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)

The purpose of this study was to purify and identify the proteinase-like substance previously recognized as responsible for the Na+/K(+)-ATPase stimulating property of plasma from insulin-dependent diabetic subjects. Anion-exchange chromatography followed by two-step heparin affinity chromatography resulted in a fraction highly enriched in both potent Na+/K(+)-ATPase stimulating activity and potent proteolytic activity. Approx. 400 micrograms of purified protein was isolated from 62 mg of starting plasma proteins. When analyzed on sodium dodecyl sulfate gels the active fraction consisted mainly of one polypeptide band with an apparent molecular mass of 66 kDa under either reducing or nonreducing conditions. The proteinase-like properties of the purified fraction were further revealed by its ability to clot plasma, split fibrinogen with production of fibrinopeptide A and induce shape change in human platelets and irreversible platelet aggregation in the presence of the stable analogue of endoperoxides U46619. Its additional capacity to affect platelet phosphoinositol metabolism was shown by the stimulation of protein kinase C-dependent phosphorylation of 47 kDa platelet membrane protein. In designing an identification protocol for the purified fraction, it was postulated that plasma proteinases are probably bound to their inhibitors, to form a stable covalently linked complex. The possibility that a proteinase-proteinase inhibitor complex was purified instead of single proteinase(s) was investigated. Neither trypsin nor neutrophil elastase were present in the active fraction whereas, among the possible plasma proteinase inhibitors tested, immunoreactivity was observed only in the presence of alpha 1-antitrypsin (alpha 1 AT) antiserum. Double immunodiffusion showed that control human alpha 1 AT and the plasma-purified fraction shared common antigens. Furthermore, both isoelectric focusing and amino acid composition analysis showed that the two substances were similar. The results obtained indicate that alpha 1 AT is apparently the only active component of the purified fraction from the plasma of insulin-dependent diabetics, thus suggesting that an altered form of the inhibitor is responsible for the broad range of proteinase-like effects elicited by the plasma-purified fraction.
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PMID:Purification of proteinase-like and Na+/K(+)-ATPase stimulating substance from plasma of insulin-dependent diabetics and its identification as alpha 1-antitrypsin. 131 11

Recruitment of inflammatory cells to the lung capillaries has been proposed as an important step in the sequence of events that lead to acute lung injury. Frequently, in the clinical setting, bacteremia and sepsis syndrome precede the acute lung failure and endotoxin priming may represent a comparable paradigm, useful for experimental pursuit. Following addition of the chemotactic tripeptide FMLP (10(-9) to 10(-6) M) to the cell-free, salt solution perfusate of isolated rat lungs, only a small degree of vasoconstriction was observed. However, in lungs isolated from rats that received 2 mg/kg intraperitoneal Salmonella enteritidis endotoxin 2 h before lung perfusion, FMLP dose dependently caused a large, transient pulmonary pressor response, edema formation, and release of large amounts of thromboxane and leukotriene B4. Since in vitro priming with endotoxin, direct vascular injury by neutrophil elastase, nor direct stimulation with FMLP of pulmonary artery rings from endotoxin-pretreated rats, mimicked the effects of in vivo endotoxin priming, we conclude that the presence of inflammatory cells in the lung capillaries accounted for the large amount of eicosanoids produced by the lungs after FMLP stimulation. In fact, by retrograde lavage of the lung circulation with a collagenase solution, previously adherent cell clumps were mobilized and identified. These cell clumps, composed of red blood cells, neutrophils, and platelets, were not seen in the vascular lavage sediment obtained from unprimed control lungs. Indomethacin, a thromboxane antagonist, AA861, a 5-lipoxygenase inhibitor, and WEB 2086, a platelet-activating factor (PAF) antagonist, reduced the thromboxane synthesis and release after FMLP (10(-7) M) in in vivo endotoxin-primed lungs. None of the inhibitors employed exclusively inhibited only one particular eicosanoid mediator but rather affected the release of several mediators, suggesting a close link between the different synthetic arachidonic acid pathways. An inhibitor of phospholipase C (2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate), NCDC, but not an inhibitor of phospholipase D (Wortmannin) or of protein kinase C (staurosporine) inhibited the FMLP-stimulated pulmonary pressure rise and eicosanoid release in endotoxin-primed lungs in vivo. Our data suggest that eicosanoids (in particular thromboxane) released from cells trapped in the lung circulation, but not from constitutive lung cells, contribute to vasoconstriction and edema formation caused by the chemoattractant FMLP in endotoxin-primed lungs.
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PMID:FMLP causes eicosanoid-dependent vasoconstriction and edema in lungs from endotoxin-primed rats. 154 53

The neutral proteinase elastase is released from polymorphonuclear (PMN) leukocytes in various physiological and pathological conditions. Aim of the present study was to gain further insight into the mechanisms which govern the liberation of this proteinase. Therefore, the effects of the calcium ionophore A23187 and of the protein kinase C activator phorbol myristate acetate (PMA) on neutrophils were investigated in human whole-blood samples. Furthermore, the inhibitory effects of the calcium channel blocker verapamil and of the calmodulin blocker trifluoperazine were followed. A23187 induced a release of elastase from neutrophils in a dose- and time-dependent manner. Complexation of extracellular calcium by ethylenediamine tetraacetate (EDTA) completely abolished the stimulatory effect of A23187. In a concentration of 10(-4) M verapamil was capable of attenuating (-49%) the A23187-induced secretion of PMN elastase. Besides the increase in intracellular calcium concentration, the activation of protein kinase C by PMA did also cause a release of neutrophil elastase. This release was strictly depending on the concentration of PMA and the time of incubation. In contrast to the stimulatory effect of A23187, the PMA-induced liberation of neutrophil elastase was attenuated, but not completely abolished, by complexation of extracellular calcium with EDTA. Both 10(-4) M verapamil (-43%) and 10(-5) M trifluoperazine (-42%) were able to reduce the PMA-induced release of neutrophil elastase. Based upon these data, we conclude that both the translocation of calcium intracellularly by A23187 and the activation of protein kinase C by PMA stimulate the release of neutrophil elastase. Verapamil and trifluoperazine were capable of suppressing the stimulation of elastase release.
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PMID:Stimulation and inhibition of elastase release from human neutrophil-dependence on the calcium messenger system. 311 99

The neutral proteinase elastase is released from polymorphonuclear (PMN) leukocytes in various physiological and pathological conditions. Aim of the present study was to gain further insight into the mechanisms which govern the liberation of this proteinase. Therefore, the effects of the calcium ionophore A23187 and of the protein kinase-C activator phorbol myristate acetate (PMA) on neutrophils were investigated in human whole-blood samples. Furthermore, the inhibitory effects of the calcium channel blocker verapamil and of the calmodulin blocker trifluoperazine were followed. A23187 induced a release of elastase from neutrophils in a dose- and time-dependent manner. Complexation of extracellular calcium by ethylenediamine tetraacetate (EDTA) completely abolished the stimulatory effect of A23187. In a concentration of 10(-4) M verapamil was capable to attenuate (-49%) the A23187-induced secretion of PMN elastase. Beside the increase in intracellular calcium concentration, the activation of protein kinase C by PMA did also cause a release of neutrophil elastase. This release was strictly depending on the concentration of PMA and the time of incubation. In contrast to the stimulatory effect of A23187, the PMA-induced liberation of neutrophil elastase was attenuated, but not completely abolished, by complexation of extracellular calcium with EDTA. Both 10(-4) M verapamil (-43%) and 10(-5) M trifluoperazine (-42%) were able to reduce the PMA-induced release of neutrophil elastase. Based upon these data, we conclude that both the translocation of calcium intracellularly by A23187 and the activation of protein kinase C by PMA stimulate the release of neutrophil elastase. Verapamil and trifluoperazine were capable to suppress the stimulation of elastase release.
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PMID:Stimulation and inhibition of elastase release from human neutrophils dependent on the calcium messenger system. 312 93

Neutrophil cathepsin G and thrombin, the only platelet agonists that are proteases, exhibit a mandatory requirement for catalytic activity to induce platelet aggregation and signal transduction. The thrombin receptor is a G-protein-coupled receptor which undergoes proteolysis to generate a tethered ligand that causes self-activation. Since cathepsin G strongly resembles thrombin in its ability to activate platelets, we have attempted to determine whether cathepsin G and thrombin function through the same or different receptors. Evidence that thrombin and cathepsin G act at different receptors was as follows: (a) an antibody directed against the thrombin receptor blocked thrombin-induced but not cathepsin G-induced platelet responses; (b) human fibroblasts responded to thrombin and to a synthetic thrombin receptor peptide (comprising residues 42-55 of the thrombin receptor) by exhibiting an elevation in cytosolic Ca2+ concentration but did not respond to cathepsin G; and (c) platelets pretreated with neutrophil elastase failed to respond to thrombin but responded when rechallenged by cathepsin G. Thrombin and cathepsin G exhibit heterologous desensitization that is potentiated by okadaic acid and is attenuated by staurosporine, indicating that phosphorylation of serine/threonine residues is important for desensitization and that protein kinase C may be involved. Since catalytic activity of cathepsin G is required for platelet stimulation, it is probable that platelet activation by cathepsin G requires receptor proteolysis and that a tethered ligand mechanism is involved, suggesting that platelets may possess a family of protease receptors.
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PMID:Cathepsin G and thrombin: evidence for two different platelet receptors. 829 30

Neutral serine proteinases such as mast cell chymase, cathepsin G, and neutrophil elastase are far more potent secretagogues for airway gland serous cells than all other agonists studied (e.g., histamine and bradykinin). To determine the mechanism of proteinase-induced secretion, we investigated the stimulation-secretion coupling in cultured bovine serous cells. Histamine stimulates degranulation of serous cells via adenosine 3', 5'-cyclic monophosphate-, protein kinase C-, and intracellular Ca2+ concentration ([Ca2+]i)-dependent pathways. Similarly, bradykinin-induced secretion involves inositol phosphates, protein kinase C, and [Ca2+]i. Degranulation caused by both agonists also depends on the activity of an endogenous metalloprotease, which is required in a late step of stimulation-secretion coupling, i.e., after Ca2+ entry. On the basis of the effect of different inhibitors, this metalloprotease is a Zn(2+)- and Ca(2+)-dependent enzyme similar to a gelatinase A synthesized by serous cells. In marked contrast to other secretagogues, degranulation induced by chymase, cathepsin G, and neutrophil elastase neither involves the classical second messengers nor the activity of the endogenous metalloprotease. These observations suggest that exogenous proteinases such as chymase, cathepsin G, and elastase may substitute for or mimic the action of an endogenous metalloprotease and directly activate degranulation, bypassing the signal transduction mechanisms necessary for secretion caused by other agonists.
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PMID:Classical second messengers are not involved in proteinase-induced degranulation of airway gland cells. 894 23

We have previously reported that the abnormally rapid down-regulation of protein kinase C (PKC) activity is responsible for the cellular dysfunction in natural killer (NK) cells and polymorphonuclear leukocytes (PMNs) from Chediak-Higashi syndrome (beige) mice. In this report, we examined whether the down-regulation of PKC is associated with giant granule formation in fibroblasts from beige mice. In cultured beige fibroblasts, the membrane-bound PKC activity declined significantly after phorbol ester stimulation. We found that E-64-d, which is a thiol proteinase inhibitor and protects PKC from calpain-mediated proteolysis, reversed the declined PKC activity and prevented giant granule formation in beige fibroblasts. Moreover, E-64-d corrected the reduced lysosomal elastase and cathepsin G activity in beige fibroblasts. In contrast, specific PKC inhibitors, chelerythrin and calphostin C, promoted giant granule formation in normal fibroblasts. We also demonstrate that ceramide production is enhanced in beige fibroblasts and is involved in the rapid down-regulation of PKC. These results suggest that the accelerated breakdown of PKC observed in beige fibroblasts is caused by enhanced ceramide production and is also responsible for giant granule formation.
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PMID:Abnormal down-regulation of PKC is responsible for giant granule formation in fibroblasts from CHS (beige) mice--a thiol proteinase inhibitor, E-64-d, prevents giant granule formation in beige fibroblasts. 1081 Oct 17

We have established that treatment of cultured human skin fibroblasts with tropoelastin or with heterogenic peptides, obtained after organo-alkaline or leukocyte elastase hydrolysis of insoluble elastin, induces a high expression of pro-collagenase-1 (pro-matrix metalloproteinase-1 (pro-MMP-1)). The identical effect was achieved after stimulation with a VGVAPG synthetic peptide, reflecting the elastin-derived domain known to bind to the 67-kDa elastin-binding protein. This clearly indicated involvement of this receptor in the described phenomenon. This notion was further reinforced by the fact that elastin peptides-dependent MMP-1 up-regulation has not been demonstrated in cultures preincubated with 1 mm lactose, which causes shedding of the elastin-binding protein and with pertussis toxin, which blocks the elastin-binding protein-dependent signaling pathway involving G protein, phospholipase C, and protein kinase C. Moreover, we demonstrated that diverse peptides maintaining GXXPG sequences can also induce similar cellular effects as a "principal" VGVAPG ligand of the elastin receptor. Results of our biophysical studies suggest that this peculiar consensus sequence stabilizes a type VIII beta-turn in several similar, but not identical, peptides that maintain a sufficient conformation to be recognized by the elastin receptor. We have also established that GXXPG elastin-derived peptides, in addition to pro-MMP-1, cause up-regulation of pro-matrix metalloproteinase-3 (pro-stromelysin 1). Furthermore, we found that the presence of plasmin in the culture medium activated these MMP proenzymes, leading to a consequent degradation of collagen substrate. Our results may be, therefore, relevant to pathobiology of inflammation, in which elastin-derived peptides bearing the GXXPG conformation (created after leukocyte-dependent proteolysis) bind to the elastin receptor of local fibroblasts and trigger signals leading to expression and activation of MMP-1 and MMP-3, which in turn exacerbate local connective tissue damage.
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PMID:Conformational dependence of collagenase (matrix metalloproteinase-1) up-regulation by elastin peptides in cultured fibroblasts. 1108 20

We have reported previously that the abnormally down-regulated protein kinase C (PKC) causes cellular dysfunction observed in natural killer (NK) cells, polymorphonuclear leucocytes (PMNs) and fibroblasts from beige mouse, an animal model of Chediak-Higashi syndrome (CHS). Here we show that the abnormal down-regulation of PKC activity also occurs in Epstein-Barr (EB) virus-transformed cell lines from CHS patients. When CHS cell lines were stimulated with concanavalin A (Con A) for 20 min, the membrane-bound PKC activity declined markedly, whereas that in control cell lines increased. We found that E-64-d, which protects PKC from calpain-mediated proteolysis, reversed the declined PKC activity and corrected the increased Con A cap formation to almost normal levels in CHS cell lines. We confirmed that the dysregulation of PKC activity also occurred in peripheral blood mononuclear leucocytes (PBMC) from CHS patients and that E-64-d corrected both the declined PKC activity and increased Con A cap formation. E-64-d also corrected the reduced lysosomal elastase and cathepsin G activity in CHS cell lines. In contrast, chelerythrin, a specific inhibitor of PKC, and C2-ceramide, which promotes PKC breakdown induced by calpain, increased Con A cap formation and inhibited both elastase and cathepsin G activity in normal cell lines. Moreover, we found that ceramide production in CHS cell lines increased significantly after Con A stimulation, which coincides with our previous observation in fibroblasts from CHS mice. These results suggest an association between ceramide-induced PKC down-regulation and the cellular dysfunctions in CHS.
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PMID:A thiol proteinase inhibitor, E-64-d, corrects the abnormalities in concanavalin A cap formation and the lysosomal enzyme activity in leucocytes from patients with Chediak-Higashi syndrome by reversing the down-regulated protein kinase C activity. 1152 21

Sepsis is a life-threatening event when it occurs in patients suffering from smoke inhalation injury. Pneumonia is one of the most frequent sources of infection in sepsis. Activated leukocytes likely play a role in the pathogenesis of sepsis. Cepharanthin is a biscoclaurine alkaloid that reportedly inhibits the activation of neutrophils. In this study, we investigated the effects of cephranthin on a post-smoke inhalation model of sepsis in sheep. Female sheep (n = 15) were surgically prepared for the study. After 5 days recovery from the operative procedures, tracheostomy was performed in all animals and 48 breaths of cotton smoke (<40 degrees C) were given via a modified bee smoker under halothane anesthesia. After smoke insufflation, Pseudomonas aeruginosa (5 x 109 cfu/kg) was instilled into the airway using a bronchoscope. All of the animals were mechanically ventilated with 100% O(2). Cepharanthin (1.3 mg/kg/h) was infused in five sheep continuously beginning 1 h after the insult and thereafter for the remainder of the 24-h study period. Control animals (n = 6) were treated with 5% dextrose as a vehicle control. Cepharanthin significantly attenuated changes in lung histology as well as in lung wet/dry weight ratio. An in vitro study revealed that cepharanthin inhibited the release of neutrophil elastase from isolated neutrophils stimulated with either formyl-methyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate with an IC(50) of 60 microM. Cepharanthin also inhibited the fMLP-induced increase in intracellular calcium levels of neutrophils. This result indicates cepharanthin inhibits protein kinase C or a more downstream signaling pathway in neutrophil activation. In conclusion, cepharanthin attenuates acute lung injury and septic shock after smoke inhalation in sheep.
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PMID:Cepharanthin, an alkaloid from Stephania cepharantha, inhibits increased pulmonary vascular permeability in an ovine model of sepsis. 1281 68


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