MacFarland R T, Zelus B D, Beavo J A
Department of Pharmacology, University of Washington, Seattle 98195.
J Biol Chem. 1991 Jan 5;266(1):136-42.
An adrenal cGMP-stimulated phosphodiesterase (cGS-PDE) has been shown to mediate atrial natriuretic peptide (ANP)-induced reductions in aldosterone secretion and cAMP levels in primary bovine glomerulosa cells. High concentrations of cGS-PDE have been localized to the zona glomerulosa cell layer of the adrenal cortex using biochemical and immunological techniques. Immunoblot analysis using an affinity-purified, isozyme-specific antiserum revealed a single band that comigrated with a purified cGS-PDE (105 kDa) (1) and that was most highly concentrated in the outermost 1-2 mm of the cortex, representing the capsule and zona glomerulosa regions. Greater than 90% of the overall phosphodiesterase activity present in tissue extracts prepared from these regions was immunoprecipitated using a solid-phase monoclonal antibody reagent, indicating the cGS-PDE as the predominant phosphodiesterase isozyme. Immunohistochemical staining experiments of frozen thin sections of intact adrenal tissue revealed that the cGS-PDE present in this region was localized in the glomerulosa cells themselves. The role of this isozyme as a mediator of ANP-induced decreases in intracellular cAMP concentrations and aldosterone production was tested in primary cultures of bovine adrenal glomerulosa cells. In cells stimulated by ACTH, ANP treatment produced dose-dependent reductions in aldosterone secretion and cellular cAMP content over the same concentration range. Increases in aldosterone production elicited by three cell-permeable cAMP derivatives (8-bromo-cAMP, 8-p-chlorophenylthio-cAMP, and N6-2'-O-dibutyryl-cAMP) were antagonized by ANP, indicating a site of action distal to adenylate cyclase for this hormone. Because the relative magnitude of the ANP effect differed depending upon the derivative used, the three derivatives were compared with respect to their relative rates of in vitro hydrolysis by adrenal cGS-PDE. A positive correlation between their rates of hydrolysis and the degree to which the steroidogenic response produced by these derivatives was antagonized by ANP was demonstrated, further suggesting an ANP-induced activation of the cGS-PDE as being responsible for this effect. The possible contribution of an additional pathway mediated by an inhibitory guanine nucleotide binding regulatory protein (Gi) acting on adenylate cyclase was tested by pretreatment of primary glomerulosa cells with pertussis toxin. Levels of pertussis toxin sufficient to inhibit subsequent in vitro ribosylation did not significantly alter the ANP effect on aldosterone production, although a partial reduction in the ANP effect on cAMP levels was observed.(ABSTRACT TRUNCATED AT 400 WORDS)
肾上腺环磷酸鸟苷刺激的磷酸二酯酶（cGS-PDE）已被证明可介导心房利钠肽（ANP）诱导的原代牛球状带细胞醛固酮分泌减少和环磷酸腺苷（cAMP）水平降低。利用生化和免疫技术已将高浓度的cGS-PDE定位到肾上腺皮质的球状带细胞层。使用亲和纯化的同工酶特异性抗血清进行的免疫印迹分析显示出一条与纯化的cGS-PDE（105 kDa）迁移一致的条带（1），且该条带在皮质最外层1 - 2毫米处浓度最高，代表被膜和球状带区域。使用固相单克隆抗体试剂可免疫沉淀从这些区域制备的组织提取物中超过90%的总磷酸二酯酶活性，表明cGS-PDE是主要的磷酸二酯酶同工酶。对完整肾上腺组织冷冻薄片进行的免疫组织化学染色实验表明，该区域存在的cGS-PDE定位于球状带细胞自身。在原代培养的牛肾上腺球状带细胞中测试了这种同工酶作为ANP诱导细胞内cAMP浓度降低和醛固酮生成减少的介质的作用。在促肾上腺皮质激素（ACTH）刺激的细胞中，ANP处理在相同浓度范围内使醛固酮分泌和细胞内cAMP含量呈剂量依赖性降低。三种细胞可渗透的cAMP衍生物（8-溴-cAMP、8-对氯苯硫基-cAMP和N6,2'-O-二丁酰-cAMP）引起的醛固酮生成增加被ANP拮抗，表明该激素的作用位点在腺苷酸环化酶的远端。由于ANP效应的相对大小因所用衍生物而异，比较了这三种衍生物在体外被肾上腺cGS-PDE水解的相对速率。结果表明它们的水解速率与这些衍生物产生的类固醇生成反应被ANP拮抗的程度呈正相关，进一步表明ANP诱导的cGS-PDE激活是造成这种效应的原因。通过用百日咳毒素预处理原代球状带细胞来测试由抑制性鸟嘌呤核苷酸结合调节蛋白（Gi）作用于腺苷酸环化酶介导的另一条途径的可能贡献。足以抑制后续体外核糖基化的百日咳毒素水平虽使ANP对cAMP水平效应部分降低，但未显著改变ANP对醛固酮生成的效应。（摘要截短至400字）
