ± 0.02,p < 0.01?;MAECs were pretreated with a specific NO scavenger,carboxy PTIO,subsequent application of SNP failed to impact activity of TRPP2/TRPV4 channel complex.The results showed that NO regulated the TRP2/TRPV4 channel activity in MAECs.In addition,NO reversed aldosterone-induced inhibition of TRPP2/TRPV4 channel in MAECs.ConclusionThe dysfunction TRPP2/TRPV4 channel complex may be one of the mechanisms induced vascular endothelial cells lesion in salt-sensitive hypertension;and the channel complex activity was regulated by aldosterone and NO.