Sirotkin A V, Grossmann R
Research Institute of Animal Production, Hlohovská 2, 949 92 Nitra, Slovakia.
Comp Biochem Physiol A Mol Integr Physiol. 2007 May;147(1):239-46. doi: 10.1016/j.cbpa.2006.12.038. Epub 2007 Jan 16.
The general aim of these in-vitro experiments was to determine whether ghrelin controls the secretory activity of chicken ovarian cells and whether its action is mediated by TK-, MAPK-, CDK- or PKA-dependent intracellular mechanisms. We postulated that particular protein kinases could be considered as mediators of ghrelin action (a) if they are controlled by ghrelin, and (b) if blockers of these kinases modify the action of ghrelin. In our in-vitro experiments we investigated whether ghrelin altered the accumulation of TK, MAPK, CDK and PKA in chicken ovarian cells and whether ghrelin, with or without blockers of MAPK, CDK and PKA, affected the secretion of progesterone (P4), testosterone (T), estradiol (E2) or arginine-vasotocin (AVT). In the first series of experiments, the influence of a ghrelin 1-18 analogue (1, 10 or 100 ng/mL) was studied on the expression of TK, MAPK and PKA in cultured chicken ovarian granulosa cells. The percentage of cells containing TK/phosphotyrosine MAPK/ERK1, 2 and PKA was determined using immunocytochemistry. Ghrelin increased the expression of both TK and MAPK. The low concentration of ghrelin (1 ng/mL) increased the accumulation of PKA in ovarian cells whilst the high concentration (100 ng/mL) decreased it. The 10 ng/mL concentration had no effect. In the second series of experiments, the effects of the ghrelin analogue combined with an MAPK blocker (PD98059; 100 ng/mL), a CDK blocker (olomoucine; 1 microg/mL), or a PKA blocker (KT5720; 100 ng/mL), were tested for their effects on the secretion of hormones by cultured fragments of chicken ovarian follicular wall. P4, T, E2 and AVT secretions were measured using RIA and EIA. Ghrelin increased T and decreased E2, but did not affect P4 or AVT secretion. The PKA blocker promoted P4 secretion and suppressed E2 and AVT, but did not affect T secretion. It prevented or even reversed the effect of ghrelin on T and E2, but did not modify its effect on AVT secretion. The MAPK blocker enhanced P4 and T and reduced AVT, but did not affect E2 secretion. It was able to prevent or reverse the effect of ghrelin on T and E, and it induced a stimulatory effect of ghrelin on AVT secretion. The CDK blocker reduced the secretion of AVT, but had no effect on steroid hormone secretion. It induced the stimulatory influence of ghrelin on the secretion of P4 and AVT, but did not modify the effect of ghrelin on other hormones. These observations clearly demonstrate that ghrelin is a potent regulator of the secretory activity of ovarian cells and of TK, MAPK and PKA. Furthermore, they suggest that MAPK-, CDK- and PKA-dependent intracellular mechanisms are involved in the control of ovarian secretion and that they mediate the effects of ghrelin on these processes.
这些体外实验的总体目的是确定胃饥饿素是否控制鸡卵巢细胞的分泌活性，以及其作用是否由依赖酪氨酸激酶（TK）、丝裂原活化蛋白激酶（MAPK）、细胞周期蛋白依赖性激酶（CDK）或蛋白激酶A（PKA）的细胞内机制介导。我们推测，如果特定的蛋白激酶受胃饥饿素调控，并且这些激酶的阻滞剂能改变胃饥饿素的作用，那么它们可被视为胃饥饿素作用的介质。在我们的体外实验中，我们研究了胃饥饿素是否改变鸡卵巢细胞中TK、MAPK、CDK和PKA的积累，以及胃饥饿素在有或没有MAPK、CDK和PKA阻滞剂的情况下，是否会影响孕酮（P4）、睾酮（T）、雌二醇（E2）或精氨酸加压催产素（AVT）的分泌。在第一系列实验中，研究了胃饥饿素1 - 18类似物（1、10或100 ng/mL）对培养的鸡卵巢颗粒细胞中TK、MAPK和PKA表达的影响。使用免疫细胞化学法测定含有TK/磷酸酪氨酸、MAPK/细胞外信号调节激酶1、2和PKA的细胞百分比。胃饥饿素增加了TK和MAPK的表达。低浓度的胃饥饿素（1 ng/mL）增加了卵巢细胞中PKA的积累，而高浓度（100 ng/mL）则使其减少。10 ng/mL的浓度没有影响。在第二系列实验中，测试了胃饥饿素类似物与MAPK阻滞剂（PD98059；100 ng/mL）、CDK阻滞剂（olomoucine；1 μg/mL）或PKA阻滞剂（KT5720；100 ng/mL）联合使用对鸡卵巢卵泡壁培养片段激素分泌的影响。使用放射免疫分析（RIA）和酶免疫分析（EIA）测定P4、T、E2和AVT的分泌。胃饥饿素增加了T的分泌并降低了E2的分泌，但不影响P4或AVT的分泌。PKA阻滞剂促进了P4的分泌并抑制了E2和AVT的分泌，但不影响T的分泌。它阻止甚至逆转了胃饥饿素对T和E2的作用，但没有改变其对AVT分泌的影响。MAPK阻滞剂增加了P4和T的分泌并减少了AVT的分泌，但不影响E2的分泌。它能够阻止或逆转胃饥饿素对T和E的作用，并诱导胃饥饿素对AVT分泌的刺激作用。CDK阻滞剂减少了AVT的分泌，但对甾体激素分泌没有影响。它诱导了胃饥饿素对P4和AVT分泌的刺激作用，但没有改变胃饥饿素对其他激素的作用。这些观察结果清楚地表明，胃饥饿素是卵巢细胞分泌活性以及TK、MAPK和PKA的有效调节剂。此外，它们表明依赖MAPK、CDK和PKA的细胞内机制参与了卵巢分泌的控制，并且它们介导了胃饥饿素对这些过程的影响。
