Prenatal and early postnatal β-adrenergic receptor-mediated increase of cyclic AMP in slices of rat brain

The levels of cyclic AMP in slices of cerebral cortex and cerebellum from newborn rats were significantly, but transiently, increased by exposure to the β-adrenergic agonist, isoproterenol. Isobutylmethyxanthine, an inhibitor of phosphodiesterase, enhanced this effect and permitted its detection in cerebral cortex obtained from the prenatal rat. These results are consistent with the possibilities that functional noradrenergic synapses are formed early in the ontogeny of the CNS, and that norepinephrine may exert cyclic AMP-mediated influences on brain development.     

Effect of morphine on catecholamine - stimulated cyclic AMP production in cortex slices from rats and mice

Morphine (10 micro M) blocked noradrenaline-stimulated cyclic AMP production in slices of cerebral cortex from normal rats but not in slices from rats pretreated with 6-hydroxydopamine (6-OHDA). In contrast, morphine failed to prevent noradrenaline-stimulated cyclic AMP production in mouse cortex slices. Levorphanol weakly antagonized the rise in cyclic AMP produced by noradrenaline in both normal and 6-OHDA-treated mouse cortex. Morphine had no effect on the adrenaline-stimulated cyclic AMP accumulation in mouse cortex but it entirely prevented the rise in cyclic AMP produced by isoprenaline. This effect was no observed in brain slices from 6-OHDA-treated mice. It is concluded that in slices of rat cortex, morphine stimulates postulated presynaptic, 6-OHDA-sensitive, opiate receptors associated with noradrenergic nerve terminals. These opiate receptors alter postsynaptic alpha - and beta - adrenoceptor activity. In the mouse, morphine appears to stimulate presynaptic opiate receptors that modify exclusively beta - adrenoceptor-mediated cyclic AMP production.     

Polarizing currents increase noradrenaline-elicited accumulation of cyclic AMP in rat cerebral cortex.

Cyclic AMP accumulation elicited by noradrenaline was determined in cerebral cortical slices of rats 24 h after an application of weak anodal direct current (anodal polarization) to the surface of the sensorimotor cortex. Noradrenaline-elicited accumulation of cyclic AMP was altered regionally by the anodal polarization in relation to the duration and intensity of the polarizing current. The cyclic AMP accumulation elicited by noradrenaline was highest in the left anterior cortical region including the polarized point under polarization conditions of 0.3 microA for 1.5 h and 3.0 microA for 30 min. Under these two polarization conditions, the cyclic AMP accumulation elicited by noradrenaline was higher than that in the non-polarized control in the same cortical region. Furthermore, the beta-adrenergic antagonist propranolol almost completely reduced the elicited accumulation of cyclic AMP by noradrenaline to the control level. These results suggest that anodal polarization enhances activity of noradrenaline-sensitive cyclic AMP generating systems through beta-adrenergic mechanisms as a function of both duration and intensity in the cerebral cortex and that one polarization event has a long-lasting aftereffect on noradrenaline-sensitive cyclic AMP generating systems in the cerebral cortex.     

Cyclic AMP-generating systems: regional differences in activation by adrenergic receptors in rat brain

Catecholamine, histamine, and adenosine-mediated accumulations of radioactive cyclic AMP were assessed in adenine-labeled slices from eight rat brain regions. 2-Fluoronorepinephrine, a selective beta-adrenergic agonist, elicited an an accumulation of cyclic AMP in cerebral cortex, cerebellum, hippocampus, striatum, superior colliculi, thalamus, hypothalamus, and medulla-pons. In cerebral cortex and most other brain regions, the beta-adrenergic-mediated response appeared to involve primarily beta 1-adrenergic receptors, while in cerebellum, there was a significant involvement of beta 2-adrenergic receptors. 6-Fluoronorepinephrine, a selective alpha-adrenergic agonist, elicited accumulations of cyclic AMP in all regions except cerebellum. Combinations of the two fluoro derivatives afforded in all brain regions an accumulation of cyclic AMP identical with that elicited by norepinephrine. In hypothalamus, the alpha- and beta-adrenergic responses were significantly greater than additive. In cerebral cortex, the alpha-adrenergic receptor-mediated response appeared to involve alpha 1-adrenergic receptors and to be nearly completely dependent on adenosine, while in other brain regions, the dependence of the alpha-adrenergic response on adenosine was less or absent. Combinations of 6-fluoronorepinephrine and histamine had greater than additive effects in cortex and hippocampus. The results indicate that the interactive control of cyclic AMP-generating systems by alpha-adrenergic, beta-adrenergic, adenosine, and histamine receptors differs significantly among rat brain regions.     

Effect of opioid peptides onl-noradrenaline-stimulated cyclic AMP formation in homogenates of rat cerebral cortex and hypothalamus

Morphine and the opioid peptides leucine-enkephalin (leu-enk), methionine-enkephalin (met-enk) and beta-endorphin had no effect on basal cyclic AMP levels in rat cerebral cortex and hypothalamus, but each inhibited noradrenaline (NA)-stimulated cyclic AMP formation in both brain regions. This inhibition was reversed by naloxone. Naloxone did not reverse phentolamine- or propranolol-induced inhibition of NA-stimulated cyclic AMP formation. The increase in cyclic AMP formation induced by NaF or MnCl2 was unaffected by met-enk or morphine. These data suggest that in rat cerebral cortex and hypothalamus opiates bind to opiate receptors and that the opiate-receptor complex interferes with noradrenergic receptor activity.     

REGULATION of the CYCLIC GUANOSINE 3''-5'' MONOPHOSPHATE SYSTEM IN HUMAN BRAIN TUMORS

Several reports have suggested that cylcic guanosine 3'-5' monophosphate (cGMP) and cyclic 3'-5' adenosine monophosphate (cAMP) are involved in the regulation of cellular proliferation. Following our previous reports on the cAMP system in human brain tumors, we decided to investigate the cGMP system in the same pathological tissues by studying the activity of guanylate cyclase and cGMP-phosphodiesterase (cGMP-PDE). We found that the activity of both enzymes is lower in neurinomas and glioblastomas than in meningiomas or in normal cerebral cortex. Furthermore, the subcellular distribution of guanylate cyclase in human cerebral cortex differs from that of neurinomas and glioblastomas. On the basis of such observations we have discussed the possibility that the regulatory mechanism of the enzymes related to the cyclic nucleotide metabolism is altered in brain tumors.     

PACAP-induced formation of cyclic AMP in the chicken brain: regional variations and the effect of melatonin

We have studied the effects of pituitary adenylate cyclase-activating polypeptide (PACAP27 and PACAP38) on cyclic AMP formation in chick brain, and the action of melatonin upon the PACAP-evoked effects. PACAP stimulated cyclic AMP production in the hypothalamus>cerebral cortex>pineal gland>optic lobes. In the hypothalamus and cerebral cortex, the rank-order of both PACAP forms and VIP in evoking the cyclic AMP response was: PACAP38 approximately PACAP27>VIP, suggesting the presence in the tested tissues of PAC1 receptors. Melatonin suppressed (IC50=19.8 nM) the PACAP27 (0.1 microM)-induced cyclic AMP response in the hypothalamus, but not in the cerebral cortex. Melatonin also suppressed the hypothalamal cyclic AMP synthesis stimulated by forskolin, but not that evoked by histamine or isoprenaline. Our observations show that PACAP is capable of potently stimulating cyclic AMP formation in some regions of the chick brain, and suggest that the hypothalamus may be a site for a functional interaction between PACAP and the pineal hormone melatonin.     

Influence of lesions of the noradrenergic locus coeruleus system on the cerebral metabolic response to bicuculline-induced seizures

The objective of the present study was to explore if lesions of the ascending noradrenergic pathways, originating in the locus coeruleus, modulate the cerebral metabolic response to bicuculline-induced seizures in rats. Bilateral noradrenergic lesions were performed by 6-hydroxydopamine injections in the caudal mesencephalon, 12–22 days before seizures were induced in animals ventilated on N₂O:O₂ (75:25). After 5 min of seizures the brain was frozen in situ and cerebral cortex and hippocampus were sampled for analysis. Labile phosphates, glycolytic metabolites, cyclic nucleotides, and free fatty acids were measured. In another series, lesioned animals were used for measurements of cerebral oxygen consumption.The noradrenergic lesions neither modified the electroencephalographically recorded seizure discharge, nor did they alter cerebral oxygen consumption or cerebral energy state. However, when compared to sham-operated animals, those with noradrenergic lesions had significantly higher (115% and 68%) glycogen concentrations and lower (50% and 52%) cyclic AMP concentrations in cerebral cortex and hippocampus, respectively, demonstrating the marked influence of noradrenergic activity on adenylate cyclase activity and glycogenolysis. The lesions failed to modulate the rise in free fatty acids in the cerebral cortex, or the cyclic GMP concentrations in the cerebral cortex and hippocampus. Thus, increased noradrenergic activity during status epilepticus does not seem responsible for lipolysis or for activation of guanylate cyclase.     

Regional changes of brain Na, K-transporting adenosine triphosphatase related to ovarian function

Synaptosomal Na+,K+-transporting ATPase activity of the mediobasal hypothalamus (MBH), the medial preoptic-suprachiasmatic (POSC) region and cerebral cortex was measured in rats at different stages of the estrous cycle and after ovariectomy and estradiol replacement. Enzyme activity of the MBH and POSC showed cyclic changes. In both regions it increased shortly before the proestrus surge of LH. However, the two areas responded to castration and estrogen treatment in an opposite fashion. No changes were detected in enzyme activity in the cerebral cortex. These findings are consistent with previous reports on cyclic changes in electrical activity and suggest that Na+,K+-ATPase activity could be a useful indicator of neural activity for the study of neuroendocrine interactions.     

Effects of topical methylene blue on cyclic GMP level,blood flow,and O2 consumption in focal cerebral ischaemia

Abstract

We hypothesized that a decrease in cyclic CMPa second messenger in the glutamate—nitric oxide pathway, would reduce oxygen consumption and improve O₂ balance in the ischaemic cerebral cortex. To test this hypothesis, a study was performed in unilateral middle cerebral artery occluded rats which were assigned to either a control or methylene blue (10^–3 m) group. Regional cerebral blood flow was determined using 14C-iodoantipyrine and regional arterial and venous O₂ saturations were determined by microspectrophotometry (n = 6). Cyclic GMP level was measured by radioimmunoassay (n = 8). Cuanylate cyclase and cyclic CMP-phosphodiesterase activities were determined in an additional set of control rats (n = 10). The cyclic CMP levels were not different between the ischaemic and contralateral areas in the control group. Compared to the cyclic CMP in the control ischaemic cortextopical methylene blue significantly decreased the cyclic CMP level by 56% in the ischaemic cortex of the methylene blue group. Ischaemia did not alter the activities of guanylate cyclase but mildly decreased cyclic GMP-phosphodiesterase. The regional cerebral blood flow and O₂ consumption in the control group were 50% and 32% lower than those in corresponding contralateral cortex. Topical methylene blue did not alter regional cerebral blood flow and O₂ consumption in the ischaemic cortex. Our data showed that cyclic CMP is not a major controller on O₂ supply or O₂ consumption in the ischaemic brain. [Neurol Res 1994; 16: 449-455]     

A biochemical and morphological study of the altered growth pattern of central catecholamine neurons following 6-hydroxydopamine

The ability of a series of adrenergic agents to displace the binding to brain membranes of [3H]WB 4101, a potent alpha-adrenergic antagonist (WB 4101 = 2-[2-(2,6-dimethoxyphenoxy)ethylaminomethyl]-1,4-benzodioxane hydrochloride), has been compared with the potency of these agents in stimulating or inhibiting the alpha-adrenergic component of cyclic AMP accumulation in rat cerebral cortex slices. [3H]WB 4101 rapidly bound to a high affinity site (KD = 2.7 nM) in membranes from cerebral cortex. Binding came to equilibrium by 2 min at 37 degrees C and was rapidly reversed in the presence of phentolamine. The potencies of adrenergic agents (WB 4101 greater than phentolamine greater than naphazoline) in displacing binding of [3H]WB 4101 were comparable to the potencies of these agents as inhibitors of the alpha-adrenergic component of norepinephrine-stimulated cyclic AMP accumulations. Phenoxybenzamine, clonidine, chlorpromazine and haloperidol were about 10--30 times more potent in inhibiting cyclic AMP accumulation than in displacing [3H]WB 4101 binding. The potency of classical alpha-adrenergic agonists in displacing WB 4101 (epinephrine greater than norepinephrine greater than methoxamine) correlated with the ability of these agonists to increase cyclic AMP levels. Overall a significant correlation (r = 0.87, P less than 0.005) was found between WB 4101 binding and alpha-adrenergically mediated cyclic AMP accumulation in brain. Several ligands bind to specific sites in brain membranes with alpha-adrenergic receptor properties. The identification of these binding sites as receptors depends on a correlation of binding with a known alpha-adrenergic receptor-mediated response in brain. These data demonstrating that WB 4101 correlates with norepinephrine-stimulated cyclic AMP accumulation suggest that WB 4101 may bind to the membrane receptor sites mediating the alpha-adrenergic accumulation of cyclic AMP in rat cerebral cortex.     

Effect of nitroprusside on regional cerebral cyclic GMP, blood flow and O2 consumption in rat

This investigation was conducted to test whether topical nitroprusside (NP), a cytosolic guanylate cyclase activator, would increase the level of cyclic GMP and alter O₂ consumption or blood flow in the cerebral cortex of rats. Male Long-Evans rats were used in a control (n = 9), low dose NP (n = 13, 10⁻³ M) or high dose NP (n = 12, 10⁻² M) group. Nitroprusside or saline was topically applied to the right side of the cerebral cortex and the left side was used as a control. The cyclic GMP level was determined in five rats in each group using a radioimmunoassay. In the o ther rats in each group, regional cerebral blood flow was measured by [¹⁴C]iodoantipyrine and regional arterial and venous O₂ saturations were determined microspectrophotometrically. Nitroprusside significantly increased the cyclic GMP level from 21.4 ± 12.0 pmol/g (contralateral cortex) to 52.2 ± 36.7 pmol/g (NP treated cortex) in low dose nitroprusside group and from 19.9 ± 22.6 pmol/g (contralateral cortex) to 58.5 ± 15.1 pmol/g (NP treated cortex) in high dose nitroprusside group. High dose nitroprusside significantly increased cerebral blood flow from 80 ± 11 ml · min⁻¹ · 100 g (contralateral cortex) to 114 ± 11 ml · min⁻¹ · 100 g (NP treated cortex). However, there was no significant difference in O₂ extraction and O₂ consumption between the NP treated cortex and contralateral cortex in either the low or the high dose NP groups. In the high dose NP group, the O₂ extraction was 8.0 ± 1.3 ml O₂ · 100 ml⁻¹ in the treated cortex and 8.8 ± 1.5 ml O₂ · 100 ml⁻¹ in the contralateral cortex, while the O₂ consumptions in the NP treated cortex and contralateral cortex were 8.1 ± 1.3 ml O₂ · min⁻¹ · 100 g⁻¹ and 7.3 ± 1.2, respectively. Thus, NP increased the cyclic GMP level without a significant change in O₂ consumption in the cerebral cortex. Our data suggested that O₂ consumption in the cerebral cortex was not affected by the increased level of cyclic GMP.     

Somatostatin binding to a fresh rat astrocyte-enriched suspension.

Since there are conflicting reports regarding the effects of somatostatin (SS) on cyclic AMP levels in astrocytes derived from rat cerebral cortex and, to date, the SS binding to mature astrocytes is unknown, the present study has determined SS binding and its effect on cyclic AMP accumulation in a fresh astrocyte-rich suspension from rat cerebral cortex. 125I-Tyr11-SS binding was inhibited by SS in a dose-dependent manner. The Scatchard analysis of binding data was linear and yielded a dissociation constant of 0.95 +/- 0.15 nM with a maximal binding capacity of 122 +/- 13 fmol/mg protein. Vasoactive intestinal peptide (VIP) stimulated cyclic AMP accumulation up to 2.3 times above the basal levels whereas SS had no effect. This effect at any of the VIP concentrations. Likewise, SS did not inhibit the stimulation of cyclic AMP accumulation provoked by other effectors such as isoproterenol and forskolin. In view of our results and those of other authors, SS receptor localized in astrocytes must be able to couple with signal transduction systems other than adenylate cyclase, in order to carry out its biological actions in the cell.     

Norepinephrine regulation of cerebral glycogen utilization during seizures and ischemia

Norepinephrine (NE) depletion of the cerebral cortex after lesion of the ipsilateral locus ceruleus (LC) causes abnormalities of cerebral oxidative metabolism when the cortex is stimulated to increased energy demand (Harik, S. I., J. C. LaManna, A. I. Light, and M. Rosenthal (1979) Science 206: 69-71; LaManna, J. C., S. I. Harik, A. I. Light, and M. Rosenthal (1981) Brain Res. 204: 87-101). These abnormalities were exhibited as decreased mitochondrial reducing equivalent flow. One possible cause of this would be the decreased availability of oxidative metabolic substrates in the NE-depleted cortex. We therefore investigated the effect of unilateral LC lesion and the resultant depletion of ipsilateral endogenous NE on glycogen and other energy metabolites in the cerebral cortex of rats under three conditions: (1) at "rest," (2) when energy demand is inncreased markedly by seizures, and (3) during total cerebral ischemia. We report no differences in cerebral metabolites between NE-depleted and control hemispheres at "rest." In seizures and ischemia, however, the increase in the level of adenosine 3':5'-monophosphate (cyclic AMP) and the breakdown of glycogen were impaired considerably in the NE-depleted cortex. The data suggest that depletion of central NE impairs cerebral glycogenolysis in response to increased energy demands and ischemia. Such impairment may be mediated via a cyclic AMP-related mechanism.     

Astrocyte cell lineage. V. Similarity of astrocytes that form in the presence of dBcAMP in cultures to reactive astrocytes in vivo

The relationship between astrocytes forming in the presence of dibutyryl cyclic AMP (dBcAMP) in culture and reactive astrocytes responding to a cerebral cortex stab wound was investigated using computerized image analysis (Zeiss IBAS 1) and immunocytochemical staining. The diameters of the nuclei of astrocytes in primary cultures of newborn mouse neopallial cells were compared to those of the nuclei of normal and reactive astrocytes in histological sections of mouse cerebral cortex. We found that the nuclei of astrocytes that formed in the presence of dBcAMP in cultures are significantly larger than those of spontaneously occurring small stellate astrocytes in culture and of normal astrocytes of the cerebral cortex in vivo but corresponded more closely to the nuclei of reactive astrocytes in the area surrounding a stab wound in the cerebral cortex. Large stellate cells formed in the presence of dBcAMP had vimentin and an increase in GFP-containing intermediate filaments. Formation of reactive astrocytes in vivo is also associated with an increase in both vimentin and GFP-containing intermediate filaments. These observations indicate a closer relationship of astrocytes formed in the presence of dBcAMP in cultures to the reactive astrocytes in the cerebral cortex than to normal astrocytes. We propose, therefore, that the large stellate astrocytes that form in the presence of dBcAMP be referred to as reactive astrocytes in culture.     

Somatostatin inhibition of VIP- and isoproterenol-stimulated cyclic AMP accumulation in dissociated cells from rat cerebral cortex

Freshly dissociated cerebral cortex cells from adult rats have been used in the present study to determine if dual regulation of cyclic AMP levels by inhibitory and stimulatory agents can be expressed in the mature brain. Somatostatin, an inhibitory agent, barely affected the basal cyclic AMP metabolism while vasoactive intestinal peptide (VIP) and isoproterenol, two stimulatory agents enhanced cyclic AMP production. However, this increase was depressed by somatostatin, which decreased the efficiency, but not the potency, of the effects of the two stimulatory agents on cyclic AMP accumulation.     

The postnatal development of norepinephrine-stimulated cyclic AMP accumulation in rat spinal cord

Norepinephrine (NE) stimulated cyclic adenosine 3',5'-monophosphate (cyclic AMP) accumulation in regional spinal cord and cortical tissue slices from postnatal rats demonstrated distinct developmental patterns. NE-concentration-response studies using a 10-min incubation period demonstrated minimal NE-stimulated cyclic AMP accumulation in whole spinal cord at PD 1-5 with maximal sensitivity on PD 12. Thereafter, sensitivity decreased to adult values at PD 30. Sensitivity changes were reflected in alterations in maximal response only since NE EC50s all averaged 10(-6) M. This agrees with no change in calculated Kd for NE with increased Vmax to PD 12 and a reduction thereafter. Studies on regional spinal cord and cerebral cortical cyclic AMP accumulation indicated peak NE sensitivity in cervical and thoracic cord occurred at PD 10, in lumbar cord at PD 15, and in cerebral cortex at PD 20. The fact that inhibition of phosphodiesterase (PPDE) produced the same percentage alteration in peak accumulation in spinal cord slices regardless of postnatal age suggests that PPDE is not the primary determinant of the ontogenic changes. The results indicate that the postnatal development of spinal NE receptors may be reflected in an increase in the responsiveness of the cyclic AMP system to NE.     

Inhibitory effects of anticonvulsant drugs on cyclic nucleotide accumulation in brain.

Veratridine causes deplorization of excitable cells and produces marked elevation of adenosine 3',5'-monophosphate (cyclic AMP) and guanosine 3',5'-monophosphate (cyclic GMP) levels in incubated slices of mouse cerebral cortex. Phenytoin, carbamazepine, phenobarbital, primidone, phensuximide, methsuximide, alpha-methyl-alpha-phenylsuccinimide, and high concentrations of clonazepam are anticonvulsant drugs that preferentially prevent maximal electroshock seizures (MES) and generalized tonic-clonic convulsions; all these agents inhibit veratridine-induced accumulation of both cyclic AMP and cyclic GMP. In contrast, ethosuximide, trimethadione, valproic acid, and low concentrations of clonazepam are anticonvulsant drugs that act predominantly against Metrazol and absence seizures; these agents are ineffective or inhibit accumulation of only cyclic GMP. The results suggest that inhibition of cyclic AMP and cyclic GMP accumulation in depolarized brain tissue is a molecular neuropharmacological action characteristic of anticonvulsant drugs that have direct effects on cellular membrane function and prevent MES. Anticonvulsant drugs that do not inhibit accumulation of both cyclic AMP and cyclic GMP in depolarized brain tissue preferentially prevent Metrazol and absence seizures and probably exert their effects by altering neurotransmission mechanisms.     

Effects of noradrenaline depletion on adrenergic and muscarinic cholinergic receptors in the cerebral cortex, hippocampus, and cerebellum

The effects of noradrenaline depletion on α- and β-adrenergic and muscarinic cholinergic receptors in the cerebral cortex, hippocampus, and cerebellum of Wistar rats were studied. Noradrenaline depletion was obtained either by chemical (6-hydroxydopamine) lesions of the locus cerulet's or by chronic reserpine treatment. Two weeks after locus ceruleus lesions and 10 days after chronic reserpine treatment, the rats were killed and the cerebral cortex, hippocampus, and cerebellum dissected. A small portion of each tissue was assayed for noradrenaline, to assess the success of locus ceruleus lesions or reserpine treatment, and the remainder was used to measure the specific binding of tritiated α- and β-adrenergic and muscarinic cholinergic receptor ligands to particulate fractions of these tissues. The effect of noradrenaline depletion on isoproterenol-induced cyclic AMP generation in cerebellar slices was also studied. Noradrenaline depletion whether by locus ceruleus lesion or chronic reserpine treatment induced reproducible and significant increases in the binding to β-adrenergic receptors in the cerebral cortex and hippocampus. Scatchard analyses revealed that this increased binding was due to an increased density of β-adrenergic receptor binding sites. In the cerebellum, however, noradrenaline depletion did not result in an increase in β-adrenergic receptor binding nor in isoproterenol-induced cyclic AMP generation. Noradrenaline depletion did not cause significant changes in the binding characteristics of α-adrenergic or muscarinic cholinergic receptors in any of the three regions of the brain that were studied.     

Effects of reserpine, propranolol, and aminophylline on seizure activity and CNS cyclic nucleotides.

Convulsant doses of pentylenetetrazol (100 mg/kg) increase levels of both cyclic adenosine monophosphate (AMP) and cyclic guanosine monophosphate (GMP in mouse cerebral cortex and hippocampus. In animals pretreated with reserpine, propranolol, or aminophylline, pentylenetetrazol seizures were more severe, cyclic AMP elevations were attenuated or blocked, and cyclic GMP increases were unaffected or augmented. These data indicate that norepinephrine, adenosine, and perhaps other biogenic amines have a regulatory effect on cyclic AMP, but not cyclic GMP, levels in epileptic brain. An increased level of cyclic AMP in brain tissue may have an antiepileptic effect leading to seizure attenuation or termination. By contrast, elevated levels of cyclic GMP may have an epileptogenic effect in initiating or maintaining seizure activity.