The resolution of dopamine and β1- and β2-adrenergic-sensitive adenylate cyclase activities in homogenates of cat cerebellum, hippocampus and cerebral cortex

The stimulation of adenylate cyclase by dopamine and various β-adrenergic agonists has been investigated in homogenates from 3 areas of cat brain: the cerebral cortex, cerebellum and hippocampus. The purpose of the study was to determine whether the β-adrenergic receptors coupled to adenylate cyclase could be classified as either β₁ and β₂ subtypes in the different regions studied.The stimulation of adenylate cyclase by the β-adrenergic agonist, (−)isoproterenol (5 × 10⁻⁶M), was completely blocked by the specific β-adrenergic antagonist, (−)alprenolol (10⁻⁵ M), but not by the dopaminergic antagonist, fluphenazine (10⁻⁵ M), whereas the stimulation of adenylate cyclase by (−)epinephrine (10⁻⁴ M) was blocked to varying extents by these two drugs in each of the 3 regions studied. The (−)epinephrine effect was always blocked in the combined presence of (−)alprenolol and fluphenazine. The adenylate cyclase stimulation by (−)epinephrine which is not blocked by (−)alprenolol was due to interaction of (−)epinephrine with a dopaminergic-sensitive adenylate cyclase which has been characterized in cerebral cortex, hippocampus and cerebellum.Regional differences in the affinity of β-adrenergic-sensitive adenylate cyclase for various agonists were investigated in the presence of fluphenazine (10⁻⁵ M). In the cerebellum the potency order was (±)protokylol> (±)hydroxybenzylisoproterenol> (±)isoproterenol> (−)epinephrine> (±)salbutamol> (−)norepinephrine, indicating the presence of a β₂-adrenergic receptor. In the cerebral cortex the potency order was (−)isoproterenol> (±)protokylol> (±)hydroxybenzylisoproterenol> (−)epinephrine= (−)norepinephrine((±)salbutamol being inactive). A similar pattern was found in the hippocampus indicating the presence of a β₁-adrenergic receptor in these two regions. (±)Salbutamol was a partial agonist in the cerebellum and a competitive antagonist in the cerebral cortex.The ratio of the antagonist potencies of (±)practolol and (±)butoxamine preferential β₁- and β₂-adrenergic antagonists respectively, to block the stimulation of adenylate cyclase was 25 in the cerebellum, compared to 0.5 in the cerebral cortex and 1.6 in the hippocampus. These results confirm the presence of a β₂ subtype of receptor coupled to adenylate cyclase in the former and β₁ subtypes in the latter two regions. The comparison between the affinities of a series of β-adrenergic agonists and antagonists for the β-adrenergic receptors coupled with an adenylate cyclase in cerebral cortex and cerebellum with their affinities for well characterized β₂-adrenergic receptors in lung and β₁-adrenergic receptor in heart substantiated this conclusion.     

Morphine-induced place preference in the CXBK mouse: characteristics of μ opioid receptor subtypes

The role of μ opioid receptor subtypes, μ₁ and μ₂, in morphine-conditioned place preference was examined using ddY and μ₁ opioid receptor-deficient CXBK mice. In ddY mice, the μ receptor agonist morphine caused a dose-related preference for the drug-associated place, but the κ agonist U-50,488H produced a dose-related place aversion. These results demonstrated that the mouse is available for place preference conditioning using opioids. Under this condition, the influence of pretreatment with selective μ₁ opioid receptor antagonist naloxonazine morphine-induced place preference was investigated in ddY mice. Although pretreatment with the selective μ₁ antagonist naloxonazine (35 mg/kg, s.c.) did not modify the morphine-induced place preference, pretreatment with the selective μ antagonist β-funaltrexamine (β-FNA 10 mg/kg, s.c.) eliminated the appetitive effect of morphine. Furthermore, morphine (1–5 mg/kg, s.c.) produced a dose-related preference for the drug-associated place in CXBK mice. These findings suggest that the morphine-induced conditioned place preference may be mediated by naloxonazine-insensitive sites (μ₂ opioid receptors). In addition, chronic infusion of the dopamine D₁ antagonist SCH23390 (1.0 mg/kg/day) during the conditioning sessions eliminated the morphine-induced place preference in CXBK mice. Similarly, morphine combined with naloxonazine failed to produce the place preference in ddY mice chronically treated with SCH23390. The blocking effect of SCH23390 on the morphine-conditioned place preference suggests that μ₂ receptors may regulate the dopaminergic system, especially dopamine D₁ receptors, and are also involved in the reinforcing effects of morphine.     

Alterations in the activity of adenylate cyclase and high affinity GTPase in Alzheimer's disease

The aim of this study was to assess the effect that Alzheimer's disease has on the functional integrity of several signal transduction proteins. The relative levels of the G-protein α subunits G_sα-L, G_sα-S, G_iα-1, G_iα-2 and G_oα were measured by western blotting and found to be unchanged in membranes prepared from Alzheimer-diseased frontal cortex or hippocampus compared to control brains. However the activity of the G-protein associated enzyme, high affinity GTPase, was found to be reduced in the frontal cortex (reduced by 25%) and by a similar magnitude in the hippocampus (reduced by 27%) of Alzheimer subjects. The same membrane preparation were also assayed for the activity of adenylate cyclase. Basal enzyme activity was not significantly altered in Alzheimer diseased hippocampus, but was markedly reduced (by 45%) in the frontal cortex. The ability of fluoride and aluminum ions to stimulate adenylate cyclase was not significantly changed in either brain region. This suggests that G-proteins, especially G_s, are still able to interact with this enzyme. These results indicate that although the presence of Alzheimer's disease does not significantly alter G-protein levels, changes have taken place in the overall activity of these proteins. However this alteration does not affect their ability to stimulate adenylate cyclase activity.     

Lewis and Fischer rat strains display differences in biochemical, electrophysiological and behavioral parameters: studies in the nucleus accumbens and locus coeruleus of drug naive and morphine-treated animals

In previous studies, we demonstrated that tyrosine hydroxylase and neurofilament proteins are regulated by chronic morphine and chronic cocaine treatments in the ventral tegmental area in Sprague-Dawley rats and that the imbred Lewis and Fischer 344 rat strains, under drug-naive conditions, show different levels of these proteins specifically in this brain region. In the current study, we compared Lewis and Fischer rats with respect to levels of adenylate cyclase, cyclic AMP-dependent protein kinase and G-proteins in the nucleus accumbens (NAc) and locus coeruleus (LC), brain regions in Sprague-Dawley rats where these proteins are regulated by chronic exposure to morphine or to cocaine. We found that levels of adenylate cyclase and cyclic AMP-dependent protein kinase activity are higher in the NAc and LC of Lewis rats compared to Fischer rats, whereas levels of G_iα and G_β were lower. These strain differences were not seen in several other brain regions analyzed and no strain differences were detected in levels of other G-protein subunits. Lewis and Fischer rats also differed in the ability of chronic morphine to regulate adenylate cyclase and cyclic AMP-dependent protein kinase in the NAc and LC. In the NAc, chronic morphine increased levels of the two enzymes in the Fischer strain only, whereas in the LC chronic morphine increased levels of the enzymes in both strains, with more robust effects seen in the Lewis rat. To understand possible physiological consequences of these strain differences in the cyclic AMP pathway, we studied LC neuronal activity under basal and chronic morphine-treated conditions. LC neurons of Lewis rats showed higher spontaneous firing rates in brain slices in vitro than those of Fischer rats and also showed greater morphine-induced increases in responsiveness to bath-applied 8-bromo-cyclic AMP. These electrophysiological findings are generally consistent with the biochemical observations. Moreover, Lewis and Fischer rats displayed very different opiate withdrawal syndromes, with different types of behaviors elicited upon precipitation of opiate withdrawal with the opiate receptor antagonist, naltrexone. The possible relationship between these behavioral findings and the biochemical and electrophysiological data is discussed. These studies provide further support for the possibility that Lewis and Fischer rat strains provide a useful model system in which some of the genetic factors that contribute to drug-related behaviors can be investigated.     

Arachidonic acid and prostaglandin D2 cooperatively accelerate desensitization of nicotinic acetylcholine receptor channel in mouse skeletal muscles

To clarify the effects of arachidonic acid (AA) and its metabolites on desensitization of nicotinic acetylcholine (ACh) receptor channel in mouse skeletal muscle cells, we investigated the time-dependent decrease in the channel opening frequency of ACh (1 μM)-activated channel currents by the cell-attached patch clamp technique. AA (30–100 μM) applied to a patched membrane or to non-patched membrane accelerated the decrease in the channel opening frequency. A cyclooxygenase inhibitor, indomethacin (10 μM), prevented the acceleration elicited by 30 μM AA, but not by 100 μM AA. A lipoxygenase inhibitor, nordihydroguaiaretic acid (10 μM), and a cytochrome P-450 inhibitor, ketoconazole (3 μM), did not affect the acceleration by 30 μM AA. Prostaglandin (PG) D₂ at 10 μM alone and at 25 nM in combination with 10 μM AA accelerated the decrease in the channel opening frequency. No acceleration was observed with PGE₂ at 10 μM alone and at 25 nM in combination with 10 μM AA. Pretreatment with a protein kinase (PK) C inhibitor, staurosporine (10 nM), but not with a PKA inhibitor, H-89 (3 μM), prevented the acceleration elicited by AA+PGD₂. These results suggest that AA, and PGD₂ of its metabolites, cooperatively accelerate desensitization of nicotinic ACh receptor channel. The activation of PKC by AA and PGD₂ may be involved in the mechanism of the cooperative acceleration of desensitization.     

Prostaglandin E2 mediates the stimulatory effect of methoxamine on in vivo luteinizing hormone-releasing hormone (LH-RH) release in the ovariectomized female rhesus monkey

Previously, we found that noradrenergic input throughα₁-receptors modulates pulsatile release of luteinizing hormone-releasing hormone (LH-RH) in ovariectomized rhesus monkeys in the absence of estrogen. In the present study, the role of prostaglandin E₂ (PGE₂) in mediating α-adrenergic stimulation of LH-RH release is investigated. In the first experiment the effects of theα₁-adrenergic agonists methoxamine (MTX) on LH-RH and PGE₂ release were examined. Push-pull perfusion of the stalk-median eminence (S-ME) was performed in conscious, ovariectomized monkeys, and perfusate samples were collected on ice. MTX (10⁻⁵ M) was infused into the S-ME through the push cannula for 10 min at 90-min intervals, and LH-RH and PGE₂ in aliquots of the same perfusate samples were measured by radioimmunoassay. Infusion of MTX significantly stimulated LH-RH release (n = 12; P < 0.01) and PGE₂ release (P < 0.05). In the second experiment, the effect of PGE₂ infusion on LH-RH release was tested. PGE₂ (10⁻⁷ M) was infused using the same protocol as above, and LH-RH was measured in the perfusates. Infusion of PGE₂ through the push cannula significantly stimulated LH-RH release (n = 23; P < 0.05). These results suggest that the stimulatory effect of MTX on LH-RH release is at least partly mediated by PGE₂, since MTX stimulated not only LH-RH but also PGE₂ release, and since PGE₂ itself stimulated LH-RH release. Therefore, PGE₂ may be an important endogenous mediator ofα₁-adrenergic input stimulating pulsatile PH-RH release. Moreover, the stimulatory effects of MTX and PGE₂ can be observed in the absence of estrogen in the rhesus monkey, unlike in rhodents. Our results also demonstrate the usefulness of the push-pull perfusion technique for studies of cellular mechanisms in neuroendocrine research.     

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.     

Cholera toxin-induced Gsα down-regulation in neural tissue: studies on the pineal gland

Cholera toxin (CT) treatment (50 μg/ ml) was used to down regulate the α subunit of the stimulatory guanine nucleotide binding protein (Gsα) in pineal glands in organ culture, as has been seen in non-neural tissue. A 15 h treatment reduces G_sα by ≈ 75% as measured using semi-quantitative Western blot technology. In contrast, this treatment does not alter the abundance of Gβ, G_iα or G_oα. This effect on G_sα was still apparent following a 36-h washout period. The 48-h CT treatment increased cyclic AMP accumulation 10- to 17-fold but blocked the norepinephrine (NE)-induced increase in cyclic AMP accumulation, presumably reflecting the loss of G_sα. This treatment did not, however, inhibit protein synthesis or stimulation of arylalkylamine N-acetyltransferase (NAT) activity produced by treatment with either DB-cyclic AMP (N⁶,2′-O-dibutyryl adenosine 3′,5′monophosphate) or 8 Br-cyclic AMP, stable cyclic AMP derivatives. This indicates that a 48-h CT treatment was not generally toxic. In contrast, this treatment blocked subsequent CT stimulation of NAT. The effects of CT treatment on the adrenergic stimulation of NAT was examined using treatments which selectively produced α- or β-adrenergic stimulation. α₁-Adrenergic activation of the pineal gland elevates [Ca²⁺]_i, which potentiates effects of cyclic AMP; in these studies the response to α-adrenergic activation was markedly increased in 48-h CT-treated glands, reflecting Ca²⁺ potentiation of the effects of elevated levels of cyclic AMP. In contrast, the effects of the selective β-adrenergic agonist isoproterenol was reduced by ≈ 75%. These studies not only establish CT-induced G_sα down-regulation as a new tool for the study of adrenergic signal transduction in the pineal gland, but indicate that this paradigm is probably useful in all neural tissue.     

GABAA and GABAB receptors modulating basal and footshock-induced nitric oxide releases in rat prefrontal cortex

Using an in vivo brain microdialysis technique, we measured extracellular levels of nitric oxide (NO) metabolites (NO_x⁻) in the medial prefrontal cortex (mPFC) upon perfusion of γ-aminobutyric acid (GABA) receptor antagonists as well as agonists, and also examined the effects of GABA receptor agonists on mild intermittent footshock-induced NO releases in the mPFC in conscious rats. Perfusion of either bicuculline methiodide, a GABA_A receptor antagonist, or saclofen, a GABA_B receptor antagonist, through a microdialysis probe resulted in dose-dependent increases in NO_x⁻ levels. Higher-dose perfusion of either muscimol (50 μM), a GABA_A receptor agonist, or baclofen (250 μM), a GABA_B receptor agonist resulted in a significant decrease in NO_x⁻ levels. The elevated levels of NO_x⁻ after mild intermittent footshock were attenuated by perfusion of either muscimol (10 μM) or baclofen (50 μM), either of which alone did not affect basal NO_x⁻ levels. These findings are likely to provide helpful clues to our understanding of the inhibitory modulation of basal and footshock-induced NO metabolites releases by GABA_A and GABA_B receptors in the mPFC.     

Characteristics of the β1- and β2-adrenergic-sensitive adenylate cyclases in glial cell primary cultures and their comparison with β2-adrenergic-sensitive adenylate cyclase of meningeal cells

The agonist specificity pattern of the β-adrenergic adenylate cyclase in glial primary cultures was not typical of either β₁- or β₂-adrenergic receptors. The dose-response curves for adrenaline did not correspond to simple mass action kinetics and their computer analysis suggests the presence of both β₁- and β₂-adrenergic-sensitive adenylate cyclase (58 ± 17% and 42 ± 17% respectively).Similar properties of β₁- and β₂-adrenergic-sensitive adenylate cyclases were found by computer analysis of the dose-response curves for isoprenaline in the presence of a constant concentration of practolol (a selective β₁ antagonist) ( 55 ± 10% and 45 ± 10% of β₁- and β₂-sensitive adenylate cyclase respectively).The curves for displacement of [³H]dihydroalprenolol by practolol confirm these results.For purpose of comparison, the β-adrenergic receptors of meningeal cells in cultures were subjected to similar analysis. The results clearly showed that these cells exclusively contained β₂-adrenergic receptors.     

Involvement of metabotropic glutamate receptors in Gi- and Gs-dependent modulation of adenylate cyclase activity induced by a novel cognition enhancer NS-105 in rat brain

The effect of a novel cognition enhancer [(+)-5-oxo-

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-prolinepiperidinamide monohydrate] (NS-105) on cAMP formation was investigated in both slices and membranes of the rat cerebral cortex. NS-105 (10⁻⁸–10⁻⁶ M) inhibited forskolin-stimulated cAMP formation in membranes, however, the compound significantly enhanced the cAMP formation in pertussis toxin-pre-treated membranes, an action that was abolished by cholera toxin. In contrast, in digitonin-permeabilized membranes, NS-105 had no influence on Mn²⁺-stimulated cAMP formation. Both of the inhibitory and facilitatory actions of NS-105 on cAMP formation were mimicked by a metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) and an adrenergic α₂ agonist UK-14,304, and blocked by a mGluR antagonist 2-amino-3-phosphonopropanoate but not by an α₂ antagonist yohimbine. In cortical slices, NS-105 (10⁻⁸–10⁻⁷ M) inhibited forskolin-stimulated cAMP accumulation but enhanced isoproterenol-stimulated cAMP accumulation, as did by a GABA_B agonist (−)baclofen. On the other hand, (−)baclofen, while it significantly inhibited cAMP accumulation in slices, did no longer inhibit cAMP accumulation, when treated with NS-105 (10⁻⁸–10⁻⁵ M). Similarly, (−)baclofen-induced inhibition of the cAMP accumulation was reversed by 1S,3R-ACPD and UK-14,304. NS-105 (10⁻⁶) increased [³⁵S]GTPγS binding in the intact but not digitonin-permeabilized cortical membranes, as produced by UK-14,304, although the compound (10⁻⁹–10⁻³ M) had no influence on various neurotransmitter receptor bindings, including α₂ receptors. These results suggest that NS-105 modulates adenylate cyclase activity by stimulating mGluRs which might coupled to both G_i/G_o and G_s. © 1997 Elsevier Science B.V. All rights reserved.     

Interactions between laudanosine, GABA, and opioid subtype receptors: implication for laudanosine seizure activity

We examined the interactions ofd,l-laudanosine, a potentially epileptogenic metabolite of the neuromuscular relaxant atracurium besylate, with γ-aminobutyric acid (GABA) and opioid binding sites, all of which have been implicated in seizure activity. Laudanosine was almost ineffective at [³H]muscimol binding to high-affinity GABA receptors (IC₅₀ = 100 μM). However, laudanosine displayed an inhibitory effect at the low-affinity GABA receptors labeled by [³H]bicuculline methochloride, with an IC₅₀ value of 10 μM. At the opioid receptor subtype, laudanosine lowered radiolabeled opioid binding at the μ₁, μ₂, δ, κ₁, and κ₃ receptors with K_i values of 2.7, 13, 5.5, 21, and 24 μM, respectively, concentrations seen clinically in blood and approaching those measured in cerebrospinal fluid. Saturation studies of μ₁, μ₂, δ, and κ₃ sites in the presence of laudanosine revealed competitive interactions, with increases in the apparent K_d values but without significant changes in the maximal numbers of binding sites. In addition, we investigated whether the in vitro laudanosine-opioid receptor interaction would also be expressed by analgesic physiologic effects. We found that laudanosine elicited a dose-dependent analgesia in mouse tail-flick assay that was attenuated by coadministration of β-funaltrexamine (μ₁- and μ₂-sselective antagonist) and of naloxonazine (μ₁ antagonist), but not by nor-binaltophimine (κ₁-selective antagonist) or naltrindole (δ-selective antagonist), indicating a μ₁ mechanism for analgesia-mediated properly of laudanosine. There is evidence suggesting μ₂ activity as well, but this is due to the ability of laudanosine to elicit analgesia when given intrathecally. We also observed cross-tolerance between laudanosine and morphine, as well as a partial effect of laudanosine on gastrointestinal transit. These results suggest an interaction between laudanosine and the low-affinity GABA receptor, as well as opioid μ₁ and μ₂ receptors.     

β-Adrenergic receptor binding in brain of alcoholics

The binding of agonists and antagonists to β-adrenergic receptors in brain tissue obtained postmortem in nonalcoholic controls and matched intoxicated and sober alcoholics was measured to assess the state of the receptors and their coupling to adenylate cyclase. Binding of antagonist, iodocyanopindolol, to cerebral cortical and cerebellar membrane preparations was not different in alcoholics compared to that in controls, suggesting that the number of β-adrenergic receptors was not affected by chronic ethanol ingestion. Agonist binding data, however, indicated the loss of the high-affinity agonist binding state of the β-adrenergic receptor, representing the receptor-guanine nucleotide binding protein (G_s) complex. Such changes were observed in cerebral cortex but not in cerebellum of intoxicated alcoholics. These data suggest that cerebral cortical β-adrenergic receptors are uncoupled from adenylate cyclase in these subjects. In cerebral cortical and cerebellar membranes of sober alcoholics both the high- and low-affinity agonist binding sites were observed. These findings are similar to those seen in animal studies and suggest that the effect of chronic ethanol ingestion on β-adrenergic receptor-adenylate cyclase coupling is brain region specific and reversible with abstinence. Ethanol-induced changes in the coupling of receptors to adenylate cyclase may contribute to the physiological and behavioral manifestations of alcohol abuse.     

Chronic neurosteroid treatment attenuates single cell GABAA response and its potentiation by modulators in cortical neurons

In previous studies we have observed that chronic neurosteroid 5α-pregnan-3α-ol-20-one (5α3α) treatment produced downregulation of the GABA_A receptors, heterologous uncoupling, and decreased heterologous efficacy at the GABA_A receptor complex in cultured mammalian cortical neurons. In this study, using whole cell recording, we examined the consequence of chronic 5α3α (1 μM; 5 days) treatment on GABA-induced currents in isolated cortical neurons. We observed that the GABA current was decreased by 78% after 5 days treatment of cortical cells with 1 μM; 5α3α. We also observed decreased pentobarbital, and 5α3α potentiation of GABA currents after chronic 5α3α treatment. These findings support the notion that GABA response, and its potentiation by pentobarbital, and neurosteroid, 5α3α, are attenuated after chronic 5α 3α treatment.     

Functional analysis of GABAA receptors in nucleus tractus solitarius neurons from neonatal rats

To gain insight into specific GABA_A receptor configurations functionally expressed in the nucleus tractus solitarius (NTS), we conducted several physiological and pharmacological assessments. NTS neurons were characterized in thin brain slices from 1–14 day old rats using whole-cell patch clamp recordings. GABA_A− receptor-mediated currents were detected in all neurons tested, with an average EC₅₀ of 22.2 μM. GABA currents were consistently stimulated by diazepam (EC₅₀=63 nM), zolpidem (EC₅₀=85 nM), loreclezole (EC₅₀=10.1 μM) and the neurosteroid 5α-pregnan-3α-hydroxy-20-one (3α-OH-DHP). In contrast, GABA-gated currents of the NTS were inhibited by the divalent cation Zn²⁺ (IC₅₀=33.6 μM) picrotoxin (IC₅₀=2.4 μM) and blockade of endogenous protein tyrosine kinase. GABA-activated currents were insensitive to furosemide (10–1000 μM) in all NTS neurons tested. Collectively, the data suggest that in neonatal rats, the predominant α subunit isoform present in GABA_A receptors of the NTS appears to be the α1 and/or α2 subunit. β2 and/or β3 subunits are the major β isoform, while the predominant γ subunit is likely γ2. Our data suggest the contribution to NTS GABA currents by α3–α6, β1, γ1 and δ subunits, if present, is minor by comparison.     

Involvement of dopamine D1 and D2 receptors in the ethanol-associated place preference in rats exposed to conditioned fear stress

The present study was designed to investigate: (1) the involvement of dopamine D₁ and D₂ receptors, and (2) the roles of these receptors and endogenous opioid systems (endorphinergic and enkephalinergic systems) in the ethanol-induced place preference in rats exposed to conditioned fear stress using the conditioned place preference paradigm. The administration of ethanol (300 mg/kg, i.p.) induced a significant place preference. The selective D₁ receptor antagonist R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H3-benzazepine)hydrochloride (SCH23390; 0.01 and 0.03 mg/kg, s.c.) and the selective D₂ receptor antagonist S(−)-5-(aminosulfonyl)-N-[(1-ethyl-2-pyrrolidinyl)-methyl]-2-methoxybenzamide (sulpiride; 20 and 40 mg/kg, s.c.) significantly attenuated the ethanol-induced place preference. The administration of ethanol (75 mg/kg, i.p.) tended to produce a place preference, but this effect was not significant. SCH23390 (0.03 mg/kg, s.c.) and sulpiride (40 mg/kg, s.c.) significantly attenuated the enhancement of the ethanol (75 mg/kg, i.p.)-induced place preference produced by the μ-opioid receptor agonist morphine (0.1 mg/kg, s.c.). In addition, SCH23390 (0.03 mg/kg, s.c.) also significantly attenuated the enhancement of the ethanol (75 mg/kg, i.p.)-induced place preference produced by the selective δ-opioid receptor agonist 2-methyl-4aα-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aα-octahydroquinolino[2,3,3,-g]isoquinoline (TAN-67; 20 mg/kg, s.c.). On the other hand, sulpiride (40 mg/kg) had no significant effect on the enhancement of the ethanol (75 mg/kg, i.p.)-induced place preference produced by TAN-67. These results suggest that D₁ and D₂ receptors may be involved in the rewarding mechanism of ethanol under psychological stress. In addition, D₁ receptors may participate in the rewarding effect of ethanol modulated by the activation of μ- and δ-opioid receptors, whereas D₂ receptors may participate in the rewarding effect of ethanol modulated by the activation of μ-opioid receptors, but not in that modulated by the activation of δ-opioid receptors.     

Evidence for 5-HT4 receptor involvement in the enhancement of acetylcholine release by p-chloroamphetamine in rat frontal cortex

The roles of endogenous serotonin (5-HT) and 5-HT receptor subtypes in regulation of acetylcholine (ACh) release in frontal cortex of conscious rats were examined using a microdialysis technique. Systemic administration (1 and 3 mg/kg, i.p.) of the 5-HT-releasing agent p-chloroamphetamine (PCA) elevated ACh output in a dose-dependent manner. Depletion of endogenous 5-HT by p-chlorophenylalanine significantly attenuated the facilitatory effect of PCA on ACh release. The PCA (3 mg/kg)-induced increase in ACh release was significantly inhibited by local application of the 5-HT₄ receptor antagonists RS23597 (50 μM) and GR113803 (1 μM), while the 5-HT_1A antagonist WAY-100135 (10 mg/kg, i.p.; 100 μM), 5-HT_1A/1B/β-adrenoceptor antagonists (−)-pindolol (8 mg/kg, i.p.) and (−)-propranolol (150 μM), 5-HT_2A/2C antagonist ritanserin (1 mg/kg, i.p.; 10 μM) and 5-HT₃ antagonist ondansetron (1 mg/kg, i.p.; 10 μM) failed to significantly modify the effect of PCA. These results suggest that PCA-induced enhancement of 5-HT transmission facilitates ACh release from rat frontal cortex at least in part through 5-HT₄ receptors.     

Pharmacological characterization of buprenorphine, a mixed agonist–antagonist with κ3 analgesia

Buprenorphine is a mixed opioid agonist/antagonist analgesic. This study was designed to determine the role of opioid receptor subtypes, especially κ₃, in buprenorphine-induced analgesia in mice. Buprenorphine, when injected systemically, revealed a potent analgesic effect by tailflick assay, with a biphasic dose–response curve, which was reversed by naloxone. The presence of analgesic cross-tolerance between buprenorphine and naloxone benzoylhydrazone (NalBzoH) and morphine indicated a role for κ₃ and μ receptor subtype in buprenorphine analgesia. Additional studies with selective opioid antagonists indicated κ₁ mechanisms of action. We did not detect any involvement of the δ receptor subtype. Low doses of buprenorphine antagonized morphine analgesia, while high doses of buprenorphine coadministered with morphine elicited increasing analgesia in a dose-dependent manner. These findings suggest that buprenorphine elicits analgesia through an interaction with κ₃ receptors and to a lesser extent with κ₁ as well as its activity as partial μ receptor agonist.     

Cardiovascular effects of central administration of clonidine in conscious cats

The effects on arterial blood pressure and heart rate after an intracerebroventricular (i.c.v.) administration of clonidine were investigated using conscious normotensive cats. Injection of clonidine (5–10 μg; 5 μl; i.c.v.) elicited a decrease in mean arterial pressure (MAP) and heart rate (HR) in a dose-dependent manner. The highest dose of 10 μg of clonidine decreased MAP and HR by 39 ± 3 mmHg and 74 ± 5 b.p.m., respectively (n = 7). Pretreatment with yohimbine, the α₂-adrenoceptor antagonist (8 μg; 5 μl; i.c.v.) blocked the cardiovascular responses to a subsequent i.c.v. injection of 10 μg clonidine (n = 7). Furthermore, preadministration of cimetidine (100 μg; 5 μl; i.c.v.), the H₂ histamine receptor antagonist with imidazoline receptor activating properties, prevented the decreases in MAP and HR to a subsequent i.c.v. injection of 10 μg clonidine (n = 7). By contrast, pretreatment with the specific I₁ imidazoline receptor blocker, efaroxan (100–500 μg; 5 μl; i.c.v.), failed to inhibit the cardiovascular effects of an i.c.v. administration of 10 μg clonidine (n = 7). These results suggest that the effects of centrally administered clonidine on MAP and HR are probably not mediated through activation of the I₁ subtype of imidazoline receptors in conscious cats. However, the cardiovascular effects elicited by i.c.v. administration of clonidine appear to result from stimulation of central α₂-adrenergic or the H₂ histaminergic-like receptors.     

Fluorescence imaging of GABAA receptor-mediated intracellular [Cl] in P19-N cells reveals unique pharmacological properties

This study describes the pharmacological properties of GABA_A receptors expressed in P19-N cells using fluorescence imaging of intracellular chloride with 6-methoxy-N-ethylquinolinium iodide (MEQ). We show that application of the GABA agonist, muscimol (10–200 μM), produces time- and concentration-dependent increases in intracellular [Cl⁻] that are blocked by bicuculline. Diazepam (10 μM) and pentobarbital (1 mM) potentiate muscimol-stimulation. These receptors exhibit novel pharmacological properties. The neurosteroid, 3α-hydroxy-5α-pregnane-20-one (1–10 μM) exhibited weak potency in enhancement of muscimol-stimulation. Ethanol (50 and 100 mM) exhibited high efficacy on muscimol responses, a 4- to 5-fold potentiation, respectively, of muscimol (10 μM) alone. GABA and muscimol allosterically modulated specific binding of [³H]flunitrazepam to differentiated P19 cells. Modulation of GABA_A receptor mediated increases in intracellular [Cl⁻] demonstrated stability in response magnitude from 7 to 15 days following removal of retinoic acid. In concert, GABA_A receptor subunit mRNA and protein expression patterns in these neuron-like cells were stable over the same period. Using RT-PCR we determined that differentiated P19 cells lack γ1, γ2L, α6 and δ subunit mRNAs while expressing α1, α2, α3, α4, α5, β1, β2, β3, γ2S and γ3. Furthermore, subunit specific antibody immunocytochemical labeling of cells with a neuronal morphology indicated the presence of α1, α2, α4, and γ2 subunits (the only subunits tested). Therefore, P19-N cells should prove useful to researchers in need of a model cell culture system in which to study function and regulation of neuronal GABA_A receptors.