Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have previously shown that 10-12 kDa N-terminal fragments of rat proopiomelanocortin (POMC) and human POMC1-76 stimulate mitosis and/or differentiation in lactotrophs of early postnatal rat pituitary. A truncated form, POMC1-26, mimics the differentiation-inducing but not the mitogenic action of the former peptides. To further characterize these two biological responses, the present study compared changes in the intracellular free calcium concentration ([Ca2+]i) in response to POMC1-76 and POMC1-26 in isolated pituitary cells from 14-day-old female rats. Calcium (Ca2+) responses were also used as a guide to determine whether the responsive cells belong to the lactosomatotroph lineage. Application of POMC1-76 or POMC1-26 induced a maintained oscillating [Ca2+]i increase in a small population of cells. Increasing doses of the peptides did not affect the magnitude and the frequency of [Ca2+]i oscillations but clearly augmented the number of responding cells. Approximately 2% of the cells responded at 0.1 nM POMC1-76 or 5 nM POMC1-26, and 11-13% of the cells responded at 10 nM and 500 nM of the respective peptides. About one-third of the cells responsive to these peptides also showed a [Ca2+]i increase in response to growth hormone-releasing peptide-6 (GHRP-6) while, in a small number of responsive cells, [Ca2+]i was depressed by dopamine, suggesting that the former cells are somatotrophs and the latter lactotrophs. This interpretation was confirmed by immunocytochemical identification of prolactin and growth hormone (GH) in the cells. Of the cells showing Ca2+ response to POMC1-76, approximately one-third contained GH and another third prolactin. The remainder contained neither GH nor prolactin. Comparable results were obtained with POMC1-26. The rise of [Ca2+]i induced by the N-terminal POMC peptides persisted after depletion of intracellular Ca2+ stores by thapsigargin. Removal of Ca2+ from the extracellular medium or addition of cadmium completely abolished both the POMC1-76- and POMC1-26-induced [Ca2+]i increase. Nifedipine inhibited the Ca2+ response to both peptides, although only in 55% of the responsive cells. Depletion of some isoforms of protein kinase C by preincubation with the phorbol ester PMA for 24 h did not modify the Ca2+ responses. In contrast, blockade of the protein kinase A pathway with Rp-cAMPs partially inhibited the POMC1-76- or POMC1-26-induced [Ca2+]i increase. The present data show that, in immature pituitary cells, POMC1-76 induces an increase in [Ca2+]i through extracellular Ca2+ influx, possibly mediated in part by protein kinase A activation. The active domain of POMC1-76 seems to comprise its N-terminal moiety. The data support the hypothesis that POMC1-76 exerts a specific function in the development of different members of the lactosomatotroph lineage and that the peptide mobilizes different subsets of cells within this lineage, by a mechanism determined by its concentration.
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PMID:Stimulation of Ca2+ entry in lactotrophs and somatotrophs from immature rat pituitary by N-terminal fragments of proopiomelanocortin. 957 10

Cocultivation of primary pig hepatocytes with RL-ET-14 cells, an endothelial-like cell line resulted in spontaneous induction of glutamine synthetase (GS)-activity in the hepatocytes by more than 10-fold within 120 h to 144 h. Hepatocyte-specific induction was confirmed by immunocytochemistry. Addition of trijodothyronine (T3) to the culture medium inhibited the induction in a concentration dependent manner. No comparable influence of T3 was seen with pure cultures of either cell type suggesting that only the spontaneous induction was affected. Other hormones such as glucagon, insulin, growth hormone, epinephrine and testosterone did not interfere with the induction. Addition of several protein kinase-inhibitors such as staurosporine and genistein were without influence. However, a strong inhibition was found after addition of okadaic acid in nanomolar concentrations indicating an involvement of protein-phosphatase 2A in the induction process.
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PMID:The spontaneous induction of glutamine synthetase in pig hepatocytes cocultured with RL-ET-14 cells is completely inhibited by trijodothyronine and okadaic acid. 961 8

There is a difference between the sheep and rat somatotrophs in the response to growth hormone-releasing peptide-2 (GHRP-2), which raises the question of what the response may be in human somatotrophs. In the present study, cells were obtained from seven human acromegalic tumours and the effects of GHRP-2 were studied. Cells were dissociated and kept in primary culture for 1-3 weeks before experimentation. Application of GHRP-2 for 30 min induced a significant increase in GH secretion from the cultured cells from all seven tumours whereas human GH-releasing hormone (hGHRH) at a dose of 10 nM induced a significant GH release in only four of seven tumours. The intracellular levels of cAMP in all seven tumours were significantly increased by both 10 nM GHRP-2 and GHRH, but the response to GHRH was significantly higher than the response to GHRP-2. The adenylyl cyclase inhibitor, MDL 12330A, blocked the effect of GHRH and GHRP-2 on intracellular cAMP levels, whereas the Ca2+ channel blocker Co2+ (0.5 mM) did not attenuate the cAMP response. For the tumours in which GH secretion was increased by GHRH and GHRP-2, the cAMP antagonist Rp-cAMP blocked the GH response to GHRH but not to GHRP-2. When a protein kinase A (PKA) inhibitor (H89) was applied, GHRH stimulated GH release was blocked, but cAMP accumulation was not affected. The response to GHRP-2 was not altered by H89. Calphostin C [a protein kinase C (PKC) inhibitor] reduced the effect of GHRP-2 on the secretion of GH but did not affect the response to GHRH. Both GHRH and GHRP-2 increased the intracellular Ca2+ concentration in a concentration-dependent manner. We conclude that (1) GHRH increases GH secretion from human GH tumours via the cAMP pathway whereas GHRP-2 increases GH secretion mainly via the PKC pathway; (2) GHRH increases cAMP (without GH release) in a subset of tumours whereas GHRP-2 increases cAMP levels (slightly) and GH secretion in all tumours; and (3) GHRP-2 and GHRH do not act on the same receptor on human somatotrophs derived from acromegalic tumours.
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PMID:Effect of growth hormone-releasing peptide-2 (GHRP-2) and GH-releasing hormone (GHRH) on the the cAMP levels and GH release from cultured acromegalic tumours. 968 50

Previous studies have demonstrated that intravenous lipopolysaccharide (LPS) will increase concentrations of growth hormone (GH). One possible explanation for this may reside in the response of the pituitary to specific cytokines. This study sought to determine the effects of recombinant bovine tumor necrosis factor alpha (TNF), recombinant ovine (ro) interleukin-1alpha (IL-1alpha), roIL-1beta, ro interleukin-2 (IL-2), and ro gamma-interferon (INT) on GH release from cultured sheep pituitary cells. Sheep were sacrificed and pituitary cells cultured in DMEM with 10% fetal bovine serum for 3 days. On day 4, cells were washed and serum-free DMEM added to cells. IL-1alpha and IL-1beta were used at 0.2, 2 and 20 ng/ml and the remaining cytokines at 2, 20 and 200 ng/ml. Neither IL-2 nor INT had effects on basal or on GH-releasing hormone (GRH)-stimulated GH release. TNF inhibited GRH-stimulated GH release (p < 0.05). Both IL-1alpha and IL-1beta stimulated GH release from cultured pituitary cells at all doses tested (p < 0.01). Neither IL-1alpha nor IL-1beta had an effect on GRH-stimulated GH release. IL-1 effects were inhibited by H-89 (p < 0.05; a protein kinase A inhibitor) and by nifedipine (p < 0.05; a calcium channel blocker). Both of these mechanisms are central signal transduction mechanisms mediating GRH-stimulated GH release. IL-1-stimulated GH release is partially inhibited (p < 0.05) by lipoxygenase pathway blockers. Phorbol myristate acetate downregulation of protein kinase C did not alter IL-1-stimulated GH release. IL-1beta increased the content of both GH and GH mRNA in cultured sheep pituitary cells. We conclude that IL-1 produces a strong stimulus to GH release, which is mediated by calcium entry and protein kinase A activation. IL-1 also activates lipoxygenase pathways. This latter pathway as well as calcium entry were shown to mediate LPS stimulation of GH release from cultured pituitary cells. The similarity between IL-1 and LPS signal transduction suggests that LPS may activate pituitary production of IL-1 to produce the stimulus to GH. The lack of inhibitory effects of INT, TNF and IL-2 as opposed to what is seen in the rat may suggest a partial mechanism to explain the different effects of LPS on GH release between sheep and that seen in cattle and rats.
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PMID:Cytokine-mediated growth hormone release from cultured ovine pituitary cells. 973 4

This study was designed to investigate the effect of follicle-stimulating hormone (FSH) on nuclear maturation, fertilization, and early embryonic development of in-vitro-matured bovine oocytes and to find out whether this effect is exerted through a cyclic adenosine monophosphate (cAMP) signal transduction pathway. In addition the effect of the combination of FSH and growth hormone (GH) on subsequent cleavage and embryo development was studied. Therefore cumulus oocyte complexes were cultured in the presence of FSH (0.05 IU/ml) and the nuclear stage of the oocytes was assessed using 4,6-diamino-2-phenyl-indole (DAPI) staining either after 16, 20, or 24 hr of in vitro maturation or 18 hr after the onset of fertilization. To assess the effect of FSH and the combination of FSH and GH added during in vitro maturation on the developmental capacity of the oocytes, cumulus oocyte complexes were incubated in the presence of either FSH (0.05 IU/ml) or FSH (0.05 IU/ml) plus GH (100 ng/ml) for 22 hr, followed by in vitro fertilization and in vitro embryo culture. To investigate whether FSH-induced oocyte maturation is exerted through the cAMP pathway, cumulus oocyte complexes were cultured in M199 supplemented with FSH (0.05 IU/ml) and H-89 (10 microM), a specific inhibitor of cAMP-dependent protein kinase A. After 16 hr of culture, the proportion of oocytes in metaphase II (MII) stage was determined. Cultures with GH and without FSH and H-89 served as controls. The percentage of MII oocytes at 16 hr of incubation was significantly lower (P < 0.001) in the presence of FSH than in the control group, while the number of MII oocytes beyond 20 hr did not differ from the control group. That points to a transient inhibition of nuclear maturation by FSH. Opposite to FSH, addition of GH during in vitro maturation significantly enhanced the number of MII oocytes after 16 hr of culture (P < 0.001), which points to the acceleration of nuclear maturation by GH. Addition of FSH during in vitro maturation significantly enhanced the proportion of normal fertilized oocytes, cleaved embryos and blastocysts (P < 0.001). Similarly, addition of GH during in vitro maturation significantly enhanced the number of cleaved embryos and blastocysts (P < 0.001); however, in vitro maturation in the presence of GH and FSH did not result in an extra enhancement of the embryo development. Both the inhibition of nuclear maturation by FSH and its acceleration by GH was completely abolished by H-89. In conclusion, in vitro maturation of bovine oocytes in the presence of FSH retards nuclear maturation via a cAMP-mediated pathway, while it enhances fertilizability and developmental ability of the oocytes. Supplementation of GH and FSH during in vitro maturation did not result in an extra increase in the number of blastocysts following in vitro fertilization and in vitro embryo culture.
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PMID:Follicle-stimulating hormone and growth hormone act differently on nuclear maturation while both enhance developmental competence of in vitro matured bovine oocytes. 977 55

The pituitary of a number of teleosts contains two gonadotropins (GtHs) which are produced in distinct populations of cells; the beta subunit of the GtH I being found in close proximity to the somatotrophs, while the II beta cells are more peripheral. In several species the GtH beta subunits are expressed at varying levels throughout the reproductive cycle, the I beta dominating in early maturing fish, after which the II beta becomes predominant. This suggests differential control of the beta subunit synthesis which may be regulated by both hypothalamic hormones and gonadal steroids. At ovulation and spawning, changes also occur in the somatotrophs, which become markedly more active, while plasma growth hormone (GH) levels increase. In a number of species, GnRH elevates either the I beta or the II beta mRNA levels, depending on the reproductive state of the fish. In tilapia, the GnRH effect on the II beta appears to be mediated through both cAMP-PKA and PKC pathways. GnRH also stimulates GH release in both goldfish and tilapia, but it increases the GH transcript levels only in goldfish; both GnRH and direct activation of PKC are ineffective in altering GH mRNA in tilapia pituitary cells. Dopamine (DA) does not alter II beta transcript levels in cultured tilapia pituitary cells, but increases GH mRNA levels in both rainbow trout and tilapia, in a PKA-dependent manner. This effect appears to be through interactions with Pit-1 and also by stabilizing the mRNA. Somatostatin (SRIF) does not alter GH transcript levels in either tilapia or rainbow trout, although it may alter GH synthesis by modulation of translation. Gonadal steroids appear to have differential effects on the transcription of the beta subunits. In tilapia, testosterone (T) elevates I beta mRNA levels in cells from immature or early maturing fish (in low doses), but depresses them in cells from late maturing fish and is ineffective in cells from regressed fish. Similar results were seen in early recrudescing male coho salmon injected with T or E2. T or E2 administered in vivo has dramatic stimulatory effects on the II beta transcript levels in immature fish of a number of species, while less powerful effects are seen in vitro. A response is also seen in cells from early maturing rainbow trout or tilapia, or regressed tilapia, but not in cells from late maturing or spawning fish. These results are substantiated by the finding that the promoter of the salmon II beta gene contains several estrogen responsive elements (EREs) which react and interact differently when exposed to varying levels of E2. In addition, activator protein-1 (AP-1) and steroidogenic factor-1 (SF-1) response elements are also found in the salmon II beta promoter; the AP-1 site is located close to a half ERE, while the SF-1 acts synergistically with the E2 receptor. The mRNA levels of both AP-1 and SP-1 are elevated, at least in mammals, by GnRH, suggesting possible sites for cross-talk between GnRH and steroid activated pathways. Reports of the effects of T or E2 on GH transcription differ. No effect is seen in vitro in pituitaries of tilapia, juvenile rainbow trout or common carp, but T does increase the transcript levels in pituitaries of both immature and mature goldfish. Reasons for these discrepancies are unclear, but other systemic hormones may be more instrumental than the gonadal steroids in regulating GH transcription. These include T3 which increases both GH mRNA levels and de novo synthesis (in tilapia and common carp) and insulin-like growth factor-I (IGF-I) which reduces GH transcript levels as well as inhibiting GH release.
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PMID:Endocrine regulation of gonadotropin and growth hormone gene transcription in fish. 982 5

Ca2+ plays an essential role in pituitary adenylate cyclase-activating polypeptide (PACAP)-stimulated growth hormone (GH) secretion from porcine somatotropes. Here, Indo-1 microfluorimetry was used to investigate the dynamics of free cytosolic Ca2+ concentration ([Ca2+]i) in single porcine somatotropes in response to PACAP38 and PACAP27. We also evaluated the relative contributions of extra- and intracellular Ca2+ sources and of cAMP-dependent protein kinase (PKA) and phospholipase C (PLC). A high proportion of somatotropes responded to PACAP38 (79.4%) and PACAP27 (68.4%) with [Ca2+]i rises that could be followed by a refractory plateau (type 1 response), or by a decrease in [Ca2+]i during which somatotropes were responsive to a subsequent PACAP pulse (type II response). Although Ca2+ profiles in response to both peptides were similar, PACAP38-induced [Ca2+]i rises were higher. Somatotrope response to PACAP38 or PACAP27 was markedly reduced by removing extracellular Ca2+, blocking Ca2+ entry through L-type voltage sensitive Ca2+ channels (VSCC), or inhibiting PKA. Conversely, Ca2+ depletion from intracellular stores or PLC inactivation did not affect the response to PACAP27 but considerably reduced maximal [Ca2+]i induced by PACAP38. We conclude that both peptides stimulate extracellular Ca2+ influx through L-type VSCC by a PKA-dependent mechanism. However, PACAP38 also triggers a PLC-mediated Ca2+ mobilization from intracellular stores, thereby indicating that the two molecular forms of PACAP activate common and distinct second messenger pathways within porcine somatotropes.
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PMID:Pituitary adenylate cyclase-activating polypeptides 38 and 27 increase cytosolic free Ca2+ concentration in porcine somatotropes through common and distinct mechanisms. 992 28

Previously we reported that a phorbol ester, phorbol 12, 13-dibutyrate (PDBu), increased the release of human growth hormone-binding protein (hGH-BP) in IM-9 cells, and that this phorbol ester-enhanced release was mediated by protein kinase Ca (PKCalpha). In the present study, the mechanisms of the phorbol ester-enhanced hGH-BP release were further investigated. Treatment of IM-9 cells with PDBu did not increase hGH-BPs (55-60 kDa) in the intracellular soluble fraction. When the cells were treated with trypsin to remove human growth hormone receptors (hGHRs) on the cell surface after stimulation, no hGH-BPs were detected in the culture supernatants, nor did treatment with bafilomycin A1 or chloroquine affect the PDBu-enhanced hGH-BP release. These results suggest that hGH-BPs released by PDBu stimulation are derived from cell surface hGHRs and not generated within the cells. Protein kinase inhibitors with broad specificities, K-252a and K-252b, inhibited the PDBu-enhanced release with almost the same dose-dependency, although only a trace amount of K-252b was incorporated into IM-9 cells than K-252a, suggesting that K-252b probably inhibits an ecto-kinase extracellularly. PDBu actually enhanced the phosphorylation of several extracellular proteins, and this enhanced phosphorylation was completely inhibited by K-252b treatment. Moreover, the PKCalpha-specific inhibitor bisindolylmaleimide III which inhibits PDBu enhanced hGH-BP release inhibited the PDBu-enhanced phosphorylation of extracellular proteins. On the other hand, the impermeable PKC inhibitor PKC inhibitor peptide 19-31 did not inhibit PDBu-enhanced release, suggesting that the target PKCalpha for PDBu is not present on the extracellular surface. Taken together, these results suggest that, in addition to intracellular PKCalpha, activation of an undefined ecto-kinase may also be involved in the PDBu-enhanced hGH-BP release.
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PMID:Role of ecto-kinase in phorbol ester-enhanced growth hormone-binding protein release from human IM-9 cells. 1043 24

Hypothalamic growth hormone-releasing hormone (GHRH) stimulates growth hormone (GH) gene expression in anterior pituitary somatotrophs by binding to the GHRH receptor, a G-protein-coupled transmembrane receptor, and by mediating a cAMP-mediated protein kinase A (PKA) signal-transduction pathway. Two nonclassical cAMP-response element motifs (CGTCA) are located at nucleotides -187/-183 (distal cAMP-response element; dCRE) and -99/-95 (proximal cAMP-response element; pCRE) of the human GH promoter and are required for cAMP responsiveness, along with the pituitary-specific transcription factor Pit-1 (official nomenclature, POU1F1). Although a role for cAMP-response element binding protein (CREB) in GH stimulation by PKA has been suggested, it is unclear how the effect may be mediated. CREB binding protein (CBP) is a nuclear cofactor named for its ability to bind CREB. However, CBP also binds other nuclear proteins. We determined that CBP interacts with Pit-1 and is a cofactor for Pit-1-dependent activation of the human GH promoter. This pathway appears to be independent of CREB, with CPB being the likely target of phosphorylation by PKA.
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PMID:CREB-independent regulation by CBP is a novel mechanism of human growth hormone gene expression. 1052 51

1. Whole-cell voltage-gated K+ currents and the K+ current response to growth hormone-releasing hormone (GHRH) were characterised in primary cultures of human acromegalic somatotropes. 2. Both delayed rectifier (IK) and transient (IA) K+ currents were recorded from human somatotropes held at -80 mV and bathed in a solution containing Cd2+ (1 mM), TTX (1 microM) and a low concentration of Ca2+ (0.5 mM). Only IK was recorded, however, when a holding potential of -40 mV was used. 3. GHRH (10 nM) immediately and significantly reduced the amplitude of both IA and IK. While the reduction in the amplitude of IA was fully reversed following the removal of GHRH, the amplitude of IK had only partially recovered 10 min after GHRH removal. In addition, GHRH shifted the voltage-dependent inactivation curve of IA by 13.5 mV in the negative direction. 4. In a low Ca2+ and Cd2+-containing solution, the Ca2+-activated K+ channel blockers apamin (100 nM and 1 microM) and charybdotoxin (1 microM) did not alter K+ currents or the effect of GHRH on the recorded K+ currents. 5. The whole-cell K+ currents and their responses to GHRH were unaffected by the application of 8-bromo-cAMP (100 microM), Rp-cAMP (100 microM) or the protein kinase A (PKA) inhibitor H89 (1 microM). In addition, intracellular dialysis of the PKA inhibitory peptide PKI (10 microM) had no effect on the K+ current response to GHRH. 6. While the application of protein kinase C (PKC) inhibitors calphostin C (100 nM) or chelerythrine (1 microM) did not affect the amplitude of the K+ currents, the K+ current response to GHRH was significantly attenuated. Downregulation of PKC with phorbol 12,13-dibutyrate (PDBu, 0.5 microM for 16 h) also abolished the K+ current response to GHRH. In addition, intracellular dialysis of somatotropes with the PKC inhibitory peptide PKC19-36 (1 microM) prevented the GHRH-induced decrease in the amplitude of the voltage-gated K+ currents. Local application of PDBu (1 microM) significantly reduced the amplitude of the voltage-gated K+ currents in a similar manner to that induced by GHRH, but without clear recovery upon removal. 7. This study demonstrates that GHRH decreases voltage-gated K+ currents via a PKC-mediated pathway in human adenoma somatotropes, rather than by the cAMP-PKA pathway that is usually implicated in the actions of GHRH.
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PMID:Human GHRH reduces voltage-gated K+ currents via a non-cAMP-dependent but PKC-mediated pathway in human GH adenoma cells. 1054 37


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