Parfenova H, Neff R A, Alonso J S, Shlopov B V, Jamal C N, Sarkisova S A, Leffler C W
Laboratory for Research in Neonatal Physiology, Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA.
Am J Physiol Cell Physiol. 2001 Dec;281(6):C1954-63. doi: 10.1152/ajpcell.2001.281.6.C1954.
Endogenous carbon monoxide (CO) contributes to vasodilator responses of cerebral microvessels in newborn pigs. We investigated the expression, intracellular localization, and activity of heme oxygenase (HO), the key enzyme in CO production, in quiescent cerebral microvascular endothelial cells (CMVEC) from newborn pigs. HO-1 and HO-2 isoforms were detected by RT-PCR, immunoblotting, and immunofluorescence. HO-1 and HO-2 are membrane-bound proteins that have a strong preference for the nuclear envelope and perinuclear area of the cytoplasm. Betamethasone (10(-6) to 10(-4) M for 48 h) was associated with upregulation of HO-2 protein by approximately 50% and inhibition of Cox-2 but did not alter HO-1 or endothelial nitric oxide synthase expression in CMVEC. In vivo betamethasone treatment of newborn pigs (0.2 and 5.0 mg/kg im for 48 h) upregulated HO-2 in cerebral microvessels by 30-60%. HO activity as (14)CO production from [(14)C]glycine-labeled endogenous heme was inhibited by chromium mesoporphyrin (10(-6) to 10(-4) M). L-Glutamate (0.3-1.0 mM) stimulated HO activity 1.5-fold. High-affinity specific binding sites for L-[(3)H]glutamate suggestive of the glutamate receptors were detected in CMVEC. Altogether, these data suggest that, in cerebral circulation of newborn pigs, endothelium-derived CO may contribute to basal vascular tone and to responses that involve glutamate receptor activation.
内源性一氧化碳（CO）有助于新生猪脑微血管的血管舒张反应。我们研究了新生猪静止脑微血管内皮细胞（CMVEC）中血红素加氧酶（HO）的表达、细胞内定位及其活性，HO是CO生成的关键酶。通过逆转录聚合酶链反应（RT-PCR）、免疫印迹和免疫荧光检测HO-1和HO-2亚型。HO-1和HO-2是膜结合蛋白，对核膜和细胞质的核周区域有很强的偏好。倍他米松（10⁻⁶至10⁻⁴M，作用48小时）使HO-2蛋白上调约50%并抑制Cox-2，但不改变CMVEC中HO-1或内皮型一氧化氮合酶的表达。对新生猪进行体内倍他米松治疗（0.2和5.0mg/kg，肌肉注射，48小时）使脑微血管中的HO-2上调30%-60%。用中卟啉铬（10⁻⁶至10⁻⁴M）抑制了由[¹⁴C]甘氨酸标记的内源性血红素产生¹⁴CO的HO活性。L-谷氨酸（0.3-1.0mM）使HO活性提高了1.5倍。在CMVEC中检测到了提示谷氨酸受体的L-[³H]谷氨酸的高亲和力特异性结合位点。总之，这些数据表明，在新生猪的脑循环中，内皮源性CO可能有助于基础血管张力以及涉及谷氨酸受体激活的反应。
