Ethanol-induced changes in cyclic guanosine 3',5'-phosphate metabolism in mouse vestibular region

The content of cyclic guanosine 3',5'-phosphate (cGMP) in the region of the vestibular nuclei of mice increased after 10–14 days of ethanol intake. The increase was preceded by an increase in guanylate cyclase activity. A second increase was noted after 4 weeks on an ethanol-containing diet at the same time as decreased cGMP-phosphodiesterase activity was noted. The early increases were less pronounced in the alcohol-rejecting DBA/2J strain but were reproduced by preincubation or addition of arachidonic acid. Cerebellectomy had no effect on early or late rises in cGMP levels.     

Stimulatory effect of ionophores on adenosine 3',5'-monophosphate content in human mononuclear leukocytes

Ionophores A23187 and bromo-lasalocid ethanolate enhanced the cyclic AMP content in human mononuclear leukocytes. The maximum effect of A23187 with a 10-min incubation was found with 0.3–1.0μM concentrations with or without l-isoproterenol (1 μM) or prostaglandin E ₁ (pge ₁) (0.3 μM). The maximum effect after 5 min of incubation at 37° was observed with 0.05, 0.2 and 1 μm A23187. The effect of ionophore A23187 was enhanced by both aminophylline (1 mM) and isobutyl-methylxanthine (1 mM). Calcium (1 mM). aspirin (1 mM) and indomethacin (100 μM) decreased the stimulatory action of A23187. Bromo-lasalocid ethanolate increased cyclic AMP content in cells maximally at a 3 μM concentration with or without 0.3 μM pge ₁.     

Dephosphorylation of nitrobenzylthioinosine 5'-monophosphate by ecto 5'-nucleotidase of HeLa cells

HeLa cells as well as human and mouse erythrocytes possess membrane sites which bind the inhibitor of nucleoside transport, nitrobenzylthioinosine (NBMPR), reversibly but tightly (K_D, 10^?9–10^?10M). Site-specific binding of the ligand correlates with inhibition of nucleoside transport. The present study showed that the 5'-phosphate of NBMPR, NBMPR-P, was not transport inhibitory. Upon exposure to [³⁵S]NBMPR-P or [G-³H]NBMPR-P, HeLa cells retained the isotopic labels virtually exclusively in the form of NBMPR. The dephosphorylation of [G-³H]NBMPR-P by HeLa cells, assayed by the production of extracellular [G-³H]NBMPR, was competitively inhibited by AMP, but was not affected by the presence of 5 μM NBMPR, a concentration sufficient to completely occupy the transport inhibitory sites. Thus, the sites at which dephosphorylation of NBMPR occurs in HeLa cells are separate from and function independently of the high affinity sites which bind NBMPR.     

Neurotoxicity of deoxycoformycin: effect of constant infusion on adenosine deaminase,adenosine, 2'-deoxyadenosine and monoamines in the mouse brain

The tight-binding adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF), was continuously infused into mice by intraperitoneal implantation of microosmotic pumps delivering the compound at a rate of 0.16 mg hr^?1 kg^?1 for up to 6 days. The activity of cerebral adenosine deaminase was nearly totally inhibited. The amount of adenosine and 2'-deoxyadenosine was determined in the brain frozen in liquid nitrogen through the intact skull bone. The concentration of adenosine was about 1 $\text{nmol}\text{g}$, and was essentially not altered following treatment with deoxycoformycin. Deoxycoformycin induced a progressive increase in cerebral content of 2'-deoxyadenosine, which after 1 day of treatment equalled the amount of adenosine. The concentrations of serotonin, dopamine and noradrenaline in the brain were not altered.     

The effects of electrical stimulation, adenosine and adenosine -5′ triphosphate (ATP) on mouse rectal muscle

The effects of electrical field stimulation and of purine compounds, adenosine and adenosine-5'-triphosphate (ATP) were examined on the mouse isolated rectum. Electrical field stimulation induced frequency-dependent contractions of mouse rectal muscles which were potentiated by physostigmine and inhibited by atropine or tetrodotoxin. Contractile amplitude at 37 degrees C was significantly (P less than 0.05) greater than at 25 degrees C, but the degree of potentiation by physostigmine was significantly (P less than 0.05) greater at 25 degrees C. ATP (1.6 x 10(-4)-1.28 x 10(-3) M) and adenosine (1.8 x 10(-4)-1.48 x 10(-3) M) inhibited in concentration-related fashion contractile responses induced by KCl (1.34 x 10(-2) M-1.07 x 10(-1) M) by acetylcholine (2.2 x 10(-7) M-1.4 x 10(-5) M) and by CaCl2 in high KCl (120 mM)-CaCl2-free Tyrode solution. Theophylline and quinidine ('purinoceptor' antagonists) antagonized ACH contractile effects and so could not be satisfactorily employed in the characterization of the purine receptors in the mouse rectum. It may be concluded from this study that in the mouse rectum, acetylcholine is an excitatory neurotransmitter and that there is a non-adrenergic, non-cholinergic inhibitory neuromuscular transmission in this tissue. Further, ATP and adenosine have been demonstrated to cause relaxation in this tissue by possibly a post-synaptic mechanism involving inhibition of Ca2+ influx into the depolarized muscle.     

Structural analogs of 5'-methylthioadenosine as substrates and inhibitors of 5'-methylthioadenosine phosphorylase and as inhibitors of human lymphocyte transformation

5'-Deoxy-5'-methylthioadenosine (MTA) phosphorylase was purified 13.4-fold from human peripheral lymphocytes. The enzyme demonstrated normal Michaelis-Menten kinetics with Km values of 26 microM and 7.5 mM for the two substrates, MTA and phosphate, respectively. The rate of MTA degradation was temperature dependent, 47 degrees being the optimum temperature. Five structural analogs served as alternative substrates with Km values ranging from 31 to 53 microM while two compounds, 5'-deoxy-5'-methylthiotubercidin (MTT) (Ki = 31 microM) and adenine (Ki = 172 microM), were inhibitory. These same analogs were examined as inhibitors of mitogen-induced human lymphocyte blastogenesis. MTT was found to be the most effective inhibitor of lymphocyte transformation with an I50 of 80 microM.     

Adenosine deaminase from human erythrocytes: Purification and effects of adenosine analogs

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) has been purified about 3000-fold from human erythrocytes. The molecular weight of the enzyme was estimated to be 33,000. With the partially purified erythrocytic adenosine deaminase, K_m and V_max values relative to adenosine were: adenosine, 25 μM, 100 per cent; formycin A, 1000 μM, 753–850 per cent; 8-aza-adenosine, 130 μM, 310 per cent; 6-chloropurine ribonuclcoside, 1000 μM, 91 per cent; 2,6-diaminopurine ribonucleoside, 74 μM, 91 per cent; 2'-deoxyadenosine. 7 μm, 60 per cent; xylosyladenine, 33 μm, 62 per cent; arabinosyi adenine, 100 μM, 47 per cent; 3'-deoxyadenosine (cordycepin), 41 μM, 100 per cent; 3'-amino3'-deoxyadenosine. 133 μM, 89 per cent: 4'-thioadenosine, 13 μM, 43 per cent; and 6-methylselenopurine ribonucleoside, 27 μM, 88 per cent. Apparent K_ti values of reaction products and some adenosine analogs using adenosine as a substrate were as follows; inosine. 116 μM; 2'-deoxyinosine, 60 μM; guanosine, 140 μM; 2-fluoroadenosine, 60 μM; 2-fluorodeoxyadenosine. 19 μM; N⁶-methyladenosine, 17 μM; N₁-methyladenosine, 275 μM; 6-thioguanosine, 92 μM; 6-thioinosine, 330 μM; 6-methylthioinosine, 270 μM; arabinosyl 6-thiopurine, 360 μM; and coformycin, 0.01 μM. Tubercidin (7-deaza-adenosine) and toyocamycin were devoid both of substrate and inhibitor activity. Also. N⁷-methylinosine, N⁷-methylguanosine and dipyridamole (Persantin®) did not inhibit the enzymic activity.     

Inhibition by nitrobenzylthioinosine of uptake of adenosine, 2′-deoxyadenosine and 9-β-d-arabinofuranosyladenine by human and mouse erythrocytes

Nitrobenzylthioinosine was previously shown to inhibit transport of uridine and thymidine across the plasma membrane of the human erythrocyte. This report shows that treatment of human and mouse erythrocytes with nitrobenzylthioinosine, nitrobenzyldeoxythioinosine or hydroxynitrobenzylthioguanosine reduced formation of metabolites from extracellular adenosine, 2′-deoxyadenosine and ara-A, apparently through inhibition of mediated transport of these compounds into the cells. With human erythrocytes, the most potent inhibitor of the three was nitrobenzyldeoxythioinosine.     

Inhibition of 5-hydroxytryptamine uptake in human platelets by antidepressant agents in vivo

The accumulation of ¹⁴C-5-hydroxytryptamine (¹⁴C-5-HT) in platelet-rich plasma (PRP) and the concentration of 5-hydroxytryptamine (5-HT) in whole blood of patients treated with the antidepressant agents zimelidine (2×100 mg. daily), desipramine (2×75 mg daily), and clomipramine (2×75 mg daily) were examined before and during the treatment. Clomipramine and zimelidine markedly reduced the accumulation of ¹⁴C-5-HT and the concentration of 5-HT in the blood. Desipramine had a weaker, but significant effect. Added to the PRP in vitro clomipramine was ten-times more potent than norzimelidine, the active metabolite of zimelidine, and 60- and 300-times more active than desipramine and zimelidine, respectively in inhibiting the accumulation of ¹⁴C-5-HT. Analysis of plasma concentrations of zimelidine and norzimelidine showed that the decreased blood 5-HT and the inhibition of ¹⁴C-5-HT accumulation in platelets was mainly produced by norzimelidine. The inhibition of the ¹⁴C-5-HT accumulation and the decrease in blood 5-HT by desipramine were significantly correlated to the log plasma concentration of desipramine. It is concluded that the decrease in blood 5-HT caused by these agents is due to the inhibition of 5-HT uptake in platelets. The half-life of the decrease in blood 5-HT after clompramine and zimelidine was about 5 days. The return to normal 5-HT level after withdrawal of the drugs was 14 days or longer. These observations might indicate that only the newly formed platelets can accumulate 5-HT.     

Inhibition of 5-hydroxytryptamine uptake by tetrahydronorharmane in vivo

Summary Injection of tetrahydronorharmane (THN) elicited a dose-dependent increase of the level of 5-hydroxytryptamine (5-HT) and a fall of 5-hydroxyindoleacetic acid in the rat brain whereas noradrenaline and dopamine levels remained unchanged. The effect on indoles was shortlasting which may be explained by the short half life of THN or its metabolization to 6-OH-THN. The 5-HT depleting action of parachloro-N-methylamphetamine (CMA) was used to study uptake inhibition in vivo. The results support the notion that THN acts as an inhibitor of the reuptake of 5-HT in vivo.Further evidence for this hypothesis was obtained by intraventricular injection of radiolabelled 5-HT. The disappearance of (³H)-5-HT from brain tissue was reduced by THN. Thus, THN may exert its central 5-HT-like effects in pharmacological experiments by preventing 5-HT from being removed from the synaptic cleft.Supported by Deutsche Forschungsgemeinschaft     

Inhibition of orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase of human erythrocytes by purine and pyrimidine nucleotides.

Orotidine-5′-phosphate decarboxylase of human hemolysates exhibits triphasic kinetics with K_m values of 33, 1.7 and 0.082 μM. Inhibition of this enzyme at low OMP concentrations (<3 μM) by several naturally occurring purine and pyrimidine nucleotides was investigated. No significant inhibition was observed with IMP, GMP, TMP, ADP, and TTP at 5 mM. Inhibition constants for CMP, AMP, and dAMP were 31 μM, 0.11 mM and 0.21 mM, respectively. The results are discussed in relation to inhibition by nucleotides of orotate phosphoribosyltransferase, previously measured with a method which depends on orotidine-5′-phosphate decarboxylase activity.     

In vivo effects of pentobarbital and halothane anesthesia on levels of adenosine 3', 5'-monophosphate and guanosine 3',5'-monophosphate in rat brain regions and pituitary

The effects of two general anesthetics, pentobarbital and halothane, on in vivo levels of cyclic AMP and cyclic GMP were examined in seventeen brain regions and the pituitary in the rat. Ventilation was controlled to produce normal values of arterial pH, pCO₂ and pO₂, to eliminate changes in cerebral perfusion and oxygen delivery which occur as a result of the respiratory depressant effect of these drugs. Arterial pressure was monitored and colonie temperature was maintained within normal limits. Pentobarbital was given as a single i.p. injection of 80mg/kg. Control animals received an equivalent volume of vehicle solution. Induction of halothane anesthesia was accomplished by placing the animals in a jar flushed with 3% halothane in air. After 3 min the animals received 2% halothane in air via a nose cone. Control animals for this experiment were placed in an air-filled jar. Experimental and control animals were killed by microwave irradiation 1 hr after the start of anesthesia. Both drugs decreased levels of cyclic GMP in virtually all regions. The largest changes occurred in the cerebellum, where cyclic GMP dropped to 7.4 per cent of control with pentobarbital and to 9.8 per cent of control following halothane. Levels of cyclic AMP significantly increased in the cerebellum, brainstem and hypothalamus after halothane, by 58, 49 and 65 per cent, respectively. Both pentobarbital and halothane markedly increased cyclic AMP levels in the pituitary (to 784 and 270 per cent of control values, respectively). These results show that halothane and pentobarbital, which modify synaptic transmission, selectively alter cyclic AMP and cyclic GMP levels in specific brain regions and the pituitary.     

Effects of pentoxifylline and theophylline on neurotransmitter uptake and release by synaptosome-rich homogenates of the rat hypothalamus.

Preincubation of synaptosome-rich homogenates of rat hypothalamus with pentoxifylline [3,7-dimethyl-1(5-oxo-hexyl)-xanthine] or theophylline resulted in significant decreases of norepinephrine, dopamine, serotonin and glutamate uptake. Methylxanthine inhibition was non-competitive; apparent K_i's ± S.E.M. for pentoxifylline and theophylline were 0.48 ± 0.03 and 0.84 ± 0.10 mM (norepinephrine), 0.20 ± 0.02 and 0.35 ± 0.05 mM (dopamine), 0.22 ± 0.03 and 0.98 ± 0.12 mM (serotonin) and 0.69 ± 0.08 and 1.13 ± 0.14 mM (glutamate). Methylxanthines did not affect the synaptosomal uptake of leucine, phenylalanine or alanine. Transmitter release evoked by increasing [K⁺] in the medium was augmented by pentoxifylline and theophylline in a dose-dependent manner. Dibutyryl cyclic AMP (1.0 mM) caused a significant increase of transmitter release following elevation of [K + ] in the medium but did not affect neurotransmitter uptake by hypothalamic synaptosomes.     

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.     

Potentiation of 5-methoxy-n,n-dimethyltryptamine-induced head-twitches by diazepam: Evidence for involvement of adenosine uptake inhibition

Previous work shows that benzodiazepines potentiate head-twitches induced by 5-HT agonists and that this action is not mediated via the GABA receptor complex. In the present study the involvement of adenosinergic mechanisms in this effect has been examined, as in addition to their actions at the GABA receptor, benzodiazepines also inhibit adenosine uptake. The adenosine antagonists caffeine (0.3-30 mg/kg ip) and 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (0.03-1 mg/kg ip) dose-dependently inhibited the ability of diazepam (4 mg/kg ip) to potentiate head-twitches induced by 5-methoxy-N,N-dimethyltryptamine (5-MeODMT; 2.5 mg/kg ip) without affecting head-twitches induced by 5-MeODMT alone at a higher dose (10 mg/kg), which induced a similar number of head-twitches to the combination of 5-MeODMT and diazepam. The adenosine uptake inhibitors papaverine, mioflazine, and dilazep all potentiated head-twitches induced by 5-MeODMT, but this effect was seen at only a single dose of each compound. The benzodiazepine antagonist flumazenil did not inhibit the potentiation of head-twitches by diazepam but did itself potentiate head-twitches at 30 mg/kg, consistent with its ability to inhibit adenosine uptake. In contrast, the adenosine uptake inhibitor dipyridamole and the peripheraltype benzodiazepine receptor antagonist Ro 5-4864, which also inhibits adenosine uptake, failed to potentiate head-twitches. The adenosine agonists N⁶-cyclohexyladenosine, 5'-(N-ethylcarboxamido-adenosine), and (–)-N⁶-(R-phenylisopropyl)adenosine were similarly without effect. These results confirm previous findings that the potentiation of head-twitches by benzodiazepines is not mediated via an action at benzodiazepine receptors and suggest that inhibition of adenosine uptake is an important component of the mechanism involved. © 1993 wiley-Liss, Inc.