| As a major risk factor for cardiovascular disease, sodium plays an significant rolein the pathogenesis and therapy of hypertension. The increase in blood pressure drivenby a salt load is characteristic of salt-sensitive hypertension, a condition affectingmore than two thirds of individuals with essential hypertension who are older than60years. It has been proposed that the kidney and the central nervous system are the2major sites for salt sensing, but the underlying molecular mechanisms are largelyunclear. As agenetic model of salt-sensitive hypertension mimicking that of humans,Dahl-salt sensitive (DS) hypertensive rats have been extensively used for the study ofmolecular mechanisms mediating increased salt sensitivity.With more than half of hypertensives being salt sensitive, increased attention tostrategies that target specifically in reducing salt sensitivity, particularly in high-riskindividuals, is urgently needed. It has been reported that salt intake restrictiondecreases systolic and diastolic blood pressures in both hypertensive andnormotensive individuals, with a bigger magnitude in the hypertensive group.However, the molecular mechanisms underlying salt-dependent regulation of bloodpressure remain to be defined. Several powerful endocrine/paracrine/autocrinesystems or factors have been implicated to be involved, which include but are notlimited to the renin-angiotensin system, smooth muscle system and the endothelinsystem in blood vessels. The data from the previous study provide the evidence thatsalt intake would descend TRPV4expression and function in the kidney, DRG andmesenteric artery of salt-sensitive rats. Our objective in the present study is toinvestigate the change of expression and function of TRPV4in the hypothalamicneurons in salt-sensitive hypertension.Methods:1Neurons were cultured from the hypothalamic region in the Dahl’S rats.2The immunofluorescence staining method is applied to detect the presence ofTRPV4channel in neurons and glias in the hypothalamic region. 3Apply the patch-clamp technique to observe the agonist, antagonist and highsalt of TRPV4channel affect the action potentials of neurons in hypothalamic regionof Dahl’S rats.4Apply the Western blot method to test the effect that high salt induced to theTRPV4channel protein expression in hypothalamic neurons.5Apply the Real-time PCR method to test the effect that high salt induced to theTRPV4channel mRNA expression in hypothalamic neurons.6Evaluate the impact PVN mircroinjection of TRPV4channel’s agonist andantagonist on circulation.Results:1.Immunohistochemistry resultsThese immunohistochemistryresults posed anatomical evidence for the notionthat TRPV4is existed on neuron and glial in hypothalamic region of SD and Dahl’Srat.2. GSK Decreases Firing Rate in Normal and High Salt Treated NeuronsSpontaneous APs were recorded in bath solution, and no significant differencehas been detected in the firing rate between SD and Dahl’S rat neurons, which displaysimilar electrophysiological properties. GSK, a potent agonist of TRPV4, did notsignificantly elicit firing rate change between NNSSD（neurons of normal salt treatedSD rats） and NHSSD（neurons of high salt treated SD rats）(0.518±0.035vs0.3.2±0.049, P>0.05, n=5). By contrast, the effects of GSK on firing rate weremarkedly greater in both NNSDS（neurons of normal salt treated Dahl’S rats） andNHSDS（neurons of high salt treated Dahl’S rats）, in which GSK can inducesignificant firing rate change between NNSDS and NHSDS (0.821±0.042vs0.596±0.024, P<0.05, n=5). The data above demonstrated that GSK can decreasefiring rate in Dahl’S and SD rat, including both normal and high salt treated neurons,especially in normal Dahl’S neurons. Furthermore, GSK can produce a moresignificant effect on normal rather than high salt treated neurons, suggesting that thehigh concentration of sodium would impair the function of TRPV4on neurons.3. RuR Increases Firing Rate in Normal and High Salt Treated NeuronsBeside the effect of TRPV4agonist, GSK, on firing rate, we further studied therole of TRPV4antagonist, RuR, on the firing rate in both normal and high salt treated neurons. It was found that RuR could increase the firing rate for both normal and highsalt treated neurons, no matter Dahl’S or SD rat, in which RuR can significantlyinduce the change between NNSDS and NHSDS (2.344±0.320v1.667±0.261, P<0.05,n=5), while the change between NNSSD and NHSSD was fair (0.6.1±0.203vs0.513±0.100, P>0.05, n=5).4. Protein Expression and change of TRPV4in hypothalamusExpression of TRPV4of normal and high salt treated in PVN tissue andhypothalamic neurons. To investigate the effects of high salt on TRPV4expression inthe hypothalamic, TRPV4protein levels were determined by Western blotting analysisin SD and Dahl’S rat. Based on the molecular weight estimation, the single bandabout100kDa represents TRPV4protein. Similar TRPV4expression levels werefound in hypothalamic neurons among the different treatments (NNSSD,0.334±0.224%, NHSSD0.295±0.310%, NNSDS,0.860±0.251%versusNHSDS0.395±0.251%of GAPDH arbitrary, n=5, p<0.05). In PVN, HS treatment hasno effect in SD rats (SDNS,0.444±0.0.056%versus SDHS,0.407±0.080%ofGAPDH arbitrary), while it can decrease TRPV4expression in Dahl’S rats (DSNS,0.615±0.096%versus DSHS,0.359±0.043%of GAPDH arbitrary, n=5, p<0.05).Therefore, the differential expression levels of TRPV4protein revealed that highconcentration of sodium could reduce the TRPV4expression in hypothalamus ofDahl’S rats but not SD rats.5. Altered mRNA level of TRPV4in hypothalamusReal-time PCR was used to assess the effect of high salt on TRPV4mRNA levelin PVN tissue and cultured neurons of Dahl’S rats and SD rats. In hypothalamicneurons, TRPV4mRNA expression was higher in normal treated condition; however,treatment of these same cells with high concentration of sodium (NHSDS:1.15±0.130%of GAPDH arbitrary; n=5, p<0.05) resulted in a significant decreasecompared with the normal treated (NNSDS:1.862±0.130%of GAPDH arbitrary; n=5,p<0.05). In the PVN tissue, TRPV4mRNA transcript level was decreased in high saltintake rats, compared with normal rats in Dahl’S rats (DSNS（Dahl’S with normal saltdiet））,3.061±0.186%versus DSHS,0.894±0.150%of GAPDH arbitrary, p<0.05).These results demonstrated that high concentration of sodium can alter the mRNAtranscription level of TRPV4in the hypothalamus in salt-sensitive rat and had nomarked effect on non salt-sensitive rat. 6. Effects of TRPV4Activated in PVN.As a TRPV4activator,100nl of GSK (1mmol/l) can open TRPV4channels anddecreased MAP and HR in all4of the groups PVN microinjection. The depressoreffect of MAP began after PVN microinjection, reached the lowest point at3-5minutes, and lasted for10minutes after GSK PVN administration. The magnitude ofthe decreases in MAP induced by GSK was biggest in DSNS (50.3±3.6mm Hg; n=5,P<0.05) rats compared with SDNS (21.3±2.0mm Hg), SDHS (15.9±2.8mm Hg) andDSHS (26.05±1.4mm Hg). The magnitude of the HR decreased by GSK PVNmicroinjection was biggest in DSNS (126.6±9.6bpm; n=5, P<0.05) rats comparedwith SDNS (47.0±3.9bpm), SDHS (41.5±2.5bpm) and DSHS (75.4±7.8bpm).7. Effects of TRPV4Blocked in PVN.In response to PVN microinjection of100nl RUR (1mmol/l), MAP waselevated immediately and can reach the peak4-5minutes after administration in allof groups. The pressor effect of RUR lasted for more than10minutes. Changes ofMAP were highest in DSNS (38.8±2.2mmHg; n=5, P<0.05) rats compared with other3groups (SDNS:18.9±2.1mm Hg; SDHS:14.3±2.1mm Hg; DSHS:21.7±2.0mmHg), and HR were also higher in DSNS (143±8.7bpm; n=5, P<0.05) rats comparedwith other3groups (SDNS:84.8±7.9bpm; SDHS:70.5±5.5; DSHS:93.3±6.4bpm).Conclusion:1. The TRPV4channel exists in hypothalamic neurons and glial cells inhypothalamic region of rats.2. High salt reduced the TRPV4channel’s sensitivity to its agonist and antagonistin Dahl’S rats, rather than the SD rats.3. High salt decreased the TRPV4channel’s protein expression in hypothalamicregion of Dahl’S rats, instead of SD rats.4. High salt decreased the TRPV4channel’s mRNA level in hypothalamic regionof Dahl’S rats, rather than the SD rats.5. The activity of TRPV4channel in PVN of s Dahl’S rats declined because ofhigh salt diet. High salt destroyed the TRPV4channel on blood pressure regulationfunction, leading to blood pressure increasing in Dahl’S rats. |
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