| Na+/K+-ATPase is widely distributed in various eukaryotic cells membrane, including the vascular smooth muscle cells. Na+/K+-ATPase is a specific transmembrane protein to maintain the Na+ and K+ concentration gradient between the inside and outside of cells and to keep the membrane potential, with the important functions of maintenance of the steady-state sodium and membrane potential, thus played an important role in the regulation of the vascular tone . This meaned that the changes of Na+/K+-ATPase activity may be a potential protective factor when ischemic cerebrovascular diseases occur. Our previous studies showed that, in either endothelium-intact or endothelium-denuded vascular rings and in either normal or hypoxic circumstances, ouabain could contract rat basilar artery concentration-dependently. The concentration-effect relationship curves could be best fitted by the model of two points binding and implied that there existed high affinity and low affinityα-isoforms of Na+/K+-ATPase in rat basilar artery. Ouabain and free-potassium liquid may reduce the hypoxia-induced constriction of the basilar artery without endothelium. The concentration -response curves of ouabain shifted to right when hypoxia occured, and it reduced the ratio of the high affinityα-isoform,which showed that Na+/K+-ATPaseα2-isoform involved in hypoxia-induced constriction of basilar artery. Not only Na+/K+-ATPase catalytic subunit with high affinity(α2 and/orα3 isoform) , but also catalytic subunit with low affinity (α1-isoform) existed in guinea pig basilar artery. Hypoxia could significantly reduce the binding affinity of twoαsubunits for ouabain, and the impact on Na+/K+-ATPaseαsubunit with high affinity was more apparent thanα-subunit with low affinity.In recent years, adenosine, as a kind of important active substance, has gradually been the focus of attention, which involved in the regulation of ischemic cerebrovascular diseases. Adenosine is an inhibitory neuromodulator of the central nervous system, and it could signal between the energy demand and energy storage in brain to reach a balance.Under normal circumstances, the extracellular adenosine concentration is maintained in 40 nmol/L400 nmol/L. When hypoxia- or ischemia occured, on the one hand, glucose and glycogen are to accelerate the decomposition of ATP to supply the energy demand, then the intracellular level of adenosine increased dramatically, and released into the outside of cells. On the other side, nerve cells would also rapidly generate and release a large number of adnosine, which made the adenosine concentration 100-1000 times higher than normal. These adenosine bind with adenosine A2a receptor and increase glycogen glycolysis, the relaxation of regional cerebral blood vessels, and the cerebral blood flow, thereby increase the cell's energy supply and play a role in cerebral protection. At the recovery phase of hypoxia, endogenous adenosine as a precursor of ATP synthesis, could be regenerated to ATP in adenosine kinase (AK) catalysis to prolong the cells'survival time. Thus, endogenous adenosine involves in the pathological process of ischemic cerebrovascular diseases through a variety of approches, and exerted neuroprotective effect in ischemic brain injury. The exogenous adenosine analogues, agonists and antagonists would result in different effects, because of their different target cells, as well as the receptor density and/or affinity.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 constrictive mechanism of 5-HT on cerebral arteries is associated with 5-HT binding to its specific receptors on VSMCS, which is to increase [Ca2+]i and contract cerebral arteries through Ca2+-dependent protein kinase C. Our previous study also found that, ouabain did not affect vasoconstriction induced by 5-HT in normal rats, but it could significantly enhance 5-HT induced constriction of cerebral arteries in hypertensive rats and made concentration -response curve of 5-HT shift to left, suggesting that bothαsubunits of Na+/K+-ATPase with high affinity and low affinity involved in the cerebrovascular contractile response induced by 5-HT in the hypertensive rats. Is there any relationship between the relaxation of cerebral blood vessels by adenosine and Na+/K+-ATPase? The purpose of the present experiments was to test the inhibitory effects of adenosine on the constriction of rat basilar artery induced by 5-HT under normoxic circumstances and further explore whether Na+/K+-ATPase involved in the role of adenosine vascular relaxation response.Objective: To observe the regulated effects of adenosine on isolated rat basilar arterial constriction induced by 5-HT and its interrelation with Na+/K+-ATPase.Method: Wistar rats (250-300 g) 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 118.99, 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 circu-mference at 100 mmHg transmural pressure. Isometric tension was expressed as millinewtons per millimeter of vessels'responce to drugs, the degrees of contractile activity of drugs were recorded. Results:1 Constrictive effects of 5-HT on isolated rat basilar arteries. The cumulative constriction values of normal isolated rat basilar arteries for 5-HT 10-9 mol/L, 10-8 mol/L, 3×10-8 mol/L, 10-7 mol/L,3×10-7 mol/L,10-6 mol/L,3×10-6 mol/L,10-5 mol/L were (mN) : 0.03±0.02, 0.11±0.09, 0.45±0.48, 1.50±1.10, 3.25±1.80, 5.14±1.56, 5.64±1.63 and 5.49±1.25. Finally 5×10-7 mol/L (EC80) is used to study.2 Inhibitory effects of adenosine on the constrictions induced by 5-HT in isolated rat basilar arteries with or without endothelium. Basilar artery was perfused with adenosine 10-9 mol/L, 10-8 mol/L, 10-7 mol/L, 10-6 mol/L, 10-5 mol/L, 10-4 mol/L or 3×10-4 mol/L in 15 minutes, then perfused with the same concentration of adenosine and 5×10-7 mol/L 5-HT. The contraction values of 5×10-7 mol/L 5-HT in basilar artery with endothelium were :0.01±0.009, 0.07±0.05, 0.28±0.15, 0.48±0.18, 0.71±0.20, 0.91±0.24 and 0.92±0.31, and EC50: 9.59E-7 and 1.34E-6; Emax 0.99 and 1.239; while the contraction values of 5×10-7 mol/L 5-HT in basilar artery without endothelium were:0.05±0.06, 0.13±0.12, 0.36±0.12, 0.47±0.23, 0.68±0.24, 0.91±0.26 and 0.92±0.28, and EC50: 1.34E-6, Emax: 1.23. There was no difference between these two groups. These results demonstrated that the vasodilatation effect of adenosine was not related to endothelium, it maybe depended on vascular smooth muscle.3 Effects of EDRF inhibitors on the effects of adenosine inhibiting constriction of isolated rat basilar arteries induced by 5-HT.L-NAME and Indomethacin were the inhibitors of Endothelium-derived relaxing factor (EDRF) . Basilar artery were perfused with 10-6 mol/L,10-5 mol/L indomethacin or 10-6 mol/L L-NAME in 30minutes, then perfused with the same concentration of indomethacin orL-NAME, and 10-4 mol/L adenosine, finally with 5×10-7 mol/L 5-HT. The contraction values of 5×10-7 mol/L 5-HT in basilar artery with endothelium were :(1) adenosine (10-4 mol/L) :0.82±0.12; (2) adenosine (10-4 mol/L) +indomethacin (10-6 mol/L) :0.79±0.14;(3) adenosine (10-4 mol/L) +indomethacin (10-5 mol/L):0.77±0.12; (4) adenosine (10-4 mol/L) +L-NAME:0.81±0.10; (5) Ach (10-6 mol/L) : 0.71±0.07; (6) Ach (10-6 mol/L) +L-NAME: 0.22±0.07. These results futher demonstrated that 10-6 mol/L and10-5 mol/L indomethacin and 10-6 mol/L L-NAME have no effect on the inhibition of adenosine on contraction induced by 5-HT in normal isolated rat basilar arteries with endothelium, but they could significantly inhibit the vasodilatation effect of PGI2 andAch on the endothelium-intact arteries. Literature also reported 10-6 mol/L indomethacin can significantly restrain the inhibition of PGI2 from endothelium on the vasoconstriction induced by 5-HT.4 Effect of adenosine receptor antagonists on the roles of adenosine inhibiting constriction of isolated rat basilar arteries induced by 5-HT DPCPX is an antagonist of the adenosine A1 receptor; SCH58261 is an antagonist of A2 receptor, and MRS1191 is an antagonist of A3 receptor. Pretreament of 10-7 mol/L DPCPX and 10-6 mol/L MRS1191 did't change the cumulative concentration-response curve of the inhibition of adenosine on basilar artery constriction by 5-HT. The contraction values of 5×10-7 mol/L 5-HT were: 0.05±0.06, 0.13±0.08, 0.25±0.11, 0.39±0.17, 0.56±0.14, 0.76±0.10 and 0.91±0.04. While the basilar arteries were pretreated with SCH58261 10-7 mol/L or 10-6 mol/L in 30 minutes, we found the cumulative concentration-response curve of the inhibition of adenosine on basilar artery constriction by 5-HT was shifted to the right, and the Emax decreased significantly. The contraction values of 5×10-7 mol/L 5-HT in SCH58261 pretreated artery were : 10-7 mol/L SCH58261:0.02±0.02, 0.05±0.05, 0.09±0.10, 0.15±0.13, 0.28±0.16, 0.37±0.15 and 0.49±0.17; 10-6 mol/L SCH58261: 0.03±0.06, 0.04±0.06, 0.08±0.09, 0.09±0.11, 0.14±0.09, 0.21±0.11 and 0.23±0.11. These results indicated that the vasodilatation effect of adenosine was mediated to adenosine A2 receptor, but not A1 or A3 receptor.5 Effects of adenosine receptor agonist on the constriction induced by 5-HT in isolated rat basilar arteriesCGS21680 is an agonist of the adenosine A2a receptor. Basilar arteries were perfused with CGS21680 10-9 mol/L, 10-8 mol/L, 10-7 mol/L, 10-6 mol/L or 10-5 mol/L in 15 minutes, then perfused with the same concentration of CGS21680 and 5×10-7 mol/L 5-HT. The contraction values of 5×10-7 mol/L 5-HT were:0.05±0.04, 0.16±0.09, 0.33±0.16, 0.54±0.21, 0.81±0.07 and 0.92±0.05; While the basilar artery was pretreated with SCH58261 10-6 mol/L in 30 minutes, we found the cumulative concentration-response curve of CGS21680 was shifted to the right and the Emax decreased significantly. The contraction values of 5×10-7 mol/L 5-HT were: 0±0, 0±0, 0±0, 0.02±0.02, 0.15±0.05 and 0.28±0.08. These results futher indicated that the vasodilatation effect of adenosine was mediated to adenosine A2a receptor.6 Effect of ouabain on the tone of rat basilar arteryOur results revealed that ouabain could constrict normal rat basilar artery in a concentration-dependent manner. The contraction values of ouabain in different concentration were: 0.02±0.03, 0.09±0.06, 0.26±0.06, 0.39±0.13, 0.47±0.13, 0.68±0.17 and 0.83±0.22, respectively. The fitting parameters in normal condition were following: the Kd value of high affinity pump (kh) :1.5078e-7 (mol/L) , the Kd value of low affinity pump (kl) :3.9832e-4 (mol/L) ; the fraction of high affinity pump (fh) : 0.5072, the fraction of low affinity pump (fl) : 0.4928, which suggest there were two different functional of Na+/K+-ATPase isoforms in rat basilar artery, the high affinityα-isoform and the low affinityα-isoform.7 Effect of adenosine on the constriction induced by ouabain in isolated rat basilar arteries.In the presence of adenosine (10-4 mol/L) the cumulative concentration-response curve of ouabain was shifted to the right and the constrictive of ouabain (10-9 mol/L, 10-8 mol/L, 10-7 mol/L, 10-6 mol/L, 10-5 mol/L, 10-4 mol/L and 10-3 mol/L) were: 0.01±0.01, 0.02±0.02, 0.03±0.03, 0.05±0.03, 0.09±0.05, 0.19±0.11 and 0.23±0.11, respectively; and the fitting parameters of concentration-response curve of ouabain were: kh:1.0966e-5 (mol/L) , kl:0.0300 (mol/L) ; fh: 0.2082, fl: 0.7918. Compared with normal condition, the sensitivities of both low and high affinityα-isoforms of Na+/K+-ATPase were decreased,the former dereased by 75-fold and the latter decreased by 73-fold, respectively. These results suggest Na+/K+-ATPase low and the high affinity pumps involve in the relaxation of adenosine on rat basilar artery contracted by ouabain.8 Effects of ouabain on the roles of adenosine inhibiting the constriction of isolated rat basilar arteries induced by 5-HT.Basilar artery was perfused with ouabain10-9 mol/L, 10-8 mol/L, 10-7 mol/L, 10-6 mol/L, 10-5 mol/L, 10-4 mol/L or 10-3 mol/L for 30 minutes, then perfused with the corresponding concentration of ouabain and adenosine (10-4 mol/L) with 5×10-7 mol/L 5-HT. The contraction value in rat basilar artery without ouabain pretreatment was:0.36±0.06, and in the presence of different concentration ouabain were: 0.37±0.05, 0.44±0.10, 0.52±0.13, 0.53±0.09, 0.54±0.09, 0.61±0.10 and 0.72±0.07 mN. Its concentration-effect relationship curve could best be fitted by the two point binding model, and kh: 5.1278e-8, kl: 1.4524e-3 (mol/L) ; fh: 0.2702, fl: 0.7298. Compared with normal condition, the sensitivity of low affinityα-isoform decreased by 3.6-fold and the high affinityα-isoform increased by 3-fold. Compared with adenosine relaxing rat basilar artery contracted by ouabain, the sensitivities of two different affinity pumps were increased, the low affinityα-isoform of 21-fold and high affinityα-isoform of 214-fold. These results suggest that the high affinityα-isoform of Na+/K+-ATPase involves in the effect of adenosine inhibiting the constriction of isolated rat artery induced by 5-HT.9 Effect of two different functional sodium pumps on the roles of adenosine inhibiting the constriction of isolated rat basilar arteries induced by 5-HT.Basilar artery was perfused with ouabain 10-6 mol/L, which inhibits the high affinity pump, or 10-3 mol/L, which inhibits the high and low affinity pumps, for 30 minutes, then perfused with the corresponding concentration of ouabain and adenosine 10-9 mol/L, 10-8 mol/L, 10-7 mol/L, 10-6 mol/L, 10-5 mol/L, 10-4 mol/L or 3×10-4 mol/L with 5×10-7 mol/L 5-HT. The contraction values in rat basilar artery with ouabain 10-6 mol/L pretreatment were:0.02±0.02, 0.10±0.09, 0.13±0.09, 0.21±0.18, 0.29±0.22, 0.47±0.34 and 0.56±0.32 mN, respectively, which were similar to those pretreated with the ouabain 10-3 mol/L: 0.04±0.05, 0.13±0.08, 0.23±0.13, 0.31±0.13, 0.45±0.15, 0.57±0.16 and 0.62±0.18 mN. These results demonstrated that ouabain 10-6 and 10-3 mol/L shifted the cumulative dose-response curve of adenosine to the right, and both significantly decreased the dilatation of adenosine on rat basilar artery. But there were no difference in the contraction value between two ouabain concentration groups above (p>0.05) , suggesting that the high affinityα-isoform of Na+/K+-ATPase involves in the effect of adenosine relaxing isolated rat basilar artery contracted by 5-HT.Conclusion:1 5-HT could concentration-dependently contract rat basilar artery, and adenosine could concentration-dependently relax the rat basilar artery contracted by 5-HT. The vasodilation of adenosine was mediated through the A2a receptor on vascular smooth muscle, but was not related to vascular endothelium and endothelial-derived relaxing factor.2 Ouabain could concentration-dependently contract rat basilar artery, and the fitting parameters of its concentration-response cuver showed that there exist both high and low affinity sodium pumps in rat basilar artery. Adenosine could relax the rat basilar artery contracted by ouabain, shift the concentration-response cuver of ouabain to the right, and de crease the affinities of high and low affinityα-isoforms to ouabain, which suggested that both high and low affinityα-isoforms of Na+/K+-ATPase involve in the relaxation of adenosine on rat basilar artery contracted by ouabain.3 Ouabain could concentration-dependently antagonist the role of adenosine relaxing isolated rat basilar artery contracted by 5-HT. Compared with adenosine relaxing rat basilar artery contracted by ouabain, the sensitivities of the high affinityα-isoform and the low affinityα-isoform in rat basilar artery were increased and the former was higher 100-fold than the latter. In addition, there were similar shifts of the concentration-response cuver of adenosine in the presence of 10-6 and 10-3 mol/L ouabain. These results indicate that the high affinityα-isoform of Na+/K+-ATPase involves in the role of adenosine relaxing isolated rat basilar artery contracted by 5-HT.
|
PDF Full Text Request