Wendy K. W. Ko

The secretogranin II-derived peptide secretoneurin stimulates luteinizing hormone secretion from gonadotrophs.

Secretoneurin (SN) is a 33- to 34-amino acid neuropeptide derived from secretogranin-II, a member of the chromogranin family. We previously synthesized a putative goldfish (gf) SN and demonstrated its ability to stimulate LH release in vivo. However, it was not known whether goldfish actually produced the free SN peptide or whether SN directly stimulates LH release from isolated pituitary cells. Using a combination of reverse-phase HPLC and mass spectrometry analysis, we isolated for the first time a 34-amino acid free gfSN peptide from the whole brain. Moreover, Western blot analysis indicated the existence of this peptide in goldfish pituitary. Immunocytochemical localization studies revealed the presence of SN immunoreactivity in prolactin cells of rostral pars distalis of the anterior pituitary. Additionally, we found that magnocellular cells of the goldfish preoptic region are highly immunoreactive for SN. These neurons send heavily labeled projections that pass through the pituitary stalk and innervate the neurointermediate and anterior lobes. In static 12-h incubation of dispersed pituitary cells, application of SN antiserum reduced LH levels, whereas 1 and 10 nM gfSN, respectively, induced 2.5-fold (P < 0.001) and 1.9-fold (P < 0.01) increments of LH release into the medium, increases similar to those elicited by 100 nM concentrations of GnRH. Like GnRH, gfSN elevated intracellular Ca(2+) in identified gonadotrophs. Whereas we do not yet know the relative contribution of neural SN or pituitary SN to LH release, we propose that SN could act as a neuroendocrine and/or paracrine factor to regulate LH release from the anterior pituitary.

Insulin-like growth factor as a novel stimulator for somatolactin secretion and synthesis in carp pituitary cells via activation of MAPK cascades

Somatolactin (SL), a member of the growth hormone/prolactin family, is a pituitary hormone unique to fish models. Although SL is known to have diverse functions in fish, the mechanisms regulating its secretion and synthesis have not been fully characterized. Using grass carp pituitary cells as a model, here we examined the role of insulin-like growth factor (IGF) in SL regulation at the pituitary level. As a first step, the antisera for the two SL isoforms expressed in the carp pituitary, SLα and SLβ, were produced, and their specificity was confirmed by antiserum preabsorption and immunohistochemical staining in the carp pituitary. Western blot using these antisera revealed that grass carp SLα and SLβ could be N-linked glycosylated and their basal secretion and cell content in carp pituitary cells could be elevated by IGF-I and -II treatment. These stimulatory effects occurred with parallel rises in SLα and SLβ mRNA levels, and these SL gene expression responses were not mimicked by insulin but blocked by IGF-I receptor inactivation. In carp pituitary cells, IGF-I and -II could induce rapid phosphorylation of IGF-I receptor, MEK1/2, ERK1/2, MKK3/6, and p38 MAPK; and SLα and SLβ secretion, protein production, and mRNA expression caused by IGF-I and -II stimulation were negated by inactivating MEK1/2 and p38 MAPK. Parallel inhibition of PI3K and Akt, however, were not effective in these regards. These results, taken together, provide evidence that IGF can upregulate SL secretion and synthesis at the pituitary level via stimulation of MAPK- but not PI3K/Akt-dependent pathways.

Grass carp prolactin: molecular cloning, tissue expression, intrapituitary autoregulation by prolactin and paracrine regulation by growth hormone and luteinizing hormone.

Prolactin (PRL), a pituitary hormone with diverse functions, is well-documented to be under the control of both hypothalamic and peripheral signals. Intrapituitary modulation of PRL expression via autocrine/paracrine mechanisms has also been reported, but similar information is still lacking in lower vertebrates. To shed light on autocrine/paracrine regulation of PRL in fish model, grass carp PRL was cloned and its expression in the carp pituitary has been confirmed. In grass carp pituitary cells, local secretion of PRL could suppress PRL release with concurrent rises in PRL production and mRNA levels. Paracrine stimulation by growth hormone (GH) was found to up- regulate PRL secretion, PRL production and PRL transcript expression, whereas the opposite was true for the local actions of luteinizing hormone (LH). Apparently, local interactions of PRL, GH and LH via autocrine/paracrine mechanisms could modify PRL production in carp pituitary cells through differential regulation of PRL mRNA stability and gene transcription.

Novel pituitary actions of TAC3 gene products in fish model: receptor specificity and signal transduction for prolactin and somatolactin α regulation by neurokinin B (NKB) and NKB-related peptide in carp pituitary cells.

TAC3 is a member of tachykinins, and its gene product neurokinin B (NKB) has recently emerged as a key regulator for LH through modulation of kisspeptin/GnRH system within the hypothalamus. In fish models, TAC3 not only encodes NKB but also a novel tachykinin-like peptide called NKB-related peptide (NKBRP), and the pituitary actions of these TAC3 gene products are still unknown. Using grass carp as a model, the direct effects and postreceptor signaling for the 2 TAC3 products were examined at the pituitary level. Grass carp TAC3 was cloned and confirmed to encode NKB and NKBRP similar to that of other fish species. In carp pituitary cells, NKB and NKBRP treatment did not affect LH release and gene expression but up-regulated prolactin (PRL) and somatolactin (SL)α secretion, protein production, and transcript expression. The stimulation by these 2 TAC3 gene products on PRL and SLα release and mRNA levels were mediated by pituitary NK2 and NK3 receptors, respectively. Apparently, NKB- and NKBRP-induced SLα secretion and transcript expression were caused by adenylate cyclase/cAMP/protein kinase A, phospholipase C/inositol 1,4,5-triphosphate/protein kinase C and Ca(2+)/calmodulin/Ca(2+)/calmodulin-dependent protein kinase II activation. The signal transduction for the corresponding responses on PRL release and mRNA expression were also similar, except that the protein kinase C component was not involved. These findings suggest that the 2 TAC3 gene products do not play a role in LH regulation at the pituitary level in carp species but may serve as novel stimulators for PRL and SLα synthesis and secretion via overlapping postreceptor signaling mechanisms coupled to NK2 and NK3 receptors, respectively.

Duck lymphocytes. VII. Selection of subpopulations using lectin-coated magnetic beads

The optimum conditions (time, concentration of duck serum, ratio of cells:beads) for binding of duck lymphocytes to magnetic beads (Dynabeads) coated with the lectins phytohaemagglutinin (PHA), wheat germ agglutinin (WGA), lentil lectin (LL) and concanavalin A (Con A) were determined. In blood and lymphoid organs, the largest population was cells binding PHA (55-80%) and the smallest was cells binding Con A (0-15%) with cells binding WGA and LL occurring at 15-58%. Most blood lymphocytes had receptors for more than one lectin, though cells with receptors for WGA were mostly a subpopulation within those possessing receptors for PHA. It is possible that, as in some other animals, lectins identify functionally important duck lymphocyte populations. Notably, PHA deserves further investigation as a marker for duck T lymphocytes, while receptors for Con A appear to be associated with duck macrophages.

Dual Role of Insulin in Spexin Regulation: Functional Link Between Food Intake and Spexin Expression in a Fish Model

Spexin (SPX), a neuropeptide discovered by the bioinformatics approach, has been recently identified as a satiety factor in a fish model. However, the functional link between feeding and SPX expression as well as the signal transduction for SPX regulation are totally unknown. In this study, we used goldfish as a model to examine the functional role of insulin as a postprandial signal for SPX regulation in bony fish. In goldfish, feeding could elevate plasma levels of glucose, insulin, and SPX with concurrent rises in insulin and SPX messenger RNA (mRNA) expression in the liver. Similar elevation in SPX mRNA level was also observed in the liver and brain areas involved in appetite control in goldfish after intraperitoneal injection of glucose and insulin, respectively. In parallel experiments with goldfish hepatocytes and brain cell culture, insulin signal induced by glucose was shown to exert a dual role in SPX regulation, namely (1) acting as an autocrine/paracrine signal to trigger SPX mRNA expression in the liver and (2) serving as an endocrine signal to induce SPX gene expression in the brain. Apparently, the peripheral (in the liver) and central actions of insulin (in the brain) on SPX gene expression were mediated by insulin receptor (to a lesser extent by insulin-like growth factor I receptor) coupled to mitogen-activated protein kinase kinase 3/6/p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin but not mitogen-activated protein kinase kinase 1/2/extracellular signal-regulated kinase 1/2 cascades. Our findings indicate that an insulin component inducible by glucose is present in the liver of the fish model and may serve as the postprandial signal linking food intake with SPX expression both in the central as well as at the hepatic level.

Plasma, cardiac tissue and brain morphine concentrations in acute and chronic morphine treated rats

1. The plasma, cardiac tissues and brain morphine concentrations in rats after acute or chronic morphine treatment were measured by high‐performance liquid chromatography.

Kisspeptin induction of somatolactin-α release in goldfish pituitary cells: functional role of cAMP/PKA-, PLC/PKC-, and Ca2+/calmodulin-dependent cascades

Although the importance of kisspeptin in the pituitary is firmly established, the signaling mechanisms for the pituitary actions of kisspeptin are still largely unknown. Somatolactin (SL), a member of the growth hormone (GH)/prolactin (PRL) family, is a pituitary hormone with pleiotropic functions in fish, but its regulation by kisspeptin has not been examined. To investigate the functional role of kisspeptin in SL regulation, expression of two paralogues of goldfish Kiss1 receptors (Kiss1ra and Kiss1rb) were confirmed in immunoidentified SLα but not SLβ cells isolated by RT-PCR coupled with laser capture microdissection. In goldfish pituitary cells prepared from neurointermediate lobe (NIL), synthetic goldfish Kiss decapeptides (gKiss1-10 and gKiss2-10) could increase SLα release. Consistent with the lack of Kiss1r expression in SLβ cells, SLβ release was not altered by kisspeptin stimulation. In parallel experiments, goldfish gKiss1-10 could elevate cyclic adenosine monophosphate (cAMP) production, upregulate protein kinase A (PKA) and protein kinase C (PKC) activities, and trigger a rapid rise in intracellular Ca(2+) levels in goldfish NIL cells. Using a pharmacological approach, cAMP/PKA and phospholipase C (PLC)/PKC pathways and subsequent activation of Ca(2+)/calmodulin (CaM)-dependent cascades were shown to be involved in SLα release induced by gKiss1-10. Apparently, the Ca(2+)-dependent cascades were triggered by extracellular Ca(2+) entry via voltage-sensitive Ca(2+) channels and mobilization of inositol trisphosphate-sensitive intracellular Ca(2+) stores. Our results demonstrate that gKiss1-10 can act directly at the pituitary level to trigger SLα release via a complex network of post-receptor signaling mechanisms.

Gene expression of luteinizing hormone receptor in carp somatotrophs differentially regulated by local action of gonadotropin and dopamine D1 receptor activation.

In grass carp, luteinizing hormone (LH) can act locally within the pituitary to regulate growth hormone expression. To test if LH receptor (LHR) expression in the carp pituitary can also serve as a target of modulation for LH actions, grass carp LHR was cloned and characterized by functional expression. In carp pituitary cells, LHR mRNA (lhr) level could be reduced by LH or human chorionic gonadotropin (hCG) but up-regulated by dopamine treatment. Dopamine-induced lhr expression occurred mainly in carp somatotrophs via the cAMP/PKA pathway coupled to pituitary D1 receptors. This stimulatory effect could be blocked by LHR activation by hCG, presumably through phosphodiesterase III activation. These findings provide evidence that lhr expression in the carp pituitary is under the differential control of LH and dopamine via modification of cAMP-dependent signaling mechanisms, which may play a role in regulating somatotroph responsiveness to the paracrine action of LH in carp species.

TAC1 Gene Products Regulate Pituitary Hormone Secretion and Gene Expression in Prepubertal Grass Carp Pituitary Cells

Tachykinin-1 (TAC1) is known to have diverse functions in mammals, but similar information is scarce in fish species. Using grass carp as a model, the pituitary actions, receptor specificity and postreceptor signaling of TAC1 gene products, namely substance P (SP) and neurokinin A (NKA), were examined. TAC1 encoding SP and NKA as well as tachykinin receptors NK1R and NK2R were cloned in the carp pituitary. The newly cloned receptors were shown to be functional with properties similar to mammalian counterparts. In carp pituitary cells, SP and NKA could trigger luteinizing hormone (LH), prolactin (PRL), and somatolactin α (SLα) secretion, with parallel rises in PRL and SLα transcripts. Short-term SP treatment (3 hours) induced LH release, whereas prolonged induction (24 hours) could attenuate LHβ messenger RNA (mRNA) expression. At pituitary cell level, LH, PRL, and SLα regulation by TAC1 gene products were mediated by NK1R, NK2R, and NK3R, respectively. Apparently, SP- and NKA-induced LH and SLα secretion and transcript expression were mediated by adenylyl cyclase/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), phospholiphase C (PLC)/inositol 1,4,5-triphosphate/protein kinase C (PKC), and Ca2+/calmodulin (CaM)/CaM-dependent protein kinase-II pathways. The signal transduction for PRL responses was similar, except for the absence of a PKC component. Regarding SP inhibition of LHβ mRNA expression, the cAMP/PKA- and PLC/PKC-dependent (but not Ca2+/CaM-dependent) cascades were involved. These results, as a whole, suggest that TAC1 gene products play a role in LH, PRL, and SLα regulation via overlapping postreceptor signaling coupled to different subtypes of tachykinin receptor expressed in the carp pituitary.