Davies W A, Berghorn K A, Albrecht E D, Pepe G J
Department of Physiology, Eastern Virginia Medical School, Norfolk, Virginia 23501.
Endocrinology. 1993 Jun;132(6):2491-7. doi: 10.1210/endo.132.6.8504752.
We have previously demonstrated that the estrogen-regulated change in transuteroplacental metabolism of cortisol (F) and cortisone (E) from preferential reduction (E to F) at midgestation to oxidation (F to E) near term results in activation of the hypothalamic-pituitary-adrenal axis of the baboon and the ontogenesis of rate-limiting steroidogenic enzymes, culminating in de novo F secretion. It is well established that transcription of messages activated by peptide-mediated binding to membrane receptors can occur via cAMP-dependent protein kinase-A (PKA) and/or phospholipid/calcium-dependent protein kinase-C (PKC). The present study was designed to determine whether basal levels of PKA and PKC in the fetal adrenal are developmentally regulated during baboon gestation and, thus, could provide the mechanism(s) by which activation of the fetal adrenal near term is mediated. Fetal adrenal glands were obtained on day 100 (n = 8) and day 165 (n = 6) of gestation (term = day 184) from untreated baboons and on day 100 after treatment of the mother with estradiol benzoate, injected sc between days 70-100 to increase estrogen production. PKA activity (picomoles of 32P incorporated into kemptide per min/mg protein) was determined by incubation of adrenal cytosol (12,000 x g; 0.3-30 micrograms protein) in reaction mixtures containing 0.25 mM ATP, 1 x 10(6) dpm [lambda-32P]ATP, and 3 micrograms kemptide in the presence or absence of 0.02 mM cAMP. PKC activity (picomoles of 32P incorporated into histone IIIS per min/mg protein) was determined in cytosol (105,000 x g) and detergent-solubilized membrane fractions after incubation with 0.02 mM ATP, 50 micrograms histone IIIS, and 1 x 10(6) dpm [lambda-32P]ATP in the presence or absence of calcium and phospholipids. Mean (+/- SE) maternal serum estradiol concentrations (nanograms per ml) were 3-fold greater (P < 0.05) at term (1.9 +/- 0.3) than at midgestation and increased (P < 0.05) after treatment with estradiol. PKA activity was greater at term (3965 +/- 546) than at midgestation (2130 +/- 240) and increased (P < 0.05) 2-fold after treatment with estrogen (3525 +/- 416) at midgestation. PKC activity was always 3- to 4-fold lower than that of PKA and was similar in the cytosol and membrane fractions of the cell. In contrast to PKA, cytosolic PKC activity was similar at mid (265 +/- 98)-and late (353 +/- 99) gestation and was not altered by treatment with estradiol (223 +/- 27).(ABSTRACT TRUNCATED AT 400 WORDS)
我们之前已经证明，雌激素调节的皮质醇（F）和可的松（E）经胎盘代谢的变化，从中期妊娠时的优先还原（E转化为F）到足月时的氧化（F转化为E），会导致狒狒下丘脑 - 垂体 - 肾上腺轴的激活以及限速类固醇生成酶的个体发生，最终导致从头合成F分泌。众所周知，由肽介导与膜受体结合激活的信使转录可通过环磷酸腺苷依赖性蛋白激酶 - A（PKA）和/或磷脂/钙依赖性蛋白激酶 - C（PKC）发生。本研究旨在确定狒狒妊娠期间胎儿肾上腺中PKA和PKC的基础水平是否受到发育调节，从而可能提供足月时胎儿肾上腺激活的介导机制。在妊娠第100天（n = 8）和第165天（n = 6）（足月为第184天）从未经处理的狒狒获取胎儿肾上腺，并在妊娠第70 - 100天之间皮下注射苯甲酸雌二醇处理母亲后于第100天获取胎儿肾上腺，以增加雌激素产生。PKA活性（每分钟每毫克蛋白质掺入凯普肽的32P皮摩尔数）通过将肾上腺胞质溶胶（12,000×g；0.3 - 30微克蛋白质）在含有0.25 mM ATP、1×10(6) dpm [λ - 32P]ATP和3微克凯普肽的反应混合物中孵育来测定，反应混合物中存在或不存在0.02 mM环磷酸腺苷。PKC活性（每分钟每毫克蛋白质掺入组蛋白IIIS的32P皮摩尔数）在胞质溶胶（105,000×g）和经去污剂溶解的膜部分中测定，在存在或不存在钙和磷脂的情况下，将其与0.02 mM ATP、50微克组蛋白IIIS和1×10(6) dpm [λ - 32P]ATP孵育后进行测定。足月时母体血清雌二醇浓度（每毫升纳克数）的平均值（±标准误）（1.9±0.3）比中期妊娠时高3倍（P < 0.05），并且在用雌二醇处理后升高（P < 0.05）。PKA活性在足月时（3965±546）高于中期妊娠时（2130±240），并且在中期妊娠时用雌激素处理后（3525±416）增加了2倍（P < 0.05）。PKC活性始终比PKA低3至4倍，并且在细胞的胞质溶胶和膜部分中相似。与PKA相反，胞质PKC活性在妊娠中期（265±98）和晚期（353±99）相似，并且未因用雌二醇处理（223±27）而改变。（摘要截断于400字）
