CYCLIC ADENOSINE 3'',5''-MONOPHOSPHATE IN GUINEA-PIG CEREBRAL CORTICAL SLICES: STUDIES ON THE ROLE OF ADENOSINE

Abstract— In guinea-pig cerebral cortical slices levels of cyclic AMP increase in response to adenosine to about 200pmol/mg protein within 10 min and stay at that level up to 30 min. In the absence of calcium ions and the presence of 1mm -EGTA in the Krebs-Ringer-bicarbonate medium the effect of adenosine is enhanced, cyclic AMP levels rise to about 600 pmol/mg protein within 30 min. In normal and calcium deficient media restimulation of cyclic AMP formation with adenosine is possible after a prior stimulation with adenosine. When slices are preincubated for various periods of time with histamine or adenosine before addition of the complementary agent i.e. adenosine or histamine cyclic AMP levels obtained are unaltered compared to levels seen when adenosine and histamine are added together. Slices which are rendered unresponsive to stimulation with histamine + noradrenaline by a prior incubation with these agents do not regain any response during a 100 min period of incubation in medium. The PDE inhibitors diazepam, SQ 66007 and isobutylmethylxanthine are capable of restoring the sensitivity of the slices to histamine + noradrenaline. This suggests an involvement of PDE in the unresponsive phase of the slices. Addition of adenosine to slices not affected by histamine + noradrenaline does reestablish the response of these slices to the neurohormones. A dose-response curve of adenosine for the interaction with histamine + noradrenaline yields an ED₅₀ of 16 μM using sensitive or desensitized slices. An adenosine concentration of only 7 μM is necessary to restore the original increase of cyclic AMP in response to histamine + noradrenaline to slices insensitive to the biogenic amines. The data are discussed in terms of a possible activation of PDE within cerebral cortical slices from guinea-pig. Adenosine may reverse this activation. The possibility of inactivation of adenylate cyclase during stimulation of cyclic AMP formation and the role of adenosine and PDE inhibitors in this process is being considered.     

The decomposition of benzodiazepines during analysis by capillary gas chromatography/mass spectrometry

A capillary gas chromatography column directly interfaced to a mass spectrometer was used for the analysis of sixteen benzodiazepines. The thermal stability of the drugs was found to be related to their chemical structure. Nine of the benzodiazepines were thermally unstable indicating that care should be taken in the interpretation of gas chromatographic data from this class of drugs. The unstable benzodiazepines were: ketazolam which decomposes to diazepam; N-4 oxides (chlordiazepoxide and demoxepam) which lose an oxygen radical; aromatic 7-nitro compounds (nitrazepam and clonazepam) which are partially reduced to the corresponding amine; alpha-hydroxy ketones (lorazepam and oxazepam) which decompose with the loss of water and N-methyl-alpha-hydroxy ketones (lormetazepam and temazepam) which partially decompose with the loss of a hydrogen molecule to produce the corresponding alpha, beta-diketones. Few problems were encountered in distinguishing the drugs by their mass spectra, the exceptions being ketazolam which decomposes to diazepam and demoxepam which decomposes to desmethyldiazepam. In general, good spectra were obtained from 20-50 ng of drug injected. However, for those compounds where the decompositions were not quantitative (nitrazepam, clonazepam, lormetazepam, temazepam) detection limits were poor.     

Inhibition of 3',5'-nucleotide phosphodiesterase in guinea pig cerebral cortical slices

Several compounds have been tested for their activity as inhibitors of 3′,5′-nucleotide phosphodiesterase in brain cortical slices from guinea pig. SQ 20,009 (1-ethyl-4-isopropylidenehydrazino)-1H-pyrazolo (3,4-b)pyridine-5-carboxylate, ethylester, hydrochloride), a very potent inhibitor of 3′,5′-nucleotide phosphodiesterase from rat and rabbit brain shows only moderate activity as 3′,5′-nucleotide phosphodiesterase inhibitor when tested in brain slices. It enhances cyclic AMP accumulation only when slices are stimulated by histamine. It does not affect cyclic AMP levels when histamine/norepinephrine are used as stimuli of cyclic AMP formation and decreases the activity of adenosine as stimulant slightly. Ro 20–1724 (4-(3-butoxy-4-methoxy)-2-imidazolidinone) a potent inhibitor of canine cerebral cortex PDE activity effectively augments the increase in cyclic AMP under all stimulating conditions mentioned, as does to a somewhat smaller extent the more water soluble Ro 20–2926 (4-(3-ethoxy-ethoxy-4-methoxy)-2-imidazolidinone). Dose-response curves for Ro 20–1724 under three stimulating conditions of increased cyclic AMP formation (0.1 mm histamine, 0.1 mm histamine/0.1 mm norepinephrine, 0.1 mm adenosine) yield an ED₅₀ of about 20 μm in all instances. A significant increase over respective controls is seen even at 1 μm Ro 20–1724 (histamine/norepinephrine). The drugs may be useful as tools for studying the regulation of cyclic AMP levels in the central nervous system.     

CYCLIC ADENOSINE 3'', 5''-MONOPHOSPHATE IN GUINEA PIG CEREBRAL CORTICAL SLICES: POSSIBLE REGULATION OF PHOSPHODIESTERASE ACTIVITY BY CYCLIC ADENOSINE 3'', 5''-MONOPHOSPHATE AND CALCIUM IONS

Cyclic adenosine 3′, 5′-monophosphate (cyclic AMP) accumulates in guinea pig cerebral cortical slices during incubation with histamine, histamine + noradrenaline and adenosine. Noradrenaline does not enhance cyclic AMP formation. In the absence of Ca²⁺ ions and presence of 1 mM-EGTA in the Krebs-Ringer bicarbonate medium the effects of histamine, histamine + noradrenaline and adenosine are significantly enhanced and noradrenaline elicits an increase in cyclic AMP over control levels. When histamine is used as stimulant, cyclic AMP levels start to decline after only 5 min. However, in the absence of calcium and in the presence of EGTA in the medium this decline is not observed and cyclic AMP levels continue to rise for a considerable period of time. In normal medium, responses to restimulation by histamine or histamine + noradrenaline are greatly reduced in magnitude after a prior stimulation by these putative neurotransmitters. In contrast, when calcium is omitted from the incubation medium and 1 mM-EGTA is included, cyclic AMP levels increase to normal values at a second stimulation with histamine or histamine + noradrenaline. When slices are preincubated for various periods of time with histamine before addition of noradrenaline, the accumulation of cyclic AMP is significantly reduced as compared to levels obtained when histamine + noradrenaline were added simultanously. This decline in the overall response to histamine + noradrenaline is not observed when preincubation with histamine and subsequent incubations with histamine + noradrenaline are performed in Ca²⁺-free, 1 mM-EGTA containing buffer. Also preincubation with noradrenaline in normal, calcium-containing medium does not affect the total amount of cyclic AMP accumulating in the brain slices. The results are discussed in terms of an activation of phosphodiesterase within the cerebral cortical slices by increased levels of intracellular, freely available calcium which is mediated by the elevation of cyclic AMP concentration following hormonal stimulation.     

1-Isoamyl-3-isobutylxanthine: a remarkably potent agent for the potentiation of norepinephrine, histamine, and adenosine-elicited accumulations of cyclic AMP in brain slices.

1-Isoamyl-3-isobutylxanthine (EC₅₀ t 5 μM) potentiates by 2 to 6-fold the accumulations of cyclic AMP elicited in guinea pig cerebral cortical slices by norepinephrine, histamine, and adenosine. In addition, the xanthine derivative causes a 2 to 3-fold elevation of basal levels of cyclic AMP. 1-Isoamyl-3-isobutylxanthine has no effect on accumulations of cyclic AMP elicited by histamine or adenosine in the presence of a potent phosphodiesterase inhibitor, ZK 62771. The xanthine derivative retards the disappearance of cyclic AMP after a prior stimulation by adenosine. The results indicate that 1-isoamyl-3-isobutylxanthine is an extremely potent and effective inhibitor of phosphodiesterases involved in the regulation of cyclic AMP levels in guinea pig cerebral cortical slices. The 1-benzyl, 1-isoamyl, and 1-isobutyl derivatives of 3-isobutylxanthine potentiate the accumulation of cyclic AMP elicited by adenosine, while the 1-methyl derivative and 1-isoamyl-3-methylxanthine are inhibitory undoubtedly because of blockade of adenosine-receptors by these compounds. Xanthines with bulky 1- and 3- substituents appear to be relatively weak adenosine-antagonists and relatively specific and potent agents for inhibition of phosphodiesterases involved in cyclic AMP metabolism in brain tissue.     

Clinical pharmacology and therapeutics of benzodiazepines.

Benzodiazepines are among the most commonly prescribed drugs in the world. In contrast to their extensive use, the therapeutic indications and potential of benzodiazepines are limited. All benzodiazepine derivatives available in Canada are similar structurally and in their pharmacologic actions. Few have specific advantages over any others. For example, no benzodiazepine has been shown to be superior to chlordiazepoxide in the treatment of acute anxiety, chronic anxiety neurosis or insomnia. Barbiturates should not be prescribed for these problems since benzodiazepines are just as effective and are safer. Persons more than 70 years old should receive initial doses of benzodiazepines 50% less than those prescribed for younger persons, and individuals with cirrhosis should receive chlordiazepoxide or diazepam in one third the usual dose; oxazepam or lorazepam should be considered for these two groups of patients. Diazepam and chlordiazepoxide should not be given intramuscularly. Benzodiazepines should be prescribed only when clearly indicated and only for the necessary length of time.     

High-performance liquid chromatographic method for the determination of benzodiazepines in plasma or serum using the column-switching technique

A column-switching high-performance liquid chromatographic method for the simultaneous determination of five frequently prescribed benzodiazepines: clonazepam, diazepam, flunitrazepam, midazolam and oxazepam was developed. A 50-μl plasma sample was directly injected into a BioTrap 500 MS (hydrophobic polymer) column. After a washing step with a mixture of phosphate buffer and acetonitrile, the retained benzodiazepines were back-flushed into a reversed-phase (LiChrospher Select B C₈) column with a mobile phase of acetonitrile–phosphate buffer. The method showed excellent linearity from 50 to 1000 ng/ml for clonazepam, flunitrazepam and midazolam and from 50 to 5000 ng/ml for diazepam and oxazepam. The recoveries were around 98% for all the benzodiazepines studied. The relative standard deviation for between- and within-day assay was <20% for low concentrations close to the values of the limit of quantification and <4% for high concentrations. The procedure described is relatively simple and rapid because no off-line manipulation of the sample is required: the total analysis time is approximately 30 min.     

Simultaneous determination of twelve benzodiazepines in human serum using a new reversed-phase chromatographic column on a 2-μm porous microspherical silica gel

A high-performance liquid chromatographic method has been developed for the simultaneous analysis of twelve frequently used benzodiazepines (BZPs) (bromazepam, clonazepam, chlordiazepoxide, estazolam, etizolam, flutazoram, haloxazolam, lorazepam, nitrazepam, oxazolam, triazolam and diazepam, internal standard) by using commerically available 2 or 5 μm particle size reversed-phase columns and a microflow cell-equipped ultraviolet detector. The separation was achieved using a C₁₈ reversed-phase column (condition 1: 100×4.6 mm I.D., particle size 2 μm, TSK gel Super-ODS; condition 2: 100×4.6 mm I.D., particle size 5 μm, Hypersil ODS-C₁₈). The mobile phase was composed of methanol-5 mM NaH₂PO₄ (pH 6) (45:55, v/v), and the flow-rate was 0.65 ml/min (conditions 1 and 2). The absorbance of the eluent was monitored at 254 nm. Retention times under condition 1 were shorter than those of condition 2. When the twelve benzodiazepines were determined, sensitivity and limits of quantification were about four to ten times better under condition 1 than under condition 2. The rate of recovery and linearity in condition 1 were approximately the same as those in condition 2. These results show that a new ODS filler with a particle size of 2 μm was more sensitive, provided better separation and was more rapid than that with conventional ODS filler.     

Benzodiazepines and Tricyclic Antidepressant Plasma Levels

Twelve patients acted successfully as subjects to study what effect if any the benzodiazepines nitrazepam, diazepam, oxazepam, and chlordiazepoxide might have on steady-state plasma levels of nortriptyline and amitriptyline. No significant detectable effect was discovered. In view of the known interaction effects of other alternative tranquillizing drugs and hypnotics it seems reasonable to choose benzodiazepines wherever possible when anxiolytics or hypnotics need to be added during treatment of depression with tricyclic antidepressants.     

Benzodiazepine-induced voraciousness in cats and inhibition of amphetamine-anorexia

The effect of different benzodiazepines on food intake was studied in cats which had been trained to eat their daily food within 3 hours, under conditions in which emotional factors were not present. When benzodiazepines were administered (at a dose ranging from 0.1 to 3 mg/Kg) before feeding, they increased both the total amount of food eaten and also the rate at which food was injested. When they were administered at the end of the feeding period, these compounds made the animals resume eating voraciously. The order of decreasing potency of the benzodiazepines tested was: oxazepam, N-methyl-lorazepam, diazepam, chlordiazepoxide, pinazepam, medazepam. Oxazepam (3 mg/Kg) stimulated maximally the food intake, even when administered up to 12 hours before feeding. Finally, oxazepam antagonized the anorexigenic effect of d-amphetamine, but did not influence amphetamine-induced hyperactivity and stereotyped behavior.     

Separation of 1,4-benzodiazepines by micellar elektrokinetic capillary chromatography

In this work the applicability of micellar elektrokinetic capillary chromatography (MECC) for the determination of benzodiazepines (BZD) has been studied. The applied method was used for the simultaneous separation of 8 BZDs (alprazolam, bromazepam, chlordiazepoxide, diazepam, flunitrazepam, medazepam, oxazepam, nitrazepam), and also for the study of stability in acidic medium. A fast and reliable method has been developed; using a separation buffer composed of sodium tetraborate 25 mM (pH 9.5), SDS (50 mM) and methanol (at least 12%) as an organic modifier.     

[3H]GLYCOGEN HYDROLYSIS IN BRAIN SLICES: RESPONSES TO NEUROTRANSMITTERS AND MODULATION OF NORADRENALINE RECEPTORS

Abstract— Different agents have been investigated for their effects on [C³H]glycogen synthesized in mouse cortical slices. Of these noradrenaline, serotonin and histamine induced clear concentration-dependent glycogenolysis.
[C³H]Glycogen hydrolysis induced by noradrenaline appears to be mediated by beta-adrenergic receptors because it is completely prevented by timolol, while phentolamine is ineffective. It seems to involve cyclic AMP because it is potentiated in the presence of isobutylmethylxanthine; in addition dibutyryl cyclic AMP (but not dibutyryl cyclic GMP) promotes glycogenolysis.
Lower concentrations of noradrenaline were necessary for [C³H]glycogen hydrolysis (EC₅₀= 0.5μM) than for stimulation of cyclic AMP accumulation (EC₅₀= 8μM).
After subchronic reserpine treatment the concentration-response curve to noradrenaline was significantly shifted to the left (EC₅₀= 0.09 ± 0.02 μM as compared with 0.49 ± 0.08 μM in saline-pretreated mice) without modifications of either the basal [C³H]glycogen level, maximal glycogenolytic effect, or the dibutyryl cAMP-induced glycogenolytic response.
In addition to noradrenaline, clear concentration-dependent [³H]glycogen hydrolysis was observed in the presence of histamine or serotonin. In contrast to the partial [³H]glycogen hydrolysis elicited by these biogenic amines, depolarization of the slices by 50 mM K⁺ provoked a nearly total [C³H)glycogen hydrolysis.     

CRF stimulates α-MSH secretion and cyclic AMP accumulation in rat pars intermedia cells

Ovine corticotropin-releasing factor (CRF) stimulates α-MSH release and cyclic AMP accumulation in rat pars intermedia cells in culture at ED₅₀ values of 1 and 6 nM, respectively. The stimulatory effect of CRF on both parameters is inhibited by the dopaminergic agonist 2-bromo-α-ergocryptine (CB-154). The present data show that CRF is a potent stimulator of peptide secretion in the intermediate lobe of the pituitary gland and suggest a role of this pituitary lobe in the response to stress. In addition, the present data clearly indicate a role of cyclic AMP as mediator of the action of CRF in pars cells.     

Correlation between the experimental and clinical effectiveness of benzodiazepines and their affinity for benzodiazepine receptors

The therapeutic effect of 6 benzodiazepine tranquilizers (diazepam, oxazepam, chlordiazepoxide, phenazepam, lorazepam, nitrazepam) was compared to the activity displayed in the most widely used experimental models. The methods of conflict situation, antagonism with thiosemicarbazide and corasole were found to be highly significant for predicting the clinical efficacy of benzodiazepines. The conditioned reflex techniques were shown untenable for estimating the therapeutic action of the tranquilizers. The correlation was discovered between integral clinical tranquilizing effects of benzodiazepines, their experimental activity and affinity to benzodiazepine receptors.     

Hormones and Neurotransmitters Control Cyclic AMP Metabolism in Choroid Plexus Epithelial Cells

The choroid plexus is a major site of CSF production. When primary cultures of bovine choroid plexus epithelial cells were exposed to 1 micrograms/ml cholera toxin, a 50-fold increase of intracellular cyclic AMP was found 1 h later. Exposure of cells to 10(-5) M isoproterenol, 10(-4) M prostaglandin E1, 10(-5) M histamine, and 10(-5) M serotonin caused increases of intracellular cyclic concentrations of 100-, 50-, 20-, and 4-fold, respectively. From 5 to 15 min were required for these maximal responses to occur. Many other molecules including prolactin, vasopressin, and corticotropin did not alter cellular cyclic AMP levels. The accumulation of cyclic AMP could be inhibited by specific antagonists: propranolol inhibited the isoproterenol-mediated stimulation while diphenhydramine and metiamide inhibited the histamine response. In addition, diphenhydramine inhibited serotonin-dependent cyclic AMP accumulation. Combinations of isoproterenol, prostaglandin E1, histamine, and serotonin elicited additive responses as measured by cyclic AMP accumulation with one exception, i.e., serotonin inhibited the histamine response. Our findings suggest that distinct receptor sites on choroid plexus epithelia exist for isoproterenol, prostaglandin E1, and histamine. Efflux of cyclic AMP into the extracellular medium was found to be a function of the intracellular cyclic AMP levels over a wide range of concentrations. Our studies provide direct evidence for hormonal regulation of cyclic AMP metabolism in epithelial cells of the choroid plexus.     

Hypnotic action of benzodiazepines: a possible mechanism

The objective of this investigation was to determine whether the effects of muscimol on benzodiazepine receptor binding relate to the hypnotic activity of nine benzodiazepines (clonazepam, triazolam, diazepam, flurazepam, nitrazepam, oxazepam, temazepam, clobazam, and chlordiazepoxide) and CL 218,872. There was no correlation between the basal receptor binding affinities of the drugs tested and their hypnotic potencies, whereas the benzodiazepine receptor agonists whose receptor bindings are strongly modulated by muscimol possess potent hypnotic activity. These results indicate that benzodiazepine receptors that couple to GABA receptors are involved in the hypnotic activity of the benzodiazepines.     

Cyclic nucleotide phosphodiesterase activities from isolated fat cells: correlation of subcellular distribution with effects of nucleotides and insulin

Cyclic nucleotide phosphodiesterasc activities were determined in fractions of fat cell homogenates, prepared either by differential centrifugation or by centrifugation on discontinuous sucrose gradients.In the supernatant fraction (150,000g supernatant in 0.25 m sucrose, or 92,000g supernatant in 0.32m sucrose): (a) there was 70% of the cyclic AMP phosphodiesterase activity of the whole homogenate, and over 90% of the cyclic GMP phosphodiesterase activity; (b) double reciprocal kinetic plots were nonlinear for both substrates; (c) cyclic (GMP, 0.02-2 μm, activated hydrolysis of 10 μm cyclic AMP; (d) 25 or 50 μm cyclic GMP noncompetitively inhibited hydrolysis of 5–20 μm cyclic AMP (K_i = 38 μm); (e) cyclic AMP, 0.1 μm, slightly activated hydrolysis of 10 μm cyclic GMP; (f) 10 or 20 μm cyclic AMP competitively inhibited hydrolysis of 5–20 μm cyclic GMP (K_i = 18 μm).In the particle fraction (1000g, 1000-16,000g, and 16,000–150,000g pellets in 0.25m sucrose, or 0.8-1.2m sucrose interface at 92,000g): (a) there was 30% of the cyclic AMP phosphodiesterase activity of whole homogenate, but less than 5% of the cyclic GMP phosphodiesterase; (b) the double reciprocal kinetic plot of hydrolysis of cyclic AMP was nonlinear; (c) cyclic GMP, 0.02-2μm, did not affect hydrolysis of 10 μm cyclic AMP; (d) 5 or 10 μm cyclic GMP competitively inhibited hydrolysis of 5–20 μm cyclic AMP (K_i = 1.9 μm).Incubation of fat cells with insulin, 40 ng/ml, increased the maximum velocity of particulate high-affinity cyclic AMP phosphodiesterase, but did not affect the supernatant activity. Addition of insulin after homogenization of the cells had no effect on any phosphodiestesterase activity.     

Corticotropin-releasing factor stimulates adenylate cyclase activity in the anterior pituitary gland

Ovine corticotropin-releasing factor (CRF) stimulates adenylate cyclase activity in rat anterior pituitary homogenate at an ED₅₀ value of 70 nM. GTP increases the stimulatory effect of CRF on [³²P] cyclic AMP formation in a rat adenohypophysial particulate fraction and in bovine anterior pituitary plasma membranes. The present data show that CRF stimulates adenylate cyclase activity in the anterior pituitary gland at least partly through a guanyl nucleotide-dependent mechanism.     

Peripheral-type benzodiazepine receptors are involved in the regulation of cholesterol side chain cleavage in adrenocortical mitochondria

In an attempt to elucidate the physiological relevance of the peripheral type of benzodiazepine receptor in adrenocortical mitochondria, we examined the effect of three different benzodiazepines (diazepam, Ro5-4864, and chlordiazepoxide) on the conversion of cholesterol to pregnenolone, the rate-limiting step in steroidogenesis, by using cholesterol-loaded mitochondria from bovine adrenal zona fasciculata. These benzodiazepines, except chlordiazepoxide, caused a dose-dependent stimulation of the cholesterol side chain cleavage in the mitochondria. The stimulatory effect of Ro5-4864 was approximately 10 times more potent than that of diazepam. No inhibitory effect of YM-684 (Ro15-1788), a potent antagonist to central-type benzodiazepine receptors, was observed in the stimulation induced by diazepam and Ro5-4864. Both external calcium ion and voltage-dependent calcium channel blocker, (+)-PN200-110, were without effect on the diazepam-induced steroidogenesis. By contrast, pretreatment of mitochondria with digitonin abolished the stimulatory effect of diazepam on the mitochondrial steroidogenesis. The present results indicate that the peripheral-type benzodiazepine receptor of adrenocortical mitochondria plays an essential role in regulating cholesterol side chain cleavage without any change of calcium channels.     

Prostacyclin analogues inhibit canine parietal cell activity and cyclic AMP formation

To assess further the mechanism by which prostacyclin inhibits acid secretion, the actions of two stable prostacyclin analogues on parietal cell function and cyclic AMP formation were tested using enzymatically dispersed cells from canine fundic mucosa. Accumulation of ¹⁴C-aminopyrine (AP) was used as an index of parietal cell response to stimulation. The 16-phenoxy derivative of PGI₂ inhibited accumulation of AP stimulated by histamine (10 μM), with 50% inhibition (ID₅₀) at 10 nM. 6_β-PGI₁ also inhibited the action of histamine (ID₅₀ 0.5μM) but failed to block stimulation by carbachol or the dibutyryl derivative of cyclic AMP (dbcAMP). In similiar concentrations to those producing inhibition of histamine-stimulated AP accumulation, the 16-phenoxy analogue and 6_β-PGI₁ inhibited histamine-stimulated cyclic AMP generation by parietal cells. At 100 fold higher concentrations, 6_β-PGI₁ stimulated cyclic AMP formation, presumably in non-parietal cells. Even in high concentrations the 16-phenoxy analogue failed to increase cyclic AMP formation by mucosal cells. These data indicate that the stable prostacyclin analogues are potent, direct inhibitors of histamine-stimulated parietal cell function and that it is the inhibition, rather than the stimulation, of cyclic AMP formation that is linked to the antisecretory actions of these prostanoid compounds.