Na~+/K~+-ATPase Involves In The Contraction Of SHRs Basilar Artery

Hypertension is a main risk factor for human with high attack rate, mutilation and mortality rate. Alterations of the structure, Ca2+ movements, and Na+ transport have been described in this disease. Hypertension is a disease that provokes important alterations in the cardiovascular and CNS system and supposes a more elevated risk of cardiovascular events in the elderly than in younger people. The Na+/K+-ATPase is a major cellular transport system that controls Na+ homeostasis and membrane potential, both key factors in the regulation of vascular tone and blood pressure, which suggests that an altered Na+/K+-ATPase could be an underlying factor in essential hypertension. In humans, a chronic high-salt diet causes the levels of cardiotonic steroids (CTSs) to rise in the plasma. Moreover, 50% of patients with essential hypertension have substantially elevated levels of endogenous ouabain. Indeed, PST2238, an ouabain antagonist, lowers blood pressure in salt-dependent hypertensive rats and in certain patients with essential hypertension.Hypertensive disease develops with the structure changes of the brain blood vessels which have alterations in reaction to vasoconstrictor. Serotonin (5-HT) is one of the most potent cerebrovascular contractors. The powerful contractive effect of 5-HT on cerebral arteries is considered to be involved in vasospasm after cerebral ischemia. The contractive effect of 5-HT on cerebral arteries is associated with Ca2+,5-HT binding to specific receptors on VSMCS, increased [Ca2+]i and contracted cerebral arteries through Ca2+-dependent protein kinase C.The purpose of the present experiments was to test the changes of isolated basilar artery reaction to stringent using SHRs. We also investigated the role that Na+/K+ -ATPase plays in the process of changes and analyzed its mechanisms.Objective: To observe the effects of 5-HT and KCl on isolated rat cerebral artery and its interactions with Na+-K+-ATPase activity.Methods: Wistar rats and SHR (250-300g) were killed. Thereafter, the cerebral arteries were removed immediately and placed in a cold physiological salt solution (4℃PSS) of the following composition (mmol/L: NaCl 119, KCl 4.7,MgSO4﹒7H2O 1.17,NaHCO3 25,KH2PO4 1.18,EDTA 0.027,Glucose 5.5). Isolated cerebral arteries were cut into rings (2-mm in length) and mounted on 40-μm stainless steel wires in the chambers of Multi Myograph System-610M (Danish Myo Technology A/S) for measurement of isometric tension; in the bath, the PSS was heated to 37℃and bubbled with 95%O2+5%CO2 mixture. The vessels were equilibrated for 1 hour in PSS before they were set to a normalized internal circumference, estimated to be 0.9 times the relaxed circumference at 100 mmHg transmural pressure. Isometric tension is expressed as millinewtons per millimeter of vessels'responsiveness to drugs, the degrees of contractile activity of drugs were recorded.Results:1 The blood pressure change of SHRsThe pressure of systolic blood pressure, diastolic blood pressure, meanarterial pressure were: 117±6.75,88.5±5.59, 96.5±4.09mmHg, respectively, hypertensive rats significantly increased blood pressure, systolic blood pressure, diastolic blood pressure, mean arterial pressure, respectively: 208.5±27.04, 154.8±36.21, 172.8±36.02 mmHg (p<0.01).2 Effect of hypertension on basilar artery contractile response induced by KClThe cumulative dose-response curve of KCl in SHRs was shifted to the right. The contraction values of SHRs for 30 mmol/L, 40 mmol/L, 60 mmol/L, 80 mmol/L, 100 mmol/L, 120 mmol/L KCl were 0.17±0.02, 0.32±0.08, 0.48±0.08, 0.64±0.08, 0.67±0.08, 0.73±0.08 mN, respectively, which were significantly lower than normal rats and were 0.42±0.152, 0.84±0.19, 1.19±0.23, 1.49±0.24, 1.50±0.22, 1.46±0.22 mN (p<0.05), These results indicated that hypertension decreasesd the basilar artery contractile response to KCl.3 Effect of hypertension on basilar artery contractile response induced by 5-HT The cumulative dose-response curve of 5-HT in SHRs was shifted to the right. The contraction values of SHRs for 3×10-8, 10-7, 3×10-7, 10-6, 3×10-6, 10-5M 5-HT were 0.14±0.02, 0.41±0.06, 0.89±0.16, 1.90±0.25, 2.05±0.29, 1.78±0.24 mN, respectively, which were significantly lower than normal rats and were 0.37±0.09, 1.50±0.21, 2.78±0.40, 4.41±0.55, 5.35±0.38, 4.90±0.98 mN(p<0.05). These results indicated that hypertension decreased the basilar artery contractile response to 5-HT.4 Potassium-induced relaxationsWe demonstrated that the vasodilatation of different KCl concentration, in the normal isolated basilar artery were: 1, 0.53±0.06, 0.21±0.05, 0.14±0.03, 0.12±0.04, 0.12±0.04, 0.14±0.04; in pretreatment of ouabain for normal rats basilar artery were: 1, 0.94±0.02, 0.82±0.03, 0.53±0.08, 0.26±0.08, 0.18±0.04, 0.18±0.04;in SHR basilar artery were: 1, 0.78±0.07, 0.53±0.07, 0.23±0.06, 0.14±0.03, 0.15±0.03, 0.17±0.05; in pretreatment of ouabain for SHR basilar artery were: 1, 0.91±0.01, 0.91±0.02, 0.90±0.03, 0.73±0.03, 0.63±0.06, 0.58±0.06. The extent of their relaxation: Normal rats>SHRs>Normal rats in pretreatment of ouabain>SHRs in pretreatment of ouabain. The relaxation induced by KCl of SHRs in pretreatment of ouabain was significantly lower than the other three groups. Differences in area under the concentration response curve (dAUC) to potassium indicated the sodium pump activity, the value of SHR were 4.60±0.25, respectively, which were higher than the normal rats (2.54±0.25 ,p<0.05).5 Effects of ouabain on basilar artery contractile response precontracted with 5-HTBasilar artery were pretreated of ouabain 10-9M, 10-8M, 10-7M, 10-6M, 10-5M, 10-4M, 10-3M in 30 minutes, then perfused with 5×10-7M 5-HT. The contraction values in normal rat of 5×10-7M 5-HT were :3.95±0.47, 4.04±0.49, 4.51±0.48, 4.64±0.40, 4.40±0.43, 4.64±0.43, 4.19±0.38mN, respectively, which were higher than SHRs were 2.01±0.19, 2.38±0.09, 3.15±0.24, 3.17±0.19, 3.52±0.38, 3.67±0.50, 3.66±0.56 mN and these values were significantly dependented on the concentrations of ouabain(r=0.9394, p<0.05). These results also indicated that hypertension decrease the basilar artery contractile response to 5-HT and further suggested that the Na+/K+-ATPase involved in the effect of 5-HT induced contraction in SHRs basilar artery.6 Effect of ouabain on the normal rats basilar artery contractile responeOur results revealed that in normal rat basilar artery, there were two different functional of Na+-K+-ATPase isoform: the high affinityα-isoform and the low affinityα-isoform. The values of Kd were kh 1.7×10-8,ki 1.6×10-5mol/L.7 Effect of ouabain on the basilar artery contractile response precontracted by KCl in SHRsOuabain (5×10-7 mol/L) caused the cumulative dose-response curve of KCL shift to the left, overturned the the role of hypertension induced the dose-response curves of KCl shifted to right. The contraction values in ouabain (5×10-7 mol/L) pretreated normal rats basilar artery for 30 mmol/L, 40 mmol/L, 60 mmol/L, 80 mmol/L, 100 mmol/L, 120 mmol/L KCl were 0.39±0.07, 0.85±0.11, 1.39±0.16, 1.63±0.18, 1.57±0.16, 1.53±0.15 mN;respectively, in ouabain (5×10-7 mol/L) pretreated SHR basilar artery were 0.48±0.12, 0.91±0.21, 1.53±0.22, 1.69±0.28, 1.56±0.31, 1.56±0.31 mN. There were no difference in the two groups above compared with the normal rats basilar artery(p>0.05), but higher than the SHRs basilar artery(p<0.05).The contraction values in ouabain (10-4 mol/L) pretreated normal rats basilar artery for 30 mmol/L, 40 mmol/L, 60 mmol/L, 80 mmol/L, 100 mmol/L, 120 mmol/L KCl were 0.61±0.19, 1.15±0.35, 1.39±0.34, 1.57±0.36, 1.49±0.29, 1.35±0.21 mN; respectively, in ouabain (10-4 mol/L) pretreated SHRs basilar artery were 0.54±0.28, 0.96±0.22, 1.46±0.34, 1.60±0.30, 1.65±0.30, 1.69±0.29mN. There were no difference in the two groups above compared with the normal rats basilar artery (p>0.05), but higher than the SHR basilar artery (p<0.05).In normal rats basilar artery, ouabain 5×10-7 mol/L and 10-4 mol/L pretreatment did not effect the contractile response induced by KCl. But, in SHR basilar artery, ouabain 5×10-7mol/L and 10-4 mol/L pretreatment increased the contractile response induced by KCl and there were no difference between ouabain 5×10-7mol/L and ouabain 10-4 mol/L. These results indicated that the high affinityα-isoform of Na+/K+-ATPase were involved in the KCl-induced vasoconstriction on SHRs basilar artery.8 Effect of ouabain on the basilar artery contractile response precontracted by 5-HT in hypertensive ratsOuabain (5×10-7 mol/L) caused the cumulative dose-response curve of 5-HT shift to the left, partially block the role of hypertension induced the dose-response curves of 5-HT shifted to right. The contraction values in ouabain (5×10-7 mol/L) pretreated normal rats basilar artery for 10-9, 10-8, 3×10-8, 10-7, 3×10-7, 10-6, 3×10-6 , 10-5M 5-HT were 0, 0.06±0.02, 0.29±0.07, 1.00±0.05, 2.46±0.37, 4.12±0.46, 5.37±0.23, 4.37±0.29 mN; respectively, in ouabain (5×10-7 mol/L) pretreatment SHR basilar artery were 0, 0.06±0.01, 0.17±0.02, 0.69±0.06, 1.44±0.22, 2.66±0.23, 3.10±0.27, 2.81±0.22 mN, The value of the two groups above were higher than the SHRs basilar artery(P<0.05).The contraction values in ouabain (10-4 mol/L) pretreatment normal rats basilar artery for 10-9, 10-8, 3×10-8, 10-7, 3×10-7, 10-6, 3×10-6 , 10-5M 5-HT were 0, 0.09±0.03, 0.30±0.06, 1.05±0.13, 2.88±0.36, 4.84±0.40, 5.56±0.29 , 4.87±0.28Mn; respectively, in ouabain (10-4mol/L) pretreatment SHRs basilar artery were 0, 0.04±0.01, 0.34±0.05, 0.74±0.06, 1.49±0.17, 3.19±0.44, 4.00±0.26, 3.65±0.20 mN, The value of the two groups above were higher than the SHRs basilar artery(P<0.05).In normal rats basilar artery, ouabain 5×10-7 mol/L and 10-4 mol/L pretreatment did not effect on the contractile response with 5-HT. But, in SHRs basilar artery, ouabain 5×10-7mol/L and 10-4 mol/L pretreatment different increased the contractile response with 5-HT. These results indicated that the high affinityα-isoform and the low affinityα-isoform of Na+/K+-ATPase were involved in the 5-HT-induced vasoconstriction on SHRs basilar artery.Conclusion: Hypertension decreased the basilar artery contractile response to KCl and 5-HT. There were two different functional of Na+-K+-ATPaseα-isoform: the high affinityα-isoform and the low affinityα-isoform in rat. The high affinityα-isoform of Na+-K+-ATPase were involved in the KCl-induced vasoconstriction on SHRs basilar artery. The high affinityα-isoform and the low affinityα-isoform of Na+-K+-ATPase were involved in the 5-HT-induced vasoconstriction on SHR basilar artery.