Identification of α-Adrenergic Receptors in Human Platelets by [3H]Dihydroergocryptine Binding
Identification of α-Adrenergic Receptors in Human Platelets by [3H]Dihydroergocryptine Binding
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通过 [3H]二氢麦角隐亭结合鉴定人血小板中的 α-肾上腺素能受体
DOI：
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发表时间：
1978
期刊：
影响因子：
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通讯作者：
R. Lefkowitz
中科院分区：
文献类型：
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作者：
Newman Kd;L. Williams;Bishopric Nh;R. Lefkowitz
[(3)H]二氢麦角隐亭与血小板裂解物的结合似乎具有与α-肾上腺素能受体结合的所有特征。在25℃时，结合在20分钟内达到平衡，当加入过量的酚妥拉明时是可逆的。结合是饱和的，每毫克蛋白质结合183+/-22fmol的[(3)H]二氢麦角隐亭，相当于每个血小板220+/-26个位点。动力学和平衡研究表明，[(3)H]二氢麦角隐亭对受体的解离常数为1-3 nM。结合部位的特异性是α-肾上腺素能受体的典型特征。儿茶酚胺激动剂以(-)肾上腺素>；(-)去甲肾上腺素>；&(-)异丙肾上腺素的效力顺序竞争[(3)H]二氢麦角隐亭结合位点。由于肾上腺素和去甲肾上腺素的(+)异构体的效力比(-)异构体低10-20倍，因此表现出立体特异性。强效的α-肾上腺素能拮抗剂酚妥拉明、苯氧苯甲胺和育亨宾能有效地竞争这些位点，而β-受体拮抗剂如心得安和二氯异丙肾上腺素则很弱。多巴胺和5-羟色胺仅在高浓度(0.1 mM)时相互竞争。[(3)H]二氢麦角隐亭结合位点也可以在完整的血小板中被证实，在那里它们表现出类似的特异性、立体特异性和饱和性。与完整血小板的饱和结合研究表明，每个血小板有220+/-45个受体，这与用血小板裂解物研究得出的值很好地一致。α-肾上腺素能激动剂抑制腺苷环化酶的能力和α-肾上腺素能拮抗剂对抗这种抑制作用的能力与与[(3)H]二氢麦角隐亭结合位点相互作用的能力直接平行。这些数据证明了用[(3)H]二氢麦角隐亭直接研究人血小板α-肾上腺素能受体结合部位的可行性。
Binding of [(3)H]dihydroergocryptine to platelet lysates appears to have all the characteristics of binding to alpha-adrenergic receptors. At 25 degrees C binding reaches equilibrium within 20 min and is reversible upon addition of excess phentolamine. Binding is saturable with 183+/-22 fmol of [(3)H]dihydroergocryptine bound per mg of protein at saturation, corresponding to 220+/-26 sites per platelet. Kinetic and equilibrium studies indicate the dissociation constant of [(3)H]dihydroergocryptine for the receptors is 1-3 nM. The specificity of the binding sites is typical of an alpha-adrenergic receptor. Catecholamine agonists compete for occupancy of the [(3)H]dihydroergocryptine binding sites with an order of potency (-)epinephrine> (-)norepinephrine>> (-)isoproterenol. Stereospecificity was demonstrated inasmuch as the (+)isomers of epinephrine and norepinephrine were 10-20-fold less potent than the (-)isomers. The potent alpha-adrenergic antagonists phentolamine, phenoxybenzamine, and yohimbine competed potently for the sites, whereas beta-antagonists such as propranolol and dichlorisoproterenol were quite weak. Dopamine and serotonin competed only at high concentrations (0.1 mM). The [(3)H]dihydroergocryptine binding sites could also be demonstrated in intact platelets where they displayed comparable specificity, stereospecificity, and saturability. Saturation binding studies with the intact platelets indicated 220+/-45 receptors per platelet, in good agreement with the value derived from studies with platelet lysates. Ability of alpha-adrenergic agonists to inhibit adenylate cyclase and of alpha-adrenergic antagonists to antagonize this inhibitory effect directly paralleled ability to interact with the [(3)H]dihydroergocryptine binding sites. These data demonstrate the feasibility of directly studying alpha-adrenergic receptor binding sites in human platelets with [(3)H]dihydroergocryptine.