Cutler L S, Christian C P, Feinstein M B
Biochim Biophys Acta. 1985 Jun 30;845(3):403-10. doi: 10.1016/0167-4889(85)90205-8.
Platelets were briefly fixed in paraformaldehyde/glutaraldehyde and then incubated with 5'-adenylyl imidodiphosphate under conditions suitable for the cytochemical detection of adenylate cyclase activity. The adenylate cyclase activity of these platelets retains the ability to respond to prostaglandins E1, D2, I2 (prostacyclin), forskolin and fluoride. Sites of stimulated adenylate cyclase activity were localized cytochemically by the reaction of lead with the reaction product imidodiphosphate to form deposits of lead imidodiphosphate that are visible in the electron microscope. Reaction product deposition was seen only in the dense tubule system of human platelets when the incubation medium contained forskolin, prostacyclin, or prostaglandin D2 at concentrations known to stimulate the enzyme in intact platelets. Epinephrine, an antagonist of adenylate cyclase inhibited the cytochemical reaction stimulated by prostacyclin. The fact that the cytochemical reaction was induced by agonists that stimulate the enzyme through two different types of prostaglandin receptors and by forskolin, which acts distal to the receptors, confirms that the method specifically detects adenylate cyclase. The presence of adenylate cyclase in the dense tubules may be significant for the regulation of intracellular Ca2+ and arachidonic acid metabolism by this membrane system.
血小板先用多聚甲醛/戊二醛短暂固定，然后在适合腺苷酸环化酶活性细胞化学检测的条件下与5'-腺苷酰亚胺二磷酸一起孵育。这些血小板的腺苷酸环化酶活性保留了对前列腺素E1、D2、I2（前列环素）、福斯高林和氟化物作出反应的能力。通过铅与反应产物亚胺二磷酸的反应，将受刺激的腺苷酸环化酶活性位点进行细胞化学定位，形成亚胺二磷酸铅沉淀，在电子显微镜下可见。当孵育培养基中含有已知能刺激完整血小板中该酶的浓度的福斯高林、前列环素或前列腺素D2时，仅在人血小板的致密小管系统中可见反应产物沉积。腺苷酸环化酶的拮抗剂肾上腺素抑制了前列环素刺激的细胞化学反应。激动剂通过两种不同类型的前列腺素受体刺激该酶，以及福斯高林在受体远端起作用，都能诱导细胞化学反应，这一事实证实了该方法能特异性检测腺苷酸环化酶。致密小管中存在腺苷酸环化酶可能对该膜系统调节细胞内Ca2+和花生四烯酸代谢具有重要意义。
