Functional Coupling of the γ-Aminobutyric AcidB Receptor with Calcium Ion Channel and GTP-Binding Protein and Its Alteration Following Solubilization of the γ-Aminobutyric AcidB Receptor

The coupling mechanism of the gamma-aminobutyric acid (GABA)B receptor, one of the subtypes of GABA receptors, with calcium ion channel and GTP-binding protein was examined using a crude synaptic membrane (P2) fraction from the bovine cerebral cortex and a fraction solubilized with sodium deoxycholate. In the P2 fraction, [3H]GABA binding to the GABAB receptor was increased significantly by the addition of calcium ion, and this enhancement was accentuated further by calcium ion channel blockers such as nicardipine and diltiazem. In contrast, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist, did not affect on the calcium ion-induced enhancement of GABAB receptor binding. These results suggest that the GABAB receptor may be functionally coupled with the calcium ion channel, which exhibits an inhibitory modulation against the receptor. On the other hand, GABAB receptor binding, which was noncompetitively inhibited by guanine nucleotides such as GTP, guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), guanosine 5'-(beta, gamma-imido)triphosphate [Gpp(NH)p], and GDP, was competitively inhibited by (-)-baclofen. Although the affinity of (-)-baclofen for the GABAB receptor was decreased in the presence of GTP, pretreatment of the P2 fraction with islet-activating protein (IAP) eliminated the effect of GTP. In addition, GABA and (-)-baclofen induced an increase of GTPase activity in the P2 fraction, and this increase was also eliminated by treatment with IAP. These results suggest that the GABAB receptor may also be functionally coupled with IAP-sensitive GTP-binding protein. Treatment of the P2 fraction with sodium deoxycholate resulted in the highest solubilization of GABAB receptor among various detergents examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

An Examination of the Involvement of Phospholipases A2 and C in the α-Adrenergic and γ-Aminobutyric Acid Receptor Modulation of Cyclic AMP Accumulation in Rat Brain Slices

Experiments were undertaken to define the role of two calcium-associated enzyme systems in modulating transmitter-stimulated production of cyclic nucleotides in rat brain. Cyclic AMP (cAMP) accumulation was examined in cerebral cortical slices using a prelabeling technique. The enhancement of isoproterenol-stimulated cAMP production by alpha-adrenergic and gamma-aminobutyric acid-B (GABAB) agonists was reduced by exposing the tissue to EGTA, a chelator of divalent cations, or quinacrine, a nonselective inhibitor of phospholipase A2. Likewise, chronic (2 weeks) administration of corticosterone decreased the alpha-adrenergic and GABAB receptor modulation of second messenger production. Neither cyclooxygenase nor lipoxygenase inhibitors selectively influenced the facilitating response of alpha-adrenergic and GABAB agonists. Other experiments revealed that although norepinephrine and 6-fluoronorepinephrine stimulated inositol phosphate (IP) production in cerebral cortical slices with potencies equal to those displayed in the cyclic nucleotide assay, selective alpha 1-adrenergic agonists were less efficacious on IP formation and were without effect in the cAMP assay. Conversely, a selective alpha 2-adrenergic receptor agonist facilitated the cAMP response to a beta-adrenergic agonist without affecting IP formation. The rank orders of potency of a series of alpha-adrenergic antagonists suggest that IP accumulation is mediated solely by alpha 1-adrenergic receptors, whereas the augmentation of cAMP accumulation is regulated by a mixed population of alpha-adrenergic sites. The results suggest that the alpha-adrenergic and GABAB receptor-mediated enhancement of isoproterenol-stimulated cAMP formation appears to be more closely associated with phospholipase A2 than phospholipase C and may be mediated by arachidonate or some other fatty acid.     

γ-Aminobutyric AcidB Receptor Activation Modifies Agonist Binding to β-Adrenergic Receptors in Rat Brain Cerebral Cortex

The interaction of isoproterenol with beta-adrenergic receptor (beta AR) binding sites was measured in membranes prepared from rat brain cerebral cortical slices previously incubated in the presence or absence of gamma-aminobutyric acid (GABA) receptor agonists. Both GABA and baclofen, but not isoguvacine, altered beta AR agonist binding by increasing the affinity of both the low- and high-affinity binding sites and by increasing the proportion of low-affinity receptors. The response to baclofen was stereoselective, and the effect of GABA was not inhibited by bicuculline. The results suggest that GABAB, but not GABAA, receptor activation modifies the coupling between beta AR and stimulatory guanine nucleotide-binding protein, which may in part explain the ability of baclofen to augment isoproterenol-stimulated cyclic AMP accumulation in brain slices.     

GABAB Receptor-Mediated Enhancement of Vasoactive Intestinal Peptide-Stimulated Cyclic AMP Production in Slices of Rat Cerebral Cortex

Basal and vasoactive intestinal peptide (VIP)-stimulated accumulations of cyclic AMP were measured in slices of rat cerebral cortex. Neither gamma-aminobutyric acid (GABA) nor the selective GABAB receptor agonist (-)-baclofen stimulated basal cyclic AMP accumulation, whereas VIP caused a large dose-dependent increase in cyclic AMP levels. However, in the presence of 100 microM (-)-baclofen, the effects of VIP on cyclic AMP accumulation were significantly enhanced, with the responses to 1 microM and 10 microM VIP being approximately doubled. The enhancing effects of (-)-baclofen was dose related (1-1,000 microM), but an enhancing effect was not observed with 100 microM (+)-baclofen. In the presence of the GABA uptake inhibitor nipecotic acid (1 mM), GABA caused a similar dose-related enhancement of the VIP response. The ability of either GABA or (-)-baclofen to augment VIP-stimulated production of cyclic AMP was not mimicked by the GABAA, agonists isoguvacine and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) and was not antagonized by the GABAA antagonist bicuculline. The putative GABAB antagonist 5-aminovaleric acid (1 mM) significantly reduced the effect of (-)-baclofen. The ability of (-)-baclofen to enhance VIP-stimulated accumulation of cyclic AMP was observed in slices of rat cerebral cortex, hippocampus, and hypothalamus. These results indicate that GABA and (-)-baclofen can enhance VIP-stimulated accumulation of cyclic AMP in rat brain slices via an interaction with specific GABAB receptors.     

Prevention of the agonist binding to gamma-aminobutyric acid B receptors by guanine nucleotides and islet-activating protein, pertussis toxin, in bovine cerebral cortex. Possible coupling of the toxin-sensitive GTP-binding proteins to receptors

Using the membranes treated with Triton X-100, we studied the interaction between gamma-aminobutyric acid (GABA)B receptors and the GTP-binding proteins which are the substrates for ADP-ribosylation by the islet-activating protein (IAP), pertussis toxin. The addition of guanine nucleotides to the membranes markedly decreased the binding of GABA to GABAB receptors. Preincubation of the membranes with IAP plus NAD caused ADP-ribosylation of the 41,000- and 39,000-Da proteins selectively and decreased GABA binding to GABAB receptors in a time- and dose-dependent manner. This decrease of binding appeared to be due to the reduction of receptor affinity for agonist. The GTP-binding proteins which are ADP-ribosylated by IAP were purified from the membrane fraction of bovine cerebral cortex. The addition of the purified GTP-binding proteins to IAP-treated membranes restored the high affinity binding of GABA to GABAB receptor. The two GTP-binding proteins which were resolved by octyl-Sepharose column chromatography showed similar efficacy in restoring GABA binding. Thus, GABAB receptors are coupled to GTP-binding proteins, IAP-specific substrates, in the brain membranes.     

Calmodulin Involvement in Stress- and Corticosterone-Induced Down-Regulation of Cyclic AMP-Generating Systems in Brain

Manipulation of the hypothalamic-pituitary-adrenal axis selectively alters alpha-adrenergic potentiation of the cyclic AMP response to beta-adrenergic receptor stimulation in rat cerebral cortex. Calcium has been implicated in this alpha-receptor-mediated response, which may involve activation of phospholipases A2 and C and/or calmodulin-dependent adenylate cyclase. We therefore investigated the effects of stress and corticosterone (CORT) on membrane calmodulin-dependent adenylate cyclase and noradrenaline-stimulated cyclic AMP accumulation in brain slices. Repeated stress for 21 days selectively attenuated the adenylate cyclase response to calcium/calmodulin in cerebral cortex membranes, without affecting basal or forskolin-stimulated enzyme activity. There was no such effect in hippocampal membranes. The same pattern of response was elicited by daily CORT injection (50 mg/kg s.c.) for 21 days, while vehicle injection had no effect. CORT in the drinking water (400 micrograms/ml) elicited the same reduction of body weight as CORT injections, but had no effect on calmodulin adenylate cyclase. In parallel with calmodulin adenylate cyclase, cyclic AMP accumulation elicited by noradrenaline in slices of cerebral cortex was suppressed by both stress and daily CORT injections, with smaller effects observed with CORT in the drinking water. Unlike calmodulin adenylate cyclase, noradrenaline-stimulated cyclic AMP accumulation in hippocampus showed the same suppression as that in cerebral cortex. These results are discussed in relation to the differential mode of coupling of alpha-adrenergic receptors to cyclic AMP-generating systems between brain regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

ACCUMULATION OF CYCLIC ADENOSINE 3', 5'-MONOPHOSPHATE IN CEREBRAL CORTICAL SLICES FROM RAT AND MOUSE: STIMULATORY EFFECT OF α- AND β-ADRENERGIC AGENTS AND ADENOSINE

Abstract— Norepinephrine, epinephrine, isoproterenol, and adenosine elicit enhanced accumulations of cyclic AMP in incubated slices of rat cerebral cortex. Combinations of norepinephrine, epinephrine, isoproterenol, or histamine with adenosine have a greater than additive effect on cyclic AMP levels. The effects of isoproterenol appear to be mediated via a classical β-adrenergic receptor whereas the effects of norepinephrine appear due to interactions with both α- and β-adrenergic receptors. The presence of the phosphodiesterase inhibitor, isobutylmethylxanthine, potentiates the effects of the catecholamines and reveals a histamine-mediated increase in cyclic AMP levels. After an initial stimulation of cyclic AMP formation with norepinephrine, followed by washing of the slices, the cyclic AMP-generating system is unresponsive to norepinephrine but does respond to an adenosine-norepinephrine combination. In mouse cerebral cortical slices, catecholamines appear to elicit an accumulation of cyclic AMP primarily via interaction with a β-adrenergic receptor.     

Activation of Protein Kinase C Suppresses the γ-Aminobutyric AcidB Receptor-Mediated Inhibition of the Vesicular Release of Noradrenaline and Acetylcholine

Modulation of the gamma-aminobutyric acidB (GABAB) receptor-mediated response by protein kinase C (PKC) was examined with regard to inhibition by stimulation of the GABAB receptor of stimulation-evoked release of noradrenaline (NA) from slices of cerebellar cortex and of acetylcholine (ACh) from strips of ileum. 12-O-Tetradecanoylphorbol 13-acetate (TPA) potentiated the high K(+)-evoked Ca2+-dependent release of NA and ACh, but not the ouabain-evoked release, even in the presence of external Ca2+. The potentiating effect was antagonized by sphingosine, thereby suggesting that PKC participates in the exocytotic-vesicular release of neurotransmitters, but does not do so in case of a nonvesicular release. GABA inhibited the high K(+)-evoked release of NA and ACh, but not the ouabain-evoked Ca(2+)-independent release. The effect of GABA was mimicked by baclofen and was antagonized by phaclofen, thereby suggesting that stimulation of the GABAB receptor inhibits the vesicular but not the nonvesicular release of neurotransmitters. TPA suppressed the GABAB receptor-mediated inhibition of high K(+)-evoked release of NA and ACh. The effect of TPA was antagonized by sphingosine. These results indicate that stimulation of the GABAB receptor inhibits the stimulation-evoked Ca(2+)-dependent release of neurotransmitters and that activation of PKC suppresses the GABAB receptor-mediated response.     

Possible interaction of alpha 1-adrenergic receptor with pertussis-toxin-sensitive guanine-nucleotide-binding regulatory proteins (G proteins) responsible for phospholipase C activation in rat liver plasma membranes

Islet-activating protein (IAP; pertussis toxin) was employed to test the hypothesis that IAP-sensitive GTP-binding regulatory proteins (G proteins) are coupled with alpha 1-adrenergic receptor in rat liver plasma membranes. The high-affinity state of the binding of alpha 2-adrenergic agonist, which is known to be coupled with IAP-sensitive G protein, was abolished in IAP-treated plasma membranes. IAP treatment of plasma membranes could also diminish the high-affinity state of the alpha 1-adrenergic receptor for the agonist. Restoration of the high-affinity state of the alpha 1-adrenergic receptor for the agonist occurred on reconstitution of the bovine brain IAP-sensitive G proteins. The alpha 1-adrenergic receptor agonist stimulated inositol triphosphate (InsP3) production from [3H]inositol-labeled liver plasma membranes in a concentration-dependent manner. IAP treatment also decreased alpha 1-adrenergic-agonist-induced InsP3 production but not completely. From these results, we concluded that there is a possibility that both IAP-sensitive and IAP-insensitive G proteins were involved in alpha 1-adrenergic-receptor-stimulated phospholipase C activation in rat liver plasma membranes.     

Effect of monoamine receptor agonists and antagonists on cyclic AMP accumulation in human cerebral cortex slices.

In human cerebral cortex slices noradrenaline, isoproterenol (a beta-adrenergic agonist), dopamine, apomorphine (a dopaminergic agonist), and serotonin stimulated cyclic AMP formation: noradrenaline greater than or equal to isoproterenol greater than dopamine = apomorphine = serotonin. Clonidine (and alpha-adrenergic agonist) was ineffective in stimulating cyclic AMP formation in temporal cortex slices. The stimulatory effect of noradrenaline and isoproterenol was blocked by propranolol (a beta-adrenergic blocker) but not by phentolamine (an alpha-adrenergic blocker). Pimozide (a selective dopaminergic antagonist) inhibited the increase of cyclic AMP formation induced by dopamine or apomorphine but not that induced by noradrenaline, isoproterenol, or serotonin. Neither propranolol or phentolamine had any effect on dopamine- or serotonin-stimulated cyclic AMP formation. Chlorpromazine blocked the increase of cyclic AMP formation induced by noradrenaline, dopamine or serotonin, while cyproheptadine, a putative central serotonergic antagonist, was ineffective. These observations suggest that there may be at least two monoamine-sensitive adenylate cyclases in human cerebral cortex which have the characteristics of a beta-adrenergic and a dopaminergic receptor, respectively, and also possibly a serotonergic receptor.     

NMDA Receptor Activation Increases Cyclic AMP in Area CA1 of the Hippocampus via Calcium/Calmodulin Stimulation of Adenylyl Cyclase

Abstract: We observed previously that activation of N -methyl- d -aspartate (NMDA) receptors in area CA1 of the hippocampus, through either NMDA application or long-term potentiation (LTP)-inducing high-frequency stimulation (HFS), results in an increase in cyclic AMP. In the present study, we performed experiments to determine the mechanism by which NMDA receptor activation causes this increase in cyclic AMP. As the NMDA receptor-mediated increase in cyclic AMP is dependent upon extracellular calcium, we hypothesized that NMDA receptors are coupled to adenylyl cyclase (AC) via calcium/calmodulin. In membranes prepared from area CA1, AC was stimulated by calcium in the presence of calmodulin, and the effect of calcium/calmodulin on AC in membranes was blocked by the calmodulin antagonists N -(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and trifluopera-zine (TFP). In intact hippocampal slices, W-7 and TFP blocked the increase in cyclic AMP levels caused by both NMDA application and HFS of Schaffer collateral fibers. Exposure of hippocampal slices to elevated extracellular potassium to induce calcium influx also caused increased cyclic AMP levels; the increase in cyclic AMP caused by high potassium was also blocked by W-7 and TFP. These data support the hypothesis that NMDA receptor activation is positively coupled to AC via calcium/calmodulin and are consistent with a role for cyclic AMP metabolism in the induction of NMDA receptor-dependent LTP in area CA1 of the hippocampus.     

Accumulations of Cyclic AMP in Adenine-Labeled Cell-free Preparations from Guinea Pig Cerebral Cortex: Role of α-Adrenergic and H1-Histaminergic Receptors

Norepinephrine, histamine, adenosine, glutamate, and depolarizing agents elicit accumulations of radioactive cyclic AMP from adenine-labeled nucleotides in particulate fractions from Krebs-Ringer homogenates of guinea pig cerebral cortex. The particulate fractions contain sac-like entities, which apparently are associated with a significant portion of the membranal adenylate cyclase. Particulate fractions from sucrose homogenates are a less effective source of such responsive entities. Activation of the adenine-labeled cyclic AMP-generating systems by norepinephrine is by means of alpha-adrenergic receptors, while activation by histamine is through H1- and H2-histaminergic receptors. Adenosine responses are potentiated by the amines and are antagonized by alkylxanthines. Glutamate and depolarizing agents appear to elicit accumulations of cyclic AMP via "release" of endogenous adenosine. It is proposed, based on the virtual absence of an alpha-adrenergic or H1-histaminergic response in the presence of a combination of potent adenosine and H2-histaminergic antagonists, that alpha-adrenergic and H1-histaminergic receptor mechanisms do not activate adenylate cyclase directly in brain slices or Krebs-Ringer particulate fractions, but merely facilitate activation by beta-adrenergic, H2-histaminergic, or adenosine receptors.     

Increase in the Number, G Protein Coupling, and Efficiency of Facilitatory Adenosine A2A Receptors in the Limbic Cortex, but not Striatum, of Aged Rats

Adenosine's effects result from a balanced activation of inhibitory A1 and facilitatory A2A receptors. Because in aged animals there is an increased number of A2A receptors, we now compared the efficiency of A2A receptors in cortical and striatal preparations of young adult (6-week-old) and aged (2-year-old) rats. In cortical, in contrast to striatal, membranes from aged rats, A2A receptors were more tightly coupled to G proteins, because 5'-guanylylimidodiphosphate (100 microM) increased by 321% the Ki of the A2A agonist CGS21680 as a displacer of binding of the A2A antagonist [3H]ZM241385 (1 nM), compared with a 112% increase in young rats. In cortical slices, CGS21680 (30-1,000 nM) was virtually devoid of effect on cyclic AMP accumulation in young rats but increased cyclic AMP accumulation with an EC50 of 153 nM in aged rats, whereas the efficiency of CGS21680 was similar in striatal slices of young and aged rats. CGS21680 (30 nM) was virtually devoid of effect on acetylcholine release from hippocampal CA1 slices of young rats but caused a 55% facilitation in aged rats. These results show that the number of A2A receptors, their coupling to G proteins, and their efficiency are enhanced in the limbic cortex of aged rats, suggesting a greater involvement of facilitation in adenosine responses.     

Characteristics of the catecholamine and histamine receptor sites mediating accumulation of cyclic adenosine 3',5'-monophosphate in guinea pig brain

—Five areas of guinea pig brain were examined to determine the properties of the receptor sites mediating increases in [³H]adenosine 3′,5′-monophosphate (cyclic AMP). Both epinephrine and histamine were effective in causing increases in cyclic AMP in slices derived from cerebral cortex, hippocampus or amygdala, but not in diencephalon or brainstem. Stimulation of slices of cerebral cortex by either epinephrine or histamine resulted in a small, but reproducible, decrease in specific radioactivity of the [³H]-cyclic AMP produced, as did stimulation of the hippocampus by epinephrine. The catecholamine receptor was an α-adrenergic receptor in all three areas where epinephrine was effective; α-adrenergic stimulation, but not β-adrenergic stimulation, increased levels of [³H]-cyclic AMP. Furthermore, α-, but not β-adrenergic blocking agents, prevented the epinephrine- induced increase of both [³H]- and total cyclic AMP in cerebral cortex and hippocampus. Only antihistaminic agents were capable of antagonizing the histamine-induced increase of both [³H]- and total cyclic AMP in these two brain areas. The catecholamine receptor in the amygdala also appeared to be an α-adrenergic receptor. The effects of histamine and epinephrine together were far greater than the sum of effects of either hormone alone in both cerebral cortex and hippocampus.     

Subtype-Selective Immunoprecipitation of the β2-Adrenergic Receptor

Most antibodies known to interact with beta-adrenergic receptors do not exhibit subtype selectivity, nor do they provide quantitative immunoprecipitation. A monoclonal antibody, G27.1 raised against a synthetic peptide corresponding to the C-terminus of the beta 2-adrenergic receptor of hamster, is selective for the beta 2 subtype. G27.1 provides nearly quantitative immunoprecipitation of the beta 2-adrenergic receptor from hamster lung that has been photoaffinity-labeled and solubilized with sodium dodecyl sulfate. Immunoprecipitation is completely blocked by nanomolar concentrations of the immunizing peptide. This antibody interacts with beta 2-adrenergic receptors from three rodent species, but not with those from humans. When C6 glioma cells, which contain both beta 1- and beta 2-adrenergic receptors, are photoaffinity-labeled in the absence or presence of subtype-selective antagonists, subtype-selective photoaffinity-labeling results. G27.1 can immunoprecipitate beta 2-, but not beta 1-, adrenergic receptors from these cells. Similar results were obtained following subtype-selective photoaffinity-labeling of membranes from rat cerebellum and cerebral cortex. The beta-adrenergic receptors from C6 glioma cells and rat cerebral cortex exist as a mixture of two molecular weight species. These species differ in glycosylation, as shown by endoglycosidase F digestion of crude and immunoprecipitated receptors.     

Desensitization of β-Adrenergic Receptor-Coupled Adenylate Cyclase in Cerebral Cortex After In Vivo Treatment of Rats with Desipramine

Continuous treatment (1-10 days) of rats with desipramine (10 mg/kg, twice per day) caused desensitization of the beta-adrenergic receptor-coupled adenylate cyclase system of cerebral cortical membranes. The decrease in the isoproterenol-stimulated adenylate cyclase activity was more rapid and greater than the decrease in the number of beta-adrenergic receptors in membranes during treatment of the membrane donor rats with desipramine, indicating that the desensitization occurring at an early stage of the treatment was not accounted for solely by the decrease in the receptor number. Neither the guanine nucleotide regulatory protein (N) nor the adenylate cyclase catalyst was impaired by the drug treatment, since there was no decrease in the cyclase activity measured in the presence or absence of GTP, guanyl-5'-yl-beta-gamma-imidodiphosphate [Gpp(NH)p], NaF, or forskolin. Gpp(NH)p-induced activation of membrane adenylate cyclase developed with a lag time of a few minutes in membranes from control or drug-treated rats. The lag was shortened by the addition of isoproterenol, indicating that beta-receptors were coupled to N in such a manner as to facilitate the exchange of added Gpp(NH)p with endogenous GDP on N. This effect of isoproterenol rapidly decreased during the drug treatment of rats. Thus, functional uncoupling of the N protein from receptors was responsible for early development of desensitization of beta-adrenergic receptor-mediated adenylate cyclase in the cerebral cortex during desipramine therapy.     

Cannabinoid Receptors and Modulation of Cyclic AMP Accumulation in the Rat Brain

The mechanism by which cannabinoid compounds produce their effects in the rat brain was evaluated in this investigation. Cannabinoid receptors, quantitated by [3H]CP-55,940 binding, were found in greatest abundance in the rat cortex, cerebellum, hippocampus, and striatum, with smaller but significant binding also found in the hypothalamus, brainstem, and spinal cord. Using rat brain slice preparations, we evaluated the effect of desacetyllevonantradol on basal and forskolin-stimulated cyclic AMP accumulation in the regions exhibiting the greatest cannabinoid receptor density. Desacetyllevonantradol (10 microM) reduced cyclic AMP levels in the hippocampus, frontal cortex, and striatum. In the cerebellum, however, the response to desacetyllevonantradol was biphasic with cyclic AMP accumulation being decreased at lower and increased at higher concentrations. Desacetyllevonantradol reduced cyclic AMP accumulation in isoproterenol-stimulated slices in the cortex and cerebellum, but not in the hippocampus. Cells that responded to vasoactive intestinal peptide with an increase in cyclic AMP accumulation in the hippocampus and cortex also responded to desacetyllevonantradol. The modulation of cyclic AMP accumulation by desacetyllevonantradol could be attenuated following stereotaxic implantation of pertussis toxin, supporting the involvement of a G protein in the cannabinoid response in the brain. However, other actions of cannabinoid compounds may also affect the cyclic AMP levels in brain slice preparations.     

β2-Adrenergic Receptors on Peripheral Nerves

We report that peripheral nerves have a functional adenylate cyclase-coupled beta-adrenergic receptor. The pharmacological specificity of this receptor is shown to be of the beta 2 subtype. Two peripheral nerves, the sciatic from the frog and rat and the vagus from the rat, responded to beta 2-agonists with 10-50-fold increases in intracellular cyclic AMP level. This increase was inhibited by the beta-adrenergic antagonist propranolol. In contrast, a central nerve tract, the corpus callosum, responded to isoproterenol with only a minimal one- to twofold increase in cyclic AMP level. These studies demonstrate that peripheral nerves have beta 2-adrenergic receptors that are responsive to exogenously applied catecholamines and suggest a role for these ligands in the previously described modulation of axonal conduction.     

ADENOSINE 3'',5''-MONOPHOSPHATE IN GUINEA PIG CEREBRAL CORTICAL SLICES: EFFECTS OF α- AND β-ADRENERGIC AGENTS, HISTAMINE, SEROTONIN AND ADENOSINE

—Norepinephrine and epinephrine, in combination with either adenosine or histamine, enhanced the accumulation of cyclic AMP in guinea pig cerebral cortical slices. Isoproterenol had only marginal effects under the same conditions. Studies with d- and l-norepinephrine and with the α- and β-adrenergic blocking agents, phenoxybenzamine, phentolamine, dihydroergokryptamine, propranolol and sotalol, indicated that the effect of catecholamines on cyclic AMP levels in this tissue was stereo-specific and was mediated primarily via interaction with a classical α-adrenergic receptor. Studies with the antihistaminics, diphenhydramine and pheniramine, and the antiserotonin agent, methysergide, indicated that guinea pig cerebral cortical slices contain receptors for histamine and serotonin, whose activation also stimulates an enhanced accumulation of cyclic AMP in the presence of adenosine.     

Lack of desensitization of muscarinic receptor–mediated second messenger signals in rat brain upon acute and chronic inhibition of acetylcholinesterase

We studied the effects of acute and chronic in vivo inhibition of acetylcholinesterase on both the density and function of brain muscarinic cholinergic receptors. Adult male rats were treated either once or multiple times over a period of 10 days with the irreversible acetylcholinesterase inhibitor diisopropylfluorophosphate (DFP). The concentration and affinity of muscarinic receptors in various brain regions were determined using radioligand binding techniques. Acute DFP treatment resulted in a significant reduction in receptor number only in the brain stem, while chronic treatment caused receptor downregulation in the brain stem, cerebral cortex, and striatum. There was no change in ligand affinity in any of the brain regions. In sharp contrast, muscarinic receptor function was fully preserved, in terms of coupling of the receptors to increased phosphoinositide hydrolysis in the cerebral cortex, hippocampus, and striatum, or inhibition of cyclic AMP formation in the cerebral cortex or striatum. Therefore, there is a marked lack or correlation between DFP-induced muscarinic receptor down-regulation and receptor desensitization.