Small GTPase control of pituitary hormone secretion: Evidence from studies in the goldfish (Carassius auratus) neuroendocrine model.

The secretion of vertebrate pituitary hormones is regulated by multiple hypothalamic factors, which, while generally activating unique receptor systems, ultimately propagate signals through interacting intracellular regulatory elements to modulate hormone exocytosis. One important family of intracellular regulators is the monomeric small GTPases, a subset of which (Arf1/6, Rac, RhoA, and Ras) is highly conserved across vertebrates and regulates secretory vesicle exocytosis in many cell types. In this study, we investigated the roles of these small GTPases in basal and agonist-dependent hormone release from dispersed goldfish (Carassius auratus) pituitary cells in perifusion experiments. Inhibition of these small GTPases elevated basal LH and GH secretion, except for Ras inhibition which only increased basal LH release. However, variable responses were observed with regard to LH and GH responses to the two goldfish native gonadotropin-releasing hormones (GnRH2 and GnRH3). GnRH-dependent LH release, but not GH secretion, was mediated by Arf1/6 GTPases. In contrast, inhibition of Rac and RhoA GTPases selectively enhanced GnRH3- and GnRH2-dependent GH release, respectively, while Ras inhibition only enhanced GnRH3-evoked LH secretion. Together, our results reveal novel divergent cell-type- and ligand-specific roles for small GTPases in the control of goldfish pituitary hormone exocytosis in unstimulated and GnRH-evoked release.

Receptor-proximal effectors mediating GnRH actions in the goldfish pituitary: Involvement of G protein subunits and GRKs.

In goldfish (Carassius auratus), two endogenous isoforms of gonadotropin-releasing hormone (GnRH) stimulate luteinizing hormone (LH) and growth hormone (GH) secretion. These isoforms, GnRH2 and GnRH3, act on a shared population of cell-surface GnRH receptors (GnRHRs) expressed on both gonadotrophs and somatotrophs, and can signal through unique, yet partially overlapping, suites of intracellular effectors, in a phenomenon known as functional selectivity or biased signalling. In this study, G-protein alpha (Gα) subunits were targeted with two inhibitors, YM-254890 and BIM-46187, to ascertain the contribution of specific G-protein subunits in GnRH signalling. Results with the Gαq/11-specific inhibitor YM-254890 on primary cultures of goldfish pituitary cells revealed the use of these subunits in GnRH control of both LH and GH release, as well as GnRH-induced elevations in phospho-ERK levels. Results with the pan-Gα inhibitor BIM-46187 matched those using YM-254890 in LH release but GH responses differed, indicating additional, non-Gαq/11 subunits may be involved in somatotrophs. BIM-46187 also elevated unstimulated LH and GH release suggesting that Gα subunits regulate basal hormone secretion. Furthermore, G-protein-coupled receptor kinase (GRK2/3) inhibition reduced LH responses to GnRH2 and GnRH3, and selectively enhanced GnRH2-stimulated GH release, indicating differential use of GRK2/3 in GnRH actions on gonadotrophs and somatotrophs. These findings in a primary untransformed system provide the first direct evidence to establish Gαq/11 as an obligate driver of GnRH signalling in goldfish pituitary cells, and additionally describe the differential agonist- and cell type-selective involvement of GRK2/3 in this system.

Nesfatin-1 stimulates the hypothalamus-pituitary-interrenal axis hormones in goldfish.

Stress in vertebrates is mediated by the hypothalamus-pituitary-adrenal (in mammals)/interrenal (in fish) (HPA/I) axis, which produces the corticotropin-releasing factor (CRF), adrenocorticotropic hormone (ACTH), and corticosteroids, respectively. Nesfatin-1, a novel anorexigenic peptide encoded in the precursor nucleobindin-2 (NUCB2), is increasingly acknowledged as a peptide that influences the stress axis in mammals. The primary aim of this study was to characterize the putative effects of nesfatin-1 on the fish HPI axis, using goldfish (Carassius auratus) as an animal model. Our results demonstrated that nucb2/nesfatin-1 transcript abundance was detected in the HPI tissues of goldfish, with most abundant expression in the pituitary. NUCB2/nesfatin-1-like immunoreactivity was found in the goldfish hypothalamus, pituitary, and interrenal cells of the head kidney. GPCR12, a putative receptor for nesfatin-1, was also detected in the pituitary and interrenal cells. NUCB2/nesfatin-1-like immunoreactivity was observed in ACTH-expressing pituitary corticotrophs. Acute netting and restraint stress upregulated nucb2/nesfatin-1 mRNA levels in the forebrain, hypothalamus, and pituitary, as well as crf and crf-r1 expression in the forebrain and hypothalamus. Intraperitoneal and intracerebroventricular administration of nesfatin-1 increased cortisol release and hypothalamic crf mRNA levels, respectively. Finally, we found that nesfatin-1 significantly stimulated ACTH secretion from dispersed pituitary cells in vitro. Collectively, our data provide the first evidence showing that nesfatin-1 is a stress responsive peptide, which modulates the stress axis hormones in fish.

Nesfatin-1 is an inhibitor of the growth hormone-insulin-like growth factor axis in goldfish (Carassius auratus).

Nesfatin-1, an 82 amino acid peptide cleaved from the N-terminal of its precursor nucleobindin-2 (NUCB2), is emerging as a multifunctional peptide in fish. The present study aimed to determine whether nesfatin-1 plays a role in fish somatic growth by modulating the growth hormone (GH)/insulin-like growth factor (IGF) axis, using a representative teleost model, the goldfish (Carassius auratus). The results demonstrated that a single i.p. injection of synthetic goldfish nesfatin-1 significantly decreased the expression of hypothalamic pacap (approximately 90%) and pituitary Gh (approximately 90%) mRNAs at 15 minutes post-injection. Serum GH levels were also reduced as a result of nesfatin-1 administration, by approximately 45% and 55% at 15 and 30 minutes post-injection, respectively. Likewise, in vitro treatment of goldfish dispersed pituitary cells with nesfatin-1 reduced Gh secretion, suggesting that nesfatin-1 acts directly on pituitary somatotrophs to inhibit Gh release. Exposure of cultured liver fragments to nesfatin-1 (0.1, 1 and 10 nmol L-1 ) led to a significant reduction in igf-1 mRNA at 120 minutes and of igf-II mRNA at 30 and 60 minutes post-incubation. Collectively, these results indicate a suppressive role for nesfatin-1 on the goldfish GH/IGF axis. Immunohistochemical studies demonstrated that NUCB2/nesfatin-1-like immunoreactivity, although present in the goldfish pituitary, is not colocalised with GH in goldfish somatotrophs. Thus, nesfatin-1 does not appear to act in an autocrine manner to regulate GH secretion. Taken together, this research found that the pituitary gland is an important source of endogenous NUCB2/nesfatin-1 and also that nesfatin-1 directly suppresses the Gh/IGF axis in goldfish.

Seasonal Related Multifactorial Control of Pituitary Gonadotropin and Growth Hormone in Female Goldfish: Influences of Neuropeptides and Thyroid Hormone.

Female reproduction is under multifactorial control of brain-pituitary-peripheral origin. The present study provides information on seasonal changes in circulating LH and GH concentrations, as well as transcript levels for a number of genes involved in the regulation of reproduction and growth in female goldfish. We also provide information on the effects of treatments with GnRH and/or GnIH, and their interaction with T3, at three stages of gonadal recrudescence. Maximum basal concentration of LH was observed at late recrudescence (Spring) while no seasonal changes in basal serum GH levels was detected. Serum LH and GH levels were stimulated by GnRH as expected, depending on the season. GnIH stimulated basal GH concentrations in gonadally regressed fish. GnIH inhibitory action on GnRH-induced LH response was observed in late, but not in mid recrudescence. T3 actions on basal and GnRH- or GnIH-induced GH secretion were generally inhibitory, depending on season. Administration of T3 attenuated GnRH-induced LH responses in mid and late stages of gonadal recrudescence, and the presence of GnIH abolished inhibitory actions of T3 in fish at mid recrudescence. Our results also demonstrated seasonal patterns in basal and GnRH- and/or GnIH-induced transcript levels for ERα, ERßI, FSHR, aromatase, TRαI, TRß, IGF-I, and Vtg in the liver and ovary. However, there were no clear correlations between changes in transcript levels and circulating levels of LH and GH. The results support the hypothesis that GnRH, GnIH, and T3 are contributing factors in complex reciprocal control of reproduction and growth in goldfish.

ß-Arrestin-dependent signaling in GnRH control of hormone secretion from goldfish gonadotrophs and somatotrophs.

In goldfish, two native isoforms of gonadotropin-releasing hormone (GnRH2 and GnRH3) stimulate luteinizing hormone (LH) and growth hormone (GH) release from pituitary cells through activation of cell-surface GnRH-receptors (GnRHRs) on gonadotrophs and somatotrophs. Interestingly, GnRH2 and GnRH3 induce LH and GH release via non-identical post-receptor signal transduction pathways in a ligand- and cell-type-selective manner. In this study, we examined the involvement of ß-arrestins in the control of GnRH-induced LH and GH secretion from dispersed goldfish pituitary cells. Treatment with Barbadin, which interferes with ß-arrestin and ß2-adaptin subunit interaction, reduced LH responses to GnRH2 and GnRH3, as well as GH responses to GnRH2; but enhanced GnRH3-induced GH secretion. Barbadin also had positive influences on basal hormone release, and basal GH release in particular, as well as basal activity of extracellular signal-regulated kinase (ERK) and GnRH-induced ERK activation. These findings indicate that ß-arrestins play permissive roles in the control of GnRH-stimulated LH release. However, in somatotrophs, ß-arrestins, perhaps by mediating agonist-selective endosomal trafficking of engaged GnRHRs, participate in GnRH-isoform-specific GH release responses (stimulatory and inhibitory for GnRH2-GnRHR and GnRH3-GnRHR activation, respectively). The correlative stimulatory influences of Barbadin on basal hormone release and ERK activation suggest that ß-arrestins may negatively regulate basal secretion through modulation of basal ERK activity. These results provide the first direct evidence of a role for ß-arrestins in hormone secretion from an untransformed primary pituitary cell model, and establish these proteins as important receptor-proximal players in mediating functional selectivity downstream of goldfish GnRHRs.

Comparative aspects of GnRH-Stimulated signal transduction in the vertebrate pituitary - Contributions from teleost model systems.

Gonadotropin-releasing hormone (GnRH) is a major regulator of reproduction through actions on pituitary gonadotropin release and synthesis. Although it is often thought that pituitary cells are exposed to only one GnRH, multiple GnRH forms are delivered to the pituitary of teleost fishes; interestingly this can include the cGnRH-II form usually thought to be non-hypophysiotropic. GnRHs can regulate other pituitary cell-types, both directly as well as indirectly, and multiple GnRH receptors (GnRHRs) may also be expressed in the pituitary, and even within a single pituitary cell-type. Literature on the differential actions of native GnRH isoforms in primary pituitary cells is largely derived from teleost fishes. This review will outline the diversity and complexity of GnRH-GnRHR signal transduction found within vertebrate gonadotropes as well as extra-gonadotropic sites with special emphasis on comparative studies from fish models. The implications that GnRHR transduction mechanisms are GnRH isoform-, function-, and cell-specific are also discussed.

The nonapeptide isotocin in goldfish: Evidence for serotonergic regulation and functional roles in the control of food intake and pituitary hormone release.

Nonapeptides are a highly conserved family of peptides synthesized in the neuroendocrine brain and acting on central and peripheral receptors to regulate physiological functions in vertebrates. While the evolution of the two gene families of oxytocin-like and vasopressin-like nonapeptides and their receptors, as well as the neuroanatomy of their independent neuronal circuits have been well-characterized across vertebrate species, comparative studies on the physiological roles across vertebrates are lagging behind. In the current study, we focused on the comparative neuroendocrine functions and regulation of isotocin, the teleost homologue of mammalian oxytocin. Specifically, we address the hypothesis that isotocin exerts opposing effects on food intake and reproduction, which are well-established effects of its homologue oxytocin in mammalian species. Using goldfish, a well-characterized model of neuroendocrine regulation of both food intake and reproduction, we here showed that isotocin acts as an anorexigenic factor while exerting stimulatory effects on pituitary luteinizing hormone and growth hormone release. Given the dual inhibitory and stimulatory roles of serotonin on food intake and pituitary release of reproductive hormone in goldfish, we also investigated the potential crosstalk between both systems using immunohistochemistry and pharmacological approaches. Results provide neuroanatomical and pharmacological evidence for serotonergic regulation of magnocellular isotocinergic neurons in the preoptic area and pituitary. Together, these findings firstly provide the basis to investigate neuroendocrine cross-talk between serotonergic and nonapeptidergic systems in the regulation of both food intake and reproduction in goldfish, and secondly point to a conserved function of oxytocin-like peptides in the differential neuroendocrine control of both physiological processes in vertebrates.

Endocrine disrupting effects of waterborne fluoxetine exposure on the reproductive axis of female goldfish, Carassius auratus.

Evidence suggests that pharmaceuticals and personal care products reach urban watersheds, bioconcentrate in fish, and potentially disrupt physiological homeostasis. These impairments often affect hormone functions. Selective serotonin reuptake inhibitors (SRRIs) are increasingly studied with regards to their endocrine disrupting effects on teleost physiological processes, including reproduction. To examine whether FLX effects on the endocrine regulation of reproductive physiology in goldfish are sex-specific, we exposed sexually recrudescent female goldfish to two waterborne concentrations of FLX (0.54µg/L and 54µg/L) using an experimental design previously used for sexually mature male goldfish. To evaluate possible endocrine disrupting effects, we quantified the gonadosomatic index, circulating hormone concentrations (luteinizing hormone, LH; growth hormone, GH; 17-ß estradiol, E2; and testosterone, T), and the expression of isotocin and vasotocin in the telencephalon, gonadotropin subunits and GH in the pituitary, and gonadotropin receptors, GH receptors, and aromatase in the ovary. Female goldfish exposed to 0.54µg/L FLX exhibited a significant decrease in circulating E2, and conversely, a significant increase in circulating LH and ovarian aromatase mRNA levels, suggesting disruption of E2-mediated feedback on LH release. These results, when compared with those previously observed in males, reveal that waterborne exposure to environmentally relevant levels of FLX sex-specifically disrupts the reproductive endocrine axis in goldfish, characterized by a decrease in E2 in females, and conversely, estrogen-like effects in males. These data emphasize the importance of studying the effect of endocrine disrupting chemicals on both sexes.

Ligand-Biased Regulation of PtdIns(3,4,5)P3-Dependent Signal Transduction in GPCR Control of Pituitary Hormone Release.

Biased signaling describes the selective activation of signal transduction cascades by structurally related ligands downstream of shared G protein-coupled receptors (GPCRs). Although class I phosphoinositide 3-kinases (PI3Ks) are important components of GPCR-controlled transduction networks, little is known regarding the potential for biased regulation of class I PI3K-dependent signaling. The full complement of class I PI3K catalytic subunits (p110α, p110ß, p110δ, and p110γ) first appears in bony fishes and, despite being associated with distinct cellular functions, all class I PI3Ks produce the lipid second-messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3]. We have previously shown that 2 endogenous gonadotropin-releasing hormones (GnRH2 and GnRH3), which both signal through shared Gαq/11-coupled receptors, selectively activate different subsets of class I PI3K isoforms in their control of hormone release from goldfish (Carassius auratus) pituitary cells. Here, we tested the hypothesis that the biased activation of class I PI3K isoforms results in the selective recruitment of PtdIns(3,4,5)P3-sensitive effectors downstream of GnRH-stabilized GPCRs using pharmacological mapping. Our results reveal that distinct PtdIns(3,4,5)P3-sensitive effectors are involved in the differential control of GnRH2- and GnRH3-stimulated, as well as basal, hormone release and implicate the participation of noncanonical PtdIns(3,4,5)P3-sensitive transduction elements. Furthermore, observations using a selective inhibitor of the shared Gßγ-effector interaction surface indicate a role for Gßγ-dependent signaling in the integrated control of pituitary hormone exocytosis. These findings add to our understanding of functional selectivity in GPCR signal transduction networks, in general, and reveal the complexity of biased signaling downstream of class I PI3K catalytic activity.

Teleost leukocyte immune-type receptors activate distinct phagocytic modes for target acquisition and engulfment.

Channel catfish (Ictalurus punctatus) IpLITRs belong to the Ig superfamily and regulate innate immune cell effector responses. This study tested the hypothesis that ITAM-dependent and ITAM-independent phagocytic pathways are engaged by different subtypes of the IpLITR family. When stably expressed in RBL-2H3 cells, the ITAM-containing fusion-construct IpLITR 2.6b/IpFcRγ-L stimulated phagocytic responses that were abrogated at suboptimal incubation temperatures and by pharmacological inhibitors of the classic signaling components of the mammalian FcR-dependent phagocytic pathway. Interestingly, the ITIM-containing receptor IpLITR 1.1b also induced phagocytosis through an actin-dependent mechanism, but this process was insensitive to the pharmacological inhibitors tested and remained functional at temperatures as low as 22°C. The IpLITR 1.1b also displayed a unique target-acquisition phenotype that consisted of complex, membranous protrusions, which captured targets in phagocytic cup-like structures but often failed to completely engulf targets. Taken together, these findings suggest that teleost immunoregulatory receptors that associate with ITAM-containing adaptors can engage conserved components of the phagocytic machinery to engulf extracellular targets akin to the classic FcR-mediated response in mammals. Alternatively, IpLITR 1.1b displays a stalled phagocytic phenotype that is likely dependent on the selective recruitment of the minimal molecular machinery required for target capture but results in incomplete target engulfment. Overall, this study demonstrates that IpLITRs can selectively engage distinct components of the phagocytic process and provides important new information regarding the target acquisition as well as internalization mechanisms involved in controlling phagocytic responses across vertebrates.

Ligand-selective signal transduction by two endogenous GnRH isoforms involves biased activation of the class I PI3K catalytic subunits p110ß, p110γ, and p110δ in pituitary gonadotropes and somatotropes.

In goldfish, 2 endogenous GnRH isoforms, GnRH2 and GnRH3, are released at the pituitary and directly stimulate LH and GH release using the same population of GnRH receptors (GnRHRs) but with GnRH-specific transduction mechanisms. Previously, we have shown that phosphoinositide 3-kinases (PI3Ks) mediate GnRH2- and GnRH3-stimulated LH and GH release. Among the 3 classes of PI3Ks, class I PI3Ks are the best characterized and consist of 4 110-kDa catalytic isoforms (p110α, p110ß, p110γ, and p110δ). Importantly, p110ß and p110γ, but not p110α or p110δ, can be directly activated by the Gßγ heterodimer of Gαßγ protein complexes. In the present study, we examined the expression of class I PI3K isoforms and the effects of selective inhibitors of p110α, p110ß, p110γ, and p110δ catalytic activity on basal, as well as acute, GnRH2- and GnRH3-stimulated LH and GH release responses using primary cultures of dispersed goldfish pituitary cells in column perifusion. Results demonstrate that p110γ and p110δ are involved in the control of basal LH and GH release, whereas p110α and p110ß only regulate basal LH secretion. However, p110ß and p110γ both participated in GnRH3- and GnRH2-stimulated GH release, whereas p110ß and p110γ mediated GnRH2- and GnRH3-induced LH release responses, respectively. GnRH2- and GnRH3-stimulated LH release, as well as GnRH3-elicited GH release, also required p110δ. These results constitute the first evidence for the differential involvement of class I PI3K catalytic subunits in GnRH actions, in general, and suggest that GnRH2 and GnRH3 binding to GnRHRs can bias the activation of class I PI3K signaling to mediate hormone release responses in 2 distinct pituitary cell types. The involvement of both class IA and IB PI3Ks implicates Gßγ subunits, as well as other known regulators of class I PI3Ks, as important components of GnRHR-mediated responses that could influence GnRH-selective signaling in other cell types.

Relationship between nitric oxide- and calcium-dependent signal transduction pathways in growth hormone release from dispersed goldfish pituitary cells.

Nitric oxide (NO) and Ca(2+) are two of the many intracellular signal transduction pathways mediating the control of growth hormone (GH) secretion from somatotropes by neuroendocrine factors. We have previously shown that the NO donor sodium nitroprusside (SNP) elicits Ca(2+) signals in identified goldfish somatotropes. In this study, we examined the relationships between NO- and Ca(2+)-dependent signal transduction mechanisms in GH secretion from primary cultures of dispersed goldfish pituitary cells. Morphologically identified goldfish somatotropes stained positively for an NO-sensitive dye indicating they may be a source of NO production. In 2h static incubation experiments, GH release responses to the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) were attenuated by CoCl2, nifedipine, verapamil, TMB-8, BHQ, and KN62. In column perifusion experiments, the ability of SNP to induce GH release was impaired in the presence of TMB-8, BHQ, caffeine, and thapsigargin, but not ryanodine. Caffeine-elicited GH secretion was not affected by the NO scavenger PTIO. These results suggest that NO-stimulated GH release is dependent on extracellular Ca(2+) availability and voltage-sensitive Ca(2+) channels, as well as intracellular Ca(2+) store(s) that possess BHQ- and/or thapsigargin-inhibited sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases, as well as TMB-8- and/or caffeine-sensitive, but not ryanodine-sensitive, Ca(2+)-release channels. Calmodulin kinase-II also likely participates in NO-elicited GH secretion but caffeine-induced GH release is not upstream of NO production. These findings provide insights into how NO actions many integrate with Ca(2+)-dependent signalling mechanisms in goldfish somatotropes and how such interactions may participate in the GH-releasing actions of regulators that utilize both NO- and Ca(2+)-dependent transduction pathways.

PI3K signalling in GnRH actions on dispersed goldfish pituitary cells: relationship with PKC-mediated LH and GH release and regulation of long-term effects on secretion and total cellular hormone availability.

Goldfish pituitary cells are exposed to two GnRHs, salmon (s)GnRH and chicken (c)GnRH-II. Phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) both participate in acute sGnRH- and cGnRH-II-stimulated LH and GH release. Using goldfish pituitary cells, we examined the relationship between PI3K and PKC in acute LH and GH secretion, and PI3K involvement in chronic hormone release and total LH and GH availability. The PI3K inhibitor LY294002 did not affect PKC agonists-induced LH or GH release, and PKC agonists did not alter PI3K p85 phosphorylation, suggesting PKC activation is not upstream of PI3K in acute hormone release. In 2, 6, 12 and 24h treatments, LY294002 did not affect LH release but stimulated total LH availability at 6h. sGnRH stimulatory actions on LH release and total availability at 12 and 24h, and cGnRH-II effects on these parameters at 6h were inhibited by LY294002. LY294002 enhanced basal GH release at 2 and 6h, but reduced total GH at 12 and 24h. Increased GH release was seen following 6, 12 and 24h of sGnRH, and 2, 6 and 24h of cGnRH-II treatment but total GH availability was only elevated by 24h cGnRH-II treatment. Whereas LY294002 inhibited GH release responses to sGnRH at 12h and cGnRH-II at 6h, it attenuated cGnRH-II-elicited, but not sGnRH-induced, effects on total GH. These results indicate that PI3K differentially modulates long-term basal and GnRH-stimulated hormone release, and total hormone availability, in a time-, cell-type-, and GnRH isoform-selective manner.

Induction of phagocytosis and intracellular signaling by an inhibitory channel catfish leukocyte immune-type receptor: evidence for immunoregulatory receptor functional plasticity in teleosts.

Immunoregulatory receptors are categorized as stimulatory or inhibitory based on their engagement of unique intracellular signaling networks. These proteins also display functional plasticity, which adds versatility to the control of innate immunity. Here we demonstrate that an inhibitory catfish leukocyte immune-type receptor (IpLITR) also displays stimulatory capabilities in a representative myeloid cell model. Previously, the receptor IpLITR 1.1b was shown to inhibit natural killer cell-mediated cytotoxicity. Here we expressed IpLITR 1.1b in rat basophilic leukemia-2H3 cells and monitored intracellular signaling and functional responses. Although IpLITR 1.1b did not stimulate cytokine secretion, activation of this receptor unexpectedly induced phagocytosis as well as extracellular signal-related kinase 1/2- and protein kinase B (Akt)-dependent signal transduction. This novel IpLITR 1.1b-mediated response was independent of an association with the FcRγ chain and was likely due to phosphotyrosine-dependent adaptors associating with prototypical signaling motifs within the distal region of its cytoplasmic tail. Furthermore, compared to a stimulatory IpLITR, IpLITR 1.1b displayed temporal differences in the induction of intracellular signaling, and IpLITR 1.1b-mediated phagocytosis had reduced sensitivity to EDTA and cytochalasin D. Overall, this is the first demonstration of functional plasticity for teleost LITRs, a process likely important for the fine-tuning of conserved innate defenses.

Differential modulation of ghrelin-induced GH and LH release by PACAP and dopamine in goldfish pituitary cells.

Ghrelin (GRLN) participates in multiple physiological processes, including the regulation of growth hormone (GH) and luteinizing hormone (LH) release. In the goldfish, neuroendocrine control of GH and LH release are multifactorial. In this system, pituitary adenylate cyclase-activating polypeptide (PACAP)-stimulated GH and LH secretion, as well as dopamine (DA)-induced GH release, are mediated by protein kinase A (PKA)-dependent, but protein kinase C (PKC)-independent, mechanisms. In addition, DA inhibits LH secretion by actions at sites along both PKA and PKC signaling pathways. Recently, goldfish GRLN (gGRLN19) has been shown to induce GH release via PKC, and LH secretion via both PKC and PKA. To further understand the neuroendocrine regulation of goldfish GH and LH release, we examined the effects of DA and PACAP on gGRLN19 actions in primary cultures of goldfish pituitary cells in perifusion and in Ca(2+)-imaging experiments. Consistent with their known intracellular signaling mechanisms in gonadotrophs, DA inhibited gGRLN19-induced LH release while cotreatment of PACAP and gGRLN19 did not produce additive LH responses. When applied prior to gGRLN19, PACAP potentiated gGRLN19-induced GH release and Ca(2+) signals within somatotrophs. In contrast, neither prior treatment with DA followed by gGRLN19 nor pretreatment with gGRLN19 prior to PACAP produced an enhanced GH release response. These observations suggest that PKA activators positively modulate gGRLN19 actions on goldfish somatotrophs in a ligand- and treatment order-specific manner. Results add to our understanding of the complexity of neuroendocrine control of GH and LH release at the pituitary cell level, and our understanding of GRLN action.

MEK1/2 differentially participates in GnRH actions on goldfish LH and GH secretion and hormone protein availability: acute and long-term effects, in vitro.

Two endogenous gonadotropin-releasing hormones (GnRHs), sGnRH and cGnRH-II, stimulate LH and GH release via protein kinase C (PKC) signaling in goldfish. In this study, extracellular signal-regulated kinase kinase 1 and 2 (MEK1/2) involvement in acute and prolonged GnRH effects on goldfish gonadotrope and somatotrope functions, as well as potential interactions with PKC in the control of LH and GH release from goldfish pituitary cells was investigated. MEK1/2 inhibitors U0126 and PD098059 significantly decreased sGnRH but not cGnRH-II-stimulated GH release from perifused goldfish pituitary cells and U0126 significantly reduced the GH, but not the LH, release responses to synthetic PKC activators. In long-term static incubations (up to 24h) with goldfish pituitary cells, U0126 generally did not affect basal LH release but attenuated sGnRH- and cGnRH-II-induced LH release, as well as the time-dependent effects of sGnRH and/or cGnRH-II to elevate total LH availability (sum of release and cell content). sGnRH and cGnRH-II reduced cellular GH content and/or total GH availability at 2, 6, and 12h while static incubation with U0126 alone generally increased basal GH release but reduced cellular GH content and/or the total amount of GH available. U0126 also selectively reduced the sGnRH-induced GH release responses at 6 and 24h but paradoxically inhibited cGnRH-II-stimulated GH secretion while enhancing sGnRH-elicited GH release at 2h. Taken together, this study reveals the complexity of GnRH-stimulated MEK1/2 signaling and adds to our understanding of cell-type- and GnRH-isoform-selective signal transduction in the regulation of pituitary cell hormone release and production.

Growth hormone-releasing hormone stimulates GH release while inhibiting ghrelin- and sGnRH-induced LH release from goldfish pituitary cells.

Goldfish GH-releasing hormone (gGHRH) has been recently identified and shown to stimulate GH release in goldfish. In goldfish, neuroendocrine regulation of GH release is multifactorial and known stimulators include goldfish ghrelin (gGRLN19) and salmon gonadotropin-releasing hormone (sGnRH), factors that also enhance LH secretion. To further understand the complex regulation of pituitary hormone release in goldfish, we examined the interactions between gGHRH, gGRLN19, and sGnRH on GH and LH release from primary cultures of goldfish pituitary cells in perifusion. Treatment with 100nM gGHRH for 55min stimulated GH release. A 5-min pulse of either 1nM gGRLN19 or 100nM sGnRH induced GH release in naïve cells, and these were just as effective in cells receiving gGHRH. Interestingly, gGHRH abolished both gGRLN19- and sGnRH-induced LH release and reduced basal LH secretion levels. These results suggest that gGHRH does not interfere with sGnRH or gGRLN19 actions in the goldfish somatotropes and further reveal, for the first time, that GHRH may act as an inhibitor of stimulated and basal LH release by actions at the level of pituitary cells.

Nitric oxide signaling in ghrelin-induced LH release from goldfish pituitary cells.

Among its many known functions, ghrelin has been proposed to participate in the regulation of reproduction; however, its effect on pituitary LH release is controversial, especially in mammals. In the goldfish, ghrelin directly stimulates pituitary LH release via increased entry of calcium through voltage sensitive channels and activation of protein kinase C. Nitric oxide (NO) is an important signaling molecule in many physiological systems including hormone regulation at the level of the pituitary. Goldfish pituitary cells and extracts have previously been reported to express immunoreactivity for inducible and neuronal NO synthase (iNOS and nNOS). In this study, we determined if NO is involved in goldfish ghrelin (gGRLN(19))-induced LH release from primary cultures of dispersed goldfish pituitary cells in column perifusion. Treatment with the NO scavenger PTIO significantly decreased gGRLN(19)-induced LH release and co-treatment with the NO donor SNP and gGRLN(19) did not induce an additive increase in LH release, suggesting that NO is critical to gGRLN(19) stimulation of LH release in goldfish pituitary cells. Further work examined the involvement of the NOS using the NOS isoform-selective inhibitors 1400W, 7-Ni, and AGH. While 1400W (selective for iNOS) and AGH (selective for iNOS and nNOS) abolished gGRLN(19)-induced LH release from goldfish pituitary cells, 7-Ni (selective for nNOS and endothelial NOS) had no significant effect on this stimulation. Our results indicate, for the first time in a teleost species, that gGRLN(19)-induced LH release from pituitary cells is NO-dependent and likely involves iNOS, adding to the understanding of GRLN intracellular signaling in general and specifically to the regulation of LH release from the pituitary.