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:3.2.1.31 (beta-glucuronidase)
7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Arachidonic acid metabolism in resident rat alveolar macrophages and in those activated with complete Freund's adjuvant (CFA) was studied. Adult Sprague-Dawley rats were injected with 0.05 ml CFA, and macrophages were harvested 10 days later. Macrophages were labeled overnight with carbon 14-labeled arachidonic acid, washed, and then stimulated with calcium ionophore A23187 (IoA), phorbol myristate acetate (PMA), or zymosan for 30 minutes. Prostaglandins, thromboxane, and leukotrienes were extracted from the medium and analyzed by radioimmunoassay or radio high-pressure liquid chromatography. Cell lipids were analyzed by radio thin-layer chromatography. Medium and cell beta-glucuronidase activity and protein kinase C activity of the membrane fraction were also assayed. We found (1) lower leukotriene B4 (LTB4) production in stimulated resident macrophages when compared with resident macrophages after IoA stimulation--the suppressed LTB4 production was reversed by PMA; (2) unchanged or higher LTB4 production in activated macrophages when compared with resident macrophages after zymosan stimulation; (3) inhibition of zymosan-stimulated LTB4 production by staurosporine, a protein kinase C inhibitor, in both groups; and (4) lower diacylglycerol (DAG) production in activated macrophages when compared with resident macrophages after IoA stimulation, but not after zymosan stimulation. These results suggest that the reduced response of activated macrophages to IoA is due to decreased production of an endogenous protein kinase C activator. This hypothesis was further supported by the observation that protein kinase C activation in response to IoA was lower in activated macrophages than in resident macrophages. In contrast, zymosan stimulation resulted in higher protein kinase C activation in activated macrophages when compared with resident cells. We hypothesize that protein kinase activation is necessary for leukotriene production and that the preserved ability of zymosan to activate PKC via DAG accounts for the high leukotriene production in zymosan-activated macrophages. We also found that stimulated thromboxane production was higher in activated than resident cells, regardless of the stimulus, and that thromboxane production was not affected by staurosporine. Thus alterations of eicosanoid metabolism in immunologically activated macrophages depend on the stimulus used and the type of eicosanoid examined. Furthermore, leukotriene biosynthesis in rat alveolar macrophages may be regulated by protein kinase C.
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PMID:Production of leukotrienes and thromboxane by resident and activated rat alveolar macrophages: a possible role of protein kinase C. 132 31

The specific inhibitor of protein kinase C, 1-O-alkyl-2-O-methylglycerol (AMG), was studied for its effect on bone resorption, measured as 45Ca-release, in fetal mouse calvariae. AMG (1 to 50 microM) had no effect on basal bone resorption. AMG inhibited parathyroid hormone (40 nM) induced bone resorption in a dose-dependent manner. Resorption induced by 1,25 (OH)2-vitamin D3 (10 nM) or prostaglandin E2 (5 microM) was also inhibited by AMG. The release of beta-glucuronidase activity paralleled the course of the 45Ca-release. The production of interleukin 6, induced by parathyroid hormone, in fetal rat calvarial osteoblasts was not affected by AMG. AMG (1 to 50 microM) had no cytotoxic effects on cells or calvariae. From these results it is concluded that protein kinase C may have an important role in the regulation of bone resorption.
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PMID:Role of protein kinase C (PKC) in bone resorption: effect of the specific PKC inhibitor 1-alkyl-2-methylglycerol. 159 Jul 94

Neutrophils from cystic fibrosis (CF) patients have been shown previously to be defective in their response (beta-glucuronidase exocytosis, NADPH oxidase activation) to the chemotactic peptide FMLP. In this work, we attempted to identify the defective step in this response. We showed that stimulated CF and control neutrophils do not differ in the formation of inositol phosphates. On the other hand, direct stimulation of protein kinase C with phorbol myristate acetate (PMA) revealed a subnormal stimulation of beta-glucuronidase exocytosis in CF neutrophils. Furthermore, retroinhibition exerted by PMA-activated protein kinase C on stimulated inositol phosphates or on beta-glucuronidase exocytosis was marginal or absent in CF neutrophils, whereas it was significant in the case of control neutrophils. Our observations suggest that the CFTR gene is expressed in neutrophils and is involved in protein kinase C-mediated actions.
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PMID:Defective protein kinase C-mediated actions in cystic fibrosis neutrophils. 189 35

We studied the effect of adenosine nucleotides on several aspects of the functional activation of human peripheral blood polymorphonuclear leukocytes (PMN). Radiolabeled ATP bound to PMN in a manner suggesting the existence of specific binding sites because: 1) binding was reversed (92 +/- 6%) by 100-fold excess concentrations of unlabeled ATP but minimally by either ADP (43 +/- 12%) or GTP (37 +/- 8%); and 2) binding saturation was achieved (i.e., specific binding did not increase) above 250 microM ATP. Binding studies revealed that significant ATP hydrolysis occurred, even at low temperatures and in the presence of phosphatase inhibitors. Adenosine nucleotides activated signal transduction mechanisms in PMN because: 1) 1 to 100 microM ATP and 5'-adenylylimidodiphosphate (AMP-PNP) stimulated increased production of 1,2-diacylglycerols; 2) ATP (0.5 to 500 microM) and ADP (0.1 to 10 mM) induced increased insoluble protein kinase (PKC) activity in a dose-dependent manner when used at concentrations greater than 50 microM; 3) ATP (greater than or equal to 50 microM) induced a shift in the solubility of phorbol receptors from mostly soluble (89% in untreated cells) to mostly insoluble (68%), whereas ADP, GTP, and GDP were effective at higher concentrations; and 4) greater than or equal to 50 microM ATP stimulated increased phosphorylation of endogenous PMN proteins. AMP-PNP induced PKC activity and phosphoprotein changes that were qualitatively similar to those observed when PMN were treated with ATP, suggesting that extracellular ATP hydrolysis is not required for signal transduction to activate PKC. Functionally, ATP stimulated the secretion of specific (but not azurophil) granules because vitamin B12-binding protein and low levels of lysozyme, but not beta-glucuronidase, were released; qualitatively similar results were obtained by using AMP-PNP. These results suggest that certain adenosine nucleotides employed at physiologically relevant concentrations stimulate increased 1,2-diacylglycerol production, PKC activity, granule secretion, and endogenous phosphoprotein formation in a manner that is independent of extracellular ATP hydrolysis.
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PMID:Extracellular adenosine nucleotides stimulate protein kinase C activity and human neutrophil activation. 215 72

Three protein kinase C (PKC) activators (PMA, mezerein, and a diacylglycerol) had bidirectional effects on human polymorphonuclear neutrophil (PMN) degranulation responses to leukotriene (LT) B4. Lower concentrations of the three agents enhanced, whereas higher concentrations inhibited, release of lysozyme and beta-glucuronidase stimulated by the arachidonic acid metabolite. Contrastingly, the activators inhibited but never enhanced LTB4-induced Ca2+ transients. We examined the causes for these varying effects. Each PKC activator reduced PMN specific binding of [3H]LTB4. Scatchard analyses revealed that PMA (greater than or equal to 0.16 nM) decreased the number of high affinity LTB4 receptors. The receptor losses correlated closely with inhibition of Ca2+ transients. PMN pretreated with 0.5 nM PMA for 5 min retained approximately 50% of their high affinity LTB4 receptors. These cells responded to 10 nM LTB4 with reduced but still substantial rises in cytosolic Ca2+, enhanced PKC mobilization, and increased granule enzyme release. The latter two effects appeared calcium-dependent because sequential exposure to PMA and LTB4 did not synergistically stimulate PKC mobilization or degranulation in PMN that were: 1) Ca2(+)-depleted; 2) challenged with 5 nM PMA; or 3) treated with LTB4 for 5 min before PMA. Each of the latter treatments completely interfered with the extent or timing of LTB4-induced Ca2+ transients. Accordingly, we suggest that the response-specific, bidirectional effects of PKC activators on LTB4 result from two opposing mechanisms. First, PKC activators down-regulate LTB4 high affinity receptors and thereby reduce those PMN responses that are not elicited by activated PKC (i.e., Ca2+ transients). Second, LTB4, by elevating cytosolic Ca2+, increases the amount of PKC mobilized by PKC activators and thereby promotes PKC-dependent responses (e.g., degranulation). The two mechanisms may be pertinent to the bidirectional effects of PKC activators on various other agonists. Furthermore, PKC, by down-regulating receptors, may serve as a physiologic stop signal for terminating function and producing a poststimulatory state of desensitization.
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PMID:Mechanisms involved in the bidirectional effects of protein kinase C activators on neutrophil responses to leukotriene B4. 215 69

Cocaine and its derivatives blunted responses of neutrophils (cell/cell aggregation, up-regulation of the receptor for C3bi (CR3, CD11b/CD18), generation of superoxide anion (O2-) and degranulation to various stimuli. The order of potency of these agents was the same as that for local anesthesia: tetracaine greater than bupivacaine greater than cocaine greater than lidocaine. Neutrophil aggregation elicited by the chemoattractant FMLP (10(-7) M) was inhibited by cocaine (10 mM) to 13.6 +/- 6% of control (p less than 0.002); the IC50 was approximately 4 mM. Cocaine and the other local anesthetics not only inhibited the upregulation of CR3 and O2- generation, but also blocked degranulation of cytochalasin B-treated cells. Cocaine (10 mM) reduced beta-glucuronidase and lysozyme secretion to 4.3 +/- 0.7 and 13 +/- 2.2% controls, respectively; its IC50 was 4 mM. Local anesthetics added after ligand/receptor engagement (FMLP) interrupted aggregation and halted generation of O2-. Moreover, local anesthetics rapidly inhibited aggregation, O2- generation, and degranulation elicited by PMA (1 microgram/ml) or the Ca ionophore A23187 (10 microM): the effects of cocaine could therefore not be attributed to unique actions at the FMLP receptor. Peak levels of intracellular Ca2+ ([Ca]i) at 5 to 10 s, and levels of [Ca]i 120 s after FMLP in Fura 2-loaded cells were significantly lower in cells treated with lidocaine, findings that could be explained by enhanced 45Ca2+ efflux from neutrophils. In cells loaded with bis(carboxyethyl)carboxyfluorescine (pH indicator) local anesthetics failed to affect the initial FMLP-induced (0 to 15 s) drop of pHi but inhibited the later (120 s) realkalinization of the cytosol (lidocaine, bupivacaine). Most remarkably, autoradiographs of SDS gels prepared from stimulated, 32P-labeled neutrophils treated with local anesthetics showed no difference from resting cells, either with respect to patterns of phosphorylation and dephosphorylation or their kinetics. Labeling of a 47-kDa protein, a component of the reduced nicotinamide-adenine dinucleotide phosphate-oxidase system, was unchanged. The effects of local anesthetics, which blunt neutrophil responses without affecting protein phosphorylation, suggest that protein phosphorylation is an insufficient signal for neutrophil activation. Inasmuch as cocaine and its derivatives affect cell functions at sites distal to activation of protein kinase C, these agents should prove useful in uncoupling protein phosphorylation from functional responses.
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PMID:Cocaine and its derivatives blunt neutrophil functions without influencing phosphorylation of a 47-kilodalton component of the reduced nicotinamide-adenine dinucleotide phosphate oxidase. 216 79

We have investigated the effect of the heat shock response on the leukotriene generation, chemotaxis, and generation of oxygen radicals of human polymorphonuclear granulocytes (PMNs) by preincubating the PMNs at 42 degrees C. Subsequently, the different test systems were performed at 37 degrees C. As we confirmed by the release of lactate dehydrogenase and beta-glucuronidase the elevated temperatures did not result in cytotoxic or degranulating processes. After heat shock treatment the generation of leukotrienes induced by the Ca(++)-ionophore A23187, fMLP or opsonized zymosan was inhibited in a time and temperature dependent manner (preincubation phase) as was measured by HPLC-analysis. In contrast, the conversion of 14C-arachidonic acid revealed the generation of LTB4, 5-HPETE and 5-HETE solely as a result of the preincubation at 42 degrees C without any further stimulation. In addition, the chemiluminescence response induced by opsonized zymosan and the chemotaxis against C5a and LTB4 was clearly inhibited after heat shock treatment. With regard to enzyme activities of the heat treated PMNs the protein kinase C activities were enhanced whereas the LTD4-dipeptidase and the LTB4-omega-hydroxylase were not affected.
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PMID:Heat shock induces alterations of the lipoxygenase pathway in human polymorphonuclear granulocytes. 251 49

Sphingoid long-chain bases (sphinganine and sphingosine) have recently been shown to inhibit protein kinase C both in vitro [Y. Hannun et al. (1986) J. Biol. Chem. 261, 12604-12609] and in intact human neutrophils, in which they block activation of the superoxide-generating respiratory burst [E. Wilson et al. (1986) J. Biol. Chem. 261, 12616-12623]. In the present study we have used sphingosine to investigate the pathways for agonist-induced secretion of neutrophil granule contents. Induction of secretion of the specific granule component lactoferrin by a variety of agonists [phorbol 12-myristate-13-acetate (PMA), formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore A23187] was completely inhibited by sphingosine with an ED50 of 6 to 10 microM. PMA-induced secretion of lysozyme (present in both the azurophilic and specific granules) was completely blocked with an ED50 of 10 microM, whereas fMLP-induced secretion was only about 50% inhibited. Secretion of the azurophilic granule proteins beta-glucuronidase and myeloperoxidase was activated by fMLP and A23187, but not by PMA, and was not affected by sphingosine. The use of A23187 in the presence of sphingosine allowed differentiation between calcium activation of protein kinase C-dependent versus-independent pathways. The effect of sphingosine was not mediated by neutralizing intracellular acidic compartments, since treatment of neutrophils with inhibitory concentrations of sphingosine did not significantly alter the uptake of labeled methylamine. We conclude that at least two mechanisms participate in the regulation of specific and azurophilic granule secretion, respectively: a protein kinase C-dependent pathway and a calcium-dependent pathway which does not involve protein kinase C.
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PMID:Protein kinase C inhibition by sphingoid long-chain bases: effects on secretion in human neutrophils. 282 97

Protein I, the major outer membrane protein of Neisseria gonorrhoeae, is a voltage-dependent anion channel which can translocate from the gonococcus into human cells. Since granule exocytosis from neutrophils is regulated by ion fluxes, we examined the effect of protein I on neutrophil activation. Pretreatment with protein I (250 nM) impaired degranulation from neutrophils: beta-glucuronidase release decreased to 27 +/- 6% S.E. of cells treated with N-f-Met-Leu-Phe (fMLP, 0.1 microM) and to 13 +/- 4% of cells treated with leukotriene B4 (LTB4, 0.1 microM); lysozyme release decreased to 52 +/- 17% of fMLP-treated cells and 22 +/- 9% of LTB4-treated cells. Morphometric analysis was consistent: control neutrophils increased their surface membrane after fMLP (43.3 +/- 5.6 microns relative perimeter versus 71.4 +/- 3.7 microns) while protein I-treated neutrophils did not (29.4 +/- 2 (S.E.) microns relative perimeter versus 34 +/- 4 microns). Enzyme release after exposure to phorbol myristate acetate was not affected (lysozyme: 86 +/- 27% of control). Cell/cell aggregation in response to fMLP was inhibited by treatment with protein I. However, generation of O2 was not affected. Protein I altered the surface membrane potential (Oxonol V): protein I evoked a transient membrane hyperpolarization which was not inhibited by furosemide. After exposure to fMLP, protein I-treated neutrophils underwent a furosemide-sensitive hyperpolarization rather than the usual depolarization. Protein I did not alter increments in [Ca]i (Fura-2) stimulated by fMLP (460 +/- 99 nM (S.E.) versus 377 +/- 44 nM) nor decrements in [pH]i (7.22 +/- 0.04 S.E. versus 7.22 +/- 0.02, bis-(carboxy-ethyl)carboxyfluorescein). The results suggest that degranulation and O2 generation have separate ionic requirements and that protein I interrupts the activation sequence proximal to activation of protein kinase C.
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PMID:Protein I, a translocatable ion channel from Neisseria gonorrhoeae, selectively inhibits exocytosis from human neutrophils without inhibiting O2- generation. 282 69

We have made mast cells and neutrophils permeable to gain access to the cytosol and thus to manipulate the composition of the cytosol. Secretion from both cell types can be triggered by elevation of cytosol Ca2+ to concentrations approaching 10-6 M; alternatively, secretion from mast cells, and of beta-glucuronidase (but not lysozyme) from neutrophils, can be triggered in the absence of Ca2+ by introducing stable analogs of GTP. We propose that GTP acts at two intracellular guanine nucleotide regulatory proteins (N proteins) in the stimulus-secretion sequence. By interaction with Np located on the inner face of the plasma membrane, it activates polyphosphoinositide phosphodiesterase to yield inositol phosphates and diacylglycerol. By interaction with Ne, situated distal to the site of action of Ca2+ and protein kinase C, it directly activates the exocytotic process without intervention of the products of polyphosphoinositide hydrolysis.
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PMID:Dual role for guanine nucleotides in stimulus-secretion coupling. 301 24


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