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    • In the second part of this

    2022-05-18

    In the second part of this study, we investigated the influence of GH on the vorapaxar of the ghrelin receptor, GHSR-1a, at the mRNA and peptide levels in cultured whole follicles. Regulation of the expression of GHSR-1a has a key role in the actions of ghrelin in target tissues. Using RT-PCR and Western blots, we demonstrated no effect of exogenous GH on either GHSR-1a protein levels or mRNA expression in the ovary (Fig. 1). The results of presented here are not in agreement with Nass et al. [21], who showed that expression of GHSR-1a mRNA was inhibited by GH in rat pituitary tumor tissue. However, it was questionable if this effect was related to direct GH effects on the pituitary or the hypothalamus or the result of an altered concentration of the natural ligand for this receptor. Inhibition of GHSR-1a mRNA expression in rat pituitary tissue by insulin growth factor (IGF) was noted by Kamegai et al. [22]. There is also data showing regulation of GHSR-1a expression by glucocorticoids [23], thyroid hormone [24] and sex steroids [25]. Another interesting finding of this study was evidence for the ability is ghrelin through the receptor GHS-R1a can modulate ovarian functions, including estradiol secretion, aromatase activity, caspase-3 activity and cell proliferation. Previously, we showed the stimulatory action of GH but not IGF-I on ghrelin synthesis and secretion by ovarian cells, suggesting the possibility of a feedback loop of GH and ghrelin but not of IGF-I and ghrelin in the ovary [12]. In order to study receptor dependent mechanism of ghrelin action in ovarian function, we used a ghrelin receptor antagonist, (d-Lys-3)-GHRP-6, in cultured ovarian cells. For many years, (d-Lys-3)-GHRP-6 has been reported as a synthetic selective antagonist of the ghrelin receptor, GHSR-1a, and its functional properties have been extensively studied both in vitro and in vivo in animals [26], [27], [28]. In cultures treated together ghrelin and (d-Lys-3)-GHRP-6, estradiol secretion, aromatase activity and cell proliferation returned to control levels (Fig. 2A, B, D), suggesting GHSR-1a receptor mediated action. On the other hand, the inhibitory action of ghrelin on caspase-3 activity seems to be independent of the ghrelin receptor, GHSR-1a, because inhibition of caspase-3 activity was not reversed by a selective antagonist of GHSR-1a (Fig. 2C). Our data are in accordance with the data of Sirotkin et al. [19], which showed that ghrelin was able to modulate the expression of several markers of apoptosis, including bax, bcl-2, caspase-3 activity and p53, while GHSR-1a expression was unnecessary. Baldanzi et al. [29] reported that cardiomyocytes ghrelin and its acylated form, des-acyl ghrelin exhibits an anti-apoptotic effect through binding to a novel, unidentified receptor that is distinct from GHSR-1a. They suggest that ghrelin anti-apoptotic activity is not mediated by GHSR-1a because no expression of GHSR-1a was detected in cardiomyocytes. Moreover, both ghrelin and des-acyl ghrelin, recognize a common high similar binding site, although only ghrelin and not des-acyl ghrelin bind to GHSR-1a receptor [1]. The putative novel receptor is expected to be highly similar to GHSR-1a, since it differs only lack of ability to distinguish between un- and estrified ghrelin peptide. Whether such receptor is encoded by alternative splicing of GHSR-1a gene or by a distinct gene still remains to be determined [29]. Granata et al. [30] showed that ghrelin receptor expression in HIT-T15 pancreatic β-cell line was no found and they suggest that ghrelin proliferative and anti-apoptotic action in these cells was mediated by a distinct and yet unidentified receptor. Similarly, Delhanty et al. [31] reported that anti-apoptotic pathway in adrenocortical tumor cells is not mediated by GHSR-1a in these cells. However, there are one data of Chung et al. [32] reported neuroprotective effect of ghrelin appears to be mediated through the activation of GHSR-1a because (d-Lys-3)-GHRP-6 completely blocks the protective effect of ghrelin against oxygen–glucose deprivation insult. We suggest that independent of GHSR-1a anti-apoptotic action of ghrelin could be due to its binding to a novel unknown receptor in the pig ovary. For example in chicken ovarian tissue three splice variants of GHSR-1a were expressed [19]. Results of our unpublished data showed that anti-apoptotic effect of ghrelin on caspase-3 activity and DNA fragmentation is mediated by intracellular signaling pathway MAPK and phosphatidylinositol (PI) 3-kinase.