大鼠尾动脉平滑肌中α肾上腺素受体的分析

采用离体血管收缩功能实验方法分析大鼠尾动脉平滑肌中α肾上腺素受体(α受体)各种亚型的组成及各自的功能意义,结果表明:尾动脉平滑肌中α_1,受体在功能上占绝对支配地位,包含α_(1A)与α_(1B)两种亚型,它们与去甲肾上腺素的亲和性相同,α_(1A)受体具有较大的储备,而α_(1B)受体无储备,前者在α_1受体激动剂致血管收缩效应中发挥主导作用,α_(1A)与α_(1B)受体之间可能存在协同效应。     

α1—肾上腺素受体拮抗剂的研究

α１－受体拮抗剂按其化学结构可分成喹唑啉类、苯并二恶烷类、芳基哌嗪类、芳烷胺类、喹啉类、缋哚烷及其药用植物的有效成分等。本文介绍上述各类具代表性的药物结构及作用特点，从而较完整地反映了目前国内外对α１－肾上腺素受体拮抗剂的最新研究成果。     

α1肾上腺素受体拮抗剂的研究进展

介绍α1肾上腺素受体拮抗剂的研究进展。α1肾上腺素受体亚型的研究进展,受体的分子生物学研究以及亚型分类的研究,随后介绍了α1肾上腺素受体拮抗剂的药理作用以及药理方面的新发展即在治疗男性良性前列腺增生中的应用。     

大鼠血管中α_1肾上腺素受体的两种亚型

α_1肾上腺素受体(α_1受体)激动引起的大鼠离体血管收缩,在主动脉可为不可逆性α_1受体拮抗剂CEC大部分阻断,而不受钙离子拮抗剂硝苯吡啶的影响;在肾动脉不受CEC阻断,却可为硝苯吡啶大部分阻断;在肠系膜动脉与门静脉则介于两者之间。竞争性α_1受体拮抗剂WB4101的pA_2值,肾动脉>肠系膜动脉>主动脉。根据已知α_1受体两种亚型的药理特征,上述结果提示大鼠血管中的α_1受体存在两种亚型,在主动脉内以α_(1b)亚型为主,在肾动脉内以α_(1a)亚型为主,在肠系膜动脉与门静脉内两种亚型的含量较为均衡。     

α1-肾上腺素受体亚型的分布及特点

近年来,运用分子生物学技术、放射配体结合技术、拮抗剂功能分析等手段,深入研究α1-肾上腺素受体亚型的分类、分布、受体蛋白结构等方面内容并取得很大进展。现将近几年研究状况综述如下。     

喹唑啉类α1-肾上腺素受体阻断药研究进展

高血压疾病严重影响着人类的健康和生活质量,目前临床用于治疗高血压的药物主要有以下几类:利尿药、β受体阻断药、钙拮抗剂、血管紧张素转换酶抑制药(ACEI)、血管紧张素Ⅱ受体拮抗剂及α1肾上腺素阻断药[1].α1肾上腺素阻断药近几年被推为第一线降压药物.其作用机制主要通过选择性作用于突触α1受体,使阻力血管和容量血管都扩张,从而使动脉血压下降.     

Nonlinear relationship between alpha 1-adrenergic receptor occupancy and norepinephrine-stimulated calcium flux in cultured vascular smooth muscle cells

To determine the relationship between vascular alpha 1-adrenergic receptor occupancy and receptor-coupled calcium flux, we have studied [3H]prazosin binding and l-norepinephrine-induced 45Ca efflux in cultured vascular smooth muscle cells isolated from the rabbit aorta. In a crude cellular homogenate, [3H]prazosin bound to a single high affinity site (Kd = 0.096 nM; Bmax = 105 +/- 15 fmol/mg of protein), whereas l-norepinephrine (NE) binding was best described by a two-site model with 43 +/- 8% of sites of high affinity (KH = 92 +/- 3 nM) and 57 +/- 7% of sites of low affinity (KL = 7460 +/- 4330 nM). NE-stimulated 45Ca efflux was concentration-dependent (EC50 = 108 nM) and potently inhibited by prazosin (IC50 = 0.15 nM), but not yohimbine (no inhibition at 10 microM). For the total receptor pool identified by [3H]prazosin binding, the relationship between receptor occupancy by NE and NE-stimulated 45Ca efflux was markedly nonlinear, such that 50% of maximum NE-stimulated efflux occurred with occupancy of only approximately 7% of receptors. Likewise, following irreversible inactivation of 69 +/- 5% of receptors by phenoxybenzamine, maximal NE-stimulated 45Ca efflux was decreased by only 8 +/- 2%. These two experimental approaches provide direct evidence for the presence in cultured rabbit aortic smooth muscle cells of a sizable pool of alpha 1-adrenergic receptors in excess of those needed for maximum NE-stimulated 45Ca efflux. This evidence of "spare" receptors, together with the finding of two affinity states of agonist binding, raises the possibility of functional heterogeneity of alpha 1-adrenergic receptors in this system.     

Nitric oxide regulates angiotensin II receptors in vascular smooth muscle cells

The objective of this study was to determine whether an enhanced generation of nitric oxide (NO) causes regulation of angiotensin II receptors in vitro using rat vascular smooth muscle cells in culture. Chronic treatment of cells with a series of NO-generating drugs, sodium nitroprusside, S-nitroso-N-acetylpenicillamine and isosorbide dinitrate for 18h dose and time-dependently decreased [¹²⁵I]-angiotensin II binding to cells without any significant change in affinity. Induction of nitric oxide synthase following lipopolysaccharide (10 and 100 ng/ml) treatment of cells for 18 h increased basal nitric oxide synthase activity with a concomitant increase of nitrite and cyclic cGMP levels in the conditioned media. LPS treatment significantly (P < 0.05) decreased [¹²⁵I]-angiotensin II binding to these cells, an effect that was significantly (P < 0.05) attenuated in the presence of N^G-nitro-L-arginine methyl ester. In contrast, treatment of cells with atrial natriuretic factor, dibutyryl cGMP, 8-bromo-cGMP, NaNO₂ or NaNO₃ failed to significantly alter the affinity or number of [¹²⁵I]-angiotensin II binding sites. These results suggest that NO regulates angiotensin II receptors in vitro through a cGMP-independent mechanism.     

Iloprost down-regulates the expression of the growth regulatory gene Cyr61 in human vascular smooth muscle cells

Prostacyclin and its mimetics have repeatedly been shown to act antiatherogenic and to inhibit neointima formation in several animal models of vascular injury. Treatment of human vascular smooth muscle cells with the prostacyclin mimetic iloprost (100 nm) drastically reduces expression of Cyr61, encoding the growth-regulatory cystein-rich angiogenic protein, without affecting the degradation rate of Cyr61 mRNA. Thrombin-induced Cyr61 expression was inhibited completely in the presence of iloprost. It is concluded that vasoprotective actions of prostacyclin in vivo may in part be due to inhibition of expression of the growth regulatory gene Cyr61 at sites of vascular lesions.     

Nitric oxide up-regulates aldose reductase expression in rat vascular smooth muscle cells: a potential role for aldose reductase in vascular remodeling

Acceleration of the polyol pathway under hyperglycemia is among the mechanisms implicated in the pathogenesis of diabetic complications. Although aldose reductase (AR), the rate-limiting enzyme in this pathway, is a target for pharmacological intervention of diabetic complications, the clinical efficacy of AR inhibitors has not been consistently proved. Because nitric oxide (NO) plays important roles in vascular hemodynamics and inflammatory responses that are affected under diabetic conditions, the interaction of NO with AR was investigated with rat aortic smooth muscle cells. Spontaneous NO donors, S-nitroso-N-acetylpenicillamine (SNAP) and 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamin e, elicited a dose-dependent increase in AR mRNA to a maximum of 7-fold in 12 h. The activity of AR was elevated after 10 h of SNAP treatment. These effects of NO donors were suppressed by the addition of 2-(trimethylammoniophenyl)-4,4,5, 5-tetramethylimidazoline-1-oxy 3-oxide, a scavenger of NO. Induction of AR mRNA by SNAP was completely abolished by actinomycin D or cycloheximide, but unaffected by guanylate cyclase inhibitors or genistein, a tyrosine kinase inhibitor. Pretreatment of the cells with N-acetyl-L-cysteine significantly suppressed the SNAP-induced up-regulation of AR mRNA. Under normal glucose conditions, inclusion of the AR inhibitor ponalrestat augmented the cytotoxic effect of SNAP on the cells. The level of AR mRNA also was elevated in a murine macrophage cell line RAW 264.7 stimulated with lipopolysaccharide and interferon-gamma. Inhibition of NO synthesis completely abolished the increase in AR mRNA in the stimulated cells. The up-regulation of AR by NO in the vascular lesions may modulate NO-induced cell death and the ensuing vascular remodeling during inflammatory responses.     

Nitric oxide stimulates elastin expression in chick aortic smooth muscle cells

Nitric oxide (NO), an endothelium-dependent relaxing factor, regulates relaxation, proliferation, and migration of smooth muscle cells (SMCs) and most likely attenuates developing vascular disease such as atherosclerosis. We investigated whether or not NO is associated with regulation of aortic elasticity. S-Nitrosoglutathione (GSNO), a NO donor, stimulated tropoelastin synthesis in cultured SMCs during both the quiescent and proliferating phases. The stimulation of tropoelastin synthesis was dose-dependent within 1-100 nM. Maximum stimulation was detected by treatment with 100 nM GSNO for 24 h. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), an exogenous cyclic GMP analog, also upregulated tropoelastin synthesis. Tropoelastin and lysyl oxidase mRNA expression, as assessed by Northern blot analysis, was also stimulated by GSNO. Administration of KT5823, a cyclic GMP-dependent protein kinase inhibitor, inhibited the GSNO-induced tropoelastin synthesis. These results indicate that the stimulatory effects of GSNO are due to cyclic GMP dependent protein kinase (PKG) activation by NO. In conclusion, NO seems to enhance aortic elasticity via tropoelastin and lysyl oxidase upregulation.     

Alpha 1-adrenergic stimulation and beta 2-adrenergic inhibition of DNA synthesis in vascular smooth muscle cells

Effects of catecholamines on DNA synthesis in vascular smooth muscle cells (VSMC) were investigated in a chemically defined medium that included insulin, transferrin, and sodium selenite. Smooth muscle-rich preparation was obtained from rat aortic media and VSMC were further purified by cell cloning. A clone that was positive for smooth muscle actin and was negative for the coagulation factor VIII was used in this study. The fetal calf serum-induced proliferation was enhanced by alpha-adrenergic and inhibited by beta-adrenergic stimulation. When cells of low passages were used, dose-response curves for norepinephrine were biphasic; when cells were subconfluent, norepinephrine stimulated DNA synthesis at as low as 1 nM and was apparently ineffective at more than 100 nM. When cells were confluent, the effect of norepinephrine was inhibitory at lower concentrations (less than 1 nM) and stimulatory at relatively higher concentrations. Cells of higher passages exhibited only inhibitory effects of the amine. Stimulatory and inhibitory effects on DNA synthesis were mediated through alpha 1- and beta 2-adrenergic receptors, respectively. Thus, the alpha 1-agonist phenylephrine was more potent than the alpha 2-agonist clonidine in stimulating DNA synthesis. An alpha 1-adrenergic antagonist, prazosin, was more effective than the alpha 2-adrenergic antagonist yohimbine in antagonizing the stimulatory effect of norepinephrine. beta-Adrenergic agonists inhibited DNA synthesis with IC50 values in the nanomolar range; the rank order of potency of agonists was isoproterenol greater than salbutamol greater than or equal to (-)-epinephrine much greater than (-)-norepinephrine, consistent with beta 2-receptor specificity. (+)-Epinephrine or (+)-norepinephrine, the stereoisomers of the catecholamines, were ineffective. The inhibitory effects of norepinephrine were reversed by beta-adrenergic antagonists, with the rank order of potency of pindolol greater than butoxamine greater than atenolol, consistent with beta 2-receptor specificity. The dose-response curves of norepinephrine, therefore, seemed to be determined by a balance between alpha 1-receptor-mediated stimulation and beta 2-receptor-mediated inhibition of DNA synthesis. Minimum time required for exhibiting alpha 1-adrenergic or beta 2-adrenergic effects was between 6 and 15 hr, suggesting that the G0 or G1 phase of the cell cycle might be the site of action. These results show that catecholamines dually modulate DNA synthesis in VSMC through specific adrenergic receptors.     

Nitric oxide from vascular smooth muscle cells: regulation of platelet reactivity and smooth muscle cell guanylate cyclase.

1. Incubation of smooth muscle cells (SMC) from bovine aorta for 3 min with human washed platelets treated with indomethacin (10 microM) promoted a cell number-related inhibition of platelet aggregation induced by thrombin (40 mu ml-1). This inhibition was not attributable to products of the cyclo-oxygenase pathway for the SMC were also treated with indomethacin (10 microM). 2. The inhibitory activity of the SMC on platelet aggregation was enhanced by incubating the SMC with E. coli lipopolysaccharide (LPS, 0.5 micrograms ml-1) for a period of 9 to 24 h. This effect was attenuated when cycloheximide (10 micrograms ml-1) was incubated together with LPS. Cycloheximide did not prevent the inhibitory activity of the non-treated cells. 3. The inhibition of platelet aggregation obtained with non-treated or LPS-treated SMC was potentiated by superoxide dismutase (SOD, 60 u ml-1) and ablated by oxyhaemoglobin (OxyHb, 10 microM). Preincubation of the SMC with NG-monomethyl-L-arginine (L-NMMA, 30-300 microM) for 60 min prevented their antiaggregatory activity. This effect was reversed by concurrent incubation with L-arginine (L-Arg, 100 microM) but not with D-arginine (D-Arg, 100 microM). 4. Exposure of the non-treated SMC (5 x 10(5) cells) to stirring (1000 r.p.m., 37 degrees C) for 10 min led to a significant increase in their levels of guanosine 3':5'-cyclic monophosphate (cyclic GMP) but not adenosine 3':5'-cyclic monophosphate (cyclic AMP). L-NMMA (300 microM) attenuated the increase in cyclic GMP induced by stirring but did not affect the basal levels of cyclic GMP in the cells.(ABSTRACT TRUNCATED AT 250 WORDS)