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)

1. The regulation of the cardiac Ca2+ release channel-ryanodine receptor (RyR) by exogenous acid phosphatase (AcPh) and purified Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) was studied in swine and rabbit sarcoplasmic reticulum (SR) vesicles using [3H]ryanodine binding and planar bilayer reconstitution experiments. 2. Addition of AcPh (1-20 U ml-1) to a standard incubation medium increased [3H]ryanodine binding in a Ca(2+)-dependent manner. Stimulation was only readily apparent in media containing micromolar Ca2+ concentrations. 3. Scatchard analysis of [3H]ryanodine binding curves revealed that AcPh enhanced binding by increasing the affinity of the receptor for [3H]ryanodine without recruiting additional receptor sites (Kd, 9.8 +/- 0.85 and 3.9 +/- 0.65 nM; Bmax (the maximal receptor density), 1.45 +/- 0.14 and 1.47 +/- 0.12 pmol mg-1 for control and AcPh, respectively). The failure of AcPh to increase Bmax suggested that the number of receptors that were 'dormant' due to phosphorylation in the SR preparation was very small. 4. At the single channel level, AcPh increased the open probability (Po) of RyR channels by increasing the opening rate and inducing the appearance of a longer open state while having no effect on single channel conductance. Thus AcPh acted directly on RyR channels or a closely associated regulatory protein. 5. CaMKII decreased both [3H]ryanodine binding and Po of RyRs when added to medium supplemented with micromolar levels of Ca2+ and calmodulin (CaM). Addition of a synthetic peptide inhibitor of CaMKII, or replacement of ATP with the non-hydrolysable ATP analogue adenylyl[beta, gamma-methylene]-diphosphate (AMP-PCP), prevented CaMKII inhibition of RyRs, suggesting that CaMKII acted specifically through a phosphorylation mechanism. 6. The inhibition of RyR channel activity by CaMKII was reversed by the addition of AcPh. Thus we showed that an in vitro phosphorylation-dephosphorylation mechanism effectively regulates RyRs. 7. The results suggest that intracellular signalling pathways that lead to activation of CaMKII may reduce efflux of Ca2+ from the SR by inhibition of RyR channel activity. The Ca2+ dependence of CaMKII inhibition suggests that the role of the phosphorylation mechanism is to modulate the RyR response to Ca2+.
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PMID:Modulation of cardiac ryanodine receptors of swine and rabbit by a phosphorylation-dephosphorylation mechanism. 854 25

Cl- and cation conductances were characterized in zymogen granules (ZG) isolated from the pancreas of wild-type mice (+/+) or mice with a homozygous disruption of the multidrug resistance P-glycoprotein gene mdr1a (-/-). Cl- conductance of ZG was assayed in isotonic KCl buffer by measuring osmotic lysis, which was induced by maximal permeabilization of ZG membranes (ZGM) for K+ with valinomycin due to influx of K+ through the artificial pathway and of Cl- through endogenous channels. To measure cation conductances, ZG (pHi 6.0-6.5) were suspended in buffered isotonic monovalent cation acetate solutions (pH 7.0). The pH gradient was converted into an outside-directed H+ diffusion potential by maximally increasing H+ conductance of ZGM with carbonyl cyanide m-chlorophenylhydrazone. Osmotic lysis of ZG was induced by H+ diffusion potential-driven influx of monovalent cations through endogenous channels and nonionic diffusion of the counterion acetate. ZGM Cl- conductances were not different in (-/-) and (+/+) mice (2.6 +/- 0.3 h-1 versus 3.1 +/- 0.2 h-1 (relative rate constant)). The nonhydrolyzable ATP analog adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP) (0.5 mM) activated the Cl- conductance both in (+/+) and (-/-) mice. However, activation of Cl- conductance by AMP-PCP was reduced in (-/-) mice as compared with (+/+) mice (5.0 +/- 0.4 h-1 versus 7.6 +/- 0.7 h-1; p < 0. 005). In contrast, ZGM K+ conductance was increased in (-/-) mice as compared with (+/+) mice (14.2 +/- 2.0 h-1 versus 8.5 +/- 1.2 h-1; p < 0.03). In the presence of 0.5 mm AMP-PCP, which completely blocks K+ conductance but leaves a nonselective cation conductance unaffected, there was no difference between (-/-) and (+/+) mice (5.3 +/- 0.7 h-1 versus 3.2 +/- 0.5 h-1). In Western blots of ZGM from wild-type mice, a polyclonal MDR1 specific antibody labeled a protein band of approximately 80 kDa. In mdr1a-deficient mice, the intensity of this band was reduced to 39 +/- 7% of the wild-type signal. This indicates that a mdr1a gene product of approximately 80 kDa enhances the AMP-PCP-activated fraction of mouse ZGM Cl- conductance and reduces AMP-PCP-sensitive K+ conductance.
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PMID:Chloride and potassium conductances of mouse pancreatic zymogen granules are inversely regulated by a approximately 80-kDa mdr1a gene product. 862 34

The regulation of capacitative Ca2+ influx in Xenopus oocytes was investigated using both the two electrode voltage-clamp (where Ca2+ is monitored through the Ca2+-dependent Cl- current) and patch-clamp techniques. Following stimulation of expressed 5-hydroxytryptamine (5-HT) receptors, capacitative Ca2+ influx deactivated in around 15 min. Following injection of [adenosine 5'-O-(3-Thiotriphosphate)] (ATP [gamma-S]), an ATP analogue that is readily used by protein kinases, capacitative Ca2+ influx activated by 5-HT application either did not deactivate or was prolonged around twofold. However, injection of adenylyl 5'-(beta,gamma-methylene)-diphosphonate (AMP-PCP), another ATP analogue that is not utilised by kinases, did not affect the time-course of Ca2+ influx. When capacitative Ca2+ influx was activated by readmission of Ca2+ to oocytes incubated in thapsigargin/0 Ca2+ solution for several hours, Ca2+ influx occurred and a weakly saturating relationship between external Ca2+ and Ca2+ influx was found. Ca2+ influx in thapsigargin-treated cells was unaffected by ATP [gamma-S]. ATP [gamma-s] and several kinases had no effect on the Ca2+-dependent Cl- current when the latter was activated by elevation of Ca2+ independent of capacitative Ca2+ influx. Protein kinase C slowly and partially inhibited the Cl- current. Outside-out patches taken from thapsigargin-treated cells failed to demonstrated any Ca2+ current or Ca2+-dependent Cl- current on reapplying high Ca2+ to the patch, despite the oocyte showing a large capacitative Ca2+ influx. The results suggest that a kinase, activated on receptor stimulation, prolongs the activation time-course of capacitative Ca2+ influx.
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PMID:Effects of protein phosphorylation on the regulation of capacitative calcium influx in Xenopus oocytes. 866 63

[3H]Ryanodine binding to, as well as functions of, ryanodine receptor intracellular Ca2+ release channel complexes are modulated by several adenosine-based compounds. In this study, we determined the effects of endogenous compounds termed diadenosine polyphosphates (ApnAs; n = 2-6 phosphate groups) on [3Hlryanodine binding to membranes prepared from rat brain and skeletal and cardiac muscle. Under low ionic strength buffer conditions, [3H]ryanodine binding to brain membranes was significantly increased by 171% with 333 microM P1,P5-di(adenosine-5') pentaphosphate (Ap5A) and by 209% with the same concentration of the metabolism-resistant ATP analogue betagamma-methyleneadenosine 5'-triphosphate (AMP-PCP) compared with control values for [3H]ryanodine binding of 9.6 +/- 1.8 fmol/mg of protein. Dose-related increases in [3H]ryanodine binding were observed for all five ApnAs tested [P1,P2-di(adenosine-5') pyrophosphate (Ap2A), P1,P3-di(adenosine-5') triphosphate (Ap3A), P1,P4-di(adenosine-5') tetraphosphate (Ap4A), Ap5A, and P1,P6-di(adenosine-5') hexaphosphate (Ap6A)] as well as AMP-PCP; oxidized salts of ApnAs stimulated [3H]ryanodine binding to a greater degree than did nonoxidized APnAs. The apparent rank order for the capacity of these agents to increase [3H]-ryanodine binding was oxidized Ap4A = oxidized Ap5A > oxidized Ap3A > Ap6A > AMP-PCP > Ap5A > AP2A. Addition of the approximate EC50 dose of oxidized Ap4A (37 microM) increased the affinity (KD) of ryanodine receptors from 34 +/- 7 to 12 +/- 2 nM, the apparent binding site density (Bmax) was not significantly different from control values of 107 +/- 33 fmol/mg of protein. Increases in [3H]-ryanodine binding by either oxidized Ap4A or nonoxidized Ap5A were not further enhanced by coincubation with AMP-PCP, which suggests a similar site of action for the ApnAs and AMP-PCP. [3H]Ryanodine binding to skeletal and cardiac muscle membranes was enhanced by addition of oxidized AP4A Ap5A, and AMP-PCP. Oxidized AP4A increased the specific binding by ninefold in skeletal muscle and by threefold in cardiac muscle. These results suggest that ApnAs, at physiologically relevant concentrations, may serve as endogenous modulators of ryanodine receptor-gated Ca2+ release channels.
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PMID:Regulation of ryanodine receptor calcium release channels by diadenosine polyphosphates. 876 82

Six per cent of rat pheochromocytoma (PC12) cells extended neurites (processes greater than one cell diameter in length) in the presence of 300 microM extracellular GTP or 300 microM guanosine for 48 hr, compared to only 2.5% of cells in control cultures. In the presence of 40 ng/ml of 2.5S NGF, about 20-35% of PC12 cells had neurites after 48 hr, and the addition of 300 microM guanosine or GTP together with NGF synergistically increased the proportion of cells with neurites to 40-65%. GTP and guanosine also increased the average number of branches per neurite, from 0.6 in NGF-treated cultures to 1.2 (guanosine) or 1.5 (GTP). Neurites formed after exposure to NGF alone had axonal characteristics as determined by immunocytochemistry with antibody, SMI-31, against axonal-specific polyphosphorylated neurofilament epitopes. Neurites generated with the addition of both guanosine or GTP had the same characteristics. GTP probably did not exert its effects via the P2X or P2Y purinoceptors because the adenine nucleotides ATP, ATP gamma S, ADP beta S, and ADP, which are all agonists of these receptors, inhibited rather than enhanced, NGF-induced neurite outgrowth. UTP also enhanced the proportion of cells with neurites, although not to the same degree as did GTP. This may indicate activity through a P2U-like nucleotide receptor. However, the response profile obtained, GTP > UTP >> ATP, does not fit the profile of any known P2Y, P2X or P2U receptor. The poorly hydrolyzable GTP analogues, GTP gamma S and GDP beta s were also unable to enhance the proportion of cells with neurites. This implied that GTP may produce its effects through a GTP-specific ectoenzyme or kinase. This idea was supported by results showing that another poorly hydrolyzable analogue, GMP-PCP, competitively inhibited the effects of GTP on neurite outgrowth. GTP did not exert its effects after hydrolysis to guanosine since the metabolic intermediates GDP and GMP were also ineffective in enhancing the proportion of cells with neurites. Moreover, the effects of GTP and guanosine were mutually additive, implying that these two purines utilized different signal transduction mechanisms. The effects of guanosine were not affected by the nucleoside uptake inhibitors nitrobenzylthioinosine (NBTI) and dipyridamole, indicating that a transport mechanism was not involved. Guanosine also did not activate the purinergic P1 receptors, because the A2 receptor antagonists, 1,3-dipropyl-7-methylxanthine (DPMX) or CGS15943, and the A1 receptor antagonist, 1,3-dipropyl-8-(2-amino-4-chloro)xanthine (PACPX) did not inhibit its reaction. Therefore guanosine enhanced neurite outgrowth by a signal transduction mechanism that does not include the activation of the P1 purinoceptors. The enhancement of the neuritogenic effects of NGF by GTP and guanosine may have physiological implications in sprouting and functional recovery after neuronal injury in the CNS, due to the high levels of nucleosides and nucleotides released from dead or injured cells.
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PMID:GTP and guanosine synergistically enhance NGF-induced neurite outgrowth from PC12 cells. 877 5

The kinetic mechanism of the pp60c-src tyrosine kinase (src TK) reaction was investigated in the forward and reverse directions. In the forward direction, initial velocities obtained by varying ATP and the peptide (FGE)3Y(GEF)2GD indicated a sequential addition of the two substrates. The peptide analog, (FGE)3F(GEF)2GD, was a competitive inhibitor versus the peptide substrate and a noncompetitive inhibitor versus MgATP. Interestingly, the tyrosine hydroxyl group imparts only a 6-fold increase in binding. AMP-PCP was a competitive inhibitor versus MgATP and a noncompetitive inhibitor versus the peptide substrate. These results prove that the addition of substrates is random. Furthermore, there appears to be little binding synergy as the KiMgATP approximately equal to 2.4KmMgATP. The phosphorylated peptide (FGE)3-pY-(GEF)2GD was a competitive inhibitor versus peptide and a noncompetitive inhibitor against MgATP, suggesting that a dead end complex can form between MgATP, the phosphorylated peptide product, and the enzyme. The reverse reaction was investigated by varying ADP and the phosphopeptide. (FGE)3-pY-(GEF)2GD. The initial velocity pattern was indicative of a sequential mechanism. There was even less binding synergy in the reverse direction as the KiMgADP approximately equal to 1.4KmMgADP. AMP-CP was a competitive inhibitor versus MgADP and a noncompetitive inhibitor versus the phosphopeptide. (FGE)3F(GEF)2GD was a competitive inhibitor versus the phosphopeptide and a noncompetitive inhibitor versus MgADP. These data prove that addition of the substrates in the reverse direction is random. (FGE)3Y(GEF)2GD was a competitive inhibitor against peptide substrate and a noncompetitive inhibitor against MgADP; therefore a dead end complex can form between MgADP, (FGE)3Y(GEF)2GD, and the enzyme. These results indicate that the src TK reaction follows a sequential bi-biequilibrium random mechanism in both directions, with dead end complexes forming when either MgATP and (FGE)3-pY-(GEF)2GD or MgADP and (FGE)3Y(GEF)2GD bind to the enzyme. The kinetic constants determined from the forward and reverse reactions were used in the Haldane equation to determine a K(eq) constant for the forward reaction of 10.1, corresponding to a delta G of -1.4 kcal/mol. This further confirms that the O-P bond of phosphotyrosine is similar in energy to that of the gamma-phosphoryl of MgATP.
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PMID:Kinetic mechanisms of the forward and reverse pp60c-src tyrosine kinase reactions. 884 69

Cholecystokinin (CCK) receptors on rat pancreatic acinar cells display two binding affinity states in the presence of adeninine and guanine triphosphates with the effect of ATP mediated by the enzyme nucleoside diphosphate kinase. To determine whether this behavior was intrinsic to a single receptor protein we studied the binding affinity of CHO cells stably transfected with a cloned rat CCKA receptor. 125I-CCK binding to intact cells at 37 degrees C revealed two affinity states for CCK of Kd values 20 pM and 2.4 nM. Membranes prepared from these cells displayed a single affinity state for CCK but two affinity states could be restored in the presence of GTP[gamma S], ATP and ATP[gamma S] but not AMP-PCP. ATP and ATP[gamma S] but not AMP-PCP were substrates for nucleoside diphosphate kinase present in CHO cell membranes and transferred their terminal phosphate to GDP. These findings indicate that the interconvertible affinity states of the CCK receptor are inherent in a single receptor protein and that nucleoside diphosphate kinase mediates the effect of ATP to regulate these two affinity states.
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PMID:Nucleotides regulate the binding affinity of the recombinant type A cholecystokinin receptor in CHO K1 cells. 885 9

We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na+/H+ antiporter, using the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pHi) by microfluorimetry. Na+/H+ exchange activity was measured as the Na(+)-driven pHi recovery from an acid load, which was imposed via the patch pipette. In Chinese hamster ovary (CHO) fibroblasts stably transfected with NHE-1, omission of ATP from the pipette solution inhibited Na+/H+ exchange. Conversely, ATP perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxy-D-glucose and oligomycin. In cells dialyzed in the presence of ATP, no "run-down" was observed even after extended periods, suggesting that the nucleotide is the only diffusible factor required for optimal NHE-1 activity. Half-maximal activation of the antiporter was obtained at approximately 5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na+/H+ exchange even when an ATP regenerating system was included in the pipette solution. High ATP concentrations are also known to be required for the optimal function of other cation exchangers. In the case of the Na/Ca2+ exchanger, this requirement has been attributed to an aminophospholipid translocase, or "flippase.". The involvement of this enzyme in Na+/H+ exchange was examined using fluorescent phosphatidylserine, which is actively translocated by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBD-PS), indicating that the flippase was inhibited. Diamide, an agent reported to block the flippase, was as potent as ATP depletion in reducing NBD-PS uptake. However, diamide had no effect on Na+/H+ exchange, implying that the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATP gamma S were as efficient as Mg-ATP in sustaining NHE-1 activity, while AMP-PNP and AMP-PCP only partially substituted for ATP. In contrast, GTP gamma S was ineffective. We conclude that ATP is the only soluble factor necessary for optimal activity of the NHE-1 isoform of the antiporter. Mg2+ does not appear to be essential for the stimulatory effect of ATP. We propose that two mechanisms mediate the activation of the antiporter by ATP: one requires hydrolysis and is likely an energy-dependent event. The second process does not involve hydrolysis of the gamma-phosphate, excluding mediation by protein or lipid kinases. We suggest that this effect is due to binding of ATP to an as yet unidentified, nondiffusible effector that activates the antiporter.
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PMID:ATP dependence of Na+/H+ exchange. Nucleotide specificity and assessment of the role of phospholipids. 904 42

Under resting conditions the mammalian distal colon is a NaCl-absorptive epithelium. NaCl absorption occurs at surface cells in colonic crypts. Intracellular Ca2+ or cAMP are important second messengers that activate NaCl secretion, a function that is most pronounced in crypt bases. In the present study we examined the effect of extracellular ATP on isolated crypts of rat distal colon using the fura-2 technique. Intracellular Ca2+ ([Ca2+]i) was measured spectrofluorimetrically either by photon counting or video imaging. ATP reversibly increased [Ca2+]i in crypt base cells with an EC50 of 4.5 micromol/l (n = 11). This [Ca2+]i increase was composed of an initial peak, reflecting intracellular store release, and a secondary plateau phase reflecting transmembrane influx. Digital video imaging revealed that agonist-induced [Ca2+]i elevations were most marked at the crypt base. In the middle part of the crypt ATP induced smaller increases of [Ca2+]i (peak and plateau) as compared to basal cells and in surface cells this [Ca2+]i transient was even further reduced. Attempts to identify the relevant P2-receptor demonstrated the following rank order of potency: 2MeS-ATP > ADP >/= ATP >> AMP > UTP > AMP-PCP > adenosine. In Ussing chamber experiments ATP (1 mmol/l) functioned as a secretagogue, increasing transepithelial voltage (Vte) and equivalent short-circuit current (Isc): Delta Isc = -36.4 +/- 5.4 microA/cm2, n = 17. Adenosine itself (1 mmol/l) induced an increase of Isc of similar magnitude to that induced by ATP: Delta Isc = -55. 1 +/- 8.4 microA/cm2, n = 9. The effect of adenosine, but not that of ATP, was fully inhibited by the A1/A2-receptor antagonist 8-(p-sulphophenyl)theophylline, 0.5 mmol/l, n = 4. Together these data indicate that: (1) basolateral ATP induces [Ca2+]i in isolated rat colonic crypts and acts as a secretagogue in the distal rat colon; (2) a basolateral P2Y-receptor is responsible for this ATP-induced NaCl secretion; (3) the ability of ATP to increase Isc in Ussing chamber experiments is not mediated via adenosine; and (4) the agonist-induced [Ca2+]i signals are mostly located in the crypt base, which is the secretory part of the colonic crypt.
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PMID:ATP increases [Ca2+]i and ion secretion via a basolateral P2Y-receptor in rat distal colonic mucosa. 909 58

Recent studies have suggested that a variety of ion channels possess a binding site for ligands such as phencyclidine (PCP), dizocilpine and certain sigma ligands and that some imidazoline compounds can also bind to this site. We have investigated whether interaction with this binding site could account for the ability of imidazolines to stimulate insulin secretion from rat islets. Neither PCP nor dizocilpine shared the insulin secretory activity of the imidazoline efaroxan in rat islets suggesting that they do not have similar actions in the pancreatic B-cell. Further, we were able to define a new antagonist, KU14R (2(2-ethyl 2,3-dihydro-2-benzofuranyl)-2-imidazole), which selectively blocks the insulin secretory response to imidazolines. The results suggest that imidazolines do not stimulate insulin secretion by causing physical blockade of the K(+)-ATP channel in pancreatic B-cells and show that their effects are not reproduced by PCP or sigma receptor ligands.
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PMID:Evidence that the ability of imidazoline compounds to stimulate insulin secretion is not due to interaction with sigma receptors. 912 45


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