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)

Sphingosine inhibition of [3H] [N3-Me-His] TRH (MeTRH) binding, previously shown to be independent of its effects on protein kinase-C, has been further characterized in GH3 cell membranes and in a partially purified, digitonin-solubilized receptor preparation. In membranes, as in intact cells, sphingosine inhibited [3H]MeTRH binding by decreasing receptor affinity, but, in contrast to its effect in intact cells, did not affect the number of available binding sites. The inhibition of binding was linear up to 75 microM sphingosine (in the presence of 100 microM BSA at 0.1 mg membrane protein/ml), yielding an apparent Ki of 51 microM. Since GTP decreases the affinity for MeTRH binding in GH3 cell membranes, we studied interactions between GTP and sphingosine. While the effects of low concentrations of GTP gamma S and sphingosine were additive, sphingosine inhibition of MeTRH binding surpassed and was not affected by the addition of maximally inhibitory concentrations of GTP gamma S. Also, sphingosine (75 microM) did not affect the ability of a maximally effective dose of TRH to stimulate the low Km GTPase (vehicle, +35 +/- 5%; sphingosine, +32 +/- 10%); there was a 25% decrease in total GTPase activity in the presence of sphingosine. MeTRH binding to digitonin-solubilized receptors, which had properties similar to those described previously by others, including no effect of GTP on binding, was inhibited by sphingosine. In solubilized receptors, as in membranes, sphingosine caused a decrease in apparent affinity without changes in the number of binding sites. These data suggest that sphingosine interacts directly with the TRH receptor [or an associated factor(s) in the receptor complex] to decrease affinity by a mechanism that does not involve uncoupling of G-proteins.
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PMID:Sphingosine interacts directly with the receptor complex to inhibit thyrotropin-releasing hormone binding. 210 32

The experimental accessibility of monolayer culture has been used to study signal transduction mechanisms in primary CNS neurons and clonal pituitary cells. Here we review results on two signals representative of the emerging diversity of mechanisms discovered in all species studied thus far. One is mediated by micromolar concentrations of the amino acid GABA at postsynaptic membranes throughout the mammalian CNS and involves transient activation of Cl- ion channels whose distribution of conducting periods accounts for the millisecond time course of the signal. This signal serves to depress the probability that the target cell will trigger an action potential. The signal intensifies as the postsynaptic membrane is depolarized and can be modulated by clinically important drugs, primarily through changes in channel kinetics. The other signal involves nanomolar concentrations of the peptide TRH, which stimulates secretion of prolactin from clonal "GH3" pituitary cells. Intracellular recordings of GH3B6 cells show that TRH triggers a complex electrical response lasting several minutes. The response consists of Ca2+-activated K+ conductance followed by Ca2+-action potential activity. Whole-cell patch recordings, which rapidly dialyze the cell, can eliminate the TRH-induced changes in membrane excitability. Inclusion of aqueous lysates of the GH3B6 clone or the soluble second messenger factors inositol trisphosphate (IP3) or protein kinase (PKC) can restore various aspects of the change in membrane excitability. Thus, TRH alters ion conductance mechanisms through a second messenger cascade likely to involve IP3-mediated mobilization of Ca2+ from the endoplasmic reticulum and transient translocation of PKC from cytoplasm to plasma membrane. These synaptic and extrasynaptic signals reflect some of the diversity of transduction mechanisms involved in intercellular communication.
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PMID:Signal transduction mechanisms in cultured CNS neurons and clonal pituitary cells. 244 68

This report presents findings pertaining to the role of protein kinase-Cs in the release of PRL and liberation of arachidonate from PRL-secreting cells. In our experiments, protein kinase-C activators increased PRL release and arachidonate liberation from anterior pituitary cells and from the PRL-secreting cell line MMQ. In cells depleted of pituitary protein kinase-Cs by chronic exposure to protein kinase-C activators, such as phorbol dibutyrate or 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, TRH, angiotensin-II, and neurotensin each increased PRL release and [3H]arachidonate liberation in a normal manner. In addition, the PRL-releasing activities of protein kinase-C activators and those of TRH appeared to be synergistic, an unexpected effect if these substances were functioning through the same intracellular pathways. It, therefore, appears that phorbol diester-sensitive protein kinase-Cs may not be involved in the increased secretion of PRL or liberation of arachidonate that is caused by TRH, angiotensin-II, or neurotensin.
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PMID:Evidence that phorbol diester-sensitive protein kinase-C(s) may not be directly involved in secretagogue-stimulated prolactin release and arachidonate liberation. 250 62

The biochemical mechanisms underlying the direct stimulatory action of dopamine (DA) withdrawal on PRL release and on the potentiation of TRH stimulation are not known. These actions can be mimicked by pretreatment of lactotrophs with the protein kinase-C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate. Previous studies have shown that administration of TRH or withdrawal of DA stimulates polyphosphoinositide breakdown, although to different degrees. We have tested whether the acute withdrawal of DA activates PKC and determined if the prior removal of DA modifies the activation of PKC by TRH. Primary cultures of dispersed anterior pituitaries from estradiol-treated rats consisting of approximately 80% lactotrophs were maintained overnight in 500 nM DA. Activation of PKC was assayed immunochemically as translocation of PKC to a membrane fraction and by in situ phosphorylation of an acid-soluble heat-stable 80K substrate. Acute withdrawal of DA induced translocation of immunoreactive PKC to the membrane fraction (25-250%) and enhanced phosphorylation (40-100%) of an 80K protein. These effects were detected within 5-15 sec of DA withdrawal and were prolonged (10-30 min). TRH induced a rapid and transient activation of both parameters. The duration and magnitude of the action of TRH were increased by prior removal of DA. These results are consistent with a role for PKC activation in transduction of the stimulation of PRL release by the withdrawal of DA. The longer lasting activation of PKC may explain at least in part the potentiation of the PRL-releasing action of TRH by the withdrawal of DA.
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PMID:Dopamine withdrawal and addition of thyrotropin-releasing hormone stimulate membrane translocation of protein kinase-C and phosphorylation of an endogenous 80K substrate in enriched lactotrophs. 250 64

Although dopamine inhibits PRL release from the normal anterior pituitary lactotroph, a conclusive demonstration of the mechanisms involved in this response has been impeded by the presence of other cell types in the anterior pituitary. To circumvent this problem, we have isolated a clonal cell line, designated MMQ, from the 7315a rat pituitary tumor. The MMQ cell is an exemplary model for our use because it only secretes PRL. Our studies show that dopamine inhibits secretagogue-induced PRL release from these cells. In addition, dopamine decreases the intracellular cAMP concentration in MMQ cells that have been exposed to forskolin, cholera toxin, or vasoactive intestinal polypeptide, each a stimulator of cAMP generation. This inhibition is, in turn, reversed by the dopamine antagonist haloperidol and by pertussis toxin, an inactivator of the GTP-binding coupling protein. Dopamine also decreases the uptake and fractional efflux of 45Ca2+ by MMQ cells that have been exposed to the calcium channel activator maitotoxin. It seems, therefore, that dopamine decreases PRL release from MMQ cells at least in part by decreasing intracellular cAMP levels and calcium uptake. In additional experiments, we have found that MMQ cells are responsive to somatostatin, estrogen, progesterone, and acetylcholine, but not to TRH, angiotensin II, neurotensin, or bombesin. Furthermore, these cells possess a functional protein kinase-C system, as evidenced by the increase in PRL release and decrease in stimulated intracellular cAMP levels that occur in response to treatment with phorbol diesters. We suggest that the MMQ cell line will prove a useful model system for study of the biochemical effects of dopamine and other factors that modify PRL release.
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PMID:Characterization of the MMQ cell, a prolactin-secreting clonal cell line that is responsive to dopamine. 284 8

The interaction between two neuropeptides, VIP and TRH, was studied. The TRH receptor binding ability was examined using intact GH3 cells and its membrane fraction. The TRH binding ability decreased when intact cells were preincubated with VIP, forskolin or db-cAMP, but not when the membrane fraction was treated with these agents. The binding was reduced only when the membrane fraction was treated with catalytic subunit of cAMP dependent protein kinase (A-kinase). These results indicate that the binding ability of TRH receptors, which are linked to inositol phospholipid metabolism, is suppressed when A-kinase increases due to activation of the adenylate cyclase or its dependent system. This, in turn, suggests the presence of a communication via a cytoplasmic factor (probably A-kinase) between the two principal second messenger systems in the cell.
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PMID:Receptor-stimulated system mediated interactions of neuropeptides in GH3 cells. 285 60

TRH, epidermal growth factor (EGF), and 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulate PRL synthesis by GH4C1 rat pituitary cells. Recent evidence suggests that TPA activates directly phospholipid- and calcium-dependent protein kinase C in other cell types and that TRH might act analogously by altering phospholipid metabolism in GH4C1 cells. To examine the pathways by which these three agents stimulate PRL synthesis, we determined their calcium dependencies as well as their combined effects on PRL production. By equilibration of GH4C1 cells in a protein-free medium for 24 h, the free cytosolic calcium concentration ([Ca2+]i) was found to increase (from 90 to 360 nM) when the extracellular calcium concentration ([Ca2+]e) was varied from 15 to 800 microM. Basal PRL production increased in parallel (from 1 to 4 micrograms/ml X 24 h). TPA-stimulated PRL production was highly calcium dependent and required 180 nM [Ca2+]i for maximal enhancement. TRH-stimulated PRL production was constant between 10 and 660 microM [Ca2+]e, whereas EGF stimulated PRL production to a similar extent as TRH at 10 microM [Ca2+]e, but continued to enhance production with increasing [Ca2+]e. TRH elevated [Ca2+]i acutely, and at [Ca2+]e greater than 100 microM caused both a burst and a plateau phase in elevated [Ca2+]i. At lower [Ca2+]e, at which TRH still caused a maximal stimulation of PRL production, only the burst phase of [Ca2+]i occurred. When cultures were treated with any combination of maximally effective concentrations of TPA, TRH, or EGF, PRL production was increased by additive increments. The additive actions of TPA and TRH could not be explained by a calcium-promoted increase in TPA-stimulated PRL production. We conclude that TPA stimulates PRL production by a highly calcium-dependent pathway and that TRH and EGF stimulate PRL production by a different pathway(s) requiring lower [Ca2+]i.
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PMID:Thyrotropin-releasing hormone and epidermal growth factor stimulate prolactin synthesis by a pathway(s) that differs from that used by phorbol esters: dissociation of actions by calcium dependency and additivity. 393 Feb 23

It has been suggested that TRH stimulation of TSH release is mediated by the adenylate cyclase-cAMP system. To determine whether cAMP is a necessary intracellular messenger for TRH stimulation of TSH release, we have performed detailed studies of the TRH effect employing a nearly homogeneous population of mouse thyrotropic tumor cells in culture. Dibutyryl cAMP, methylisobutylxanthine, and cholera toxin caused an increase in TSH release which was additive to that of TRH. TRH stimulated TSH release in a dose-dependent fashion; half-maximal stimulation occurred at approximately 0.6 nM but had no effect on total intracellular cAMP levels measured in the presence or absence of methylisobutylxanthine. There was no correlation between total intracellular cAMP levels and TSH release after 1 h. Moreover, there was no effect of TRH on protein kinase-bound or total intracellular cAMP levels at 1, 5, or 60 min of incubation. Lastly, TRH had no effect on adenylate cyclase activity in homogenates of thyrotropic cells in the presence or absence of guanylylimidodiphosphate. These results suggest that stimulation of TRH release by TRH from these cells does not involve cAMP as an intracellular messenger.
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PMID:Thyrotropin-releasing hormone (TRH) action in mouse thyrotropic tumor cells in culture: evidence against a role for adenosine 3',5'-monophosphate as a mediator of TRH-stimulated thyrotropin release. 615 19

The effects and interactions of GnRH, TRH, a cAMP analog, a protein kinase-C (PKC) activator, a calcium ionophore, and a calcium channel blocker on pituitary glycoprotein hormone free alpha-subunit secretion and intracellular free alpha-subunit content were investigated. Treatment of dispersed rat pituitary cells with GnRH (100 nM) effected a time-dependent release of alpha-subunit, reaching a 4.5-fold increase (P < 0.05) at 24 h. Smaller effects were observed with TRH (10 nM). A rapid and progressive fall in intracellular alpha-subunit content was observed for 8 h after stimulation with GnRH (61% decrease; P < 0.05) or TRH (55% decrease; P < 0.05), which then remained constant at 24 h. The cAMP analogue 8-bromo-cAMP augmented a late release of alpha-subunit (4.5-fold increase at 24 h; P < 0.05) without affecting levels of alpha-subunit within the cells. Co-addition of 8-bromo-cAMP with GnRH or TRH arrested the marked fall in intracellular alpha-subunit seen with GnRH or TRH alone. These results suggest that although cAMP is capable of stimulating alpha-subunit secretion and maintaining cell content in the face of GnRH- and TRH-stimulated secretion, it does not mediate their effects on alpha-subunit. Like GnRH, the PKC activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) rapidly stimulated alpha-subunit secretion (1.7-fold increase at 4 h; P < 0.05) and progressively lowered cell content over 24h (73% decrease; P < 0.01). This similarity of action and the lack of demonstration of additive effects of TPA with GnRH or TRH imply a role for PKC as a mediator of GnRH and TRH action on alpha-subunit. Using verapamil (50 microM) to block L-type calcium channels had no effect on either basal or GnRH-stimulated alpha-secretion over 24 h. The calcium ionophore A23187 (3 microM) blocked the stimulatory effects of GnRH on alpha-subunit release and alone inhibited free alpha-subunit secretion (28% decrease at 24 h; P < 0.05). Our results suggest that neither cAMP nor an influx of extracellular calcium mediates the effects of GnRH or TRH on free alpha-subunit secretion. Accordingly, we postulate that PKC is involved in the actions of GnRH and TRH on alpha-subunit in rat pituitary cells, although further studies are required in PKC-depleted cells to confirm this hypothesis.
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PMID:Regulation of glycoprotein hormone free alpha-subunit secretion and intracellular alpha-subunit content in primary pituitary cells. 750 33

The D2 dopamine agonist, bromocriptine, has been used as treatment for human PRL-secreting pituitary adenomas. The result of bromocriptine treatment is often a substantial reduction of tumor mass, suggesting that the dopamine agonist is acting as an antiproliferative agent. This action can be observed with a clonal pituitary tumor cell line. The agonist activation of the D2 dopamine receptor inhibits the growth of GH4ZR7 cells, a GH4C1 cell line stably transfected with the cDNA encoding the short form of the D2 dopamine receptor. This effect of dopamine was not sensitive to overnight treatment with 100 ng/ml pertussis toxin. Treatment of GH4ZR7 cells with the phorbol ester 4 beta-phorbol 12,13-didecanoate resulted in the loss of dopaminergic inhibition of growth, whereas treatment with 4 alpha-phorbol 12,13-didecanoate had no effect. Inhibitors of protein kinase-C (PKC), such as staurosporine and H7, also blocked the effect of dopamine. Down-regulation of cellular PKC by phorbol ester treatment resulted in a complete loss of dopaminergic inhibition of growth. Long term treatment of GH4ZR7 cells with TRH results in a specific down-regulation of the epsilon form of PKC and abolished the ability of dopamine to inhibit growth. These results suggest that PKC epsilon is involved in mediating the antiproliferative effects of dopamine. This mediation of growth appears to be through a novel signaling pathway for the D2 dopamine receptor.
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PMID:The D2 dopamine receptor mediates inhibition of growth in GH4ZR7 cells: involvement of protein kinase-C epsilon. 750 37


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