The effects of phenobarbitone, leptazol, dexamphetamine, iproniazid, imipramine, LSD, chlorpromazine, reserpine and hydroxyzine on the in vivo levels of adenine nucleotides and phosphocreatine in the rat brain

1. Enzymic and ion-exchange chromatographic analyses were used to measure adenosine triphosphate (ATP), diphosphate (ADP) and monophosphate (AMP) in brain extracts from rats treated with a wide range of centrally acting drugs. Phosphocreatine (PC) was assayed by the acid molybdate method.2. An anaesthetic dose of phenobarbitone caused an increase in brain levels of ATP and PC, and a reduction in ADP and AMP. A convulsant dose of leptazol gave rise to precisely opposite effects. Subanaesthetic (hypnotic) and subconvulsive doses of the two drugs, respectively, produced no alterations in brain nucleotide levels.3. Among the psychotropic drugs, dexamphetamine, LSD and hydroxyzine, at the doses used, caused no changes in brain levels of the adenine nucleotides. Iproniazid and imipramine caused slight increases in the ATP level and ATP / ADP ratio, respectively. Chlorpromazine failed to give rise to any effect in the nucleotides 3 hr after administration, but produced a rise in brain ATP after 6 hr. Reserpine, on the other hand, caused a fall in the ATP/ADP ratio 6 hr after injection.4. These results indicate that some psychotropic drugs can cause small changes in the rat brain ATP/ADP ratio but do not support claims by certain workers that such changes correlate closely with the behavioural effects of these drugs.     

Pathways of nucleotide metabolism in Schistosoma mansoni. IV. Incorporation of adenosine analogs in vitro

The incorporation in vitro of adenine or adenosine analogs into schistosome nucleotides is demonstrated. Tubercidin, 2-fluoroadenosine and 2-fluoroadenine were all shown to be converted into analog triphosphate nucleotides. Since tubercidin and 2-fluoroadenosine are not substrates for adenosine deaminase or purine nucleoside phosphorylase and are not susceptible to degradation to the free base level, it is assumed that they are converted to nucleotides by reaction with adenosine kinase. The incorporation of 2-fluoroadenine into the nucleotide pools indicates that it serves as a substrate for adenine phosphoribosyltransferase.Tubercidin, added to the culture medium, interferes with the maintenance of normal ATP levels. When the concentration of the analog greatly exceeded that of adenine or adenosine in the medium, virtual shutdown of adenosine triphosphate synthesis followed. It is suggested that stoichiometric competition for enzyme sites may determine the relative amounts of nucleotides formed.     

The influence of calcium antagonists on the adenine nucleotide metabolism in the guinea-pig working heart during ischaemia and reperfusion

Summary With the aim of gaining more insight into the metabolism of adenine nucleotides in working normoxic guinea-pigs and in hearts subjected to 45 min of global ischaemia and subsequent reperfusion for 25 min, we evaluated the effect of nifedipine, verapamil, diltiazem, bepridil, CERM 11956, lidoflazine, mioflazine and dipyridamole on the adenine nucleotide catabolite levels in these hearts. The drugs were applied at the concentrations that reduced the aortic dP/dt of normoxic working hearts by 10% (EC₁₀) and 30% (EC₃₀).In globally ischaemic hearts there was a large accumulation of adenine nucleotide catabolites. Inosine proved to be the major catabolite. The drugs, with the exception of bepridil, CERM 11956 and dipyridamole (3 mol/l), decreased the accumulation of catabolites. In hearts treated with mioflazine and dipyridamole the amount of adenosine increased. A deficit in the balance between adenine nucleotides and catabolites indicated that in globally ischaemic hearts there was a large accumulation of inosine monophosphate. Indeed, a substantial amount of inosine monophosphate was determined in untreated hearts, and hearts treated with nifedipine (EC₃₀) and mioflazine (EC₁₀). During the first 5 min of reperfusion a large quantity of catabolites, mainly inosine, was washed out. During 20 min of subsequent reperfusion in untreated hearts and in nifedipine and mioflazine-treated hearts the efflux of catabolites returned to normoxic values. Similar to the effect in ischaemic hearts, in early perfusate from lidoflazine, mioflazine and dipyridamoletreated hearts the adenosine/inosine ratio was increased. An incomplete balance of adenine nucleotides and catabolites, as well as the presence of a considerable quantity of inosine monophosphate in mioflazine-treated hearts, suggests that adenosine suppresses the breakdown of inosine monophosphate upon reperfusion.A reduced efflux of catabolites and the presence of a considerable amount of inosine monophosphate in lidoflazine, mioflazine and dipyridamole-treated hearts does not lead to an increased synthesis of adenine nucleotides during 25 min of reperfusion. Calcium antagonists without nucleoside transport inhibitory activity exert only limited influence on the adenine nucleotide catabolism in globally ischaemic and reperfused guineapig hearts. The ineffectiveness of all drugs to enhance the adenine nucleotide levels in reperfused hearts seems to results from the incapacity of the adenine nucleotide metabolism to convert inosine monophosphate, nucleosides and hypoxanthine into adenine nucleotides. Send offprint requests to J. G. Hugtenburg at the above address     

Effects of antibiotic antitumor drugs on nucleotide levels in cultured tumor cells: an exploratory method to distinguish the mechanisms of antitumor drug action based on targeted metabolomics

Nucleotide pools in mammalian cells change due to the influence of antitumor drugs, which may help in evaluating the drug effect and understanding the mechanism of drug action. In this study, an ion-pair RP-HPLC method was used for a simple, sensitive and simultaneous determination of the levels of 12 nucleotides in mammalian cells treated with antibiotic antitumor drugs (daunorubicin, epirubicin and dactinomycin D). Through the use of this targeted metabolomics approach to find potential biomarkers, UTP and ATP were verified to be the most appropriate biomarkers. Moreover, a holistic statistical approach was put forward to develop a model which could distinguish 4 categories of drugs with different mechanisms of action. This model can be further validated by evaluating drugs with different mechanisms of action. This targeted metabolomics study may provide a novel approach to predict the mechanism of action of antitumor drugs.Abbreviations: ADP, adenosine diphosphate; AMP, adenosine monophosphate; ANOVA, analysis of variance; ATP, adenosine triphosphate; AUC, area under the curve; CDP, cytidine diphosphate; CTP, cytidine triphosphate; dATP, deoxyadenosine triphosphate; dCDP, deoxycytidine diphosphate; dCTP, deoxycytidine triphosphate; dGMP, deoxyribonucleic monophosphate; dGTP, deoxyguanosine triphosphate; DMEM, Dulbecco׳s modified eagle׳s cell culture media; DMSO, dimethyl sulfoxide; DNA, deoxyribonucleic acid; dUDP, deoxyuridine diphpsphate; dUTP, deoxyuridine triphosphate; EC, energy charge; EDTA, ethylene diamine tetra-acetic acid; FCS, fetal calf serum; GDP, guanosine diphosphate; GMP, guanosine monophosphate; GTP, guanosine triphosphate; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate buffered saline; PCA, principal component analysis; RNA, ribonucleic acid; ROC, receiver operating characteristic; RPMI-1640, Roswell Park Memorial Institute-1640; TBAHS, tetrabutylammonium hydrogen sulfate; TCA, trichloroacetic acid; UDP, uridine diphosphate; UTP, uridine triphosphateKEY WORDS: Nucleotides, Targeted metabolomics analysis, Tumor cells, Potential biomarkers, Mechanisms of antitumor drug action, Antibiotic anticancer drugs, Principal component analysis, Ion-pair HPLC     

Potentiation of adenosine triphosphate-induced contractile responses of the guinea-pig isolated vas deferens by adenosine monophosphate and adenosine 5''-monophosphorothioate.

The effects of incubating the guinea-pig isolated vas deferens in the presence of adenine nucleotides (adenosine triphosphate, ATP; adenosine diphosphate, ADP; and adenosine monophosphate, AMP), or in the presence of their phosphorothioate analogues (adenosine 5'-O-(3-thiotriphosphate), ATP gamma S; adenosine 5'-O-(2-thiodiphosphate), ADP beta S; and adenosine 5'-monophosphorothioate, AMP alpha S), on contractile responses to ATP were compared. After challenge with a low (1 microM) or high (300 microM) concentration of ATP to obtain control responses, one vas deferens of a pair was incubated for 5 min with one of the adenine nucleotides, while the contralateral preparation was incubated with the corresponding phosphorothioate analogue. At the conclusion of the incubation the preparations were challenged again with ATP. Incubation with AMP or AMP alpha S resulted in a transient potentiation of responses to 1 microM and 300 microM ATP. The potentiation following incubation with AMP alpha S was larger than that produced by AMP. After incubation with ADP, ADP beta S, ATP and ATP gamma S, responses to 1 microM ATP were decreased, while those to 300 microM ATP were unaffected. Thus, incubation with AMP and AMP alpha S results in potentiation, rather than inhibition, of ATP-induced responses. On the other hand, 5'-diphosphate, 5'-triphosphate, 5'-O-(2-thiodiphosphate) and 5'-O-(3-thiotriphosphate) moieties on adenosine have no effect or cause autoinhibition. These results indicate that AMP exerts a potentiating effect on reactivity to exogenous ATP. AMP arising from the enzymatic degradation of ATP might modulate the level of response to ATP released endogenously as a cotransmitter.     

Effect of trimetazidine on the nucleotide profile in rat kidney with ischemia-reperfusion injury.

Ischemia-reperfusion injury is often responsible for delayed graft function after transplantation. Trimetazidine (TMZ) is an antioxidant agent used to protect grafts from ischemia-reperfusion injury. The aim of the study was to examine the effect of TMZ on nucleotide profile in rat kidney with ischemia-reperfusion injury. The study was carried out on Wistar rats divided into two groups: animals treated with TMZ and control group receiving placebo. TMZ 10mg/kg/day was administrated for 30 days. Concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine (Ado), guanosine triphosphate (GTP), guanosine diphosphate (GDP), guanosine monophosphate (GMP), guanosine (Guo), inosine monophosphate (IMP), inosine (Ino), hypoxanthine (Hyp), xanthine (Xan), uric acid (UA), uridine (Urd), nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) were determined in kidney tissues after ischemia-reperfusion using HPLC. The total adenine nucleotide concentration (TAN) and adenylate energy charge (AEC) were also determined. Moreover the kidneys were evaluated histologically. Tissue concentrations of ATP, ADP, AMP, TAN and AEC were significantly increased in kidneys from rats treated with TMZ in comparison with rats receiving placebo. Concentrations of products of nucleotide degradation: inosine (Ino), guanosine (Guo) and uridine (Urd), as well as oxypurines: Hyp and Xan, were significantly decreased in rats treated with trimetazidine. Moreover, significantly less pronounced acute tubular necrosis was observed in kidneys of rats treated with TMZ. These results suggest that trimetazidine protects against dephosphorylation of nucleotides and ischemic damage.     

The effects of adenine nucleotides on cutaneous afferent nerve activity.

1 The activity produced by the adenine nucleotides adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) and by potassium, acetylcholine (ACh), 5-hydroxytryptamine (5-HT) and bradykinin when applied to an exposed blister base on the ear of anaesthetized rabbits or intra-arterially to anaesthetized cats was investigated in multiple strands dissected from the auricular-temporal and saphenous nerves of rabbits and cats, respectively. 2 In the rabbit preparation potassium and the adenine nucleotides produced activity in the nerve fibres. The effects of these substances were produced in comparable dose ranges; threshold effects being produced by potassium at a concentration of 13 mM and by ADP at a concentration of 4 mM. ACh, 5-HT and bradykinin were inactive at similar or higher concentrations. 3 In the cat preparation all the substances tested produced activity in the nerve fibres. The adenine nucleotides were comparatively less potent than ACh, 5-HT or bradykinin, but had greater potency than potassium. 4 It was concluded that the adenine nucleotides do possess effects on afferent nerve terminals or fibres and thus resemble other known algogenic substances such as potassium, ACh, 5-HT and bradykinin.     

Changes of platelets’ function in preeclampsia

Increased aggregation of platelets during preeclampsia was shown in several studies, yet several others reported no change. The aim of our study was to investigate platelet aggregation in a group of patients suffering from preeclampsia. In a cross-sectional study blood samples were taken from 89 hospitalized patients in the third trimester of pregnancy: 38 were suffering from mild to moderate preeclampsia and 51 patients were without preeclampsia. From the blood samples platelet aggregation, secretion of adenine nucleotides from platelets, concentration of energy-rich adenine compounds and levels of cyclic adenosine-mono-phosphate and cyclic guanosine mono-phosphate in platelets were measured. In the patients with preeclampsia, the adenosine diphosphate threshold for biphasic aggregation [odds ratio (OR):.75; 95% Confidence Interval (CI): 0.55–1.02; p<0.05], total adenine nucleotides concentration in the metabolic pool of platelets (OR:0.99; CI: 0.62–1.57; p<0.01) and cyclic adenosine-mono-phosphate (OR:0.81; CI: 0.57, 1.14; p<0.05) and cyclic guanosine mono-phosphate (OR:.78; CI: 0.55–1.09; p<0.05) levels in platelets were decreased in comparison with the control group, while adenylate energy charge in the metabolic pool of platelets (OR: >100.00; CI: 0.00->100.00; p<0.05) and secretion of adenosine triphosphate (OR:.13; CI: 0.00–14.26; p<0.05) and adenosine diphosphate (OR:.77; CI: 0.08–36.79; p<0.05) were increased. The results of our study show increased activation and aggregation of platelets in pregnant females with preeclampsia.     

8-Cyclopentyl-1,3-dimethylxanthine enhances effectiveness of antidepressant in behavioral tests and modulates redox balance in the cerebral cortex of mice

The objective of our study was to investigate whether 8-cyclopentyl-1,3-dimethylxanthine (CPT), associated with the adenosine system, enhances the antidepressant efficacy of antidepressant. All experiments were carried out on Albino Swiss mice. Following drugs: CPT (3?mg/kg) and imipramine (15?mg/kg) were administered intraperitoneally (ip), 60?min before tests. Two behavioral tests on antidepressant capability – a forced swim test (FST) and a tail suspension test (TST) – were performed. To examine whether co-administration of CPT with antidepressants affects the redox balance, the lipid peroxidation products (LPO), glutathione (GSH), glutathione disulfide (GSSG), nicotinamide adenine dinucleotide phosphate (NADP+), and reduced nicotinamide adenine dinucleotide phosphate (NADPH) were determined in the cerebral cortex. The results have demonstrated a CPT-induced enhancement of the antidepressant-like effect of imipramine both in the FST and TST, which may indicate that the adenosine system may be involved in the increasing the effect of antidepressant. Co-administration of CPT with imipramine, such as imipramine alone, decreased the NADP+ and LPO concentrations and increased the GSH/GSSG ratio in comparison to the control, which may confirm beneficial – but comparable to imipramine – effect on redox balance under environmental stress conditions. An increase in the concentration of GSSG in the cortex of animals treated with imipramine in ineffective dose compared to control and no such changes after combined administration of both drugs may suggest a favorable oxidation-reduction potential resulting from their synergistic antidepressant effect.     

Adenosine triphosphate-evoked vascular changes in human skin: Mechanism of action

Adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine, adenine and inosine were injected intradermally into the backs of human volunteers. ATP, ADP and AMP evoked weal and flare responses in the skin in a dose dependent manner. The rank order of potency was ATP>ADP>AMP; other metabolites were apparently inactive. The potency of ATP was approximately 0.002 times that of histamine. In the forearm, cross tachyphylaxis was demonstrated between ATP and histamine weals; also the flare due to injected ATP spead beyond a band which was applied to prevent diffusion, indicating that the flare is neurogenic. Injections of ATP and high doses of ADP produced a sensation of persistent pain, unlike histamine which produced transient pain or itch on some occasions, and saline which was without effect. The possible involvement of histamine, mast cells and prostaglandins in the response was examined. The inhibitory actions of systemic pretreatment with diphenhydramine suggests that the erythema and wealing responses to ATP are at least partly due to ATP-evoked histamine release. Indomelhacin, doxantrazole and cimetidine did not alter the ATP reaction.     

Antagonism of tetrodotoxin- and procaine-induced axonal blockade by adenine nucleotides in the frog sciatic nerve

The effects of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine on compound action potentials were investigated in de-sheathed frog-sciatic nerve preparations. ATP and ADP but not adenosine antagonized the inhibitory action of tetrodotoxin (TTX) on nerve conduction. AMP had little or no antagonistic effect on TTX-induced axonal block. ATP was more effective than ADP. The effects of the nucleotides were related to the degree of the TTX-induced inhibition and were more evident where the blockade was more intense. ATP and ADP but not adenosine antagonized the procaine-induced axonal blockade which, in some experiments, was completely reversed by these nucleotides. ATP and ADP were of similar potency. The axonal blockade induced by pentobarbitone was not antagonized by ATP, ADP, AMP or adenosine. The possibility that ATP stimulates a TTX-sensitive sodium channel is discussed.     

Behavioural effects of d-amphetamine alone and in combination with antidepressants,antihistamines or other psychotropic drugs in young chicks

Antidepressants, antihistamines or other psychotropic drugs were administered before injection of d-amphetamine in 3–5 day old chicks. This pretreatment completely protected some animals against the characteristic behavioural changes seen after d-amphetamine 6 mg/kg i.p. In addition chlorpheniramine, chlorimipramine, imipramine, cocaine and diphenhydramine were also able to antagonize a dose of 10 mg/kg i.p. It was further noticed that well developed d-amphetamine symptoms were interrupted by subsequent treatment with imipramine.     

Pathways of nucleotide metabolism in Schistosoma mansoni--VII. Inhibition of adenine and guanine nucleotide synthesis by purine analogs in intact worms.

Twelve analogs of adenosine or its related purines have been tested as single agents or in combinations as possible antischistosomal compounds. Measurements were made of the effect of these drugs on the anabolism and catabolism of [8-¹⁴C]adenosine by Schistosoma mansoni in vitro. Based on the degree of inhibition of biosynthesis of adenine and guanine nucleotides by intact worms. the best currently available purine analogs are 7-deaza-adenosine (tubercidin) and N⁶-phenyladenosine. These drugs reduced the total synthesis of nueleotides to 30 and 25 per cent, respectively, of controls. Blockade of the catabolic pathway (adenosine deaminase) by coformycin resulted in significantly increased synthesis of adenine nucleotides rather than the expected decrease. Thus, adenosine kinase must play a more prominent role in nucleotide synthesis than had been previously estimated. The implications of these findings in the development of new anti-schistosomal drugs are discussed.     

An arginine/glutamine difference at the juxtaposition of transmembrane domain 6 and the third extracellular loop contributes to the markedly different nucleotide selectivities of human and canine P2Y11 receptors.

The recently cloned canine P2Y11 receptor (cP2Y11) and its human homolog (hP2Y11) were stably expressed in Chinese hamster ovary cells (CHO-K1) and 1321N1 human astrocytoma cells, and their agonist selectivities and coupling efficiencies to phospholipase C and adenylyl cyclase were assessed. Adenosine triphosphate nucleotides were much more potent and efficacious at the hP2Y11 receptor than their corresponding diphosphates in promoting both inositol phosphate and cyclic AMP accumulation. In contrast, adenosine diphosphate nucleotides were considerably more potent at the cP2Y11 receptor than their corresponding triphosphate analogs. The tri- versus diphosphate specificity of the two receptors was further confirmed in studies using Ca(2+) mobilization as a measure of receptor activation under conditions that minimized nucleotide degradation. Moreover, 2-methylthioadenosine-5'-triphosphate and 2-methylthioadenosine-5'-diphosphate were 58- and 75-fold more potent than ATP and ADP, respectively, at the cP2Y11 receptor compared with only 2- to 3-fold more potent at the hP2Y11 receptor. Mutational analysis revealed that the change of Arg-265, which is located at the juxtaposition of transmembrane domain 6 and the third extracellular loop in the hP2Y11 receptor, to glutamine in the cP2Y11 receptor is at least partly responsible for the diphosphate selectivity but not the increased sensitivity to 2-thioether-substituted adenine nucleotides at the canine receptor. These results imply a key role for a positively charged arginine residue in contributing to the recognition of extracellular nucleotides by the P2Y11 receptor and perhaps other P2Y receptors.     

Inhibitory action of adenosine on synaptic transmission in the hippocampus of the guinea pig in vitro

Thin hippocampal slices were prepared from guinea pig brains. the postsynaptic field potential elicited in the pyramidal cell layer of CA3 region by mossy fiber stimulation was reversibly inhibited by application of adenosine to the perfusion medium. Among purine and pyrimidine derivatives, only adenosine and adenine nucleotides depressed the field potential with similar dose-response curves at concentrations of 10(-5).10(-3) M. In order to elucidate the mechanism of the inhibitory action of these agents, the effects of adenosine and adenine nucleotides on membrane events in pyramidal neurons were studied using intracellular recording techniques. Application of adenosine and adenine nucleotides hyperpolarized membrane potential and markedly depressed the EPSPs (excitatory postsynaptic potentials) elicited in the pyramidal cell by granular cell activation. However the spike generating mechanism of the neuron was not interfered with and membrane conductance was not increased by adenosine and adenine nucleotides. 4-Aminopyridine counteracted the inhibitory action of adenosine. These findings indicate that the mechanism of the inhibitory action of adenosine and adenine nucleotides is different from that of conventional inhibitory neurotransmitters such as gamma-aminobutyric acid and suggest a presynaptic action of adenosine and adenine nucleotides.     

Distribution and HPLC study of chromium-51 binding sites in Chinese hamster ovary cells

Chinese hamster ovary cells were cultured with chromium-51 chromate to study the site of chromium interaction with cell biomolecules. After incubation, cells were homogenized and separated into nuclear, mitochondrial, microsomal, and cytosolic fractions. Greater than 75% of the radioactivity was found in the cytosolic fraction. The supernatant from the centrifuged cell homogenate, which contained greater than 90% of the chromium radioactivity, was subjected to chromatographic investigation. The combination of anion exchange and ion-pair high-performance liquid chromatography (HPLC) showed that at least three different molecular species interact with chromate or its reduced derivative, Cr(III). These species are glutathione, the nucleotides cytosine triphosphate, uridine triphosphate, guanine triphosphate, adenosine triphosphate, and adenosine diphosphate, plus an as yet unknown species of protein or peptide. Preliminary data for the specific activity of nucleoside triphosphates range from 6000 to 18,000 cpm/micrograms ribonucleoside triphosphate. The glutathione accounted for 50% of the observed radioactivity, the nucleotides for 30%, and the metalloprotein accounted for the remainder.     

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 NaF

1. A method for measuring the rate of production of ¹⁴C-labelled adenine nucleotides, including cyclic adenosine 3′,5′monophosphate (cyclic AMP), from [¹⁴C]-adenosinetriphosphate (ATP) was developed and used to study the effects of ATP, adenosine diphosphate (ADP) and adenosine 5′-monophosphate (AMP) on the rate of accumulation of cyclic AMP in cell-free preparations of adenyl cyclase from rat brain.2. The mechanism by which NaF increases cyclic AMP accumulation was studied by comparing its effect on adenine nucleotide metabolism with that of an ATP regenerating system.3. ADP and ATP are potent inhibitors of phosphodiesterase (PDE) and it is the sum of the concentrations of these two nucleotides which controlled the rate of destruction of cyclic AMP. The effect of these nucleotides was significant even in the presence of 6·7 mM theophylline; theophylline itself inhibited PDE only 50-60%.4. Fluoride ion had no direct effect on PDE but it inhibited the rate of hydrolysis of ADP and ATP and thus indirectly inhibited PDE. The effect of fluoride ion on cyclic AMP accumulation can be explained, at least in part, by this indirect inhibition of PDE.5. Studies on a more purified preparation of adenyl cyclase clearly demonstrated a direct action of NaF on adenyl cyclase.     

Removal of interfering nucleotides from brain extracts containing substance p. Effect of drugs on brain concentrations of substance p

Several methods for removing interfering nucleotides, adenosine-5'-monophosphate and adenosine 5'-triphosphate from brain extracts have been studied. An enzymic method, using adenylic acid deaminase, has been found suitable. This deaminates adenosine monophosphate to 5'-inosinic acid, an inactive compound which does not influence the estimations of substance P. Owing to the adenosine triphosphatase content of the enzyme extract, adenosine triphosphate was also inactivated. For the estimation of adenosine monophosphate-deaminase activity, a simple colorimetric method is described which measures the ammonia liberated from adenosine monophosphate. Substance P in mouse brain extracts was estimated after treatment of the animals with various drugs, and after the enzymic removal of interfering nucleotides from the brain extracts. The drugs had no effect on the substance P content of mouse brain. The effect of drugs on the contractions of the guinea-pig ileum induced by substance P was also investigated, and the effect of drugs on the estimations of substance P in brain extracts is discussed.     

Autoradiography of P2x ATP receptors in the rat brain.

1. Binding of a P2x receptor specific radioligand, [3H]-alpha,beta-methylene adenosine triphosphate ([3H]-alpha,beta-MeATP) to sections of rat brain was reversible and association/dissociation parameters indicated that it consisted of two saturable components. Non-specific binding was very low (< 7% at 10 nM ligand concentration). 2. The binding was completely inhibited by suramin (IC50 approximately 14-26 microM) but none of the ligands specific for P2y receptors such as 2-methylthio-adenosine triphosphate (2-methyl-S-ATP) and 2-chloro-adenosine triphosphate (2-C1-ATP) nor 2-methylthio-adenosine diphosphate (2-methyl-S-ADP) a ligand for the P2 receptor on blood platelets ('P2T' type) produced strong inhibitions except for P1,P4-di(adenosine-5')tetraphosphate (Ap4A). 3. Inhibitors of Na+,K(+)-dependent adenosine triphosphatase (ATPase) ouabain, P1-ligand adenosine and an inhibitor of transport of, respectively, adenosine and cyclic nucleotides, dilazep, had no effect. 4. The highest density of P2x binding sites was found to be in the cerebellar cortex but the binding sites were present in all major brain regions, especially in areas known to receive strong excitatory innervation.     

Effects of betaxolol, a cardioselective beta-adrenoceptor antagonist, on ischemic myocardial energy and carbohydrate metabolism in dogs

The effect of betaxolol, a beta 1-adrenoceptor antagonist, on ischemic myocardial metabolism was studied in dog hearts subjected to an occlusion of the left anterior descending coronary artery for 10 or 30 min. Betaxolol (0.1 or 0.3 mg/kg) was injected i.v. 5 min before ischemia. Betaxolol decreased heart rate, (+)dp/dt, coronary flow and blood pressure. Coronary occlusion decreased the levels of creatine phosphate, adenosine triphosphate, total adenine nucleotides and energy charge potential in the ischemic myocardium. Ten minutes after ischemia, betaxolol significantly diminished these impairments of energy metabolism. Even 30 min after ischemia, a higher dose of betaxolol significantly inhibited the depletion of total adenine nucleotides. Myocardial ischemia produced a breakdown of glycogen, an accumulation of lactate and an inhibition of glycolytic flux through the phosphofructokinase reaction. Betaxolol also reduced these alterations of carbohydrate metabolism 10 min after ischemia. These results indicate that betaxolol delays the onset of myocardial metabolic change from aerobic to anaerobic during ischemia and hence reduces the severity of myocardial ischemic injury.