n(6)-cyclohexyladenosine and 1-3-diethyl-8-phenylxanthine

Adenosine analogs were used to investigate the cellular mechanisms by which adenosine may alter renal tubular function. Cultured rabbit cortical collecting tubule (RCCT) cells, isolated by immunodissection, were treated with 5'-N-ethylcarboxamideadenosine (NECA), N6-cyclohexyladenosine (CHA), and R-N6-phenylisopropyladenosine (PIA). All three analogs produced both dose-dependent inhibition and stimulation of RCCT cell cyclic AMP (cAMP) production. Stimulation of cAMP accumulation occurred at analog concentrations of 0.1 microM to 100 microM with the rank order of potency NECA greater than PIA greater than CHA. Inhibition occurred at concentrations of 1 nM to 1 microM with the rank order of potency CHA greater than PIA greater than NECA. These effects on cAMP production were inhibited by 1,3-diethyl-8-phenylxanthine and isobutylmethylxanthine. CHA (50 nM) blunted AVP- and isoproterenol-stimulated cAMP accumulation. This modulation of hormone-induced cAMP production was abolished by pretreatment of RCCT cells with pertussis toxin. Prostaglandin E2 production was unaffected by 0.1 mM CHA. These findings indicate the presence of both inhibitory (A1) and stimulatory (A2) receptors for adenosine in RCCT cells. Moreover, occupancy of the A1 receptor causes inhibition of both basal and hormone-stimulated cAMP formation through an action on the inhibitory guanine nucleotide-binding regulatory component, Ni, of the adenylate cyclase system.

Topics: 1-Methyl-3-isobutylxanthine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylate Cyclase Toxin; Animals; Arginine Vasopressin; Cyclic AMP; Isoproterenol; Kidney Cortex; Kidney Tubules; Kidney Tubules, Collecting; Pertussis Toxin; Phenylisopropyladenosine; Rabbits; Receptors, Purinergic; Virulence Factors, Bordetella; Xanthines

The inhibitory effects of adenosine as well as its related analogues on the contractile response of the rat vas deferens to field stimulation were compared in the absence and in the presence of nitrobenzylthioguanosine (NBTGR), a potent adenosine uptake inhibitor. In the presence of NBTGR, the order of potency was N6-cyclohexyladenosine (CHA) greater than or equal to L-N6-phenylisopropyladenosine (L-PIA) greater than 2-chloroadenosine greater than D-N6-phenylisopropyladenosine (D-PIA) greater than or equal to adenosine greater than 2'-deoxyadenosine. The inhibitory effect of adenosine but not that of clonidine, beta-endorphin and somatostatin was blocked by 1,3-diethyl-8-phenylxanthine (DPX, pA2 = 7.2), a potent P1-purinergic antagonist. The results suggest that adenosine inhibited the electrically evoked contractions of the rat vas deferens via the activation of the A1 subtype of P1-purinergic receptors.

Topics: Adenosine; Animals; Electric Stimulation; Guanosine; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth; Phenylisopropyladenosine; Rats; Rats, Inbred Strains; Synaptic Transmission; Thionucleosides; Vas Deferens; Xanthines

Caffeine, a potent central stimulant, is known to competitively inhibit the specific binding of both adenosine and benzodiazepine receptor ligands to brain membranes in vitro. In mice receiving a diet containing non-toxic doses of caffeine (200 or 400 mg/kg diet) for periods up to 40 days, a dose-related increase in the number of binding sites for [3H]-CHA and [3H] DPX was observed in whole brain membranes without modifications of the receptors' affinity. Furthermore, a transitory increase in the number of [3H]-DZP binding sites was observed. These preliminary data seem to confirm the involvement of the adenosine receptors in the mode of action of caffeine and may be relevant to the development of both tolerance and dependence to some of the central effects of this compound.

Topics: Adenosine; Animals; Brain; Caffeine; Diazepam; Diet; Mice; Receptors, Cell Surface; Receptors, Purinergic; Xanthines

The binding properties of N6-cyclohexyl [3H]adenosine ( [3H]CHA) and 1,3-diethyl-8-[3H]phenylxanthine ( [3H]DPX) in rat forebrain membrane are compared. The kinetic parameters of binding for each ligand are quite distinct, with [3H]CHA displaying two populations of binding sites (KD = 0.4 +/- 0.05 nM and 4.2 +/- 0.3 nM; Bmax = 159 +/- 17 and 326 +/- 21 fmol/mg protein), whereas [3H]DPX yielded monophasic Scatchard plots (KD = 13.9 +/- 1.1 nM; Bmax = 634 +/- 27 fmol/mg protein). The metals copper, zinc, and cadmium are potent inhibitors of [3H]CHA binding, with respective IC50 concentrations of 36 microM, 250 microM, and 70 microM. Copper is a much less potent inhibitor of [3H]DPX binding (IC50 = 350 microM). The inhibitory effect of copper on both [3H]CHA and [3H]DPX binding is apparently irreversible, as membranes pretreated with copper cannot be washed free of its inhibitory effect. The inhibitory effect of both copper and zinc on [3H]CHA binding was reversed by the guanine nucleotide Gpp(NH)p. [3H]DPX binding is only partially inhibited by zinc and cadmium (60% of specific binding remains unaffected), suggesting that this adenosine receptor ligand binds to two separate sites. Guanine nucleotides had no effect on the inhibition of [3H]DPX binding by either copper or zinc. Differential thermal and proteolytic denaturation profiles are also observed for [3H]CHA and [3H]DPX binding, with the former ligand binding site being more labile in both cases. Stereospecificity is observed in the inhibition of both [3H]CHA and [3H]DPX binding, with L-N-phenylisopropyladenosine (PIA) being 50-fold more potent than D-PIA in both cases. Evidence is therefore provided that adenosine receptor agonists and antagonists have markedly different binding properties to brain adenosine receptors.

Topics: Adenosine; Animals; Binding, Competitive; Brain; Captopril; Cell Membrane; Kinetics; Male; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Purinergic; Structure-Activity Relationship; Xanthines

Guanine nucleotides, divalent cations, and sodium differentially regulate agonist and antagonist binding to adenosine A1 receptors in brain membranes. Guanine nucleotides decrease the binding of the adenosine A1 receptor agonist [3H]N6-cyclohexyladenosine ([3H]CHA) to guinea pig and bovine brain membranes by about 50% at 1--3 microM, while not affecting binding of the antagonist [3H]1,3-diethyl-8-phenylxanthine ([3H]DPX) to A1 receptors in bovine brain. GTP decreases the potency of agonists competing for [3H]DPX binding by 3--6 times, without altering the potency of antagonists. This effect can be used to grade experimental substances along an adenosine agonist-antagonist continuum. The 66% inhibition of [3H]CHA binding by 1 mM EDTA, with no change in [3H]DPX binding, suggests that endogenous divalent cations may regulate adenosine receptor interactions. Removal of endogenous divalent cations by EDTA treatment greatly increases the enhancement of [3H]CHA binding by divalent cations. Specific binding of [3H]CHA to guinea pig brain is increased 150--170% by 0.3--1.0 mM Mn2+, Mg2+, and Ca2+ following EDTA preincubation, secondary to an increase in apparent affinity and receptor number. Sodium ions also selectively regulate the binding of [3H]CHA. Sodium decreases [3H]CHA binding 40%, whereas lithium and potassium are ineffective. Sodium does not affect [3H]DPX binding.

Topics: Adenosine; Animals; Brain; Cations; Guanine Nucleotides; Guinea Pigs; In Vitro Techniques; Kinetics; Membranes; Receptors, Cell Surface; Receptors, Purinergic; Sodium; Xanthines

The binding of various adenosine receptor ligands and of [3H]diazepam, as well as their inhibition of methylxanthines, have been studied in human brain cerebral cortex membranes. Caffeine and theophylline competitively inhibit binding of [3H]cyclohexyladenosine, [3H]diethylphenylxanthine, [3H]phenylisopropyladenosine and [3H]diazepam. Both caffeine and theophylline are more potent as inhibitors of adenosine receptor ligand binding compared to [3H]diazepam binding. Theophylline was more potent than caffeine in its ability to compete with adenosine receptor ligand binding while the reverse was true for [3H]diazepam binding. The meaning of these results for the mode of action of methylxantine is discussed.

Topics: Adenosine; Binding, Competitive; Brain; Caffeine; Diazepam; Humans; Phenylisopropyladenosine; Receptors, Cell Surface; Receptors, Drug; Receptors, GABA-A; Receptors, Purinergic; Theophylline; Xanthines

Topics: Adenosine; Animals; Brain; Cattle; Guinea Pigs; Humans; Male; Rabbits; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Purinergic; Species Specificity; Temperature; Thermodynamics; Tritium; Xanthines

Several adenosine analogs were evaluated for their effects on rate and contractility in guinea pig isolated atria. Among adenosine agonists, (-)-N-(1-methyl-2-phenylethyl) adenosine (l-phenylisopropyladenosine; l-PIA) and N-cyclohexyladenosine (CHA), decreased rate and force at nanomolar concentrations, whereas 2-chloroadenosine, N,N-dimethyladenosine (N6-dimethyladenosine) and (+)-N-(1-methyl-2-phenylethyl)adenosine (d-phenylisopropyladenosine; d-PIA) were less potent cardiac depressants. The degree and order of potency of these agonists suggest that the cardiac depressant effects of adenosine are mediated via A1-receptors. The cardiac depressant effects of CHA and l-PIA were antagonized by theophylline and 1,3-diethyl-8-phenylxanthine (DPX).

Topics: 2-Chloroadenosine; Adenosine; Animals; Depression, Chemical; Guinea Pigs; Heart Atria; Male; Myocardial Contraction; Phenylisopropyladenosine; Theophylline; Xanthines

N6-Cyclohexyl[3H]adenosine ([3H]CHA) and 1,3-diethyl-8-[3H]phenylxanthine ([3H]DPX) to bind to adenosine receptors in brain membranes. The agonist [3H]CHA has high affinity in both bovine and guinea pig brain (Kd, 0.7 nM and 6 nM, respectively). [3H]CHA binding kinetics are slow (dissociation t1/2;60 min); binding is much higher at 25 degrees C than at 0 degrees C and is inhibited by guanine nucleotides. Potencies of nucleosides and xanthines in competing for [3H]CHA sites indicate that specific binding is entirely to A1 adenosine receptors. In bovine brain, the antagonist [3H]DPX exhibits high-affinity binding (Kd, 5 nM) to the same A1 receptors that bind [3H]CHA. Binding kinetics are rapid (dissociation t1/2, 1 min), and binding is moderately higher at 0 degrees C than at 25 degrees C. In guinea pig brain, [3H]DPX binding has only moderate affintiy (Kd 50 nM), and about 60% of specific binding is to sites that resemble A2 adenosine receptors.

Topics: Adenosine; Adenosine Deaminase; Animals; Brain; Cattle; Guanine; Guinea Pigs; Membranes; Nucleosides; Purines; Receptors, Cell Surface; Receptors, Purinergic; Species Specificity; Xanthines