| Adenosine is an inhibitory neuromodulator of the nervous system, cardiovascular system, digestive system, and respiratory system. In physiological condition adenosine is released in low concentration. Adenosine outside cell conducts its biological effects through combination with adenosine receptors. It has been shown that there are 4 adenosine receptor subtypes, A1, A2a, A2b and A3. A1 and A3 are Gi/Go-protein-coupled receptor, A2a and A2b are Gs-protein-coupled receptors[1-3]. Under normal circumstances, the maintenance of extracellular adenosine concentration is 40 nmol/L400 nmol/L. When hypoxia/ischemia occures, on the one hand, glucose and glycogen are to accelerate the decomposition of ATP to supply the energy demand, then ATP degrades so that the intracellular adenosine increases dramatically and releases into the cells outside. On the other side, nerve cells would also rapidly generate and release a large number of adnosine, which make the adenosine concentration 100-1000 times higher than normal[4]. The actions of this nucleoside on the vasculature are most prominent when oxygen demand is high and there is a reduction in oxygen tension at the site in question. Therefore, adenosine has been shown to be an important regulator of blood vessel tone under hypoxic conditions[5]. 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.Na/K pump (Na+/K+-ATPase) is widely distributed in various eukaryotic cells membrane, including the vascular smooth muscle cells. It has been shown that there are 4 Na/K pump receptor subtypes. depressive of Na/K pump activity may increase Ca2+ within a cell,and upgrade the constriction of blood vessel. Our previous studies showed that, 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 glycoside affinity and low glycoside affinityα-isoforms of Na/K pump in rat basilar artery. Compared with adenosine relaxing rat basilar artery contracted by ouabain, the sensitivities of the high glycoside affinityα-isoform and the low glycoside 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 glycoside affinityα-isoform of Na/K pump involves in the role of adenosine relaxing isolated rat basilar artery contracted by 5-HT.5-HT is one of the most potent cerebral vascular contractors. This powerful contractive effect of 5-HT on cerebrovascular is considered to be involved in vasospasm and obstruction of blood flow after cerebral ischemia. The contractive effect of 5-HT on cerebrovascular is associated with Ca2+, 5-HT binding to specific receptors on VSMCS, increased [Ca2+] and contracted cerebrovascular through Ca2+-dependent protein kinase C. Ouabain could concentration-dependently upgrade the effect of 5-HT on basilar artery of hypertension rat, shifts the concentration-response curve to the left. These results indicate that Na/K pump involves in the role of effect in isolated rat basilar artery contracted by 5-HT.What is the effect of hypoxia on the relaxation induced by adenosine on isolated rat basilar arteries which is contract by 5-HT? 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 hypoxia circumstances and further explore whether Na/K pump involved in the role of adenosine's vascular relaxation response in hypoxia circumstances.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 in hypoxia circumstances. Method: SD rats (220-280g) were killed. Thereafter,the basilar arteries were removed immediately and placed in a cold physiological salt solution (4℃, PSS) of the following composition (mmol/L: KCl 4.7, NaCl 118.99, EDTA 0.027, Glucose 5.5, MgSO4﹒7H2O 1.17, KH2PO4 1.18,NaHCO3 25). Isolated basilar arteries were cut into rings (2-mm in length) and mounted on 40-μm stainless steel wires in the bath 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 was expressed as millinewtons per millimeter of vessels'responce to drugs, the degrees of contractile activity of drugs were recorded.Results:1 Effect of 5-HT on contraction of norm isolated rat basilar arteries.The cumulative contraction values of norm isolated rat basilar arteries for 5-HT 10-9 mol/L, 3×10-9, 10-8 mol/L, 3×10-8 mol/L, 10 -7 mol/L, 3×10 -7 mol/L, 10 -6 mol/L, and 3×10 -6 mol/L were (mN): 0.03±0.02, 0.07±0.03, 0.19±0.12, 0.47±0.21, 1.87±0.56, 3.37±0.61, 4.86±0.63, and 4.92±0.63, respectively. Finally 5×10-7mol/L (EC80) of 5-HT is used to other experiments.2 Effect of hypoxia on contraction of isolated rat basilar arteries induced by 5-HT.Setting the contraction value of 5×10-7 mol/L 5-HT in basilar artery with normoxia as 100%, then the contraction values (percentage) in basilar artery were: 101.30±11.75, 107.13±17.85, and112.79±12.55 after 40min, 80min, and 120min with hypoxia . Finally 40min of hypoxia is used to other experiments.3 Effect of hypoxia on adenosine-induced dilatation in isolated rat basilar arteries contracted by 5-HT.Setting the dilatation value of 10-3 mol/L adenosine in basilar artery contracted by 5×10-7 mol/L 5-HT as 100%,the percentages of dilatation of the adenosine 10-8 mol/L, 10-7mol/L, 10-6mol/L, 10-5mol/L, 10-4mol/L, and 10-3mol/L were measured and calculated. The percentages of dilatation of adenosine in basilar artery contracted by 5×10-7 mol/L 5-HT were: 3.33±4.22, 11.42±8.15, 24.45±14.83,49.85±9.97, 94.57±9.48, and 100±9.92 in the group of normoxia, and 2.66±6.90, 8.21±4.85, 16.58±10.47, 37.99±12.53, 69.16±11.74, and 78.99±13.38 in the group of hypoxia, respectively. Effect of 10-5mol/L, 10-4mol/L and 10-3mol/L adenosine in normoxia circumstancewere more potent than those in hypoxia circumstance(P<0.05).Their EC50 in normoxia and hypoxia circumstance were 2.594E-5 and 6.791E-6, and their Emaxwere1.00 and 0.79,respectively. These results indicate hypoxia could significantly inhibit the vasodilatation of adenosine on rat basilar arteries.4 Effect of adenosine receptor antagonists on adenosine-induced dilatation in isolated rat basilar arteries contracted by 5-HT in normoxia and hypoxia circumstance.DPCPX, SCH58261, and MRS1191 are the antagonists of the adenosine A1, A2, and A3 receptors, respectively. Setting the dilatation value of 10-4 mol/L adenosine in basilar artery contracted by 5×10-7 mol/L 5-HT as 100%, the percentages of dilatation of 10-4 mol/L adenosine in basilar artery contracted by 5×10-7 mol/L 5-HT were 98.35±16.06, 60.28±14.08, and 96.66±14.48 after incubation with 10-6mol/L DPCPX, 10-6mol/L SCH58261 and 10-6mol/L MRS1191, respectively. In hypoxia circumstance, the dilatation values of 10-4 mol/L adenosine in basilar artery contracted by 5×10-7 mol/L 5-HT were respectively 75.39±7.98,42.37±11.25,72.38±8.21 after incubation with 10-6mol/L DPCPX, 10-6mol/L SCH58261 and 10-6mol/L MRS1191. Either in normoxia or hypoxia circumstance, DPCPX and MRS1191 have no remarked effect on the dilatation of lisolated rat basilar arteries induced by adenosine, while SCH58261 can restrain it notable (p<0.05).These results indicate that the vasodilatation effect of adenosine on the rat basilar arteries was mediated by adenosine A2 receptor.5 Influence of hypoxia on the inhibitory effects of adenosine A2 receptor antagonist on the vasodilatation of adenosine.Setting the dilatation value of 10-4 mol/L adenosine in basilar artery contracted by 5×10-7 mol/L 5-HT as 100%, Adenosine A2 receptor antagonist SCH58261 10-9mol/L,10-8 mol/L,10-7mol/L,and 10-6mol/L decreased the dilatation percentages of 10-4 mol/L adenosine in basilar artery contracted by 5×10-7 mol/L 5-HT . to 91.12±8.53, 84.12±9.15, 71.88±9.89, and 60.28±14.08 in normoxia circumstance, and to 68.72±6.75, 64.92±10.53, 56.62±8.42, and 42.37±11.25 in hypoxia circumstance, respectively. In the present experiment, we can find that hypoxia can significantly enhance the roles of 10-9mol/L, 10-8 mol/L, 10-7mol/L, 10-6mol/L SCH58261 inhibiting the rat basilar artery dilatation induced by 10-4mol/L adenosine,indicating the inhibition of hypoxia on the vasodilatation of adenosine is mediated via adenosine A2 receptors.6 Effect of hypoxia on the vasodilatation of the adenosine A2 receptor agonist in the isolated rat basilar arteries.Adenosine A2 receptors can be divided into two subtypes, A2a and A2b. CGS21680 is an agonist of the adenosine A2a receptor. In the hypoxia, the dilatation role of 10-6mol/L CGS21680 in the rat basilar arteries can be inhibited. Setting the dilatation value of 10-6mol/L CGS21680 in basilar artery contracted by 5-HT as 100%, the dilatation percentages of 10-6mol/L CGS21680 in basilar artery contracted by 5-HT were 99.77±9.30 and 69.52±12.97 in normoxia or hypoxia condition, respectively. There are significant differences between the two group(sP<0.05). This further suggests that hypoxia depresses the vasodilatation of adenosine via inhibiting adenosine A2 receptors.7 Effects of ouabain on the inhibition of hypoxia on adenosine-induced dilatation in isolated rat basilar arteries contracted by 5-HT.In hypoxia condition, the dilatation percentages of 10-4mol/L adenosine in basilar artery contracted by 5-HT were 73.13±12.41, 57.73±11.07, 55.21±10.93 in the groups of 10-4mol/L adenosine alone, 10-6mol/L ouabain that could block the high glycoside affinityα2-isoform of Na/K pump , and 10-3mol/L ouabain that could block the low glycoside affinityα1-isoform of Na/K pump, respectively. The present study showed that in hypoxia condition 10-6mol/L ouabain and 10-3mol/L ouabain can significantly inhibit the dilatation of isolated rat basilar arteries induced by adenosine, but there was no difference in inhibiting the vasodilatation of adenosine between 10-6mol/L and 10-3mol/L ouabain groups. These results suggest that the Na/K pump, mainly the high glycoside affinityα2-isoform of Na/K pump, may involve in the effect of hypoxia in inhibiting the dilatation of isolated rat basilar arteries induced by adenosine. In normoxia condition, the dilatation percentages in basilar artery induced by adenosine were 0.73±0.10 and 0.59±0.13in the 10-6mol/L and 10-3mol/L ouabain groups, respectively; while in hypoxia condition, the dilatation percentages were 72.64±9.77and 57.73±11.07 , respectively. These results showed that in hypoxia condition the effect of ouabain in inhibiting the dilatation of isolated rat basilar arteries induced by adenosine can be enhanced markedly. This suggests thatthat the hypoxia can depress the vasodilatation of adenosine on rat basilar arteries by inhibiting the activity of high glycoside affinity Na/K pump.Conclusion:1 In hypoxia condition, adenosine could concentration- dependently relex rat basilar artery contracted by 5-HT, and hypoxia could antagonist the vasodilatation role of adenosine,which is mediated through the A2a receptor on vascular smooth muscle.2 In hypoxia condition, ouabain could antagonist the role of adenosine relaxing isolated rat basilar artery contracted by 5-HT, indicating that the Na/K pump, mainly through its high glycoside affinityα2-isoform, involves in the role of hypoxia on adenosine relaxing isolated rat basilar artery.
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