The Effects Of Na～+-K～+-ATPase On Isolated Rat Cerebral Arteries Evoked By 5-HT During Anoxia

Cerebral ischemia is a common disease, which may induce to brain injury. 5-HT is one of the most potent cerebral vasoconstrictors. When cerebral ischemia happens, the concentration of 5-HT outside of cells increases. The powerful contractive effect of 5-HT on cerebral arteries is considered to be involved in vasospasm after cerebral ischemia. The vasocontrictive effect of 5-HT on cerebral arteries is associated with 5-HT binding to specific receptors on vascular smooth muscle cells (VSMC_S) through which the increases in [Ca²⁺]_i is mediated and the cerebral artery contraction is induced through Ca²⁺-dependent protein kinase C. It is also found the concentration of ouabain in serum remarkably increased when cerebral ischemia occurred. Ouabain can cause artery contraction, and its mechanism may be associated with which it can inhibit the cellular membrane Na+-K+-ATPase activity. In addition, it has been also reported that the activity of Na⁺-K⁺-ATPase decreased during the cerebral ischemia. Na⁺-K⁺-ATPase is an intrinsic enzyme in the plasma membrane, which transports three Na+ out of the cell and two K⁺ in and establishes the normal transmembrane gradients of Na⁺ and K⁺ in most animal cells for maintenance of cell-specific function. When cerebral ischemia occurred, Na⁺-K⁺-ATPase activity decreased, which causes the increased cytosolic Na+ and the activation of the Na⁺-Ca²⁺ exchanger, leading to influx of Ca²⁺ and a further increase in [Ca²⁺]_i . But it is unclear whether ouabain and Na+-K+-ATPase are involved in the vasocontrictive effect of 5-HT on cerebral arteries or not. The present study was initiated to determine the interactive effects of ouabain and Na⁺-K⁺-ATPase on the contractile effect of 5-HT on cerebral arteries. Objective: To observe the effects of 5-HT on isolated rat cerebral arteries and its interactions with Na⁺-K⁺-ATPase. Methods: Sprague-Dawley rats (200-250g) 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,MgSO₄ï¹’7H2O 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 relaxedcircumference at 100 mmHg transmural pressure. Isometric tension is expressed as millinewtons per millimeter of vessels'responses to drugs, the degrees of contractile activity of drugs were recorded. Results: 1) 5-HT caused a concentration-dependent contraction in artery rings of rat in the presence or absence of oxygen. The diameters of basilar artery, vertebral artery and middle cerebral artery were 101.6±1.8μm, 91.72±1.8μm and 81.58±1.6μm, respectively. Normally, the 50% effective concentrations (EC₅₀) of BA, VA and MCA contractions by 5-HT in the presence of oxygen were 2.22±0.8 μmol/L, 4.25±0.1 μmol/L and 10.5±6.2 μmol/L respectively. During hypoxia the EC₅₀ of them were 0.099±0.004μmol/L, 0.125±0.085μmol/L and 0.458±0.014μmol/L, respectively. The extents of several cerebral arterial contraction evoked by 5-HT showed significant differences (P<0.01). The orders of three cerebral arterial sensitivity for 5-HT were BA >VA>MCA. Furthermore, hypoxia can shift the 5-HT concentration-response curve to the left. Considering the isolated BA was the most sensitive to 5-HT, the latter experiments all chose BA. 2) Under hypoxic condition, Ouabain (1 mmol/L) shifted the 5-HT concentration-response curve to the left and increased the maximum response. The EC₅₀ of 5-HT with and without ouabain for BA contraction were 93.6±10 nmol/L and 61.8±2.4 nmol/L, respectively. Ouabain can significantly enhance the contraction of 5-HT on BA (P<0.01).3) Under hypoxia condition, the full inhibition of Na⁺-K⁺-ATPase activity through the infusion of K+-free PSS solution could shift the 5-HT concentration-response curve to the left in the presence of ouabain. The EC50 of 5-HT in ouabain-contained PSS solutions with or without K⁺ for BA contraction were 71.8±10nmol/L and 30.8±4nmol/L. The inhibition of Na⁺-K⁺-ATPase activity can significantly enhance 5-HT contraction on BA (P<0.01). 4) Under hypoxic condition, dipfluzine (15 μmol/L), a calcium channel blocker, could abolish the 5-HT induced contraction on BA rings, indicating the contraction of BA induced by 5-HT also involved in Ca2+ influx. 5) Under hypoxic condition, Ca2+-free PSS solution in which ouabain could concentration-dependently contract the BA rings shifted the concentration-response curve of ouabain to the right compared to normal PSS solution, and decreased the maximum response of BA to ouabain. The EC50 of ouabain for BA contraction in the normal or Ca²⁺-free PSS solutions were 28.8±5μmol/L and 71.9±10μmol/L. Ca²⁺ can remarkably enhance the contraction of ouabain on BA (P<0.01). 6) Under hypoxia condition, dipfluzine (15 μmol/L) decreased the response on BA rings induced by ouabain not only in normal medium, but also in the Ca2+-free medium. Conclusions: (1) 5-HT causes a concentration-dependent contraction in artery rings of rat in the presence or absence of oxygen. The basilar artery is the most sensitive to 5-HT. The...