Neuropeptide-Y Enhances Luteinizing Hormone (LH)-Releasing Hormone-Induced LH Release and Elevations in Cytosolic Ca²⁺in Rat Anterior Pituitary Cells: Evidence for Involvement of Extracellular Ca²⁺Influx through Voltage-Sensitive Channels*

Abstract

The present studies were designed to investigate the mechanism by which neuropeptide-Y (NPY) augments the effect of LHRH to stimulate the release of LH from cultured rat anterior pituitary cells. Anterior pituitary cells from ovariectomized rats were enzymatically dispersed, cultured for 3 days, and then exposed to various secretagogues during 3-h incubations. As reported by this laboratory previously, NPY alone (100 nM) did not affect LH release, but significantly enhanced the LH response to 1 nM LHRH. This facilitatory action of NPY was mimicked by the dihydropyridine Ca2+ channel agonist Bay K 8644 (1 microM), and the enhancement of LHRH-induced LH release by either NPY or Bay K 8644 was prevented by the dihydropyridine antagonist nitrendipine (1 microM). Nitrendipine alone reduced the response to LHRH by approximately 25%, but did not affect basal LH release. In contrast, NPY failed to amplify the release of PRL in response to TRH, another Ca2(+)-mobilizing hormone. To test whether NPY also enhances the increase in cytosolic Ca2+ induced by LHRH, anterior pituitary cells were acutely dispersed into single cell suspensions, loaded with the fluorescent Ca2+ probe Indo-1 AM, and analyzed with a UV laser in an EPICS-753 flow cytometer at a rate of 500 cells/sec for 200 sec. The ratio of intracellular fluorescence resulting from Ca2+ bound to the Indo-1 to the fluorescence from Indo-1 alone (Indo-1 ratio), which is an index of the concentration of free cytosolic Ca2+, was determined for each cell. Approximately 7% of anterior pituitary cells responded to LHRH (1 or 10 nM) with significant increases in Indo-1 ratios, indicative of an increase in the concentration of free cytosolic Ca2+. EGTA (2.5 mM) reduced the basal Indo-1 ratios and attenuated, but did not abolish, the initial increase in response to LHRH, consistent with the initial extracellular Ca2+ influx-independent phase of the response to LHRH. NPY alone (100 nM) did not affect the Indo-1 ratios in anterior pituitary cells, but pretreatment with the peptide for 10 min before the scans significantly augmented the Indo-1 ratio response to 10 nM LHRH. This effect of NPY was also blocked by EGTA. Taken together, these biochemical and pharmacological studies suggest that NPY enhances the release of LH stimulated by LHRH by increasing extracellular Ca2+ entry, possibly by selectively affecting that component of the response involving dihydropyridine-sensitive L-type voltage-sensitive Ca2+ channels during the initial stages of the cellular response to LHRH.

Topography and associations of luteinizing hormone-releasing hormone and neuropeptide Y-immunoreactive neuronal systems in the human diencephalon

“…In mammals, two major sites of NPY actions have been identified: at the level of the hypophysiotropic LHRH neurons NPY enhances LHRH release (Crowley and Kalra, 1988;Khorram et al, 1988;Pau et al, 1991;Pau and Spies, 1997;Kasuya et al, 1998;Sahu and Kalra, 1998) and at the level of the gonadotroph cells of the pituitary NPY stimulates LH discharge. The stimulation of LH secretion can be direct (McDonald et al, 1987;Chabot et al, 1988;Crowley and Kalra, 1988) or indirect, via the potentiation of LHRH-induced LH release Crowley et al, 1990;Leupen et al, 1997;Sahu and Kalra, 1998;Leupen and Levine, 1999). These synergistic actions of NPY and LHRH on LH secretion have also been reported in humans (Watanobe et al, 1994).…”

Stimulatory effect of PYY-(3–36) on gonadotropin secretion is potentiated in fasted rats

“…Acute administration of NPY stimulates GnRH/LH release (4,5,14), whereas continuous NPY receptor activation results in suppression of gonadotropin secretion (9,15,47), which explains that, in experimental conditions that upregulate NPY synthesis and release, such as diabetes, the reproductive function is also impaired (31). Acute and chronic food deprivation and undernutrition stimulate hypothalamic NPY synthesis, storage, and release in the hypothalamus (31,46,61).…”

“…Acute intracerebroventricular (icv) administration of NPY stimulated LH release in ovariectomized rats primed with ovarian steroids (4). These excitatory effects have been shown to be the consequence of an increase in secretion and effectiveness of GnRH (5,14). In contrast, chronic treatment with the peptide decreased FSH, LH, and testosterone secretion (47, 9).…”

Release of Luteinizing Hormone from the Anterior Pituitary Gland in vitro Can Be Concurrently Regulated by at Least Three Peptides: Gonadotropin-Releasing Hormone, Oxytocin and Neuropeptide Y

“…It is unknown if the cAMP-reducing abilities of NPY [29, 30]are involved in the regulation of LH release. NPY is known to affect GnRH receptors [31, 32], and the effect on GnRH-stimulated LH release is mediated by Ca 2+ [33]and protein kinase C [4]. …”