Melanin-concentrating hormone reduces somatolactin release from cultured goldfish pituitary cells.

Melanin-concentrating hormone (MCH)-containing neurons directly innervate the adenohypophysis in the teleost pituitary. We examined immunohistochemically the relationship between MCH-containing nerve fibres or endings and somatolactin (SL)-producing cells in the goldfish pituitary. Nerve fibres or endings with MCH-like immunoreactivity were identified in the neurohypophysis in close proximity to the adenohypophysial cells showing SL-like immunoreactivity. We also examined the effect of MCH on SL release from cultured goldfish pituitary cells and SL synthesis using a cell immunoblot and a real-time PCR method. Treatment of individually dispersed pituitary cells with MCH 10(-7) M for 3 h decreased the area of SL-like immunoreactivity on immunoblots, and MCH-induced reductions in SL release were blocked by treatment with the mammalian MCH receptor (MCHR) antagonist, compound-30, at a concentration of 10(-5) M. Treatment with 10(-7) M MCH for 3 h did not affect sl-alpha and -beta (smtla and -b as given in the Zfin Database) mRNA expression levels. These led us to explore the signal transduction mechanism leading to the inhibition of SL release, for which we examined whether MCH-induced reductions in SL release are mediated by the G(i) or G(q) protein-coupled signalling pathway. The MCH-induced reductions in SL release were abolished by treatment with the G(i/o) protein inhibitors, NF023 (10(-5) M) or pertussis toxin (260 ng/ml), but not by the phospholipase C inhibitor, U-73122 (3x10(-6) M). These results indicate that MCH can potentially function as a hypothalamic factor suppressing SL release via the MCHR, and subsequently through the G(i) protein to inhibit the adenylate cyclase/cAMP/protein kinase A-signalling pathway in goldfish pituitary cells.

Pituitary adenylate cyclase-activating polypeptide induces somatolactin release from cultured goldfish pituitary cells.

In the goldfish pituitary, nerve fibers containing pituitary adenylate cyclase-activating polypeptide (PACAP) are located in close proximity to somatolactin (SL)-producing cells, and PACAP enhances SL release from cultured pituitary cells. However, there is little information about the mechanism of PACAP-induced SL release. In order to elucidate this issue, we used the cell immunoblot method. Treatment with PACAP at 10(-8) and 10(-7)M, but not with vasoactive intestinal polypeptide (VIP) at the same concentrations, increased the immunoblot area for SL-like immunoreactivity from dispersed pituitary cells, and PACAP-induced SL release was blocked by treatment with the PACAP selective receptor (PAC(1)R) antagonist, PACAP(6-38), at 10(-6)M, but not with the PACAP/VIP receptor antagonist, VIP(6-28). PACAP-induced SL release was also attenuated by treatment with the calmodulin inhibitor, calmidazolium at 10(-6)M. This led us to explore the signal transduction mechanism up to SL release, and we examined whether PACAP-induced SL release is mediated by the adenylate cyclase (AC)/cAMP/protein kinase A (PKA)- or the phospholipase C (PLC)/inositol 1,4,5-trisphosphate (IP(3))/protein kinase C (PKC)-signaling pathway. PACAP-induced SL release was attenuated by treatment with the AC inhibitor, MDL-12330A, at 10(-5)M or with the PKA inhibitor, H-89, at 10(-5)M. PACAP-induced SL release was suppressed by treatment with the PLC inhibitor, U-73122, at 3 x 10(-6)M or with the PKC inhibitor, GF109203X, at 10(-6)M. These results suggest that PACAP can potentially function as a hypophysiotropic factor mediating SL release via the PAC(1)R and subsequently through perhaps the AC/cAMP/PKA- and the PLC/IP(3)/PKC-signaling pathways in goldfish pituitary cells.

Effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on prolactin and somatolactin release from the goldfish pituitary in vitro.

Pituitary adenylate cyclase-activating polypeptide (PACAP) plays a role in mediating growth hormone and gonadotropin release in the teleost pituitary. In the present study, we examined the immunohistochemical relationship between PACAP nerve fibers and prolactin (PRL)- and somatolactin (SL)-producing cells in the goldfish pituitary. Nerve fibers with PACAP-like immunoreactivity (PACAP-LI) were identified in the neurohypophysis in close proximity to cells containing PRL-LI or SL-LI. Several cells with PRL-LI or SL-LI showed PACAP receptor (PAC(1)R)-LI. The cell immunoblot assay method was used to examine the effect of PACAP on PRL and SL release from dispersed goldfish pituitary cells. Treatment with PACAP increased the immunoblot area for PRL- and SL-LI from individual pituitary cells in a dose-dependent manner. The effect of PACAP on the expression of mRNAs for PRL and SL in cultured pituitary cells was also tested. Semiquantitative analysis revealed that the expression of SL mRNA, but not PRL mRNA, was increased significantly by the treatment with PACAP. The effect of PACAP on intracellular calcium mobilization in isolated pituitary cells was also investigated using confocal laser-scanning microscopy. The amplitude of Ca(2+) mobilization in individual cells showing PRL- or SL-LI was increased significantly following exposure of cells to PACAP. These results indicate that PACAP can potentially function as a hypophysiotropic factor mediating PRL and SL release in the goldfish pituitary.