Caffeine intensifies taste of certain sweeteners: role of adenosine receptor

Caffeine, a potent antagonist of adenosine receptors, potentiates the taste of some but not all sweeteners. It significantly enhances the taste of acesulfam-K, neohesperidin dihydrochalcone, d-tryptophan, thaumatin, stevioside, and sodium saccharin. Adenosine reverses the enhancement. Caffeine has no effect on aspartame, sucrose, fructose, and calcium cyclamate. These results suggest that the inhibitory A1 adenosine receptor plays an important local role in modulating the taste intensity of certain sweeteners and that several transduction mechanisms mediate sweet taste.     

Adenosine antagonism by purines,pteridines and benzopteridines in human fibroblasts

Theophylline (K_i 5 μM) is a competitive inhibitor of the increase in cyclic AMP caused by adenosine in the VA13 fibroblast line. More than 100 purine bases and structurally related heterocycles were tested as adenosine antagonists. Three families of adenosine antagonists were found: xanthines, benzo[g]pteridines and 9-substituted adenines. For the xanthines, the optimal group at the 1-position was butyl (5-fold improvement versus methyl), at the 7-position was 2-chloroethyl (5-fold improvement versus hydrogen) and at the 8-position was p-bromophenyl (100-fold improvement versus hydrogen). The receptors appeared to have butyl- and phenyl-sized “pockets” at the 1- and 8-positions, respectively, since compounds with larger groups had greatly reduced activity.     

Effect of cyclic adenosine monophosphate, 5'-(N-ethylcarboxyamido)-adenosine and methylxanthines on the release of thromboxane and lysosomal enzymes from human alveolar macrophages and peripheral blood monocytes in vitro.

We have investigated the effect of the manipulation of intracellular cyclic adenosine monophosphate (cyclic AMP) and the stimulation of adenosine receptors on the function of human alveolar macrophages in vitro. Human alveolar macrophages harvested by bronchoalveolar lavage were stimulated by opsonised zymosan 1 mg/ml in the presence of N6,2'0-dibutyryladenosine 3':5' cyclic monophosphate (dibutyryl cyclic AMP) 5 x 10(-6) to 5 x 10(-3) M,8-bromoadenosine 3':5'-cyclic monophosphate (8-bromo cyclic AMP) 5 x 10(-6) to 5 x 10(-3) M, 5'-(N-ethylcarboxamido)-adenosine (NECA) 10(-7) to 10(-4) M, adenosine 10(-7) to 10(-4) M, theophylline 5 x 10(-6) to 5 x 10(-3) M and enprofylline 5 x 10(-8) to 5 x 10(-4) M. The subsequent release of thromboxane B2 (TXB2) and N-acetyl-beta-D-glucosaminidase (NAG) activity was monitored. In addition, the release of TXB2 and NAG from zymosan stimulated human monocytes incubated in the presence of NECA 10(-7) to 10(-4) M was measured. The TXB2 release from alveolar macrophages were inhibited by dibutyryl cyclic AMP and 8-bromo cyclic AMP and to a lesser extent by NECA, theophylline and enprofylline. However, adenosine had no effect. None of the agents studied altered NAG release. In addition, monocytes showed greater sensitivity to the inhibitory effects of 5-N-ethylcarboxamido adenosine than alveolar macrophages. In conclusion, the alveolar macrophage was inhibited by stable analogues of cyclic AMP and xanthines at supratherapeutic concentrations but have no functional excitatory adenosine receptors and only a residual inhibitory adenosine receptor function compared to the precursor monocyte.     

Structure-activity relationships at human and rat A2B adenosine receptors of xanthine derivatives substituted at the 1-, 3-, 7-, and 8-positions

In the search for improved selective antagonist ligands of the A2B adenosine receptor, which have the potential as antiasthmatic or antidiabetic drugs, we have synthesized and screened a variety of alkylxanthine derivatives substituted at the 1-, 3-, 7-, and 8-positions. Competition for 125I-ABOPX (125I-3-(4-amino-3-iodobenzyl)-8-(phenyl-4-oxyacetate)-1-propylxanthine) binding in membranes of stably transfected HEK-293 cells revealed uniformly higher affinity (<10-fold) of these xanthines for human than for rat A2B adenosine receptors. Binding to rat brain membranes expressing A1 and A2A adenosine receptors revealed greater A2B selectivity over A2A than A1 receptors. Substitution at the 1-position with 2-phenylethyl (or alkyl/olefinic groups) and at N-3 with hydrogen or methyl favored A2B selectivity. Relative to enprofylline 2b, pentoxifylline 35 was equipotent and 1-propylxanthine 3 was >13-fold more potent at human A2B receptors. Most N-7 substituents did not enhance affinity over hydrogen, except for 7-(2-chloroethyl), which enhanced the affinity of theophylline by 6.5-fold to 800 nM. The A2B receptor affinity-enhancing effects of 7-(2-chloroethyl) vs 7-methyl were comparable to the known enhancement produced by an 8-aryl substitution. Among 8-phenyl analogues, a larger alkyl group at the 1-position than at the 3-position favored affinity at the human A2B receptor, as indicated by 1-allyl-3-methyl-8-phenylxanthine, with a K(i) value of 37 nM. Substitution on the 8-phenyl ring indicated that an electron-rich ring was preferred for A2B receptor binding. In conclusion, new leads for the design of xanthines substituted in the 1-, 3-, 7-, and 8-positions as A2B receptor-selective antagonists have been identified.     

Structure-activity relationships of 8-cycloalkyl-1,3-dipropylxanthines as antagonists of adenosine receptors.

8-Substituted xanthines currently represent the most potent class of adenosine-receptor antagonists. A series of 8-substituted 1,3-dipropylxanthines was prepared and their potency as antagonists of A1 and A2 adenosine receptors of human platelets and rat adipocytes, respectively, were determined. No agents studied were as potent as 8-cyclopentyl-1,3-dipropylxanthine as antagonists of the A1 adenosine receptor, but 8-(2-methylcyclopropyl)-1,3-dipropylxanthine was at least 1000-fold more potent as an antagonist of A1 than of A2 adenosine receptors. While most substitutions on the 8-cycloalkyl moiety caused decreased potency to inhibit both A1 and A2 adenosine receptors, 8-[trans-4-(acetamidomethyl)cyclohexyl]-1,3-dipropylxanthine was nearly equipotent as an antagonist of the two receptors and appeared to be the most potent antagonist of A2 adenosine receptors reported to date.     

Apparent affinity of some 8-phenyl-substituted xanthines at adenosine receptors in guinea-pig aorta and atria.

1 Some 8-phenyl-substituted, 1,3 dipropyl xanthines have previously been demonstrated to have a 20-400 fold greater affinity for A1 binding sites in rat CNS membranes than for A2 adenosine receptors in intact CNS cells from guinea-pigs. In the present study these compounds (1,3, dipropyl-8-phenylxanthine: DPPX; 1,3 dipropyl-8-(2 amino-4-chlorophenyl) xanthine: PACPX; 8-(4-(2-amino-ethyl)amino) carbonyl methyl oxyphenyl)-1,3-dipropylxanthine: XAC; and D-Lys-XAC) together with two that have not been reported to exhibit A1-receptor selectively (8-(p-sulphophenyl)theophylline: 8-PST; 8-(4-carboxy methyl oxyphenyl)-1,3-dipropylxanthine: XCC) have been evaluated as antagonists of the effects of 2-chloroadenosine in two isolated cardiovascular tissues. 2 The isolated tissues used were guinea-pig atria (bradycardic response) and aorta (relaxation), which are thought to possess A1 and A2 adenosine receptors, respectively. 3 All the xanthines antagonized responses evoked by 2-chloroadenosine in both tissues but did not affect responses evoked by acetylcholine (atria) or sodium nitrite (aorta). 4 The xanthines, 8-PST, XAC, D-Lys XAC, XCC and DPPX appeared to be competitive antagonists of the effects of 2-chloroadenosine, as Schild plot slopes did not differ significantly from unity. The 1,3-dipropyl substituted compounds had pA2 values from 6.5 to 7.4 and were more potent than the 1,3 dimethyl substituted 8-PST (pA2 4.9 to 5). 5 For individual xanthines, there was no difference between pA2 values obtained in the atria and in the aorta.(ABSTRACT TRUNCATED AT 250 WORDS)     

The actions of some beta-receptor agonists and xanthines on isolated muscle strips from the human oesophago-gastric junction

Isolated preparations from the circular muscle layer of the human oesophago-gastric junction were mounted in organ baths and isometric tension recorded. During an equilibration period, active resting tension developed suggesting that the preparations were representing the lower oesophageal sphincter. Active tension was abolished by exposing the preparations to Ca(++)-free medium. The two xanthines theophylline and enprofylline almost equipotently relaxed the preparations in a concentration-dependent manner (10(-7)-10(-3) M). Within therapeutic concentrations, theophylline inhibited active resting tension by 30-60%, while enprofylline lowered tension by less than 20%. Inhibitory actions of adenosine were demonstrated, and this suggests that adenosine antagonism is not the mechanism of action for xanthines in the oesophagus. Non-selective beta-receptor stimulation with isoprenaline inhibited active tension by 70% (10(-7) M), while beta 2-receptor stimulation with terbutaline inhibited tension by 47% (10(-5) M). Dobutamine, believed to preferentially stimulate beta 1-receptors, inhibited active tension in a concentration-dependent manner (10(-7)-10(-4) M). Metoprolol (10(-6) M), a selective beta 1-receptor antagonist, shifted the concentration-response curve for isoprenaline to the right, but left the maximal response unchanged. It is concluded that xanthines and beta-receptor agonists have inhibitory actions on circular muscle from the human oesophagogastric junction. The experimental data suggest the presence of beta 1- as well as beta 2-receptors, both mediating inhibition of active resting tension.     

XANTHINES INHIBIT HUMAN PLATELET AGGREGATION INDUCED BY PLATELET-ACTIVATING FACTOR

1. Platelet-activating factor (PAF) may be involved in the pathogenesis of asthma, and therefore the effects of the anti-asthma drugs theophylline and enprofylline on human platelet aggregation and adenosine triphosphate (ATP) release induced by PAF and adenosine diphosphate (ADP) were studied. 2. Enprofylline (50% inhibitory concentration [IC50] = 94.8 +/- 13.2 mumol/L) was more potent than theophylline (IC50 = 934.1 +/- 40.1 mumol/L) as an inhibitor of PAF-induced aggregation, and the xanthines were twice as potent as inhibitors of PAF-induced aggregation when compared with ADP-induced aggregation. ATP release was 1.4 times more sensitive to inhibition by the xanthines than aggregation. 3. Although high concentrations of xanthines inhibited platelet aggregation and ATP release induced by PAF, therapeutic concentrations are unlikely to inhibit PAF-induced effects.     

New 8-substituted xanthiene derivatives as potent bronchodilators

The synthesis and structure determination of 8-aryl /alkyl aryl 1, 3-dimethyl-3, 7-dihydropurin-2, 6-dione derivatives (1-13), was carried out in this study. Bronchodilator activity is investigated using isolated guinea-pig tracheal strips, pre-contracted by acetylcholine and histamine. Spasmolytic activity of the compounds was compared to theophylline. Synthesized compounds (1-13) did not inhibit the acetylcholine-induced pre-contractions except compound (8) at 10(-5) M concentration. In contrast, some of the compounds, especially (7), (11), (12) at 10(-5) M and (3), (4), (9) and (11) in 10(-4) M displayed inhibitory activity on the tracheal strips pre-contracted by histamine. The potency of the compounds at human adenosine receptors was evaluated using radioligand binding assay and a cyclic AMP functional assay in CHO cells expressing these receptors. Compound (11) displayed the greatest activity against radioligand binding of specific agonists to A2A and A2B receptors. The compounds were relatively selective for both A2A and A2B compared with A1 and A3 receptors. All compounds were also tested for the inhibition of NECA-induced cAMP accumulation mediated by the A2B adenosine receptor and compound (11) was found to be the most effective. Our results showed that these compounds are acting as selective adenosine antagonists, especially for adenosine A2B receptors, and are promising as potent anti-inflammatory agents rather than bronchodilator for the treatment of asthma.     

Pharmacodynamics and pharmacokinetics of enprofylline and theophylline in the isolated rabbit heart

The direct effects of theophylline and enprofylline, a new anti-asthma xanthine derivative without adenosine receptor blocking action, were studied in the isolated, spontaneously beating rabbit heart. At increasing concentrations from 2 x 10(-5)-5 x 10(-4) M both drugs produced increases in heart rate up to 143% and 162% and in contractility up to 135% and 147% from control values (100%), respectively. At concentrations higher than 10(-3) M contractures and heart block occured. Enprofylline showed a potency about 2.3 times higher than theophylline. Coronary flowrate did not increase. Myocardial oxygen consumption was moderately increased by the drugs. The myocardial pharmacokinetics showed two-compartment characteristics for both drugs. Half-times of the two phases of accumulation were for theophylline 0.28 and 0.98 min. and for enprofylline 0.31 and 0.78 min. The terminal disposition half-time for enprofylline was, however, 2.7 times higher than that of 0.91 min. for theophylline, apparently due to a stronger binding of the former drug intracellularly. Both xanthines accumulated against a concentration gradient showing myocardial tissue-perfusion liquid ratios of about 2.9 for theophylline and 3.7 for enprofylline. The drugs seem to exert their primary action on sarcolemmal binding sites which probably are unrelated to adenosine receptors.     

Dual effect of (-)-N6-phenylisopropyl adenosine on guinea-pig trachea.

The effect of (-)-N6-phenylisopropyl adenosine (PIA), a metabolically stable P1-receptor agonist, was investigated on guinea-pig isolated trachea. PIA showed two opposite effects: contraction, evident at low concentrations (10(-7) to 2-5 X 10(-6) M), and relaxation at higher doses. Relaxation by PIA was antagonized in an apparently competitive manner by two antagonists of extracellular (P1) adenosine receptors: theophylline (Theo) and 8-phenyltheophylline (PT). Contraction by PIA was not inhibited by methylxanthines and was not mediated by stimulation of cholinergic or histaminergic systems. Inhibitors of arachidonic acid cascade acting at different levels, i.e. indomethacin, nordihydroguaiaretic acid (NDGA) and BW755C, all inhibited the contraction by PIA, while they potentiated the relaxation in a concentration-dependent manner. Mepacrine, an inhibitor of phospholipase A2, inhibited the contraction by PIA, but did not affect the relaxation. These results indicate that the contractile effect induced by PIA is supported by an indirect mechanism involving the release of arachidonic acid derivatives (via P2-purinoceptor?). Thus the balance between the two opposite effects of adenosine on tracheal tone is possibly modulated by the prostaglandin turnover.     

Attenuation of the P2Y receptor-mediated control of neuronal Ca2+ channels in PC12 cells by antithrombotic drugs

1. In PC12 cells, adenine nucleotides inhibit voltage-activated Ca(2+) currents and adenylyl cyclase activity, and the latter effect was reported to involve P2Y(12) receptors. To investigate whether these two effects are mediated by one P2Y receptor subtype, we used the antithrombotic agents 2-methylthio-AMP (2-MeSAMP) and N(6)-(2-methyl-thioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene-ATP (AR-C69931MX). 2. ADP reduced A(2A) receptor-dependent cyclic AMP synthesis with half maximal effects at 0.1-0.17 micro M. In the presence of 30 micro M 2-MeSAMP or 100 nM AR-C69931MX, concentration response curves were shifted to the right by factors of 39 and 30, indicative of pA(2) values of 6.1 and 8.5, respectively. 3. The inhibition of Ca(2+) currents by ADP was attenuated by 10-1000 nM AR-C69931MX and by 3-300 micro M 2-MeSAMP. ADP reinhibited Ca(2+) currents after removal of 2-MeSAMP within less than 15 s, but required 2 min to do so after removal of AR-C69931MX. 4. ADP inhibited Ca(2+) currents with half maximal effects at 5-20 micro M. AR-C69931MX (10-100 nM) displaced concentration response curves to the right, and the resulting Schild plot showed a slope of 1.09 and an estimated pK(B) value of 8.7. Similarly, 10-100 micro M 2-MeSAMP also caused rightward shifts resulting in a Schild plot with a slope of 0.95 and an estimated pK(B) of 5.4. 5. The inhibition of Ca(2+) currents by 2-methylthio-ADP and ADPbetaS was also antagonized by AR-C69931MX, which (at 30 nM) caused a rightward shift of the concentration response curve for ADPbetaS by a factor of 3.8, indicative of a pA(2) value of 8.1. 6. These results show that antithrombotic drugs antagonize the inhibition of neuronal Ca(2+) channels by adenine nucleotides, which suggests that this effect is mediated by P2Y(12) receptors.     

Effects of propentofylline on adenosine A1 and A2 receptors and nitrobenzylthioinosine-sensitive nucleoside transporters: quantitative autoradiographic analysis.

Previous studies have demonstrated that the xanthine compound, propentofylline, has beneficial effects in models of cerebral ischemia and can enhance some and exhibit other effects of adenosine. We investigated the in vitro effects of propentofylline and its hydroxy metabolite, A72,0287, on the binding of [3H]cyclohexyladenosine ([3H]CHA), [3H]2-[p-(2-carbonyl-ethyl)-phenylethyl-amino]-5'-N- ethylcarboxamido adenosine ([3H]CGS 21680) and [3H]nitrobenzylthioinosine ([3H]NBMPR) to adenosine A1 and A2 receptors and NBMPR-sensitive nucleoside transporters, respectively, in 10-microns coronal rat brain sections. Both xanthines had micromolar affinity for each of these sites with approximately 10-fold lower affinity for A2 receptors than for A1 receptors and [3H]NBMPR binding sites. Saturation analysis of [3H]CHA or [3H]CGS 21680 binding in the presence of increasing concentrations of propentofylline produced significant increases in KD values without affecting Bmax values; thus propentofylline is a competitive inhibitor at A1 and A2 receptors. The effects on A2 receptors apparently require higher concentrations (Ki approximately 200 microM) than the effects on A1 receptors (Ki approximately 20 microM). Propentofylline was also found to be a competitive inhibitor of [3H]NBMPR binding. Therefore we conclude that propentofylline interacts with adenosine-responsive systems to increase interstitial adenosine concentrations and to selectively inhibit A1 receptors.     

The Actions of Some β-Receptor Agonists and Xanthines on Isolated Muscle Strips from the Human Oesophago-Gastric Junction

Isolated preparations from the circular muscle layer of the human oesophago-gastric junction were mounted in organ baths and isometric tension recorded. During an equilibration period, active resting tension developed suggesting that the preparations were representing the lower oesophageal sphincter. Active tension was abolished by exposing the preparations to Ca⁺⁺-free medium. The two xanthines theophylline and enprofylline almost equipotently relaxed the preparations in a concentration-dependent manner (10^-7-10^-3M). Within therapeutic concentrations, theophylline inhibited active resting tension by 30–60%, while enprofylline lowered tension by less than 20%. Inhibitory actions of adenosine were demonstrated, and this suggests that adenosine antagonism is not the mechanism of action for xanthines in the oesophagus. Non-selective β-receptor stimulation with isoprenaline inhibited active tension by 70% (10^-7M), while β₂-receptor stimulation with terbutaline inhibited tension by 47% (10^-5M). Dobutamine, believed to preferentially stimulate β₁-receptors, inhibited active tension in a concentration-dependent manner (10^-7-10^-4M). Metoprolol (10^-6M), a selective β₁-receptor antagonist, shifted the concentration-response curve for isoprenaline to the right, but left the maximal response unchanged. It is concluded that xanthines and β-receptor agonists have inhibitory actions on circular muscle from the human oesophagogastric junction. The experimental data suggest the presence of β₁- as well as β₂-receptors, both mediating inhibition of active resting tension.     

Enprofylline and theophylline on small human placental arteries: studies of in vitro effects and mode of action

Vascular effects of theophylline and enprofylline, a novel xanthine derivative lacking adenosine receptor antagonism, were studied comparatively in tubular preparations of small human placental arteries mounted in an isometric myograph. Both xanthines produced concentration-dependent (10(-7)-3 X 10(-3) M) relaxation of arteries contracted by PGF2 alpha or PGE2 in both normal Ca2+-medium and in Ca2+-depleted medium. Enprofylline was about three times more potent than theophylline. Also in vasopressin-contracted arteries enprofylline was a more potent vasodilator in both media. In contrast the xanthines were equally potent in relaxation of the tonic as well as the phasic part of a K+-induced contraction, but less potent than in relaxation of PG-induced contractions. Propranolol, phentolamine, atropine, indomethacin or tetrodotoxin did not influence the xanthine relaxations. It is concluded that the theophylline-induced relaxation of small human placental arteries is not due to adenosine receptor antagonism. A common important mechanism of action, in which enprofylline is more potent than theophylline, seems to be interference with intracellular Ca2+-binding/mobilisation processes. Some decrease in cellmembrane Ca2+-permeability produced by the xanthines seems to take part in the mechanism of relaxation.     

Behavioral and physiological effects of xanthines in nonhuman primates

 Caffeine and related xanthines can have significant behavioral effects on measures of locomotor activity, schedule-controlled behavior, drug self-administration, and learning and memory. Xanthines also produce numerous physiological effects including positive inotropic and chronotropic effects on the heart, decreased airway resistance in the lung, and respiratory stimulation. Due to the widespread use of xanthines as constituents of food and beverages and as therapeutic drugs, identification of mechanisms that mediate their pharmacological effects has considerable relevance for drug development and therapeutics. Two primary mechanisms involving the cyclic nucleotide system have been implicated as the bases for the effects of xanthines in the CNS. Many xanthines bind to specific adenosine recognition sites and block the actions of adenosine. Xanthines also inhibit cyclic nucleotide phosphodiesterases, the enzymes responsible for the hydrolytic inactivation of cyclic AMP and cyclic GMP. Recent research in nonhuman primates has characterized the behavioral, respiratory and cardiovascular effects of a number of xanthines and related drugs differing in affinity at different subtypes of adenosine receptors and in capacity to inhibit different molecular forms of PDE. The behavioral-stimulant effects of xanthines appear to be mediated principally by their adenosine-antagonist actions and may be limited by PDE inhibition. The respiratory-stimulant and cardiac effects of xanthines, on the other hand, appear to be linked more closely to their PDE- inhibiting actions than to adenosine antagonism. Converging lines of evidence suggest that adenosine A₂ and cAMP-specific (possibly type IV) PDE mechanisms play especially prominent roles in mediating the behavioral and physiological effects of xanthines in nonhuman primates. Received: 6 March 1996 / Final version: 11 September 1996     

Vasodilation produced by forskolin compared with that produced by adenosine in rabbit coronary artery

We compared the relaxing action of forskolin with that of adenosine in rabbit coronary artery and analyzed the pharmacological properties of this effect of forskolin. In preparations of this artery precontracted by 10-16 mM KCl, the addition of forskolin (10(-9)-10(-5) M) and adenosine (10(-9)-10(-4) M) to the organ bath produced dose-dependent relaxations. The mean EC50 for the relaxing action of forskolin was 4.5 X 10(-8) M and that of adenosine was 3.6 X 10(-7) M, forskolin being 10 times more potent than adenosine. Relaxation produced by forskolin was not affected by treatment with propranolol (10(-5) M), atropine (10(-6) M), or 8-phenyltheophylline (10(-6) M), but was competitively inhibited by ouabain (10(-6) M). The relaxation produced by adenosine was inhibited by 8-phenyltheophylline (10(-6) M) competitively and by ouabain (10(-6) M) noncompetitively. In preparations precontracted by a higher concentration of KCl, 40 mM, forskolin produced full relaxation with a shift of the dose-response curve to the right; adenosine did not produce full relaxation. Both forskolin and adenosine attenuated the maximum contraction produced by Ca2+. These findings indicate that the vasorelaxing effect of forskolin was not due to activation of beta-adrenoceptors, muscarinic receptors, or adenosine receptors, whereas that of adenosine was due to activation of adenosine receptors. Increased availability of intracellular Ca2+ competitively inhibited the relaxation induced by forskolin and noncompetitively inhibited the relaxation induced by adenosine. Both forskolin and adenosine noncompetitively inhibited contraction induced by Ca2+.     

Interactions between prostaglandin E2 and inhibitors of platelet aggregation which act through cyclic AMP.

Prostaglandin (PG) E2 potentiates platelet aggregation at low concentrations (10(-8)-10(-6) M). It also inhibits aggregation at a higher concentration (10(-5) M), probably by acting through cyclic adenosine 3',5'-monophosphate (cyclic AMP). The mechanism of this biphasic effect of PGE2 and its implications for thrombosis are not clearly understood. Using a sensitive cyclic AMP assay, in conjunction with platelet aggregation studies, we have examined the interactions between PGE2 and other inhibitors of platelet aggregation which act through cyclic AMP. Low concentrations of PGE2 reversed the inhibition of platelet aggregation and increase in cyclic AMP levels induced by PGI2, PGD2 and adenosine (which stimulate adenylate cyclase (AC) through separate and specific platelet receptors). In contrast, low concentrations of PGE2 added to the inhibition of platelet aggregation and increase in cyclic AMP levels induced by forskolin (which stimulates AC directly) and AH-P 719 and DN-9693 (which inhibit cyclic AMP phosphodiesterase (PDE]. These results suggest that the biphasic effect of PGE2 may be mediated by interaction with two separate platelet receptors. Low concentrations appear to potentiate aggregation by acting at a receptor which is directly coupled to an inhibitory guanine nucleotide-binding protein (Gi), possibly the putative PG endoperoxide receptor. High concentrations of PGE2 appear to inhibit aggregation by acting at an additional receptor, probably the PGI2 receptor. The ease with which PGE2 reverses the effects of PGI2, PGD2 and adenosine, but adds to the effects of AH-P 719 and DN-9693, suggests that PDE inhibitors might offer greater potential than these AC stimulators as an anti-thrombotic strategy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of benzodiazepines on the response of the guinea-pig isolated trachea to the contractile action of adenosine.

The effects of diazepam and other agonists of central or peripheral benzodiazepine receptors were studied on the contractile action of adenosine, 2-chloroadenosine and R-PIA (N6-(L-2-phenylisopropyl)-adenosine) on the guinea-pig isolated trachea. These effects were compared to those of dipyridamole. Diazepam 10(-7) to 10(-5) M potentiated the efficacy of adenosine; the maximal contractile effect of adenosine (% vs. acetylcholine 10(-3) M) was 20.4 +/- 4.2 (n = 21) in control conditions and 45.5 +/- 3.7 (n = 6; P less than 0.001) in the presence of diazepam 10(-5) M. Ro5-4864 (10(-7) to 10(-5) M) or alpidem (10(-7) to 10(-5) M), both agonists of peripheral benzodiazepine receptors, potentiated the contractile effects of adenosine to the same extent as diazepam. Clonazepam and zopiclone, both agonists of central benzodiazepine receptors, did not modify these effects. Antagonists of central (flumazenil) or peripheral (RP 52028) benzodiazepine receptors had no influence on the interaction between diazepam or Ro5-4864 and adenosine. Conversely, dipyridamole significantly reduced (10(-7) M) or suppressed (10(-6) M) the contractile effects of adenosine. The contractile effects of 2-chloroadenosine and R-PIA were weakly affected in presence of high concentrations of diazepam and dipyridamole. Epithelium removal potentiated the contractile effect of adenosine on the guinea-pig isolated trachea and increased the potentiating effect of diazepam. It is concluded that benzodiazepines and related compounds can potentiate the contractile effect of adenosine on the guinea-pig isolated trachea through the activation of a peripheral receptor for the benzodiazepines and the resulting inhibition of adenosine uptake.     

Absence of (-) [3H]desmethoxyverapamil binding sites on human platelets and lack of evidence for voltage-dependent calcium channels

The two major pathways for Ca2+ entry into cells are potential-sensitive channels and receptor-operated channels. The main object of this investigation was to identify which mechanism regulates Ca2+ entry into human platelets. Platelet stimulation with thrombin, adenosine diphosphate, platelet activating factor and arachidonic acid resulted in a concentration-dependent 2.5-3-fold increase in cytoplasmic free calcium concentration over the basal levels (140 +/- 32 nM or 104 +/- 21 respectively) as measured with the fluorescent dyes Quin-2 and Fura-2. Adrenaline and collagen had no effect in promoting intracellular Ca2+ increase as measured with Quin-2 and little effect when measured with Fura-2. Incubation of Quin-2-loaded platelets with the calcium antagonists verapamil and diltiazem, which are known to inhibit Ca2+ entry from voltage-gated channels in many types of cells, over the concentration range 10(-8) - 10(-4) M did not alter significantly either the resting or the cytoplasmic free Ca2+ after stimulation of platelets by several agonists. Moreover, the calcium antagonists exhibited little or no effect on aggregation and 5-hydroxytryptamine secretion induced by platelet activating factor, adenosine diphosphate, collagen or arachidonic acid in whole blood, platelet-rich plasma or washed platelets when employed at concentration ranges as above. Similar results were obtained in washed thrombin-stimulated platelets. High doses of verapamil (but not diltiazem) inhibited platelet aggregation and secretion in response to adrenaline. Direct radioligand binding studies with (-)[3H]desmethoxyverapamil showed that platelet membranes have no receptors for this drug, suggesting that Ca2+ entry occurs in human platelets via a pathway different from potential-sensitive Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)