Goldfish Calmodulin: Molecular Cloning, Tissue Distribution, and Regulation of Transcript Expression in Goldfish Pituitary Cells

Abstract Calmodulin (CaM) is a Ca2+-binding protein essential for biological functions mediated through Ca2+-dependent mechanisms. In the goldfish, CaM is involved in the signaling events mediating pituitary hormone secretion induced by hypothalamic factors. However, the structural identity of goldfish CaM has not been established, and the neuroendocrine mechanisms regulating CaM gene expression at the pituitary level are still unknown. Here we cloned the goldfish CaM and tested the hypothesis that pituitary expression of CaM transcripts can be the target of modulation by hypothalamic factors. Three goldfish CaM cDNAs, namely CaM-a, CaM-bS, and CaM-bL, were isolated by library screening. These cDNAs carry a 450-bp open reading frame encoding the same 149-amino acid CaM protein, the amino acid sequence of which is identical with that of mammals, birds, and amphibians and is highly homologous (≥90%) to that in invertebrates. In goldfish pituitary cells, activation of cAMP- or PKC-dependent pathways increased CaM mRNA levels, whereas the opposite was true for induction of Ca2+ entry. Basal levels of CaM mRNA was accentuated by GnRH and pituitary adenylate cyclase-activating polypeptide but suppressed by dopaminergic stimulation. Pharmacological studies using D1 and D2 analogs revealed that dopaminergic inhibition of CaM mRNA expression was mediated through pituitary D2 receptors. At the pituitary level, D2 activation was also effective in blocking GnRH- and pituitary adenylate cyclase-activating polypeptide-stimulated CaM mRNA expression. As a whole, the present study has confirmed that the molecular structure of CaM is highly conserved, and its mRNA expression at the pituitary level can be regulated by interactions among hypothalamic factors.

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

Endocrinology ◽

10.1210/en.2016-1740 ◽

2017 ◽

Vol 158 (6) ◽

pp. 1776-1797 ◽

Cited By ~ 8

Author(s):

Guangfu Hu ◽

Mulan He ◽

Wendy K. W. Ko ◽

Anderson O. L. Wong

Keyword(s):

Protein Kinase ◽

Mrna Expression ◽

Grass Carp ◽

Messenger Rna ◽

Hormone Secretion ◽

Adenosine Monophosphate ◽

Pituitary Hormone ◽

Neurokinin A ◽

Pituitary Cells ◽

Gene Products

Abstract 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.

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Signal Transduction Mechanisms for Glucagon-Induced Somatolactin Secretion and Gene Expression in Nile Tilapia (Oreochromis niloticus) Pituitary Cells

Frontiers in Endocrinology ◽

10.3389/fendo.2020.629077 ◽

2021 ◽

Vol 11 ◽

Author(s):

Chaoyi Zhang ◽

Anji Lian ◽

Yue Xu ◽

Quan Jiang

Keyword(s):

Gene Expression ◽

Oreochromis Niloticus ◽

Nile Tilapia ◽

Primary Cultures ◽

Hormone Secretion ◽

Hek293 Cells ◽

Luciferase Reporter ◽

Pituitary Hormone ◽

Pituitary Cells ◽

Mrna Levels

Glucagon (GCG) plays a stimulatory role in pituitary hormone regulation, although previous studies have not defined the molecular mechanism whereby GCG affects pituitary hormone secretion. To this end, we identified two distinct proglucagons, Gcga and Gcgb, as well as GCG receptors, Gcgra and Gcgrb, in Nile tilapia (Oreochromis niloticus). Using the cAMP response element (CRE)-luciferase reporter system, tilapia GCGa and GCGb could reciprocally activate the two GCG receptors expressed in human embryonic kidney 293 (HEK293) cells. Quantitative real-time PCR analysis revealed that differential expression of the Gcga and Gcgb and their cognate receptors Gcgra and Gcgrb was found in the various tissues of tilapia. In particular, the Gcgrb is abundantly expressed in the neurointermediate lobe (NIL) of the pituitary gland. In primary cultures of tilapia NIL cells, GCGb effectively stimulated SL release, with parallel rises in the mRNA levels, and co-incubation with the GCG antagonist prevented GCGb-stimulated SL release. In parallel experiments, GCGb treatment dose-dependently enhanced intracellular cyclic adenosine monophosphate (cAMP) accumulation with increasing inositol 1,4,5-trisphosphate (IP3) concentration and the resulting in transient increases of Ca2+ signals in the primary NIL cell culture. Using selective pharmacological approaches, the adenylyl cyclase (AC)/cAMP/protein kinase A (PKA) and phospholipase C (PLC)/IP3/Ca2+/calmodulin (CaM)/CaMK-II pathways were shown to be involved in GCGb-induced SL release and mRNA expression. Together, these results provide evidence for the first time that GCGb can act at the pituitary level to stimulate SL release and gene expression via GCGRb through the activation of the AC/cAMP/PKA and PLC/IP3/Ca2+/CaM/CaMK-II cascades.

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Effects of a novel hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, on pituitary hormone release in rats

Journal of Endocrinology ◽

10.1677/joe.0.1340033 ◽

1992 ◽

Vol 134 (1) ◽

pp. 33-41 ◽

Cited By ~ 103

Author(s):

G. R. Hart ◽

H. Gowing ◽

J. M. Burrin

Keyword(s):

Cyclic Amp ◽

Adenylate Cyclase ◽

Time Course ◽

Pituitary Hormone ◽

Pituitary Cells ◽

Hormone Release ◽

Α Subunit ◽

Time Period ◽

Pituitary Adenylate Cyclase ◽

Pretreatment Time

ABSTRACT We have demonstrated that the novel hypothalamic peptide pituitary adenylate cyclase-activating poly-peptide (PACAP-38; 0·1–100 nmol/l) caused an increase in the release of GH, ACTH, LH and α-subunit and accumulation of intracellular cyclic AMP from dispersed rat anterior pituitary cells in static culture for 24 h. There were no significant effects on TSH or prolactin release over the same time-period. PACAP-38 (10 nmol/l) increased the release of GH by 1·3-fold (P<0·05), ACTH by 1·9-fold (P<0·05), LH by 3·5-fold (P<0·001) and α-subunit by 2·0-fold (P< 0·005) and the accumulation of intracellular cyclic AMP by >2-fold (P<0·001) after 24 h. However, the time-course for the effect of PACAP-38 (1 mmol/l) on hormone release and intracellular cyclic AMP levels showed a temporal dissociation. The effect of PACAP-38 on GH and ACTH levels did not reach significance until 24 h whereas the effect of PACAP-38 on LH and α-subunit release reached significance after 4 h implying a different mechanism of action for their release. To investigate the PACAP-induced secretion of LH and α-subunit further, we examined the effects of PACAP after down-regulation of protein kinase C (PKC). PACAP-38 at a dose maximal for the stimulation of LH and α-subunit release (10 nmol/l) added together with the PKC activator, 12-0-tetradecanoyl-phorbol-13-acetate (TPA; 0·1 μmol/l) had no greater effect on LH and α-subunit release than TPA alone over a 4 h incubation period. Increasing the pretreatment time with TPA (0–5 h) at a dose (0·1 μmol/l) known to deplete PKC activity substantially, reduced the ability of PACAP-38 to stimulate LH and α-subunit release and intracellular cyclic AMP levels significantly. We conclude that the stimulatory actions of PACAP on LH and α-subunit relies in part on PKC activity. Journal of Endocrinology (1992) 134, 33–41

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Grass carp somatolactin: II. Pharmacological study on postreceptor signaling mechanisms for PACAP-induced somatolactin-α and -β gene expression

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.90386.2008 ◽

2008 ◽

Vol 295 (2) ◽

pp. E477-E490 ◽

Cited By ~ 21

Author(s):

Quan Jiang ◽

Mulan He ◽

Xinyan Wang ◽

Anderson O. L. Wong

Keyword(s):

Gene Expression ◽

Mrna Expression ◽

Grass Carp ◽

Pharmacological Study ◽

Primary Cultures ◽

Pituitary Hormone ◽

Pituitary Cells ◽

Mrna Levels ◽

Fish Model ◽

Subsequent Activation

Somatolactin (SL), the latest member of the growth hormone/prolactin family, is a novel pituitary hormone with diverse functions. However, the signal transduction mechanisms responsible for SL expression are still largely unknown. Using grass carp as an animal model, we examined the direct effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on SL gene expression at the pituitary level. In primary cultures of grass carp pituitary cells, SLα and SLβ mRNA levels could be elevated by PACAP via activation of PAC-I receptors. With the use of a pharmacological approach, the AC/cAMP/PKA and PLC/inositol 1,4,5-trisphosphate (IP3)/PKC pathways and subsequent activation of the Ca2+/calmodulin (CaM)/CaMK-II cascades were shown to be involved in PACAP-induced SLα mRNA expression. Apparently, the downstream Ca2+/CaM-dependent cascades were triggered by extracellular Ca2+ ([Ca2+]e) entry via L-type voltage-sensitive Ca2+ channels (VSCC) and Ca2+ release from IP3-sensitive intracellular Ca2+ stores. In addition, the VSCC component could be activated by cAMP/PKA- and PLC/PKC-dependent mechanisms. Similar postreceptor signaling cascades were also observed for PACAP-induced SLβ mRNA expression, except that [Ca2+]e entry through VSCC, PKC coupling to PLC, and subsequent activation of CaMK-II were not involved. These findings, taken together, provide evidence for the first time that PACAP can induce SLα and SLβ gene expression in fish model via PAC-I receptors through differential coupling to overlapping and yet distinct signaling pathways.

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Pituitary Actions of EGF on Gonadotropins, Growth Hormone, Prolactin and Somatolactins in Grass Carp

Biology ◽

10.3390/biology9090279 ◽

2020 ◽

Vol 9 (9) ◽

pp. 279

Author(s):

Qiongyao Hu ◽

Qinbo Qin ◽

Shaohua Xu ◽

Lingling Zhou ◽

Chuanhui Xia ◽

...

Keyword(s):

Growth Hormone ◽

Mrna Expression ◽

Grass Carp ◽

Hormone Secretion ◽

Pituitary Hormones ◽

Vital Role ◽

Pituitary Hormone ◽

Pituitary Cells ◽

Neurokinin Receptor

In mammals, epidermal growth factor (EGF) plays a vital role in both pituitary physiology and pathology. However, the functional role of EGF in the regulation of pituitary hormones has rarely reported in teleost. In our study, using primary cultured grass carp pituitary cells as an in vitro model, we examined the effects of EGF on pituitary hormone secretion and gene expression as well as the post-receptor signaling mechanisms involved. Firstly, we found that EGF significantly reduced luteinizing hormone (LHβ) mRNA expression via ErbB1 coupled to ERK1/2 pathway, but had no effect on LH release in grass carp pituitary cells. Secondly, the results showed that EGF was effective in up-regulating mRNA expression of growth hormone (GH), somatolactin α (SLα) and somatolactin β (SLβ) via ErbB1 and ErbB2 and subsequently coupled to MEK1/2/ERK1/2 and PI3K/Akt/mTOR pathways, respectively. However, EGF was not effective in GH release in pituitary cells. Thirdly, we found that EGF strongly induced pituitary prolactin (PRL) release and mRNA expression, which was mediated by ErbB1 and subsequent stimulation of MEK1/2/ERK1/2 and PI3K/Akt/mTOR pathways. Interestingly, subsequent study further found that neurokinin B (NKB) significantly suppressed EGF-induced PRL mRNA expression, which was mediated by neurokinin receptor (NK2R) and coupled to AC/cAMP/PKA signal pathway. These results suggested that EGF could differently regulate the pituitary hormones expression in grass carp pituitary cells.

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Paracrine regulation of growth hormone gene expression by gonadotrophin release in grass carp pituitary cells: functional implications, molecular mechanisms and signal transduction

Journal of Molecular Endocrinology ◽

10.1677/jme.1.01629 ◽

2005 ◽

Vol 34 (2) ◽

pp. 415-432 ◽

Cited By ~ 30

Author(s):

Hong Zhou ◽

Yonghua Jiang ◽

Wendy K W Ko ◽

Wensheng Li ◽

Anderson O L Wong

Keyword(s):

Gene Expression ◽

Growth Hormone ◽

Adenylate Cyclase ◽

Mrna Expression ◽

Grass Carp ◽

Janus Kinase ◽

Growth Hormone Gene ◽

Pituitary Cells ◽

Mrna Levels ◽

Gh Gene

Growth hormone (GH) is known to stimulate luteinizing hormone (LH) release via paracrine interactions between somatotrophs and gonadotrophs. However, it is unclear if LH can exert a reciprocal effect to modulate somatotroph functions. Here we examined the paracrine effects of LH on GH gene expression using grass carp pituitary cells as a cell model. LH receptors were identified in grass carp somatotrophs and their activation by human chorionic gonadotropin (hCG) increased ‘steady-state’ GH mRNA levels. Removal of endogenous LH by immunoneutralization using LH antiserum inhibited GH release and GH mRNA expression. GH secretagogues, including gonadotrophin releasing hormone (GnRH), pituitary adenylate cyclase-activating polypeptide (PACAP) and apomorphine, were effective in elevating GH mRNA levels but these stimulatory actions were blocked by LH antiserum. In pituitary cells pretreated with actinomycin D, the half-life of GH mRNA was not affected by hCG but was enhanced by LH immunoneutralization. Treatment with LH antiserum also suppressed basal levels of mature GH mRNA and primary transcripts. hCG increased cAMP synthesis in carp pituitary cells and hCG-induced GH mRNA expression was mimicked by forskolin but suppressed by inhibiting adenylate cyclase and protein kinase A. Similarly, the stimulatory actions of hCG and forskolin on GH mRNA expression were blocked by inhibiting Janus kinase 2 (JAK2) and MAP kinase (MAPK), including P42/44MAPK and P38 MAPK. These results suggest that LH is essential for the maintenance of GH release, GH gene expression, and somatotroph responsiveness to GH-releasing factors. The paracrine actions of LH on GH mRNA expression are mediated by a concurrent increase in GH gene transcription and GH mRNA turnover, probably through JAK2/MAPK coupled to the cAMP-dependent pathway.

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Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) as a Growth Hormone (GH)-Releasing Factor in Grass Carp. I. Functional Coupling of Cyclic Adenosine 3′,5′-Monophosphate and Ca2+/Calmodulin-Dependent Signaling Pathways in PACAP-Induced GH Secretion and GH Gene Expression in Grass Carp Pituitary Cells

Endocrinology ◽

10.1210/en.2005-0294 ◽

2005 ◽

Vol 146 (12) ◽

pp. 5407-5424 ◽

Cited By ~ 39

Author(s):

Anderson O. L. Wong ◽

Wensheng Li ◽

Ching Yu Leung ◽

Longfei Huo ◽

Hong Zhou

Keyword(s):

Protein Kinase ◽

Adenylate Cyclase ◽

Grass Carp ◽

Pituitary Cells ◽

Cam Kinase Ii ◽

Mrna Levels ◽

Cam Kinase ◽

Gh Secretion ◽

Pituitary Adenylate Cyclase ◽

Gh Gene

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the glucagon/secretin peptide family, has been recently proposed to be the ancestral GH-releasing factor. Using grass carp as a model for bony fish, we examined the mechanisms for PACAP regulation of GH synthesis and secretion at the pituitary level. Nerve fibers with PACAP immunoreactivity were identified in the grass carp pituitary overlapping with the distribution of somatotrophs. At the somatotroph level, PACAP was shown to induce cAMP synthesis and Ca2+ entry through voltage-sensitive Ca2+ channels (VSCC). In carp pituitary cells, PACAP but not vasoactive intestinal polypeptide increased GH release, GH content, total GH production, and steady-state GH mRNA levels. PACAP also enhanced GH mRNA stability, GH promoter activity, and nuclear expression of GH primary transcripts. Increasing cAMP levels, induction of Ca2+ entry, and activation of VSCC were all effective in elevating GH secretion and GH mRNA levels. PACAP-induced GH secretion and GH mRNA expression, however, were abolished by inhibiting adenylate cyclase and protein kinase A, removing extracellular Ca2+ or VSCC blockade, or inactivating calmodulin (CaM)-dependent protein kinase II (CaM kinase II). Similar sensitivity to VSCC and CaM kinase II blockade was also observed by activating cAMP production as a trigger for GH release and GH gene expression. These results suggest that PACAP stimulates GH synthesis and secretion in grass carp pituitary cells through PAC1 receptors. These stimulatory actions probably are mediated by the adenylate cyclase/cAMP/protein kinase A pathway coupled to Ca2+ entry via VSCC and subsequent activation of CaM/CaM kinase II cascades.

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Stimulation of the hypothalamic ventromedial nuclei by pituitary adenylate cyclase-activating polypeptide induces hypophagia and thermogenesis

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00334.2011 ◽

2011 ◽

Vol 301 (6) ◽

pp. R1625-R1634 ◽

Cited By ~ 36

Author(s):

Jon M. Resch ◽

Joanne P. Boisvert ◽

Allison E. Hourigan ◽

Christopher R. Mueller ◽

Sun Shin Yi ◽

...

Keyword(s):

Adenylate Cyclase ◽

Mrna Expression ◽

Energy Homeostasis ◽

Uncoupling Protein ◽

Core Body Temperature ◽

Type I ◽

Mrna Levels ◽

Brown Adipose ◽

Pituitary Adenylate Cyclase ◽

Acute Injection

Numerous studies have demonstrated that the hypothalamic ventromedial nuclei (VMN) regulate energy homeostasis by integrating and utilizing behavioral and metabolic mechanisms. The VMN heavily express pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptors (PAC1R). Despite the receptor distribution, most PACAP experiments investigating affects on feeding have focused on intracerebroventricular administration or global knockout mice. To identify the specific contribution of PACAP signaling in the VMN, we injected PACAP directly into the VMN and measured feeding behavior and indices of energy expenditure. Following an acute injection of PACAP, nocturnal food intake was significantly reduced for 6 h after injections without evidence of malaise. In addition, PACAP-induced suppression of feeding also occurred following an overnight fast and could be blocked by a specific PAC1R antagonist. Metabolically, VMN-specific injections of PACAP significantly increased both core body temperature and spontaneous locomotor activity with a concurrent increase in brown adipose uncoupling protein 1 mRNA expression. To determine which signaling pathways were responsive to PACAP administration into the VMN, we measured mRNA expression of well-characterized hypothalamic neuropeptide regulators of feeding. One hour after PACAP administration, expression of pro-opiomelanocortin mRNA was significantly increased in the arcuate nuclei (ARC), with no changes in neuropeptide Y and agouti-related polypeptide mRNA levels. This suggests that PAC1R expressing VMN neurons projecting to pro-opiomelanocortin neurons contribute to hypophagia by involving melanocortin signaling. While the VMN also abundantly express PACAP protein, the present study demonstrates that PACAP input to the VMN can influence the control of energy homeostasis.

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