α-Noradrenergic potentiation of neurotransmitter-stimulated cAMP production in rat striatal slices

The present study examines the possible involvement of α-adrenergic receptors in catecholamine-stimulated cAMP production in intact slices of rat striatum. Norepinephrine (NE) produces a greater stimulation of cAMP levels than does the ß-adrenergic agonist, isoprotererenol (ISO), and the NE response is inhibited by both the ß-adrenergic antagonist, propranolol, and the α-adrenergic antagonist, phentolamine. The α-adrenergic agonist, 6-fluoronorepinephrine (6-FNE), has little or no effec on basal cAMP levels; however, 6-FNE causes a marked potentiation of the cAMP responses to ISO. Hence, NE stimulation of cAMP levels in striatal slices appears to involve a synergistic interaction between α- and ß-adrenergic receptors.α-Receptors also potentiate adenosine stimulation of cAMP levels in striatal slices. However, in contrast to results previously reported in cerebral cortical slices, the α-adrenergic component of the NE response in striatal slices is not dependent on endogenous adenosine. Finally, 6-FNE interactions with adenylate cyclase in striatal homogenates differ from those observed in the slice preparation. In homogenates, 6-FNE appears to directly stimulate adenylate through a D-1 receptor. D-1 receptor involvement in catecholamine responses in the striatal slice preparation, on the other hand, appears to minimal.     

A procedure for measuring α2-adrenergic receptor-mediated inhibition of cyclic AMP accumulation in rat brain slices

The α₂-adrenergic receptor regulation of cyclic adenosine monophosphate (cAMP) accumulation in rat brain slices was examined using a prelabeling technique for measuring second messenger production. The mixed α-adrenergic agonist 6-fluoronorepinephrine, as well as the more selective α₂-agonists clonidine and UK-14,304, caused a concentration-dependent inhibition of forskolin-stimulated cAMP accumulation in cerebral cortical slices, whereas phenylephrine, a selective α₁-adrenergic agonists,had no inhibitory effect in this system. Moreover, α₂-adrenergic receptor antagonists were more potent than α₁-adrenergic antagonists in blocking the inhibitory response to UK-14,304. Neither α₁-norα₂-adrenergic antagonists displayed any inhibitory effect when cAMP accumulation was stimulated by isoproterenol, vasoactive intestinal peptide or 2-chloroadenosine. The results provide further evidence that some α₂-adrenergic receptors are negatively coupled to adenylate cyclase in brain, and yield a procedure for studying this phenomenon in intact central nervous system tissue.     

Extracellular cAMP accumulation and degradation in rat cerebral cortex in dissociated cell culture

Norepinephrine (NE) stimulated the accumulation of cAMP in embryonic rat cerebral cortex in dissociated cell culture. After exposure to NE for 10 min, the intracellular cAMP content of these cultures went from 22 +/- 12 to 202 +/- 75 pmol/mg protein. Using selective culturing techniques, evidence was obtained supporting the hypothesis that NE-stimulated production of cAMP is a property associated with the glial rather than the neuronal component of these cultures. Beta adrenergic agonist stimulation of cortical cultures also resulted in the efflux of cAMP into the medium. At the peak of extracellular accumulation of cAMP (following a 40-min exposure to isoproterenol), 180 pmol cAMP/mg protein had been transported into the extracellular medium. The fate of extracellular cAMP was investigated using thin-layer chromatography. Extracellular cAMP was degraded to AMP and adenosine; this degradation did not seem to be due to the presence of serum or serum components, suggesting the existence of an extracellular phosphodiesterase. In response to NE stimulation of glia, in particular astrocytes, cAMP or its metabolites may accumulate at high enough concentrations in the extracellular space in cerebral cortex to affect neuronal function, possibly via adenosine receptors.     

α-Adrenergic modulation of cyclic AMP formation in rat CNS: highest level in olfactory bulb

The cyclic adenosine monophosphate response to catecholamines in the rat brain is mediated by β-adrenergic receptors which activate adenylate cyclase and by α-adrenergic receptors which potentiate the response to β-stimulation. We have found that the α-potentiation effect in the olfactory bulb is 2–3× greater than in other forebrain areas. This correlates with the extremely high density of α-receptors in this brain region and makes it a useful model for the study of α-receptor function.     

Adenosine stimulates stellation of cultured rat cortical astrocytes

We investigated the effect of adenosine on astrocyte morphology by using cell cultures prepared from the cerebral cortices of neonatal rats. Cultured rat cortical astrocytes exhibited flattened, polygonal morphology in the absence of stimulation, but differentiated into process-bearing stellate cells in response to adenosine (1–1000 μM). Adenosine-induced astrocyte stellation was abolished by treatment with microtubule inhibitors, colchicine and paclitaxel, indicating the involvement of cytoskeletal elements. The effect of adenosine was mimicked by other adenosine receptor agonists, and blocked by adenosine receptor antagonists and guanosine 5′-O-(2-thiodiphosphate), indicating that the effect of adenosine is mediated by G protein-coupled adenosine receptors. Although adenosine receptors are known to be linked to adenylate cyclase or phospholipase C, adenosine did not change intracellular cyclic AMP level nor intracellular Ca²⁺ concentration in astrocytes. Alternatively, adenosine-induced stellation was abolished by tyrosine phosphatase inhibitors, orthovanadate and phenylarsine oxide, suggesting that adenosine causes astrocyte stellation through tyrosine dephosphorylation. Adenosine may function as a factor regulating astrocyte differentiation.     

Desensitization of β-receptors on primary astrocyte cultures by norepinephrine but not by tricyclic antidepressants

Primary astrocyte cultures from neonatal rat cerebral hemispheres were treated chronically for up to 3 weeks with the tricyclic antidepressants amitryptyline (AMT) or desipramine (DMI), or acutely with AMT and DMI added at the same time as the agonist, norepinephrine (NE). AMT and DMI were added at concentrations from 10⁻⁹ to 10⁻⁵ M. Both types of treatment did not decrease the increase in cyclic AMP (cAMP) content of these cells in response to a 10 min exposure to 10⁻⁵ M NE. Chronic exposure to the antidepressants also did not affect stimulation of cAMP by isoproterenol (iso) in both rat and mouse primary astrocyte cultures. In contrast to the lack of effect of the tricyclic antidepressants pretreatment of the cultures with 10⁻⁵ M NE resulted in total inhibition of the cAMP response after 2 h, with a 50% decrease occurring in about 45 min. This is similar to the agonist-induced desensitization of the β-receptor-adenylate cyclase system seen in many other cells. This effect could, in part, be a direct response to increased intracellular cAMP since pretreatment with 0.25 and 1.0 mM N⁶-2′-O-dibutyryl cAMP (DBcAMP) also resulted in total inhibition of the cAMP response after 4 h. Receptor labelling experiments using [¹²⁵I]cyanopindolol showed no decreases in apparent binding sites up to 3 h after exposure to 10⁻⁵ M norepinephrine, suggesting that the rapid desensitization of the cAMP response was primarily due to an uncoupling of the receptor from the adenyl cyclase. These results suggest that if the desensitization of the β-receptor mediated cAMP increase observed after chronic treatment with antidepressants in vivo is partly due to an effect on β-receptors in astrocytes it is more likely a secondary response, perhaps due to a delayed increase in extracellular NE levels.     

Effect of long-term clorgyline administration on human platelet alpha-adrenergic receptor binding and platelet cyclic AMP responses

Platelet α₂-adrenergic receptor number and physiologic responsiveness, as well as plasma norepinephrine (NE), were evaluated in psychiatric patients with major depressive disorder before and during chronic clorgyline treatment. The α₂-adrenergic receptor number was determined by measuring the binding of tritiated dihydroergocriptine (³H-DHE) to platelet membranes. Physiologic responsiveness was determined by measuring the response of cyclic adenosine 3'-5' monophosphate (cAMP) to prostaglandin E₁ (PGE₁), and the inhibition of the PGE₁-stimulated cAMP response by NE in intact platelets. No significant differences from pretreatment values were observed in platelet α₂-adrenergic binding or responsiveness during clorgyline treatment. Baseline platelet cAMP production and plasma NE levels were significantly decreased after chronic clorgyline treatment. Previous studies on animals and humans have suggested that brain α₂-adrenergic receptor responsiveness decreases during chronic clorgyline treatment. The present findings therefore suggest that such changes may represent adaptations induced by long-term clorgyline administration which may differ between the brain and the platelet, thus illustrating potential limitations of the study of platelet α₂-adrenergic receptors as a model for central α₂-adrenergic receptor adaptation.     

C-terminal tail of β-adrenergic receptors: immunocytochemical localization within astrocytes and their relation to catecholaminergic neurons in N. tractus solitarii and area postrema

β-Adrenergic receptors (βAR) in the medial nuclei of tractus solitarii (m-NTS) and area postrema (AP) may bind to catecholamines released from neurons, whereas only the AP has fenestrated capillaries allowing access to circulating catecholamines. Since varied autonomic responses are seen following βAR activation of the dorsal vagal complex, including the m-NTS and AP, we hypothesized that there might be a cellular basis for varied responses to βAR stimulation that depends pn the differential access to circulating catecholamines. Therefore, we comparatively examined the ultrastructural localization of the βAR in relation to catecholaminergic neurons in these regions. An antibody directed against the C-terminal tail (amino acids 404–418) of hamster β-adrenergic receptor (βAR404) was used in this study. The localization of βAR404 was achieved by the avidin-biotin peroxidase complex (ABC) technique in combination with a pre-embed immunogold labeling method to localize tyrosine hydroxylase (TH), the catecholamine-synthesizing enzyme. Within m-NTS and at subpostremal border, labeling for βAR404 was evident along the intracellular surface of plasma membranes of small, apparently distal, astrocytic processes. Astrocytic processes with βAR404-immunoreactivity formed multiple, thin lamellae around TH-labeled and non-TH neuronal cell bodies and dendrites. βAR404-immunoreactive astrocytes also extended end-feet around blood vessels and surrounded groups of axon terminals that were directly juxtaposed to each other. Some, but not all, of these axons demonstrated TH-immunoreactivity. Fewer βAR404-immunoreactive astrocytes were detected in AP, regardless of their proximity to catecholaminergic processes or blood vessels. The present astrocytic localization of βAR404, together with the earlier, neuronal localization of βAR's third intracellular loop, suggest that the βAR may be substantially different between neurons and astrocytes. The regional difference in the prevalence of βAR404-immunoreactive astrocytes suggests that these receptive sites may either: (i) be preferentially activated by catecholamines released from terminals rather than circulating catecholamines; or (ii) be down-regulated in AP due to blood-born substances, such as catecholamines. The extensive localization of βAR in the border between m-NTS and AP also suggests that catecholaminergic activation of these astrocytes may dictate the degree of diffusion of catecholamines which are of neuronal or vascular origin. The specific localization of βAR404-immunoreactivity to the more distal portions of astrocytes suggests the possibility that astrocytes have restrictive distributions of βAR and that the β-adrenergic activation lead to morphological or chemical changes that are also localized to the distal portions of astrocytes. Additionally, the detection of βAR404 in astrocytes contacting non-TH-immunoreactive neurons suggests the possibility for catecholaminergic modulation of non-catecholaminergic neurons via the activation of astrocytes.     

α2-Adrenergic receptors mediate inhibition of cyclic AMP production in neurons in primary culture

The actions of adrenergic agents on the intracellular production of cyclic adenosine monophosphate (AMP) was examined in intact cortical and striatal neurons in primary culture, generated from the fetal mouse brain. Exposure of striatal neurons to the β-adrenergic agonist isoproterenol (10 μM) resulted in a 5-fold increase in intraneuronal cyclic AMP; norepinephrine (100 μM), alone or in combination with isoproterenol, produced only a 3-fold increase in cyclic AMP levels. However, in the presence of yohimbine (10 μM), cyclic AMP productions due to norepinephrine or isoproterenol plus norepinephrine were identical to isoproterenol alone. When striatal or cortical neurons were exposed to pertussis toxin (100 ng/ml) overnight, there was no detectable difference between isoproterenol- and norepinephrine-stimulated cyclic AMP production. These data suggest thatα₂-adrenergic receptors mediate the attenuation of cyclic AMP production in neurons and do so via the inhibitory guanine nucleotide regulatory protein of adenylate cyclase.     

Adenosine-dependent regulation of cyclic AMP accumulation in primary cultures of rat astrocytes and neurons.

The regulation of intracellular cyclic AMP (cAMP) formation by adenosine (Ado) and its analogues has been examined in primary cultures of rat-brain astrocytes and neurons. In the presence of the phosphodiesterase inhibitor, Ro 20-1724, basal levels of cAMP ranged from 40-120 pmol/mg protein in both cell types. Levels were not altered by treating the cells with Ado deaminase, which suggested that they did not produce appreciable amounts of endogenous Ado under standard culture conditions. In the astrocytes, microM quantities of agonists increased cAMP up to 30-fold higher than basal values; the relative potencies were typical of an A2 Ado receptor (NECA greater than Ado greater than R-PIA). Neuron-enriched cultures exhibited a maximum fourfold increase in cAMP in response to NECA; this was decreased a further eightfold when the cultures had prolonged exposure to the antimitotic agent, c-Ara, to eliminate greater than 98% of the nonneuronal cells. Low (nM) amounts of the Ado agonists inhibited cAMP formation in both cell types. In the astrocytes, the order of potency of inhibition of isoproterenol-stimulated cAMP formation was typical of an A1 receptor (R-PIA greater than Ado greater than NECA); maximum inhibition was 55-65%. Isoproterenol did not increase cAMP in the neuronal cultures. However, forskolin-stimulated formation was effectively (approximately 50%) inhibited by A1 Ado agonists; inhibition was not affected by prolonged treatment with c-Ara. From this study we tentatively concluded that rat astrocytes and neurons both contain inhibitory A1 Ado receptors, but that the stimulatory "A2" subtype is localized mainly on astrocytes.     

Noradrenergic potentiation of cerebellar Purkinje cell responses to GABA: evidence for mediation through the β-adrenoceptor-coupled cyclic AMP system

Previous in vivo studies from our laboratory have consistently shown that iontophoretically applied norepinephrine (NE) can potentiate γ-aminobutyric acid (GABA)-induced depressant responses of cerebrocortical, cerebellar and hypothalamic neurons. Additional experiments have further suggested that this noradrenergic facilitating action is specific for GABA and results from the activation of a β-type adrenoceptor. The goal of the present studies was to determine if the cAMP second messenger system might also be a component of the mechanism responsible for this NE modulatory action on GABA-mediated inhibition. In one set of in vitro experiments, we examined cerebellar neuronal responses to GABA before, during and after iontophoretic application of NE, 8-bromo3′,5′-cylic AMP (BcAMP) or 3-isobutyl-1-methyl xanthine (IBMX) or bath application of forskolin (10–30 μM). In a second group of in vivo studies, extracellularly recorded responses of individual cerebellar Purkinje (P) cells to iontophoretic pulses of GABA or β-alanine were examined before, during and after NE or BcAMP microiontophoresis. In 20 of 25 cerebellar cells recorded from tissue slices, iontophoretically applied NE markedly enhanced responses to GABA in a manner similar to that observed previously in vivo. In these in vitro preprarations, bath application of forskolin was also capable of potentiating GABA-induced inhibition in each of 4 cases tested whereas dideoxy-forskolin was not. Iontophoretic application of IBMX further enhanced the facilitating effects of NE on GABA-induced inhibition in 10 of 11 cases tested. Furthermore, under in vitro conditions, BcAMP augmented inhibitory responses to GABA in all cerebellar neurons tested. In the intact rat brain, iontophoretic administration of BcAMP caused a marked NE-like augmentation of P-cell responses to GABA in 73% of the cells tested. As with NE, BcAMP was ineffective in enhancing P-cell inhibitory responses to β-alanine, an agent which like GABA causes hyperpolarization, by increasing Cl⁻ conductance. In summary, these results indicate that a membrane permeant analog of cAMP, a phosphodiesterase inhibitor and an agent which directly activates adenyl cyclase can mimic the previously observed GABA-potentiating actions of NE. Thus, these findings provide further support for the contention that noradrenergic enhancement of GABA inhibition results from a cascade of transmembrane events which includes β-receptor activation, adenyl cyclase stimulation and increased intracellular production of cAMP.     

Stimulation of cyclic AMP accumulation by pentylenetetrazol.

The convulsant agent, pentylenetetrazol (PTZ), increased the content of cyclic AMP (cAMP) in rat cortical slices incubated in vitro in physiologic media. This increase was partially reversed by theophylline. The addition of PTZ to maximally effective concentrations of either adenosine or 2-chloroadeosine resulted in a significantly greater than additive augmentation of cAMP accumulation, suggesting that PTZ may produce its effect by enhancing the action of endogenous adenosine. The PTZ response was not antagonized by either diphenylhydantoin, phenobarbital or ethosuximide.     

Schwann cell responses to cyclic AMP: Proliferation, change in shape, and appearance of surface galactocerebroside

Surface galactocerebroside (galC) was induced on cultured Schwann cells by two analogues of cyclic adenosine 3',5'-monophosphate (cAMP), dibutyryl cAMP and 8-bromo cAMP (as previously reported by Sobue and Pleasure) and also by forskolin, a potent adenylate cyclase activator. These reagents also induced a morphological transition of many of the Schwann cells, from an elongated spindle shape to flattened cells extending fenestrated cytoplasmic sheets. Surface galC and these changes in Schwann cell shape were not elicited by raising the extracellular cAMP concentration, nor by many compounds known to promote the differentiation of other cell types, suggesting that intracellular cAMP is the unique signal for their induction. The cAMP analogues also induced Schwann cell proliferation (as previously reported by Raff et al.), as did forskolin. The concentrations of cAMP analogues and forskolin eliciting largest increases in numbers of Schwann cells in the cultures were 10-fold lower than the concentrations required for optimal induction of Schwann cell surface galC.     

Characterization of the glycogenolysis elicited by vasoactive intestinal peptide, noradrenaline and adenosine in primary cultures of mouse cerebral cortical astrocytes.

In recent years evidence has accumulated indicating the presence of functional receptors for most neurotransmitters on astrocytes. In particular, receptors coupled to adenylate cyclase have been demonstrated, in primary astrocyte cultures, for vasoactive intestinal peptide (VIP), noradrenaline (NA) and adenosine. Here we provide, in primary cultures of cerebral cortical astrocytes prepared from neonatal mice, a detailed characterization of a cAMP-dependent process elicited by VIP, NA and adenosine, i.e. the hydrolysis of glycogen. The EC50s for the glycogenolytic effect of VIP, NA and adenosine are 3, 20 and 800 nM, respectively. The initial rate of glycogen hydrolysis is, in nmol/mg prot/min, 9.1 for VIP and 7.5 for NA. The effect of NA is predominantly mediated by beta-adrenoceptors, although an alpha 1-adrenergic component, acting most likely through protein kinase C activation, is also present. The action of VIP is mimicked by peptides sharing sequence homologies such as PHI and secretin. Glutamate, GABA, carbachol and the peptides NPY and somatostatin do not influence glycogen levels. The glycogen content of the cultures can be markedly increased by anabolic factors present in fetal calf serum, by high (e.g. 25 mM) glucose in the medium and by 48-h pretreatment of the cultures with dibutyryl cAMP. These results indicate that the glycogen content of astrocytes is under the dynamic control of various factors, including certain neurotransmitters. They also further stress the notion of a functional interaction between neurons and glial cells aimed at maintaining local energy metabolism homeostasis.     

Modulation of corticotropin-releasing hormone stimulated cyclic adenosine monophosphate production by brain cells

Corticotropin-releasing hormone (CRH) is believed to have a role as an important brain neuroregulator acting through specific receptors coupled to adenylate cyclase in addition to its major role in regulating pituitary adrenocorticotropin synthesis and secretion. To study the potential modulatory effects of various regulators and the central effects of CRH, we studied the effects of phorbol ester myristate acetate (PMA), arginine vasopressin (AVP), corticosterone, dexamethasone, and progesterone on CRH stimulation of cyclic adenosine monophosphate (cAMP) production in extrahypothalamic forebrain cell cultures derived from day 17 gestation fetal rats. These cultures contain CRH receptors with similar characteristics as those in anterior pituitary and brain. CRH (10⁻⁹ − 10⁻⁷ M) stimulated cAMP in a dose-dependent fashion and maximal stimulation was clearly seen at 10⁻⁷ M CRH. Incubation of the cells with PMA (10⁻⁷ M), a protein kinase C (PKC) agonist, had no effect on basal cAMP, but potentiated CRH-stimulated cAMP. AVP (10⁻⁸, 10⁻⁷ M) had no effect on basal nor CRH-stimulated cAMP accumulation. Corticosterone (10⁻⁷, 10⁻⁶ M) or dexamethasone (10⁻⁹−10⁻⁷ M) pre-incubation for 18 h did not diminish basal cAMP levels nor inhibit CRH-induced stimulation of cAMP. However, corticosterone inhibited CRH-induced cAMP production in anterior pituitary cells. Neither did exposure to progesterone (2×10⁻⁸ M) modulate basal cAMP, CRH-induced cAMP production nor the potentiation of CRH stimulation by PMA. The data demonstrate that CRH receptors in dissociated fetal extrahypothalamic forebrain cell cultures are coupled to an adenylyl cyclase/cAMP second messenger system similarly as shown in studies with anterior pituitary membranes. The interaction between CRH and other regulators is similar to that in anterior pituitary with regard to synergism by a PKC activator such as PMA, but differs in the lack of glucocorticoid inhibition or AVP potentiation of CRH-stimulated cAMP production. The ability of CRH to stimulate cAMP in fetal brain cells additionally suggests that coupling of the receptor to adenylyl cyclase occurs at an early stage of development.     

Cyclic AMP accumulation alters calmodulin localization in SK-N-SH human neuroblastoma cells.

In SK-N-SH human neuroblastoma cells, the muscarinic agonist carbachol promotes polyphosphoinositide (PPI) hydrolysis via M3 receptors and increases cyclic AMP levels through an unidentified mechanism. Activation of PPI hydrolysis by carbachol elicits a robust translocation of CaM from membranes into cytosol which was previously shown to be mimicked by the addition of the calcium ionophore ionomycin and the phorbol ester TPA28. The effect of agonist-stimulated second messenger production on CaM localization was determined by activating receptors that increase and decrease adenylyl cyclase activity on SK-N-SH cells. VIP (10 microM), prostaglandin E1 (30 microM) and forskolin (10 microM) all increased adenylyl cyclase activity 8- to 10-fold above the activity with 1 microM GTP. Carbachol (100 microM) did not stimulate adenylyl cyclase activity. The alpha 2-adrenergic agonist UK 14,304 (0.1 microM) and the delta and mu opioid DPDPE (10 microM) and DAMGO (10 microM) inhibited forskolin-stimulated cyclic AMP formation by 27-32%. CaM did not stimulate adenylyl cyclase activity. Incubation of cells with vasoactive intestinal polypeptide (VIP), dibutyryl cyclic AMP and forskolin, resulted in 30% decrease in membrane CaM and an increase in cytosolic CaM of 40-50%. The CaM translocation with the combination of an agent that elevates cyclic AMP levels and a low dose of carbachol was not different from that observed with either agent alone. UK 14,304, DPDPE and DAMGO potentiated carbachol-stimulated increases in cytosolic CaM. Upon the addition of carbachol, a 5-fold increase in intracellular calcium concentration measured with fura-2 fluorescence was observed. VIP and UK 14,304 elevated intracellular calcium concentrations 2 to 3 fold, while forskolin (10 microM) had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

17β-Estradiol depolarization of hypothalamic neurons is mediated by cyclic AMP

The process by which 17β-estradiol rapidly modulates the excitability of neurons in the ventromedial hypothalamus, a facilitation center of female sexual behavior and satiety center of feeding behavior, through mediation by cyclic nucleotides, was investigated by intracellular recording from the guinea pig brain slice preparations. Two types of short-term responses were produced by depolarization with decreased K⁺ conductance and hyperpolarization with increased K⁺ conductance. These two responses were enhanced by the phosphodiesterase inhibitor, isobutylmethylxanthine. However, the specific adenylate cyclase activator, forskolin, enhanced only the depolarization. The analogue of cyclic adenosine 3′,5′-monophosphate (cAMP), 8-bromo-cAMP, induced only depolarization, the ionic mechanism of which was similar to that of 17β-estradiol. In addition, the possibility of non-specific effects of cyclic nucleotides was precluded by an experiment using an analogue of cyclic guanosine 3′,5′-monophosphate (cGMP), 8-bromo-cGMP, which hyperpolarized neurons. Thus, the present study strongly suggests that the production of depolarizing responses of neurons in the hypothalamus produced by estradiol is specifically mediated through cAMP.     

Inhibition of long‐term potentiation by valproic acid through modulation of cyclic AMP

Purpose: Valproic acid (VPA) is widely used clinically in epilepsy, bipolar disorder, and migraine. In experimental models, it has also been shown to have neuroprotective and antiepileptogenic effects. Its mechanisms of action in these diverse conditions are, however, unclear, but there is some evidence indicating an effect of VPA upon protein kinase A (PKA) activity. We, therefore, asked whether VPA modulates cyclic adenosine monophosphate (cAMP)/PKA–dependent synaptic plasticity and whether this mode of action could explain its anticonvulsant effect. Methods: We first tested the effects of VPA on PKA‐dependent synaptic plasticity at mossy fiber to CA3 synapses in rat hippocampus slices following very high‐frequency stimulation or application of the adenylyl cyclase activator forskolin. Using biochemical assays, we then tested whether VPA had a direct effect on PKA activity or an indirect effect through modulating cAMP production. Lastly, VPA and inhibitors of adenylyl cyclase (SQ22536) and PKA (H89) were tested in in vitro models of epileptiform activity induced in hippocampal–entorhinal cortex slices using either pentylenetetrazol (2 mM ) or low magnesium. Results: VPA (1 mm ) inhibited PKA‐dependent long‐term potentiation of mossy fiber to CA3 pyramidal cell transmission. However, VPA did not directly modulate PKA activity but rather inhibited the accumulation of cAMP. In acute in vitro seizure models, the anticonvulsant activity of VPA is not mediated through modulation of adenylyl cyclase or PKA. Conclusions: These results indicate that VPA through an action on cAMP accumulation can inhibit synaptic plasticity, but this cannot fully explain its anticonvulsant effect.     

Effects of ethanol on basal and adenosine-induced increases in β-endorphin release and intracellular cAMP levels in hypothalamic cells

Recently we have shown that the cAMP system is involved in ethanol-regulated β-endorphin (β-EP) release from rat hypothalamic neurons in primary cultures. The cascade of events that leads to activation of cAMP following ethanol treatment in hypothalamic β-EP neurons is not apparent. In this study the role of adenosine, a cAMP regulator, in ethanol-regulated β-EP release was determined by measuring the cellular incorporation of [³H]adenosine, intracellular cAMP levels and media immunoreactive (IR) β-EP levels in cultures of rat hypothalamic cells following ethanol treatments in the presence and absence of an adenosine agonist and antagonist. Acute exposure to a 50 mM dose of ethanol for a period of 1 h increased media levels of IR-β-EP and cellular contents of cAMP, but the ethanol treatment decreased [³H]adenosine uptake. Constant exposure to a 50 mM dose of ethanol for a period of 48 h, failed to alter media levels of IR-β-EP, cell content of cAMP and [³H]adenosine uptake. The media level of IR-β-EP was elevated following treatment with adenosine receptor agonist phenyl-isopropyl adenosine (PIA) and was reduced following treatment with adenosine receptor antagonist isobutylmethylxanthine (IBMX) or with adenosine uptake inhibitor adenosine deaminase. The level of cellular cAMP was also increased by PIA but was decreased by IBMX and adenosine deaminase. The stimulatory actions of the adenosine agonist PIA on IR-β-EP release and on cAMP production were potentiated by simultaneous incubation with ethanol for 1 h. However, chronic ethanol exposure reduced PIA-induced IR-β-EP release and cAMP production. Additionally, both IBMX and adenosine deaminase reduced ethanol-induced IR-β-EP release and cAMP levels. These results suggest that ethanol inhibits adenosine uptake in IR-β-EP neurons in the hypothalamus, thereby increasing extracellular levels of adenosine and leading to activation of membrane adenosine receptors, cAMP production and IR-β-EP secretion from these neurons. Chronic ethanol desensitizes the adenosine-regulated cAMP production and IR-β-EP release from hypothalamic neurons.