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)

We assessed the role of cyclic nucleotides in modulating lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-alpha) generation in human peripheral blood monocytes. Exposure of monocytes to LPS (3 ng/ml) evoked a delayed, time-dependent generation of TNF-alpha that reached a maximum level 5-6 hr after LPS challenge and remained constant for up to 24 hr. This effect was concentration dependent and resulted in a 20-40-fold increase in the release of TNF-alpha that was sensitive to actinomycin D and cycloheximide. Treatment of monocytes with agents reputed to activate the cAMP/cAMP-dependent protein kinase (PKA) cascade in general inhibited LPS-induced TNF-alpha generation. Thus, the beta 2-adrenoceptor agonists albuterol and procaterol partially (approximately 40%) suppressed TNF-alpha generation in a propranolol-sensitive manner. Furthermore, 8-bromo-cAMP, cholera toxin, prostaglandin E2, and a number of drugs (i.e., rolipram (ZK 62711), denbufylline (BRL 30892), Ro 20-1724, benafentrine (AH 21-132), that inhibit the phosphodiesterase (PDE) 4 isoenzyme family abolished cytokine generation. In contrast, forskolin, inhibitors of PDE3 and PDE5, and activators of soluble and particulate guanylyl cyclase were essentially inactive. Interestingly, rolipram failed to potentiate the inhibitory effect of albuterol on LPS-induced TNF-alpha biosynthesis but, paradoxically, synergized with albuterol in the generation of cAMP and in the activation of PKA. When PGE2 was used to activate adenylyl cyclase, however, rolipram potentiated cAMP accumulation, PKA activation, and inhibition of TNF-alpha generation. In contrast, forskolin did not increase the cAMP content of monocytes in the absence or presence of rolipram. Collectively, these data suggest that LPS-induced TNF-alpha generation by human peripheral blood monocytes is due to increased transcription and subsequent translation of the TNF-alpha gene and that these effects are suppressed by a range of agents that activate the cAMP/PKA cascade. However, the failure of rolipram to potentiate the inhibitory effect of albuterol and procaterol on TNF-alpha generation suggests that beta 2-adrenoceptor agonists may affect gene expression and/or post-transcriptional regulatory processes by, at least in part, a cAMP-independent mechanism(s).
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PMID:Suppression of lipopolysaccharide-induced tumor necrosis factor-alpha generation from human peripheral blood monocytes by inhibitors of phosphodiesterase 4: interaction with stimulants of adenylyl cyclase. 747 3

To help define essential interactions of cGMP with the catalytic site, we tested a series of cGMP analogs as competitive inhibitors of each cyclic nucleotide phosphodiesterase (PDE) family known to hydrolyze cGMP (PDE1, PDE2, PDE3, PDE5, and PDE6). IC50 values, relative to cGMP, were used to predict which functional groups of cGMP contribute to binding by the catalytic sites of each isozyme. The results indicate that the N1-nitrogen of cGMP contributes to binding at the catalytic site of all PDEs, probably as a hydrogen donor. All PDEs tested, with the exception of PDE2, also use the 6-oxo group, probably as a hydrogen acceptor. In contrast to other cGMP-binding enzymes, the 2-amino and 2'-hydroxyl groups of cGMP are not major requirements for binding to any PDE. The 8-bromo- and 8-p-chlorophenylthio-substituted analogs inhibit PDE1, PDE2, and PDE6 activity with high relative affinities, suggesting that these PDEs are not sterically hindered with bulky 8-position substitutions and that they do not preferentially bind the anti-conformation of cGMP. PDE3 and PDE5 have reduced apparent affinity for these analogs and therefore either are sterically hindered with these substitutions or bind cGMP in the anti-conformation. Overall, the data show substantial differences in structural requirements for cGMP binding to the catalytic sites of the different PDE families. Comparisons with published data show different structural requirements for binding to the catalytic, compared with noncatalytic, binding domains of PDEs. Even larger differences are seen between the requirements for binding to PDE catalytic sites and those for the cGMP-dependent protein kinase and the cGMP-gated cation channel.
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PMID:Characterization of cyclic nucleotide phosphodiesterases with cyclic GMP analogs: topology of the catalytic domains. 787 41

cGMP-binding phosphodiesterases contain two homologous allosteric cGMP-binding sites (sites a and b) that are arranged in tandem; they constitute a superfamily of mammalian cyclic nucleotide receptors distinct from the cyclic nucleotide-dependent protein kinases/cation channels family. The functional role of each of these two sites in the phosphodiesterases is not known. The cGMP-binding sites of one of these phosphodiesterases, the cGMP-binding cGMP-specific phosphodiesterase (cGB-PDE, PDE5), have been analysed by using site-directed mutagenesis. Mutations that affect cGMP binding to either one or both allosteric sites do not influence cGMP hydrolysis in the catalytic site under the conditions used. However, compared with wild-type enzyme, the D289A, D478A and D289A/D478A mutants, which are defective in cGMP binding to either site a or site b, or both allosteric sites, require much higher cGMP concentrations for the allosteric stimulation of phosphorylation by the catalytic subunit of cAMP-dependent protein kinase. The cGMP effect is on the cGB-PDE rather than on the catalytic subunit of the protein kinase because the latter enzyme does not require cGMP for activity. The D289N mutant, which has higher binding affinity for cGMP than does the wild-type enzyme, is phosphorylated at lower concentrations of cGMP than is the wild-type enzyme. It is concluded that cGMP binding to the allosteric sites of cGB-PDE does not directly affect catalysis, but binding to both of these sites regulates phosphorylation of this enzyme.
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PMID:Binding of cGMP to both allosteric sites of cGMP-binding cGMP-specific phosphodiesterase (PDE5) is required for its phosphorylation. 944 76

Guanosine 3',5'-cyclic monophosphate (cGMP)-binding, cGMP-specific phosphodiesterase (PDE5) is abundant in vascular smooth muscle, and this enzyme is a potent substrate for cGMP-dependent protein kinase (PKG) in vitro. Binding of cGMP to the allosteric sites of PDE5 is required for this phosphorylation to occur. Vascular smooth muscle cells (VSMC) were used to determine if PDE5 is phosphorylated in intact cells when cGMP is increased. With the use of anti-PDE5 antibodies, a phosphorylated 93-kDa protein band was immunoprecipitated from early passaged primary cultures of VSMC that had been preincubated with 32(Pi) to label cellular ATP and then treated with atrial natriuretic factor (ANF). In the absence of ANF, there was no detectable incorporation of radiolabeled phosphate into this band. Phosphorylation of the 93-kDa protein was augmented by pretreating cells with 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) to activate PKG before addition of ANF. 8-BrcGMP, which interacts poorly with the allosteric sites of PDE5, had no effect on PDE5 phosphorylation in the absence of ANF. Phosphorylation of PDE5 in response to treatment of cells with ANF was associated with a two- to fourfold increase in PDE activity in immunoprecipitates. Multiple-passaged VSMC, which are deficient in PKG but retain PDE5, demonstrated no ANF-dependent increase in phosphorylation or catalytic activity of PDE5. However, incubation of immunoprecipitated PDE5 from these cells with purified PKG, cGMP, and a phosphorylation mixture containing [gamma-32P]ATP resulted in 32(Pi) incorporation into PDE5 that was correlated with increased catalytic activity. These studies are the first to demonstrate phosphorylation of PDE5 in intact cells, thus suggesting a physiological role for this enzyme in smooth muscle regulation.
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PMID:ANF elicits phosphorylation of the cGMP phosphodiesterase in vascular smooth muscle cells. 948 47

1. CD19+ B lymphocytes were purified from the peripheral blood of normal and atopic subjects to analyse and compare the phosphodiesterase (PDE) activity profile, PDE mRNA expression and the importance of PDE activity for the regulation of B cell function. 2. The majority of cyclic AMP hydrolyzing activity of human B cells was cytosolic PDE4, followed by cytosolic PDE7-like activity; marginal PDE3 activity was found only in the particulate B cell fraction. PDE1, PDE2 and PDE5 activities were not detected. 3. By cDNA-PCR analysis mRNA of the PDE4 subtypes A, B (splice variant PDE4B2) and D were detected. In addition, a weak signal for PDE3A was found. 4. No differences in PDE activities or mRNA expression of PDE subtypes were found in B cells from either normal or atopic subjects. 5. Stimulation of B lymphocytes with the polyclonal stimulus lipopolysaccharide (LPS) induced a proliferative response in a time- and concentration-dependent manner, which was increased in the presence of interleukin-4 (IL-4). PDE4 inhibitors (rolipram, piclamilast) led to an increase in the cellular cyclic AMP concentration and to an augmentation of proliferation, whereas a PDE3 inhibitor (motapizone) was ineffective, which is in accordance with the PDE profile found. The proliferation enhancing effect of the PDE4 inhibitors was partly mimicked by the cyclic AMP analogues dibutyryl (db) cyclic AMP and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-cyclic monophosphorothioate, Sp-isomer (dcl-cBIMPS), respectively. However, at concentrations exceeding 100 microM db-cyclic AMP suppressed B lymphocyte proliferation, probably as a result of cytotoxicity. Prostaglandin E2 (PGE2, 1 microM) and forskolin (10 microM) did not affect B cell proliferation, even when given in combination with rolipram. 6. Inhibition of protein kinase A (PKA) by differentially acting selective inhibitors (KT 5720, Rp-8-Br-cyclic AMPS) decreased the proliferative response of control cells and reversed the proliferation enhancing effects of rolipram. 7. Importantly, PDE4 activity in LPS/IL-4-activated B lymphocytes decreased by about 50% compared to unstimulated control values. 8. We conclude that an increase in cyclic AMP, mediated by down-regulation of PDE4 activity, is involved in the stimulation of B cell proliferation in response to LPS/IL-4. B cell proliferation in response to a mitogenic stimulus can be further enhanced by pharmacological elevation of cyclic AMP.
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PMID:Phosphodiesterase profile of human B lymphocytes from normal and atopic donors and the effects of PDE inhibition on B cell proliferation. 955 83

1. The activation of neutrophils with particulate stimuli such as zymosan induces the generation of the C-X-C chemokine interleukin (IL)-8. There is evidence that neutrophil derived IL-8 plays an important role in human diseases such as the adult respiratory distress syndrome. In the present study, we examined the effects of cyclic AMP elevating agents on the ability of human neutrophils to generate IL-8 in response to zymosan particles. 2. The PDE4 inhibitor rolipram had limited effect on zymosan-induced IL-8 generation. In contrast, the PDE4 inhibitors RP 73401 and SB 207499 concentration-dependently suppressed IL-8 generation. The potency of these inhibitors was RP 73401 > SB 207499 > rolipram which is correlated with their rank order of potency at inhibiting the catalytic site of purified neutrophil PDE4. Pretreatment of neutrophils with the PDE3 inhibitor ORG 9935 or the PDE5 inhibitor zaprinast had no effect on IL-8 generation. 3. The prostanoids prostaglandin E1 (PGE1) and PGE2 inhibited zymosan-induced IL-8 release from neutrophils in a dose-dependent manner, in response to 10(-5) M PGE1 and PGE2 inhibiting IL-8 generation by 89% and 75%, respectively. Similarly, the beta2-adrenoceptor agonist salbutamol also inhibited IL-8 generation, but it was less effective than the prostanoids. 4. Significant synergism between prostanoids or salbutamol and the PDE4 inhibitors to inhibit IL-8 generation was observed. In contrast, there was no significant synergism between PGE2 and the PDE3 inhibitor ORG 9935 or the PDE5 inhibitor zaprinast. 5. In order to evaluate the potential role of protein kinase A in mediating the inhibitory effects of cyclic AMP-elevating agents, we used the protein kinase A inhibitors, H 89 and KT 5720. Pretreatment of neutrophils with these drugs completely reversed the inhibitory effects of a combination treatment with rolipram and PGE2 on zymosan-induced IL-8 release. 6. Microscopic examination revealed that most neutrophils contained one or more zymosan particles and that combination treatment with rolipram and PGE2 noticeably reduced the number of ingested particles. Moreover, there was a significant reduction in the percentage of neutrophils which ingested three or more zymosan particles. 7. Thus, our results demonstrate that cyclic AMP-elevating agents modulate the ability of neutrophils to generate IL-8 in response to a particulate stimulus. However, these agents also modulate the ability of neutrophils to phagocytose zymosan particles. Whether this effect will translate into inhibition of the ability of neutrophils to deal with infectious agents needs to be investigated further.
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PMID:Effect of PDE4 inhibitors on zymosan-induced IL-8 release from human neutrophils: synergism with prostanoids and salbutamol. 955 13

This study addressed the role of guanylyl cyclase (GC) and phosphodiesterase (PDE) in interleukin (IL)-1 activation of human articular chondrocytes. The GC inhibitors LY83583 and methylene blue dose-dependently inhibited IL-1-induced nitric oxide (NO) production, inducible NO synthase (iNOS) protein, and mRNA expression. These effects of GC inhibition were consistent with the rapid induction of cGMP by IL-1, which reached maximal levels after 5 min. The effects of GC inhibitors were selective as they did not reduce IL-1-induced cyclooxygenase II protein and mRNA. An inhibitor specific for soluble GC did not affect IL-1-induced NO production, and activators of soluble GC did not induce NO. However, the expression of iNOS mRNA was induced by atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP), activators of particulate GC, indicating that particulate rather than soluble guanylyl cyclases were involved in iNOS induction. The expression of iNOS mRNA and the production of NO were induced by a slowly hydrolyzable analog of cGMP, 8-bromo-cGMP, but not by nonhydrolyzable analog, dibutyryl cGMP, suggesting that PDE rather than cGMP-dependent protein kinase mediates the cGMP effects. Chondrocytes contained extensive cGMP PDE activity. This had PDE5 biochemical features and an inhibitor profile consistent with PDE5. Furthermore, the nonisoformspecific PDE inhibitor IBMX and PDE5-specific inhibitors suppressed IL-1-induced NO release and iNOS mRNA expression. PDE5 mRNA was constitutively expressed in chondrocytes. In addition to increasing PDE5 activities, IL-1 treatment reduced the sensitivity of PDE5 to several pharmacological inhibitors by up to 50-fold. In summary, inhibitors of either GC or PDE5 prevented IL-1 induction of iNOS; IL-1 increased the rates of both cGMP generation and hydrolysis; and exogenous PDE hydrolyzable cGMP analog induced iNOS and NO. These results suggest that increased cGMP metabolic flux is sufficient to induce iNOS, and GC and PDE5 activities are required for IL-1 induction of iNOS expression via increases in coupled cGMP synthesis and hydrolysis.
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PMID:Cyclic GMP and cGMP-binding phosphodiesterase are required for interleukin-1-induced nitric oxide synthesis in human articular chondrocytes. 976 78

Investigations of recent years revealed that isozymes of cyclic-3', 5'-nucleotide phosphodiesterase (PDE) are a critically important component of the cyclic-3',5'-adenosine monophosphate (cAMP) protein kinase A (PKA) signaling pathway. The superfamily of cyclic-3', 5'-phosphodiesterase (PDE) isozymes consists of at least nine gene families (types): PDE1 to PDE9. Some PDE families are very diverse and consist of several subtypes and numerous PDE isoform-splice variants. PDE isozymes differ in molecular structure, catalytic properties, intracellular regulation and location, and sensitivity to selective inhibitors, as well as differential expression in various cell types. A number of type-specific "second-generation" PDE inhibitors have been developed. Current evidence indicates that PDE isozymes play a role in several pathobiologic processes in kidney cells. In rat mesangial cells, PDE3 and PDE4 compartmentalize cAMP signaling to the PDE3-linked cAMP-PKA pathway that modulates mitogenesis and PDE4-linked cAMP-PKA pathway that modulates generation of reactive oxygen species. Administration of selective PDE isozyme inhibitors in vivo suppresses proteinuria and pathologic changes in experimental anti-Thy-1.1 mesangial proliferative glomerulonephritis in rats. Increased activity of PDE5 (and perhaps also PDE9) in glomeruli and in cells of collecting ducts in sodium-retaining states, such as nephrotic syndrome, accounts for renal resistance to atriopeptin; diminished ability to excrete sodium can be corrected by administration of the selective PDE5 inhibitor zaprinast. Anomalously high PDE4 activity in collecting ducts is a basis of unresponsiveness to vasopressin in mice with hereditary nephrogenic diabetes insipidus. Apparently, PDE isozymes apparently also play an important role in the pathogenesis of acute renal failure of different origins. Administration of PDE isozyme-selective inhibitors suppresses some components of immune responses to allograft transplant and improves preservation and survival of transplanted organ. PDE isozymes are a target for action of numerous novel selective PDE inhibitors, which are key components in the design of novel "signal transduction" pharmacotherapies of kidney diseases.
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PMID:Cyclic-3',5'-nucleotide phosphodiesterase isozymes in cell biology and pathophysiology of the kidney. 989 13

1. The objective of the present paper was to evaluate the relevance of neuronal balance of cyclic AMP and cyclic GMP concentration for functional regulation of nociceptor sensitivity during inflammation. 2. Injection of PGE2 (10-100 ng paw-1) evoked a dose-dependent hyperalgesic effect which was mediated via a cyclic AMP-activated protein kinase (PKA) inasmuch as hyperalgesia was blocked by the PKA inhibitor H89. 3. The PDE4 inhibitor rolipram and RP73401, but not PDE3 and PDE5 inhibitors potentiated the hyperalgesic effects of PGE2. The hyperalgesic effect of dopamine was also enhanced by rolipram. Moreover, rolipram significantly potentiated hyperalgesia induced by carrageenan, bradykinin, TNF alpha, IL-1 beta, IL-6 and IL-8. This suggests that neuronal cyclic AMP mediates the prostanoid and sympathetic components of mechanical hyperalgesia. Moreover, in the neuron cyclic AMP is mainly metabolized by PDE4. 4. To examine the role of the NO/cyclic GMP pathway in modulating mechanical hyperalgesia, we tested the effects of the soluble guanylate cyclase inhibitor, ODQ. This substance counteracts the inhibitory effects of the NO donor, SNAP, on the hyperalgesia induced by PGE2. 5. The ODQ potentiated hyperalgesia induced by carrageenan, bradykinin, TNF alpha, IL-1 beta, IL-6 and IL-8. In contrast, ODQ had no significant effect on the hyperalgesia induced by PGE2 and dopamine. This indicates that the hyperalgesic cytokines may activate soluble guanylate cyclase, which down-regulate the ability of these substances to cause hyperalgesia. This event appears not to be mediated by prostaglandin or dopamine. 6. In conclusion, the results presented in this paper confirm an association between (i) hyperalgesia and elevated levels of cyclic AMP as well as (ii) antinociception and elevated levels of cyclic GMP. The intracellular levels of cyclic AMP that enhance hyperalgesia are controlled by the PDE4 isoform and appear to result in activation of protein kinase A whereas the intracellular levels of cyclic GMP results from activation of a soluble guanylate cyclase.
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PMID:Pharmacological modulation of secondary mediator systems--cyclic AMP and cyclic GMP--on inflammatory hyperalgesia. 1040 57

Stable toxin (ST) peptides are the causative agents for a severe form of watery diarrhea. These peptides bind to a membrane-associated form of guanylyl cyclase, guanylyl cyclase C. The result is an accumulation of cyclic guanosine monophosphate (cGMP) in the intestinal cell, regulating protein kinase activity and the phosphorylation of a number of proteins involved in ion transport across the intestine. Using the human T84 colonic cell line as a model system, we show that cGMP accumulation in these cells after ST application is regulated by the activity of the cGMP-binding, cGMP-specific phosphodiesterase (PDE5). The presence of human PDE5 in this cell line was confirmed by Western blot analysis, using an antibody raised to the bovine enzyme, and by the observation that cGMP hydrolytic activity detected in T84 cell lysates was almost completely inhibited by low concentrations of zaprinast, a specific inhibitor of PDE5. An increase in activity of PDE5 was observed in T84 cell lysates on exposure to the ST peptide and prolonged exposure of T84 cells to the ST peptide led to the induction of cellular refractoriness in these cells, which was largely contributed in terms of an increased rate of degradation of cGMP in desensitized cells as a result of PDE5 activation. This activation was correlated with an increase in the affinity of the enzyme for the substrate cGMP, as well as an increased affinity for zaprinast. We provide evidence for the first time that cGMP levels in the human colonocyte are regulated by the cGMP-hydrolytic activity of PDE5 and suggest that the expression and regulation of PDE5 in the intestine could therefore be important in controlling cGMP-mediated signaling in this tissue.
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PMID:Expression and regulation of the cGMP-binding, cGMP-specific phosphodiesterase (PDE5) in human colonic epithelial cells: role in the induction of cellular refractoriness to the heat-stable enterotoxin peptide. 1067 26


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