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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

CHO-K1 cells were examined for their cellular responses to the P2 receptor agonist, 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (DbATP), and for the presence of mRNA for P2X receptors. Reverse transcriptase-polymerase chain reactions, using primers directed against the rat P2X subunits, detected the presence of P2X7 but not P2X1-P2X6 subunits. DbATP (EC50 approximately equal to 100 microM) evoked non-desensitizing inward currents which reversed at approximately equal to 0 mV, suggesting activation of a non-selective cation channel. ATP also evoked inward currents but was less potent than DbATP. DbATP also stimulated the accumulation of 45calcium (45Ca2+) and the DNA binding dye, YO-PRO-1, in CHO-KI cells. Both responses were inhibited by NaCl and MgCl2. In 280 mM sucrose buffer, 45Ca2+ accumulation was measurable within 10-20 s of agonist addition, whereas YO-PRO-1 accumulation was only detectable after 8 min. ATP and ATPgammaS were also agonists but were less potent than DbATP, while UTP, 2-methylthio ATP, ADP and (alphabeta)methylene ATP were inactive at concentrations up to 100 microM. DbATP increased lactate dehydrogenase release from CHO-K1 cells, suggesting cell lysis, although this effect was only pronounced after 60-90 min. These data suggest that CHO-K1 cells express an endogenous P2X7 receptor which can be activated by DbATP to cause a rapid inward current and accumulation of 45Ca2+. Prolonged receptor activation results in a delayed, increased permeability to larger molecules such as YO-PRO-1 and ultimately leads to cell lysis. Importantly, the presence of an endogenous P2X7 receptor should be considered when these cells are used to study recombinant P2X receptors.
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PMID:Identification and characterization of an endogenous P2X7 (P2Z) receptor in CHO-K1 cells. 986 47

Several organic anions are actively extruded from intestinal epithelial cells into the lumen and vascular sides. To examine the role of the multidrug resistance-associated protein (MRP) family in the intestinal efflux of organic anions, the function and expression of these proteins were investigated with Caco-2, a human adenocarcinoma cell line that retains many of the characteristics of normal enterocytes. [(3)H]2,4-Dinitrophenyl-S-glutathione (DNP-SG) and [(3)H]17beta-estradiol 17-beta-D-glucuronide (E(2)17betaG), typical substrates for MRP1 and cMOAT (canalicular multispecific organic anion transporter)/MRP2, were taken up into brush-border membrane vesicles (BBMVs) from Caco-2 in an ATP-dependent manner, with K(m) values of 16.9 +/- 7.2 and 9.4 +/- 1.2 microM, respectively. The uptake of [(3)H]DNP-SG into BBMVs was osmotically sensitive and stimulated to some extent by other nucleotide triphosphates (GTP, CTP, and UTP) but not by ADP or AMP. An ATPase inhibitor, vanadate, inhibited the ATP-dependent uptake of [(3)H]DNP-SG to some extent. Reverse-transcriptase polymerase chain reaction resulted in the amplification of MRP1, MRP3, and MRP5. Northern blot analysis indicated extensive expression of cMOAT/MRP2 and MRP3 and only minimal expression of MRP1 and MRP5. Although cMOAT/MRP2 was continuously expressed throughout the culture period, MRP3 was not expressed immediately after the confluent state was reached. Collectively, the presence of ATP-dependent transport systems for DNP-SG and E(2)17betaG was demonstrated in Caco-2 cells. Because cMOAT/MRP2 and MRP3 may be expressed on brush-border and basolateral membranes in epithelial cells, respectively, the transport activity associated with BBMVs may result from the function of cMOAT/MRP2.
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PMID:Function and expression of multidrug resistance-associated protein family in human colon adenocarcinoma cells (Caco-2). 1060 57

Extracellular ATP can function as a glial trophic factor as well as a neuronal transmitter. In astrocytes, mitogenic signalling by ATP is mediated by metabotropic P(2Y) receptors that are linked to the extracellular signal regulated protein kinase (Erk) cascade, but the types of P(2Y) receptors expressed in astrocytes have not been defined and it is not known whether all P(2Y) receptor subtypes are coupled to Erk by identical or distinct signalling pathways. We found that the P(2Y) receptor agonists ATP, ADP, UTP and 2-methylthioATP (2MeSATP) activated Erk and its upstream activator MAP/Erk kinase (Mek). cRaf-1, the first kinase in the Erk cascade, was activated by 2MeSATP, ADP and UTP but, surprisingly, cRaf-1 was not stimulated by ATP. Furthermore, ATP did not activate B-Raf, the major isoform of Raf in the brain, nor other Mek activators such as Mek kinase 1 (MekK1) and MekK2/3. Reverse transcriptase-polymerase chain reaction (RT - PCR) studies using primer pairs for cloned rat P(2Y) receptors revealed that rat cortical astrocytes express P(2Y(1)), a receptor subtype stimulated by ATP and ADP and their 2MeS analogues, as well as P(2Y(2)) and P(2Y(4)), subtypes in rats for which ATP and UTP are equipotent. Transcripts for P(2Y(6)), a pyrimidine-preferring receptor, were not detected. ATP did not increase cyclic AMP levels, suggesting that P(2Y(11)), an ATP-preferring receptor, is not expressed or is not linked to adenylyl cyclase in rat cortical astrocytes. These signal transduction and RT - PCR experiments reveal differences in the activation of cRaf-1 by P(2Y) receptor agonists that are inconsistent with properties of the P(2Y(1)), P(2Y(2)) and P(2Y(4)) receptors shown to be expressed in astrocytes, i.e. ATP=UTP; ATP=2MeSATP, ADP. This suggests that the properties of the native P(2Y) receptors coupled to the Erk cascade differ from the recombinant P(2Y) receptors or that astrocytes express novel purine-preferring and pyrimidine-preferring receptors coupled to the ERK cascade.
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PMID:P(2Y) purinoceptor subtypes recruit different mek activators in astrocytes. 1069 92

1. The mobilization of Ca2+ by purinoceptor activation and the relative contributions of intra- and extracellular sources of Ca2+ were investigated using microfluorimetric measurements of fura-2 loaded in cultured neurones from rat intracardiac ganglia. 2. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed expression of mRNA for the G protein-coupled P2Y2 and P2Y4 receptors. 3. Brief application of either 300 microM ATP or 300 microM UTP caused transient increases in [Ca2+]i of 277 +/- 22 nM and 267 +/- 39 nM, respectively. Removal of external Ca2+ did not significantly reduce these [Ca2+]i responses. 4. The order of purinoceptor agonist potency for [Ca2+]i increases was ATP = UTP > 2-MeSATP > ADP >> adenosine, consistent with the profile for P2Y2 purinoceptors. ATP- and UTP-induced rises in [Ca2+]i were completely and reversibly blocked by 10 microM PPADS (a P2 purinoceptor antagonist) and partially inhibited by 100 microM suramin (a relatively non-specific purinoceptor antagonist). 5. In the presence of the endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (10 microM) in Ca2+-free media, the [Ca2+]i responses evoked by ATP were progressively decreased and abolished. 6. ATP- and UTP-induced [Ca2+]i rises were insensitive to pertussis toxin, caffeine (5 mM) and ryanodine (10 microM) but were significantly reduced by U-73122, a phospholipase C (PLC) inhibitor. 7. In fura-2-loaded cells, perforated patch whole-cell recordings show that ATP and UTP evoked slow outward currents at -60 mV, concomitant with the rise in [Ca2+]i, in approximately 30 % of rat intracardiac neurones. 8. In conclusion, these results suggest that in r intracardiac neurones, ATP binds to P2Y2 purinoceptors to transiently raise [Ca2+]i and activate an outward current. The signalling pathway appears to involve a PTX-insensitive G protein coupled to PLC generation of IP3 which triggers the release of Ca2+ from a ryanodine-insensitive Ca2+ store(s).
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PMID:P2Y purinoceptor activation mobilizes intracellular Ca2+ and induces a membrane current in rat intracardiac neurones. 1089 18

The effects of extracellular ATP, ADP, AMP and adenosine on cAMP accumulation have been studied in freshly isolated B-lymphocytes from patients with chronic lymphocytic leukemia. Extracellular ATP and several nucleotide analogs stimulated cAMP accumulation with the following order of potency: ATP (EC(50)=120+/-20 microM)>ADP>>AMP. ADP was less effective than ATP and may be a partial agonist. AMP exhibited variable but generally weak activity. The stable analog of ATP, alpha,beta-methylene ATP (EC(50)=110+/-15 microM) also stimulated cAMP accumulation and exhibited similar efficacy to ATP. The P2Y(2) receptor agonist, UTP had no effect on intracellular cAMP levels. Adenosine and the A(2A)/A(2B) receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) also stimulated cAMP accumulation in CLL lymphocytes. Adenosine deaminase inhibited the cAMP response to adenosine but had no effect on the ATP-induced cAMP response. On the other hand, the AMP analog, adenosine 5'-thiomonophosphate, (AMPS; 1.0 mM) inhibited ATP-induced and alpha,beta-methylene ATP-induced cAMP production but had no effect on adenosine-induced cAMP production. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the presence of P2Y(11) receptor as well as A(2A) and A(2B) receptor mRNA in chronic lymphocytic leukemia lymphocytes. However, A(2B) receptors would appear to be relatively ineffective because the A(2A) selective agonist, CGS-21680 exhibited comparable efficacy to NECA. Furthermore, the A(2A)-selective antagonist 8-(3-chlorostyryl)-caffeine (CSC) right-shifted the concentration-response curve for NECA. Taken together, the data indicate that ATP induces cAMP accumulation via the activation of P2Y(11) receptors whereas adenosine induces cAMP accumulation via the activation of A(2A) receptors. Coordinate activation of P2Y(11) and A(2A) receptors may influence the developmental fate of normal B-lymphocytes.
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PMID:P2Y(11) receptor expression by human lymphocytes: evidence for two cAMP-linked purinoceptors. 1152 39

In the search for P2-receptors modulating the stimulation-evoked entry of calcium at processes of PC12 cells differentiated in the presence of nerve growth factor and neurotrophin-3, electrically evoked increases in free calcium were assessed by fura-2 microfluorimetry. Omission of calcium and addition of cadmium (100 microM) or the N-type calcium channel blocker omega-conotoxin GVIA (0.5 microM) abolished or markedly reduced the evoked responses. The P2Y-receptor agonists 2-methylthio adenosine 5'-diphosphate (2-methylthio-ADP), ADP, and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) inhibited the electrically evoked entry of calcium without any changes in basal calcium concentrations. 2-Methylthio-ADP was the most potent agonist. Adenosine, P(1),P(4)-di(adenosine-5')-tetraphosphate (Ap4A), UDP, and UTP (30 microM each) had no effect. The effect of ADPbetaS (30 microM) was abolished by the P2-antagonists reactive blue 2 (3 microM), suramin (100 microM), 2-methylthio-AMP (10 microM), p-chloromercuriphenyl sulfonic acid (1 microM), and AR-C 69931MX [N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene adenosine 5'-triphosphate] (300 nM). In contrast, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (10 microM), the selective P2Y1-receptor antagonist MRS 2179 (N(6)-methyl-2'-deoxyadenosine 3',5'-bisphosphate; 10 microM), as well as the adenosine A(1)-receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine; 100 nM), caused no change. Pretreatment with pertussis toxin abolished the effect of ADPbetaS. Reverse transcriptase-polymerase chain reaction revealed the presence of mRNA for P2Y12-receptors in nondifferentiated and differentiated PC12 cells. The results indicate that processes of differentiated PC12 cells possess P2Y12-receptors coupling to pertussis toxin-sensitive G-proteins and mediating an inhibition of the stimulation-evoked entry of calcium through omega-conotoxin GVIA-sensitive calcium channels. This suggests a role of P2Y12-receptors in neuromodulation in addition to their involvement in platelet aggregation.
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PMID:P2Y-receptors mediating an inhibition of the evoked entry of calcium through N-type calcium channels at neuronal processes. 1238 31

The contribution of P2 receptors to vasoconstriction of mouse mesenteric arteries was determined using wild-type (WT) and P2X(1) receptor-deficient (KO) animals. alpha,beta-methylene ATP (alpha,beta-meATP) and ATP evoked transient inward currents and constrictions of WT mesenteric arteries. In contrast, alpha,beta-meATP (100 microM) and ATP (100 microM) failed to evoke responses in KO arteries from a range of vascular beds. Nerve stimulation (100 pulses at 10 Hz) evoked constrictions of mesenteric arteries. For WT arteries, the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2'-5'-disulfonate (PPADS) (30 microM) reduced the amplitude of response by approximately 50%; the residual constriction was abolished by prazosin (0.1 microM). In KO mice, vasoconstriction induced by nerve stimulation was reduced in amplitude by approximately 50%, unaffected by PPADS, but was abolished by prazosin. ADP (1 mM) (a P2Y(1), P2Y(12), and P2Y(13) receptor agonist) was ineffective. Because ATP had no effect on mesenteric artery tone from KO mice, this rules out the contribution of P2Y(2) receptors. The P2Y(4) receptor agonist ITP also failed to contract mesenteric arteries. However, UTP and UDP evoked sustained contractions of mesenteric arteries with similar potency (EC(50) approximately 10 microM). Complementary studies using reverse-transcriptase polymerase chain reaction showed that mesenteric arteries express P2Y(1), P2Y(2), and P2Y(6) receptors. These results demonstrate that homomeric P2X(1) receptors underlie the artery smooth muscle P2X receptor phenotype and contribute approximately 50% to sympathetic neurogenic vasoconstriction and indicate the presence of a UTP- and UDP-sensitive P2Y(6)-like receptor, but not vasoconstrictor P2Y(2) or P2Y(4) receptors, on mouse mesenteric arteries.
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PMID:P2X(1) receptor-deficient mice establish the native P2X receptor and a P2Y6-like receptor in arteries. 1243 12

1 We have investigated increases in cytosolic Ca(2+) in response to nucleotides in mixed rat cerebrocortical cultures (neurons and glia in similar numbers) and in essentially neuron-free glial cultures. 2 In both cultures, the agonist-response profile was 2-methylthioADP(2MeSADP)>2-methylthioATP(2MeSATP)>ADP>ATP>adenosine 5'-O-(3-thiotriphosphate), consistent with a P2Y(1) receptor. The maximal responses to 2MeSADP, 2MeSATP and ADP were identical, but that to ATP was higher. 3 Suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid, reactive blue 2 (RB2), and adenosine biphosphate (A3P5P) were antagonists with apparent pA(2) values of 5.5 for suramin, 6.4 for RB2, and 4.7 for A3P5P. 4 Single cell imaging divided the cells from the mixed neuronal-glial cultures into two populations: responsive (neurons) and unresponsive (glial cells) to high [K(+)]. The response of cells to nucleotides was almost exclusively limited to those not responsive to high K(+). 5 In the presence of extracellular Mn(2+), the response of the mixed cultures to 30 mM K(+) and 20 micro M Bay K 8644 was attenuated. However, when 2MeSADP was added there was no reduction in response in cultures previously loaded with Mn(2+). This further indicated that the 2MeSADP response was not in the neurons. 6 Reverse transcriptase-polymerase chain reaction studies detected transcripts for P2Y(1), P2Y(4) and P2Y(6) in RNA preparations from embryonic rat cortex, and from both mixed and glial cultures. P2Y(2) transcripts were not detected in the embryonic cortex. 7 Based on this and previous work, it is proposed that the principal P2Y influences in the brain are on cytosolic Ca(2+) in glial cells and presynaptic sites on neurons.
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PMID:P2Y receptor regulation of cultured rat cerebral cortical cells: calcium responses and mRNA expression in neurons and glia. 1277 Sep 33

Stroking the mucosal lining of the guinea pig colon with a brush elicits an intestinal neural reflex, and an increase in short-circuit current (Isc) indicative of chloride secretion. We tested whether endogenous and exogenous nucleotides are physiologic regulators of mucosal reflexes that modulate chloride secretion. The basal Isc was augmented by 6-N,N-diethyl-beta,gamma-dibromomethylene-D-adenosine-5'-triphosphate (ARL67156) inhibition of nucleotide breakdown or adenosine A1 receptor blockade and reduced by apyrase inactivation of nucleotidases, P2 receptor antagonists, tetrodotoxin (TTX), or piroxicam. ARL67156 augmented, and apyrase inhibited, stroking-evoked Isc responses. TTX and atropine inhibited nucleotide-evoked Isc responses. The agonist potency profile for Isc, 2-methylthioadenosine-diphosphate (2MeSADP) = 2-methioadenosine-triphosphate >> 5'adenosine-triphosphate (ATP) > or = 5'adenosine-diphosphate > 5'uridine-triphosphate > or = 5'uridine-diphosphate, supports a P2Y1 receptor (R). The P2 receptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2'4'-disulfonic acid, reduced stroking responses (36%) and their effects were additive. The selective P2Y1 R antagonist, 2'deoxy-N6-methyl adenosine 3',5'-diphosphate diammonium salt, reduced stroking (54%) and 2MeSADP (70%) responses at P2Y1 Rs. The P2X1/3 R agonist, alpha,betaMeATP, increased Isc. A desensitizing dose of alpha,betaMeATP reduced stroking Isc responses but did not prevent the 2MeSADP-evoked Isc response. Reverse transcriptase polymerase chain reaction analysis revealed mRNAs for P2Y1 R, P2Y2 R, P2Y4 R, P2Y6 R, and P2Y12 R in submucosa. The expression of P2Y R immunoreactivity (ir) in cell bodies of submucous neurons followed the order of P2Y1 = P2Y2 >> P2Y4 R ir; P2Y1 Rs and P2Y2 R ir were abundant (21-50% of neurons). P2Y1 R ir was abundant in cholinergic secretomotor neurons and fewer than 2% of neuropeptide Y (NPY)/choline acetyltransferase secretomotor neurons, and P2Y2 R ir was expressed in virtually all NPY secretomotor neurons and approximately 30% of calbindin/intrinsic primary afferent neurons. P2Y4 R ir was present in NPY-positive neurons. P2Y ir was rare or absent in varicose nerve fibers. The functional data support the hypothesis that mechanical stimulation with a brush releases nucleotides that act predominantly at P2Y1 Rs and to a lesser extent on P2X1/3 Rs to mediate reflex chloride secretion. A separate P2Y2 R neural circuit pathway exists that is not activated by mechanical forces. Other receptors including P2Y4, P2Y6, P2Y12, or P4 Rs cannot be excluded.
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PMID:Mechanical stimulation releases nucleotides that activate P2Y1 receptors to trigger neural reflex chloride secretion in guinea pig distal colon. 1468 70

The arginine deiminase system (ADS) is of critical importance in oral biofilm pH homeostasis and microbial ecology. The ADS consists of three enzymes. Arginine is hydrolyzed by AD (ArcA) to generate citrulline and ammonia. Citrulline is then converted to ornithine and carbamoylphosphate via ornithine carbamoyltransferase (ArcB). Finally, carbamate kinase (ArcC) transfers a phosphate from carbamoylphosphate to ADP, yielding ATP. Ammonia production from this pathway protects bacteria from lethal acidification, and ATP production provides a source of energy for the cells. The purpose of this study was to initiate a characterization of the arc operon of Streptococcus rattus, the least cariogenic and sole ADS-positive member of the mutans streptococci. Using an arcB gene fragment obtained by degenerate PCRs, the FA-1 arc operon was identified in subgenomic DNA libraries and sequence analysis was performed. Results showed that the genes encoding the AD pathway in S. rattus FA-1 are organized as an arcABCDT-adiR operon gene cluster, including the enzymes of the pathway, an arginine-ornithine antiporter (ArcD) and a putative regulatory protein (AdiR). The arcA transcriptional start site was identified by primer extension, and a sigma(70)-like promoter was mapped 5' to arcA. Reverse transcriptase PCR was used to establish that arcABCDT could be cotranscribed. Reporter gene fusions and AD assays demonstrated that the operon is regulated by substrate induction and catabolite repression, the latter apparently through a CcpA-dependent pathway.
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PMID:Characterization of the arginine deiminase operon of Streptococcus rattus FA-1. 1500 49


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