Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Histologic and clinical improvement of sun-exposed skin following topical treatment with retinoic acid has been reported. Daily application of retinoic acid typically results within 2-5 d in an erythematous scaling reaction, which lessens with continued usage. The cellular, immunologic, and biochemical basis of this retinoid reaction and its role in the repair of photodamaged skin are not known. To investigate the retinoid reaction in man, we have treated non-sun-exposed skin with 0.1% retinoic acid cream (Retin-A, Ortho Pharmaceutical Corporation, Raritan, NJ) under occlusion for 4 d to induce erythema and then examined changes in 1) histology, 2) expression of cell-surface molecules, 3) the enzymes and second messengers of the phospholipase C/protein kinase C signal-transduction system, 4) levels of eicosanoids, and 5) levels of interleukin-1 protein and mRNA. These parameters were chosen for measurement both because they are indicators of epidermal function and previous studies suggest they may be responsive to retinoic acid treatment. Epidermal cell growth as judged by increased epidermal thickness and mitotic figures was significantly increased in retinoic acid-treated skin compared to vehicle-treated controls. Increased numbers of CD4+ T cells accompanied by prominence of dermal dendrocytes in the papillary dermis and focal keratinocyte expression of intercellular adhesion molecule-1 were observed in retinoic acid-treated biopsies. Phosphoinositide-specific phospholipase C activity and 1,2-diacylglycerol content were also elevated in retinoic acid-treated epidermis. Protein kinase C activity was reduced by one third in both the soluble and membrane fraction, suggesting down-regulation. Surprisingly, in view of the inflammatory nature of the retinoid reaction, no increases were observed in arachidonic acid, its metabolites, interleukin-1 alpha, or interleukin-1 beta. To examine the specificity of the retinoid reaction, subjects were treated with the irritant sodium lauryl sulfate, under conditions that resulted in a reaction clinically similar to that observed with retinoic acid. The histologic alterations induced by sodium lauryl sulfate were found to be indistinguishable from those induced by retinoic acid. These data indicate that, although a wide range of cellular and molecular alterations occur in retinoic acid-treated skin, these changes may not be necessarily specific or unique for retinoic acid.
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PMID:Cellular, immunologic and biochemical characterization of topical retinoic acid-treated human skin. 167 98

Increased procoagulant activity of vascular endothelial cells may be an important component in the pathogenesis of intravascular coagulation associated with gram-negative bacterial diseases. Two bovine endothelial cell (BEC) lines isolated from pulmonary arteries (ENS-2 and ENT-18) were used in this study to investigate procoagulant signal transduction pathways of endotoxin (lipopolysaccharide, LPS)--stimulated BECs. The endothelial cell line ENS-2 was sensitive to LPS as demonstrated by tissue factor (TF) expression, but in contrast, the ENT-18 endothelial cell line was unusually resistant to the effects of LPS. No remarkable quantitative difference in binding of radiolabeled LPS was detected between the two endothelial cell lines. A protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate, PMA) failed to induce TF expression in either cell line at concentrations ranging from 0.05 to 1.00 microM when used as a sole stimulus for the endothelial cells. However, when PMA was used in combination with LPS, PMA enhanced the stimulatory effect of LPS on the endothelial cells. In parallel experiments, PKC inhibitors (H-7 and GF 109203X) interfered with the stimulatory effect of LPS on the cells by decreasing tissue factor expression. We also found that an activator of adenylate cyclase, forskolin, similarly inhibited LPS-induced tissue factor activity. In contrast, protein tyrosine kinase inhibitors (genistein, lavendustin A) had no inhibitory effect on LPS-induced endothelial cell tissue factor expression. Our results collectively suggest that activation of PKC is an important step in stimulation of endothelial cells by LPS, and that LPS and phorbol esters may synergize to produce an enhanced stimulatory effect. Our results also suggest participation of cAMP in controlling LPS-mediated stimulation of endothelial cells, but fail to demonstrate a role for protein tyrosine kinase activity.
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PMID:Signal transduction pathways of bacterial lipopolysaccharide-stimulated bovine vascular endothelial cells. 807 Sep 6

The reorganization of extracellular matrix (ECM) is an important function in many biological and pathophysiological processes. Culture of fibroblasts in a three-dimensional collagenous environment represents a suitable system to study the underlying mechanisms resulting from cell-ECM interaction, which leads to reprogramming of fibroblast biosynthetic capacity. The aim of this study was to identify receptors that transduce ECM signals into cellular events, resulting in reprogramming of connective tissue metabolism. Our data demonstrate that in human skin fibroblasts alpha 1 beta 1 and alpha 2 beta 1 integrins are the major receptors responsible for regulating ECM remodeling: alpha 1 beta 1 mediates the signals inducing downregulation of collagen gene expression, whereas the alpha 2 beta 1 integrin mediates induction of collagenase (MMP-1). Applying mAb directed against different integrin subunits resulted in triggering the heterodimeric receptors and enhancing the normal biochemical response to receptor ligation. Different signal transduction inhibitors were tested for their influence on gel contraction, expression of alpha 1(I) collagen and MMP-1 in fibroblasts within collagen gels. Ortho-vanadate and herbimycin A displayed no significant effect on any of these three processes. In contrast, genistein reduced lattice contraction, and completely inhibited induction of MMP-1, whereas type I collagen down-regulation was unaltered. Calphostin C inhibited only lattice contraction. Taken together, these data indicate a role of tyrosine-specific protein kinases in mediating gel contraction and induction of MMP-1, as well as an involvement of protein kinase C in the contraction process. The data presented here indicate that different signaling pathways exist leading to the three events discussed here, and that these pathways do not per se depend upon each other.
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PMID:Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins. 855 56

Using SK-N-SH cells, we observe that muscarinic acetylcholine receptor activation by methacholine (MCh) rapidly and selectively diminishes l-NE transport capacity (Vmax) with little or no change in norepinephrine (NE) Km and without apparent effects on membrane potential monitored directly under current clamp. Over the same time frame, MCh exposure reduces the density of [3H]nisoxetine binding sites (Bmax) in intact cells but not in total membrane fractions, consistent with a loss of transport capacity mediated by sequestration of transporters rather than changes in intrinsic transport activity or protein degradation. Similar changes in NE transport and [3H]nisoxetine binding capacity are observed after phorbol ester (beta-PMA) treatment. Inhibition of PKC by antagonists and downregulation of PKC by chronic treatment with phorbol esters abolishes beta-PMA-mediated effects but produce only a partial blockade of MCh-induced effects. Neither muscarinic acetylcholine receptor nor PKC activation require extracellular Ca++ to diminish NET activity. In contrast, treatment of cells with the Ca++/ATPase antagonist, thapsigargin in Ca++-free medium, eliminates the staurosporine-insensitive component of MCh regulation. These findings were further corroborated by the ability of [1, 2-bis(o-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester application in Ca++-free medium to abolish NET regulation by MCh. Although they may contribute to basal NET expression, we could not implicate CaMKII-, PKA- or nitric oxide-linked pathways in MCh regulation. Together, these findings 1) provide evidence in support of G-protein coupled receptor-mediated regulation of catecholamine transport, 2) reveal intracellular Ca++-sensitive, PKC-dependent and -independent pathways that serve to regulate NET expression and 3) indicate that the diminished capacity for NE transport evident after mAChR and PKC activation involves a redistribution of NET protein.
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PMID:Acute regulation of norepinephrine transport: I. protein kinase C-linked muscarinic receptors influence transport capacity and transporter density in SK-N-SH cells. 980 4

Norepinephrine (NE) transporters (NETs) found in the neuronal plasma membrane mediate the removal of NE from the extracellular space, limiting the activation of adrenoceptors at noradrenergic synapses. Our previous studies with the noradrenergic neuroblastoma SK-N-SH have revealed a muscarinic receptor-triggered regulation of NET surface density and transport capacity, mediated in part by protein kinase C activation. Low abundance of NET proteins in this native cell model, however, preclude direct confirmation of altered trafficking of NET proteins. In our study, we monitored the activity and surface distribution of human NET proteins in transient and stably-transfected cell lines after application of kinase activators and inhibitors. Using hNET stably transfected HEK-293 and LLC-PK1 cells, as well as transiently transfected COS-7 cells, we demonstrate that PKC-activating phorbol esters, beta-PMA or beta-PDBu selectively diminish l-NE transport capacity (Vmax) with little change in NE Km. Effects of phorbol esters are rapid, stereospecific and blocked by protein kinase C inhibitors, staurosporine and bisindolylmaleimide I. As in SK-N-SH cells, beta-PMA induces a reduction in intact cell [3H]nisoxetine binding sites with no change in nisoxetine Kd or total membrane NET density. Cell-surface biotinylation and confocal immunofluorescence techniques confirm that protein kinase C-dependent reductions in NE transport capacity and whole-cell antagonist binding density are accompanied by reductions in cell-surface human NET protein expression. Together these findings argue for kinase-modulated protein trafficking as a potential route for acute regulation of antidepressant-sensitive NE clearance.
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PMID:Acute regulation of norepinephrine transport: II. PKC-modulated surface expression of human norepinephrine transporter proteins. 980 5

Presynaptic transporter proteins regulate the clearance of extracellular biogenic amines after release and are important targets for multiple psychoactive agents, including amphetamines, cocaine, and antidepressant drugs. Recent studies reveal that dopamine (DA), norepinephrine (NE), and serotonin (5-HT) transporters (DAT, NET, and SERT, respectively) are rapidly regulated by direct or receptor-mediated activation of cellular kinases, particularly protein kinase C (PKC). With SERTs, PKC activation results in activity-dependent transporter phosphorylation and sequestration. Protein phosphatase 1/2A (PP1/PP2A) inhibitors, such as okadaic acid (OA) and calyculin A, also promote SERT phosphorylation and functional downregulation. How kinase, phosphatase, and transporter activities are linked mechanistically is unclear. In the present study, we found that okadaic acid-sensitive phosphatase activity is enriched in SERT immunoprecipitates from human SERT stably transfected cells. Moreover, blots of these immunoprecipitates reveal the presence of PP2A catalytic subunit (PP2Ac), findings replicated using brain preparations. Whole-cell treatments with okadaic acid or calyculin A diminished SERT/PP2Ac associations. Phorbol esters, which trigger SERT phosphorylation, also diminish SERT/PP2Ac associations, effects that can be blocked by PKC antagonists as well as the SERT substrate 5-HT. Similar transporter/PP2Ac complexes were also observed in coimmunoprecipitation studies with NETs and DATs. Our findings provide evidence for the existence of regulated heteromeric assemblies involving biogenic amine transporters and PP2A and suggest that the dynamic stability of these complexes may govern transporter phosphorylation and sequestration.
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PMID:Cocaine and antidepressant-sensitive biogenic amine transporters exist in regulated complexes with protein phosphatase 2A. 1102 16

Polychlorinated biphenyls (PCBs) are persistent, bioaccumulative, toxic, and widely distributed environmental chemicals. There is now both epidemiological and experimental evidence that PCBs cause cognitive deficits; however, the underlying cellular or molecular mechanism(s) is not known. We have hypothesized that altered signal transduction/second messenger homeostasis by PCBs may be associated with these effects since second messengers in signal transduction pathways, such as calcium, inositol phosphates (IP), and protein kinase C (PKC), play key roles in neuronal development and their function. In vitro studies using cerebellar granule neurons and isolated organelle preparations indicate that ortho-PCBs increase intracellular free Ca2+ levels by inhibiting microsomal and mitochondrial Ca2+ buffering and the Ca2+ extrusion process. Ortho-PCBs also increase agonist-stimulated IP accumulation and cause PKC translocation at low micromolar concentrations where no cytotoxicity is observed. On the other hand, non-ortho-PCBs are not effective in altering these events. Further SAR studies indicate that congeners with chlorine substitutions favoring non-coplanarity are active in vitro, while congeners favoring coplanarity are relatively inactive. Subsequent in vivo studies have shown that repeated exposure to a PCB mixture, Aroclor 1254, increases PKC translocation and decreases Ca2+ buffering in the brain, similar to in vitro studies. These changes in vivo are associated with elevated levels of non-coplanar ortho-PCB congeners at levels equivalent to 40-50 microM in brain, the concentrations that significantly inhibited second messenger systems in neuronal cultures in vitro. Current research is focusing on PCB-induced alterations in second messenger systems following developmental exposure.
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PMID:Neurochemical effects of environmental chemicals: in vitro and in vivo correlations on second messenger pathways. 1108 2

Presynaptic, cocaine- and antidepressant-sensitive norepinephrine (NE) transporters (NETs) dictate levels of extracellular NE after vesicular release. Recent studies suggest that G protein-coupled receptors linked to protein kinase C (PKC) down-regulate cell surface NET protein levels and diminish NE uptake capacity. We identified distinct phosphatidylinositol 3-OH kinase (PI3K)-linked pathways supporting basal and insulin-triggered NE transport in the human noradrenergic neuroblastoma, SK-N-SH. Acute (0-60 min) insulin treatments produced a time- and concentration-dependent stimulation of NE transport, resolved in kinetic studies as an enhancement of NE transport capacity (Vmax) without an alteration in NE Km. Basal and insulin-modulated NET activities were reduced by the tyrosine kinase inhibitor genistein and the PI3K inhibitors wortmannin and LY-294002, but not by the PKC inhibitor staurosporine. PI3K activation was found to support phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK). However, basal and insulin-stimulated NET activities were differentiated by their reliance on p38 MAPK activation. Thus, the p38 MAPK inhibitor SB203580 and SB202190 abolished insulin activation of NE transport yet failed to impact basal NET activity. Moreover, p38 MAPK activation and insulin activation of NETs were found to be sensitive to external Ca2+ depletion, blockade of voltage-sensitive Ca2+ channels, and inhibition of protein phosphatase 2A. Effects of tyrosine kinase and PI3K inhibitors on basal NET uptake appear to arise from a loss of cell surface NET protein, whereas the p38 MAPK-dependent enhancement of NE transport occurs without a detectable enhancement of surface NET. Our findings establish two distinct pathways for regulation of NE uptake involving PI3K, one linked to transporter trafficking and a second linked to Ca2+-dependent, p38 MAPK phosphorylation that promotes activation of cell surface NETs.
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PMID:Trafficking-dependent and -independent pathways of neurotransmitter transporter regulation differentially involving p38 mitogen-activated protein kinase revealed in studies of insulin modulation of norepinephrine transport in SK-N-SH cells. 1160 80

The muscarinic agonist carbachol stimulated phospholipase D (PLD) in rat submandibular gland (RSMG) ductal cells in a time and concentration-dependent manner. This effect was inhibited by chelation of extracellular calcium with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). PLD could also be activated by epinephrine and AlF(4)(-), two polyphosphoinositide-specific phospholipase C (PPI-PLC) activators, and by the phorbol ester o-tetradecanoylphorbol 13-acetate (TPA) which activates protein kinase C (PKC). Ionomycin and thapsigargin only slightly increased PLD activity. Ortho-vanadate, a tyrosine phosphatase inhibitor, also stimulated PLD activity. Both carbachol and o-vanadate increased the formation of inositol phosphates and the tyrosine phosphorylation of at least two proteins (55-60 and 120 kDa). Calphostin C (a PKC inhibitor), U73122 (a PPI-PLC inhibitor) and genistein (a tyrosine kinase inhibitor) blocked the activation of PLD, of PLC and the phosphorylation of tyrosyl residues in response to carbachol and vanadate. Taken together, these results suggest that rat submandibular gland ductal cells express a calcium-dependent PLD activity. This enzyme is regulated by carbachol via a PLC-PKC-tyrosine kinase pathway.
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PMID:Regulation of phospholipase D by muscarinic receptors in rat submandibular ductal cells. 1240 25

Cocaine, methylphenidate and other drugs that block dopamine transport indirectly promote immediate early gene expression, via dopamine-mediated activation of D1 dopamine receptors. Increased expression of the immediate early gene (IEG) c-fos, initiates a cascade of intracellular events that may underlie neuroadaptive changes following repeated exposure to the drugs. We investigated whether substrates (dopamine, norepinephrine) of the human dopamine (hDAT) and norepinephrine (hNET) transporters can directly induce c-Fos protein in HEK-293 (HEK) cells transfected with the hDAT and hNET and whether PKC modulators affect this process. Dopamine and norepinephrine robustly induced c-Fos immunofluorescence in both hDAT and hNET cells, but not in untransfected HEK-293 cells, demonstrating that catecholamine-induced c-Fos induction was DAT- and NET-dependent. The PKC activator PMA induced c-Fos in hDAT, hNET and HEK cell lines indicating that PKC stimulated c-Fos independent of transporters. The PKC inhibitor bisindolylmaleimide I (BIS) significantly increased c-Fos expression in hDAT cells, but not in hNET or HEK-293 cells, suggesting that inhibition of DAT-mediated phosphorylation results in c-Fos induction. BIS pretreatment abolished norepinephrine-induced c-Fos expression hNET but not dopamine-induced c-Fos induction in hDAT cells. In conclusion, induction of c-Fos by dopamine and norepinephrine requires the presence of hDAT and hNET but the contributions of hDAT and hNET to c-Fos induction is distinguishable on the basis of differing responses to a PKC inhibitor. These findings present a cell system and methodology for investigating the potential contribution of monoamine transporters to pre-synaptic neuroadaptation.
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PMID:Dopamine and norepinephrine transporter-dependent c-Fos production in vitro: relevance to neuroadaptation. 1576 38


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