Neuropeptide glutamic-isoleucine (NEI) specifically stimulates the secretory activity of gonadotrophs in primary cultures of female rat pituitary cells

Abstract

The neuropeptide EI (NEI) is derived from proMCH. It activates GnRH neurons, and has been shown to stimulate the LH release following intracerebroventricular administration in several experimental models. The aim of the present paper was to evaluate NEI actions on pituitary hormone secretion and cell morphology in vitro. Pituitary cells from female rats were treated with NEI for a wide range of concentrations (1–400 × 10⁻⁸ M) and time periods (1–5 h). The media were collected and LH, FSH, PRL, and GH measured by RIA. The interaction between NEI (1, 10 and 100 × 10⁻⁸ M) and GnRH (0.1 and 1 × 10⁻⁹ M) was also tested. Pituitary cells were harvested for electron microscopy, and the immunogold immunocytochemistry of LH was assayed after 2 and 4 h of NEI incubation. NEI (100 × 10⁻⁸ M) induced a significant LH secretion after 2 h of stimulus, reaching a maximum response 4 h later. A rapid and remarkable LH release was induced by NEI (400 × 10⁻⁸ M) 1 h after stimulus, attaining its highest level at 2 h. However, PRL, GH and FSH were not affected. NEI provoked ultrastructural changes in the gonadotrophs, which showed accumulations of LH-immunoreactive granules near the plasma membrane and exocytotic images, while the other populations exhibited no changes. Although NEI (10 × 10⁻⁸ M), caused no action when used alone, its co-incubation with GnRH (1 × 10⁻⁹ M), promoted a slight but significant increase in LH. These results demonstrate that NEI acts at the pituitary level through a direct action on gonadotrophs, as well as through interaction with GnRH.

Introduction

The rat neuropeptide EI (NEI) is a peptide derived from the proMCH located in neurons from the lateral hypothalamus and zona incerta, which have widespread projections throughout the central nervous system [3], [10]. The structures of NEI and MCH are highly conserved among vertebrates, indicating that these peptides may have important physiological functions [13], [20], among which, are the control of water and electrolyte behavior [26]. Although there is little information about the neurobiological functions of NEI, it has been demonstrated that NEI immunoreactivity (NEI-IR) can be modified by ovariectomy and treatment with 17-β estradiol benzoate [30], and it has been shown that this peptide is secreted from cultured rat hypothalamic cells [25]. Furthermore, it was demonstrated that both NEI and MCH can suppress the TRH release from hypothalamic cultures, with MCH being able to reduce the TRH-induced TSH secretion from the dispersed pituitary cell cultures [16]. In addition, we previously demonstrated that an intracerebroventricular (i.c.v.) injection of NEI, given to male and chronically ovariectomized (OVEX) female rats treated with estradiol benzoate plus progesterone, induced an increase in the serum LH levels compared to controls injected with artificial cerebrospinal fluid (ACSF) [2]. In order to identify the anatomical substrate underlying this effect, we used combined immunohistochemistry methods to analyze the forebrains of female rats. In the afternoon of the proestrus day, the NEI fibers were found to be in close apposition with the anteroventral periventricular nucleus and the GnRH neurons expressing c-Fos. In the median eminence, NEI varicosities and terminal like-structures were in near proximity to blood vessels and GnRH fibers. Related to this, it has previously been described that NEI can induce LH secretion, by direct release into the median eminence, through the modulation of GnRH neurons located in the preoptic area, or by the modulation of GnRH terminals located in the median eminence [10].

Leading on from these results, in the present study we decided to investigate whether NEI can act directly at the pituitary level by modulating hormone secretion. To this purpose, the effect of NEI in pituitary cell cultures from female rats on the release of several pituitary hormones such as LH, FSH, GH and prolactin was studied. Furthermore, the pituitary cell morphology was evaluated by electron microscopy and immunocytochemistry, and finally, the ability of NEI to potentiate GnRH-induced LH release was tested.