Alteration Of NO And NOS In Lung And Aorta In Rats Exposed To Hypoxia And The Effect Of Exogenous NO On Kv2.1

Background and objectiveHypoxic pulmonary hypertension (HPH) is usual clinical pathophysiologic process.It is also an important component element to many diseases such as chronic obstructive pulmonary disease (COPD),chronic cor pulmonale,et al.It is indicated that hypoxia causes the alteration of substances which are secreted by vascular endothelial cells and the alteration contributes to pulmonary vasoconstriction and pulmonary vascular remodeling,at last cause the pulmonary artery hypertension (PAH).Chronic hypoxia results in PAH,but seldom causes hypertension,it is may be the different response to hypoxia between pulmonary artery and aorta and its mechanism is poorly understood.NO is endothelium-derived relax factor (EDRF), which is produced from L-arginine by three nitric oxide synthase (NOS) isoforms: two constitutive forms, i.e., neuronal-NOS (nNOS) and endothelial-NOS (eNOS) and inducible-NOS (iNOS). It is reported that the reduction of NO content caused by the decrease of NOS expression may be one of mechanisms to HPH.In PASMCs, there are mainly three types of K⁺channels : voltage-gated K⁺ channels(Kv),Ca²⁺-activated K⁺ channels (KCa), ATP-sensitive K⁺ channels (KATP). Hypoxia down-regulates expression of Kv channels ,decreases IK and causes membrane depolarization,therefore, activates voltage-dependent, long-lasting type (L-type) Ca2+ channels,and allows for an influx of Ca2+ into the cell, thereby increasing [Ca2+]cyt, triggers hypoxic pulmonary vasoconstriction(HPV).Many studies have indicated that Kv is more relative to HPH than other potassium channels and predominately responsible for the maintenance of the Em in PASMCs .Hypoxia inhibits Kv,then causes a decrease in transmembrane IK(V), which results in a less negative Em,leading to cellular membrane depolarization.This experiment observes the NO content and NOS activity in lung, aorta and blood from plasma of pulmonary artery (PA) and thoracic aorta (TA) in HPH rats.In order to demonstrate the effect of NO and NOS on the different response between pulmonary and arota expose to hypoxia. In vitro,we observe the effect of sodium nitroprusside(SNP), which is exogenous donor of NO, on the expression of Kv2.1 in PASMCs.一Alteration of NO and NOS in lung and aorta in hypoxic pulmonary hypertension rats1 MethodsTwenty–four male SD rats were randomly divided into four groups (n=6)in each group:normoxic 2 weeks group,normoxic 3 weeks group,hypoxic 2 weeks group and hypoxia 3 weeks group; The animal model of hypoxia pulmonary hypertension was established by intermittent hypoxia (imitation of highland 5 000m).The maximum pulmonary artery pressure (PAP) and blood pressure (BP) were measured by right cardiac catheterization and by cannulating the left common carotid artery (LCCA),respectively.The right ventricle(RV) and left ventricle plus septum (LV + S) of rat were weighed.The method of nitrate reductase was used to determine the changes of NO and NOS activity in the plasma and tissue.The immunohistochemistry and Western blot were used to observe eNOS expression in pulmonary and aorta.2 ResultsIn the hypoxic 2 and 3 weeks groups,PAP(43.4±4.4 mmHg,51.8±4.2 mmHg) and the [RV/(LV + S)]% (32.3±1.0,37.0±1.6) were increased significantly compared with those in the control groups (20.8±2.4mmHg,21.8±3.9mmHg, P<0.01) and (21.3±1.0,20.3±1.2,P<0.01).And the longer time exposed to hypoxia the higher of those parameters (P<0.01),but there is no difference in BP between hypoxic groups and normoxic groups. NO content in plasma of PA and TA in HPH rats decreased significantly compared with their correspondingly control groups (P<0.01). Compared with correspondingly control group,NO content, NOS activity in lung homogenate and the expression of eNOS in pulmonary of HPH rats declined too (P<0.01);such alteration hasn't been found in aorta among the four groups.But NO content , NOS activity and the expression of eNOS in aorta is higher than in pulmonary in hypoxic groups.3 SummaryNO content and NOS activity in lung in HPH rats decreased significantly compared with control groups,but there was no difference in aorta among those groups.The alteration in lung and aorta in HPH rats may explain why hypoxia often results HPH but no hypertension. 二The effect of SNP to Kv2.1 in PASMCs1 MethodsPASMCs were divided into normoxic/hypoxic group, normal/hypoxic group with SNP and normoxic/hypoxic with SNP and MB group.Proliferation of PASMCs was investigated by methyl thiazolyl tetrazolium (MTT) assay. Using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot, we measured mRNA and channel protein of Kv2.1 in each group.2 ResultsThe OD value of PASMCs of the hypoxic 24h group was significantly higher than that of the normoxic group (P<0.01), it indicates that hypoxia promote the proliferation of PASMCs.Compared with hypoxic 24h group,the OD value of PASMCs with diffenent concentration SNP decreased remarkably(P<0.05 or P<0.01),so SNP can inhibit the proliferation of PASMCs regardless normoxic or hypoxic grouop.Compared to the normoxic 24h group,Kv2.1 mRNA and channel protein in PASMCs in the hypoxic 24h group remarkably decreased ;by offering SNP,the expression of Kv2.1 increased ;but when add with MB the expression of Kv2.1 declined again.This illustrates that SNP can up-regulate the expression of Kv2.1 through cGMP pathway.3 SummaryBy influencing the expression of Kv, SNP inhibits the proliferation of PASMCs effectively and then relieves HPV.This result provides basis for SNP to therapy HPH.Conclusion The inhibition of NO system in pulmonary circulation when exposed to hypoxia may be one of the mechanisms to HPH. This study observed that exogenous NO up-regulate the expression of Kv2.1 by cGMP pathway, and it may be explain how does NO inhibit the proliferation and constriction of PASMCs.