Effect of desmethyldiazepam and chlordesmethyldiazepam on 3′,5′-cyclic guanosine monophosphate levels in rat cerebellum

Three different benzodiazepines (diazepam, its pharmacologically active metabolite desmethyldiazepam, and the derivative chlordesmethyldiazepam) have been compared in our study for their effects on 3,5-guanosine monophosphate (cGMP) cerebellar levels. Desmethyldiazepam and chlordesmethyldiazepam are several-fold more potent that diazepam in decreasing rat cyclic cGMP cerebellar concentrations. None of the three drugs induces detectable changes of cerebellar cyclic 3,5-adenosine monophosphate (cAMP).On the other hand, the three compounds did not modify the levels of cGMP in cerebellum of newborn rats, where Purjinje cell and dendrites lack synaptic contacts. However, injection of gamma aminobutyric acid (GABA) in the newborn is still able, as in the adult, to decrease cGMP concentration in cerebellum. Our data support the hypothesis that cGMP cerebellar concentrations may be a reliable biochemical marker of the clinical activity of benzodiazepines.     

Effect of dopamine on adenosine 3′,5′-cyclic monophosphate levels in human platelets

1. The dopamine effect on intraplatelet adenosine 3′,5′-cyclic monophosphate (cAMP) levels was evaluated in healthy subjects.2. Dopamine levels over 30 nmol/l increased cAMP concentration in a concentration-dependent way.3. The platelet preincubation with propranolol (β-adrenoceptor antagonist) prevented the dopamine effect, whereas phentolamine (α-adrenoceptor antagonist) failed to modify the platelet response to this catecholamine. Dopamine receptor antagonist domperidone directly increased cAMP levels and it enhanced the dopamine effects.4. Our results indicate that dopamine effect is mainly mediated by β-adrenoceptor stimulation.     

Distribution and excretion of 2,3,6,2′,3′,6′- and 2,4,5,2′,4′,5′-hexachlorobiphenyl in senescent rats

The disposition of two symmetrical [¹⁴C]hexachlorobiphenyls (HCBs), 2,3,6,2′,3′,6′-HCB (236) and 2,4,5,2′,4′,5′-HCB (245), was studied in 24-month-old male Sprague-Dawley rats after iv treatment. Because body composition changes with age, complete dissections were performed on all rats to determine the size of the skin and adipose tissue depots. More than 50% of 236 was metabolized and excreted via the bile into the feces within 2 days. In contrast, 245 redistributed from the liver, muscle, and skin to adipose tissue where it accumulated without being metabolized. Only 2% of the total dose of 245 was excreted primarily in the feces within 21 days. The data obtained in this study were compared to results previously obtained from 2- to 3-month-old rats in this laboratory (Matthews and Tuey, 1980, Toxicol. Appl. Pharmacol.53, 377–388). Although the general pattern of HCB disposition did not change with age, i.e., metabolism and excretion of 236 versus persistence of 245, there were differences in the rates of elimination and in the tissue levels. There was enhanced metabolite retention in the muscle, skin, and adipose tissue of older animals which suggested an age-related decrease in tissue clearance. The large volume of adipose tissue in these older Sprague-Dawley rats could in part explain this observation. In general, there were few changes in decay rates from tissues or in biliary excretion. Age had a greater effect on the disposition of the persistent 245 than on the metabolizable 236. Thus, changes in body composition seemed to play a major role in age-related changes in the distribution and excretion of polychlorinated biphenyls.     

Effects of arecoline and cholinesterase inhibitors on cyclic guanosine 3′,5′-monophosphate and adenosine 3′,5′-monophosphate in mouse brain

Summary In mice, Arecoline in vivo dose-dependently increased the cGMP concentrations of the cerebellum and the cerebrum (= parts of cortex, hippocampus, hypothalamus, thalamus, striatum and midbrain) without influencing the cAMP levels. The cholinesterase inhibitors paraoxon and physostigmine caused an elevation only in cerebrum, whereas the cGMP content of the cerebellum even decreased.Pretreatment with atropine prevented the rise in cGMP levels as well as the symptoms of cholinergic stimulation elicited by arecoline or paraoxon. Diazepam reduced cGMP levels below control values and blocked the effect of arecoline, while typical symptoms due to arecoline, e.g., tremor and salivation remained unaffected. The tripeptide prolyl-leucyl-glycinamide (MIF) had no effect on either cGMP values or the peripheral signs of cholinergic stimulation elicited by arecoline.The results show that elevation of cGMP in the central nervous system caused by cholinomimetic agents can be prevented not only by cholinolytics, blocking muscarinic receptors but also by influencing other mechanisms to be discussed.     

Disposition of 2′, 3′-dideoxyadenosine and 2′, 3′-dideoxyinosine in mice

Summary Mice were dosed with [³H]2,3-dideoxyadenosine ([³H]ddA) in three procedures: intravenously, intraperitoneally, and interperitoneally following a dose of 2 -deoxycoformycin (dCF). For mice dosed intravenously, the content of radioactivity in plasma and tissue samples were essentially constant after 30 min. Of the radioactivity in plasma and brain samples collected between 30 min and 24 hr, more than 94% was present as ³H₂O, indicating that most of the tritium from [³H]ddA had exchanged with water. No intact ddA was detected, and the deamination product, 2,3 -dideoxyinosine (ddI), was present only transiently. In the urine, the major radioactive material was [³H]ddI. Also detected were ³H₂O and small amounts of [³H]hypoxanthine and [³H]ddA. Following intraperitoneal doses to mice, levels of radioactivity in plasma, liver, and kidney increased to a maximum by 15–30 min after dosing but dropped to essentially constant levels thereafter, again indicating that the tritium had exchanged with water. At 5, 15, and 30 min after dosing, ddI was the major radioactive component in plasma. Only small amounts of ddA were present. When dCF was administered 24 hr prior to intraperitoneal [³H]ddA, levels of radioactivity in plasma, liver, and kidney reached a maximum at 30 to 60 min after dosing and decreased to essentially constant levels thereafter. The dCF transiently inhibited the deamination of ddA to ddI, since, in plasma, [³H]ddA was the main radioactive component at 5 and 15 min after dosing. Comparison of HPLC assays based on radioactivity detection and UV absorbance showed that they were equivalent for measuring ddA and ddI in samples derived from dosed mice. Therefore, exchange of tritium must have occurred at a metabolic step beyond ddI.For mice dosed intravenously and orally with unlabeled ddI, there was evidence of a saturated process. Nevertheless, for the high and low intravenous doses of ddI, the percent of dose excreted in the urine as unchanged drug was the same.     

Energy-dependent extrusion of cyclic 3′,5′-adenosine-monophosphate

Summary In reticulocyte-rich suspensions of red blood cells from rats extrusion of cAMP as a regulatory mechanism of intracellular cAMP was investigated.In response to isoprenaline and/or the phosphodiesterase inhibitors Ro 20-1724 and rolipram extrusion of cAMP increases dependent on the concentration of the drugs and time of exposure. However, these drugs exert their effects on the extrusion of cAMP only indirectly, i.e. via increased intracellular levels of cAMP, since the respective EC₅₀-values of the drugs for intracellular accumulation and extrusion of cAMP are identical (isoprenaline: 50 nM; rolipram: 1 M; Ro 20-1724: 15 M).The dependence of the rate of extrusion on intracellular levels of cAMP is characterized by a typical concentration-effect relationship from which a maximal capacity of cAMP extrusion of 3–6 nmol/10 min/10⁹ cells and a half maximal effective intracellular cAMP concentration of 40–50 nmol/10⁹ cells can be derived. This relationship has been inferred from either kinetic or steady-state approaches. At rapidly changing intracellular levels of cAMP an apparent time lag of extracellular cAMP accumulation is obligatorily conditioned by this relationship. Vasodilating drugs which lower the ATP content of the cells as well as the uncoupler of oxidative phosphorylation, FCCP, inhibit the extrusive process (papaverine > FCCP > dipyridamole > dilazep hexobendine carbocromene) leading to a 3–5-fold increase of the intrato extracellular concentration gradient of cAMP.It is concluded that extrusion of cAMP is a saturable and energy-dependent process which regulates the intracellular cAMP concentration independent of the activities of adenylate cyclase and phosphodiesterase.This work was supported by a grant from the Deutsche Forschungsgemeinschaft     

Regulation of guanosine 3′:5′-cyclic monophosphate in ovine tracheal epithelial cells

1. Guanosine 3′:5′-cyclic monophosphate (cyclic GMP) is an important second messenger mediating the effects of nitric oxide (NO) and natriuretic peptides. Cyclic GMP pathways regulate several aspects of lung pathophysiology in a number of airway cells. The regulation of this system has not been extensively studied in pulmonary epithelial tissue.
2. We have studied the production of cyclic GMP by suspensions of ovine tracheal epithelial cells in response to activators of soluble guanylyl cyclase (sodium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP) and particulate guanylyl cyclase (atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP) and E. coli heat stable enterotoxin (STa)).
3. Both 10⁻⁷10⁻³ M and 10⁻⁷10⁻³ M SNAP generated a concentration-dependent marked elevation in cyclic GMP production when incubated with 10⁻³ M 3-isobutyl-1-methylxanthine (IBMX) (both greater than 25×baseline values with highest drug concentration).
4. The increase in production of cyclic GMP in response to 10⁻⁶ M SNP and 10⁻⁵ M SNAP was markedly inhibited by both 5×10⁻⁵ M haemoglobin (102% and 92% inhibition) and 5×10⁻⁵ M methylene blue (82% and 84% inhibition).
5. The increase in cyclic GMP in response to 10⁻³ M SNP was measured following co-incubation with the phosphodiesterase inhibitors 10⁻⁷10⁻³ M IBMX, 10⁻⁷10⁻⁴ M milrinone and 10⁻⁷10⁻⁴ M SKF 96231. Only 10⁻⁴10⁻³ M IBMX significantly increased cyclic GMP levels.
6. Cyclic GMP production was also significantly elevated from baseline by 10⁻⁵ M ANP, 10⁻⁵ M BNP, 10⁻⁵ M CNP and 200 iu ml⁻¹ of E. coli STa toxin in the presence of 10⁻³ M IBMX. Increases with these natriuretic peptides and STa toxin were smaller in magnitude (24 fold) than those seen with SNP and SNAP. CNP was the most potent of the natriuretic peptides studied suggesting type B membrane bound guanylate cyclase is the predominant form expressed.
7. These results suggest that ovine tracheal epithelial cells contain active guanylyl cyclases. The more marked response to SNP and SNAP than to natriuretic peptides suggests that soluble guanylyl cyclase predominates.

     

Metabolism and presynaptic inhibitory activity of 2′,3′ and 5′-adenine nucleotides in rat vas deferens

Summary In the isolated rat vas deferens stimulated at 0.2 Hz, [¹⁴C]labelled 5-AMP, 5-ADP and 5-ATP (10 M) inhibited twitch responses, were broken down to [¹⁴C]adenosine in the medium and incorporated into [¹⁴C]adenine ribonucleotides in the tissue. Pretreatment of tissues with 6-(2-hydroxy-5-nitrobenzyl)-thioguanosine (NBTGR), a potent inhibitor of adenosine transport, potentiated the presynaptic inhibitory action of these 5 nucleotides and reduced their incorporation in [¹⁴C]adenine nucleotides, but did not alter the appearance of [¹⁴C]adenosine in the medium.A series of 2, 3 and 5-substituted adenine nucleotides (10 M) inhibited the twitch responses of the vas deferens stimulated at 0.2 Hz. This effect was potentiated by NBTGR. Addition of exogenous adenosine deaminase very significantly reduced the inhibitory actions of adenosine, 5-AMP, 5-ADP and 5-ATP and also reduced those of 2, 5-ADP, NAD⁺ and dePCoA. The inhibitory actions of the other 2, 3 and 5 adenine nucleotides studied were not altered by exogenous adenosine deaminase.These results indicated that the presynaptic inhibitory actions of 5-AMP, 5-ADP and 5-ATP in rat vas deferens predominantly result from their prior hydrolysis to adenosine whereas the 2, 3 and 5-substituted adenine nucleotides appear to act mainly directly to inhibit transmitter release.Abbreviations. The following abbreviations are used 5-ADP 5-adenosine diphosphate - 2,5-ADP 2,5-adenosine diphosphate - 3,5-ADP 3,5-adenosine diphosphate - 2,3 or 5-AMP 2,3 or 5-adenosine monophosphate - 5-ATP 5-adenosine triphosphate - CoA coenzyme A - 2,3-cAMP 2,3-cyclic adenosine monophosphate - cNADP⁺ -nicotinamide dinucleotide 2,3-cyclic monophosphate - dePCoA dephosphocoenzyme A - NAD⁺ -nicotinamide adenine dinucleotide - NADP⁺ -nicotinamide adenine dinucleotide phosphate - NBTGR 6-(2-hydroxy-5-nitrobenzyl)-thioguanosine - oxid CoA oxidized-coenzyme A     

Hydrolysis of the Prodrug, 2′, 3′, 5′ -Triacetyl-6-azauridine

Purpose. The purposes were to study the kinetics of hydrolysis of 2,3,5-triacetyl-6-azauridine ( 1 ) in aqueous solution (µ = 0.5) and to identify the main intermediates and products of the reaction. Methods. A stability indicating isocratic LC assay was used to study the rate of degradation of 1 A gradient LC assay was used to study the time courses of the degradants. The products of hydrolysis were isolated by preparative liquid chromatography and identified by ¹H-NMR and CI-MS. The pK_a value was obtained by potentiometric titration. Results. At 36.8°C, the pH-rate profile of 1 in water was adequately described by a four-term rate equation. The intermediates were identified as the primary and secondary di-acetates, and the primary and secondary mono-acetates. The final product was 6-azauridine. Conclusions. A simplified kinetic scheme could be used to describe the concentration-time profiles of 1, the intermediates and the final product.     

The facile and efficient synthesis of 8-chloroadenosine 3′,5′-cyclic monophosphate by phosphorylative cyclization of 8-chloroadenosine and its characterization by1H and13C NMR spectroscopy

Purine nucleosides were chlorinated by the reaction of acyl chloride in DMF with MCPBA under mild conditions with moderate yields. And, satisfactory method for the synthesis of ribonucleoside-3′,5′-cyclic phosphates and its characterization by¹H and¹³C nmr spectroscopy is described.     

Synthesis and Pharmacological Properties of 1-Aryl-4-(3′4′,5′-trimethoxybenzoyl)piperazines

A series of 1-aryl-4-(3′,4′,5′-trimethoxybenzoyl)piperazines (IIa - IIg) having structures containing 3,4,5-trimethoxybenzoyl fragments were synthesized and characterized. Compounds IIa and IIc - IIg demonstrated weak anxiolytic properties and moderately decreased the spontaneous motor activity in rats. Compound IIb exhibited anxiolytic activity comparable with that of buspirone but, in contrast to this reference drug, increased the motor activity of test animals. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 39, No. 5, pp. 12 – 14, May, 2005.     

Differential involvement of 3′, 5′-cyclic adenosine monophosphate-dependent protein kinase in regulation of Fos and tyrosine hydroxylase expression in the heart after naloxone induced morphine withdrawal

We previously demonstrated that morphine withdrawal induced hyperactivity of the heart by the activation of noradrenergic pathways innervating the left and right ventricle, as evaluated by noradrenaline (NA) turnover and Fos expression. We investigated whether cAMP-dependent protein kinase (PKA) plays a role in this process by estimating changes in PKA immunoreactivity and the influence of inhibitor of PKA on Fos protein expression, tyrosine hydroxylase (TH) immunoreactivity levels and NA turnover in the left and right ventricle. Dependence on morphine was induced by a 7-day s.c. implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by an injection of naloxone (5 mg/kg). When opioid withdrawal was precipitated, an increase in PKA immunoreactivity and phospho-CREB (cyclic AMP response element protein) levels were observed in the heart. Moreover, morphine withdrawal induces Fos expression, an enhancement of NA turnover and an increase in the total TH levels. When the selective PKA inhibitor HA-1004 was infused, concomitantly with morphine pellets, it diminished the increase in NA turnover and the total TH levels observed in morphine-withdrawn rats. However, this inhibitor neither modifies the morphine withdrawal induced Fos expression nor the increase of nonphosphorylated TH levels. The present findings indicate that an up-regulated PKA-dependent transduction pathway might contribute to the activation of the cardiac catecholaminergic neurons in response to morphine withdrawal and suggest that Fos is not a target of PKA at heart levels.     

3′,5′-环化腺苷酸的生产

近几年来,3’,5’—环化腺苷酸(cAMP)作为第二信使物质来研究它的作用机理颇为活跃。许多研究结果表明,cAMP的作用已远远超过了激素传导物质的含意,它不但对代谢、核酸、蛋白质的合成调节,而且对分泌、运动、神经刺激的传导以及组织的形成、分化、癌变等领域皆有密切关系。使得凡从事生命科学及医学科学的人们不得不重视对它的研究。为此对摸索一条合适的生产cAMP的工艺来满足理论研究需要及试制针药提供临床试验已日显重要。     

Synthesis and biological evaluation of novel 2′,4′,5′-trimethoxyflavonol derivatives as anti-inflammatory and antimicrobial agents

A series of novel 3-hydroxy-2-(2,4,5-trimethoxyphenyl)-4H-chromen-4-one (flavonol) derivatives (2a–u) of biological interest have been prepared via CLAISEN–SCHMIDT condensation followed by ALGAR–FLYNN–OYAMADA reaction and to search for the potent nonsteroidal anti-inflammatory agents from this novel series. All the synthesized compounds have been screened for their in vitro proinflammatory cytokines tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) inhibitory activity along with antimicrobial activity. As many as three compounds viz. 2h, 2l, and 2q from this novel series were found to be potent TNF-α and IL-6 inhibitor (up to 72–81 % TNF-α and 86–92 % IL-6 inhibitory activity) but at 10 μM concentration as compared with the standard dexamethasone (71 % TNF-α and 84 % IL-6 inhibitory activities at 1 μM concentration). While the compounds 2d, 2m, 2n, and 2s were found to be potent antimicrobial agent showing even 2–2.5-fold more potency than that of standard ciprofloxacin and miconazole at the same MIC value of 10 μg/mL.     

Elevated guanosine 3′:5′-cyclic monophosphate mediates the depression of nitrovasodilator reactivity in endothelium-intact blood vessels

Summary The influence of endothelium-derived nitric oxide (EDNO) on relaxation induced by the nitrovasodilators, sodium nitroprusside and sodium nitrite was assessed in phenylephrine-stimulated hamster thoracic aortas, a preparation that displays significant basal release of EDNO. Removal of the endothelium or treatment with the NO synthase inhibitors, N^G-nitro-l-arginine (L-NAG, 10–30 µM) or N^G-methyl-l-arginine (L-NMMA; 100 µM) increased the potency and, except for sodium nitroprusside in endothelium-denuded segments, also increased the efficacy of the nitrovasodilators. Removal of the endothelium had no effect on relaxations induced by isoproterenol, an indication that these effects were specific for the nitrovasodilators. Removal of the endothelium, treatment of endothelium-intact preparations with L-NAG or L-NMMA, or exposure of these vessels to the guanylate cyclase inhibitor, methylene blue (10 µM) increased reactivity of the aortas to the guanosine 3:5-cyclic monophosphate (cGMP) analogue, 8-Br cGMP. Measurement of cGMP revealed that endothelium-intact segments had a 6.5 fold higher level of cGMP than endothelium-denuded preparations and that sodium nitroprusside increased cGMP in both preparations by similar amounts in a concentration-dependent fashion. Exposure of endothelium-denuded or L-NAG-treated segments to sodium nitroprusside, to mimic the effects of basally released EDNO, depressed sodium nitrite and 8-Br cGMP reactivity in a manner similar to endothelium-intact segments. These data indicate that EDNO increases cGMP levels in vascular smooth muscle and that the elevated cGMP levels depress nitrovasodilator and 8-Br cGMP reactivities. Send offprint requests to W. F. Jackson at the above address     

Inhibition of 3,3′,4,4′,5-Pentachlorobiphenyl-Induced Chicken Embryotoxicity by 2,2′,4,4′,5,5′-Hexachlorobiphenyl

3,3′,4,4′,5-Pentachlorobiphenyl (pentaCB) caused a dose-dependent induction of chicken embryolethality, malformations, edema, and liver lesions at doses ranging from 0.5 to 12.0 μg/kg. In contrast, no embryotoxicity was observed after treatment with 10, 25, or 50 mg/kg 2,2′,4,4′,5,5′-hexaCB. In eggs cotreated with 2.0 μg/kg, 3,3′,4,4′,5-pentaCB plus 10, 25, or 50 mg/kg 2,2′,4,4′,5,5′-hexaCB, there was significant protection from 3,3′,4,4′,5-pentaCB-induced embryo malformations, edema, and liver lesions, whereas no inhibition of embryolethality was observed. These results further extend the response-specific nonadditive interactions of binary mixtures of polychlorinated biphenyls (PCBs) and should be considered in the development of approaches for hazard assessment of PCB mixtures and related compounds.     

Long circulating liposomes of 2′,3′-dideoxyinosine: Formulation and stability

Abstract

2′,3′-Dideoxyinosine (ddI), an anti–human immunodeficiency virus (HIV) agent, was encapsulated in liposomes. The influence of the phospholipid/cholesterol ratio, concentration of phospholipid (PL), and chain length of PL on the encapsulation of ddI in multilamellar vesicles (MLVs), frozen and thawed multilamellar vesicles (FAT MLVs), and large unilamellar vesicles (LUVs) was studied. An optimum formulation was then selected to prepare long circulating liposomes. Stability studies at 4, 25, and 37°C and under certain stress conditions were performed. Release characteristics in phosphate buffer (pH 7.4) at 37°C were studied. Results show an increase in encapsulation efficiency (EE) with increasing amounts of cholesterol, a decrease in EE and increase in encapsulation yield (EY) with increasing concentrations of PL, and an increase in EE with increases in PL chain length, in both MLVs and LUVs. Freezing and thawing of MLVs had no influence on EE at a PL concentration of 10 mg/mL but increased EE at higher concentrations of PL. Various stability tests showed the formulation to be stable to leakage of entrapped drug when stored at 4, 25, and 37°C for 6 months, when subjected to mechanical stress, and on exposure to human serum. The release studies indicated that 70% of ddI was released over a period of 72 h.