Cannabinoid Receptor-Regulated Cyclic AMP Accumulation in the Rat Striatum

The present study demonstrates that desacetyllevonantradol, a synthetic cannabinoid analog, reduces cyclic AMP levels in rat striatal slices stimulated with vasoactive intestinal peptide or SKF 38393, a D1-dopamine agonist. Desacetyllevonantradol and the D2 agonist LY 171555 both inhibited D1-stimulated cyclic AMP accumulation in the striatum. Spiperone, a specific D2-dopamine antagonist, fully reversed the inhibitory effect of LY 171555 but not that of desacetyllevonantradol, indicating that this cannabinoid response is not occurring through a D2-dopaminergic mechanism. Morphine also inhibited cyclic AMP accumulation in striatal slices stimulated with either SKF 38393 or vasoactive intestinal peptide. Naloxone, an opioid antagonist, fully reversed the effect of morphine but not that of desacetyllevonantradol, indicating that cannabinoid drugs are not acting via a mechanism involving opioid receptors. The response to maximally effective concentrations of desacetyllevonantradol was not additive to that of maximally effective concentrations of either morphine or LY 171555, suggesting that dopaminergic, opioid, and cannabinoid receptors may be present on the same populations of cells.     

Changes in the cannabinoid receptor binding, G protein coupling, and cyclic AMP cascade in the CNS of rats tolerant to and dependent on the synthetic cannabinoid compound CP55,940

Chronic exposure to CP55,940 produced a significant down-regulation of cannabinoid receptors in the striatum, cortex, hippocampus, and cerebellum of rat brain. At 24 h after SR141716-precipitated withdrawal, we observed a tendency to return to basal levels in the striatum and cortex, whereas the specific binding remained lower in the hippocampus and cerebellum. When we surveyed cannabinoid receptor-activated G proteins, in chronic CP55,940-treated rats the guanosine 5'-O:-(3-[(35)S]thiotriphosphate) ([(35)S]GTPgammaS) binding assay revealed a decrease of activated G proteins in the striatum, cortex, and hippocampus, whereas no significant changes were seen in the cerebellum. At 24 h after the SR141716-precipitated withdrawal, [(35)S]GTPgammaS binding increased compared with that of rats chronically exposed to CP55,940, attaining the control level except for cerebellum, where we observed a trend to overcome the control amounts. Concerning the cyclic AMP (cAMP) cascade, which represents the major intracellular signaling pathway activated by cannabinoid receptors, in the cerebral areas from rats chronically exposed to CP55,940 we found alteration in neither cAMP levels nor protein kinase A activity. In the brain regions taken from CP55, 940-withdrawn rats, we only observed a significant up-regulation in the cerebellum. Our findings suggest that receptor desensitization and down-regulation are strictly involved in the development of cannabinoid tolerance, whereas alterations in the cAMP cascade in the cerebellum could be relevant in the mediation of the motor component of cannabinoid abstinence.     

Regional increase of cyclic GMP by atrial natriuretic factor in rat brain: markedly elevated response in spontaneously hypertensive rats

Atrial natriuretic factor (ANF)-responsive areas in rat brain were examined by measuring ANF-stimulated cyclic GMP production in rat brain slice preparations. The medulla oblongata, thalamus, and pituitary gland responded most sensitively, the septum, hypothalamus, pons, midbrain and olfactory bulb responded moderately, but neocortex, cerebellum, striatum and hippocampus were unresponsive to ANF. The most responsive regions in spontaneously hypertensive rats brains showed 2 to 5 times higher cyclic GMP production than those from the control Wistar-Kyoto rats. These findings provide evidence for biological action of ANF on brain tissues, and indicate the action of ANF produced in the brain.     

Histamine Increases Phospholipid Methylation and H2-Receptor-Adenylate Cyclase Coupling in Rat Brain

Histamine stimulated the enzymatic synthesis of phosphatidylcholine from phosphatidylethanolamine in crude synaptic membranes of rat brain containing the methyl donor S-adenosyl-L-methionine (SAM). In the presence of, but not in the absence of SAM, histamine increased cyclic AMP accumulation at the concentrations that stimulate phospholipid methylation. S-Adenosyl-L-homocysteine, an inhibitor of phospholipid methyltransferases, inhibited histamine-stimulated phospholipid methylation and histamine-induced cyclic AMP accumulation in the presence of SAM in a concentration-dependent manner. Histamine-induced [3H]methyl incorporation into phospholipids exhibited a marked regional heterogeneity in rat brain in the order of cortex greater than medulla oblongata greater than hippocampus greater than striatum greater than midbrain greater than hypothalamus. The regional distribution of histamine-induced cyclic AMP accumulation exactly paralleled histamine-stimulated [3H]methyl incorporation in rat brain. Histamine-induced cyclic AMP accumulation was inhibited by the addition of cimetidine or famotidine, but not by mepyramine or diphenhydramine. The accumulation of cyclic AMP in the presence of SAM was observed by the addition of impromidine or dimaprit, but not by 2-pyridylethylamine. These results indicate that phospholipid methylation is induced by histamine and may participate in H2-receptor-mediated stimulation of adenylate cyclase in rat brain.     

Preparation and properties of a cell-free, hormonally responsive adenylate cyclase from guinea pig brain

—The accumulation of cyclic adenosine 3′,5′-monophosphate (cyclic AMP) was studied in cell-free homogenates of guinea pig brain. Homogenates, prepared in Krebs-Ringer buffer, responded markedly to the addition of neurohormones with an increased rate of cyclic AMP synthesis; preparations from cerebellum, cerebral cortex, and hippocampus responded to a degree approximating that achieved with slices of these areas of guinea pig brain. Adenylatc cyclase activity was seen only when cyclic AMP was measured by a [³H]adenine prelabelling technique or when total cyclic AMP was measured by radioimmunoassay; [³²P]ATP did not serve as a substrate for this preparation of the enzyme. The adenylate cyclase was paniculate and required a Krebs Ringer buffer; use of tris, or tris with Mg²⁺ and Ca²⁺, resulted in a preparation totally devoid of hormonal stimulation. Digestion by purified beef heart cyclic nucleotide phosphodiesterase, Dowex chromatography, solubility in Ba(OH)₂-ZnSO₄ mixtures, and two thin layer chromatographic systems demonstrated that the product of the hormonally stimulated adenylate cyclase preparation was cyclic AMP. The selectivity of hormonal stimulation and the adrenergic character of the hormonal receptors from different brain areas were maintained in the cell-free preparation. However, simultaneous stimulation with two different neurohormones resulted in additive responses, rather than in the potentiation observed in preparations of slices of brain.     

Heterogeneity of β-Adrenoceptors in Guinea-Pig Brain: Radioligand Binding and Cyclic Nucleotide Generation

Abstract: In this report, we have examined the radioligand binding and second messenger signalling characteristics of β-adrenoceptors in the guinea-pig brain. [¹²⁵I]lodocyanopindolol ([¹²⁵I]ICYP)-labelled sites in the cerebellum and cerebral cortex were of similar densities ( B _max 34 and 24 fmol·mg⁻¹) and affinities ( K _D 20 and 55 p M ), respectively. Analysis of competition for [¹²⁵I]ICYP binding in the cerebellum was compatible with the presence of a β₂-adrenoceptor. In this tissue, isoprenaline evoked a cyclic AMP stimulation, and also potentiated cyclic GMP accumulations evoked in the presence of a nitric oxide donor, consistent with mediation via a β₂-adrenoceptor. The [¹²⁵I]ICYP binding profile in the cerebral cortex did not comply with those previously described for β-adrenoceptor subtypes, and isoprenaline failed to alter significantly cyclic AMP accumulation in the cerebral cortex, hippocampus, or neostriatum, even in the presence of forskolin or a phosphodiesterase inhibitor. Isoprenaline was also without effect on cyclic GMP accumulation or phosphoinositide turnover in the cerebral cortex. These results suggest that the guinea-pig cerebellum expresses a functional β₂-adrenoceptor coupled to cyclic AMP generation, and potentiation of cyclic GMP accumulation. However, the guinea-pig cerebral cortex displays binding sites that exhibit β-adrenoceptor-like pharmacology but fail to show functional coupling to cyclic AMP, cyclic GMP, or phosphoinositide signalling systems.     

Cyclic nucleotide levels in brains of control and hypertensive rats.

Adenosine 3′, 5′-monophosphate (cyclic AMP) and guanosine 3′,5- monophosphate (cyclic GMP) levels were measured in seven brain areas of spontaneously hypertensive rats (SHR) and two groups of control rats. In cerebral cortex, hypothalamus, pons-medulla oblongata and cerebellum cyclic AMP levels were higher in SHR than in Wistar-Kyoto controls. Cyclic GMP levels were higher in SHR than in Wistar-Kyoto rats in all brain areas except for the striatum and hippocampus where the levels were lower. There were also some differences in cyclic nucleotide levels between Wistar-Kyoto and Wistar-Charles River controls.     

PGF2 alpha, thromboxane B2 and HETE levels in gerbil brain cortex after ligation of common carotid arteries and decapitation.

The effects of ligation of both common carotid arteries in the gerbil on the levels of PGF2 alpha, TXB2, HETE and of energy metabolites in brain cortex, have been investigated. Also, in the same experimental conditions the changes of cyclic AMP in brain cortex, cerebellum, striatum and hippocampus have been monitored. ATP, glycogen, glucose and phosphocreatine decrease whereas, lactate and cyclic AMP are enhanced in the ischemic brain, as previously reported. In contrast, levels of arachidonic acid metabolites are not modified. During ischemia following decapitation, instead, PGF2 alpha, and TXB2, show considerable increase.     

CYCLIC NUCLEOTIDES IN MURINE BRAIN: THE TEMPORAL RELATIONSHIP OF CHANGES INDUCED IN ADENOSINE 3',5'-MONOPHOSPHATE AND GUANOSINE 3',5'-MONOPHOSPHATE FOLLOWING MAXIMAL ELECTROSHOCK OR DECAPITATION

–Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) levels increase about 5-fold in the cerebral cortex and 2-fold in the cerebellum following electroconvulsive shock (ECS). The peak levels of cyclic AMP occur at 45 s after ECS in the cerebral cortex, and at 15 s in the cerebellum. In the cerebral cortex, ECS produces twice the cyclic AMP accumulation as does decapitation in a comparable time period; however, the relative effect of a number of neurotropic agents on the cyclic AMP accumulation is essentially the same, whether stimulated by decapitation or by ECS. In the cerebellum, the levels of guanosine 3′,5′-cyclic monophosphate (cyclic GMP) also increase following ECS. The cyclic GMP levels are greatest at 60 s after ECS during the postictal depression. An association between elevated cerebellar cyclic GMP and depression seems unlikely, since CNS depressants either lowered or had no effect on cyclic GMP levels. From these results, cyclic nucleotide profiles following treatments such as ECS or decapitation may be useful in elucidating the molecular events involved in seizures, brain injury and ischemia.     

Selective changes of receptor binding in brain regions of aged rats

Binding to several receptors was compared in brain regions of 3 and 21-23 month-old rats. In crude membrane preparations of aged rats the number of dopamine antagonist receptors in striatum was much reduced (-53%). beta-Noradrenergic receptors (cortex) and benzodiazepine receptors (hippocampus and cerebellum) were less but significantly reduced and serotonergic receptors, alpha 1 noradrenergic receptors (both in cortex) and dopamine agonist receptors (striatum) were unchanged. For each receptor binding the KD values were the same in young and old animals. GABA receptor binding (hippocampus and cerebellum) evaluated at only one 3H-GABA concentration (8 nM) was similar in both groups when expressed per protein content but significantly reduced in aged rats when expressed per tissue wet weight because of the partial purification of the synaptic membranes used for 3H-GABA binding. In our experimental conditions age-related changes of specific binding sites in the central nervous system were selective for some receptors studied and did not seem to be due to general non-specific modification of brain tissue composition.     

Differential inhibition of cyclic AMP and cyclic GMP hydrolysis in rat renal cortex.

Adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP) metabolism in rat renal cortex was examined. Athough the cyclic AMP and cyclic GMP phosphodiesterases are similarly distributed between the soluble and particulate fractions following differential centrifugation, their susceptibility to inhibition by theophylline, dl-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724), and 1-methyl-3-isobutylxanthine (MIX) are quite different. Ro 20-1724 selectively inhibited both renal cortical-soluble and particulate cyclic AMP degradation, but had little effect on cyclic GMP hydrolysis. Theophylline and MIX effectively inhibited degradation of both cyclic nucleotides, with MIX the more potent inhibitor. Effects of these agents on the cyclic AMP and cyclic GMP content of cortical slices corresponded to their relative potency in broken cell preparations. Thus, in cortical slices, Ro 20-1724 (2 mm) had the least effect on basal (without agonist), carbamylcholine, and NaN₃-stimulated cyclic GMP accumulation, but markedly increased basal and (parathyroid hormone) PTH-mediated cyclic AMP accumulation, MIX (2 mm) which was as effective as Ro 20-1724 in potentiating basal and PTH-stimulated increases in cyclic AMP also mediated the greatest augmentation of basal, carbamylcholine, and NaN₃-stimulated accumulation of cyclic GMP. By contrast, theophylline (10 mm) which was only 12% as effective as Ro 20-1724 in increasing the total slice cyclic AMP content in the presence of PTH was much more effective than Ro 20-1724 in potentiating carbamylcholine and NaN₃-mediated increases in cyclic GMP. These results demonstrate selective inhibition of cyclic nucleotide phosphodiesterase activities in the rat renal cortex and support the possibility of multiple cyclic nucleotide phosphodiesterases in this tissue. Furthermore, both cyclic nucleotides appear to be rapidly degraded in the renal cortex.     

CYCLIC AMP-DEPENDENT PROTEIN KINASE ACTIVITY AND SYNAPTOSOMAL PROTEIN PHOSPHORYLATION IN THE BRAINS OF AGED RATS

The activity of soluble protein kinase and phosphorylation of endogenous synaptosomal proteins were studied in vitro, in the hippocampus and cerebral cortex of rats 3, 12, or 24 months of age. No between-age differences in the activity of cyclic AMP-dependent or independent protein kinase were detected in either brain region. The degree of stimulation by cyclic AMP and the apparent K_a, for cyclic AMP were similar at all stages. Cyclic AMP stimulated the phosphorylation of synaptosomal proteins from the cerebral cortex, hippocampus, caudate nucleus, and cerebellum of rats at all ages. There were no significant differences across age in the extent of phosphorylation of any membrane proteins in any brain region. The number and staining density of synaptosornal proteins separated by polyacrylamide gel electrophoresis were also similar at all ages. These studies indicate that the cyclic AMP-dependent phosphorylation system in the rat brain does not change during advanced aging.     

Enhancement of anandamide formation in the limbic forebrain and reduction of endocannabinoid contents in the striatum of delta9-tetrahydrocannabinol-tolerant rats

Recent studies have shown that the pharmacological tolerance observed after prolonged exposure to synthetic or plant-derived cannabinoids in adult rats is accompanied by down-regulation/desensitization of brain cannabinoid receptors. However, no evidence exists on possible changes in the contents of the endogenous ligands of cannabinoid receptors in the brain of cannabinoid-tolerant rats. The present study was designed to elucidate this possibility by measuring, by means of isotope dilution gas chromatography/mass spectrometry, the contents of both anandamide (arachidonoylethanolamide; AEA) and its biosynthetic precursor, N-arachidonoylphosphatidylethanolamine (NArPE), and 2-arachidonoylglycerol (2-AG) in several brain regions of adult male rats treated daily with delta9-tetrahydrocannabinol (delta9-THC) for a period of 8 days. The areas analyzed included cerebellum, striatum, limbic forebrain, hippocampus, cerebral cortex, and brainstem. The same regions were also analyzed for cannabinoid receptor binding and WIN-55,212-2-stimulated guanylyl-5'-O-(gamma-[35S]thio)-triphosphate ([35S]GTPgammaS) binding to test the development of the well known down-regulation/desensitization phenomenon. Results were as follows: As expected, cannabinoid receptor binding and WIN-55,212-2-stimulated [35S]GTPgammaS binding decreased in most of the brain areas of delta9-THC-tolerant rats. The only region exhibiting no changes in both parameters was the limbic forebrain. This same region exhibited a marked (almost fourfold) increase in the content of AEA after 8 days of delta9-THC treatment. By contrast, the striatum exhibited a decrease in AEA contents, whereas no changes were found in the brainstem, hippocampus, cerebellum, or cerebral cortex. The increase in AEA contents observed in the limbic forebrain was accompanied by a tendency of NArPE levels to decrease, whereas in the striatum, no significant change in NArPE contents was found. The contents of 2-AG were unchanged in brain regions from delta9-THC-tolerant rats, except for the striatum where they dropped significantly. In summary, the present results show that prolonged activation of cannabinoid receptors leads to decreased endocannabinoid contents and signaling in the striatum and to increased AEA formation in the limbic forebrain. The pathophysiological implications of these findings are discussed in view of the proposed roles of endocannabinoids in the control of motor behavior and emotional states.     

In situ imaging of specific binding of [3H]isatin in rat brain

Isatin is an endogenous indole that influences a range of processes both in vivo and in vitro. It has a distinct and discontinuous distribution in the brain, as well as in other mammalian tissues and body fluids. However, the distribution of isatin binding sites in the brain is not known. Using a real-time beta-imager we have investigated the distribution of [3H]isatin-specific binding in rat brain sections. The highest labeling was found in hypothalamic nuclei and in the cortex, hippocampus, and cerebellum. Administration of the mechanism based monoamine oxidase inhibitor, pargyline, reduced but did not abolish the specific binding of [3H]isatin in the rat brain. The distribution became cortex, cerebellum, hypothalamus > hippocampus > brain stem > thalamus approximately striatum.     

Effects of decapitation, ether and pentobarbital on guanosine 3′,5′-phosphate and adenosine 3′,5′-phosphate levels in rat tissues

Cyclic GMP and cyclic AMP levels in eight different rat tissues were examined after animlas were immersed in liquid nitrogen. In order of decreasing concentration, cerebellu, kidney, lung and cerebral cortex contained the greatest quantities fo cyclic GMP. These tissues also contained relatively high concentrations of cyclic AMP. Compared to values in animals which were sacrificed in liquid nitrogen, levels of both nucleotides in many of the tissues examined were altered by decapitation or anesthesia with ether and pentobarbital. Decapitation increased the levels of both cyclic GMP and cyclic AMP in cerebellum, lung, heart, liver and skeletabl muscle. However, decapitation increased only cyclic AMP in cerebral cortex and kidney. Our previously reported high level of cyclic GMP in lung was attributed to ether anesthesia and surgical removal which increased the cyclic GMP content in lung, heart, testis and skeletal muscle. The effect of ether on cyclic GMP levels in lung and heart was blocked by pretreatment of animals with atropine which indicated that cholinergic agents increase cyclic GMP content in these tissues. Acetylcholine and carbachol in the presence of theophylline increased the accumulation of cyclic GMP in incubations of rat lung minces. Increases in cyclic GMP and cyclic AMP levels in cerebellum with ether anesthesia were prevented if rats were immersed in liquid nitrogen after anesthesis with ether. Anesthesia with pentobarbital decreased the levels of cyclic GMP in cerebellum and kidney and increased the nucleotide in heart, liver, testis and skeletal muscle compared to levels in tissues from animals immersed in liquid nitrogen. However, pentobarbital increased cyclic AMP levels in cerebellum and cerebral cortex and decreased the nucleotide in liver, kidney, testis and skeletal muscle. These studies provide a possible explanation for the variability in in vivo levels of cyclic GMP and cyclic AMP which have been previously reported. In addition, these studies support the hypothesis that the synthesis and degradation of cyclic AMP and cyclic GMP are regulated independently and not necessarily in a parallel or reciprocal manner. These studies also suggest that the increase accumulation of one cyclic nucleotide has no major effect on the synthesis and/or metabolism of the other; however, such interactions cannot be entirely excluded from the results of this study.     

Characterization of CB1 Receptors on Rat Neuronal Cell Cultures: Binding and Functional Studies Using the Selective Receptor Antagonist SR 141716A

Abstract: This study was undertaken to characterize further the central cannabinoid receptors in rat primary neuronal cell cultures from selected brain structures. By using [³H]SR 141716A, the specific CB1 receptor antagonist, we demonstrate in cortical neurons the presence of a high density of specific binding sites ( B _max = 139 ± 9 fmol/mg of protein) displaying a high affinity ( K _D = 0.76 ± 0.09 n M ). The two cannabinoid receptor agonists, CP 55940 and WIN 55212-2, inhibited in a concentration-dependent manner cyclic AMP production induced by either 1 µ M forskolin or isoproterenol with EC₅₀ values in the nanomolar range (4.6 and 65 n M with forskolin and 1.0 and 5.1 n M with isoproterenol for CP 55940 and WIN 55212-2, respectively). Moreover, in striatal neurons and cerebellar granule cells, CP 55940 was also able to reduce the cyclic AMP accumulation induced by 1 µ M forskolin with a potency similar to that observed in cortical neurons (EC₅₀ values of 3.5 and 1.9 n M in striatum and cerebellum, respectively). SR 141716A antagonized the CP 55940- and WIN 55212-2-induced inhibition of cyclic AMP accumulation, suggesting CB1 receptor-specific mediation of these effects on all primary cultures tested. Furthermore, CP 55940 was unable to induce mitogen-activated protein kinase activation in either cortical or striatal neurons. In conclusion, our results show nanomolar efficiencies for CP 55940 and WIN 55212-2 on adenylyl cyclase activity and no effect on any other signal transduction pathway investigated in primary neuronal cultures.     

Analysis of brain regional distribution of aluminium in rats via oral and intraperitoneal administration

In the present work, accumulation and distribution of aluminium in the rat brain following both intraperitoneal and oral administration were studied. Electrothermal atomic absorption spectrometry was used to determine aluminium concentration in different brain areas (cerebellum, ventral midbrain, cortex, hippocampus, and striatum). Most of the brain areas showed accumulation of aluminium, but a greater and more significant increase was noted in the group receiving aluminium via intraperitoneal administration. Aluminium distribution was also dependent on the administration route.     

PGF2α, thromboxane B2 and hete levels in gerbil brain cortex after ligation of common carotid arteries and decapitation

The effects of ligation of both common carotid arteries in the gerbil on the levels of PGF_2α, TXB₂, HETE and of energy metabolites in brain cortex, have been investigated. Also, in the same experimental conditions the changes of cyclic AMP in brain cortex, cerebellum, striatum and hippocampus have been monitored. ATP, glycogen, glucose and phosphocreatine decrease whereas, lactate and cyclic AMP are enhanced in the ischemic brain, as previously reported. In contrast, levels of arachidonic acid metabolites are not modified. During ischemia following decapitation, instead, PGF_2α, and TXB₂, show considerable increase.     

RATE OF ACCUMULATION OF ACETYLCHOLINE IN DISCRETE REGIONS OF THE RAT BRAIN AFTER DICHLORVOS TREATMENT

Abstract– The time course for accumulation of acetylcholine was measured in rat brain regions after treatment with 15 mg/kg, i.v., dichlorvos. With this dose of dichlorvos 84-96% of the brain cholinester-ase is inhibited within 1 min. After killing and concomitant enzyme inactivation through microwave irradiation, the acetylcholine levels were measured by pyrolysis-gas chromatography. In the brain regions studied, the striatum had the highest rate of accumulation of acetylcholine and the cerebellum had the lowest. The calculated turnover time in minutes for the regions of the brain were cerebral cortex 0.9; hippocampus 1; striatum 1.4; cerebellum 1.7; medulla-pons 2.2; midbrain 4.5; thalamus 5.6.