Demonstration of Ri-type adenosine receptors in bovine myocardium by radioligand binding

Summary Adenosine has been shown to have negative inotropic, chronotropic and dromotropic effects on the heart. The pharmacological profiles of these effects suggest that they are mediated via R_i (A₁) adenosine receptors, but a direct demonstration of these receptors is still missing. In the present study we report direct labelling of these receptors with (-)N⁶-[¹²⁵I]-p-hydroxyphenylisopropyladenosine ([¹²⁵I]HPIA). The radioligand bound in a saturable and reversible manner to a crude membrane preparation, the B _max-value was 30.5 fmol/mg protein and the K _D-value 1.1 nmol/l. A similar affinity of the ligand was obtained in kinetic and competition experiments. Competition experiments with a variety of adenosine analogues gave a pharmacological profile characteristic of R_i adenosine receptors with high affinities of N⁶-substituted derivatives and a marked stereospecificity for N⁶-phenylisopropyladenosine (PIA). Purification of the membrane preparation by density gradient centrifugation resulted in a 30-fold increase in the number of binding sites which was paralleled by a similar increase in the number of binding sites for [³H]ouabain. Guanine nucleotides decreased binding of [¹²⁵I]HPIA in a dose-dependent manner, but the IC₅₀-values were considerably higher than those reported in other tissues. Finally, binding of [¹²⁵I]HPIA appeared to be entropy-driven which has been shown to be characteristic of agonist binding to R_i adenosine receptors. These results suggest the presence of R_i adenosine receptors in ventricular myocardium which may be responsible for the mediation of the effects of adenosine and its analogues.Abbreviations [¹²⁵I]HPIA (-)N⁶-[¹²⁵I]-p-hydroxyphenylisopropyladenosine - (-)IHPIA (-)N⁶-iodo-p-hydroxyphenylisopropyladenosine - (+)/(-)PIA (+)/(-)N⁶-phenylisopropyladenosine - CHA N⁶-cyclohexyladenosine - NECA 5-N-ethylcarboxamidoadenosine - App(NH)p 5-adenylylimidodiphosphate - Gpp(NH)p 5-guanylylimidodiphosphate     

Pharmacological characterization of A1 adenosine receptors in isolated rat ventricular myocytes

Summary The aim of the present study was the characterization of adenosine receptors in isolated rat ventricular myocytes. The CAMP-levels of rat ventricular myocytes in the presence of 1 mol/l isoprenaline were reduced by up to 48% by adenosine analogues; the rank order of potency was: R-N⁶-phenylisopropyladenosine (IC₅₀ 60 nmol/1), 5-N-ethylcarboxamidoadenosine (IC₅₀ 360 nmol/l) and S-N⁶-phenylisopropyladenosine (IC₅₀ 16 ol/l). The adenosine receptor antagonist XAC (xanthine amine congener) antagonized the effect of R-N⁶-phenylisopropyladenosine in a concentration-dependent manner with a K_i-value of 20 nmol/l. The A₁ receptor-selective radioligand R-N⁶-¹²⁵I-p-hydroxyphenylisopropyladenosine bound to membranes prepared from rat ventricular myocytes in a saturable manner with a B _max of 17.7 fmol/mg protein and a K _D-value of 1.1 nmol/l. Adenosine analogues competed for the binding with the same rank order of potency as for the inhibition of the isoprenaline-induced cAMP-increase. GTP inhibited radioligand binding with an IC₅₀-value of 73 ol/l. These results suggest the presence of A₁ adenosine receptors on rat ventricular myocytes, which mediate an inhibition of adenylate cyclase. The receptors may be responsible for the effects of adenosine and its analogues on the heart.Abbreviations ¹²⁵I-HPIA R-N⁶-¹²⁵I-p-hydroxyphenylisopropyladenosine - PIA N⁶-phenylisopropyladenosine - NECA 5-N-ethyl-carboxamidoadenosine - XAC 8-4-[([(2-aminoethyl)aminocarbonyl]methyl)oxy]phenyl-1,3-dipropylxanthine (xanthine amine congener) - Ro 20-1724 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone - ScAMPTME 2-O-monosuccinyladenosine-3,5-cyclic monophosphate tyrosyl methyl ester - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - GTP guanosine-5-tri-phosphate Send offprint requests to D. Martens     

Pharmacologic specificity of tolerance to caffeine-induced stimulation of locomotor activity

The pharmacologic specificity of tolerance to caffeine-induced stimulation of locomotor activity was studied in adult male rats that were given access to either caffeine solution (0.5 or 1.0 mg/ml) or plain water for 10 min every 6 h on a chronic daily basis; daily caffeine intake averaged 41 and 62 mg/kg, respectively. Dose-effect curves were determined for behavioral stimulant and depressant drugs in control and caffeine-treated groups. Drugs were injected IP and locomotor activity was measured for 30 min beginning 35 min later. Rats tolerant to stimulation of locomotor activity by caffeine were also tolerant to theophylline and 7-(2-chloroethyl)theophylline, but not to any of six nonxanthine stimulants, including cocaine, methylphenidate, and d-amphetamine. The adenosine analogs, R(–)-N⁶-2-(phenylisopropyl)adenosine(R(–)-PIA) and 5-(N-ethyl)carboxamidoadenosine (NECA), decreased locomotor activity of control and caffeine-treated (0.5 mg/ml) rats; dose-effect curves in rats consuming caffeine chronically were displaced to the right of the control curves by 10-fold for R(–)-PIA and 100-fold for NECA. Dose-effect curves for the nonadenosine behavioral depressants chlorpromazine and diazepam were unchanged by chronic treatment with caffeine, but the curve for pentobarbital, which is thought to inhibit adenosine receptor binding, was shifted to the right by a factor of 3. Rats withdrawn from chronic caffeine for 24 h were still completely tolerant to caffeine-induced stimulation of locomotor activity. Dose-effect curves for R(–)-PIA and d-amphetamine in rats withdrawn from chronic caffeine for 24 h were not different from curves in control animals. These results indicate that tolerance to caffeine-induced stimulation of locomotor activity is specific to the methylxanthine class of stimulants and is not a property of nonxanthine psychomotor stimulants. Furthermore, the adenosine-antagonist activity of caffeine remains evident even in rats completely tolerant to the stimulant effect of caffeine. These results provide no support for the view that caffeine tolerance is due to enhanced sensitivity of central adenosine systems.A preliminary report of this work appeared in The Pharmacologist (28:140, 1986)     

Ra Adenosine receptors in human platelets

Summary Adenosine receptors in human platelet membranes have been characterized by radioligand binding and measurement of adenylate cyclase activity. Binding of 5-N-ethylcarboxamido[³H]adenosine ([³H]NECA) was rapid, reversible and dependent on protein concentration, pH and temperature. Due to a rapid rate of dissociation (t _1/2 approximately 20 s) binding was highest at 0° C. Adenosine deaminase and GTP alone did not influence [³H]NECA binding, whereas several divalent cations decreased binding. Saturation experiments revealed two different binding sites for [³H]NECA, with K _d values of 0.16 and 2.9 mol/l and B _max values of 8.4 and 33.4 pmol/mg of protein. In competition experiments NECA was the most potent adenosine agonist (IC₅₀ 0.5 mol/l), followed by 2-chloroadenosine (IC₅₀ 6.3 mol/l) and adenosine (IC₅₀ 12mol/l). A similar rank order of potencies was observed for the stimulatory effect of adenosine analogues on platelet adenylate cyclase. NECA stimulated adenylate cyclase activity with an EC₅₀ value of 0.5 mol/l and was approximately 4-fold more potent than (–)N⁶-phenylisopropyladenosine [(–)PIA]. However, (–)PIA and N⁶-cyclohexyladenosine did not significantly affect [³H]NECA binding, an observation not consistent with the stimulatory effect on adenylate cyclase. The adenosine antagonists 3-isobutyl-1-methylxanthine, theophylline and caffeine showed IC₅₀ values between 98 and 5,600 mol/l. [³H]PIA bound to platelet membranes with very low affinity and was not displaced by NECA. The [³H]NECA binding to human platelet membranes satisfies essential criteria for R _a adenosine receptors and, with some limitations, should be of value for the characterization of adenosine receptors in R _a subtype selective cells.     

[125I] N6-p-hydroxyphenylisopropyladenosine,a new ligand for Ri adenosine receptors

Summary N⁶-p-Hydroxyphenylisopropyladenosine (HPIA) has been labelled with carrier-free Na[¹²⁵I] to very high specific activity (2,175 Ci/mmol) and used as an agonist ligand to characterize R_i adenosine receptors in rat cerebral cortex membranes. The binding is saturable, reversible, stereospecific and dependent on protein concentration. The specific binding at 37°C was of high affinity with an equilibrium dissociation constant K_D of 0.48 nmol/l and was saturable with 0.23 pmol of [¹²⁵I]HPIA per mg of protein. The rate constant of association, k₁, was 3.25×10⁸ l mol^–1 min^–1 and that of dissociation, k₂, 0.0110 min^–1 yielding a t_1/2 of 63 min. In competition experiments the (–)isomer of N⁶-phenylisopropyladenosine (PIA) was 16-fold more potent than the (+)isomer in competing for the binding sites. Specific binding was most effectively displaced by N⁶-cyclohexyladenosine (CHA, k_i=0.26 nmol/l), (–)PIA (k_i=0.33 nmol/l) and HPIA (k_i=0.52 nmol/l), whereas 5-N-ethylcarboxamidoadenosine (NECA, k_i-1.42 nmol/l) was less effective. The methylxanthines 3-isobutyl-1-methylxanthine (IBMX), theophylline and caffeine which have been classified as adenosine antagonists had k_i values between 5–34 mol/l. Binding of [¹²⁵I]HPIA was regulated by guanine nucleotides and divalent cations. The results indicate that [¹²⁵I]HPIA labels R_i adenosine receptors in rat brain membranes.     

Specific binding of 3H-adenosine to rat brain membranes

Summary The binding of ³H-adenosine to rat brain membranes was studied by a microcentrifugation technique. Specific binding of ³H-adenosine was rapid, reversible, saturable and dependent on pH and temperature. Scatchard plots of equilibrium binding data were nonlinear suggesting the existence of two different binding sites for adenosine. The dissociation constants (K _d) were 1.7 M and 13.6 M and the maximal number of binding sites (B _max) 31 and 165 pmol adenosine bound per mg of membrane protein. Ten adenosine derivatives were studied for their ability to compete with ³H-adenosine binding. The phosphorylated adenosine compounds 5-AMP, cyclic AMP and ATP were most potent in displacing ³H-adenosine from its binding sites and the IC₅₀-values ranged from 11–25 M. N⁶-Phenylisopropyladenosine produced only partial inhibition (30%) of ³H-adenosine binding and no stereospecific difference between the (–)-and (+)isomer was observed. Several methylxanthines known as adenosine antagonists competed for the ³H-adenosine binding sites parallel with their pharmacological potency. The results offer a first approach for the study of adenosine binding sites in brain membranes.     

Action of methylxanthines and imidazole on the contractility of the terminal ileum of the guinea pig

Methylxanthines (aminophylline and caffeine) and imidazole, substances with an opposite action on phosphodiesterase (PDE), were found to contract the terminal ileum and to potentiate nerve-mediated responses — contractions elicited by electrical stimulation (ES) at 3 Hz and 30 Hz. Imidazole-induced contractions which were partly reduced by atropine, potentiated both responses to ES to about the same extent, and enhanced contractility of the preparation to histamine and potassium. The action of imidazole on the terminal ileum could be related to its influence on PDE in the smooth muscle.The effects of aminophylline and caffeine were found to be more complex, possibly involving some mechanisms other than inhibition of PDE. They produced atropine-sensitive contractions of the terminal ileum, which were potentiated by physostigmine and strongly depressed by hemicholinium. In the presence of atropine, they potentiated ES-induced contractions, particularly those elicited by ES at 30 Hz, which are thought to be of adrenergic orig origin. Both actions appeared to be due to presynaptic effects—activation of cholinergic and adrenergic neurons in the intestinal wall, possibly by enhanced influx of calcium, and facilitated release of acetylcholine and noradrenaline. Aminophylline, in concentrations which potentiated nerve-mediated contractions elicited by ES, did not affect direct smooth muscle-contracting action of drugs. Higher concentrations of aminophylline, above 0.1 mM, were found to inhibit histamine- and noradrenaline-induced contractions presumbly due to inhibition of PDE in the smooth muscle and subsequent elevation of cAMP levels.     

Long-lasting resistance to haloperidol-induced catalepsy in male rats chronically treated with caffeine

Chronic caffeine consumption has been inversely associated with the risk of developing Parkinson's disease. Here we assessed whether chronic caffeine treatment increases the resistance of male Wistar rats to haloperidol (1 mg/kg, s.c.)-induced catalepsy, measured in the bar test at 15 min intervals during 3 h. Caffeine (5 mg/kg/day) was delivered for 6 months via drinking water. Control rats received only tap water. Treatments began when animals were 3–4 months old. In order to unveil long-lasting catalepsy refractoriness not attributable to the presence of caffeine in the brains of rats, they were evaluated from day 18 to day 27 after caffeine withdrawal, a time that is far in excess for the full excretion of a caffeine dose in this species. The average cataleptic immobility measured in caffeine-treated rats (n = 23) was 1148 ± 140 s, a value 34 ± 8% lower than that recorded in control animals (n = 20), whose mean immobility was 1736 ± 137 s (P = 0.0026, t-test). The percentage of catalepsy reduction measured in caffeine-treated rats evaluated on days 18–20 after caffeine discontinuation (−32 ± 13%, n = 12, P < 0.05) was comparable to the catalepsy decrease recorded in those animals tested on days 21–27 (−36 ± 10%, n = 11, P < 0.02), a finding compatible with the notion that the effect was indeed mediated by enduring changes of brain functioning and not by the physical presence of caffeine or its metabolites. Caffeine-treated rats also had higher catalepsy latency scores compared with control rats (P < 0.01, U-test). The present findings show that chronic consumption of caffeine produces perdurable resistance to catalepsy induced by dopamine receptor blockade, possibly through enhancement of dopamine transmission, giving further support to the epidemiological results indicating that prolonged caffeine consumption affords neuroprotection against Parkinson's disease.     

Synthesis of some novel substituted purine derivatives as potential anticancer, anti-HIV-1 and antimicrobial agents

In search of novel purine antimetabolites, a series of 8-substituted methylxanthine derivatives was prepared in order to explore their in vitro anticancer, anti-HIV-1 and antimicrobial activities. The target compounds include: 8-[(3-substituted-4-oxo-thiazolidin-2-ylidene)hydrazino]-1,3-dimethyl (or 1,3,7-trimethyl)-3,7-dihydropurine-2,6-diones 5a-e, 8-[(3,4-disubstituted 2,3-dihydrothiazol-2-ylidene)hydrazino]-1,3,7-trimethyl-3,7-dihydropurine-2,6-diones 6a-d and 8-(5-amino-3-arylpyrazol-1-yl)-1,3-dimethyl- (or 1,3,7-trimethyl)-3,7-dihydropurine-2,6-diones 7a-g. The in vitro anticancer results revealed that compound 5d exhibited a super sensitivity profile towards leukemia K-562 with a GI(50) value of <0.01 microM. Compound 7c showed significant activity against colon cancer HCT-15 and renal cancer CAKI-1 (GI(50) values of 0.47 and 0.78 microM, respectively). Compound 7a displayed high activity against colon cancer HCT-15 (GI(50 )= 0.8 microM). The anti-HIV-1 results indicated that compound 6b displayed a good reduction of viral cytopathic effect (56.69%). The antimicrobial results showed that compound 5a was four times more active than ampicillin against P. aerugenosa (MIC =or< 25 microg/mL), compound 5b had twice the activity of ampicillin, while compounds 5d, 7c and 7f were equipotent to ampicillin. On the other hand, compound 7a was equipotent to ampicillin against P. vulgaris (MIC = 50 microg/mL).     

Effect of the growing area on the methylxanthines and flavan-3-ols content in cocoa beans from Ecuador

Nacional is a worldwide recognized Ecuadorian cocoa variety. However, little is known regarding its phytochemical profile and how the production area influences its nutritional quality. The contents of methylxanthines (caffeine and theobromine), total polyphenols, flavan-3-ols monomers (catechin and epicatechin) and main proanthocyanidins (procyanidins B2 and C1) in beans of Nacional cocoa were evaluated. A comprehensive sampling was carried out along Ecuador, including 85 farms from three different geographical levels (region, province, and canton). Theobromine and caffeine contents were higher in cocoas from the Amazonian region. According to the caffeine/theobromine ratio, all samples were classified as Trinitario cocoa. The flavan-3-ols amount was not clearly associated to a geographical area. However, when principal component analysis was applied, common patterns were found for some provinces. These results may be useful to further characterize the composition of cocoa beans for quality and authenticity purposes and the development of chocolate products rich in bioactives.