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:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We studied the role of the phosphatidylinositol system in the action of growth hormone-releasing factor (GRF). We asked whether GRF stimulates the activity of phospholipase C by determining GRF-induced changes in 32P labeling of the individual phosphoinositides and inositol phosphates in purified rat somatotrophs. The somatotrophs were challenged with GRF (10(-7)M) for 0.33, 1, 3, 10, 30, and 90 min. GRF did not significantly or consistently alter 32P incorporation into phosphatidylinositol bisphosphate (PIP2), phosphatidylinositol monophosphate (PIP), or phosphatidylinositol (PI), except for a small reduction in PIP labeling at 90 min. In general the level of 32P incorporation into the inositol phosphates did not increase but instead decreased with GRF. There was a small but significant reduction of labeling of inositol trisphosphate (IP3) at 90 min of GRF incubation. There were also small but significant decreases in 32P incorporation into inositol bisphosphate (IP2) at 0.33, 3, and 30 min. GRF did not significantly alter 32P labeling of inositol monophosphate (IP). These results indicate that GRF does not stimulate phospholipase C activity in somatotrophs. We conclude that the phosphatidylinositol second messenger system does not play an essential role in the action of GRF.
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PMID:Effect of growth hormone-releasing factor on phosphoinositide hydrolysis in somatotrophs. 196 57

The pathophysiology of mammosomatotroph adenomas remains unclear. We studied a mammosomatotroph adenoma removed from an 8-year old boy with a 5-year history of growth acceleration and acromegalic gigantism at presentation. Elevated basal GH (mean 28 micrograms/l) and PRL (mean 120 micrograms/l) plasma levels were observed, as well as paradoxical responses of GH to L-dopa, TRH and oral glucose administration; PRL was reduced by L-dopa and slightly increased by TRH; GHRH stimulated release of both GH and PRL. Two operations were required to remove the very large tumour and the patient was treated with bromocriptine before the second. Hormonal secretion by tumour explants in culture was evaluated under basal conditions and after stimulation or inhibition. High levels of GH and PRL were secreted for up to 24 days. Furthermore, GHRH and TRH caused a dose-related stimulation of both hormones, while somatostatin and dopamine were effective in suppressing either basal or stimulated hormone release only at very high (microM) concentrations. Intracellular events were studied by determination of the guanosine triphosphate binding (G) protein levels and adenylate cyclase (AC) activity in the tumour tissue. Before bromocriptine treatment, AC activity was very high in the tumour and could be further stimulated by various agents; very high levels of the AC-stimulatory G protein alpha subunit Gs alpha and very low amounts of the AC-inhibiting G protein alpha subunit Gi3 alpha and of the phospholipase C-stimulating G protein alpha subunit Gq alpha were found in the tumour. After bromocriptine, baseline AC activity was normalized and could no longer be stimulated; Gs alpha and Gi3 alpha levels were unchanged while those of Gq alpha were normalized. Screening of tumour DNA after amplification by polymerase chain reaction followed by single-strand conformational polymorphism analysis did not reveal any mutations in the hot spots of G protein alpha subunits (alpha s, alpha i2, alpha o2 and alpha 11) genes or in the H-ras and p53 genes. Gs alpha and GH transcription factor-1 (pit-1) expression were evaluated by amplification of cDNA. While the mRNA expression of pit-1 decreased after bromocriptine treatment, that of Gs alpha increased. These data suggest the possibility of an oncogenic process involving overexpression of Gs alpha, resulting in chronic activation of adenylate cyclase. Furthermore, our results suggest that the anti-secretory and anti-proliferative effects of bromocriptine may be mediated through a decrease in Pit-1 secondary to the inhibition of adenylate cyclase activity.
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PMID:Mammosomatotroph adenoma causing gigantism in an 8-year old boy: a possible pathogenetic mechanism. 762 75

Calcium and phosphorus metabolism is mainly regulated by PTH through its actions on kidney and bone. PTHrP, which is associated with the hypercalcemia of malignancy syndrome, binds to and activates the same receptor that PTH does. cDNA clones of PTH/PTHrP receptors from rat osteosarcoma (ROS 17/2.8) and opossum kidney (OK) cells are highly homologous and are members of a novel G protein-linked receptor family that includes calcitonin, glucagon, GLP-1, GHRH, VIP, and secretin receptors. Analysis of the protein sequence predicts a receptor with 7 transmembrane domains, a 155 amino acids (aa) extracellular (EC) N-terminal, and 130aa intracellular C-terminal domaina. The extracellular domain has 6 conserved cysteines and 4 potential glycosylation sites. When transfected in COS cells, both receptors are able to bind PTH and PTHrP active fragments with equal affinity. Likewise, agonists activate both adenylate cyclase and phospholipase C efficiently. The N-terminal EC domain and the first EC loop seem to determine the receptor binding capacity with the agonists. Activation of adenylate cyclase and phospholipase C might involve multiple sites between the 3rd helix and the C-terminal tail. Partial characterization of the rat PTH/PTHrP receptor gene demonstrates the existence of at least 15 exons. The first six transmembrane domains are encoded by separated exons. The PTH/PTHrP receptor mRNA is expressed mainly in kidney and bone, and also is widely expressed in many tissues, but not all. A major 2.3-2.5 kb transcript is observed in all these tissues. Nevertheless, 2 larger transcripts are observed in kidney and liver, and multiple smaller mRNA species are observed in kidney, skin, and testis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Mode of action of parathyroid hormone (PTH) and PTH-related peptide (PTHrP) in target organs]. 785 77

In vitro studies have been performed to demonstrate and characterize specific binding sites for synthetic GH secretagogues (sGHS) on membranes from pituitary gland and different human brain regions. A binding assay for sGHS was established using a peptidyl sGHS (Tyr-Ala-hexarelin) which had been radioiodinated to high specific activity at the Tyr residue. Specific binding sites for 125I-labelled Tyr-Ala-hexarelin were detected mainly in membranes isolated from pituitary gland and hypothalamus, but they were also present in other brain areas such as choroid plexus, cerebral cortex, hippocampus and medulla oblongata with no sex-related differences. In contrast, negligible binding was found in the thalamus, striatum, substantia nigra, cerebellum and corpus callosum. The binding of 125I-labelled Tyr-Ala-hexarelin to membrane-binding sites is a saturable and reversible process, depending on incubation time and pH of the buffer. Scatchard analysis of the binding revealed a finite number of binding sites in the hypothalamus and pituitary gland with a dissociation constant (Kd) of (1.5 +/- 0.3) x 10(-9) and (2.1 +/- 0.4) x 10(-9) mol/l respectively. Receptor activity is sensitive to trypsin and phospholipase C digestion, suggesting that protein and phospholipids are essential for the binding of 125I-labelled Tyr-Ala-hexarelin. The binding of 125I-labelled Tyr-Ala-hexarelin to pituitary and hypothalamic membranes was displaced in a dose-dependent manner by different unlabelled synthetic peptidyl (Tyr-Ala-hexarelin, GHRP2, hexarelin, GHRP6) and non-peptidyl (MK 0677) sGHS. An inhibition of the specific binding was also observed when binding was performed in the presence of [D-Arg1-D-Phe5-D-Trp7,9-Leu11]-substance P, a substance P antagonist that has been found to inhibit GH release in response to sGHS. In contrast, no competition was observed in the presence of other neuropeptides (GHRH, somatostatin, galanin or Met-enkephalin) which have a known influence on GH release. In conclusion, the present data demonstrate that sGHS have specific receptors in human brain and pituitary gland and reinforce the hypothesis that these compounds could be the synthetic counterpart of an endogenous GH secretagogue involved in the neuroendocrine control of GH secretion and possibly in other central activities.
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PMID:Specific receptors for synthetic GH secretagogues in the human brain and pituitary gland. 961 63

Somatotropes comprise two morphologically and functionally distinct subpopulations of low (LD) and high (HD) density cells. We recently reported that GRF induces different patterns of increase in the cytosolic free Ca2+ concentration in single porcine LD and HD somatotropes, which for LD cells required not only Ca2+ influx but also intracellular Ca2+ mobilization. This suggested that GRF may activate multiple signaling pathways in pig LD and HD somatotropes to stimulate GH secretion. To address this question, we first assessed the direct GRF effect on second messenger activation in cultures of LD and HD cells by measuring cAMP levels and [3H]myo-inositol incorporation. Secondly, to determine the relative importance of cAMP- and inositol phosphate (IP)-dependent pathways, and of intra- and extracellular Ca2+, GRF-induced GH release from cultured LD and HD somatotropes was measured in the presence of specific blockers. GRF increased cAMP levels in both subpopulations, whereas it only augmented IP turnover in LD cells. Accordingly, adenylate cyclase inhibition by MDL-12,330A abolished GRF-stimulated GH release in both subpopulations, whereas phospholipase C inhibition by U-73122 only reduced this effect partially in LD cells. Likewise, blockade of Ca2+ influx with Cl2Co reduced GRF-stimulated GH secretion in both LD and HD somatotropes, whereas depletion of thapsigargin-sensitive intracellular Ca2+ stores only decreased the secretory response to GRF in LD cells. These results demonstrate that GRF specifically and differentially activates multiple signaling pathways in two somatotrope subpopulations to stimulate GH release. Thus, although the prevailing signaling cascade employed by GRF in both subpopulations is adenylate cyclase/cAMP/extracellular Ca2+, the peptide also requires activation of the phospholipase C/IP/intracellular Ca2+ pathway to exert its full effect in porcine LD somatotropes.
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PMID:Growth hormone (GH)-releasing factor differentially activates cyclic adenosine 3',5'-monophosphate- and inositol phosphate-dependent pathways to stimulate GH release in two porcine somatotrope subpopulations. 1009 12

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel member of the secretin-glucagon peptide family. In mammals, this peptide has been located in a wide range of tissues and is involved in a variety of biological functions. In lower vertebrates, especially fish, increasing evidence suggests that PACAP may function as a hypophysiotropic factor regulating pituitary hormone secretion. PACAP has been identified in the brain-pituitary axis of representative fish species. The molecular structure of fish PACAP is highly homologous to mammalian PACAP. The prepro-PACAP in fish, however, is distinct from that of mammals as it also contains the sequence of fish GHRH. In teleosts, the anterior pituitary is under direct innervation of the hypothalamus and PACAP nerve fibers have been identified in the pars distalis. Using the goldfish as a fish model, mRNA transcripts of PACAP receptors, namely the PAC1 and VPACI receptors, have been identified in the pituitary as well as in various brain areas. Consistent with the pituitary expression of PACAP receptors, PACAP analogs are effective in stimulating growth hormone (GH) and gonadotropin (GTH)-II secretion in the goldfish both in vivo and in vitro. The GH-releasing action of PACAP is mediated via pituitary PAC1 receptors coupled to the adenylate cyclase-cAMP-protein kinase A and phospholipase C-IP3-protein kinase C pathways. Subsequent stimulation of Ca2+ entry through voltage-sensitive Ca2+ channels followed by activation of Ca2+-calmodulin protein kinase II is likely the downstream mechanism mediating PACAP-stimulated GH release in goldfish. Although the PACAP receptor subtype(s) and the associated post-receptor signaling events responsible for PACAP-stimulated GTH-II release have not been characterized in goldfish, these findings support the hypothesis that PACAP is produced in the hypothalamus and delivered to the anterior pituitary to regulate GH and GTH-II release in fish.
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PMID:Pituitary adenylate cyclase activating polypeptide as a novel hypophysiotropic factor in fish. 1094 84

Three classes of mammalian phosphoinositide-specific phospholipase C (PLC) have been characterized, PLCbeta, PLCgamma and PLCdelta, that are differentially regulated by heterotrimeric G-proteins, tyrosine kinases and calcium. Here we describe a fourth class, PLCepsilon, that in addition to conserved PLC domains, contains a GTP exchange factor (GRF CDC25) domain and two C-terminal Ras-binding (RA) domains, RA1 and RA2. The RA2 domain binds H-Ras in a GTP-dependent manner, comparable with the Ras-binding domain of Raf-1; however, the RA1 domain binds H-Ras with a low affinity in a GTP-independent manner. While G(alpha)q, Gbetagamma or, surprisingly, H-Ras do not activate recombinant purified protein in vitro, constitutively active Q61L H-Ras stimulates PLC(epsilon) co-expressed in COS-7 cells in parallel with Ras binding. Deletion of either the RA1 or RA2 domain inhibits this activation. Site-directed mutagenesis of the RA2 domain or Ras demonstrates a conserved Ras-effector interaction and a unique profile of activation by Ras effector domain mutants. These studies identify a novel fourth class of mammalian PLC that is directly regulated by Ras and links two critical signaling pathways.
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PMID:Phospholipase C(epsilon): a novel Ras effector. 1117 19

GHRH stimulates GH secretion from somatotroph cells of the anterior pituitary via a pathway that involves GHRH receptor activation of adenylyl cyclase and increased cAMP production. The actions of GHRH to release GH can be augmented by the synthetic GH secretagogues (GHS), which bind to a distinct G protein-coupled receptor to activate phospholipase C and increase production of the second messengers calcium and diacylglycerol. The stomach peptide ghrelin represents an endogenous ligand for the GHS receptor, which does not activate the cAMP signaling pathway. This study investigates the effects of GHS and ghrelin on GHRH-induced cAMP production in a homogenous population of cells expressing the cloned GHRH and GHS receptors. Each epitope-tagged receptor was shown to be appropriately expressed and to functionally couple to its respective second messenger pathway in this heterologous cell system. Although activation of the GHS receptor alone had no effect on cAMP production, coactivation of the GHS and GHRH receptors produced a cAMP response approximately twice that observed after activation of the GHRH receptor alone. This potentiated response is dose dependent with respect to both GHRH and GHS, is dependent on the expression of both receptors, and was observed with a variety of peptide and nonpeptide GHS compounds as well as with ghrelin-(1-5). Pharmacological inhibition of signaling molecules associated with GHS receptor activation, including G protein betagamma-subunits, phospholipase C, and protein kinase C, had no effect on GHS potentiation of GHRH-induced cAMP production. Importantly, the potentiation appears to be selective for the GHRH receptor. Treatment of cells with the pharmacological agent forskolin elevated cAMP levels, but these levels were not further increased by GHS receptor activation. Similarly, activation of two receptors homologous to the GHRH receptor, the vasoactive intestinal peptide and secretin receptors, increased cAMP levels, but these levels were not further increased by GHS receptor activation. Based on these findings, we speculate that direct interactions between the GHRH and GHS receptors may explain the observed effects on signal transduction.
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PMID:Ghrelin and growth hormone (GH) secretagogues potentiate GH-releasing hormone (GHRH)-induced cyclic adenosine 3',5'-monophosphate production in cells expressing transfected GHRH and GH secretagogue receptors. 1244 84

The effects of a GH secretagogue, L-692,585 (L-585), and human GH-releasing hormone (hGHRH) on calcium transient and GH release were investigated in isolated porcine pituitary cells using calcium imaging and the reverse hemolytic plaque assay (RHPA). Somatotropes were functionally identified by the application of hGHRH. All cells that responded to hGHRH responded to L-585 application. Perfusion application of 10 microM hGHRH and L-585 for 2 min resulted in an increase in intracellular calcium concentrations ([Ca(2+)](i)) of 53+/-1 nM (mean+/-S.E.M.) (P < 0.01) and 68+/-2 nM (P < 0.01) respectively. The L-585 response was characterized by an initial increase in [Ca(2+)](i) followed by a decline to a plateau level above the baseline. Concurrent calcium imaging with RHPA indicated that the L-585-evoked increase in [Ca(2+)](i) coincided with GH release. L-585 significantly increased the percentage of plaque-forming cells (24+/-3 vs 40+/-6%; P < 0.05) and mean area of plaques (1892+/-177 vs 3641+/-189 micro m(2); P < 0.01) indicating increased GH release. Substance P (SP) analogue ([d -Arg(1),d -Phe(5),d -Trp(7,11)]-SP) blocked, and the hGHRH receptor antagonist ((Phenylac-Tyr(1),d -Arg(2), p-chloro-Phe(6), Homoarg(9), Tyr (Me)(10), Abu(15), Nle(27),d -Arg(28), Homoarg(29))-GRF (1-29) amide) decreased the stimulatory effect of hGHRH. These failed to block the stimulatory effect of L-585, suggesting a different receptor for L-585 from the GHRH receptor. The hGHRH-induced calcium transients and initial peak increase induced by L-585 were significantly decreased by removal of calcium from the bathing medium or the addition of nifedipine, an L-calcium channel blocker. The plateau component of L-585-induced calcium change was abolished by removal of calcium and nifedipine. These results suggest an involvement of calcium channels in GH release. Either SQ-22536, an adenylate cyclase inhibitor, or U73122, a phospholipase C (PLC) inhibitor, blocked the stimulatory effects of hGHRH and L-585 on [Ca(2+)](i) transient, indicating the involvement of adenylate cyclase-cAMP and PLC-inositol triphosphate pathways. These results further suggested that calcium mobilization from internal stores during the first phase of the L-585 response induced an increase in [Ca(2+)](i) whereas calcium influx during the second phase is a consequence of somatotrope depolarization.
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PMID:Mechanism of action of the growth hormone secretagogue, L-692,585, on isolated porcine somatotropes. 1247 74

Ghrelin is a newly discovered peptide that binds the receptor for GH secretagogues (GHS-R). The presence of both ghrelin and GHS-Rs in the hypothalamic-pituitary system, together with the ability of ghrelin to increase GH release, suggests a hypophysiotropic role for this peptide. To ascertain the intracellular mechanisms mediating the action of ghrelin in somatotropes, we evaluated ghrelin-induced GH release from pig pituitary cells both under basal conditions and after specific blockade of key steps of cAMP-, inositol phosphate-, and Ca2+-dependent signaling routes. Ghrelin stimulated GH release at concentrations ranging from 10-10 to 10-6 m. Its effects were comparable with those exerted by GHRH or the GHS L-163,255. Combined treatment with ghrelin and GHRH or L-163,255 did not cause further increases in GH release, whereas somatostatin abolished the effect of ghrelin. Blockade of phospholipase C or protein kinase C inhibited ghrelin-induced GH secretion, suggesting a requisite role for this route in ghrelin action. Unexpectedly, inhibition of either adenylate cyclase or protein kinase A also suppressed ghrelin-induced GH release. In addition, ghrelin stimulated cAMP production and also had an additive effect with GHRH on cAMP accumulation. Ghrelin also increased free intracellular Ca2+ levels in somatotropes. Moreover, ghrelin-induced GH release was entirely dependent on extracellular Ca2+ influx through L-type voltage-sensitive channels. These results indicate that ghrelin exerts a direct stimulatory action on porcine GH release that is not additive with that of GHRH and requires the contribution of a multiple, complex set of interdependent intracellular signaling pathways.
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PMID:Intracellular signaling mechanisms mediating ghrelin-stimulated growth hormone release in somatotropes. 1296 33


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