Adenosine-homocysteine interactions in controlling cyclic AMP in rat brain cortical slices

Preincubation of rat brain cortical slices with homocysteine resulted in a reduction of the cyclic AMP response to subsequently added noradrenaline or isoproterenol. There was no effect of homocysteine on the adenosine response, nor of added S-adenosyl homocysteine on any of the responses studied. The results are interpreted in terms of binding of endogenous adenosine by the enzyme S-adenosylhomocysteinase, and provide further evidence for the controlling role of adenosine on cerebral cyclic AMP production.     

Effects of lithium in vitro on noradrenaline-induced cyclic AMP accumulation in rat cortical slices after reserpine-induced supersensitivity

Lithium in vitro at concentrations of 1-2 mM and above inhibited cyclic AMP accumulation due to noradrenaline in cerebral cortical slices prepared from both control rats and rats rendered supersensitive for the noradrenaline response by intraperitoneal injection of reserpine. There was no difference in the concentration-dependence of lithium inhibition between control and reserpine-treated rats.     

Adaptation of cyclic AMP generating system in rat cerebral cortical slices during chronic ethanol treatment and withdrawal

Basal and norepinephrine-stimulated (1–100 μM) production of cAMP in rat cerebral cortical slices was determined during chronic ethanol treatment and withdrawal employing a treatment procedure which rapidly produces a hyperactive withdrawal reaction. Basal and norepinephrine-stimulated cAMP production was increased above control values, in slices, following 88 hr of ethanol treatment. After 72 hr of treatment with ethanol, maximal increases in cAMP were found after 16–18 hr of withdrawal. These increases were essentially the same as those found in animals continuously treated for 88 hr. By 24 hr after the removal of ethanol, cAMP production returned to control values. Hyperactive behaviour during the withdrawal period quantitatively paralleled the observed changes in cAMP production. It is proposed that the sensitivity of cerebral cortical adenylate cyclase rapidly adapts subsequent to changes in neuronal input during and after ethanol treatment but that adaptation in the cortical adrenergic system is not a primary event in the development of physical dependence on ethanol.     

Effects of PACAP, VIP and related peptides on cyclic AMP formation in rat neuronal and astrocyte cultures and cerebral cortical slices

The effects of pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), peptide histidine-isoleucine (PHI) and peptide histidine-methionine (PHM) on cyclic AMP formation were studied in parallel on rat cerebral cortical slices, primary neuronal cultures and primary glial (astrocyte) cultures. PACAPappeared to be the most potent agent in all biological systems. The rank order of the peptides' potency was as follows: PACAP > VIP > PHI = PHM for cortical slices and neuronal cell cultures, and PACAP > PHM approximately VIP > PHI for glial cell cultures. The cyclic AMP responses to the tested peptides, especially to PACAP, were distinctly larger in glial cell cultures than in neuronal cell cultures or brain slices. In an additional study, the cyclic AMP response to helodermin and secretin, as well as isoprenaline, histamine and forskolin, were tested in parallel on glial and neuronal cell cultures, and directly compared with the actions of PACAP. Helodermin and isoprenaline showed clearly stronger activity in glial cell cultures, yet their activity was much weaker than that of PACAP, whereas the effect of forskolin was only 2 times larger in glial cells than in neuronal cultures; histamine had no effect in any cell culture, while secretin produced a small but significant effect only in glial cells. The obtained results suggest that the astrocyte compartment of the rat brain may be the main target for such peptides as PACAP, VIP, or structurally related PHI/PHM or helodermin.     

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

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

Effects of flavonoids on cyclic AMP phosphodiesterase and lipid mobilization in rat adipocytes.

Thirty-one flavonoids were tested for their effects on low Km phosphodiesterase with cyclic AMP as the substrate. Quercetin, luteolin, scutellarein, phloretin and genistein showed inhibitory potencies comparable to or greater than 3-isobutyl-2-methylxanthine (EC50 30-50 microM). Only four compounds namely, catechin, epicatechin, taxifolin and fustin stimulated the enzyme activity (stimulatory EC50 130-240 microM). The most potent phosphodiesterase (PDE) inhibitors were aglycones that had a C2.3 double bond, a keto group at C4 and hydroxyls at C3' and/or C4'. However, when the C-ring is opened then the requirement for the C2.3 double bond is eliminated. The same series of flavonoids were also tested for their lipolytic activity. The structural features required for effective synergistic lipolysis (with epinephrine) were generally similar to that required for potent PDE inhibition except that, for lipolytic activity, an intact C-ring was necessary. Fisetin and quercetin having the above-mentioned structure showed a dose- and time-dependent increase in lipolysis which was synergistic with epinephrine. Only butein and hesperetin showed inhibition of epinephrine-induced lipolysis, and their effect was dose-dependent. A time-course study indicated that hesperetin was able to delay the lipolytic action of epinephrine. It is most likely that the lipolytic effects of these compounds were not a result of PDE inhibition, as the orders of potency for the two activities had poor correlation. Apparently, the effective lipolytic flavonoids were also potent PDE inhibitors but not all the PDE inhibitors were able to induce lipolysis.     

Differences in beta-adrenergic regulation of cyclic AMP formation in cerebral cortical slices of the rat and spiny mouse--Acomys cahirinus

In both the rat and Acomys cahirinus the adrenergic cyclic AMP generating system in the brain is dependent not only on beta-, but also on alpha-adrenoceptors. The relative role of alpha-adrenoceptors is much greater in the Acomys cahirinus. This feature makes the Acomys an interesting animal model for investigating the role of alpha-beta-adrenoceptor coupling in generation of cyclic AMP and the mechanism of action of antidepressant treatment.     

Effects of vasopressin on noradrenaline-induced cyclic AMP accumulation in rat brain slices

Addition of arginine vasopressin (AVP) or 1-desamino-8-D-arginine vasopressin (DDAVP) to rat cortical slices resulted in significant inhibition of the rise in cyclic AMP produced by incubation with 50 microM noradrenaline. A single injection of DDAVP (20 micrograms/rat) produced a reduced response to noradrenaline in derived cortex and caudate slices. In animals pretreated at day 5 of life with IP desipramine and intracisternal 6-hydroxydopamine (6-OHDA), both acute and chronic treatments with DDAVP resulted in a reduction in response in derived cortical, caudate and hippocampal slices. The 6-OHDA pretreated animals also showed reduced open-field behavioural activity after both acute and chronic DDAVP, while animals which were not pretreated responded to acute treatment only. The relationship between the effects of vasopressin on noradrenaline-induced cyclic AMP accumulation and its action on learning and memory is discussed.     

The role of calcium in the cyclic AMP response to histamine in rabbit cerebral cortical slices.

The effect of calcium on the H1- and H2-receptor components of the cyclic AMP response to histamine in rabbit cerebral cortical slices has been investigated. Removal of calcium ions from the incubation medium during the preparation, preincubation and final incubation of brain slices significantly reduced the cyclic AMP responses to adenosine, histamine and the H2-selective agonist, impromidine. Removal of calcium ions from the incubation medium during only the final incubation with agonists did not influence the responses to adenosine, histamine, impromidine and the H1-selective agonist, 2-thiazolylethylamine. Final incubation of rabbit cerebral cortical slices in calcium-free buffer containing EGTA (1 mM) however, selectively reduced the cyclic AMP responses to the H1-agonists histamine and 2-thiazolylethylamine without affecting the response to impromidine or adenosine. These latter incubation conditions significantly reduced the maximal extent of the augmentation of impromidine- or adenosine-stimulated cyclic AMP accumulation produced by H1-receptor stimulation, without affecting the EC50 values of the H1-agonists. Calcium-free/EGTA conditions did not, however, alter the dose-response parameters for the response to the H2-agonist, impromidine. These data provide further evidence that the two histamine receptor systems affect cyclic AMP accumulation in rabbit cerebral cortical slices by different mechanisms.     

Characterization of histamine receptors mediating the stimulation of cyclic AMP accumulation in rabbit cerebral cortical slices.

The characteristics of histamine-stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in slices of rabbit cerebral cortex have been investigated. The selective H2-receptor antagonists, cimetidine, tiotidine, metiamide and ranitidine appeared to antagonize the stimulation of cyclic AMP accumulation elicited by histamine in a competitive manner consistent with an interaction with histamine H2-receptors. The H1-receptor antagonist mepyramine (0.8 microM) produced only a weak inhibition of the response to histamine. The inhibition appeared to be non-competitive producing a decrease in the maximal response with little effect on the EC50 value. The specific H2-receptor agonist, impromidine, produced a maximum response of only 31 +/- 2% of that obtained with histamine. Studies with histamine and impromidine in combination indicated that impromidine was not acting as a partial agonist. 2-Thiazolylethylamine, a selective H1-agonist, produced only a weak response (EC50 approximately 1mM) yielding a relative potency with respect to histamine (= 100) of 2.5. In the presence of a supramaximal concentration of impromidine, histamine and 2-thiazolylethylamine further elevated the response to impromidine. In these conditions the relative potency of 2-thiazolylethylamine was increased to 59 (histamine = 100), a value which was comparable with that reported for H1-receptor-mediated contractions of guinea-pig ileum. The H1-receptor antagonists mepyramine, promethazine, triprolidine and chlorpheniramine competitively antagonized the potentiation of impromidine-stimulated cyclic AMP accumulation elicited by histamine and 2-thiazolylethylamine in rabbit cerebral cortex without affecting the response to impromidine alone. (+)-Chlorpheniramine was some 150 fold more potent than the (-)-isomer in this respect. Histamine and adenosine in combination had a much greater than additive effect on the accumulation of cyclic AMP in rabbit cerebral cortical slices. The potentiation of the adenosine response could be partially but not completely antagonized by either cimetidine or mepyramine. In the presence of H2-receptor blockade with 0.02 mM tiotidine, histamine elicited a significant potentiation (EC50 44 microM) of the response to adenosine. This response was antagonized competitively by mepyramine yielding a KB value of 0.05 microM similar to that obtained from inhibition of the potentiation of impromidine-stimulated accumulation of cyclic AMP (0.02 microM). These results suggest that there are two components in the response to histamine in rabbit cerebral cortical slices.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alpha 2-adrenoceptor-mediated inhibition of histamine release from rat cerebral cortical slices.

1. Depolarization of rat cerebral cortical slices, prelabelled with [3H]-histidine, in high potassium (40 mM KCl) medium stimulated the release of [3H]-histamine. The K+-evoked release of [3H]-histamine was attenuated by incubation in calcium-free medium and prevented by prior incubation of brain slices with the selective histidine decarboxylase inhibitor S-(alpha)-fluoromethylhistidine. 2. The K+-evoked release of [3H]-histamine was significantly (P less than 0.001) reduced following stimulation of histamine H3-receptors with R-(alpha)-methylhistamine (1 microM) and this effect was antagonized by the H3-antagonist thioperamide (1 microM). 3. Noradrenaline and the alpha 2-selective adrenoceptor agonists clonidine and UK-14,304 inhibited the K+-evoked release of [3H]-histamine in a concentration-dependent manner yielding EC50 values of 2.5, 0.8 and 1.2 microM, respectively. However, the maximum response to clonidine was only 52 +/- 8% of that obtained with noradrenaline. 4. The inhibitory effect of noradrenaline was antagonized by the non-selective alpha-antagonist phentolamine and by the selective alpha 2-antagonists yohimbine and idazoxan. However, the response to noradrenaline was not inhibited by the alpha 1-antagonist prazosin at concentrations up to 1 microM. 5. These results suggest that both histamine H3-receptors and alpha 2-adrenoceptors are present on histamine-containing nerve terminals in rat cerebral cortex and can exert an inhibitory influence on neurotransmitter release.     

Effects of phosphodiesterase inhibition on cortical spreading depression and associated changes in extracellular cyclic GMP

Cortical spreading depression (CSD) is a temporary disruption of local ionic homeostasis that propagates slowly across the cerebral cortex, and may contribute to the pathophysiology of stroke and migraine. Previous studies demonstrated that nitric oxide (NO) formation promotes the repolarisation phase of CSD, and this effect may be cyclic GMP (cGMP)-mediated. Here, we have examined how phosphodiesterase (PDE) inhibition, either alone or superimposed on NO synthase (NOS) inhibition, alters CSD and the associated changes in extracellular cGMP. Microdialysis probes incorporating an electrode were implanted into the frontoparietal cortex of anaesthetised rats for quantitative recording of CSD, pharmacological manipulations, and dialysate sampling for cGMP measurements. CSD was induced by cathodal electrical stimulation in the region under study by microdialysis. Extracellular cGMP increased, but only slightly, during CSD. Perfusion of either zaprinast or sildenafil through the microdialysis probe, at concentrations that inhibited both PDE5 and PDE9 (and possibly other PDE), increased significantly extracellular cGMP. Unexpectedly, these levels remained high when NOS was subsequently inhibited with N(omega)-nitro-l-arginine methyl ester hydrochloride (l-NAME, 1mM). The most interesting pharmacological effect on CSD was obtained with sildenafil. This drug altered neither CSD nor the subsequent characteristic effect of NOS inhibition, i.e. a marked widening of CSD. The fact that NOS inhibition still widened CSD in the presence of the high extracellular levels of cGMP associated with PDE inhibition, suggests that NO may promote CSD recovery, independently of cGMP formation.     

Dibenzocycloalkenimine (MK-801) stimulates phosphoinositide hydrolysis in rat cerebral cortical slices)

Dibenzocycloalkenimine (MK-801), a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, induced a concentration-dependent accumulation of inositol monophosphate in rat cerebral cortical slices. This effect appears unrelated to antagonism of NMDA receptors as AP-7 and PCP did not cause comparable effects. In addition, the MK-801-induced response was unaffected by NMDA or glycine, alone or in combination, Zn2+, or the removal of Mg2+ from the buffer. These results indicate a novel site of action for MK-801 associated with activation of phosphoinositide hydrolysis.     

Modulation by cyclic AMP and phorbol myristate acetate of cephaloridine-induced injury in rat renal cortical slices

Intracellular signaling pathways of cAMP and protein kinase C (PKC) have been suggested to modulate the generation of free radicals. We investigated the effects of cAMP and phorbol myristate acetate (PMA), a PKC activator, on cephaloridine (CER)-induced renal cell injury, which has been reported to be due to the generation of free radicals. Incubation of rat renal cortical slices with CER resulted in increases in lipid peroxidation and lactate dehydrogenase (LDH) release and in decreases in gluconeogenesis and p-aminohippurate (PAH) accumulation in rat renal cortical slices, suggesting free radical-induced injury in slices exposed to CER. A derivative of cAMP ameliorated not only the increase in lipid peroxidation but also the renal cell damage induced by CER. This amelioration by a cAMP derivative of lipid peroxidation and renal cell damage caused by CER was blocked by KT 5720, a protein kinase A (PKA) inhibitor. Lipid peroxidation and the indices of cell injury were increased by PMA. PMA also enhanced CER-induced lipid peroxidation and cell damage in the slices. This enhancement by PMA of CER-induced injury was blocked by H-7, a PKC inhibitor. These results indicated that intracellular signaling pathways of cAMP and PKC modulate free radical-mediated nephrotoxicity induced by CER.     

Tissue selective inhibition of cyclic nucleotide phosphodiesterase by denbufylline

The effects of the novel alkylxanthine denbufylline (1,3-d-n-butyl-7-(2'-oxopropyl)-xanthine), theophylline and 3-isobutyl-1-methyl-xanthine (IBMX), on the breakdown of cyclic AMP in homogenates of rat erythrocytes, abdominal aorta, adipocytes and cardiac and skeletal muscle were studied. Theophylline and IBMX inhibited cyclic nucleotide phosphodiesterase in all tissue extracts. In contrast, denbufylline was a tissue selective inhibitor of cyclic nucleotide phosphodiesterase. In skeletal muscle and erythrocytes, denbufylline (10 mumol/l) inhibited cyclic nucleotide phosphodiesterase activity by at least 80%. In these tissues, denbufylline was 10-30 and 100-300fold more potent than IBMX and theophylline, respectively. In adipocytes and cardiac and smooth muscle, denbufylline was not an effective inhibitor of cyclic AMP breakdown. Hofstee analysis of phosphodiesterase activity revealed that denbufylline selectively inhibited high affinity cyclic AMP phosphodiesterase in erythrocytes and skeletal muscle. In adipocytes, cardiac and vascular smooth muscle, denbufylline did not effectively inhibit the composite cyclic nucleotide phosphodiesterase activities either with high or with low affinity for cyclic AMP.     

Effects of 1-benzylxanthines on cyclic AMP phosphodiesterase 4 isoenzyme

Based on our results regarding the structure-activity relationships of alkylxanthines and imidazo[2,1-i]purines as phosphodiesterase 4 (PDE4) inhibitors, we designed new 1-benzylxanthines and investigated their PDE4 inhibitory activities. 3,7-Dihydro-7-acetonyl-1-(2,4-dichlorobenzyl)-3-propyl-1H-purine-2,4-dione (2h) exhibited both more selective and more potent PDE4 inhibitory activity than rolipram and XT-611.     

Effect of serotonin on cyclic AMP level in rat hypothalamus slices during development.

The effect of serotonin (5-HT) on cyclic AMP levels in rat hypothalamic slices was age-dependent. The sensitivity of the cyclic AMP system to 5 X 10(-5) M 5-HT already existed in the foetus at 21 days of pregnancy. It reached a maximum on the 7th postnatal day and then decreased with age. In adult tissue the response was still present and was antagonized by two serotonergic antagonists (methiothepin and metergoline). When compared to the progressive increase of 5-HT level in the hypothalamus the data suggest a different evolution of serotonergic innervation and of 5-HT receptors.     

Comparison of cyclic nucleotide phosphodiesterase isoforms from rat heart and bovine aorta. Separation and inhibition by selective reference phosphodiesterase inhibitors

The resolution as well as the biochemical properties of the multiple molecular forms of cyclic nucleotide phosphodiesterase, in a given tissue, may be strongly dependent upon experimental conditions of preparation (extraction of crude enzyme from tissues and fractionation procedures). In the present study, we compare the different molecular forms of cardiac (rat heart ventricle) and vascular (bovine aorta) phosphodiesterase isolated from crude extracts prepared either in sucrose medium or in hypotonic medium (in the presence of protease inhibitors and ion chelators) using two different fractionation procedures: isoelectric focusing on flat gel bed and DEAE-Trisacryl anion exchange chromatography. Both the calmodulin-dependent and the cAMP-specific forms exhibited close IEF and chromatographic patterns and showed similar sensitivities towards reference inhibitors regardless of the tissue of origin. In marked contrast, the cGMP-specific isoform notably differed from one to another tissue with respect to its biochemical properties (only the cardiac tissue being capable of stimulation by cGMP) and sensitivities to xenobiotics. Thus the possibility exists that pharmacological agents may modulate phosphodiesterase activity differently in cardiac and vascular target tissues.