For full fertility in the female ovulation is necessary, which is dependent on the production of a surge of LH. An understanding of the processes which result in the high levels of LH requires delineation of the activities of individual component cells. In this study the responses of gonadotrophs to two signalling hypothalamic peptides, GnRH and oxytocin, were investigated. A cell immunoblot method was used to identify and distinguish between cells which secrete LH and those which contain LH but do not secrete the glycohormone. Rats were killed on the morning of pro-oestrus, the pituitary collected and the cells dispersed onto a protein-binding membrane for study. Cells were then incubated with GnRH and oxytocin, after which the membranes including the attached cells were stained by immunocytochemistry for LH. GnRH increased the total number of immunopositive cells which were present in a concentration-dependent manner. The most prominent change after 2 h incubation was in the number of secreting cells, whereas after 4 h there was also a marked increase in numbers of nonsecreting cells. Oxytocin also increased the total number of immunopositive cells in a concentration-responsive manner, however the profile of action of oxytocin was different from that observed for GnRH. Oxytocin had a relatively greater effect on numbers of immunopositive nonsecreting cells. Thus, the results reveal the potential for gonadotrophs to be flexibly and appropriately modulated by selected hypothalamic peptides. When cells were preincubated with oxytocin prior to GnRH there was not an additive increase in the numbers of immunopositive cells, suggesting that the two agonists act, in a nonidentical manner, on similar cells. The increase in the total number of immunopositive cells implies that there was a production of LH or post-translational processing, induced by exposure to GnRH or oxytocin. The results confirmed the heterogeneity of gonadotrophs and the existence of functionally distinguishable subpopulations, and revealed a difference between the effects of GnRH and oxytocin on expression and secretion of LH.
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JJ Evans, AH Youssef, MM Abbas, and J Schwartz
JJ Evans, AH Youssef, TG Yandle, LK Lewis, and MG Nicholls
Regulation of cardiovascular system activity involves complex interactions amongst numerous factors. Three of these vasoactive factors are adrenomedullin, C-type natriuretic peptide (CNP) and endothelin-1 (ET-1), each of which is claimed to have important local effects. To investigate paracrine/autocrine regulation of the secretion of these peptides we used a cell immunoblot method. We postulated that basal release of adrenomedullin and CNP by endothelial cells is modulated by ET-1. Dispersed human aortic endothelial cells were attached to a protein binding membrane and incubated for 1 or 4 h with control medium or with ET-1, endothelin receptor antagonists or antibody to ET-1, and then submitted to immunohistochemical staining. Peptides (adrenomedullin, CNP and ET-1) within individual cells were stained, as was peptide secreted and adjacent to the cell. It was demonstrated that adrenomedullin, CNP and ET-1 can be contained within the same cell. In addition, we observed that individual endothelial cells can secrete all three peptides. The endothelin ET-A/ET-B receptor antagonist, bosentan, the ET-B receptor antagonist, BQ-788, and anti-ET-1 serum decreased the percentage of endothelial cells that secreted adrenomedullin and CNP relative to control. Conversely, the addition of ET-1 induced an increase in the number of endothelial cells that secreted adrenomedullin and CNP. These results provide strong evidence that endogenous ET-1, from human vascular endothelial cells, acts in a paracrine/autocrine manner to modulate the basal release of adrenomedullin and CNP. Our observations of this modulation suggest that vascular endothelial cells of humans constitute an important component of a self-responsive vasoregulatory system.