Solubilization and Characterization of σ-Receptors from Guinea Pig Brain Membranes

The sigma-receptor, a distinct binding site in brain tissue that may mediate some of the psychotomimetic properties of benzomorphan opiates and phencyclidine, has been solubilized using the ionic detergent sodium cholate. Binding assays were performed with the solubilized receptor using vacuum filtration over polyethyleneimine-treated glass fiber filters. The pharmacological specificity of the solubilized binding site for sigma-receptor ligands is nearly identical to the membrane-bound form of the receptor, with the order of potencies for displacement of the selective sigma-ligand [3H]di-o-tolylguanidine ([3H]DTG) closely correlated. The stereoselectivity for (+)-benzomorphan opiate enantiomers was retained by the solubilized receptor. The soluble receptor retained high affinity for binding of [3H]DTG (KD = 28 +/- 0.5 nM) and (+)-[3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [(+)-[3H]3-PPP] (KD = 36 +/- 2 nM). Photoaffinity labeling of the solubilized receptor by [3H]p-azido-DTG, a sigma-selective photoaffinity label, resulted in labeling of a 29-kilodalton polypeptide identical in size to that labeled in intact membranes. Estimation of the Stokes radius of the [3H]DTG binding site was obtained by Sepharose CL-6B chromatography in the presence of 20 mM cholate and calculated to be 8.7 nm. This value was identical to the molecular size found for the binding sites of the sigma-selective ligands (+)-[3H]3-PPP and (+)-[3H]SKF-10,047, supporting the hypothesis that all three ligands bind to the same macromolecular complex.     

Effects of Oxygen Depletion on Norepinephrine- and Carbachol-Stimulated Phosphoinositide Turnover in Rat Brain Slices

We examined the effects of in vitro anoxia and in vivo hypoxia (8% O2/92% N2) on norepinephrine (NE)- and carbachol-stimulated phosphoinositide (PI) turnover in rat brain slices. The formation of 3H-labeled polyPI in cortical slices was impaired by in vitro anoxia and fully restored by reoxygenation. Accumulation of 3H-labeled myo-inositol phosphates (3H-IPs) stimulated by 10(-5) M NE was significantly reduced by anoxia (control at 60 min, 1,217 +/- 86 cpm/mg of protein; anoxia for 60 min, 651 +/- 82 cpm/mg; mean +/- SEM; n = 5; p less than 0.01), and reoxygenation following anoxia resulted in overshooting of the accumulation (control at 120 min, 1,302 +/- 70 cpm/mg; anoxia for 50 min plus oxygenation for 70 min, 1,790 +/- 126 cpm/mg; n = 5; p less than 0.01). The underlying mechanisms for the two phenomena--the decrease caused by anoxia and the overshooting caused by reoxygenation following anoxia--seemed to be completely different because of the following observations. (a) Although the suppression of NE-stimulated accumulation at low O2 tensions was also observed in Ca2+-free medium, the overshooting in response to reoxygenation was not. (b) Carbachol-stimulated accumulation was significantly reduced by anoxia and was restored by reoxygenation only to control levels. Thus, the postanoxic overshooting in accumulation of 3H-IPs seems to be a specific response to NE. (c) The decrease observed at low O2 tensions was due to a decrease in Emax value, whereas the postanoxic overshooting was due to a decrease in EC50 value.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological Characteristics and Distributions of σ and Phencyclidine Receptors in the Animal Kingdom

The phylogenetic distributions ofσ- and phencyclidine receptors in neural tissues of 13 species and the pharmacological characteristics of these receptors in whole sea anemone and neural tissues of the guinea pig, chicken, and frog were studied. Specific binding of [³H]haloperidol and [³H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine, ligands that bind with high affinity to σ- and phencyclidine receptors, respectively, was detected in all organisms examined. The order of potencies of various ligands to inhibit 1 nM [³H]haloperidol binding in brains of frogs and guinea pigs or 1 nM [³H]N-[1-(2-thienyl)cyclohexyl]-3,4–piperidine in chicken or guinea pig brain homogenates was very similar. However, the characteristics and stereospecificity of binding of the two radioligands in sea anemone were different than in higher organisms. The results suggest that σ– and phencyclidine binding sites are evolutionarily old, as the characteristics of the two sites are well preserved over a range of vertebrate phyla.     

Electroconvulsive Shock Increases α1b-but Not α1a-Adrenoceptor Binding Sites in Rat Cerebral Cortex

Repeated administration of electroconvulsive shock (ECS) increases [3H]prazosin binding to alpha 1-adrenoceptors in rat cerebral cortex. In contrast, [3H]WB4101 binding in cortex has been reported to be unchanged after ECS. [3H]Prazosin labels two alpha 1-adrenoceptor subtypes, termed alpha 1a and alpha 1b, whereas [3H]WB4101 labels the alpha 1a subtype preferentially. The purpose of this study was to determine whether ECS increases one or both alpha 1-adrenoceptor subtypes in rat cerebral cortex. We found that treatment of rats with ECS once daily for 10-12 days increased [3H]prazosin binding in cortex by about 25% but did not significantly alter [3H]WB4101 binding to alpha 1-adrenoceptors. Measurement of alpha 1a and alpha 1b receptors by competition analysis of the selective alpha 1a antagonist 5-methylurapidil against [3H]prazosin and measurement of [3H]prazosin binding in homogenates preincubated with chlorethylclonidine, which alkylates alpha 1b binding sites, also indicated that the ECS-induced increase in alpha 1-adrenoceptors is confined to the alpha 1b subtype. In contrast to its effect on [3H]prazosin binding, ECS did not increase phosphoinositide hydrolysis as measured by [3H]inositol 1-phosphate accumulation in slices of rat cerebral cortex stimulated by either norepinephrine or phenylephrine. The failure of ECS to increase [3H]inositol 1-phosphate accumulation stimulated by phenylephrine, which is a partial agonist for this response, suggests that spare receptors do not account for the apparent absence of effect of ECS on alpha 1-adrenoceptor-mediated phosphoinositide hydrolysis.     

Desensitization of Muscarinic Receptor-Coupled Phosphoinositide Hydrolysis in Rat Hippocampus: Comparisons with the α1-Adrenergic Response

In the presence of lithium, carbamylcholine chloride (carbachol) and epinephrine increase the accumulation of inositol monophosphate severalfold in hippocampal slices from the rat. The stimulation by carbachol (EC50, 31 microM) is mediated by muscarinic receptors, whereas the effects of epinephrine (EC50, 2 microM) are due to activation of alpha 1-adrenergic receptors. The responses of epinephrine and carbachol are additive, even under conditions that significantly reduce the levels of phosphoinositides and free inositol, suggesting that the muscarinic and adrenergic receptors may be located on separate cells. At concentrations that saturate their respective receptors, epinephrine induces an increase in inositol monophosphate that is linear with time to at least 60 min, whereas the response to carbachol begins to reach a plateau by 20-40 min. When hippocampal slices are preincubated with saturating concentrations of carbachol, the subsequent response to carbachol is reduced by 42%. However, preincubation with carbachol or epinephrine has no effect on the subsequent response to epinephrine. Despite the lack of adrenergic desensitization by this paradigm, preexposure of hippocampal slices to the tumor-promoting phorbol ester, phorbol 12,13-dibutyrate, reduces the response to epinephrine to a significantly greater degree (57%) than it reduces the muscarinic response (25%). These studies indicate that, although they utilize the same second messenger, the muscarinic and alpha 1-adrenergic receptors of hippocampal slices have different characteristics and regulatory mechanisms.     

Functional and Binding Properties of σ Receptors in Rat Cerebellum

Abstract: Autoradiographic studies have shown that σ receptors are enriched in the locus coeruleus, the origin of noradrenergic projections to the cerebellum, as well as in the Purkinje, molecular, and granular layers and the interpositus cerebellar nucleus of the cerebellum itself. In contrast, the cerebellum is relatively poor in phencyclidine (PCP) binding sites, which have been historically confused with σ sites. The high ratio of σ to PCP receptors in cerebellum is advantageous for discriminating σ-mediated physiological effects. σ agonists and antagonists have been shown to regulate N -methyl- d -aspartate (NMDA)-stimulated norepinephrine release in hippocampus, which is innervated by locus coeruleus projections. We now report that σ drugs also regulate norepinephrine release from cerebellum. In contrast to findings in the hippocampus, where regulation is via σ₁ and σ₂ receptors, σ-mediated regulation in cerebellum seems to be primarily via σ₁ receptors. In radioligand binding studies, we find that σ receptors primarily of the σ₁ type are present in the cerebellum. We further report that binding to σ receptors in cerebellum is not affected by the addition of NMDA or glycine or by the presence of NMDA antagonists, suggesting that σ receptors are not located within the NMDA-operated cation channel in this brain region.     

In Vivo Determination of 5-Hydroxytryptamine Receptor-Stimulated Phosphoinositide Turnover in Rat Brain

Phosphoinositide turnover stimulated by 5-hydroxytryptamine (5-HT) receptors in the intact rat brain was studied using an in vivo method. Phosphoinositides in the rat brain were prelabeled with [3H]inositol injected into the lateral cerebral ventricles. The rats were killed by microwave irradiation after 48 h and the contents in the frontal cortex of 3H-inositol phosphates, [3H]inositol-1-monophosphate [( 3H]IP1), [3H]inositol-1,4-bisphosphate [( 3H]IP2), and a mixture of [3H]inositol-1,4,5-trisphosphate and [3H]inositol-1,3,4-trisphosphate [( 3H]IP3) were assayed by HPLC. Lithium treatment (10 mEq/kg, i.p., 2 h before) increased the content of [3H]IP1 and [3H]IP2. 5-Methoxy-N,N-dimethyltryptamine (5-MeODMT) and quipazine, 5-HT agonists, significantly increased the amount of 3H-inositol phosphates under lithium pretreatment. The response to 5-MeODMT was inhibited by ritanserin, a 5-HT2 antagonist, but not by (-)-propranolol, a 5-HT1 antagonist. These results suggest that phosphoinositide turnover in the rat frontal cortex in vivo is stimulated by 5-HT2 receptor activation. It is considered that this method will be useful for measurement of 5-HT2 receptor-stimulated phosphoinositide turnover in vivo to examine the in vivo effects of various psychotropic drugs such as antidepressants.     

Potassium Ion Facilitation of Phosphoinositide Turnover Activation by Muscarinic Receptor Agonists in Rat Brain

In rat hippocampal slices kept in Krebs-Henseleit medium, an increase of K+ ions to 12 mM potentiates the stimulation of phosphoinositide turnover elicited by carbachol and (+/-)-cis-methyldioxolane. Oxotremorine is inactive if tested in Krebs-Henseleit medium but it stimulates by 220% the phosphoinositide turnover when K+ is increased to 12 mM. The K+ facilitation of the carbachol stimulation of phosphoinositide turnover was blocked by pirenzepine, a muscarinic antagonist. This drug was equally potent in inhibiting the carbachol stimulation of phosphoinositide turnover both in normal and 12 mM K+ Krebs medium. This facilitatory effect of K+ appears to be preferential for muscarinic receptors, since it failed to increase the activation of phosphoinositide breakdown induced by norepinephrine and histamine. The K+ potentiation of the muscarinic stimulation of phosphoinositide turnover is not mediated by a release of one of the endogenous neurotransmitters stored in these slices because such a facilitation occurs in Ca2+-deprived Krebs-Henseleit medium and failed to occur following a depolarizing dose of veratrine. Our experiments excluded that K+ facilitates carbachol stimulation of phosphoinositide turnover because it modifies the binding characteristics of muscarinic receptors; however, they cannot exclude that K+ acts at the receptor transducer coupling.     

Effects of Systemically Administered Lithium on Phosphoinositide Metabolism in Rat Brain, Kidney, and Testis

A single subcutaneous dose of 10 mEq/kg LiCl gives rise to an increase in the cerebral cortex level of myo-inositol-1-P (I1P) that closely follows cortical lithium levels and, at maximum, is 40-fold above the control value. Kidney and testis show smaller increases in I1P level following LiCl administration. The I1P level is still sixfold greater than that of untreated rat cortex 72 h later. In cortex, parallel increases also occur in myo-inositol-4-P (I4P) and myo-inositol 1,2-cyclic-P (cI1,2P), whereas myo-inositol-5-P (I5P) remains unchanged. The cortical increases in I1P and I4P levels are partially reversed by administering 150 mg/kg of atropine 22 h after the LiCl, treatment that does not affect cI1,2P. When doses of LiCl from 2 to 17 mEq/kg are given, the cerebral cortex levels of I1P and myo-inositol, measured 24 h later, are found to reach a plateau at about 9 mEq/kg of LiCl, whereas cortical lithium levels continued to increase with greater LiCl doses. Levels of all three of the brain phosphoinositides are unchanged by the 10 mEq/kg LiCl dose, as is the uptake of 32Pi into these lipids. Chronic dietary administration of LiCl for 22 days showed that the effects of lithium on I1P and myo-inositol levels persist for that period. Over the course of the chronic administration of the lithium, levels of I1P, myo-inositol, and of lithium in cortex remained significantly correlated. We believe that these increases in inositol phosphates result from endogenous phosphoinositide metabolism in cerebral cortex and that lithium is capable of modulating that metabolism by reducing cellular myo-inositol levels. The size of the effect is a function of both lithium dose and the degree of stimulation of receptor-linked phosphoinositide metabolism. This property of lithium may explain part of its ability to moderate the symptoms of mania. Our chronic study suggests that prolonged administration of LiCl does not result in compensatory changes in myo-inositol-1-P synthase or myo-inositol-1-phosphatase.     

Effects of Human Type 1α Metabotropic Glutamate Receptor Expression Level on Phosphoinositide and Ca2+ Signalling in an Inducible Cell Expression System

Abstract: Stable expression of the human type 1α metabotropic glutamate (mGlu1α) receptor was achieved in Chinese hamster ovary cells using an isopropyl-β- d -thiogalactoside (IPTG)-repressible expression system. Treatment of the cells with IPTG resulted in a time- and concentration-dependent induction of receptor expression. Maximal expression was obtained after treatment of the cells with 100 µ M IPTG for 20 h, leading to a marked increase in receptor immunoreactivity detected by western blot, >30-fold stimulation of ³H-labelled inositol phosphate (³H-InsP) production, and a robust increase in intracellular calcium concentration in single cells after stimulation with 20 µ M quisqualate. The basal level of ³H-InsP accumulation in cells induced with IPTG was increased by two- to threefold as compared with control cells; however, this basal activity was found to be dependent on glutamate released by the cells into the incubation medium. Following IPTG treatment, stable expression of the mGlu1α receptor was maintained for at least 1 week. Taken together, these results clearly indicate the advantages of working with an inducible expression system when studying the biochemical and pharmacological properties of the human mGlu1α receptor in transfected cells.     

Modulation of Rat Brain Cortical α-Adrenoceptors by Treatment with Hydrocortisone for 10 Days

The role of glucocorticoids in the modulation of central alpha 2-receptor mechanisms was investigated by in vitro receptor binding studies. [3H]Clonidine and [3H]idazoxan were used as radioligands. The alpha 2-receptor subtypes and guanine nucleotide sensitivity were studied in homologue and heterologue displacement experiments following hydrocortisone treatment (25 mg/kg s.c.) for 10 days. High and low agonist affinity states of the alpha 2-receptor could be identified in 3H-antagonist-agonist and 3H-agonist-antagonist displacement experiments, which may correspond to different regulatory protein-nucleotide associated forms of the receptor. In the presence of 10 microM GTP, the high-affinity binding was depressed. Following hydrocortisone treatment, there was no detectable change either in the affinity or the binding site concentration of clonidine in homologue displacement ("cold saturation") experiments. The affinity of idazoxan, however, was depressed. The effect of GTP was similar to the controls in this experimental arrangement. In contrast, in heterologue binding studies the high-affinity binding site was not demonstrable and the amount of low-affinity binding increased following the hydrocortisone treatment. The high-affinity site reappeared in the presence of GTP. The change in GTP sensitivity suggests that the nucleotide regulatory system may be involved in the action of adrenal steroids on central alpha 2-receptoral mechanisms.     

Rat Brain α-Mannosidase: Purification, Properties, and Interaction with Its Antibodies

Abstract: α - d -Mannosidase (EC 3.2.1.24.) was purified to homogeneity from adult rat brain. The enzyme, of apparent molecular weight 397,000, appears to be formed of subunits of molecular weight 120,000 made of two protomers (62,000) bound by disulfide bridges. Isoelectric focusing gives two bands, of pi 5.40 and 5.15. Both isoenzymes seem to have the same pH curve (a small peak of activity at pH 4.5 and a maximum of activity around pH 6.0). These two isoenzymes are immunologically related.     

Similarity Between Rat Brain Nicotinic α-Bungarotoxin Receptors and Stably Expressed α-Bungarotoxin Binding Sites

Abstract: The present results demonstrate stable expression of α-bungarotoxin (α-BGT) binding sites by cells of the GH₄C₁ rat pituitary clonal line. Wild-type GH₄C₁ cells do not express α-BGT binding sites, nor do they contain detectable mRNA for nicotinic receptor α2, α3, α4, α5, α7, β2, or β3 subunits. However, GH₄C₁ cells stably transfected with rat nicotinic receptor α7 cDNA (α7/GH₄C₁ cells) express the transgene abundantly as mRNA, and northern analysis showed that the message is of the predicted size. The α7/GH₄C₁ cells also express saturable, high-affinity binding sites for ¹²⁵I-labeled α-BGT, with a K_D of 0.4 nM and B_max of 3.2 fmol/10⁶ intact cells. ¹²⁵I-α-BGT binding affinities and pharmacological profiles are not significantly different for sites in membranes prepared either from rat brain or α7/GH₄C₁ cells. Furthermore, K_D and K_i values for ¹²⁵I-α-BGT binding sites on intact α7/GH₄C₁ cells are essentially similar to those for hippocampal neurons in culture. Sucrose density gradient analysis showed that the size of the α-BGT binding sites expressed in α7/GH₄C₁ cells was similar to that of the native brain α-BGT receptor. Chronic exposure of α7/GH₄C₁ cells in culture to nicotine or an elevated extracellular potassium concentration induces changes in the number of α-BGT binding sites comparable to those observed in cultured neurons. Collectively, the present results show that the properties of α-BGT binding sites in transfected α7/GH₄C₁ cells resemble those for brain nicotinic α-BGT receptors. If the heterologously expressed α-BGT binding sites in the present study are composed solely of α7 subunits, the results could suggest that the rat brain α-BGT receptor has a similar homooligomeric structure. Alternatively, if α-BGT binding sites exist as heterooligomers of α7 plus some other previously identified or novel subunit(s), the data would indicate that the α7 subunits play a major role in determining properties of the α-BGT receptor.     

α1-Adrenergic Receptors in Neuronal Cultures from Rat Brain: Increased Expression in the Spontaneously Hypertensive Rat

Neuronal cells in primary culture from 1-day-old brains of normotensive, Wistar-Kyoto strain (WKY) and spontaneously hypertensive (SH) rats have been utilized to study the expression of alpha 1-adrenergic receptors. Binding of a selective alpha 1 antagonist, [125I]2-[beta-(4-hydroxy-3-iodophenyl)-ethylaminomethyl]-tetralone ([125I]HEAT) to neuronal membranes prepared from primary brain cultures of WKY and SH rats was 75-80% specific, rapid, and time-dependent although the binding was 1.5-2 times higher in neuronal membranes from SH rat brain cultures. Kinetic analysis of the association and dissociation data demonstrated no significant differences between rat strains. Competition-inhibition experiments provided IC50 values for various antagonists and agonists in the following order: prazosin less than phentolamine less than yohimbine less than phenylephrine less than norepinephrine less than propranolol, suggesting that [125I]HEAT bound selectively to alpha 1-adrenergic receptors. Scatchard analysis of the binding data provided straight lines for both strains of rats, indicating the presence of a homogeneous population of binding sites. It also showed that the increase in the binding in neuronal cells from SH rat brains over those from normotensive WKY controls was a result of an increase in the number of alpha 1-adrenergic receptors. Incubation of neuronal cultures from both strains of rats with phenylephrine, an alpha 1-adrenergic agonist, caused a time- and dose-dependent decrease in the binding of [125I]HEAT. This decrease was due to a decrease in the number of alpha 1-adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of [3H](–)-SKF-10,047 with Brain σ Receptors: Characterization and Autoradiographic Visualization

The sigma opiates differ from other opiates in their stimulatory and psychotomimetic actions. The sigma opiate [3H](-)-SKF-10,047 has been used to characterize sigma receptors in rat nervous tissue. Binding of [3H](-)-SKF-10,047 to rat brain membranes was of high affinity, saturable, and reversible. Scatchard analysis revealed the apparent interaction of this drug with two distinct binding sites characterized by affinities of 0.03 and 75 nM (5 mM Tris-HCl buffer, pH 7.4, at 4 degrees C). Competition analyses involving rank order determinations for a series of opiates and other drugs indicate that the high-affinity binding site is the mu opiate receptor. The lower-affinity site (revealed after suppression of mu and delta receptor binding) has been identified as the sigma opiate/phencyclidine receptor. In vitro autoradiography has been used to visualize neuroanatomical patterns of receptors labeled using [3H](-)-SKF-10,047 in the presence of normorphine and [D-Ala2,D-Leu5]enkephalin to block mu and delta interactions, respectively. Labeling patterns differ markedly from those for mu, delta, or kappa receptors. The highest densities (determined by quantitative autoradiography) are found in the medial portion of the nucleus accumbens, amygdaloid nucleus, hippocampal formation, central gray, locus coeruleus, and the parabrachial nuclei. Receptors in these structures could account for the stimulatory, mood-altering, and analgesic properties of the sigma opiates. Although not the most selective sigma opiate ligand, [3H](-)-SKF-10,047 binds to sigma opiate receptors in brain, and this interaction can be readily distinguished from its interactions with other classes of brain opiate receptors.     

3α-Hydroxysteroid Oxidoreductase in Rat Brain

Abstract: We describe a simple procedure for the microassay of 3α-hydroxysteroid oxidoreductase in homogenates of rat brain. This enzyme converts dihydrotestosterone to 3α-androstandiol. We have mapped the distribution of the enzymatic activity in 14 regions of the rat brain. The highest activities were observed in homogenates of olfactory bulb (51/nmol/mg protein/h) and olfactory tubercle (29 nmol/mg protein/h). Substantially lower values were seen in the other brain regions, including thalamus, caudate nucleus, frontal cortex, hippocampus, hypothalamus, and preoptic area (6–20 nmol/mg protein/ h).     

Ontogenesis of α2-Adrenoceptor Coupling with GTP-Binding Proteins in the Rat Telencephalon

The ontogenesis of alpha 2-adrenoceptors and GTP-binding proteins and their coupling activity were investigated in telencephalon membranes of developing rats. The manganese-induced elevation of [3H]clonidine binding was increased in an age-dependent manner but the guanosine 5'-O-(3-thio)triphosphate-induced decrease in binding did not change. The extent of the binding of [3H]clonidine at 15 nM (saturable concentration) increased in an age-dependent manner and reached the adult level at 4 days after birth. Cholera toxin and pertussis toxin catalyzed ADP-ribosylation of proteins of 46 and 41/39 kilodaltons (kDa) in solubilized cholate extracts of the membranes. The 41/39-kDa proteins ADP-ribosylated by pertussis toxin (Gi alpha + Go alpha) were increased with age and reached the adult level at day 12, whereas the 46-kDa protein (Gs alpha) reached its peak on day 12 and then decreased to the fetal level at the adult stage. The immunoblot experiments of the homogenates with antiserum (specific antibody against alpha- and beta-subunit of GTP-binding proteins) demonstrated that the 39-kDa alpha-subunit of (Go alpha) and the 36-kDa beta-subunit of GTP-binding protein (beta 36) increased with postnatal age. In contrast, 35-kDa beta-subunit (beta 35) did not change. From these results, it is suggested that the coupling activity of alpha 2-adrenoceptor with GTP-binding protein gradually develops in a manner parallel with the increase of alpha 2-adrenoceptor and pertussis toxin sensitive GTP-binding proteins, Gi, and that alpha 39 beta 36 gamma may be related to the differentiation and/or growth of nerve cells in rat telencephalon.     

Cloning, Characterization, and Distribution of a μ-Opioid Receptor in Rat Brain

Abstract: We report the isolation and characterization of a rat cDNA clone encoding a μ-opioid receptor. This receptor, a 398 amino acid protein, shares 59% overall identity with the mouse Δ-and K -opioid receptors. Transient expression of the receptor in COS cells revealed high-affinity binding of μ-selective opioid antagonists and agonists, with a K _D for naloxone ∼1.5 n M , and for [D-Ala², N -Me-Phe⁴, Gly⁵-ol]-enkephalin (DAMGO) and morphine at the high-affinity site of 2–4 n M , confirming a μ-opioid pharmacological profile. Northern blotting and in situ hybridization histoohemistry revealed that the μ-opioid receptor mRNA was expressed in many brain regions, including cerebral cortex, caudate putamen, nucleus accumbens, olfactory tubercle, septal nuclei, thalamus, hippocampus, and medial habenular nucleus, in keeping with the known distribution of the μ-opioid receptor.     

Regional Variations in α1-Adrenergic Receptor Subtypes in Rat Brain

alpha 1-Adrenergic receptor subtypes were differentiated by their affinities for the competitive antagonist WB 4101 and their sensitivities to inactivation by chlorethylclonidine (CEC) in eight rat brain regions. WB 4101 showed low Hill coefficients for inhibition of specific 125I-[2-beta-(4-hydroxyphenyl)ethylaminomethyl]tetralone (125IBE) binding in all regions. Nonlinear regression analysis showed that there were two binding sites with different affinities for WB 4101 in each region. The proportions of these sites varied among regions, although the affinity of WB 4101 for each site remained constant. Thalamus and cerebral cortex had the highest proportion of low-affinity sites, whereas hippocampus and pons-medulla had the highest proportion of high-affinity sites. Pretreatment with CEC in hypotonic buffer significantly reduced the density of 125IBE binding sites in all brain regions. Cerebral cortex and cerebellum had the highest proportion of CEC-sensitive sites, whereas hippocampus and spinal cord had the highest proportion of CEC-insensitive sites. There was a significant correlation between the proportion of binding sites with a low affinity for WB 4101 and those sensitive to inactivation by CEC.     

Regulation of α2A-Adrenergic Receptor Expression by Epinephrine in Cultured Astroglia from Rat Brain

Abstract: Epinephrine (Epi) mediates various physiological effects via α_2A-adrenergic receptors (α_2A-ARs). Studies in mice with a point mutation in the gene for α_2A-AR have shown that these receptors are responsible for the centrally mediated depressor effects of α₂-AR agonists. These studies underscore the importance of understanding the basic cellular mechanisms involved in the expression of α_2A-ARs, of which little is known. We use astroglia cultured from the hypothalamus and brainstem of adult Sprague-Dawley rats as a model system in which to study factors that regulate α_2A-AR expression. These cells contain α₂-ARs, which are predominately of the α_2A-AR subtype. Our studies have shown that Epi causes a dose- and time-dependent decrease in steady-state levels of α_2A-AR mRNA and number of α_2A-ARs, effects that are mediated via α₁- and β-adrenergic receptors (α₁-ARs and β-ARs). These effects of Epi on α_2A-AR mRNA and α_2A-AR number are mimicked by activation of protein kinase C or increases in cellular cyclic AMP, which are intracellular messengers activated by α₁-ARs and β-ARs, respectively. Taken together, these results indicate that expression of α_2A-ARs is regulated in a heterologous manner by Epi, via α₁-AR- and β-AR-mediated intracellular pathways.