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:3.4.16.2 (PCP)
3,761 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new receptor based assay is described for the determination of classes of drugs which have high affinities for the acetylcholine receptor. The method is based upon the inhibition of the enzyme activity of an enzyme-drug conjugate by the binding to the receptor protein, and competition between free drugs and the enzyme-drug conjugate for a limited number of receptor sites. The activity of the enzyme marker system, glucose-6-phosphate dehydrogenase covalently conjugated to desipramine, is monitored by colorimetric detection of the rate of NADH formation at 340 nM. The procedure proposed is designed to provide a simple drug screen which can be done in a minimally equipped laboratory while achieving the required sensitivity. The technique is illustrated for three acetylcholine channel binding compounds: the hallucinogen phencyclidine (PCP) and the antipsychotic agents chlorpromazine and trifluoperazine. This procedure yields calibration curves with detection limits at nanomolar levels of drug, with binding responses dependent on the amounts of receptor and enzyme-labeled drug used. Aspecific binding responses of unlabeled enzyme to drug or receptor to compounds with low affinity for the receptor are shown to have minimal effect on the assay.
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PMID:Enzyme-amplified receptor assay screening test for chlorpromazine, trifluoperazine, and phencyclidine. 169 Feb 78

The binding of the competitive antagonist alpha-bungarotoxin (alpha-Btx) and the noncompetitive inhibitor phencyclidine (PCP) to a synthetic peptide comprising residues 172-227 of the alpha-subunit of the Torpedo acetylcholine receptor has been characterized. 125I-alpha-Btx bound to the 172-227 peptide in a solid-phase assay and was competed by alpha-Btx (IC50 = 5.0 x 10(-8) M), d-tubocurarine (IC50 = 5.9 X 10(-5)M), and NaCl (IC50 = 7.9 x 10(-2)M). In the presence of 0.02% sodium dodecyl sulfate, 125I-alpha-Btx bound to the 56-residue peptide with a KD of 3.5 nM, as determined by equilibrium saturation binding studies. Because alpha-Btx binds to a peptide comprising residues 173-204 with the same affinity and does not bind to a peptide comprising residues 205-227, the competitive antagonist and hence agonist binding site lies between residues 173 and 204. After photoaffinity labeling, [3H]PCP was bound to the 172-227 peptide. [3H]PCP binding was inhibited by chlorpromazine (IC50 = 6.3 x 10(-5)M), tetracaine (IC50 = 4.2 x 10(-6)M), and dibucaine (IC50 = 2.7 x 10(-4)M). Equilibrium saturation binding studies in the presence of 0.02% sodium dodecyl sulfate showed that [3H]PCP bound at two sites, a major site of high affinity with an apparent KD of 0.4 microM and a minor low-affinity site with an apparent KD of 4.6 microM. High -affinity binding occurred at a single site on peptide 205-227 (KD = 0.27 microM) and was competed by chlorpromazine but not by alpha-Btx.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Binding sites for alpha-bungarotoxin and the noncompetitive inhibitor phencyclidine on a synthetic peptide comprising residues 172-227 of the alpha-subunit of the nicotinic acetylcholine receptor. 185 49

We have shown previously that the lipophilic photoreagent 3-(trifluoromethyl)3-m-([125I]iodophenyl)-diazirine ([125I]TID) photolabels all four subunits of the Torpedo nicotinic acetylcholine receptor (AChR) and that greater than 70% of this photoincorporation is inhibited by cholinergic agonists and some noncompetitive antagonists, including histrionicotoxin (HTX), but not phencyclidine (PCP; White, B.H., and Cohen, J.B. (1988) Biochemistry 27, 8741-8751). We have now examined the effects of nonradioactive TID on (a) AChR photoincorporation of [125I]TID, (b) AChR-mediated ion transport, and (c) AChR binding of several cholinergic ligands. We find that TID inhibits [125I]TID photoincorporation into the AChR to the same extent as carbamylcholine. The saturable component of [125I]TID photolabeling is half-maximal at 4 microM [125I]TID with 0.5 mol specifically incorporated per mol of AChR after 30 min photolysis with 60 microM [125I]TID. Repeated labeling of membranes at a fixed [125I]TID concentration gave results consistent with a maximal incorporation of one [125I]TID molecule per AChR. Nonradioactive TID also noncompetitively inhibits agonist-stimulated 22Na+ efflux from Torpedo vesicles with an IC50 of 1 microM. Furthermore, TID inhibits allosterically the binding of [3H]HTX, decreasing its affinity for the AChR 5-fold both in the presence and absence of agonist. In contrast, TID has little effect on [3H]PCP binding in the absence of agonist but completely inhibits it in the presence of agonist. TID enhances the cooperativity of [3H]nicotine binding. [125I]TID is thus a photoaffinity label for a novel noncompetitive antagonist binding site on the AChR that is linked allosterically to the binding sites of both agonists and other noncompetitive antagonists. The [125I]TID site is presumably located within the central pore of the AChR.
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PMID:The hydrophobic photoreagent 3-(trifluoromethyl)-3-m-([125I] iodophenyl) diazirine is a novel noncompetitive antagonist of the nicotinic acetylcholine receptor. 193 89

Histrionicotoxin, a spiropiperidine alkaloid, and twenty-two analogs inhibited binding of [3H]perhydrohistrionicotoxin [( 3H]H12-HTX) and of [3H]phencyclidine [( 3H]PCP) to sites on the acetylcholine receptor-ion complex of Torpedo electroplax membranes. Structural alterations to the nitrogen (secondary amine) or oxygen (alcohol) functions or to the five carbon and four carbon side chain of histrionicotoxin altered the potency versus [3H]H12-HTX and [3H]PCP binding measured in the presence or absence of a receptor agonist, carbamylcholine. Histrionicotoxin itself was 3-fold more potent versus [3H]PCP binding than versus [3H]H12-HTX binding. N-Methylation or O-acetylation increased this difference, while alterations to the side chains either slightly decreased or markedly increased this difference. Histrionicotoxin was some 3.5-fold more potent versus [3H]H12-HTX binding in the presence of carbamylcholine than in its absence. O-Acetylation increased this selectivity for the carbamylcholine-activated state of the receptor channel complex, while alterations in the side chains either reduced or increased the selectivity. Histrionicotoxin was some 2.2-fold more potent versus [3H]PCP binding in the presence of carbamylcholine than in its absence. N-Methylation of O-acetyl-histrionicotoxin greatly increased this selectivity, while alterations in the side chains either reduced or had no effect on selectivity.
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PMID:Binding of [3H]perhydrohistrionicotoxin and [3H]phencyclidine to the nicotinic receptor-ion channel complex of Torpedo electroplax. Inhibition by histrionicotoxins and derivatives. 241 60

The actions of phencyclidine [1-(1-phenylcyclohexyl)piperidine, PCP] and its morpholine analog [1-(1-phenylcyclohexyl)morpholine, PCM] on ionic currents of nicotinic acetylcholine receptors were studied at the neuromuscular junction of frog skeletal muscle and on embryonic rat muscle cells in tissue culture. PCP and PCM reduced the peak amplitude and the decay time constant of the endplate current (EPC). PCP produced a voltage-dependent curvature and a time-dependent hysteresis loop at negative potentials (at potentials from -50 to -150 mV). In contrast, PCM caused a depression of EPC peak amplitude, but the current-voltage relationship (+60 to -150 mV) remained linear. When PCP-modified EPCs were elicited in trains at hyperpolarized potentials the amplitudes of successive events were progressively decreased and the magnitude of the decrease was dependent on the level of hyperpolarization. At positive potentials the process was reversed; the amplitude increased with successive stimulations. The EPC decayed exponentially in the presence of PCP and PCM, with a shortened time constant of decay that was less dependent on membrane potential than control. PCP and PCM caused only a 20% decrease of the amplitude of the iontophoretically evoked acetylcholine potential, which was significantly different from that induced by the desensitizing alkaloid perhydrohistrionicotoxin. Both PCP and PCM reduced by 50% the mean channel open time obtained from rat myoballs, giving a potency ratio for PCP to PCM of 2.5. This relative potency was correlated with that obtained for the reduction in the decay time constant of the EPC (ratio = 2.2). The effects of PCP on the peak amplitude of the EPC seem to be related to a conformational change of the acetylcholine receptor occurring before channel activation and not to a receptor desensitization.
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PMID:Voltage- and time-dependent effects of phencyclidines on the endplate current arise from open and closed channel blockade. 242 53

The nicotinic effects of a novel antiparkinsonian compound, diprobutine were investigated on the acetylcholine receptor (AChR) from Torpedo marmorata electric organ and on rat brain membranes by a variety of techniques including stopped flow measurements. On the nicotinic AChR from Torpedo, diprobutine behaved as a typical noncompetitive blocker: it inhibited the agonist-regulated 22Na+ efflux from excitable microsacs; it shifted in the ms-s time-range the conformation of the AChR towards a high affinity state for agonists; it competed with [3H]PCP bound to its high affinity 'allosteric' site. On rat brain membrane, it displaced [3H]PCP bound to its high affinity site. The pharmacological properties of diprobutine are discussed in the context of its biochemical effects.
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PMID:Allosteric effects of diprobutine on acetylcholine receptors. 300 Aug 3

The effects of phencyclidine(PCP) on the post-tetanic potentiation(PTP) of twitch tension were studied on the isolated mouse phrenic nerve diaphragm preparation. Phencyclidine increased directly elicited twitch tension while it decreased post-tetanic potentiation of the indirectly elicited twitch tension. The maximal depression effect of the PTP was found after higher frequencies and longer durations of stimulation. After repetitive stimulation, the amplitude of endplate potential was potentiated. Phencyclidine decreased the post-tetanic potentiation of the amplitude of endplate potential while the quantal content of the endplate potential was not affected. 4-Aminopyridine increased both directly and indirectly elicited twitch tension while it did not inhibit the post-tetanic potentiation of the twitch tension. It is concluded that phencyclidine suppressed the post-tetanic potentiation of the indirectly elicited twitch tension. The depressant effect may be mainly due to its effect on the acetylcholine receptor-ionic channel complex of the motor endplate.
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PMID:Effect of phencyclidine on post-tetanic twitch tension of the mouse diaphragm preparation. 303 6

The effect of trifluoperazine (TFP) and phencyclidine (PCP) on acetylcholine receptor (AChR) function was studied in rat muscles differentiated in cell culture. While both drugs exerted an inhibitory effect on carbamylcholine (CCh)-induced Na+ or Ca2+ influx (I50 = 5-7 microM), alpha-bungarotoxin binding was not affected. The inhibitory effect of both drugs was independent of CCh concentration, which deems it unlikely that these drugs enhanced desensitization. The mutual inhibitory effect of TFP and PCP on Ca2+ influx was analyzed using three alternative models of interaction between the two drugs: competitive, additive and synergistic inhibition models. Our results are in accordance with a synergistic interaction between the drugs. This synergistic interaction between the drugs provides a biochemical rationale to the phenothiazine contraindication in the treatment of PCP psychosis.
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PMID:Trifluoperazine and phencyclidine inhibit synergistically carbamylcholine-induced cation influx in muscle cultures. 343 92

Purified Torpedo nobiliana electric organ acetylcholine receptor (AChR) was reconstituted into membranes containing natural phospholipids supplemented with cholesterol (25% w/w). The reconstituted system facilitates the study of the effects of drugs on the regulation of the AChR channel complex under both resting and carbachol (carb)-stimulated conditions. Neostigmine (Neo) was the only carbamate to induce activation of [3-H]-phencyclidine ([3-H]-PCP) binding to the channel sites, acting as a weak agonist. The activation of [3-H]-PCP binding is dependent upon the nature of the reconstituted systems, with carb/Neo activation ratios of 8, 3, and 1 for the intact purified AChR vesicles fraction (PVF), the PVF reconstituted in phospholipid/cholesterol (CRPVF), and the PVF reconstituted in phospholipid (RPVF), respectively. The carbamates Neo, physostigmine (Physo), and pyridostigmine (Pyrido) inhibited carb-activated [3-H]-PCP binding with Ki values of 10, 20, and 1,600 microM, respectively. The inhibition was mixed competitive-noncompetitive in nature. The characteristic response of CRPVF to carb-stimulated [22-Na] influx was inhibited by the three carbamates, with IC-50 values of 6, 50, and 1,000 microM for Neo, Physo, and Pyrido, respectively. The quaternary ammonium organophosphate ecothiophate (Eco) inhibited carb-stimulated [22-Na] influx with potency similar to that of Neo. Preincubation of AChR preparation with the carbamates and ecothiophate caused a reduction in the binding of [125-I]-alpha-bungarotoxin ([125-I]-alpha-BGT) with the following decreasing order of potency: Neo less than Physo less than Eco less than Pyrido. Calcium has a direct modulatory role on the time-course inhibition of [125-I]-alpha-BGT binding by these drugs. While we observed a high potency of Neo and Physo in inhibiting [125-I]-alpha-BGT binding, it was undetectable for the carbamate insecticide 2-methyl-2-(methylthio)propionaldehyde-O-(methylcarbamoyl)oxime (aldicarb). These data suggest that the potent anticholinesterase carbamate agents interact differently with the AChR and its ionic channel. Their interactions with the nicotinic AChR channel system can be described as (a) weakly agonist, (b) directly acting on the open conformation of the channel, and (c) blocking the AChR-binding sites.
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PMID:Biochemical interactions of carbamates and ecothiophate with the activated conformation of nicotinic acetylcholine receptor. 350 76

The effect of trifluoperazine (TFP) and phencyclidine (PCP) on acetylcholine receptor (AChR) function was studied in rat myotubes differentiated in vitro. While both drugs exerted an inhibitory effect on carbamylcholine (CCh)-induced Na+ or Ca2+ flux (I50 = 5-9 microM), alpha-bungarotoxin (alpha-Bgt) binding was not affected. The inhibitory effect of both drugs was independent of CCh concentration. The mutual inhibitory effect of TFP and PCP on Ca2+ influx was analyzed using three alternative models of interaction between the two drugs: competitive, additive and synergistic inhibition models. Our results are in accord with a synergistic interaction between the drugs probably not through desensitization. This synergistic interaction between the drugs provides a biochemical rationale to the phenothiazine contraindication in the treatment of PCP psychosis.
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PMID:Synergistic inhibition by trifluoperazine and phencyclidine of carbamylcholine-induced cation influx in muscle cultures. 360 41


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