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:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The subtype of muscarinic receptor which mediates cAMP attenuation is not established. Therefore, several selective muscarinic antagonists were used to characterize the subtype of muscarinic receptor coupled to the inhibition of hormone-stimulated cAMP accumulation using NG108-15 neuroblastoma x glioma hybrid cells. These cells were prelabeled with [2-3H]-adenine, washed, and resuspended in a culture medium containing the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM). The labeled cells were preincubated with the different antagonists 12-15 min. before they were challenged with agonists. The formation of [3H]-cAMP was activated by PGE1 (1 microM) or forskolin (1 microM). In all cases, [3H]-cAMP formed was separated and measured. Carbachol (100 microM) and McN-A343 (10 mM) were used as standard muscarinic agonists. These studies gave the following results: a) McN-A343 (10 mM), an M1 receptor agonist, was only a partial agonist causing 40% inhibition of cAMP accumulation indicating that this effect was not mediated by an M1 receptor; b) The M1-selective antagonist, pirenzepine, exhibited low affinity (pA2 6.2) further suggesting that an M1 receptor was not coupled to the attenuation of cAMP accumulation; c) Two selective M2 antagonists (AF-DX 116 and methoctramine) and M3 antagonist (HHSiD) were used to further characterize these muscarinic receptors. The order of all antagonists based on their affinities (pA2 values) could be arranged in the following order: atropine (9.0) > methoctramine (7.6) > HHSiD (6.9) > AF-DX 116 (6.6) > pirenzepine (6.2). HHSiD exhibits the same degree of affinity to M2 receptors of other tissues as it does to those of NG cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Subtype of muscarinic receptor coupled to the attenuation of hormone-stimulated cAMP accumulation in NG108-15 neuroblastoma x glioma hybrid cells. 128 46

1. The pharmacological profile of the inhibitory 5-hydroxytryptamine (5-HT) receptor in rat oesophageal smooth muscle has been characterized by means of a series of agonists active at 5-HT1-, 5-HT2-, 5-HT3- and 5-HT4-receptor sites, and a broad range of antagonists. The possible involvement of cyclic nucleotides in the 5-HT response was also examined. 2. Under conditions of tone induced by muscarinic receptor activation, the upper two-thirds (proximal segment) of the oesophageal smooth muscle tunic was more sensitive to the inhibitory effects of 5-HT receptor agonists when compared with the distal region. 3. The inhibitory response to 5-HT was blocked by MDL 72222 (5-HT3 antagonist) and ICS 205-930 (5-HT3/5-HT4 antagonist) but not by antagonists active at 5-HT1- or 5-HT2-receptors. 4. The phosphodiesterase inhibitor, 3-isobutyl-methyl-xanthine (IBMX) enhanced oesophageal smooth muscle inhibitory response to 5-HT, isoprenaline and forskolin, but not that elicited by the potassium channel opener, BRL 34915. 5. 5-HT increased tissue cyclic AMP content over basal levels in proximal and distal segments of oesophageal smooth muscle. However, 5-HT had no significant effect on basal cyclic GMP levels in both segments. 6. We conclude that the inhibitory 5-HT receptor in rat oesophageal smooth muscle may represent a high affinity subtype which is sensitive to 5-HT3/5-HT4 antagonists and is coupled to the cyclic AMP pathway.
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PMID:Pharmacological profile of the 5-hydroxytryptamine receptor that mediates relaxation of rat oesophageal smooth muscle. 132 46

1. [3H]-adenosine 3':5'-cyclic monophosphate ([3H]-cyclic AMP) responses were studied in primary cultures of human tracheal smooth muscle cells derived from explants of human trachealis muscle and in short term cultures of acutely dissociated trachealis cells. 2. Isoprenaline induced concentration-dependent [3H]-cyclic AMP formation with an EC50 of 0.2 microM. The response to 10 microM isoprenaline reached a maximum after 5-10 min stimulation and remained stable for periods of up to 1 h. After 10 min stimulation, 1 microM isoprenaline produced a 9.5 fold increase over basal [3H]-cyclic AMP levels. The response to isoprenaline was inhibited by ICI 118551 (10 nM), (apparent KA 1.9 x 10(9) M-1) indicating the probable involvement of a beta 2-adrenoceptor in this response in human cultured tracheal smooth muscle cells. However, with 50 nM ICI 118551 there was a reduction in the maximum response to isoprenaline. Prostaglandin E2 also produced concentration-dependent [3H]-cyclic AMP formation (EC50 0.7 microM, response to 1 microM PGE2 6.4 fold over basal). 3. Forskolin (1 nM - 100 microM) induced concentration-dependent [3H]-cyclic AMP formation in these cells. A 1.6 fold (over basal) response was also observed following stimulation with NaF (10 mM). 4. The nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (0.1 mM) and the type IV, cyclic AMP selective, phosphodiesterase inhibitor rolipram (0.1 mM) both elevated basal [3H]-cyclic AMP levels by 1.8 and 1.5 fold respectively. IBMX (1-100 microM) and low concentrations of rolipram (< 10 microM), also potentiated the response to 1 microM isoprenaline. Inhibitors of the type III phosphodiesterase isoenzyme (SK&F 94120 and SK&F 94836) were without effect upon basal or isoprenaline-stimulated cyclic AMP responses in these cells.5. Carbachol (1 nM-I 00 microM) produced concentration-dependent inhibition of the [3H]-cyclic AMP response to 1 microM isoprenaline in human cultured tracheal smooth muscle cells (IC50 0.24 JM). Carbachol(1 JM) inhibited the [3H]-cyclic AMP response to 1 JM isoprenaline by 60%. This effect of carbachol was itself inhibited by atropine (50 nM) (KA 2.3 x 109 M-') indicating the involvement of a muscarinic receptor.6. These results show that primary cultures of human tracheal smooth muscle cells demonstrate cyclic AMP responses to direct receptor stimulation, adenylyl cyclase activation and inhibition with nonselective and type IV-selective cyclic AMP phosphodiesterase isoenzyme inhibitors, and that the cyclic AMP response to isoprenaline can be inhibited by muscarinic receptor stimulation.
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PMID:Control of cyclic AMP levels in primary cultures of human tracheal smooth muscle cells. 138 13

Calmodulin (CaM) mediates the Ca(2+)-dependent activation of many enzyme systems in accordance with its cellular localization. We have described previously a muscarinic receptor-mediated translocation of CaM from membranes into the cytosol of SK-N-SH human neuroblastoma cells. To explore the potential targets (CaM-binding proteins, CaMBP) for CaM upon translocation, a photoreactive CaM derivative was introduced into living SK-N-SH cells using a scrape-loading technique. Scrape-loading incorporated rhodamine isothiocyanate-labeled CaM with an efficiency of 38%. CaM-diazopyruvamide (CaM-DAP), a Ca(2+)-dependent and CaM-specific probe, was also introduced into the cells. The muscarinic agonist carbachol stimulated a translocation of CaM from membranes into cytosol in CaM-DAP-loaded SK-N-SH cells. Upon photochemical cross-linking, cross-linked adducts of CaM-CaMBP were detected by immunoblotting with anti-CaM antibody. Carbachol stimulated increased photoaffinity labeling of three proteins with relative adduct molecular masses of 70, 120, and 180 kDa. The time course of labeling for the 70- and 120-kDa adducts showed maximal increased by 15-30 min. The 180-kDa adduct displayed a slower time course of maximal labeling, with increases maintained for 2-4 h. Subtracting the molecular mass of CaM, carbachol stimulated binding to CaMBPs of 55, 105, and 163 kDa. Predominant cellular CaMBP were identified using a biotinylated CaM overlay procedure. Western blot analysis indicated the expression of specific CaM-dependent enzymes such as calcineurin, phosphodiesterase, the beta-isoform (rat brain) of CaM kinase II, and Ca(2+)-ATPase. Numerous cytoskeletal CaMBP were expressed such as microtubule-associated protein-2, spectrin, tubulin, caldesmon, adducin, and neuromodulin. Of the CaMBP expressed, phosphodiesterase, calcineurin, caldesmon, and adducin cross-linked with CaM-DAP in the loaded SK-N-SH cells. Carbachol stimulated the time-dependent CaM-DAP labeling of calcineurin and adducin. This study demonstrates the novel incorporation of a photoreactive CaM derivative into living cells, as well as muscarinic receptor-activated CaM-DAP interaction with several cellular CaMBP. We postulate that carbachol-stimulated CaM translocation in SK-N-SH cells may affect the activity of CaM-dependent enzymes and may alter aspects of cytoskeletal function.
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PMID:Carbachol stimulates binding of a photoreactive calmodulin derivative to calmodulin-binding proteins in intact SK-N-SH human neuroblastoma cells. 155 1

Cyclic AMP regulation by muscarinic and adenosine receptors was investigated in isolated canine ventricular myocytes. Both the muscarinic receptor agonist, carbachol, and the adenosine receptor agonist, phenylisopropyladenosine, decreased isoproterenol-stimulated cyclic AMP accumulation in a concentration-dependent manner. Carbachol was more potent than phenylisopropyladenosine and had a greater inhibitory effect. At 10(-6) M, carbachol reduced isoproterenol-stimulated cyclic AMP by 73 +/- 5% while 10(-3) M phenylisopropyladenosine was required to decrease cyclic AMP accumulation by 54 +/- 8%. Pretreatment of myocytes with pertussis toxin to inactivate the inhibitory guanine nucleotide binding protein, Gi, completely abolished the effect of phenylisopropyladenosine to reduce cyclic AMP stimulation. In comparison, pertussis toxin treatment blunted the response to carbachol and shifted the dose-effect curve to the right but did not eliminate the inhibitory action of carbachol. In toxin-treated myocytes, 10(-3) M carbachol produced a 26 +/- 6% reduction of isoproterenol-induced cyclic AMP accumulation. This pertussis toxin-insensitive action of carbachol was antagonized by atropine and pirenzepine and was prevented when either of two different phosphodiesterase inhibitors. RO-20-1724 or isobutylmethylxanthine, was included in the incubation medium. The results indicate that adenosine receptor-mediated inhibition of hormone-stimulated cyclic AMP accumulation in ventricular myocytes occurs by a single, Gi-dependent mechanism while muscarinic inhibition appears to involve both Gi-dependent and Gi-independent mechanisms. The Gi-independent mechanism may reflect enhanced phosphodiesterase activity which results from the activation of muscarinic receptors.
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PMID:Differential effect of pertussis toxin on adenosine and muscarinic inhibition of cyclic AMP accumulation in canine ventricular myocytes. 164 26

Although recent studies indicate that MCI-154 exerts novel positive inotropic actions in heart muscle, the chronotropic properties of this new drug remain undefined. The present study compared the inotropic/chronotropic profile of MCI-154 with those of a nonselective beta 1/beta 2-agonist (isoproterenol, Iso) and a type III phosphodiesterase inhibitor (imazodan, CI-914) in cardiac preparations isolated from guinea pigs. The inotropic efficacy of MCI-154 was approximately equal to that of Iso and CI-194 in electrically paced (1 Hz) atrial muscle, with each agent increasing isometric contractile tension approximately 170% above basal (predrug) values. The inotropic EC50 for Iso (2.9 +/- 0.7 x 10(-9) M) was several orders of magnitude less than that for MCI-154 (1.4 +/- 0.4 x 10(-4) M) and CI-914 (1.1 +/- 0.2 x 10(-4) M). The inotropic potency of MCI-154 was equivalent in atrial and left ventricular myocardium. Both Iso and CI-194 substantially increased spontaneous beating frequency of sinoatrial preparations, and the inotropic/chronotropic potency ratio for each was unity. In contrast, MCI-154 exerted a slight negative chronotropic action on basal sinoatrial rate. Moreover, the negative chronotropic influence of MCI-154 was increased several-fold in the presence of Iso-stimulated maximal increases in heart rate (HR), and this inhibitory chronotropic action of MCI-154 was not prevented by muscarinic receptor blockade with atropine. These findings indicate that MCI-154 has a unique inotropic/chronotropic profile in cardiac tissues of guinea pigs in that this drug (a) efficaciously increased myocardial contractility, (b) had minimal effect on basal sinoatrial automaticity and yet (c) markedly inhibited sympathetically mediated sinus tachycardia.
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PMID:Inotropic and chronotropic profile of MCI-154: comparison with isoproterenol and imazodan in guinea pig cardiac preparations. 169 67

The ability of the beta-adrenoceptor agonist isoprenaline to inhibit agonist-stimulated phosphoinositide metabolism was examined in bovine tracheal smooth muscle slices prelabelled with [3H]inositol. Accumulation of [3H]inositol phosphates was enhanced by the muscarinic agonists carbachol, oxotremorine and pilocarpine although the latter were only partial agonists for this response. Histamine stimulation of [3H]inositol phosphates was sensitive to mepyramine but maximal responses were only comparable to those of pilocarpine. Preincubation of tracheal slices with isoprenaline reduced the maximal phosphoinositide response to histamine and pilocarpine but the responses to carbachol and oxotremorine were unaffected. The inhibitory effect of isoprenaline (IC50 = 0.04 microM) was reversed competitively by 1 microM propranolol. The non-selective phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) (1 mM) resulted in a more severe suppression of the histamine and pilocarpine responses and also produced a significant suppression of the maximal response to oxotremorine and a small shift in the carbachol dose-response curve. The different susceptibility of agonist-stimulated phosphoinositide hydrolysis to isoprenaline and IBMX are discussed in relation to the relative intrinsic activity of the agonists and/or the role of different muscarinic receptor subtypes.
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PMID:Beta-adrenoceptor induced inhibition of muscarinic receptor-stimulated phosphoinositide metabolism is agonist specific in bovine tracheal smooth muscle. 171 79

Although adenosine is known to activate K+ conduction in atrial tissue, there is still debate as to the involvement of cAMP-dependent mechanisms. In isolated adult guinea pig atrial myocytes, we demonstrate that the highly A1-selective adenosine receptor agonist 2-chloro-N6-cyclopentyladenosine reduced basal cAMP levels by 30-40% in the absence and presence of the nonxanthine phosphodiesterase inhibitor Ro 20-1724. Isoprenaline caused a concentration-dependent increase in cAMP levels, which was more pronounced in the presence of the phosphodiesterase inhibitor. Several adenosine derivatives suppressed the isoprenaline-induced cAMP increase by approximately 80%. The rank order of potency was 2-chloro-N6-cyclopentyladenosine (IC50, 93 nM) greater than (R)-N6-phenylisopropyladenosine (IC50, 309 nM) greater than 5'-N-ethylcarboxamidoadenosine (IC50, 813 nM) much greater than (S)-N6-phenylisopropyladenosine (IC50, 26,300 nM). A similar but complete suppression of the isoprenaline-induced cAMP increase was produced by the muscarinic receptor agonist carbachol (IC50, 398 nM), which like adenosine is known to activate atrial K+ channels. The A1-adenosine receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine antagonized the effect of 2-chloro-N6-cyclopentyladenosine concentration-dependently, with a KB value of 9.6 nM. In atrial myocytes isolated from guinea pigs pretreated with pertussis toxin, the inhibitory effects of adenosine analogs on basal and isoprenaline-stimulated cAMP accumulation were markedly attenuated. It is concluded that the adenosine receptor in guinea pig atrial myocytes, which is known to be linked to K+ channels, is also coupled to adenylate cyclase via a pertussis toxin-sensitive guanine nucleotide-binding protein and shows the characteristics of the A1-adenosine receptor subtype.
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PMID:Pharmacological characterization of the adenylate cyclase-coupled adenosine receptor in isolated guinea pig atrial myocytes. 216 17

Agonist occupation of muscarinic cholinergic receptors of 1321N1 human astrocytoma cells results in an activation of phosphodiesterase and a resultant 50-75% attenuation of isoproterenol-stimulated cyclic AMP accumulation. The effects of a series of phosphodiesterase inhibitors on muscarinic receptor-mediated inhibition of cyclic AMP accumulation and on the activities of partially purified, soluble phosphodiesterase have been compared to determine which form of phosphodiesterase activity is regulated by muscarinic receptors. The phosphodiesterase inhibitors (50 microM) 1-methyl-3-isobutylxanthine (MIX), 1-methyl-3-isobutyl-7-benzylxanthine (7-BzMIX), 1-methyl-3-isobutyl-8-methoxymethylxanthine (8-MeOMeMIX), and 2-O-propoxyphenyl-8-azapurin-6-one (MB 22948) blocked the effect of muscarinic receptor activation. However, 1-isoamyl-3-isobutylxanthine (IIX) and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724) did not block muscarinic receptor-mediated effects but enhanced isoproterenol-stimulated cyclic AMP accumulation. Three forms of soluble phosphodiesterase activity were resolved by DEAE-cellulose chromatography and sucrose density gradient centrifugation. A calmodulin-stimulated phosphodiesterase activity was inhibited by MIX, 7-BzMIX, 8-MeOMeMIX, and MB 22948 (IC50 values = 1-10 microM) but was not inhibited by IIX and Ro 20-1724. The similar relative capacities of the phosphodiesterase inhibitors for blocking both the muscarinic receptor-mediated attenuation of cyclic AMP accumulation and the calmodulin-stimulated phosphodiesterase activity in vitro suggest that it is this form of enzyme that is regulated by muscarinic receptor stimulation.
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PMID:Identification of the phosphodiesterase regulated by muscarinic cholinergic receptors of 1321N1 human astrocytoma cells. 242 35

The interactions between dopamine and muscarinic receptor subtypes coupled to adenylate cyclase in superfused rat neostriatal slices were investigated using the efflux of cyclic AMP, in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, as a highly sensitive parameter of cyclic AMP production. Cyclic AMP efflux induced by simultaneous activation of (stimulatory) D-1 and (inhibitory) D-2 dopamine receptors by dopamine was reduced profoundly by the muscarinic receptor agonist oxotremorine and by inhibition of acetylcholinesterase with physostigmine, but not by the M-1 muscarinic receptor agonist McN-A-343. In contrast, upon blockade of D-2 receptors with (-)-sulpiride, dopamine-stimulated cyclic AMP efflux was inhibited by oxotremorine and physostigmine as well as by McN-A-343. Cyclic AMP efflux induced by isoprenaline, adenosine or vasoactive intestinal peptide was not affected by oxotremorine. The M-1 receptor-selective antagonist pirenzepine, unlike the nonselective antagonist atropine, was about 10 times less potent in antagonizing the inhibitory effects of (a near-maximally effective concentration of) oxotremorine upon simultaneous D-1 and D-2 receptor activation that upon selective D-1 receptor activation (i.e., upon blockade of D-2 receptors). In the latter case, pirenzepine was about 5 times more effective as an antagonist when muscarinic receptors were activated by McN-A-343 than upon exposure of the slices to oxotremorine or physostigmine, whereas the potency of atropine was independent of the agonist used.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:M-1 and M-2 muscarinic receptor-mediated inhibition of dopamine-sensitive adenylate cyclase in rat neostriatum: a permissive role for D-2 dopamine receptors. 245 77


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