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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Effects of a putative A2-adenosine receptor agonist 2-[(p-2-carboxyethyl)-phenethylamino]-5'-N-ethyl-carboxamide-adeno sine (CGS 21680C) on force of contraction, protein phosphorylation, cyclic adenosine monophosphate (cAMP) content, and the activity of phosphodiesterase (PDE) isoenzymes in guinea pig ventricular (GPV) preparations were studied. CGS 21680C (1-100 microM) did not affect force of contraction in isolated electrically driven papillary muscles and was ineffective in increasing phosphorylation of phospholamban (PLB) and the inhibitory subunit of troponin (TnI) in [32P]-labeled GPV cardiomyocytes. However, under the same conditions, CGS 21680C (10 microM) increased cAMP content from 4.3 +/- 0.2 to 13.0 +/- 0.6 pmol/mg protein, and this effect was completely abolished by A2-adenosine receptor antagonist 9-chloro-2-(2-furanyl)-5,6-dihydro-1,2,4-triazolo-(1,5-c)quinazolin++ +-5-imine (CGS 15943A). CGS 21680C (10 microM) inhibited PDE isoenzymes I, II, III, IV by 7.0, 8.3, 4.7, and 23.2%, respectively. Similarly, rolipram (100 microM), a selective PDE IV inhibitor, increased cAMP content from 4.4 +/- 0.3 to 7.2 +/- 0.3 pmol/mg protein without affecting the phosphorylation state of PLB and TnI. We conclude that CGS 21680C increases cAMP content in GPV cardiomyocytes by activation of adenylyl cyclase or in part by inhibition of PDE IV activity. The increase in cAMP content by CGS 21680C or rolipram is ineffective in increasing phosphorylation of PLB and TnI. These results support the concept of compartments for cAMP or protein kinase A or both in cardiomyocytes that are not coupled to phosphorylation and contractility.
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PMID:Characterization of biochemical effects of CGS 21680C, an A2-adenosine receptor agonist, in the mammalian ventricle. 943 14

1. The present study was undertaken to determine the role of adenosine in mediating the cellular responses to hypoxia in rat phaeochromocytoma (PC12) cells, an oxygen-sensitive clonal cell line. 2. Reverse transcriptase polymerase chain reaction studies revealed that PC12 cells express adenosine deaminase (the first catalysing enzyme of adenosine degradation) and the A2A and A2B adenosine receptors, but not the A1 or A3 adenosine receptors. 3. Whole-cell current- and voltage-clamp experiments showed that adenosine attenuated the hypoxia-induced membrane depolarization. The hypoxia-induced suppression of the voltage-sensitive potassium current (IK(V)) was markedly reduced by adenosine. Furthermore, extracellularly applied adenosine increased the peak amplitudes of IK(V) in a concentration-dependent manner. This increase was blocked by pretreatment not only with a non-specific adenosine receptor antagonist, 8-phenyltheophylline (8-PT), but also with a selective A2A receptor antagonist, ZM241385. 4. Ca2+ imaging studies using fura-2 acetoxymethyl ester (fura-2 AM) revealed that the increase in intracellular free Ca2+ during hypoxic exposure was attenuated significantly by adenosine. Voltage-clamp studies showed that adenosine inhibited the voltage-dependent Ca2+ currents (ICa) in a concentration-dependent fashion. This inhibition was also abolished by both 8-PT and ZM241385. 5. The modulation of both IK(V) and ICa by adenosine was prevented by intracellular application of an inhibitor of protein kinase A (PKA), PKA inhibitor fragment (6-22) amide. In addition, the effect of adenosine on either IK(V) or ICa was absent in PKA-deficient PC12 cells. 6. These results indicate that the modulatory effects of adenosine on the hypoxia-induced membrane responses of PC12 cells are likely to be mediated via activation of the A2A receptor, and that the PKA pathway is required for these modulatory actions. We propose that this modulation serves to regulate membrane excitability in PC12 cells and possibly other oxygen-sensitive cells during hypoxia.
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PMID:Adenosine modulates hypoxia-induced responses in rat PC12 cells via the A2A receptor. 949 Aug 23

Using receptor-selective agonists and antagonists, the possible presence of both A2a and A2b adenosine receptor subtypes coupled to activation of adenylyl cyclase was investigated in NG108-15 neuroblastoma x glioma hybrid cells. The relatively non-selective adenosine receptor agonist 5'-(N-ethyl carboxamido)-adenosine (NECA; 1 nM-300 microM) produced a biphasic increase in adenylyl cyclase activity in cell homogenates, best fitted to two components with high (EC50 0.7 microM) and low (EC50 16.0 microM) potency, respectively. The selective adenosine A2a receptor agonist CGS-21680 (1 nM-300 microM) also produced a biphasic increase in adenylyl cyclase. The NECA-dependent increase in adenylyl cyclase activity was almost completely inhibited by the non-selective adenosine receptor antagonist xanthine amine congener (XAC; 30 microM), but only partially inhibited by the selective A2a adenosine antagonist 8-(3-chlorostyryl)caffeine (CSC; 1 microM). Experiments were also performed to investigate the time course of NECA-induced desensitization of putative A2a and A2b receptor responses. The A2a-response was quantified using 10 microM CGS-21680, whilst the A2b response was quantified using 100 microM NECA in the presence of 1 microM CSC. The t0.5 for desensitization for each subtype was found to be around 20 min. Neither activation (with dibutyryl cAMP; 1 mM) nor inhibition (with H-89; 10 microM) of cyclic AMP-dependent protein kinase altered the ability of NECA pretreatment to desensitize A2a or A2b receptor-activated adenylyl cyclase. However zinc (200 microM), an inhibitor of G-protein coupled receptor kinase 2 (GRK2), significantly reversed the agonist-induced desensitization of A2a and A2b receptor-activated adenylyl cyclase. These experiments suggest the co-existence of A2a and A2b receptors coupled in a stimulatory fashion to adenylyl cyclase in NG108-15 cells. Furthermore desensitization of A2a and A2b responses occurs at the same rate and may involve a G-protein-coupled receptor kinase.
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PMID:Evidence for co-expression and desensitization of A2a and A2b adenosine receptors in NG108-15 cells. 951 70

1. The effects of adenosine triphosphate (ATP), adenosine diphosphate (ADP), alpha,beta-methylene-ATP (alpha,beta-MeATP) and 2-methylthio-ATP (2-MeSATP) on longitudinally orientated smooth muscle strips from marmoset urinary bladder were investigated by use of standard organ bath techniques. 2. After being mounted in superfusion organ baths, 66.7% (n=249) of marmoset detrusor smooth muscle strips developed spontaneous tone, 48.2% of all strips examined developed tone equivalent to greater than 0.1 g mg(-1) of tissue and were subsequently utilized in the present investigation. 3. On exposure to ATP, muscle strips exhibited a biphasic response, a rapid and transient contraction followed by a more prolonged relaxation. Both responses were found to be concentration-dependent. ADP and 2-MeSATP elicited a similar response (contraction followed by relaxation), whereas application of alpha,beta-MeATP only produced a contraction. The potency order for each effect was alpha,beta-MeATP> >2-MeSATP> ATP>ADP (contractile response) and ATP=2-MeSATP> or = ADP> > alpha,beta-MeATP (relaxational response). 4. Desensitization with alpha,beta-MeATP (10 microM) abolished the contractile phase of the response to ATP, but had no effect on the level of relaxation evoked by this agonist. On the other hand, the G-protein inactivator, GDPbetaS (100 microM) abolished only the relaxation response to ATP. Suramin (general P2 antagonist, 100 microM) shifted both the contractile and relaxation ATP concentration-response curves to the right, whereas cibacron blue (P2Y antagonist, 10 microM) only antagonized the relaxation response to ATP. In contrast, the adenosine receptor antagonist, 8-phenyltheophylline (10 microM), had no effect on the relaxation response curve to ATP. 5. Incubation with tetrodotoxin (TTX, 3 microM) or depolarization of the muscle strip with 40 mM K+ Krebs failed to abolish the relaxation to ATP. In addition, neither Nomega-nitro-L-arginine (L-NOARG, 10 microM) nor methylene blue (10 microM) had any effect on the relaxation response curve. However, tos-phe-chloromethylketone (TPCK, 3 microM), an inhibitor of cyclicAMP-dependent protein kinase A (PKA), significantly (P<0.01) shifted the curve for the ATP-induced relaxation to the right. 6. It is proposed that marmoset detrusor smooth muscle contains two receptors for ATP, a classical P2X-type receptor mediating smooth muscle contraction, and a P2Y (G-protein linked) receptor mediating smooth muscle relaxation. The results also indicate that the ATP-evoked relaxation may occur through the activation of cyclicAMP-dependent PKA.
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PMID:Purinoceptor subtypes mediating contraction and relaxation of marmoset urinary bladder smooth muscle. 960 64

Adenosine increased the DNA synthesis rate and the percentage of S-phase cells 2-3-fold in mouse mammary epithelial cells (NMuMG), with an optimum concentration of 10-100 microM. This effect was not mimicked by adenosine metabolites adenine, hypoxanthine, or inosine. N-ethylcarboxamidoadenosine (NECA, a relatively nonselective adenosine receptor agonist) and 2-p-(2-carboxyethyl) phenethylamino-5'-N ethylcarbox-amidoadenosine (CGS-21680, an A2 selective agonist) also increased DNA synthesis by mammary epithelial cells. However, N6-cyclohexyladenosine (CHA, an agonist for A1 type receptors) decreased DNA synthesis. The A1 selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) had no effect on basal or adenosine-induced DNA synthesis, whereas the A2 selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) decreased adenosine-induced DNA synthesis. Similar effects were observed in another nontumorigenic mouse mammary epithelial line, HC11, as well as the nontumorigenic human lines MCF-10A and 184.A1. Binding studies indicated that NMuMG cells contained approximately 3200 A1 receptors and about 5300 A2 receptors per cell. Both CGS-21680 and CHA increased GTPase activity in isolated cell membranes, whereas only CGS-21680 increased activity of adenylyl cyclase. Adenosine and CGS-21680 increased expression of a cyclic AMP responsive reporter gene. In addition, the protein kinase A inhibitor H-89 blocked the ability of adenosine and CGS-21680 to induce DNA synthesis, but did not affect EGF-induced DNA synthesis. These results indicate that adenosine appears to be a possible growth promoting agent in mammary tissue, and this effect may be mediated by extracellular receptors of the A2 type.
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PMID:Adenosine stimulation of DNA synthesis in mammary epithelial cells. 971 78

The role of the A2A adenosine receptor in regulating voltage-sensitive calcium channels (VSCCs) was investigated in PC12 cells. Ca2+ influx induced by membrane depolarization with 70 mM K+ could be inhibited with CGS21680, an A2A receptor-specific agonist. Both L- and N-type VSCCs were inhibited by CGS21680 treatment. Effects of adenosine receptor agonists and antagonists indicate that the typical A2A receptor mediates inhibition of VSCCs. Cholera toxin (CTX) treatment for 24 h completely eliminated the CGS21680 potency. Similar inhibitory effects on VSCCs were obtained by membrane-permeable activators of protein kinase A (PKA). These effects were blocked by Rp-adenosine-3',5'-cyclic monophosphothioate, a PKA inhibitor. The data suggest that activation of the A2A receptor leads to inhibition of VSCCs via a CTX-sensitive G protein and PKA. ATP pretreatment caused a reduction in subsequent rise in cytosolic free Ca2+ concentration induced by 70 mM K+, presumably by inactivation of VSCCs. Simultaneous treatment with ATP and CGS21680 produced significantly greater inhibition of VSCCs than treatment with CGS21680 or ATP alone. Furthermore, the CGS21680-induced inhibition of VSCCs was not affected by the presence of reactive blue 2. CGS21680 still significantly inhibited ATP-evoked Ca2+ influx without VSCC activity after cobalt or 70 mM K+ pretreatment. These data suggest that the A2A receptor-sensitive VSCCs differ from those activated by ATP treatment. Although A2A receptors induce inhibition of VSCCs as well as ATP-induced Ca2+ influx, the two inhibitory effects are clearly distinct from each other.
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PMID:Inhibition of voltage-sensitive calcium channels by the A2A adenosine receptor in PC12 cells. 972 51

1. Extracellular ATP (EC50=146+/-57 microM) and various ATP analogues activated cyclic AMP production in undifferentiated HL-60 cells. 2. The order of agonist potency was: ATPgammaS (adenosine 5'-O-[3-thiotriphosphate]) > or = BzATP (2'&3'O-(4-benzoylbenzoyl)-adenosine-5'-triphosphate) > or = dATP > ATP. The following agonists (in order of effectiveness at 1 mM) were all less effective than ATP at concentrations up to 1 mM: beta,gamma methylene ATP > or = 2-methylthioATP > ADP > or = Ap4A (P1, P4-di(adenosine-5') tetraphosphate) > or = Adenosine > UTP. The poor response to UTP indicates that P2Y2 receptors are not responsible for ATP-dependent activation of adenylyl cyclase. 3. Several thiophosphorylated analogs of ATP were more potent activators of cyclic AMP production than ATP. Of these, ATPgammaS (EC50=30.4+/-6.9 microM) was a full agonist. However, adenosine 5'-O-[1-thiotriphosphate] (ATPalphaS; EC50=45+/-15 microM) and adenosine 5'-O-[2-thiodiphosphate] (ADPbetaS; EC50=33.3+/-5.0 microM) were partial agonists. 4. ADPbetaS (IC50=146+/-32 microM) and adenosine 5'-O-thiomonophosphate (AMPS; IC50=343+/-142 microM) inhibited cyclic AMP production by a submaximal concentration of ATP (100 microM). Consistent with its partial agonist activity, ADPbetaS was estimated to maximally suppress ATP-induced cyclic AMP production by about 65%. AMPS has not been previously reported to inhibit P2 receptors. 5. The broad spectrum P2 receptor antagonist, suramin (500 microM), abolished ATP-stimulated cyclic AMP production by HL-60 cells but the adenosine receptor antagonists xanthine amine congener (XAC; 20 microM) and 8-sulpho-phenyltheophylline (8-SPT; 100 microM) were without effect. 6. Extracellular ATP also activated protein kinase A (PK-A) consistent with previous findings that PK-A activation is involved in ATP-induced differentiation of HL-60 cells (Jiang et al., 1997). 7. Taken together, the data indicate the presence of a novel cyclic AMP-linked P2 receptor on undifferentiated HL-60 cells.
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PMID:Pharmacological profile of a novel cyclic AMP-linked P2 receptor on undifferentiated HL-60 leukemia cells. 972 74

1. Adenosine has been shown to decrease Ca2+ current (ICa) and attenuate the hypoxia-induced enhancement of intracellular free Ca2+ ([Ca2+]i) in oxygen-sensitive rat phaeochromocytoma (PC12) cells. These effects are mediated via the adenosine A2A receptor and protein kinase A (PKA). The current study was undertaken to determine the effects of adenosine on Ca2+ current and hypoxia-induced change in [Ca2+]i during chronic hypoxia. 2. Whole cell patch-clamp studies revealed that the effect of adenosine on ICa was significantly reduced when PC12 cells were exposed to hypoxia (10 % O2) for 24 and 48 h. 3. Ca2+ imaging studies using fura-2 revealed that the anoxia-induced increase in [Ca2+]i was significantly enhanced when PC12 cells were exposed to 10 % O2 for up to 48 h. In contrast, the inhibitory effects of adenosine on anoxia-induced elevation of [Ca2+]i was significantly blunted in PC12 cells exposed to hypoxia for 48 h. 4. Northern blot analysis revealed that mRNA for the A2A receptor, which is the only adenosine receptor subtype expressed in PC12 cells, was significantly upregulated by hypoxia. Radioligand binding analysis with [3H]CGS21680, a selective A2A receptor ligand, showed that the number of adenosine A2A receptor binding sites was similarly increased during exposure to 10% O2 for 48 h. 5. PKA enzyme activity was significantly inhibited when PC12 cells were exposed to 10% O2 for 24 and 48 h. However, we found that hypoxia failed to induce change in adenosine- and forskolin-stimulated adenylate cyclase enzyme activity. Chronic hypoxia also did not alter the immunoreactivity level of the G protein Gsalpha, an effector of the A2 signalling pathway. 6. Whole cell patch-clamp analysis showed that the effect of 8-bromo-cAMP, an activator of PKA, on ICa was significantly attenuated during 48 h exposure to 10% O2.7. We conclude therefore that the reduced effect of adenosine on ICa and [Ca2+]i in PC12 cells exposed to chronic hypoxia is due to hypoxia-induced downregulation of PKA. This mechanism may serve to reduce the negative feedback on ICa and [Ca2+]i by adenosine and therefore maintain enhanced membrane excitability of PC12 cells during long-term hypoxia.
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PMID:Chronic hypoxia reduces adenosine A2A receptor-mediated inhibition of calcium current in rat PC12 cells via downregulation of protein kinase A. 976 26

Effects of YT-146 [2-(1-octynyl) adenosine], an adenosine A2 receptor agonist, on cAMP production and noradrenaline (NA) release were investigated in PC12 cells. YT-146 caused a concentration-dependent cAMP accumulation (EC50: 1.2+/-0.9 nM). In [3H]NA-prelabeled cells, YT-146 increased the basal NA release and enhanced ATP-evoked NA release in a concentration-dependent manner (EC50: 0.23+/-0.15 nM). YT-146 augmented the maximal response to ATP without affecting the EC50 value of ATP. These effects of YT-146 were inhibited by several adenosine receptor antagonists with a characteristic of adenosine A2A receptor subtype. The effects of YT-146 were mimicked by forskolin, dibutylyl cAMP and Sp-cAMPS, and inhibited by H-89, a cAMP-dependent protein kinase inhibitor. YT-146 had little effect on ATP-induced increase in intracellular Ca2+ concentration. YT-146 enhanced the NA release induced by several different stimuli including Ca2+ ionophore A23187. The present results suggest that YT-146 is a potent agonist on adenosine A2A receptors in PC12 cells and causes a cAMP-dependent enhancement of NA release by affecting the exocytosis process at a point downstream of the intracellular Ca2+ increase.
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PMID:Effects of YT-146 [2-(1-octynyl) adenosine], an adenosine A2A receptor agonist, on cAMP production and noradrenaline release in PC12 cells. 986 60

1. Adenosine influences the vectorial transport of Na+ and HCO3- across kidney epithelial cells. However, its action on effector proteins, such as the Na+-H+ exchanger NHE3, an epithelial brush border isoform of the Na+-H+ exchanger (NHE) gene family, is not yet defined. 2. The present study was conducted in Xenopus laevis distal nephron A6 epithelia which express both an apical adenosine receptor of the A1 type (coupled to protein kinase C (PKC)) and a basolateral receptor of the A2 type (coupled to protein kinase A (PKA)). The untransfected A6 cell line expresses a single NHE type (XNHE) which is restricted to the basolateral membrane and which is activated by PKA. 3. A6 cell lines were generated which express exogenous rat NHE3. Measurements of side-specific pHi recovery from acid loads in the presence of HOE694 (an inhibitor with differential potency towards individual NHE isoforms) detected an apical resistant Na+-H+ exchange only in transfected cell lines. The sensitivity of the basolateral NHE to HOE694 was unchanged, suggesting that exogenous NHE3 was restricted to the apical membrane. 4. Stimulation of the apical A1 receptor with N 6-cyclopentyladenosine (CPA) inhibited both apical NHE3 and basolateral XNHE. These effects were mimicked by the addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and partially prevented by the PKC inhibitor calphostin C which also blocked the effect of PMA. 5. Stimulation of the basolateral A2 receptor with CPA inhibited apical NHE3 and stimulated basolateral XNHE. These effects were mimicked by 8-bromo-cAMP and partially prevented by the PKA inhibitor H89 which entirely blocked the effect of 8-bromo-cAMP. 6. In conclusion, CPA inhibits rat NHE3 expressed apically in A6 epithelia via both the apical PKC-coupled A1 and the basolateral PKA-coupled A2 adenosine receptors.
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PMID:Adenosine inhibits the transfected Na+-H+ exchanger NHE3 in Xenopus laevis renal epithelial cells (A6/C1). 1006 8


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