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Previous studies have shown that CT-like immunoreactive peptide(s) (pit-CT) is synthesized by the anterior pituitary (AP) gland, and exogenously added salmon(s) CT inhibits PRL release and PRL gene transcription in cultured AP cells. Anti-sCT serum, which immunoreacts with pit-CT, stimulates PRL secretion, suggesting pit-CT is a physiologically relevant PRL-inhibiting hormone. Using proliferating cell nuclear antigen (PCNA) staining and 5-bromo-2'deoxyuridine (BrdU) incorporation into newly replicated DNA, the effect of calcitonin (CT) on cellular proliferation in the rat anterior pituitary gland (AP) was examined. CT significantly attenuated PCNA-immunopositive as well as BrdU-positive AP cell populations in dispersed rat AP cells. A second series of experiments tested the effects of CT on AP cell proliferation in vivo. OVX + E2 rats were injected with 200 microg CT (iv), the rats killed at various time points, and the APs were processed for BrdU staining. CT inhibited BrdU incorporation at all time points up to 15 h after the injection, and this inhibitory effect was reversed at later time points. The effect of CT was concentration dependent, and a maximal inhibition was observed 10 h after the CT injection. Subsequent experiments identified CT-responsive AP cell populations using double immunofluorescence for BrdU and either PRL or FSH. The number of BrdU-labeled lactotropes in the AP gland declined by 74% in the CT-treated rats. Neutralization of endogenous pit-CT by passive immunization with anti-sCT serum caused a 2-fold increase in AP cell proliferation. These results suggest an important role for the endogenous pit-CT in regulation of lactotrope population of the AP gland.

This study examined the effect of salmon calcitonin (sCT) on hypothalamic tyrosine hydroxylase (TH) activity and evaluated the cellular signaling mechanisms involved in the response. Fetal hypothalamic cells were cultured in a defined medium and treated with sCT and/or specific protein kinase inhibitors on day 14 in vitro. sCT (0.1-10 nM) increased both TH activity and cellular cAMP content in a concentration-dependent manner. sCT (10 nM) increased TH activity to 150-175% of control values and resulted in a 10-fold increase in cellular cAMP content. Both the C1a and C1b CT receptor isoforms were present in the cultures, as assessed by RT-PCR. Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), a cAMP antagonist, and H-8, a cyclic nucleotide kinase inhibitor, blocked the sCT-induced increase in TH activity, with complete abolition of the response observed at concentrations of 1 mM and 5 microM, respectively. sCT (10 nM) increased radiolabeled phosphate incorporation into TH protein to 169% of control values and 1 mM Rp-cAMPS completely blocked this effect. In contrast, neither Calphostin C, a protein kinase C inhibitor, nor U-73122, a phospholipase C inhibitor, significantly altered the ability of sCT to increase TH activity. Likewise, the sCT-induced increase in TH activity was observed after pretreating the cells with either BAPTA/AM, an intracellular calcium chelator, or thapsigargin, an inhibitor of the endoplasmic reticulum calcium pump. These data indicate that sCT has a profound stimulatory effect on TH activity in fetal hypothalamic cells and that enhanced phosphorylation of TH coincides with the sCT-induced increase in enzyme activity. Moreover, CT receptors, which are linked to cAMP production, are expressed in the hypothalamic cells and a cAMP-dependent mechanism mediates the sCT-induced activation and phosphorylation of TH.

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.

Calcitonin (CT) and related peptides, such as CT gene-related peptide and salmon CT (sCT)-like peptide, are present in the rat nervous system and the pituitary gland, and sCT markedly inhibits basal and TRH-stimulated PRL release from anterior pituitary (AP) cells. Because TRH-induced PRL release is known to involve increases in cytosolic free Ca2+ derived from both extracellular and intracellular sources, the objective of the present study was to test whether sCT interferes with this effect. Secretogogue-induced elevations of cytosolic free Ca2+ ([Ca2+]i) in acutely dispersed AP cells were monitored using the fluorescent Ca2+ indicator Indo-1 AM and flow cytometry. AP cells were enzymatically dispersed to single cell suspensions and loaded with 20 microM Indo-1 AM for 30 min. Indo-1-loaded AP cells were scanned at a rate of approximately 500 cells/sec for 200-300 sec in a flow cytometer, and the ratio of fluorescence due to Ca2+ bound to Indo-1 to free Indo-1 (Indo-1 ratio), which is an index of [Ca2+]i, was determined for each cell. Under basal conditions, AP cells showed stable Indo-1 ratios during the scans, and 100% of the cells responded to the Ca2+ ionophore ionomycin with increases in the Indo-1 ratio. Approximately 25-30% of the AP cells responded to a 1 microM pulse of TRH with marked increases in the Indo-1 ratio, indicative of increases in [Ca2+]i, with the response consisting of two phases, an initial rapid rise that was unaffected by the presence of EGTA in the extracellular environment, followed by a decrease to a sustained secondary phase that was completely eliminated by EGTA. In a normal extracellular Ca2+ environment, pretreatment with 100 nM sCT almost totally inhibited the response to 1 microM TRH. In EGTA-pretreated AP cells, the initial EGTA-insensitive phase of the TRH-induced [Ca2+]i increase was also abolished by prior exposure to sCT. These results suggest that sCT inhibits TRH-stimulated PRL release in AP cells by attenuating the TRH-induced increase in [Ca2+]i, an effect that probably occurs as a consequence of inhibition of the stimulatory effect of TRH on the Ca2+/phospholipid messenger system.