| Cerebral ischemia is an ischemic condition where the brain or parts of the brain do not receive enough blood flow to maintain normal neurological function. The cerebral vascular is very important for supplying enough oxygen and glucose through the blood flow to maintain the brain normal metabolism. Therefore, vascular dysfunction is a key factor in occurrence and development of cerebral ischemia.Smooth muscle in the vascular wall has been implicated as potential target site of action for hypoxia. For example, low Po2 may induce vasoconstriction via altered Ca2+ homeostasis in pulmonary smooth muscle cells. Also, a role for altered function of the Na+, K+-ATPase in the hypoxic response may be suggested.Na+, K+-ATPase is a transport ATPase in the plasma membrane which establishes and maintains the low cytosolic Na+ and high cytosolic K+ concentrations in all animal cells. By utilizing the energy from the hydrolysis of one molecule of ATP, it transports three Na+ out of the cell and two K+ into the cell. The electrochemical gradient the Na+, K+-ATPase generates is critical in maintaining the resting membrane potential and excitable properties of cells.When cerebral ischemia happened, the activity of Na+, K+-ATPase is reduced because of ATP exhaustion, resulting in intracellular Na+ concentration increasing quickly. As a result, the raise of the intracellular Na+ activates the reverse Na+-Ca2+ exchange which contributes to Ca2+ overload and in turn causes the cell damage. Therefore, Na+, K+-ATPase plays an important role in occurrence and development of cerebral ischemia.Previous studies about vascular injury induced by hypoxia are focused on pulmonary artery. However, the role of Na+, K+-ATPase in cerebral vascular smooth muscle during hypoxia remains uncertain. Therefore, the present study used the rat isolated basilar artery and cultured basilar artery smooth muscle cell to explore the role of Na+, K+-ATPase in basilar artery hypoxic contraction.1 Effect of hypoxia on basilar artery contractionObjective: To study the effect of hypoxia on the constriction of rat basilar artery.Methods: The contractile responses induced by KCl or U46619 were measured in isolated rat basilar artery rings with or without the intact endothelium under normoxic and hypoxic conditions using Multi Myograph. Also, the contractile response of basilar artery before and after addition of different calcium blockers was measured by Multi Myograph under hypoxic conditions. The cultured rat basilar artery smooth muscle cells were prepared and identified by immunohistochemistry and light microscopy. The changes of [Ca2+]i were measured by confocal microscopy.Results:(1) Effect of hypoxia on basilar artery contractile response precontracted with KClUnder hypoxic conditions, the contractile response induced by KCl was increased both in the presence or absence of endothelium and it reached the most potent at 10 min after hypoxia.(2) Effect of hypoxia on dose-response curve of KCl-induced vasoconstrictionIn rat basilar artery rings with intact endothelium, the cumulative dose-response curve of KCl in normoxia was shifted to the left after hypoxia. The contraction values in hypoxia for 40mmol/L, 60mmol/L, 70mmol/L, 80mmol/L, 90mmol/L and 100mmol/L KCl were 0.09±0.04, 0.421±0.08, 0.55±0.12, 0.62±0.14, 0.68±0.15 and 0.73±0.16 mN/mm, respectively, which were significantly higher than those in normoxia and were 0.04±0.03, 0.20±0.09, 0.26±0.08, 0.33±0.08, 0.38±0.08 and 0.43±0.07 mN/mm, respectively. These results indicated that the hypoxia increased the basilar artery contractile response to KCl.Hypoxia also caused the cumulative dose-response curve of KCl shift to the left in deduced arteries and the increases of Emax (from 0.392±0.052 to 0.773±0.106 mN/mm, p<0.01) and pD2 (from 1.23±0.05 to 1.32±0.03, p<0.01).(3) Effect of hypoxia on basilar artery contractile response precontracted with U46619Hypoxia induced an increase in vascular tension in deduced arteries precontracted with U46619. And the contractile response to hypoxia reached the maximal at 5 min after hypoxia. Although the magnitude of contraction was slightly decreased relative to the maximal response as the hypoxic time prolonged, it was still more potent than the contraction in normoxia.(4) Effect of hypoxia on dose-response curve of U46619-induced vasoconstrictionIn deduced rings of rat basilar artery, the cumulative dose-response curve of U46619 in normoxia was shifted to the left after hypoxia. The contraction values in hypoxia for 40mmol/L, 60mmol/L, 70mmol/L, 80mmol/L, 90mmol/L and 100mmol/L KCl were 0.106±0.018, 0.248±0.042, 0.620±0.108 and 0.670±0.084 mN/mm, respectively, which were significantly higher than those in normoxia and were 0.060±0.024, 0.102±0.012, 0.312±0.070 and 0.340±0.076 mN/mm, respectively (p<0.01).(5) Effect of nimodipine on basilar artery hypoxic contractionIn intact endothelium arteries, the contraction magnitude induced by 60mM KCl was increased from 0.18±0.065mN/mm to 0.38±0.061mN/mm after exposing to hypoxia. Nimodipine (10-8M) alleviated the hypoxic contraction magnitude to 0.31±0.10mN/mm. In deduced arteries the 60mM KCl-induced contraction were 0.26±0.047mN/mm and 0.54±0.049mN/mm before and after hypoxia, respectively. Incubation of nimodipine also attenuated the hypoxic contraction. However, under normoxic conditions nimodipine had no effect on the KCl-induced contraction of basilar artery rings with or without intact endothelium. (6) Effect of ryanodine on the basilar artery hypoxic contractionThe contraction magnitudes induced by KCl were increased after hypoxia both in intact endothelium artery rings (0.19±0.043→0.38±0.061mN/mm) and deduced artery rings (0.19±0.010→0.54±0.049mN/mm), which were moderated by pretreatment of ryanodine (10-5M) to 0.25±0.066mN/mm in intact endothelium artery rings and to 0.30±0.083mN/mm in deduced rings. However, the ryanodine did not alter the KCl-induced contraction of both basilar arteries with or without intact endothilium in normoxic condition.(7) Effect of Ni2+ on basilar artery hypoxic contractionIn intact endothelium and deduced artery rings, pretreatment of Ni2+ (10-3M) decreased the contraction magnitudes of basilar arteries induced by 60 mM KCl under hypoxic conditions (E+: 0.38±0.06→0.23±0.07 mN/mm; E-: 0.54±0.05→0.23±0.08 mN/mm) , but did not under normoxic conditions.(8) Effect of hypoxia on [Ca2+]i①The morphologic change of vascular smooth muscle cell (VSMC)After 3 days of incubation, primary cultures of rat basilar artery smooth muscle cells began to stick to the wall of the incubation dishes. It could be observed that great amount of cells had grown on the bottom after being cultivated 5 days. The cultured cells showed the typical"peak and valley"morphological appearance under microscope. After being stained by means of anti-α-actin immunohistochemistry, great amount of buffy actin could be observed beneath the cell membrane of the fourth generation cell.②Effect of hypoxia on [Ca2+]i in cultured VSMCThe fluorescence represented the [Ca2+]i of cultured VSMC was significantly increased from 39.8±6.5 to 66.4±13.8 after perfusion with normoxic solution (P <0.01).The [Ca2+]i began to increase at 3h and reach the maximal at 12h after hypoxia. After that, the [Ca2+]i didn't further increase with the prolonged hypoxia.Conclusion: Hypoxia induces a significant increase in contractile response in basilar artery rings with or without endothelium. The basilar artery hypoxic contraction is attenuated by nimodipine, ryanodine or Ni2+, indicating that hypoxic contraction is associated with [Ca2+]i increase induced by Ca2+ influx through sarcolemmal L-type Ca2+ channels, Ca2+ release from sarcoplasmic reticulum mediated via ryanodine receptor, or/and Na+-Ca2+ exchanger mediated by Na+, K+-ATPase inhibition. Hypoxia increases the [Ca2+]I of basilar artery smooth muscle cells.2 Effect of hypoxia on Na+, K+-ATPase function in rat basilar artery Objective: To study the effect of hypoxia on Na+, K+-ATPase of basilar artery smooth muscle cellsMethods: The activity of Na+, K+-ATPase was assessed through the double enzymic method. The amount ofαisoform message and protein was assessed by RT-PCR and Western blot, respectively. The distribution ofαisoform was determined with immunofluorescence assay. The pump current was recorded by whole cell patch clamp.Results:(1) Effect of hypoxia on the activity of Na+, K+-ATPase①Effect of hypoxia on the activity of Na+, K+-ATPase in isolated basilar arteryThe Na, K-ATPase activity in isolated basilar artery was decreased with prolonged hypoxia. Their activities at 0min, 2min, 5min, 10min, 15min, and 20min exposed to hypoxia were 33±5.4, 31±6.2, 26±5.2, 17±3.4, 20±3.7 and 21±3.6μmolPi/mg protein/h, respectively, and reached the lowest at 10 min after hypoxia (p<0.01).②Effect of hypoxia on the activity of Na+, K+-ATPase in cultured basilar artery smooth muscle cellsExposure of cell to hypoxia induced a significant decrease in Na+,K+-ATPase activity. It was decreased at 3h of exposure to hypoxia compared with normoxic cells (36.86±7.35 vs 52.72±8.69μmolNADH/mg protein/h, respectively, P<0.01), and reached the lowest at 6h after hypoxia (22.67±8.38μmolNADH/mg protein/h, P<0.01).③Effect of hypoxia on pump current of basilar artery smooth muscle cellsThe pump current density of basilar artery smooth muscle cell was significantly decreased from 0.177±0.0257 pA/pF to 0.101±0.0217 pA/pF after 24h exposure to hypoxia (P<0.01).(2) Effect of hypoxia on the expression ofαisoform mRNAThe mRNA ofα1 isoform began to decrease at 24h after hypoxia (P<0.05), whileα2 mRNA was decreased at 12h after hypoxia. Hypoxia didn't alterα3 mRNA.(3) Effect of hypoxia on the expression ofαisoform proteinThe expression ofα2 isoform protein was decreased at 12h (P<0.01) after hypoxia, while the protein expression ofα1 isoform showed no change during hypoxia.(4) Effect of hypoxia on distribution of Na+,K+-ATPaseαisoformUnder normoxic conditions,α1 isoform is distributed ubiquitously over the surface of the nucleus and myocyte, little was detected in the cytoplasm. In contrastα1,α2 was confined to a reticular distribution within the cytoplasm or over surface of myocyte, whileα3 exhibited an organized reticular pattern in the cytoplasm. There was no change in different distribution ofαisoforms before and after hypoxia 24h.Conclusion:The Na+,K+-ATPase activity gradually decreases with prolonged hypoxia. Theα1 isoform mRNA decreases but the amount of protein doesn't change at 24h after hypoxia. The amount ofα2 mRNA and protein level is decreased at 12h, whileα3 mRNA remains the same during the hypoxic period. The pump current is decreased at 24h after compared with normoxia. There is no change in different distribution ofαisoforms before and after hypoxia 24h.3 Na+, K+-ATPase is involved in basilar artery hypoxic contractionObjective: To study the mechanism and the role of Na, K-ATPase in hypoxic basilar artery contractionMethods: The contractile response of basilar artery before and after addition of ouabain and K-free solution under hypoxic conditions was measured by Multi Myograph. The activity of Na+, K+-ATPase was assessed by test kit. The changes of [Ca2+]i were measured by confocal microscopy.Results:(1) Effect of ouabain on basilar artery contractile response precontracted with KCl during hypoxiaIn artery rings with intact endothelium, ouabain (5×10-7mol/L) had no effect on KCl-induced contractile response in hypoxia, but it reduced the magnitude of contraction in artery rings deduced endothelium in hypoxia. Furthermore, under normoxic conditions, ouabain had no effect on the contractile response inducd by KCl both in intact endothelium and deduced basilar artery rings.(2) Effect of ouabain on dose-response curve of KCl-induced vasoconstriction before and after hypoxiaIn rat basilar artery rings with intact endothelium, the cumulative dose-response curve of KCl in normoxia was shifted to the left after hypoxia. Pretreatment of ouabain increased the pD2 (p<0.01) in hypoxia, but Emax value in hypoxia and the cumulative dose-response curve of KCl in normoxia were not altered in basilar artery rings with intact endothelium.Hypoxia also caused the cumulative dose-response curve of KCl shift to the left in deduced arteries and the increases of Emax (from 0.392±0.052 to 0.773±0.106 mN/mm, p<0.01) and pD2 (from 1.23±0.05 to 1.32±0.03, p<0.01). In the same way, incubation of deduced arteries with ouabain reduced the Emax and pD2, which caused the KCl dose-response curve shift to right in hypoxia.(3) Effect of K-free solution and ouabain on basilar artery contractile response precontracted with U46619 during hypoxiaHypoxia induced an increase in vascular tension in deduced arteries precontracted with U46619. And the contractile response to hypoxia reached the maximal at 5 min after hypoxia. Pretreatment of ouabain or K-free solution both attenuated the hypoxic contractions, but didn't completely inhibit them.(4) Effect of ouabain and K-free solution on dose-response curve of U46619-induced vasoconstriction before and after hypoxia In deduced rings of rat basilar artery, the cumulative dose-response curve of U46619 in normoxia was shifted to the left after hypoxia. The contraction values in hypoxia for 40mmol/L, 60mmol/L, 70mmol/L, 80mmol/L, 90mmol/L and 100mmol/L KCl were 0.106±0.018, 0.248±0.042, 0.620±0.108 and 0.670±0.084 mN/mm, respectively. And in the presence of ouabain the hypoxic contraction was decreased to 0.085±0.019, 0.143±0.031, 0.463±0.103, 0.513±0.085 mN/mm, respectively. Similarly, in the presence of K-free solution, the hypoxic contraction was attenuated to 0.083±0.022, 0.160±0.027, 0.444±0.091, 0.503±0.077 mN/mm, respectively. Under hypoxic conditions, ouabain and K-free solution had no effect on the cumulative dose-response curve of U46619, but reduced the pD2 in hypoxia (p<0.05).(5) Effect of hypoxia on the dose-response curve of vasoconstriction induced by ouabainIn rat basilar artery rings with and without intact endothelium, the cumulative dose-response curve of ouabain in normoxia was shifted to the right after hypoxia. Hypoxia could reduce the ratio of the high affinityα-isoform in basilar artery (fh) both in intact endothelium artery rings (62.0%→46.6%) and deduced rings( 48.4%→8.8%), while increase the ratio of the low affinityα-isoform (fl) ( E+: 38.0%→53.4%; E-: 51.6%→91.2%).(6) Effect of ouabain and hypoxia on [Ca2+]i in cultured basilar artery smooth muscle cellThe fluorescence represented the [Ca2+]i in cultured basilar artery smooth muscle cell was significantly increased from 38.9±6.2 to 47.0±7.6 after perfusion with ouabain (5×10-7mol/L) in normoxic solution (P <0.01). And the raised fluorescence of Ca2+ by ouabain was further increased followed the hypoxic perfusion contained ouabain (P <0.01). The fluorescence of Ca2+ induced by hypoxic perfusion was increase, compared with that of by hypoxic perfusion contained ouabain (p<0.01).(7) Effect of ouabain on the activity of Na+, K+-ATPase in isolated basilar arteryThe Na+, K+-ATPase activity in isolated basilar artery was decreased at 10 min exposed to hypoxia. Pretreatment of ouabain decreased their activities of both in hypoxia and in normoxia and there was no significant difference between them (P>0.05).Conclusion: Ouabian and K-free solution reduce the magnitude of hypoxic contraction in deduced basilar artery rings. Hypoxia can reduce the ratio of the high affinityα-isoform in basilar artery with and without endothelium. Ouabain increases the [Ca2+]i, while decreases the Na+, K+-ATPase activity. The high affinityα2-isoform of Na+, K+-ATPase is involved in basilar artery hypoxic contraction. |
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