Richard M. Jobin

Morphological identification of live gonadotropin, growth-hormone, and prolactin cells in goldfish (Carassius auratus) pituitary-cell cultures

To better understand neuroendocrine regulation and the intracellular mechanisms mediating pituitary-hormone release, it is necessary to study the physiology of identified single cells. We have developed a system to identify gonadotropin, growth-hormone, and prolactin cells in primary cultures of goldfish pituitary cells. Using Nomarski differential interference-contrast microscopy, the unique morphologies of discrete subpopulations of cells were characterized. To aid in the initial characterization of different pituitary-cell types, a discontinuous Percoll density-gradient cell-separation technique was developed. This method provided fractions enriched with functional gonadotropin, growth-hormone, and prolactin cells. The morphology of each cell type was initially characterized in enriched fractions of immunofluorescently labelled cells using differential interference-contrast microscopy. The cell type-specific morphologies were then confirmed in live pituitary-cell cultures. Gonadotropin, growth-hormone, and prolactin cells were correctly identified in live pituitary-cell cultures. Gonadotropin, growth-hormone, and prolactin cells were correctly identified in live mixed cultures in 92, 94, and 100% of the trials, respectively. The ability to directly identify cells in primary cultures allows the physiological study of identified single cells with minimal pretreatment.

Differences in Extracellular Calcium Involvement Mediating the Secretion of Gonadotropin and Growth Hormone Stimulated by Two Closely Related Endogenous GnRH Peptides in Goldfish Pituitary Cells

Two endogenous gonadotropin-releasing hormone (GnRH) peptides, salmon GnRH (sGnRH) and chicken GnRH II (cGnRH II), stimulate gonadotropin (GtH) and growth hormone (GH) secretion in the goldfish. The extracellular calcium (e-Ca2+) dependence of the GtH and GH response to the two GnRH peptides were compared using static incubations of dispersed goldfish pituitary cells. Incubation with Ca(2+)-depleted medium (without the addition of Ca2+ salts and in the presence of EGTA) did not alter basal GtH secretion, but reduced the GtH response to sGnRH, and abolished the cGnRH II-induced GtH release. Blockade of e-Ca2+ entry by low concentrations of CoCl2 had no effect on basal GtH secretion but reduced cGnRH II and sGnRH stimulated GtH release when applied at 0.1 and 0.5 mM concentrations, respectively. In general, treatments with voltage-sensitive Ca2+ channel (VSCC) antagonists, verapamil, nifedipine and nicardipine, did not alter basal GtH release but attenuated GnRH-stimulated GtH responses. cGnRH II-induced GtH release was decreased by 10 nM verapamil and 1 nM nifedipine, whereas the reduction of GtH responses to sGnRH required 100 times higher concentrations of these VSCC antagonists. cGnRH II but not sGnRH stimulation of GtH secretion was also abolished by 10 microM nicardipine. In contrast to GtH release, exposure to Ca(2+)-depleted medium reduced basal GH release and abolished the GH responses to both GnRH peptides. sGnRH and cGnRH II-stimulated GH responses were both abolished by 0.1 mM CoCl2, decreased by 1 nM verapamil, and reduced by 10 nM nicardipine. Addition of 0.1 and 10 microM nifedipine inhibited the GH responses to sGnRH and cGnRH II, respectively. Basal GH release was not affected by the VSCC antagonists tested. Results from this study indicate that entry of e-Ca2+, in part through VSCC, is involved in GnRH stimulation of GtH and GH release from goldfish gonadotropes and somatotropes; however, the e-Ca2+ dependence of the GtH and GH responses to the two endogenous GnRHs differ. The stimulatory effects of cGnRH II on GtH secretion is more dependent on and sensitive to e-Ca2+ than sGnRH. Whereas the sensitivity of GH responses to manipulations of e-Ca2+ availability is, in most instances, similar for both GnRH peptides. These results further suggest that basal secretion of GH is more sensitive to e-Ca2+ than basal GtH release; however, VSCC are not involved in the maintenance of basal release of either hormone.

Possible involvement of protein kinase C in gonadotropin and growth hormone release from dispersed goldfish pituitary cells

Static incubation with tumor-promoting 4 beta-phorbol esters, activators of the Ca2(+)- and phospholipid-dependent protein kinase C enzyme (PKC), caused dose-dependent increases in gonadotropin (GTH) and growth hormone (GH) secretion in primary cultures of dispersed goldfish pituitary cells. The estimated half-maximal effective doses (ED50) for stimulating GTH and GH release were 0.35 +/- 0.17 and 0.32 +/- 0.13 nM 12-O-tetradecanoyl phorbol 13 acetate (TPA), 3.71 +/- 1.30 and 1.37 +/- 0.76 nM 4 beta-phorbol 12,13-dibutyrate, 6.90 +/- 4.84 and 1.89 +/- 0.25 nM 4 beta-phorbol 12,13-dibenzoate, and 455 +/- 258 and 311 +/- 136 nM 4 beta-phorbol 12,13-diacetate, respectively. In contrast, treatments with up to 10 microM of the inactive 4 alpha-phorbol 12,13-didecanoate ester did not alter GTH and GH release. Additions of the synthetic diacylglycerol, dioctanoyl glycerol, also enhanced GTH and GH secretion in a dose-dependent manner and with ED50s of 1.73 +/- 0.83 and 1.73 +/- 1.19 microM, respectively. The GTH and GH responses to stimulation by TPA were attenuated by incubation with Ca2(+)-depleted medium containing EGTA or by treatment with the Ca2+ channel blocker verapamil. Coincubation with the PKC inhibitor H7 reduced the GTH and GH responses to TPA. As in previous studies, additions of salmon gonadotropin-releasing hormone (sGnRH) or chicken GnRH-II (cGnRH-II) induced GTH and GH release; these hormone responses to sGnRH and cGnRH-II were also decreased by the addition of H7. These results indicate that activation of PKC may stimulate GTH and GH release in goldfish and suggest that sGnRH and cGnRH-II actions on goldfish pituitary GTH and GH secretion are also mediated, at least partially, by PKC.

Agonist-specific Ca2+ signaling systems, composed of multiple intracellular Ca2+ stores, regulate gonadotropin secretion.

Ca2+ signals regulate many cellular functions, including hormone secretion. Agonist-specific Ca2+ signaling may arise from the differential mobilization of multiple Ca2+ stores. Although they act through the same receptor subtype, two gonadotropin-releasing hormones (sGnRH and cGnRH-II) generate quantifiably different Ca2+ signals in goldfish gonadotropes, suggesting that their Ca2+-dependent signaling cascades may differ. We combined electrophysiology, Ca2+ imaging, and radioimmunoassay detection of gonadotropin (GTH-II) secretion to determine the role of intracellular Ca2+ stores in GnRH-stimulated exocytosis. Our findings suggest that voltage-gated Ca2+ channels do not mediate acute GnRH-signaling. Instead, both sGnRH- and cGnRH-II-stimulated GTH-II releases are dependent on Ca2+ mobilized from TMB-8/CPA-sensitive compartments. However, sGnRH, but not cGnRH-II, utilizes intracellular stores sensitive to caffeine and xestospongin C. We also identified a homeostatic mechanism where reduced extracellular Ca2+ availability increase GTH-II release by mobilizing Ca2+ stores. Our results are the first to suggest that several classes of intracellular Ca2+ stores differentially participate in agonist signaling and homeostasis in gonadotropes.

GnRH signaling in goldfish pituitary cells.

In goldfish, maturational gonadotropin (GTH) and growth hormone (GH) release are stimulated by two native GTH-releasing hormones (sGnRH and cGnRH-II). Both GnRHs stimulate GTH and GH release via activation of phospholipase C, protein kinase C, Ca2+ entry through voltage-sensitive channels and calmodulin. However, sGnRH-induced GTH release also involves arachidonic acid and intracellular Ca2+ components absent from its action on GH, as well as from cGnRH-II action on GTH and GH secretion. The relative roles and interactions of these signaling pathways in mediating sGnRH and cGnRH-II action on acute and prolonged GTH and GH release are compared. How two GnRHs bind to similar receptors but induce similar and dissimilar transduction mechanisms in two cell types and within one cell type is unknown.