Aldosterone Impacts On ENaC Expression In Rat Aortic Depressor Nerve

BackgroundHeart failure(HF),often referred to as congestive heart failure.Heart failure is the end stage of various heart diseases.Decompression reflex,or baroreflex is one of the important mechanisms of cardiovascular regulation.The sensory nerve endings in the aortic arch and carotid sinus detect blood pressure changes,and then transfer the signal to the central nervous system.By balancing the activity of the sympathetic nerve and the vagus nerve,the cardiovascular function is regulated.In 1969 Smyth et al.first measured the braroreflex sensitivity(BRS)by the Smyth method.In heart failure patients and animals,baroreflex is significantly reduced,which disturbs the autonomic nervous balance hallmarked by decreased parasympathetic and increased sympathetic nerve activity,and increased morbidity and mortality.Although lowered impulse activity of baroreflex afferents is involved in the blunted baroreflex sensitivity in HF patients and animals,the mechanism(s)that impair baro-afferent input are still unclear.It is reported that HF-induced decrease in Na+ channel function inhibits the electrical activity of the aortic depressor nerves.There are mechanically sensitive ion channels that detect blood pressure changes in the aortic depressed nerve endings.Heart failure may have altered the mechanical sensitivity of the ion channel,which reduces the baroreceptor sensitivity to blood pressure changes.Epithelial sodium channel(ENaC)is a membrane bound ion channel that is selectively permeable to Na+ ENaC belongs to the ENaC / Deg superfamily with amiloride sensitivity that is a heterotrimer consisting of ? or ?,?,and ? subunits,mainly distributed in the kidney and other epithelial tissue,the function to maintain water and sodium balance.The aldosterone binds to the intracellular aldosterone receptor,increases the expression of the ENaC channel on the cell membrane,regulates ENaC-mediated Na+ cell transport.In non-epithelial tissues such as ganglion,ENaC is a mechanically sensitive ion channel.Whether aldosterone can reduce ENaC expression in peripheral neurons has not been reported.In the event of heart failure(HF),aldosterone is significantly elevated,and elevating aldosterone in normal animals or human blood significantly reduces the sensitivity of baroreflex.It has been found that ENaC channel protein expresses in aortic depressor nerve endings and cell bodies,and it is unclear whether aldosterone affects ENaC channels in aortic depressor nerves.ObjectiveThis study is to explore whether aldosterone modulates the expression of ENaC channels in aortic depressor nerve and attenuates baroreceptor sensitivity.Methods1.The method of continuous infusion of aldosterone: osmotic pump was used to induce rat model of high plasma aldosterone.Alzet 2004 micro-osmotic pump were implanted subcutaneously in SD male rats(180 g~200 g)anesthetized with 10% hydrated chloral hydrate(0.3 ml / 100 g).Thirty healthy male Sprague-Dawley rats were f randomly divided into 5 groups:(1)normal control group(CON,n=6): normal diet and drinking water.(2)vehicle control group(VEH,n=6): normal diet and drinking water,subcutaneously implanted with 5% methanol osmotic pump.(3)spironolactone group(SPI,n=6): normal diet and drinking,teartment with 25 mg/kg spironolactone daily by oral gavage.(4)aldosterone group(ALD,n=6): normal diet and drinking water,subcutaneous implantation osmotic pump pre-installed in 5% methanol-dissolved aldosterone(0.75 ?g/h).(5)aldosterone + spironolactone group(ALD + SPI,n=6): rats were treated with aldosterone(0.75 ?g/h)and spiroonolactone(25 mg/kg/day).Measure the plasma aldosterone concentration.After successful preparation of the model,the arterial blood of each rat was collected(1.5-2 ml)and centrifuged,1000 g,15 minutes,the supernatant was kept in-80 ?for measurement of plasma aldosterone.The concentration of aldosterone in plasma was measured with rat aldosterone kit.2.Blood pressure and heart rate measurements.Rat was anesthetized with 20% urethane(0.5 ml/kg,i.p.),catheters were implanted into the femoral artery and vein for arterial blood pressure and heart rate(or cardiac cycle,CC)measurements and drug administration,respectively.3.Measurement of baroreflex sensitivity(BRS).Animals in each group were tested for baroreflex sensitivity according to the improved Smyth method after 4 weeks of treatments.Baroreflex sensitivity was measured using reflex changes in cardiac cycle in response to changes in blood pressure.The arterial blood pressure was decreased to about to 50 mm Hg by sodium nitroprusside(SNP,30 ?g,i.v.),and then increased by Phenylephrine(PE,10 ?g,i.v).BRS was defined as the change in interbeat interval in millisecond per unit change in systolic blood pressure(?SBP/?CC).4.The distribution of ENaC-? and ENaC-? in nodose ganglion(NG)was detected by immunofluorescence staining with specific antibodies.5.The expression of ENaC-? and ENaC-? in nodular ganglion(NG)were detected by Western blot.6.Statistics analysis: All data were presented as means ± SD.Graph Pad Prism5.0 was used for data analysis.One-way ANOVA with post hoc Bonferroni test and t-test were used to determine statistical significance.Statistical significance was accepted when p?0.05.Results1.After 28 days of aldosterone administration with osmotic pump,the plasma aldosterone was elevated(550.9 ± 7.63 pg/ml,P <0.001)compared with that in control group(193.2 ± 4.95 pg/ml).2.After the aldosterone concentration was increased,blood pressure of the rats(98.7 ± 3.76 mm Hg),heart rate(357.8 ± 8.32 beat / min)and body weight(412.7 ± 7.65 g)were significantly higher than those in the control group(94.65 ± 3.97 mm Hg,345.7 ± 11.59 beta / min,416.8 ± 10.3 g),no significant change(P>0.05).3.After treatment with aldosterone,BRS was significantly decreased(0.225 ± 0.05 ms/mm Hg)compared with that in control group(1.113 ± 0.04 ms/mm Hg)(P<0.001).Aldosterone receptor blocker,spironolactone abolished the effect of aldosterone on BRS(0.973 ± 0.05 ms/mm Hg),compared to ALD group(P>0.05).It suggests that Aldosterone blunts BRS by receptor dependent pathway.4.The ENaC-? and ENaC-? proteins were expressed in NeuN antibody-labeled neurons(nodose ganglion).5.After the aldosterone concentration was increased,the expression of ENaC-? and ENaC-? protein in the ALD group were 31.51±3.33 and 7.10±3.52(P<0.001),respectively,by western blot compared with 100 in the normal group,which was blocked by spironolactone.The expression levels of ? / ?ENaC in the combination of spironolactone were 97.08±10.08 and 98.05±11.14,compared with 100 in the normal group(P>0.05),respectively.The results showed that aldosterone receptor antagonist spironolactone blocked the depression of aldosterone on ENaC.ConclusionNormal rat given aldosterone decreased ENaC expression in aortic depressor nerve and blunted baroreflex sensitivity,which were blocked with spironolactone.The results suggest that aldosterone reduces ENaC expression through receptor dependent pathway in aortic depressor nerve and then blunts baroreflex.