The xanthine derivative 1-(5'-oxohexyl)-3-methyl-7-propyl xanthine (HWA 285) enhances the actions of adenosine

The effect of two closely related xanthine derivatives, pentoxifylline and HWA 285, on cyclic AMP accumulation in rat hippocampal slices and on adenosine uptake in erythrocytes was examined. Pentoxifylline was a weak competitive antagonist of adenosine effects on cyclic AMP accumulation. HWA 285, by contrast, had a small stimulatory effect per se and also potentiated the effect of adenosine (10-30 microM). Neither pentoxifylline nor HWA 285 significantly affected the cyclic AMP accumulation induced by the stable adenosine analogue NECA or by alpha- or beta-adrenoceptor activation. HWA 285 was a much more potent inhibitor of adenosine uptake into human erythrocytes than pentoxifylline and other examined xanthines including thiocaffeine, 8-p-sulphophenyltheophylline, theophylline, caffeine and enprofylline. It is suggested that HWA 285 may potentiate, rather than antagonize, the effects of endogenous as well as exogenous adenosine, partly as a consequence of adenosine uptake inhibition.     

The effect of alkylxanthines and other phosphodiesterase inhibitors on adenosine-receptor mediated decrease in lipolysis and cyclic AMP accumulation in rat fat cells

Cyclic AMP accumulation and glycerol release were studied in isolated rat fat cells. Both processes were inhibited by R-site specific adenosine analogues (L-PIA greater than NECA greater than 2-chloro-adenosine greater than D-PIA), but poorly or not at all by the P-site selective analogue SQ 22,536. The effect of a series of xanthine derivatives and of some structurally unrelated phosphodiesterase inhibitors as inhibitors of 2-chloro-adenosine induced inhibition of NA stimulated cyclic AMP accumulation and lipolysis was subsequently examined. The 2-chloroadenosine effect on cyclic AMP accumulation was antagonized by the xanthines with the following order of potency: DPX greater than 8-phenyl-theophylline greater than 8-p-sulpho-phenyl-theophylline greater than verrophylline greater than IBMX greater than theophylline greater than HWA 285 greater than pentoxiphylline greater than caffeine greater than 7-benzyl IBMX greater than theobromine greater than enprofylline greater greater than ZK 62,711. The rank order potency of xanthines against the antilipolytic effect of 2-chloro-adenosine was the same with two notable exceptions: the two potent phosphodiesterase inhibitors 7-benzyl-IBMX and ZK 62,711 were more than 20 times more potent as inhibitors of the antilipolytic effect of 2-chloro-adenosine. The results show that antagonism of adenosine analogue-induced antilipolytic effects is a convenient assay for adenosine antagonistic potency of drugs, except for drugs with a high potency as phosphodiesterase inhibitors. The lipolytic potency of the xanthine derivatives was also studied. The ability of the xanthines to stimulate basal and noradrenaline stimulated lipolysis was generally in agreement with their potency as adenosine antagonists. Adenosine deaminase induced lipolysis was stimulated by potent phosphodiesterase inhibitors.     

The effects of theophylline and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (ro 20-1724) on protein secretion from rat parotid gland

1 The effects of two chemically distinct cyclic nucleotide phosphodiesterase (PDE) inhibitors on protein secretion from superfused rat parotid gland were studied.2 In the presence of 1.0 mM Ca(2+), Ro 20-1724 (10 muM), an imidazolidinone derivative, increased the secretory response to isoprenaline 100% and the isoprenaline-dependent accumulation of adenosine cyclic 3',5'-monophosphate (cyclic AMP) 300-400%. At this concentration Ro 20-1724 alone did not cause protein secretion, accumulation of cyclic AMP or significantly inhibit PDE activity in cell-free preparations from parotid gland.3 In the absence of added Ca(2+) and in the presence of 1.0 mM EGTA, Ro 20-1724 inhibited the secretory response to isoprenaline 65% while increasing isoprenaline-dependent cyclic AMP accumulation 200%.4 In the presence of Ca(2+), theophylline (10 mM) stimulated protein secretion but did not cause the accumulation of cyclic AMP. When combined with isoprenaline the rate of secretion was greater than the sum of the effects of the individual drugs but there was no effect of theophylline on the isoprenaline-dependent accumulation of cyclic AMP.5 Theophylline-stimulated protein secretion is increased by omitting Ca(2+) from the superfusion medium without any detectable change in cyclic AMP accumulation. Under these conditions Ro 20-1724 inhibits theophylline-stimulated protein secretion and the maximum rate of protein secretion in the presence of isoprenaline and theophylline is no greater than that seen with either agent alone.6 It is concluded that the theophylline effects do not result from inhibition of PDE. It is suggested that the primary action of both drugs on parotid gland acinar cells is to alter the distribution of intracellular Ca(2+). Ro 20-1724 may also inhibit Ca(2+)/calmodulin activated enzymes such as PDE.     

Interaction between adenosine and catecholamines on cyclic AMP accumulation in guinea pig ventricular myocardium.

Adenosine stimulated cyclic AMP accumulation in guinea pig ventricular slice preparations Stimulation produced by adenosine and isoproterenol combined was essentially the sum of the two individual responses. In contrast, the response produced by adenosine and epinephrine or adenosine and norepinephrine was lower than that produced by the nucleoside alone. In the absence or presence of adenosine, propranolol decreased epinephrine- or norepinephrine-mediated accumulation of cyclic AMP below control levels, whereas phentolamine increased these responses. Methoxamine reduced cyclic AMP accumulation mediated by isoproterenol or adenosine. The results are interpreted to indicate that alpha and beta adrenergic agents have opposing effects on cyclic AMP formation in this preparation, alpha receptor stimulation acting to decrease cyclic AMP levels. The effect of alpha receptor activation was particularly pronounced when cyclic AMP levels were elevated by adenosine.     

Propentofylline and a hydroxy metabolite augment A2-receptor mediated cyclic AMP accumulation and attenuate A1-receptor mediated inhibition of acetylcholine release in the rat hippocampus

In contrast to the adenosine receptor antagonist theophylline, the xanthine derivative propentofylline (HWA 285) is reported to protect against ischaemic cell damage. We examined the effect of propentofylline on two adenosine actions in the rat hippocampus; the A2-mediated stimulation of 3H-cAMP accumulation and the A1-mediated inhibition of 3H-ACh release. Propentofylline (0.5-1 mM) increased the adenosine (3 and 30 microM) -induced 3H-cAMP accumulation. This effect was shared by its metabolite A 72 0287. The mechanism may be a decreased inactivation of adenosine, since the effect of the stable adenosine derivative NECA was not altered. In contrast, the inhibitory effect of adenosine on evoked 3H-ACh release was inhibited by propentofylline and by its metabolite A 72 0287, but enhanced by the uptake inhibitor dipyridamole. The effect of the stable, A1-receptor selective analogue R-PIA (1 microM) was also blocked by propentofylline and by A 72 0287. In addition, propentofylline (0.5 mM) blocked the presynaptic inhibitory effect of carbachol (1 and 50 microM). Thus, propentofylline and one of its metabolites inhibits adenosine A1-receptor mediated presynaptic inhibition while it enhances adenosine A2-mediated cAMP accumulation in the rat hippocampus differently. This selectivity in action can only partly be explained by receptor subtype selectivity, and effects at sites other than the adenosine receptor are of major importance.     

Effects of the novel anti-ulcer agent 1-(5'-oxohexyl)-3-methyl-7-propyl xanthine on experimental ulcers and gastric secretion in rats

The effects of 1-(5'-oxohexyl)-3-methyl-7-propyl xanthine (HWA 285) on various experimentally induced ulcers and gastric acid secretion were investigated in rats. HWA 285 (10-50 mg/kg, p.o.) inhibited restraint and water-immersion-induced stress, ulcers, indometacin- and absolute ethanol-induced gastric ulcers and mepirizole-induced duodenal ulcers in rats in a dose-dependent manner. HWA 285 (10-25 mg/kg i.d.) had inhibitory effects on acetylsalicylic acid-induced ulcers. The healing of acetic acid-induced chronic ulcers was significantly accelerated by HWA 285 (25 mg/kg p.o.) when it was given twice daily for 7 consecutive days. When given orally (twice a day, 11 doses in total) before the induction of gastric ulcers by stress, cimetidine at 100 mg/kg aggravated the ulcers, whereas, HWA 285 at 25 mg/kg had not such an effect. In conscious pylorus-ligated rats, HWA 285 (10-100 mg/kg i.p.) showed a dose-dependent inhibition on basal and desglugastrin- and 2-deoxy-D-glucose (2-DG)-stimulated gastric acid secretion. In stomach-lumen perfused rats, HWA 285 (30 mg/kg i.v.) inhibited 2-DG-stimulated gastric acid secretion but not carbachol-stimulated gastric acid secretion. These results suggest that the anti-ulcer effects of HWA 285 are produced by cytoprotective and central anti-secretory activity without peripheral anti-cholinergic properties. Whether the central anti-secretory effects of HWA 285 play thereby the key role, have to be clarified in further investigation.     

Is adenosine involved in inhibition of forskolin-stimulated cyclic AMP accumulation by caffeine in rat brain?

Caffeine potently inhibited forskolin-stimulated cyclic AMP accumulation in slices of rat cerebral cortex, with an IC50 of 21 +/- 3 microM. Because caffeine competitively blocks adenosine receptors, we examined whether the action of forskolin involved endogenous adenosine or whether caffeine was acting through some novel mechanism. Inhibition by caffeine was observed at all forskolin concentrations examined, although the degree of inhibition decreased at higher concentrations of forskolin. The effect of caffeine was not blocked by the presence of a phosphodiesterase inhibitor but was mimicked by several other methylxanthines. The most potent of these was 8-(p-sulfophenyl)-theophylline, which does not readily cross cell membranes, arguing for an extracellular site of action. Addition of either adenosine or the adenosine uptake blocker dipyridamole potentiated the forskolin response, suggesting that forskolin and adenosine act synergistically in increasing cyclic AMP accumulation. The nonxanthine adenosine receptor antagonist CGS 15943 potently blocked cyclic AMP responses to forskolin, adenosine, and combinations. 3-Isobutyl-1-methylxanthine potently blocked the response to adenosine but caused little or no inhibition of the response to forskolin. Adenosine deaminase (ADA) was added to eliminate contributions of endogenous adenosine. ADA inhibited the response to both adenosine and forskolin; however, 200 times as much enzyme was necessary to inhibit the forskolin response. Inhibition of added ADA with 2'deoxycoformycin dramatically increased the concentration of ADA required to inhibit the adenosine response, without altering the concentration required to inhibit the forskolin response. These results suggest that forskolin-stimulated cyclic AMP accumulation may be partially dependent on endogenous adenosine but that the inhibition observed with caffeine is not solely due to blockade of adenosine receptors.     

The effect of 1-(5'-oxohexyl)-3-methyl-7-propyl-xanthine on the respiratory activity of the rat brain mitochondria

The effect of 1-(5'-oxohexyl)-3-methyl-7-propyl-xanthine (HWA 285) on the respiration and oxidative phosphorylation in mitochondria isolated from normal (CM), ischemic (IsM) and postischemic (PIsM) rat brain was investigated. After the administration of 10 mg/kg HWA 285 p.o. daily for 14 days the mitochondrial ATPase activity was significantly increased, whereas O2-consumption and the respiratory control rate (RCR) were decreased. In IsM the RCR was increased, if they consumed glutamate and malate as substrates (from 3.7 +/- 0.8 to 5.0 +/- 0.75) as consequence of increased oxygen consumption in status 3. The pretreatment of the rats with 10 mg/kg HWA 285 p.o. induced a normalization of RCR in mitochondria from ischemic brains. The RCR in PIsM was apparently not influenced by HWA 285 but the oxidative phosphorylation was slightly increased. These results are consistent with the assumption that HWA 285 exerts a modulative effect on the rat brain mitochondria dependent on their functional status.     

Inhibition by amiloride of both adenylate cyclase activity and the Na+/H+ antiporter in fish erythrocytes

In fish erythrocytes isoproterenol stimulates cellular accumulation of cyclic adenosine 3':5'-monophosphate (cyclic AMP) and produces a large increase in sodium permeability which corresponds to the activation of Na+/H+ exchanges and chloride-dependent sodium uptake. The stimulation of sodium transport by isoproterenol was reproduced by adding cyclic AMP or forskolin to the medium and was blocked by propranolol. This increase in sodium permeability was completely inhibited by amiloride at the relatively high levels (0.1-1 mM) of the diuretic required to inhibit the activity of the Na+/H+ exchanger under physiological conditions in various biological systems. It was shown that amiloride inhibited cyclic AMP accumulation. This effect, which was reversible and dose-dependent (ED50 6 X 10(-6) M-maximal effect 0.5 mM), resulted from the inhibition of the catalytic unit of adenylate cyclase. Amiloride also directly inhibited the sodium entry system but the Na transporter was less sensitive than adenylate cyclase to amiloride (ED50 6 X 10(-5) M). It appears from the data presented in this report that the inhibition of sodium permeability observed in fish erythrocytes in the presence of amiloride can result either from the effect of the diuretic on the adenylate cyclase system or from the effect on the sodium transport system, depending on the conditions in which amiloride is used. Thus, caution is required when interpreting amiloride action in terms of inhibition of specific transport processes.     

G protein-coupled receptor kinase 6 (GRK6) selectively regulates endogenous secretin receptor responsiveness in NG108-15 cells

1. To determine the role of G protein-coupled receptor kinases (GRKs) in the regulation of endogenous secretin receptor responsiveness, we have transiently overexpressed both wild-type (WT) and dominant negative mutant (DNM) GRKs in NG108-15 mouse neuroblastoma x rat glioma hybrid cells and investigated the effects of this on agonist-stimulated adenylyl cyclase activity. 2. Overexpression of WT GRK6 selectively inhibited secretin-stimulated cyclic AMP accumulation (fold stimulation of cyclic AMP above basal following 15 min incubation with 100 nM secretin was 12.1+/-2.0 and 6.2+/- 0.8 in control and WT GRK overexpressing cells, respectively) without affecting cyclic AMP responses mediated by the adenosine A(2) receptor agonist 5'-(N-ethylcarboxamido) adenosine (NECA) or the prostanoid-IP receptor agonist iloprost, or the direct activator of adenylyl cyclase, forskolin. On the other hand DNM GRK6 (Lys(215)Arg) overexpression produced the opposite effect--a selective increase in the secretin-stimulated cyclic AMP response was observed in cells overexpressing DNM GRK6 compared to plasmid-transfected cells (fold stimulation of cyclic AMP above basal following 15 min incubation with 100 nM secretin was 12.6+/-2.7 and 29.6+/-5.6 for control and DNM GRK6-overexpressing cells, respectively). 3. Overexpression of WT GRK5 likewise inhibited the secretin-stimulated cyclic AMP response, however, this effect was not as selective as with GRK6, since adenosine A(2) receptor responsiveness was also suppressed by GRK5 overexpression. Unlike DNM GRK6, overexpression of DNM GRK5 failed to modulate secretin or A(2) adenosine receptor signalling suggesting that endogenous GRK5 is unlikely to regulate desensitization of these receptors in NG108-15 cells. 4. Overexpression of WT GRK2 did not affect secretin-stimulated cyclic AMP accumulation. Instead, GRK2 overexpression selectively inhibited A(2) adenosine receptor responsiveness, confirming our previous findings. 5. Together these results suggest a selective role of endogenous GRK6 in regulating secretin receptor responsiveness in NG108-15 cells. In addition, these data indicate that GRKs exert a surprising degree of selectivity in the regulation of natively expressed GPCR responses.     

Potentiation of antiaggregating activity of adenosine by a phosphodiesterase inhibitor, EG626 (oxagrelate), in human platelets in vitro

EG626 (oxagrelate), a specific inhibitor of cyclic AMP phosphodiesterase, produced in vitro a concentration-dependent inhibition of platelet aggregation induced by collagen and ADP in human platelets. When adenosine was added to the platelet rich plasma (PRP) in the presence of a threshold concentration of EG626, the potency of adenosine in inhibiting platelet aggregation was markedly potentiated. This potentiating effect of EG626 proved to be synergistic, but not additive and was accompanied by a marked accumulation of cyclic AMP in the platelets. The antiaggregating and cyclic AMP increasing activities of adenosine were little affected by S-(p-nitrobenzyl)-6-thioguanosine (6TG), an uptake inhibitor of adenosine, or 2'-deoxycoformycin, an inhibitor of adenosine deaminase. The incorporation of adenosine into platelets was abolished by 6TG. These observations indicate that incorporation of adenosine into platelets is not required for inhibition of aggregation or an increase in cyclic AMP and that the site of action of adenosine is probably extracellular. It also appears that the synergistic action by EG626 is not the result of an inhibition of adenosine uptake and/or adenosine deaminase. This speculation is supported in part by the finding that EG626 also potentiates the antiaggregating activity of 2-chloroadenosine. Antiaggregating activity of prostaglandin E1, an activator of adenylate cyclase, was markedly potentiated in combination with EG626. Dibutyryl cyclic AMP showed a time-dependent inhibition of the platelet aggregation, and the inhibitory action was markedly potentiated by EG626. Qualitatively similar results were obtained with another phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). All these data suggest that the synergistic potentiation of the antiaggregating activity of adenosine by EG626 might be due to the synergistic accumulation of cyclic AMP in the platelets. This action is mediated through activation of adenylate cyclase by adenosine in combination with the inhibition of cyclic AMP phosphodiesterase by EG626.     

Effects of 3-methyl-1-(5-oxohexyl)-7-propylxanthine on the in vivo release of monoamines studied by intracerebral dialysis

The effect of a xanthine derivative, 3-methyl-1-(5-oxohexyl)-7-propylxanthine (HWA 285, 10 mg/kg i.p.), on the release of endogenous monoamines and their metabolites from the rat striatum and hippocampus are examined in vivo by intracerebral dialysis technique. Using this method, which is based on the dialysis principle, a small-calibered (0.5 mm) dialysis tube is inserted into the striatum or hippocampus transversally and is perfused with Ringer solution. The amounts of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in the perfusates are measured by high performance liquid chromatography system with electrochemical detection. HWA 285 (10 mg/kg) decreased monoamine levels in hippocampal perfusates after its i.p. administration. However, there were no consistent changes in monoamine release in striatal perfusates after the drug administration. These findings suggest that HWA 285 exerts an inhibitory influence on hippocampal dopaminergic and serotonergic systems.     

Stereospecific influence of oxazepam hemisuccinate on cyclic AMP accumulation elicited by adenosine in cerebral cortical slices.

The effects of the d-, dl- and l-isomers of the water-soluble benzodiazepine, oxazepam sodium hemisuccinate, on cyclic AMP levels in superfused slices of guinea-pig and rat cerebral cortex were investigated. At 100 μM these drugs decreased the accumulation of cyclic AMP elicited by adenosine in a manner closely correlated to their stereostructure and relative anticonvulsant and anxiolytic potencies in vivo. The drugs inhibited uptake of low concentrations of radioactive adenosine into the slices in a similar manner. Addition of theophylline or adenosine deaminase to the superfusion medium sharply decreased both cyclic AMP basal levels and levels elicited by the d-isomer, suggesting mediation by adenosine of the effects of benzodiazepines on cyclic AMP and hence their psychotropic action.     

The relation of adenyl cyclase to the activity of other ATP utilizing enzymes and phosphodiesterase in preparations of rat brain; mechanism of stimulation of cyclic AMP accumulation by adrenaline, ouabain and Mn++

1. The mechanism of stimulation of cyclic adenosine 3',5'-monophosphate (cyclic AMP) accumulation by adrenaline and ouabain and the effect of Mn(++) substitution for Mg(++) as the metal ion requirement of this system was studied in cell-free preparations of adenyl cyclase from rat brain.2. In the rat cerebral cortex preparation, substitution of Mn(++) for Mg(++) significantly increased cyclic AMP accumulation while significantly inhibiting adenosine triphosphate (ATP) and adenosine diphosphate (ADP) hydrolysis and adenosine 5'-monophosphate (AMP) accumulation. In the synaptic membrane preparation, in the absence of NaF, the highest amount of ATP hydrolysis was obtained in tissue prepared with Mn(++) and incubated with Mg(++); under these conditions cyclic AMP accumulation was equal to that produced under any other condition and significantly higher than that observed in the presence of Mg(++) prepared and Mg(++) incubated tissue.3. Preparation and/or incubation of tissue with Mn(++) significantly reduced phosphodiesterase (PDE) activity compared to that observed in Mg(++) prepared tissue.4. Adrenaline and ouabain both significantly increased cyclic AMP accumulation in the rat cerebral cortex preparation but did not inhibit ATP or ADP hydrolysis. In the synaptic membrane preparation, in the presence of 0.01 mM Ca(++), adrenaline but not ouabain significantly increased cyclic AMP accumulation. Phenoxybenzamine (0.1 mM) and pronethalol (0.1 mM) significantly inhibited adrenaline-induced cyclic AMP accumulation in both these preparations.5. Ouabain and adrenaline both failed to stimulate cyclic AMP accumulation in the presence of Mn(++) prepared and/or incubated tissue.6. Ouabain and adrenaline had no effect on PDE activity in either of these preparations.7. It was concluded that Mn(++) increased cyclic AMP accumulation in part by indirect inhibition of ATP and ADP hydrolysis which provides inhibitors of cyclic AMP destruction, by direct stimulation of adenyl cyclase and by inhibition of cyclic AMP destruction in a way unrelated to nucleotide inhibition of PDE. Adrenaline and ouabain appeared tp stimulate cyclic AMP accumulation in a more direct manner.     

Behavioral effects of propentofylline (HWA 285) on ambulatory activity, discrete avoidance response and passive avoidance response in mice

Behavioral effects of propentofylline (HWA 285) were investigated by means of ambulatory activity, discrete lever-press avoidance and step-through type passive avoidance response in mice. Single administration of HWA 285 produced no marked change in the bodily condition and also produced no changes in ambulatory activity at 1.25-20 mg/kg, s.c.; the discrete avoidance response at 2.5-40 mg/kg, s.c.; and the passive avoidance response at 10-30 mg/kg, s.c. However, 5-20 mg/kg of HWA 285 attenuated the ambulation-increasing effect of methamphetamine (2 mg/kg, s.c.) and scopolamine (0.5 mg/kg, s.c.). HWA 285 tended to attenuate the avoidance-suppressing effect of chlorpromazine (2 mg/kg, s.c.) and physostigmine (0.1 mg/kg, s.c.) at 2.5 mg/kg, while it enhanced the effect of chlorpromazine at 10-40 mg/kg. The mice treated with HWA 285 (10-30 mg/kg) at 30 min before or immediately after the acquisition trial did not show a marked change in the passive avoidance response when the retention trial was done 24 hr after the acquisition trial. The treatment with scopolamine (2 mg/kg, s.c.) at 30 min before the acquisition trial suppressed the passive avoidance response, eliciting a marked shortening of the step-through latency and decrease in % of mice to the 300 sec criterion of latency. The effect of scopolamine was attenuated by combined administration of HWA 285 (30 mg/kg) and treatment with HWA 285 (30 mg/kg) after the end of the acquisition trial. The present results suggest that HWA 285 demonstrates complex behavioral effects which vary dependently on the doses and types of behaviors.     

Effect of adenosine receptor agonists and other compounds on cyclic AMP accumulation in forskolin-treated hippocampal slices

The effect of adenosine analogues and some putative neurotransmitters have been studied on cyclic AMP accumulation in rat hippocampal slices treated with the adenylate cyclase activator forskolin. The effects of PGE2 and histamine were potentiated by forskolin (0.1 microM). Isoprenaline and NECA had essentially additive effects with 0.1 microM forskolin and serotonin (above 10(-4) M) inhibited forskolin-stimulated cyclic AMP accumulation. The A1-adenosine receptor selective adenosine analogue R-PIA inhibited forskolin stimulated cyclic AMP accumulation in low doses and stimulated in high. NECA, adenosine and 2-chloroadenosine uniformly stimulated cyclic AMP accumulation. 2',5'-dideoxyadenosine inhibited, but only at high concentrations. Both the stimulatory and the inhibitory effects of R-PIA were antagonized by 8-phenyltheophylline (10 microM). Enprofylline (100 microM) selectively inhibited the stimulatory effect. In the presence of enprofylline both 2-chloroadenosine showed an inhibitory effect on cyclic AMP accumulation. It is concluded that the forskolin-treated rat hippocampal slice is a useful preparation to study both stimulatory and inhibitory effects of transmitters and modulators on adenylate cyclase. The results also show that the rat hippocampus has both A1-receptors that are linked to inhibition of cyclic AMP accumulation and A2-receptors that are linked to stimulation. Furthermore, enprofylline is shown to selectively antagonize the stimulatory response, revealing inhibitory effects of compounds such as 2-chloroadenosine and adenosine.     

Biochemical mechanisms for the inotropic effect of the cardiotonic drug milrinone

Milrinone is a new inotropic agent for the treatment of refractory congestive heart failure. Our understanding of the mechanisms(s) of action of this synthetic cardiotonic drug is incomplete. We examined the effects of milrinone and the parent compound amrinone on sarcoplasmic reticulum function (45Ca-uptake and Ca-ATPase); radioligand binding to adenosine, beta-adrenergic, and cholinergic muscarinic receptors; cyclic AMP accumulation; and inhibition of various forms of cyclic AMP phosphodiesterases. Comparisons were made to observe how these effects correlate with the inotropic response of heart. Milrinone was shown to be a potent phosphodiesterase inhibitor that was 40 times more potent than amrinone and 10 times more potent at inhibiting the high-affinity (Km = 0.23 microM) form (Ki = 22 microM) than the low-affinity (Km = 140 microM) form (Ki = 225 microM) of cyclic AMP phosphodiesterase in heart. The potency of milrinone as a phosphodiesterase inhibitor was the same in the presence and absence of calcium. Concentrations of milrinone that increased cyclic AMP accumulation also produced positive inotropy. A comparison of milrinone with amrinone and methylxanthines revealed the order of potency to be isobutylmethylxanthine greater than milrinone greater than theophylline greater than caffeine greater than amrinone. Milrinone and amrinone had no effect on 45Ca-uptake or Ca-ATPase activity in myocyte sarcoplasmic reticulum. However, milrinone did bind weakly to adenosine receptors (KD = 466 microM) but not to cholinergic muscarinic or beta-adrenergic receptors. Also, in combination with isoproterenol high concentrations of milrinone blocked the negative inotropic response to the adenosine agonist phenylisopropyladenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

J.D.P. Graham

The effect of both isomers of niguldipine, a highly selective alpha 1-adrenoceptor antagonist and dihydropyridine calcium channel blocker, on noradrenaline-stimulated inositol phosphate (IP) accumulation and adenosine 3':5'-cyclic monophosphate (cyclic AMP) potentiation was examined. Both isomers inhibited noradrenaline-stimulated IP accumulation. (+)-Niguldipine was 100 fold more potent than (-)-niguldipine. Potentiation of beta-adrenoceptor-stimulated cyclic AMP by noradrenaline was only partially inhibited by both isomers. The dihydropyridine, israpidine, did not inhibit either second messenger response. This study provides further evidence that the alpha 1-adrenoceptors mediating IP accumulation and cyclic AMP potentiation are different.     

Studies on the receptor mediating cyclic AMP-independent enhancement by adenosine of IgE-dependent mediator release from rat mast cells.

Adenosine produced a concentration-related enhancement of antigen-induced 5-hydroxytryptamine (5-HT) release from rat serosal mast cells. This potentiation was maximal following the simultaneous addition of adenosine with antigen. Enhancement of 5-HT release was accompanied by potentiation of the adenosine 3':5'-cyclic monophosphate (cyclic AMP) response to challenge. The cyclic AMP response, which was antagonized by 8-phenyltheophylline, was characterized as an A2-purinoceptor-mediated effect by the use of 5'-N-ethylcarboxamideadenosine (NECA) and L-N6-phenylisopropyladenosine (L-PIA). Enhancement of 5-HT release, conversely, was not blocked by 8-phenyltheophylline suggesting it to be mediated by a cyclic AMP-independent mechanism. The effect of adenosine on 5-HT release was not reduced by the inhibition of the facilitated uptake of adenosine with dipyridamole, hexobendine or p-nitrobenzylthioguanosine, therefore, suggesting it to be mediated by a cell surface receptor. The receptor mediating enhancement of 5-HT does not appear to belong to the P2-purinoceptor subtype as adenosine was more potent than both adenosine monophosphate (AMP) and adenosine diphosphate (ADP) and alpha, beta-methylene ATP was inactive. Furthermore, the effects of AMP were blocked by alpha, beta-methylene ADP, which inhibits the conversion of AMP to adenosine. Adenosine, NECA, L- and D-PIA were all of equal potency in enhancing 5-HT release. Inosine and 3-deazaadenosine were also active. The rank order of potency of these adenosine analogues is not consistent with an effect at A1- or A2-purinoceptors. There appear to be two adenosine receptors on rat mast cells, an A2-purinoceptor which stimulates adenylate cyclase and a separate purinoceptor, stimulation of which produces enhancement of mediator release by an unknown mechanism. The effects mediated by these receptors appear to be independent of each other.     

Effects of acute and chronic ethanol on cyclic AMP accumulation in NG108-15 cells: differential dependence of changes on extracellular adenosine.

1. This study investigated the effects of acute and chronic ethanol on basal, agonist- and forskolin-stimulated cyclic AMP formation in NG108-15 mouse neuroblastoma x rat glioma hybrid cells, and examined the role of changes in extracellular adenosine concentrations on the effects observed. 2. NG108-15 cells incubated acutely with ethanol (1-200 mM) displayed concentration-dependent increases in basal and iloprost-stimulated (300 nM; a prostanoid IP receptor agonist) cyclic AMP accumulation but a concentration-dependent decrease in forskolin-stimulated (10 microM) accumulation. 3. Cells treated chronically with ethanol (200 mM) for 48 h displayed increases over control in basal, iloprost- (0.001-10 microM) and forskolin (0.01-100 microM)-stimulated cyclic AMP formation. However, chronic ethanol did not affect [3H]-iloprost binding to cell membranes. 4. Inclusion of adenosine deaminase (ADA; 1 unit ml-1) during the incubation period to measure cyclic AMP accumulation completely abolished the increase in basal accumulation following chronic ethanol, but did not affect the increase in iloprost stimulation. On the other hand ADA partially reversed the increase in forskolin stimulation following chronic ethanol, but even in the presence of high concentrations of ADA (5 units ml-1) the forskolin stimulation remained elevated above control. 5. Cells treated chronically with the adenosine receptor agonist 5'-(N-ethylcarboxamido)-adenosine (NECA; 10 microM for 48 h) displayed a reduction in subsequent NECA- and forskolin-stimulated cyclic AMP accumulation, but iloprost stimulation was not affected. ADA included acutely during the incubation period to measure cyclic AMP accumulation abolished the reduction in forskolin but not NECA stimulation produced by the chronic NECA pretreatment.(ABSTRACT TRUNCATED AT 250 WORDS)