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)

Experiments have been performed to characterize guinea-pig peritoneal eosinophil cyclic nucleotide phosphodiesterase (PDE) activity and establish whether it is involved in regulating superoxide (.O2-) generation. Eosinophils were found to contain a predominantly membrane-bound cAMP PDE(s) (92.5 +/- 2.4% of total activity) which was resistant to solubilization with Triton X-100 (1%). This particulate PDE exhibited complex kinetics (Km = 1.3 and 31.4 microM) and was unaffected by cGMP (IC50 greater than 100 microM) or CaCl2 (2 mM) + calmodulin (10 units/mL). Little cGMP PDE activity was detected in either the soluble or particulate fractions. Inhibitors of the Ro-20-1724-inhibited (Type IV) cAMP PDE, namely Ro-20-1724 (IC50 = 0.92 +/- 0.43 microM), rolipram (IC50 = 0.20 +/- 0.04 microM) and denbufylline (IC50 = 0.20 +/- 0.01 microM), potently inhibited the particulate cAMP PDE, as did the non-selective inhibitors trequinsin (IC50 = 0.11 +/- 0.02 microM) and AH-21-132 (IC50 = 2.57 +/- 0.02 microM). Eosinophil cAMP PDE was resistant to SK&F 94120 (IC50 greater than 1000 microM), the cGMP-inhibited (Type III) cAMP PDE inhibitor, and the cGMP PDE (Type I) inhibitor, zaprinast, was only weakly active (IC50 = 35.33 +/- 10.74 microM). .O2- release from resting cells was potently inhibited by rolipram (IC50 = 0.05 +/- 0.03 microM) and denbufylline (IC50 = 0.06 +/- 0.04 microM) but surprisingly, in view of its potent cAMP PDE inhibitory activity, was only weakly decreased by trequinsin (IC50 = 8.0 +/- 2.7 microM). AH-21-132 (IC50 greater than 10 microM), SK&F 94120 (IC50 greater than 10 microM) and zaprinast (IC50 greater than 10 microM) were without effect. Rolipram and denbufylline alone exerted little effect on cAMP in intact cells but, in the presence of 10 microM isoprenaline, potently increased intracellular accumulation (EC50 = 0.45 +/- 0.16 and 0.28 +/- 0.08 microM, respectively). Trequinsin and AH-21-132 only weakly enhanced isoprenaline-stimulated cAMP accumulation. Although it induced a marked rise in cAMP only in the presence of isoprenaline, rolipram (50 microM) alone was able to increase the activity ratio of cAMP-dependent protein kinase from 0.24 to 0.84. The results suggest that Ro-20-1724-inhibited cAMP PDE plays a role in regulating eosinophil .O2- generation. The poor correlation between the PDE inhibitory actions of certain compounds and their effectiveness in elevating cAMP and inhibiting .O2- suggests the existence of a barrier impeding access to the enzyme.
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PMID:Characterization of guinea-pig eosinophil phosphodiesterase activity. Assessment of its involvement in regulating superoxide generation. 165 Oct 83

1. The effect of rolipram (ME3176) on ADP- and IP3-induced repetitive IK(Ca) in rat megakaryocyte was investigated by use of the nystatin perforated patch and conventional whole-cell patch-clamp techniques. 2. The ADP-induced IK(Ca) was depressed by treatment with rolipram in a concentration-dependent manner. The inhibition by rolipram disappeared after treatment with a cyclic nucleotide-dependent protein kinase inhibitor, H-8. The inhibition of IK(Ca) was also observed in the presence of cyclic AMP accumulating agents such as forskolin and isobutylmethylxanthine (IBMX). 3. Rolipram enhanced the inhibitory action of forskolin, suggesting that rolipram facilitates the accumulation of cyclic AMP by blocking its breakdown. Similar results was obtained with adenosine, an endogenous adenylate cyclase activator. 4. Intracellular application of inositol trisphosphate (IP3) induced repetitive IK(Ca) in megakaryocytes. The induced IK(Ca) was also inhibited by rolipram and by other cyclic AMP accumulating agents. 5. It was concluded that megakaryocytes possess rolipram-sensitive phosphodiesterase (PDE), which was not detected in platelets, but plays a distinct modulatory role in megakaryocytes for generating ADP-induced IK(Ca).
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PMID:Existence of rolipram-sensitive phosphodiesterase in rat megakaryocyte. 769 62

In several cell types, the expression of the proenkephalin (PEnk) gene is enhanced after activation of protein kinase A. In the present study, astroglial cells cultured from rat cortex were used to investigate whether protein kinases A and C can act in a synergistic manner on the endogenous proenkephalin gene. The activator of protein kinase C tetradecanoylphorbolacetate (0.001-1 microM) increased the level of proenkephalin-mRNA in a concentration dependent manner. When used together with the phosphodiesterase inhibitor Rolipram (1 microM), the effect of tetradecanoylphorbolacetate (0.01 microM) was potentiated. 8-Bromoadenosine 3',5'-cyclic monophosphate (0.01-1 mM) also enhanced the expression of the proenkephalin gene. When used together with tetradecanoylphorbolacetate (0.01 and 0.1 microM), respectively, both agents had additive effects. Inhibition of protein synthesis with cycloheximide (35 microM) significantly changed the effects of both agents. While the effect of 8Br.cAMP (1 mM) on PEnk-mRNA was enhanced, that of tetradecanoylphorbolacetate (0.1 microM) was abolished. The results provide evidence for a synergistic effect of protein kinase A and C on the expression of the proenkephalin gene in astroglial cells. However, the protein kinases seem to act via different transcription factors on the expression of the proenkephalin gene.
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PMID:Interaction of protein kinases A and C in their effects on the proenkephalin gene in astroglial cells. 782 71

Several independent studies indicate that synthetic inhibitors of cyclic-3',5'-nucleotide phosphodiesterase (PDE) isozymes, especially inhibitors of PDE-IV, are potent agents which suppress generation of reactive oxygen metabolites (ROM) by NADPH oxidase in leukocytes. Recent studies also show that NADPH oxidase is present in all cell types populating glomeruli. In view of this, we investigated PDE isozymes and their relation to ROM in isolated rat glomeruli. Glomeruli have the capacity to hydrolyze cAMP by isozymes PDE-II, PDE-III and PDE-IV, whereas cGMP is hydrolyzed by PDE-I and PDE-V. Inhibitor of PDE-IV rolipram inhibited significantly (cca 40 to 50%) ROM generation in response to stimulation by phorbol myristate acetate (PMA). Inhibitor of PDE-III cilostamide had only minor suppressive effects and inhibitors of other PDE isozymes did not influence ROM generation. Rolipram (3 microM) suppressed ROM generation without detectable increase in cAMP content. Incubation of glomeruli with forskolin, which increased cAMP content in glomeruli tenfold, inhibited ROM generation to a similar degree as rolipram. The suppression of ROM generation by rolipram was prevented by Rp-cAMPS, a specific inhibitor of protein kinase A (PKA) activity. Further, incubation of glomeruli with rolipram elicited marked in situ activation of PKA (+ 100%), as documented by increase in the (-cAMP/+cAMP) PKA activity ratio. We suggest that selective inhibitor of PDE-IV rolipram acted via the cAMP-signaling pathway and suppressed ROM generation possibly via phosphorylating ras-type GTP-binding protein component of NADPH oxidase and thereby blocking assembly of functional NADPH oxidase complex.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Formation of reactive oxygen metabolites in glomeruli is suppressed by inhibition of cAMP phosphodiesterase isozyme type IV. 793 46

1. The possible role of cyclic AMP phosphodiesterase (PDE) in the inhibitory actions of ibudilast on tracheal smooth muscle contractility and eosinophil thromboxane generation was investigated. 2. Ibudilast was a non-selective inhibitor of partially purified cyclic nucleotide PDE isoenzymes from pig aorta and bovine tracheal smooth muscle, exhibiting only moderate potency against bovine tracheal PDE IV (IC50 = 12 +/- 4 microM, n = 3). Similar or slightly lower potencies were displayed against PDEs I, II, III and V. In contrast, rolipram exhibited selectivity for PDE IV (3 +/- 0.5 microM, n = 3). 3. Ibudilast (IC50 = 0.87 +/- 0.37 microM, n = 3), like rolipram (IC50 = 0.20 +/- 0.04 microM, n = 3), was a more potent inhibitor of membrane-bound PDE IV from guinea-pig eosinophils than of partially purified PDE IV from bovine tracheal smooth muscle. The potency of ibudilast increased when the eosinophil enzyme was solubilised with deoxycholate and NaCl (IC50 = 0.11 +/- 0.05 microM, n = 3) or exposed to vanadate/glutathione complex (V/GSH) (IC50 = 0.11 +/- 0.02 microM, n = 3). The potency of rolipram was also increased by solubilization (IC50 = 0.012 +/- 0.003, n = 3) or V/GSH (IC50 = 0.012 +/- 0.003, n = 3). 4. In intact eosinophils, ibudilast (0.032 microM-20 microM) potentiated isoprenaline-induced cyclic AMP accumulation in a concentration-dependent manner, being approximately 20 fold less potent than rolipram. Little or no effect on basal cyclic AMP levels was observed with either compound. The cyclicAMP-dependent protein kinase activity ratio was significantly increased following incubation of eosinophils with either ibudilast (20 MicroM) or rolipram (20 MicroM) in the absence or presence of isoprenaline.5. Leukotriene B4 (300 nM)-induced thromboxane generation from guinea-pig eosinophils was inhibited by ibudilast (IC50 = 11.3 +/- 3.7 MicroM, n = 5) and rolipram (IC50 = 0.280 +/- 0.067 MicroM, n = 5) in a concentration-dependent manner.6. Ibudilast (10 nM-1 MicroM), whilst generally less potent than rolipram (1 nM- 1 MicroM), produced concentration-dependent relaxation of spasmogen (methacholine, histamine, LTD4)-induced tone in the guinea pig isolated tracheal strip. Ibudilast was less potent in reversing the methacholine (IC50 = 1.95 +/- 0.40 JM,n =6)-induced contraction than those of histamine (IC50 = 0.18 +/- 0.70 MicroM, n =6) or leukotriene D4(LTD4, IC50 = 0.12 +/- 0.05 MicroM, n = 6). Rolipram also exhibited a similar pattern of activity, although the difference in potency against methacholine (IC50 = 0.1 +/- 0.01 MicroM, n = 6) compared with the other two spasmogens, histamine (IC50 = 0.034 +/- 0.017 MicroM, n = 7) and LTD4 (IC50 = 0.026 +/- 0.008 MicroM, n = 7), was not as great.7. These results demonstrate that ibudilast, like rolipram, has several biological actions on the eosinophil and airways smooth muscle which may be attributed to inhibition of cyclic AMP PDE. These actions may account, at least in part, for the recently reported anti-asthma effects of ibudilast.
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PMID:Possible role of cyclic AMP phosphodiesterases in the actions of ibudilast on eosinophil thromboxane generation and airways smooth muscle tone. 803 94

To elucidate the mechanism causing the transient accumulation of intracellular cAMP in the FRTL-5 thyroid cell line, the short-term effect of thyroid-stimulating hormone (TSH) on phosphodiesterase (PDE) activity was studied. Together with an increase in cAMP levels, TSH produced a significant increase in total PDE activity as early as 3 min, with a maximal stimulation reached after 15 min. This short-term increase in PDE activity was dependent on the TSH concentration (ED50 = 4 x 10(-11) M TSH). Forskolin and dibutyryl cAMP produced an even larger stimulation than that produced by TSH, suggesting that the effect of TSH is mediated by cAMP. To determine the properties of the PDE forms activated by TSH, antibodies specific for the cAMP-PDEs were used to immunoprecipitate the PDEs present in control cells, and cells incubated for 15 min in the presence of 10 nM TSH. Comparison of the activity recovered in the immunoprecipitation pellets demonstrated that TSH produced more than a 2.5-fold increase in the cAMP-PDE form(s) recognized by this antibody. Conversely, the activity remaining in the supernatants was not affected by the TSH treatment. Most of the activity recovered in the immunoprecipitation pellets (90%) was inhibited by 10 microM Rolipram, an inhibitor specific for the high affinity cAMP-PDEs. No TSH stimulation of the Rolipram-insensitive PDE activity could be observed under these conditions. Western blot analyses with two different cAMP-PDE specific antibodies showed that a 15-min stimulation with TSH induced the appearance of a new band with electrophoretic mobility slower than the polypeptide present in unstimulated cells. The appearance of this band did not require ongoing protein synthesis because it occurred in the presence of cycloheximide. Metabolic [32P]orthophosphate labeling of intact FRTL-5 cells indicated that the TSH treatment caused an increased 32P incorporation into a polypeptide that co-purified with the stimulated PDE activity and had an electrophoretic mobility identical to that of the cAMP-PDE. Okadaic acid, a potent inhibitor of protein phosphatase 1 and protein phosphatase 2A, elicited a potentiation of the TSH-stimulated PDE activity. The stimulating of a PDE with the same immunological properties and Rolipram sensitivity as the cAMP-PDE stimulated by TSH in the intact cells was reproduced, in a cell-free system, by incubating soluble extracts from FRTL-5 cells with the catalytic subunit of cAMP-dependent protein kinase. These data provide evidence that TSH produces a rapid activation of a cAMP-PDE in the FRTL-5 cells through a cAMP-dependent phosphorylation.
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PMID:The short-term activation of a rolipram-sensitive, cAMP-specific phosphodiesterase by thyroid-stimulating hormone in thyroid FRTL-5 cells is mediated by a cAMP-dependent phosphorylation. 813 62

We examined the sequential alterations in the binding of selective cyclic adenosine monophosphate (cAMP)-phosphodiesterase (PDE) and cAMP-dependent protein kinase (cAMP-DPK) in the gerbil brain following transient cerebral ischemia using in vitro quantitative autoradiography. [3H]Rolipram, a cAMP-PDE inhibitor, and [3H]cAMP were used to label cAMP-PDE and cAMP-DPK, respectively. Gerbils were subjected to 2-min or 6-min ischemia. Two-minute ischemia, which caused no morphological neuronal damage, produced no significant changes in either [3H]rolipram or [3H]cAMP binding throughout the recirculation period. The reduction of [3H]rolipram binding in the CA1 subfield of the hippocampus began 6 h after 6-min ischemia. Seventy percent of [3H]rolipram binding was preserved at 4 days, at which time almost all CA1 pyramidal cells had been destroyed. On the other hand, the reduction of [3H]cAMP-binding sites in the CA1 subfield began 1 day after 6-min ischemia. At 4 days, 47% of [3H]cAMP-binding sites in the CA1 subfield were preserved. Furthermore, we observed a transient reduction of [3H]cAMP binding in the dentate gyrus, which is resistant to ischemia, at 1 day and 4 days. These results indicate that marked alterations of cAMP-PDE and cAMP-DPK precede neuronal death in the hippocampal CA1 subfield, and the dentate gyrus also showed a transient alteration of cAMP-DPK.
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PMID:Sequential alterations of [3H]rolipram and [3H]cyclic adenosine monophosphate binding in the gerbil brain following transient cerebral ischemia. 838 73

Using [3H]MK-801, [3H]muscimol, [3H]cyclic AMP, and [3H]rolipram, we performed quantitative in vitro autoradiography to determine sequential alterations in the binding of N-methyl-D-aspartate and GABAA receptors, particulate cyclic AMP-dependent protein kinase, and cyclic AMP-selective phosphodiesterase, respectively, in the gerbil hippocampus following repeated brief ischemic insults. Changes from 1 h to 28 days after three 2-min ischemic insults at 1-h intervals were compared with those after 2 and 6 min of ischemia. We observed no alterations in the binding of all the four ligands throughout the observation period following 2 min of ischemia which produced no histological neuronal damage except for transient reductions in [3H]cyclic AMP binding during the early reperfusion period. [3H]Cyclic AMP binding in the CA1 subfield decreased one day after 6 min of ischemia and four days after three 2-min ischemic insults, and 62-65% of the binding was lost after 28 days. [3H]Rolipram binding in the CA1 subfield decreased one day after 6 min of ischemia and the binding was reduced by 45-50% after four and 28 days. Following three 2-min ischemic insults, [3H]rolipram binding decreased in the CA1 at one day, and decreased by 25-33% after 28 days. Both [3H]MK-801 and [3H]muscimol binding was preserved during the early reperfusion period after 6 min of ischemia and three 2-min ischemic insults. Reductions in [3H]MK-801 binding in CA1 were observed four days after ischemic insults when CA1 neuronal destruction was seen. After one month, approximately 50% of [3H]MK-801 binding was lost in CA1 in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Alterations in [3H]MK-801, [3H]muscimol, [3H]cyclic AMP, and [3H]rolipram binding in the gerbil hippocampus following repeated ischemic insults. 838 18

The effect of various phosphodiesterase (PDE) inhibitors on anti-CD3 induced interleukin-(IL)-4 and IL-5 production of the murine T helper cell clone of type 2 phenotype D10.G4.1 (D10) has been investigated in vitro. D10 cells were incubated in the presence of drugs and anti-CD3 mAb for 16 h before measurement of cytokines in the cell supernatants by ELISA. Whereas all PDE inhibitors tested exerted minimal effects on anti-CD3 induced IL-4 production, a marked increase in IL-5 production by the non-selective PDE inhibitors IBMX, theophylline and enprofylline was observed. The action of these non-selective PDE inhibitors was mimicked by the PDE IV-selective inhibitor rolipram and in part by the PDE III-selective inhibitors motapizone and milrinone, whereas the PDE V-selective inhibitor zaprinast was inactive. Rolipram and motapizone enhanced IL-5 production in a synergistic fashion. In support of the functional importance of PDE III and IV for IL-5 synthesis in intact murine D10 cells, we have found PDE III and IV to be the predominant isoenzyme activities in corresponding cell lysates. The stimulatory effect of rolipram on IL-5 production was almost totally reversed by the protein kinase A inhibitor KT-5720. In addition, the membrane-permeable cAMP analogue 8-bromo-cAMP mimicked the stimulatory effect of PDE inhibitors on IL-5 production while leaving IL-4 levels unaffected. Both results support the view that the action of the PDE inhibitors on murine D10 cells is mediated via an elevation of intracellular cAMP.
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PMID:Effect of phosphodiesterase inhibition on IL-4 and IL-5 production of the murine TH2-type T cell clone D10.G4.1. 855 18

Monocytes and macrophages produce tumor necrosis factor-alpha (TNF alpha) in response to microbial products including endotoxin. TNF alpha is a potent primer of neutrophil (PMN) oxidative activity. Certain xanthine phosphodiesterase (PDE) inhibitors such as pentoxifylline have been shown to inhibit stimulated oxidative activity in PMN. In the present study, the non-xanthine PDE type IV inhibitor rolipram (4-[3'-cyclopentyloxy-4'-methoxyphenyl]-2-pyrrolidone) alone and in combination with adenosine is examined as a potential modulator of TNF alpha-primed PMN oxidative activity. Attainable in vivo concentrations of rolipram and physiological concentrations of adenosine alone and together synergistically decreased rhTNF alpha-primed suspended PMN oxidative activity stimulated by the chemoattractant f-met-leu-phe. The rolipram effect was reversible by washing, and rolipram had a comparable effect if added before or after priming, indicating that its effect was on the primed response rather than on priming per se. In addition, rolipram especially when combined with adenosine, decreased rhTNF alpha-stimulated PMN adherence to a fibrinogen-coated surface, and the oxidative burst of rhTNF alpha-stimulated adherent PMN. The specific adenosine A2a receptor agonists CGS 21680 and WRC-0474 had comparable activity to adenosine in these experiments. Adenosine (or CGS 21680) combined with rolipram synergistically increased f-met-leu-phe-stimulated PMN cAMP content. The effects of both adenosine and rolipram with adenosine could be only partly counteracted by treatment of the PMN with the protein kinase A inhibitor KT 5720, indicating that protein phosphorylation is only partially involved. Rolipram activity was about 1000 x (by molar concentration) greater than pentoxifylline in comparable assays. Thus, rolipram, especially when combined with adenosine, has potent modulating effects on PMN activation and may be useful in decreasing inflammatory tissue damage in patients with sepsis.
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PMID:The specific type IV phosphodiesterase inhibitor rolipram combined with adenosine reduces tumor necrosis factor-alpha-primed neutrophil oxidative activity. 870 44


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