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

Immunocytochemical localization of protein kinase C (PKC) in rabbit ciliary processes was investigated using anti-PKC monoclonal antibodies (MAbs) against rabbit Types 1, 2, and 3 PKC. Specific immunolabeling was observed in nonpigmented epithelial (NPE) cells and in the capillaries of the ciliary processes with anti-Types 2 and 3 MAbs. No apparent staining was seen with anti-Type 1 MAbs. Immunoelectron microscopy of Types 2 and 3 MAbs revealed a diffuse distribution of immunoreactive PKC in the cytoplasm, in the nucleus, and on the plasma membrane in the NPE cells. When incubated with phorbol 12-myristate 13-acetate (PMA), the distribution of PKC was basically similar to that of the untreated group. However, the labelling density on the plasma membrane at basolateral interdigitation increased considerably for anti-Types 2 and 3 PKC MAbs. In addition, the enzyme cytochemical activity of Na-K-ATPase (ouabain-sensitive K-NPPase) and its change after PMA administration in the ciliary processes were observed. An intense reaction was seen on the basolateral plasma membrane of the NPE cells. In the PMA-treated group, the enzyme activity of Na-K-ATPase apparently was decreased. These findings provide evidence that PKC plays a crucial role in the function of the NPE cells of the ciliary processes, possibly in aqueous humor production.
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PMID:Cytochemistry of protein kinase C and Na-K-ATPase in rabbit ciliary processes treated with phorbol ester. 133 Sep 69

Swelling activates and protein kinase C (PKC) downregulates Cl- channels in cultured nonpigmented ciliary epithelial (NPE) cells. We now report that the PKC inhibitor staurosporine upregulates whole cell Cl- currents isosmotically. The kinetics and current-voltage relationship are similar to those of volume-activated Cl- channels of these cells. These properties are inconsistent with cloned ClC-0, ClC-1, ClC-2, and MDR1 channels but could reflect the cystic fibrosis transmembrane conductance regulator (CFTR) channel or the Cl- channel regulator pICln. CFTR mRNA was undetectable by Northern analysis of cultured NPE cells or ciliary body tissue. In contrast, a human pICln probe obtained by polymerase chain reaction cloning and showing 90% identity with the rat cDNA clone detected high levels of transcripts in NPE cells. The level was low in tissue, where the NPE message was diluted by RNA from other cells. We conclude that NPE cells display staurosporine-activated Cl- channels [gSt(Cl)] likely identical with the volume-activated channels. The same cells expressing gSt(Cl) transcribe mRNA for a novel homologue (pHCBICln) of pICln that may regulate Cl- transport into the aqueous humor.
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PMID:PKC-sensitive Cl- channels associated with ciliary epithelial homologue of pICln. 753 80

The epithelium of the ciliary body is the site of aqueous humor secretion in the eye. We have begun to study ion transport in this tissue by investigating the mechanisms of K(+)-transport in fetal human pigmented ciliary epithelial (PE) cells using 86Rb+ as a tracer for K+. In PE three mechanisms were found: (1) ouabain-sensitive, bumetanide-insensitive 86Rb+ uptake (approximately 70% of total); (2) bumetanide-sensitive, ouabain-insensitive 86Rb+ uptake (approximately 15% of total); and (3) K+ channel blocker-sensitive 86Rb+ uptake (approximately 15% of total). Evidence that the ouabain-insensitive component of 86Rb+ uptake includes a Na+, K+, Cl- cotransporter is the following: (1) bumetanide inhibited 86Rb+ uptake with an IC50 of 0.6 microM and (2) bumetanide-sensitive 86Rb+ uptake was substantially reduced in media lacking Na+ or Cl-, suggesting that both extracellular Na+ and Cl- are required for optimal 86Rb+ uptake via this mechanism. Treatment of cells for 15 min with phorbol 12-myristate, 13-acetate (PMA) caused a dose-dependent inhibition of bumetanide-sensitive 86Rb+ uptake. No other 86Rb+ uptake mechanism was affected. Efflux studies revealed that efflux via the bumetanide-sensitive mechanism was likewise inhibited by PMA. Treatment with other phorbol esters or an analog of diacylglycerol inhibited bumetanide-sensitive 86Rb+ uptake, while the protein kinase C inhibitor staurosporine blocked inhibition by phorbol esters. These data suggest that a Na+, K+, Cl- cotransporter in human PE cells is inhibited activation of protein kinase C.
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PMID:Protein kinase C regulation of a Na+, K+, Cl- cotransporter in fetal human pigmented ciliary epithelial cells. 815 22

A cell line was derived from rabbit non-pigmented ciliary epithelium. The non-pigmented ciliary epithelium is one of the two cell layers which secrete aqueous humor into the eye and concentrate ascorbic acid in the newly-formed fluid. The cultured non-pigmented epithelial cells accumulated ascorbic acid at a rate of 3-5 pmol/micrograms protein per h. As in freshly-isolated native tissue, the ascorbate uptake mechanism was sodium-dependent and could be inhibited by phloretin (apparent Ki = 2-10(-5) M). Phorbol 12,13-dibutyrate (PDBu), a protein kinase C activator, reduced the ascorbate uptake rate. The PDBu effect was concentration-dependent; at a concentration of 10(-6) M, PDBu reduced the ascorbate uptake rate to 65% of the control value. PDBu reduced the maximal rate of ascorbate uptake (determined at 200-500 microM external ascorbate) but caused no detectable change in the Km for ascorbic acid (approx. 80 microM). The PDBu-induced inhibition of ascorbate uptake persisted in the presence of ouabain and in low sodium (25 mM Na) medium, suggesting that the effect is not secondary to a change in the sodium gradient. Furthermore, no detectable elevation of cell sodium content was seen in cells equilibrated with 22Na prior to PDBu treatment. The PDBu-induced inhibition of ascorbate uptake was apparently mediated by protein kinase C because the effect was not observed in the presence of staurosporine (10(-6) M), a protein kinase C inhibitor, or in cells in which protein kinase C was downregulated. These observations suggest that activation of protein kinase C causes inhibition of the ascorbate transporter in this cultured cell line.
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PMID:Studies on regulation of the ascorbic acid transporter in a cell line derived from rabbit non-pigmented ciliary epithelium. 839 16

Cells (ODM/SV40) derived from human non-pigmented ciliary epithelial cells were studied by electronic cell sizing. After transiently suspending the cells in hypotonic solution, isotonicity was restored by addition of sucrose. The cell volumes (vc) initially fell below those of control isotonic suspensions, and subsequently increased towards the baseline level. This secondary increase in vc is termed the regulatory volume increase (RVI). Results obtained with ionic substitutions and transport inhibitors indicate that four ionic mechanisms can support the RVI in these cells: coupled Na+/H+ and Cl-/HCO3- antiports, a Na+/Cl- symport, a Na+/K+/2Cl- symport, and a Na+ channel in parallel with a Cl-/HCO3- antiport. Arachidonic acid metabolites regulate the RVI very differently from their effects on the regulatory volume response (RVD) of the same cells to cell swelling. Prostaglandin E2 (PGE2), leukotriene (LTD4) and the PKC-inhibitor staurosporine all inhibit the RVI. Blockade of the cyclooxygenase, lipoxygenase and epoxygenase pathways of arachidonic acid metabolism [with 5,8,11,14-eicosatetraynoic acid (ETYA)] produces a net acceleration of the RVI. In contrast, PGE2 and staurosporine stimulate, LTD4 has no effect, and ETYA inhibits the RVD. We suggest that knowledge of the ionic mechanisms and intracellular signalling underlying the RVI phenomenon may provide a basis for reducing the rate of net aqueous humor formation by increasing the rate of reabsorption of fluid from the aqueous humor into the non-pigmented ciliary epithelial cells.
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PMID:Regulatory volume increase of human non-pigmented ciliary epithelial cells. 898 45

Complementary DNAs (cDNAs), corresponding to the human proteinases cathepsins D and O and proteinase inhibitors alpha2-macroglobulin and PP5/TFPI-2, have recently been isolated and identified from a subtractive human ciliary body library. In the present study we determined: (i) their pattern of expression in the human eye; (ii) the ability of the ciliary body and/or ciliary epithelial cells to synthesize and secrete cathepsin D and alpha1-antitrypsin in vitro; and (iii) whether alpha1-antitrypsin expression in cultured ciliary epithelial cells is modulated by protein kinase C activation. Northern analysis demonstrated that the ciliary body expresses high levels of cathepsins D and O, alpha2-macroglobulin, alpha1-antitrypsin and PP5/TFPI-2 transcripts. Western blot analysis and immunoprecipitation experiments with cathepsin D and alpha1-antitrypsin antibodies indicated that metabolically labeled ciliary body explants and/or ciliary epithelial cells in vitro with 35S-methionine, synthesize and secrete these proteins. Cultured nonpigmented ciliary epithelial ODM-2 cells, in response to phorbol-12-myristate 13-acetate (PMA), but not to the non-protein kinase C binding phorbol ester 4 alpha-phorbol didecanoate (PDBu), elicited up-regulation (up to 5-fold) of transcription, synthesis and secretion of alpha1-antitrypsin. These results provide in vitro evidence that the ciliary epithelium synthesizes and secretes a selective group of proteinases and proteinase inhibitors detected also in aqueous humor. The expression of at least of one of the proteinase inhibitors, alpha1-antitrypsin, can be modulated in response to phorbol ester.
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PMID:Gene expression of proteases and protease inhibitors in the human ciliary epithelium and ODM-2 cells. 926 97

The molecular mechanisms for regulating water balance in many tissues are unknown. Like the kidney, the eye contains multiple water channel proteins (aquaporins) that transport water through membranes, including two (AQP1 and AQP4) in the ciliary body, the site of aqueous humor production. However, because humans with defective AQP1 are phenotypically normal and because the ocular application of phorbol esters reduce intraocular pressure, we postulated that the water channel activity of AQP4 may be regulated by these agents. We now report that protein kinase C activators, phorbol 12,13-dibutyrate, and phorbol 12-myristate 13-acetate strongly stimulate the phosphorylation of AQP4 and inhibit its activity in a dose-dependent manner. Phorbol 12,13-dibutyrate (10 microM) and phorbol 12-myristate 13-acetate (10 nM) reduced the rate of AQP4-expressing oocyte swelling by 87 and 92%, respectively. Further, phorbol 12,13-dibutyrate significantly increased the amount of phosphorylated AQP4. These results demonstrate that protein kinase C can regulate the activity of AQP4 through a mechanism involving protein phosphorylation. Moreover, they suggest important potential roles for AQP4 in several clinical disorders involving rapid water transport such as glaucoma, brain edema, and swelling of premature infant lungs.
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PMID:Regulation of aquaporin-4 water channels by phorbol ester-dependent protein phosphorylation. 949 12

Endothelins (ET) are potent vasoactive peptides present in many ocular structures and are formed from precursor Big endothelins (Big ET-1) by the action of an endothelin-converting enzyme (ECE). ET-1 is thought to decrease intraocular pressure by contracting the ciliary muscle thus enhancing the outflow of aqueous humor through the Canal of Schlemm and trabecular meshwork. However, the mechanisms involved in the regulation of endothelin-1 (ET-1) synthesis and release in ocular tissues have not been fully characterized. In this study we examined the effect of tumor necrosis factor-alpha(TNF-alpha; 10 nm), a proinflammatory cytokine, on the cellular mechanisms leading to ET-1 synthesis and release in SV-40 transformed human ciliary non-pigmented epithelial cells (HNPE). ET-1 and Big endothelin-1 (Big ET-1) immunoreactivity was time-dependently increased following TNF-alphatreatment. Phorbol esters (PMA), activators of PKC, also raised the immunoreactive levels of ET-1 and Big ET-1 while, staurosporine, a PKC inhibitor (20 nm), decreased ET-1 levels in TNF-alpha-stimulated cells. Pre-treatment with phosphoramidon (1 micron) an ECE-inhibitor, followed by TNF-alpha stimulation, decreased ir-ET-1 levels. Cycloheximide (9 micron), a protein synthesis inhibitor, decreased TNF-alpha-stimulated levels for ir-ET-1 and ir-Big ET-1, suggesting that TNF-alpha may be directly regulating ET-1 expression at the ET-1 gene. Our data indicates that TNF-alpha regulates ET-1 levels in HNPE cells possibly by activating PKC either to stimulate protein synthesis and/or to enhance ET-1 secretion. These results suggest that ET-1 released from the ciliary body may play an important role in aqueous humor dynamics following cytokine activation.
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PMID:Regulation of endothelin-1 in human non-pigmented ciliary epithelial cells by tumor necrosis factor-alpha. 953 26

Atrial natriuretic peptide (ANP) stimulates aqueous humor formation in primates, but the membrane-bound receptors which mediate this effect have not been well studied in the eye. Endocytosis of [125I]ANP bound to natriuretic peptide C receptors was characterized in fetal human nonpigmented ciliary epithelial (NPE) cells. [125I]ANP which bound to cells at 4 degreesC was detected in the cell interior after a temperature shift to 37 degreesC. Appearance of ligand within the cell peaked at 5 min, and then declined towards zero over 20 min. The endocytosis inhibitor phenylarsine oxide blocked the appearance of internalized ligand, whereas the lysosomotropic drug chloroquine had no effect on internalization but blocked subsequent loss of internalized ligand. Chloroquine also blocked the accumulation of degraded ligand in the extracellular medium. Treatment with phorbol 12-myristate, 13-acetate accelerated the loss of internalized ligand from cells and increased the accumulation of ligand in the extracellular medium. Ligand in the medium was also increased by dioctanoylglycerol but not by 4alpha phorbol didecanoate, an isomer which does not activate protein kinase C. The protein kinase inhibitors staurosporine and bisindolylymaleimide blocked the increase in ligand. Phorbol ester-stimulated loss of internalized ligand occurred in the presence of chloroquine. TCA precipitation of ligand in the extracellular medium showed that both degraded and undegraded [125I]ANP were present. However, in the presence of chloroquine only, undegraded ANP was detected in the medium, and phorbol esters stimulated its rate of appearance by approximately 2 fold. A similar stimulation occurred when cells containing internalized ligand, but stripped of membrane-bound ligand, were exposed to phorbol esters. The data suggest that ANP bound to natriuretic peptide C receptors on NPE cells is endocytosed, and that protein kinase C activates a non-lysosomal pathway for ANP retroendocytosis in these cells.
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PMID:Non-lysosomal cycling pathway for atrial natriuretic peptide activated by protein kinase C in human NPE cells. 987 17

Current models of aqueous humor outflow no longer treat trabecular meshwork (TM) as an inert tissue passively distended by the ciliary muscle (CM). Instead, ample evidence supports the theory that trabecular meshwork possess smooth muscle-like properties and is actively involved in the regulation of aqueous humor outflow and intraocular pressure. In this model, trabecular meshwork and ciliary muscle appear as functional antagonists, with ciliary muscle contraction leading to a distension of trabecular meshwork with subsequent reduction in outflow. and with trabecular meshwork contraction leading to the opposite effect. Smooth-muscle relaxing substances would therefore appear to be ideal candidates for glaucoma therapy with the dual goal of reducing intraocular pressure via the trabecular meshwork and of improving vascular perfusion of the optic nerve head. However, for such substances to effectively lower intraocular pressure, the effect on the ciliary muscle would have to he minimal. For this reason, more information is needed on the signalling processes involved in regulating trabecular meshwork and ciliary muscle contractility. This review attempts to outline current knowledge of signal transduction pathways leading to relaxation and contraction of ciliary muscle and trabecular meshwork. Pathways can be classified as involving or not involving changes of membrane voltage and of requiring or not requiring external calcium: possibly, other pathways exist. These different pathways involve different ion channels and isoforms of PKC and are expressed to a differing degree in ciliary muscle and trabecular meshwork, leading to differential responses when exposed to relaxing or contracting pharmacological agents. Some of these agents. like tyrosine kinase inhibitors and inhibitors of PKC. have been shown to relax trabecular meshwork while leaving ciliary muscle comparatively unaffected. This profile makes these substances appear as ideal drugs for simultaneously improving ocular outflow and retinal circulation, parameters that determine the time course of visual deterioration in glaucoma.
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PMID:The regulation of trabecular meshwork and ciliary muscle contractility. 1074 78


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