Comparison of the vascular effects of adenosine in isolated mouse heart and aorta.
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The present study was designed to characterize and compare the vascular effects of adenosine and its analogs in the murine heart and aorta. Mouse hearts perfused under constant pressure in standard Langendorff fashion demonstrated concentration-dependent increases in coronary flow to adenosine, 2-chloradenosine (CAD), 5'-(N-ethyl-carboxamido)-adenosine (NECA), and 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxam-idoadenosine (CGS-21680). All agonists produced comparable increases in coronary flow with the following order of potency: CGS-21680 = NECA >> CAD > or = adenosine. In l-phenylephrine hydrochloride (phenylephrine) precontracted aortic rings, all nonselective agonists (NECA, CAD, and adenosine) produced marked concentration-dependent relaxation, whereas the adenosine A(2A) selective agonist CGS-21680 did not. Adenosine receptor agonists were >100 times more potent for coronary vasodilation than aortic vasorelaxation. The selective A(2A) receptor antagonist 5-amino-7-(beta-phenylethyl)-2-(8-furyl)pyrazolo-[4,3-e]-1,2,4-triazolo-[1,5-c]py rimidine (SCH-58261) blocked both CGS-21680- and NECA-induced increases in coronary flow, whereas the A(2B) receptor antagonist benzo[g]pteridine-2,4(1H,3H)-dione (alloxazine) inhibited NECA-induced aortic relaxation. These data indicate a differential response to adenosine agonists in murine coronary vasculature and aorta where coronary vasodilation is mediated predominantly by activation of A(2A) adenosine receptors. (+info)
Effect of adenosine receptor agonists on neurodegenerative and convulsive activity of mitochondrial toxin, 3-nitropropionic acid.
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3-Nitropropionic acid (3-NPA) is a mitochondrial toxin inhibiting the activity of succinate dehydrogenase. Its experimental application in rodents causes lesions of the striatum resembling the course of Huntington's disease in humans. Recently, we have shown that 3-NPA is also a potent convulsive and proconvulsive agent. This study investigated the effects of adenosine receptor agonists on neurodegeneration and convulsions induced by 3-NPA. Adenosinergic agonists prevented seizures but not striatal neuronal loss evoked by 3-NPA, what suggests that different mechanisms might contribute to these pathologies associated with application of mitochondrial toxin. (+info)
Actions of the selective protein kinase C inhibitor PKC412 on B-chronic lymphocytic leukemia cells in vitro.
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BACKGROUND AND OBJECTIVES: The staurosporine derivative PKC412 (CGP41251) is a more selective inhibitor of the conventional isoforms of protein kinase C (PKC) than is the parent compound. In addition to its growth inhibitory properties, PKC412 reverses the efflux function of the multidrug resistance (MDR)-1 gene product, P-glycoprotein (P-gp). DESIGN AND METHODS: The in vitro actions of PKC412 were investigated in peripheral blood lymphocytes (PBL) from 4 normal volunteers, B-cell isolates from 3 normal tonsils and 31 patients with B-cell chronic lymphocytic leukemia (B-CLL). Following incubation with PKC412 for 2 days, the viability of B-CLL cells was decreased relative to that of controls (63+/-23% at 1 micromole/L; 52+/-30% at 10 micromole/L; n=20). Normal PBL were significantly more resistant to the drug (91+/-5% viable cells at 1 micromole/L; 73+/-18% at 10 micromole/L; n=4). Thirteen of the B-CLL patients were treated with oral PKC412 in a phase II trial. RESULTS: PKC activity in malignant cells from these patients showed a reduction post-treatment of 25-96% of their respective pre-treatment levels. Morphologic analysis, as well as in situ assay for DNA strand breaks (TUNEL assay) showed that B-CLL cells were killed by an apoptotic mechanism. In B-CLL cells the mean IC50, for PKC412, as measured by the reduction of 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), was 2.1 micromol/L in 16 samples in which the IC50 were below the maximum concentration of PKC412 used for the assay. In tonsillar B-cells, the mean IC50 was 11 micromol/L whereas PBL cells were resistant. Four of eight and 1/3 B-CLL samples that were resistant to chlorambucil and fludarabine, respectively, were sensitive to PKC412. In 15/31 B-CLL samples a dose-dependent reversal of P-gp-mediated drug efflux by PKC412 was observed. A statistically significant correlation (p<0.001) was observed between P-gp protein expression as measured by FACScan analysis and the reversal of efflux activity by either PKC412 or verapamil. PKC412 increased the sensitivity of B-CLL cells to 2'-chlorodeoxyadenosine and chlorambucil. INTERPRETATION AND CONCLUSIONS: This study establishes the in vitro cytotoxic and multidrug resistance (MDR) modulatory properties of PKC412 towards malignant cells from B-CLL patients. The direct antitumor activity combined with the potential for P-gp modulation make PKC412 an attractive drug for the treatment of malignancies expressing the MDR phenotype, or in combination with conventional drugs. (+info)
Differential desensitization of responses mediated by presynaptic and postsynaptic A1 adenosine receptors.
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G-protein-coupled receptors (GPCRs) often desensitize during continuous activation, but it is not known whether desensitization is influenced by subcellular location. In hippocampal neurons, activation of adenosine A1 receptors (A1Rs) or GABA(B) receptors on synaptic terminals inhibits neurotransmitter release, whereas activation of the same receptors on cell bodies and dendrites decreases excitability by activating inwardly rectifying potassium (GIRK) channels. Here we report that responses mediated by presynaptic A1Rs desensitize more slowly than responses mediated by postsynaptic (somatodendritic) A1Rs in cultured neurons. Agonist treatment for 2 hr has no effect on adenosine-induced presynaptic inhibition, whereas such treatment nearly abolishes adenosine-induced activation of postsynaptic GIRK channels. Agonist treatment for longer periods (>12 hr) eventually desensitizes A1R-mediated presynaptic inhibition. Presynaptic and postsynaptic responses both recover from desensitization after agonist removal, but recovery of presynaptic inhibition requires more time. Desensitization of postsynaptic responses apparently occurs at the level of the receptor, because postsynaptic G-proteins and GIRK channels appear to be fully functional. Inhibition of voltage-gated calcium channels by postsynaptic A1Rs also desensitizes rapidly, although this desensitization is less complete than is observed for activation of postsynaptic GIRK channels. Comparison of concentration-response curves for presynaptic and postsynaptic responses suggests that a receptor reserve exists for presynaptic inhibition, but that the magnitude of this reserve is insufficient to account for the absence of presynaptic desensitization after brief agonist exposure. These results suggest that agonist-induced desensitization of responses mediated by neuronal GPCRs may depend on the subcellular location of the receptors. (+info)
2-Chloroadenosine but not adenosine induces apoptosis in rheumatoid fibroblasts independently of cell surface adenosine receptor signalling.
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1. The apoptotic effect of adenosine and its analogues was studied in fibroblast-like synoviocytes derived from rheumatoid arthritis patients (RA-FLSs). Evoked cell death was quantitatively examined by assessing DNA fragmentation using an enzyme-liked immunosorbent assay and by measuring phosphatidylserine exposure through flow cytometric analysis of annexin V binding. 2. Exposing cells for 24 h to 2-chloroadenosine (2-CADO), a nonspecific, adenosine deaminase (ADA)-resistant, adenosine receptor (AdoR) agonist, induced DNA fragmentation, and thus apoptosis, in RA-FLSs at concentrations > or =50 microM. By contrast, incubation with adenosine for up to 72 h did not evoke DNA fragmentation, even in the presence of ADA inhibitor coformycin and nucleoside transporter inhibitor nitrobenzylmercaptopurin (NBMPR). Transcription of all four AdoR isoforms was detected in RA-FLSs; nevertheless selective AdoR agonists similarly failed to induce DNA fragmentation. 3. DNA fragmentation evoked by 2-CADO was inhibited by NBMPR and by 5'-iodotubercidin, an adenosine kinase inhibitor, but not by xanthine amine congener, an A(1) and A(2) receptor antagonist, or by selective AdoR antagonists. 4. The nonspecific caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone abolished the apoptotic effect of 2-CADO. 5. These results suggest that 2-CADO induces apoptosis in RA-FLSs independently of AdoR-mediated signalling. Instead, 2-CADO, but not adenosine, is taken up into RA-FLSs via human equilibrative nucleoside transporter-1, where it is phosphorylated by adenosine kinase. The resultant phospho-2-CADO induces DNA fragmentation by activating a caspase pathway. (+info)
Inhibition of spontaneous acetylcholine secretion by 2-chloroadenosine as revealed by a protein kinase inhibitor at the mouse neuromuscular junction.
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1. Previous studies have reported discrepancies in the potencies of A(1) adenosine receptor agonists at mouse motor nerve terminals. In addition, conflicting results on the role of protein kinase A (PKA) in mediating the inhibitory effects of A(1) receptor agonists have been published. We thus decided to investigate the possibility of endogenous control of adenosine receptor sensitivity by protein kinases, using a variety of protein kinase inhibitors in conjunction with the adenosine receptor agonist 2-chloroadenosine (CADO). 2. CADO, at the concentration employed previously to study spontaneous ACh release in the mouse (1 microM), did not inhibit spontaneous ACh release in our experiments. However, a higher concentration of CADO (10 microM) produced highly statistically-significant reductions in spontaneous ACh release. 3. In the presence of the non-selective protein kinase inhibitor, H7 (50 microM), the potency of CADO was increased such that 1 microM CADO now reduced spontaneous quantal ACh release to approximately 63% of control. 4. Both H7, and the selective PKA inhibitor, KT5720 (500 nM) prevented increases in ACh release produced by CPT cyclic AMP (250 microM), suggesting these kinase inhibitors were blocking PKA. In contrast to H7, however, KT5720, did not reveal an inhibitory effect of 1 microM CADO. A number of other non-selective PKA inhibitors also failed to increase the potency of CADO. 5. The results suggest that an endogenous H7-sensitive process modulates the sensitivity of the mouse A(1) adenosine receptor and that the inhibitory effects of CADO are independent of cyclic AMP accumulation or PKA inhibition. (+info)
Coronary function and adenosine receptor-mediated responses in ischemic-reperfused mouse heart.
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OBJECTIVES: To assess the impact of ischemia-reperfusion (I/R) on coronary function, and the role of endogenous adenosine in modifying post-ischemic vascular function in asanguinous hearts. METHODS: Vascular function was studied in Langendorff perfused mouse hearts subjected to 20-25-min ischemia and 30-min reperfusion. RESULTS: Ischemia altered the dependence of flow on work-rate observed in normoxic hearts, and inhibited reflow by mechanisms additional to diastolic compression. Coronary responses were selectively reduced: 2-chloroadenosine and ADP dilated with pEC(50)s of 8.4+/-0.1 and 7.4+/-0.1 in non-ischemic hearts versus 7.7+/-0.1 and 7.1+/-0.1 after 20-min ischemia (P<0.05). Sensitivity was further reduced after 25-min ischemia. Responses to nitroprusside were unaltered. NO-synthase antagonism (50 microM nitro-L-arginine methylester) reduced sensitivities to 2-chloroadenosine and ADP up to 10-fold, and eliminated inhibitory effects of I/R. K(ATP) blockade with 5 microM glibenclamide impaired sensitivity pre- and post-ischemia, not eliminating the inhibitory effects of I/R. A(1) adenosine receptor antagonism with 100 nM 8-cyclopentyl-1,3-dipropylxanthine worsened effects of ischemia on sensitivity. A(2A) adenosine receptor antagonism with 100 nM 8-(3-chlorostyryl)caffeine reduced post-ischemic flow by 50%, yet paradoxically enhanced post-ischemic contractile recovery. CONCLUSIONS: Ischemia modifies vascular control and impairs NO- versus K(ATP)-dependent coronary dilation in an asanguinous model. Endogenous adenosine protects against vascular dysfunction via A(1) receptors, and determines coronary reflow via A(2A) receptors. However, intrinsic A(2A) activation apparently worsens contractile dysfunction. (+info)
alpha,beta-Methylene ATP elicits a reflex pressor response arising from muscle in decerebrate cats.
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In part, the exercise pressor reflex is believed to be evoked by chemical stimuli signaling that blood supply to exercising muscles is not adequate to meet its metabolic demands. There is evidence that either ATP or adenosine may function as one of these chemical stimuli. For example, muscle interstitial concentrations of both substances have been found to increase during exercise. This finding led us to test the hypothesis that popliteal arterial injection of alpha,beta-methylene ATP (5, 20, and 50 microg/kg), which stimulates P2X receptors, and 2-chloroadenosine (25 microg/kg), which stimulates P1 receptors, evokes reflex pressor responses in decerebrate, unanesthetized cats. We found that popliteal arterial injection of the two highest doses of alpha,beta-methylene ATP evoked pressor responses, whereas popliteal arterial injection of 2-chloroadenosine did not. In addition, the pressor responses evoked by alpha,beta-methylene ATP were blocked either by section of the sciatic nerve or by prior popliteal arterial injection of pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (10 mg/kg), a selective P2-receptor antagonist. We conclude that the stimulation of P2 receptors, which are accessible through the vascular supply of skeletal muscle, evokes reflex pressor responses. In addition, our findings are consistent with the hypothesis that the stimulation of P2 receptors comprises part of the metabolic error signal evoking the exercise pressor reflex. (+info)