Regulation Of Low Dose Ouabain On Guinea Pig's Basilar Artery Under Hypoxia

Cerebrovascular accident (CVA), or stroke, which results in a prolonged neurological disability in adults, leads to a heavy public-health and economic burden to our society every year, and it is the third most common cause of death in most countries in the world. Approximately 20% of CVA patients die within one month; about 50% of survivors need care and supervision because of their permanent and considerable disabilities; and the remaining 30% are capable of leading an independent life, despite neurological deficits. Almost 90% of the patients are victims of ischemic CVA, and the rests are of hemorrhagic CVA, maybe because the hemorrhagic is with high lethality.Ischemic CVA is always caused by the lack of O2. The interrupted blood flow can't deliver essential oxygen and glucose to the brain who doesn't store nutrient substances is the received pathogenic mechanism of CVA. Stroke will lead to neurological ischemia, energetic failure and neurological symptoms, followed by tissue damage within minutes if ischemia continues. The reperfusion after ischemia advances this tissue damage and induces hemorrhagic CVA.As a key enzyme in maintaining basic physiologic function of cells, more and more researches are focused on the effect of Na⁺, K⁺-ATPase on the progress of hypoxia. When theαisoform of Na⁺, K⁺-ATPase is inhibited, the Na⁺ will be retained inside the membrane, followed by an exchange of two Ca²⁺ from extra mediator and three Na⁺ from intercellular via Na⁺/Ca²⁺ exchanger type 1 (NCX1). This progress elevates [Ca²⁺]i and initiates an increase of myogenic tone.Whether Na⁺, K⁺-ATPase participates in regulating the generation of myogenic tone on cerebral basilar arteries (CBAs), and whether this myogenic tone overrides hypoxic dilation, are what we want to tell firstly. And secondly, whether the activity of Na⁺, K⁺-ATPase could be influenced by 1 nM ouabain and whether this could help the CBAs generating myogenic tone.Objective: Our study is to identify the effects of ouabain in different concentration on Na⁺, K⁺-ATPase form Guinea Pigs' Basilar Artery with Pressure Myogragh System Model 120CP, to identify the effects of hypoxia on basilar arteries, and to identify the role of Na⁺, K⁺-ATPase in the regulation of myogenic tone under hypoxia.Methods: After the isolation of the basilar arteries of Guinea pigs acutely, 2.5 mm arteries without branches were cannulated on to two very tiny glass probes, and secured gently with two fine nylon sutures. Luminal pressure was hold at 70mmHg. After the equilibration period, each kind of solution will be administered by superfusion. And then, the effects of ouabain on guinea pigs' basilar arteries in diameter, character of Na⁺, K⁺-ATPase from basilar arteries, effects of hypoxia on basilar arteries, effects of 1 nM ouabain on hypoxic dilation and myogenic tone and effects of 1 nM ouabain on arteries when vasoconstrictor is present were tested.Results:1 Test of stability of guinea pig basilar arteryAccording to the results, the passive diameter of intact and endothelium-denuded arteries, which superfused with PSS for 60 minutes, were changed by 0.3±1.2μm and 0.3±1.4μm (P>0.05). And the effects of 40mM K⁺-PSS on diameters prior to and post the one hour superfusion contracted the intact arteries by 51.1±5.8% and 49.6±7.4% separately and contracted the endothelium-denuded arteries by 52.3±6.2% and 49.8±6.5% separately. And there is no significance between them (P>0.05). This indicates that the stability of basilar arteries could be maintained at least 1 hour.2 Effects of hypoxia on diameter of guinea pig basilar arteriesAccording to the results, After the 60 minutes of superfusion with PSS or O2 free PSS, the passive diameter of arteries from intact control, endothelium-denuded control, intact and endothelium-denuded groups are increased by 0.3±1.2μm, 0.3±1.4μm, 4.8±1.7μm and 5.2±1.9μm respectively. The increased passive diameters of intact and endothelium-denuded arteries are much higher than the match control arteries (P<0.05), but there is no significant difference between them (P>0.05). This indicates that, hypoxia could induce significant dilation on basilar arteries under 70mmHg luminal pressure and this is not endothelium dependent.3 Effects of 1nM ouabain on hypoxic dilation of guinea pig basilar arteriesAccording to the results, after 0.5 hour of hypoxia, the diameters of CBAs increased by 2.5±1.0μm,which is much higher than intact normaxia group (-0.2±1.2μm, P<0.01). Even 1 nM ouabain was added into the O2 free solution after 30 minutes of hypoxia, this could not be changed (3.0±1.1μm, P<0.01). This indicates that, 1nM ouabain has no effects on hypoxic dilation.4 Effects of 1nM ouabain on diameter of guinea pig basilar arteriesAccording to the results, during an hour of superfusion, intact arteries could contract from 333.5±23.43μm to 323.33±22.93μm when 1nM ouabain is present. And when there is an incubation with 1nM ouabain prior to hypoxia, the intact arteries contracted from 338.9±11.4μm to 302.6±14.9μm (P<0.05). But in the endothelium-denuded group, arteries contracted only from 337.0±19.2μm to 328.8±23.4μm (P>0.05). This indicates that, 1nM ouabain could help CBAs generate myogenic tone not only under normaxia, but also under hypoxia, but this happened on intact basilar arteries, which presents endothelium dependence.5 Effects of 1 nM ouabain on basilar artery contraction induced by O⁺2-free K⁺-PSSWhen a high concentration of K⁺ was present, the contraction went further along with the superfusion in about 4 minutes. At the first, second, third and forth minute of superfusion, basilar arteries in control group contracted by 30±17.8μm, 54.4±21.9μm, 68±23.7μm and 72.8±25.1μm, separately. And the basilar arteries in hypoxia group contracted by 40.6±16.9μm, 74.8±22.7μm, 86.8±21.9μm and 83.6±23.4μm. This is a faster and higher contraction than control group. In ouabain-hypoxia group, basilar arteries contracted by 61.4±20.8μm, 97.2±20.4μm, 102.6±22.8μm and 103.6±23.1μm. This is a much faster and higher contraction both than control group and hypoxia group. This indicates that, hypoxia could help hypoxic arteries increase the sensitivities to vasoconstrictor K⁺-PSS, and 1 nM ouabain could further this effect.6 Effect of hypoxia on the affinities of Na⁺, K⁺-ATP aseαisoforms in guinea pig basilar arteryAccording the effects of ouabain in different concentration on passive diameter of basilar arteries under normaxia, the amplitude of contraction went further when the concentration of ouabain went higher. The contraction effects were calculated by two isoforms with two combine domain formula. The results of the calculation indicates that, there are two kinds ofαsubunits, which are with high affinity (α₂ and/orα₃) or low affinity (α₁), present in Guinea pigs'basilar arteries. But the kD ofαisoforms are different between under normaxia and hypoxia: kD ofαisoforms with high affinity are 5.3×10^-10M and 1.57×10^-7M respectively, and kD ofαisoforms with low affinity are 1.26×10^-6 M and 9.89×10^-6 M respectively. This indicates that: 30min of hypoxia could inhibit the affinity of allαsubunits, and the inhibition onαsubunits with high affinity (α₂ and/orα₃) is more significantly.Conclusion: The isoforms ofαsubunit from guinea pigs' CBAs areα₁ with low sensitivity andα₂ &/orα₃ with high sensitivity. 1 nM ouabain could not influence hypoxia dilation, but it could help the CBAs generate myogenic tone if there is a pre-incubation. hypoxia could help hypoxic arteries increase the sensitivities to vasoconstrictor K⁺-PSS, and 1 nM ouabain could further this effect. Hypoxia induces hypoxic dilation and depress the activities of allαisoforms from Na⁺, K⁺-ATPase, especially theαisoform with high affinity.