Kaminska B, Ciereszko R E, Opalka M, Dusza L
Department of Animal Physiology, University of Warmia and Mazury in Olsztyn, Oczapowskiego 5, 10-718 Olsztyn, Poland.
Domest Anim Endocrinol. 2002 Nov;23(4):475-91. doi: 10.1016/s0739-7240(02)00173-x.
Prolactin (PRL) was found to have a stimulatory effect on adrenal steroidogenesis in vivo and in vitro in several species including pigs. PRL signal transduction pathways, however, in adrenocortical cells are poorly recognized. Therefore, the goal of this paper is to ascertain the involvement of protein kinase C (PKC) and tyrosine kinases in PRL signaling in porcine adrenal cortex. Adrenals were harvested from locally slaughtered mature gilts. Cortical cells were dispersed by sequential treatment with collagenase. The cells were seeded into 24-well culture plates at a density of 3 x 10(5)/mL. Cells were incubated with or without PRL (500 ng/mL), ACTH (5 nM--a positive control), tyrosine kinase inhibitor--genistein (1; 2.5 or 5 microM), PKC inhibitor--sphingosine (20-1000 nM) and PKC activators--diacylglycerol (DiC8; 10-100 microM) and phorbol ester (PMA; 1-1000 nM). All incubations were performed for 8 h (95% air and 5% CO(2), 37 degrees C). PRL and ACTH (P < 0.05) increased cortisol and androstenedione (A(4)) secretion. DiC8 and PMA mimicked the stimulatory effect of PRL. Sphingosine (P < 0.05) suppressed basal and PRL-stimulated steroid secretion. Genistein inhibited (P < 0.05) PRL-stimulated cortisol secretion and enhanced (P < 0.05) basal and PRL-stimulated A(4) secretion. Moreover, PKC activation was assessed by measuring the specific association of [3H]phorbol dibutyrate ([3H]PDBu) with adrenocortical cells after treatment with PRL or ionomycin (a positive control). PRL (within 2-3 min) and ionomycin (within 2-5 min) increased (P < 0.05) specific binding of [3H]PDBu to the porcine adrenocortical cells. In addition, PRL did not augment the cortisol and A(4) secretion by PKC-deficient adrenocortical cells. In conclusion, presented results support the hypothesis that PKC and tyrosine kinases are involved in PRL signaling in adrenocortical cells in pigs. Moreover, activation of PKC is associated with the increased secretion of cortisol and A(4).
在包括猪在内的多个物种中，已发现催乳素（PRL）在体内和体外对肾上腺类固醇生成具有刺激作用。然而，肾上腺皮质细胞中PRL信号转导途径却鲜为人知。因此，本文的目的是确定蛋白激酶C（PKC）和酪氨酸激酶在猪肾上腺皮质PRL信号传导中的作用。从当地屠宰的成年母猪中采集肾上腺。通过用胶原酶顺序处理使皮质细胞分散。将细胞以3×10⁵/mL的密度接种到24孔培养板中。将细胞与有或无PRL（500 ng/mL）、促肾上腺皮质激素（ACTH，5 nM——阳性对照）、酪氨酸激酶抑制剂——染料木黄酮（1、2.5或5 μM）、PKC抑制剂——鞘氨醇（20 - 1000 nM）以及PKC激活剂——二酰基甘油（DiC8，10 - 100 μM）和佛波酯（PMA，1 - 1000 nM）一起孵育。所有孵育均进行8小时（95%空气和5%二氧化碳，37℃）。PRL和ACTH（P < 0.05）增加了皮质醇和雄烯二酮（A₄）的分泌。DiC8和PMA模拟了PRL的刺激作用。鞘氨醇（P < 0.05）抑制基础和PRL刺激的类固醇分泌。染料木黄酮抑制（P < 0.05）PRL刺激的皮质醇分泌，并增强（P < 0.05）基础和PRL刺激的A₄分泌。此外，在用PRL或离子霉素（阳性对照）处理后，通过测量[³H]佛波醇二丁酸酯（[³H]PDBu）与肾上腺皮质细胞的特异性结合来评估PKC的激活。PRL（在2 - 3分钟内）和离子霉素（在2 - 5分钟内）增加（P < 0.05）了[³H]PDBu与猪肾上腺皮质细胞的特异性结合。此外，PRL并未增加PKC缺陷的肾上腺皮质细胞的皮质醇和A₄分泌。总之，所呈现的结果支持以下假设：PKC和酪氨酸激酶参与猪肾上腺皮质细胞中的PRL信号传导。此外，PKC的激活与皮质醇和A₄分泌增加有关。
