Impact Of Chronic Hypoxia In Utero On Salt-sensitive Hypertension Of Male Offspring Rat And Its Vascular Ion Channel Mechanism

Backgroud:Substantially evidence demonstrated that the fetus exposed to chronic hypoxia in utero may predispose to developing cardiovascular diseases,including hypertension,in later life.Hypertension,increasing the risk of stroke,cardiomyocytes hypertrophy,and renal failure,the leading causes of death worldwide;which give a larger economic burden on government and people.It is well known that substantially increasing dietary intake of salt plays a critical role in the pathogenesis of hypertension in the worldwide.It is has long been revealed that there is a salt sensitive and salt-resistant phenotype among individuals.Chronic hypoxia in utero is a common clinic complication in pregnancy,which could arise from numerous maternal pathologic factors.However,the impact of chronic fetal hypoxia from the early gestation specifically on the future susceptibility to develop salt-sensitive hypertension is still unknown.In addition,as the vascular Ca2+ channels and K+ channels play an important role in regulation of vascular tone and blood pressure.Thus,The present study using rat femoral artery catheterization,patch clamp and molecular biology methods to examine the effect of chronic prenatal hypoxia on male offspring rat blood pressure during intake of a high-salt diet;especially the related ion channel mechanism of vascular smooth muscle cells.Part 1 Effects of high salt diet intake on birth weight and blood pressure of male chronic prenatal hypoxia offspringObjective:To determine the effect of chronic prenatal hypoxia on offspring birth weight and male offspring rat blood pressure during intake of a high-salt diet.Methods:On day 5 of pregnancy,pregnant rats were were randomized to either the normoxia control(Control n=12)or housed inside a hypoxic chamber continuously flushed with nitrogen gas to maintain an oxygen concentration of 10.5%(Hypoxia,n=12).Rats were removed from the hypoxic chamber,and allowed to give birth in a normoxia environment at gestational day 21.The litter sizes and birth weights of Control and Hypoxia pups were recorded within 3-8h after delivery.After weaning(4 weeks),male offspring from each experimental group(Control,n=24;Hypoxia,n=24)were randomly allocated to receive either a normal-salt(NS)diet containing 1%NaCl or a high-salt(HS)diet containing 8%NaCl.Therefore,4 groups were created:Control offspring receiving NS diet(CNS,n=12 from 12 litters),Control offspring receiving HS diet(CHS,n =12 from 12 litters),Hypoxia offspring receiving NS diet(HNS,n=12 from 12 litters),and Hypoxia offspring receiving HS diet(HHS,n= 12 from 12 litters).The offsprings were receiving their respective diets for 8 weeks and were studied at 12 weeks of age.In 12 weeks,recording the daily water drinking and food intake in each group,and using the femoral artery catheterization technique to measurement of blood pressure and heart rate in offspring rats from each group in vivo.Results:The birth weight of the hypoxia rats(5.72 ± 0.11 g)was significantly reduced compared to the control rats(6.31 ± 0.08 g)at birth(P<0.001).At 12 weeks old,the body weight was not significantly different among CNS(379.1 ± 7.94 g),HNS(375.3± 8.35 g),CHS(382.0 ± 9.01 g),and HHS(371.4 ± 7.96)rats(n=8 per group).After 8 weeks of normal salt(NS)or high salt(HS)feeding,the heart rate did not differ among the four groups.SBP,DBP,and PP were the same among CNS,HNS,and CHS rats.However,SBP,DBP,and PP were higher in HHS offspring than that in CNS,HNS,and CHS.Conclusion:Chronic prenatal hypoxia induce fetal intrauterine growth restriction and lead to salt-sensitive hypertension in young adult offspring.Part 2 The mechanism of vascular ion channels involved in chronic prenatal hypoxia induced salt sensitive hypertension in male offspring ratsObjective:To examine the effect of chronic high salt diet intake on salt balalance,plasma volume and the ion channels functions in mesenteric arterial vascular myocytes of male chronic gestational hypoxia offspring.Methods:Blood electrolytes concentrations and plasma osmolarity of offspring rats were determined using GEM premier 3000 and the Fisk Micro-Osmometer of model 210,respectively.Arterial myocytes were enzymatically dissociated from third-order mesenteric arteries for patch clamp expriment.Single and whole-cell currents were recorded by inside-out or conventional whole cell patch-clamp configuration,respectively.Resting membrane potentials(Em)was measured by current clamp(1=0 pA)in the whole cell mode using conventional whole cell patches.we determined the protein expression of the pore-forming ?1C-subunit of Cav1.2 channels,subunits of Kv1.2 and Kv2.1,BK channel a and ?1 subunits in the mesenteric arterial myocytes from four groups using Western blot analysis.Results:After 8 weeks of feeding with either normal salt or high salt diets.No significant differences were observed in serum electrolytes(Na+,K+),plasma osmolarity,and haematocrit among the four groups.The mean cell capacitance were the same among the four groups.The maximal of peak Cav1.2 currents density was markly decreased in HHS cells relative to other groups,whereas the current densities were similar in other groups at-60 to +60 mV.Although BayK 8644(a specific Cav1.2 channel agonist,5?mol/L.)increased the amplitude of the Cav1.2 current evoked by depolarization were similar in CNS,HNS,and CHS cells,it did not equalize the current amplitude in HHS cells.The nifedipine(a specific Cav1.2 channel blocker,1 ?mol/L.)insensitive current densities were similar in groups.In addition,analysis of steady-state activation and inactivation curves of Cav1.2 channel revealed no statistical differences in half-maximal voltages(Vh),slope factors(k)and current inactivation rates(?)among the four groups.The resting Em was more depolarized in mesenteric arterial myocytes of HHS relative to CNS,HNS and CHS cells,and IKv values were smaller in HHS cells than in that of the others at higher voltages tested(-10 to +70 mV).The mean open dwell times of BK channels in the cells from HHS were decreased compared to CNS,HNS,and CHS cells(P>0.05,n=7 animals per group.).We found no difference in the average number of BK channels per patch and single-channel slope conductance in CNS,HNS,CHS,and HHS myocytes.Down-regulation in ?1C-subunits,Kv2.1 and ?1 subunits protein expression in HHS compared to CNS,HNS,and CHS offspring.Howerver,no significant difference in Kv1.5 and BK channel a subunit protein expression was observed in mesenteric artery myocytes among CNS,HNS,CHS,and HHS groups.Conclusion:Resting membrane potential depolarized and down-regulation of Cavl.2,Kv and BK channels in mesenteric arterial myocytes from salt-loading prenatal hypoxia offsring rats,which may be contribution to development salt-sensitive hypertension in male offspring rats exposed to chronic prenatal hypoxia.