Influences of different adenosine receptor subtypes on catalepsy in mice

The effects of adenosine A₁ and A₂ receptors on catalepsy were studied in mice. The adenosine agonists 5-N-ethylcarboxamide-adenosine (NECA), N⁶-phenylisopropyladenosine (PIA) and N⁶-cyclohexyladenosine (CHA) induced dose dependent catalepsy. The A₁ adenosine antagonist 8-phenyltheophylline (8-PT) potentiated catalepsy induced by NECA, R-PIA and CHA. However, theophylline did not potentiate but inhibited the responses induced by NECA, R-PIA and CHA. Neither 8-PT nor theophylline alone has any effect on catalepsy in mice. It is concluded that catalepsy induced by the adenosine agonists may be due to A₂ receptor stimulation and that the A₁ antagonism may potentiate the response.     

Effector coupling of stably transfected human A3 adenosine receptors in CHO cells

CHO cells stably transfected with adenosine receptors are widely utilized models for binding and functional studies. The effector coupling of human A3 adenosine receptors expressed in such a cellular model was characterized. Inhibition of adenylyl cyclase via a pertussis toxin-sensitive G protein was confirmed and exhibited a pharmacological profile in accordance with agonist binding data. The agonist potency was dependent on the assay system utilized to measure cyclase inhibition. Agonists were more potent in a cell-based assay than in experiments where cyclase inhibition was measured in a membrane preparation suggesting that receptor-effector coupling might be more efficient in intact cells. In addition to the modulation of cyclase activity, stimulation of A3 receptors elicited a Ca2+ response in CHO cells with agonist potencies corresponding to the values for the whole cell cAMP assay. The Ca2+ signal was completely eliminated by pertussis toxin treatment suggesting that it is mediated via betagamma release from a heterotrimeric G protein of the Gi/o family. These results show that cAMP and Ca2+ signaling characteristics of the A3 adenosine receptor are comparable to the ones found for the A1 subtype.     

Induction of interleukin 8 release from the HMC-1 mast cell line: synergy between stem cell factor and activators of the adenosine A(2b) receptor

The HMC-1 mast cell line has both adenosine A(3) and A(2b) receptors on its surface, but only agonists of the A(2b) receptor are effective at releasing interleukin 8. Object of this study was to look for co-factors for adenosine A(2b) receptor activation. There was a powerful and statistically significant synergy for release of IL-8, both at the mRNA level (measured after 4 hr) and protein level (measured after 24 hr), between adenosine A(2b) receptor agonists and stem cell factor (SCF). Suitable concentrations for showing synergy were 100 ng/mL SCF and 3 microM 5'-N-ethylcarboxamidoadenosine (NECA). At these concentrations, the IL-8 released into the culture medium after SCF and NECA together was typically 3-5-fold greater in amount than the sum of the amounts of IL-8 released after exposure to the same concentrations of NECA and SCF separately. Since mast cells may be exposed to both adenosine and stem cell factor in the diseased lung, the synergy observed in this model system may have implications for asthma.     

Role of endothelial adenosine receptor-mediated vasorelaxation in ethanol-induced hypotension in hypertensive rats

Our previous findings showed that chronic ethanol feeding lowers blood pressure in spontaneously hypertensive rats. The present study investigated the role of the adenosine receptor-endothelial nitric oxide (NO) pathway in the hypotensive response to ethanol. Changes in blood pressure were evaluated in radiotelemetered pair-fed rats receiving liquid diet with or without ethanol (2.5% or 5%, w/v) for 12 weeks. The vasorelaxant activity of the adenosine analogue 5'-N-ethylcarboxamidoadenosine (NECA) in isolated aortic rings obtained from ethanol and control rats were evaluated. Ethanol (2.5% and 5%) lowered blood pressure in a dose-dependent manner. The hypotension started at week 1, reached its maximum at week 4 and remained so thereafter. In aortas with intact endothelium, NECA (10(-10) to 10(-4) M) produced a concentration-dependent relaxation of the phenylephrine-precontracted aortas. Compared with control rats, ethanol (2.5% and 5%) caused significant and concentration-related increases in NECA responses. This effect of ethanol was attenuated by the adenosine receptor antagonist 8-sulfophenyltheophylline and the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA). Further, endothelium denudation abolished the ethanol-evoked enhancement of NECA responses. The vasorelaxant responses to acetylcholine or sodium nitroprusside in aortic rings were not influenced by ethanol. In conclusion, the present findings suggest that chronic ethanol enhances the NO-dependent vasorelaxant responses to adenosine receptor activation and this may explain, at least partly, the mechanism of the hypotensive effect of ethanol in spontaneously hypertensive rats.     

Phenyl‐substituted N6‐phenyladenosines and N6‐phenyl‐5′‐N‐ethylcarboxamidoadenosines with high activity at human adenosine A2B receptors

A series of phenyl‐substituted N⁶‐phenyladenosines and N⁶‐phenyl‐5′‐N‐ethylcarboxamidoadenosines were synthesized and tested at adenosine receptor subtypes. EC₅₀ values were determined for cyclic AMP production in CHO cells expressing human A_2B receptors. Binding affinities were determined for rat A₁ and A_2A receptors and human A₃ receptors. N⁶‐phenyladenosine displayed an EC₅₀ value at A_2B receptors of 6.3 μM. Several N⁶‐phenyladenosine derivatives were more active than N⁶‐phenyladenosine, while two analogs were also more potent than 5′‐N‐ethylcarboxamidoadenosine (NECA, 0.76 μM), i.e., the 4‐iodophenyl ( 10 , 0.37 μM) and the 4‐aminosulfonylphenyl ( 20 , 0.44 μM) derivatives. N⁶‐phenyl‐NECA derivatives were as active as their analogous adenosine derivatives. Drug Dev. Res. 49:85–93, 2000. © 2000 Wiley‐Liss, Inc.     

Adenosinergic system is involved in development of diazepam tolerance in mice

In the present study the effect of adenosinergic system on the development of diazepam tolerance to motor disturbances in mice was investigated. Diazepam tolerance was obtained by administration of diazepam at a dose of 5.0 mg/kg, s.c. for ten consecutive days. On the 1st and the 10th day of the experiment motor impairments were measured in two behavioural tests: rota-rod and chimney test. We showed that acute diazepam injection produced significant motor impairments in mice and that effect was decreased by repeated diazepam treatment, confirming the development of tolerance to the motor impairing effect of diazepam. We demonstrated that adenosine A₁ and/or A_2A receptor agonists: CPA (0.025 and 0.05 mg/kg, i.p.), CGS 21680 (0.1 and 0.2 mg/kg, i.p.), NECA (0.005 and 0.01 mg/kg, i.p.) pretreatment with diazepam were able to attenuate the development of diazepam tolerance and adenosine receptor antagonists: DPCPX (1.0 and 3.0 mg/kg, i.p.), DMPX (3.0 and 6.0 mg/kg, i.p.) and caffeine (10.0 and 20.0 mg/kg, i.p.) induced the opposite effect. The most apparent effects were obtained by non-selective agonist (NECA) and antagonist (caffeine) of adenosine receptors. We conclude that adenosinergic system plays an important role in mechanisms underlying the development of benzodiazepine tolerance.     

Past, present and future of A(2A) adenosine receptor antagonists in the therapy of Parkinson's disease

Several selective antagonists for adenosine A_2A receptors (A_2AR) are currently under evaluation in clinical trials (phases I to III) to treat Parkinson's disease, and they will probably soon reach the market. The usefulness of these antagonists has been deduced from studies demonstrating functional interactions between dopamine D₂ and adenosine A_2A receptors in the basal ganglia. At present it is believed that A_2AR antagonists can be used in combination with the dopamine precursor L-DOPA to minimize the motor symptoms of Parkinson's patients. However, a considerable body of data indicates that in addition to ameliorating motor symptoms, adenosine A_2AR antagonists may also prevent neurodegeneration. Despite these promising indications, one further issue must be considered in order to develop fully optimized antiparkinsonian drug therapy, namely the existence of (hetero)dimers/oligomers of G protein-coupled receptors, a topic that is currently the focus of intense debate within the scientific community. Dopamine D₂ receptors (D₂Rs) expressed in the striatum are known to form heteromers with A_2A adenosine receptors. Thus, the development of heteromer-specific A_2A receptor antagonists represents a promising strategy for the identification of more selective and safer drugs.     

Pharmacological characterization of novel adenosine ligands in recombinant and native human A2B receptors

The present study was designed to evaluate the effects of novel and recognised compounds at human recombinant A(2B) adenosine receptors expressed in Chinese hamster ovary (hA(2B)CHO), in human embryonic kidney 293 (hA(2B)HEK-293) and at endogenous A(2B) receptors in human mast cells (HMC-1). Saturation binding experiments performed using the new high affinity A(2B) adenosine radioligand [(3)H]-N-benzo[1,3]dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetra hydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]-acetamide ([(3)H]-MRE 2029F20) revealed a single class of binding sites in hA(2B)CHO, hA(2B)HEK-293 and HMC-1 cells with K(D) (nM) of 1.65+/-0.18, 2.83+/-0.34, 2.62+/-0.27 and B(max) (fmol/mg protein) of 36+/-4, 475+/-50 and 128+/-15, respectively. The pharmacological profile of new compounds, determined in inhibition binding experiments in hA(2B)HEK-293 cells using [(3)H]-MRE 2029F20, showed a rank order of potency typical of the A(2B) receptors with K(i) values in the range 3.2-28nM. In functional assays, recognised agonists and antagonists were studied by evaluating their capability to modulate the cAMP production in hA(2B)CHO and in HMC-1 cells. Novel compounds were able to decrease NECA-stimulated cAMP production in hA(2B)CHO and in HMC-1 cells showing a high potency. New compounds were also able to inhibit cAMP levels in the absence of NECA and in the presence of forskolin stimulation in hA(2B)CHO and in HMC-1 cells. In HEK-293 cells MRE 2029F20 reduced cAMP basal levels with an IC(50) value of 2.9+/-0.3nM. These results suggest that novel compounds are antagonists with an inverse agonist activity in recombinant and native human A(2B) receptors.     

Effects of indomethacin on changes in renal blood flow induced by adenosine and its analogues in conscious dogs

Summary The effects of indomethacin on changes in renal blood flow induced by adenosine, NECA (adenosine-5-N-ethyl-carboxamide) and 2,3-dinitro-NECA were investigated in 6 chronically instrumented conscious dogs. Adenosine (187.5, 375 and 750 nmol/kg, i.v.) induced a dose-dependent initial decrease, followed by a reactive increase in renal blood flow. NECA (1.5 nmol/kg, i.v.) also induced an initial decrease, which was, however, followed by a prolonged reactive increase in renal blood fow. 2,3-dinitro-NECA (50 nmol/kg, orally) induced only an increase in renal blood flow. Indomethacin (27.9 mol/kg, i.v.) caused no relevant change of the initial decrease and a significant attenuation of the reactive increase in renal blood flow induced by adenosine. NECA-induced changes in blood flow were affected by indomethacin in the same direction but to a greater extent than were adenosine-induced changes in blood flow. Indomethacin reversed the increase to a decrease in renal blood flow induced by 2,3-dinitro-NECA. Thus, prostaglandins seem to be involved in mediating the response of renal blood flow to adenosine, NECA and 2,3-dinitro-NECA.Part of this study was presented at the fall meeting of the German Pharmacological Society, September 1982 in Vienna, Austria     

The critical role of intracellular zinc in adenosine A2 receptor activation induced cardioprotection against reperfusion injury

Exogenous zinc can protect cardiac cells from reperfusion injury, but the exact roles of endogenous zinc in the pathogenesis of reperfusion injury and in adenosine A₂ receptor activation-induced cardioprotection against reperfusion injury remain unknown. Adenosine A₁/A₂ receptor agonist 5′-(N-ethylcarboxamido) adenosine (NECA) given at reperfusion reduced infarct size in isolated rat hearts subjected to 30 min ischemia followed by 2 h of reperfusion. This effect of NECA was partially but significantly blocked by the zinc chelator N,N,N′,N′-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN), and ZnCl₂ given at reperfusion mimicked the effect of NECA by reducing infarct size. Total tissue zinc concentrations measured with inductively coupled plasma optical emission spectroscopy (ICPOES) were decreased upon reperfusion in rat hearts and this was reversed by NECA. NECA increased intracellular free zinc during reperfusion in the heart. Confocal imaging study showed a rapid increase in intracellular free zinc in isolated rat cardiomyocytes treated with NECA. Further experiments revealed that NECA increased total zinc levels upon reperfusion in mitochondria isolated from isolated hearts. NECA attenuated mitochondrial swelling upon reperfusion in isolated hearts and this was inhibited by TPEN. Similarly, NECA prevented the loss of mitochondrial membrane potential (ΔΨm) caused by oxidant stress in cardiomyocytes. Finally, both NECA and ZnCl₂ inhibited the mitochondrial metabolic activity. NECA-induced cardioprotection against reperfusion injury is mediated by intracellular zinc. NECA prevents reperfusion-induced zinc loss and relocates zinc to mitochondria. The inhibitory effects of zinc on both the mPTP opening and the mitochondrial metabolic activity may account for the cardioprotective effect of NECA.