Rat Cerebral Cortical Membranes Research Articles

Multiple components of the A1 adenosine receptor-adenylate cyclase system are regulated in rat cerebral cortex by chronic caffeine ingestion.

The effects of chronic caffeine on the A1 adenosine receptor-adenylate cyclase system of rat cerebral cortical membranes were studied. Caffeine treatment significantly increased the number of A1 adenosine receptors as determined with the A1 adenosine receptor antagonist radioligand [3H]xanthine amine congener (XAC). R-PIA (agonist) competition curves constructed with [3H]XAC were most appropriately described by a two affinity state model in control membranes with a KH of 2.1 +/- 0.8 and a KL of 404 +/- 330 nM with 50 +/- 4% of receptors in the high affinity state (%RH). In contrast, in membranes from treated animals, there was a marked shift towards the high affinity state. In three of seven animals all of the receptors were shifted to a unique high affinity state which was indistinguishable from the KH observed in membranes from control animals. In four of seven animals the %RH increased from 50 to 69% with KH and KL indistinguishable from the control values. Thus, the agonist specific high affinity form of the receptor was enhanced following caffeine treatment. Maximal inhibition of adenylate cyclase activity in cerebral cortical membranes by R-PIA (1 microM) was significantly increased by 28% following caffeine treatment, consistent with an increased coupling of receptor-Gi protein with adenylate cyclase. Importantly, the quantity of Gi (alpha i) in rat cerebral cortex, determined by pertussis toxin-mediated labeling, was also increased to 133% of control values by this treatment. Thus, multiple components and interactions of the A1 adenosine receptor-adenylate cyclase complex are regulated by caffeine. These changes are likely compensatory measures to offset blockade of A1 receptors in vivo by caffeine and lead to a sensitization of this inhibitory receptor system.

Himbacine recognizes a high affinity subtype of M 2 muscarinic cholinergic receptors in the rat cerebral cortex

The in vitro receptor binding properties of a muscarinic antagonist himbacine have been studied in rat cerebral cortical, cardiac and ileal membranes. Himbacine displayed high affinity ( K H = 2.94 nM) 19%, and low affinity ( K L = 71.2 nM) for the remaining muscarinic receptors in rat cerebral cortex. This high affinity of himbacine agrees with its affinity for the 62% of cerebral cortical [ 3H]AF-DX 116 binding sites ( K H = 2.30 nM). The affinity of himbacine for cardiac receptors ( K i = 9.06 nM) and ileal receptors ( K i = 12.4 nM) was the same. Therefore, himbacine appears to be a high-affinity M 2-selective ligand which recognizes a subtype of M 2 receptors in the cerebral cortex.

Effect of long-term rolipram administration on the sensitivity of [formula omitted] in rat brain

The effects of single and chronic doses of rolipram on the sensitivity of α 2- adrenoceptors have been compared with the phosphodiesterase inhibitors, isobutylmethylxanthine (IBMX) and ICI 63,197, and the antidepressant, desipramine. While pretreatment with a single dose of rolipram, ICI 63,197 or IBMX administered either 1 or 24 h prior to clonidine (0.1 mg/kg) enhanced clonidine-induced hypothermia and hypoactivity, chronic dosing (twice daily for 14 days) with desipramine (10 mg/kg) or rolipram (5 mg/kg) antagonized these behavioural effects. In contrast, chronic dosing with IBMX or ICI 63,197 failed to antagonize clonidine-induced hypothermia and hypoactivity. In binding studies neither ICI 63,197, IBMX, rolipram nor desipramine induced changes in the binding of 3H-labelled clonidine to rat cerebral cortical membranes following chronic administration. The failure of ICI 63,107 and IBMX to antagonize clonidine-induced hypothermia and hypoactivity suggests that the antidepressant effect of rolipram is independent of its phosphodiesterase inhibitor property.

Effects of choline and inositol on 3H-dihydroalprenolol and 125I-iodocyanopindolol bindings to beta-adrenergic receptors of the guinea pig cerebral cortical membranes.

This experiment was designed to examine the effects of choline and inositol in Eagle's minimal essential medium (MEM), because the values of specific binding were increased when the 3H-dihydroalprenolol (3H-DHA) binding assay of β-adrenoceptors in the guinea pig and rat cerebral cortical membranes was carried out in Eagle's MEM instead of 60 mM Tris-HCl, 20 mM MgCl2 buffer (pH 7.4). All chemicals contained in the MEM were added to the 3H-DHA binding mixture and the values of the specific binding were compared with the control values obtained in the absence of the chemicals. The presence of choline (0.1-10 μM) or inositol (0.1-10 μM) induced an increase of the values of the specific binding. These effects of choline and inositol were observed after preinclubation for 20 and 1 min, respectively. After 30 min of incubation, no changes were observed upon removal of these chemicals. Furthermore, choline and inositol significantly increased the β-adrenoceptor density (β ) when 3H-DHA and I-iodocyanopindolol were used as the radioligands, as determined by Scatchard analysis, but no significant change was observed in the value of the dissociation constant (Kd) in 3H-DHA binding. These results suggest that choline and inositol in the membranes could have a crucial role in drug-receptor interaction.

3H-substance P binding to solubilized rat cerebral cortical membranes: Effects of GTP and cations

3H-substance P binding to solubilized rat cerebral cortical membranes: Effects of GTP and cations

Hydrodynamic properties of adenosine Ri receptors solubilized from rat cerebral-cortical membranes.

Adenosine Ri receptors and inhibitory guanine-nucleotide-regulatory components were solubilized from rat cerebral-cortical membranes with sodium cholate. (-)-N6-Phenylisopropyl[2,8-3H]adenosine [( 3H]PIA) binds with high affinity to the soluble receptors, which retain the pharmacological specificity of adenosine Ri receptors observed in membranes. The binding is regulated by bivalent cations and guanine nucleotides. Bivalent cations increase [3H]PIA binding by increasing both the affinity and the apparent number of receptors. Guanine nucleotides decrease agonist binding by increasing the dissociation of the ligand-receptor complex. Adenosine agonists stabilize the high-affinity form of the soluble receptor. The hydrodynamic properties of the adenosine receptor were determined with cholate extracts of membranes that were treated with [3H]PIA. Sucrose-gradient-centrifugation analysis indicates that the receptor has a sedimentation coefficient of 7.7 S. The receptor is eluted from Sepharose 6B columns with an apparent Stokes radius of 7.2 nm. Labelling of either sucrose-gradient or gel-filtration-column fractions with pertussis toxin and [32P]-NAD+ reveals that both the 41,000- and 39,000-Mr substrates overlap with the receptor activity. These studies suggest that the high-affinity adenosine-receptor-binding activity in the cholate extract represents a stable R1-N complex.

Specific binding of [ 3H]inositol trisphosphate to rat cerebral cortical microsomal membranes

Specific binding sites for inositol-(1,4,5)-trisphosphate (IP 3) were demonstrated in rat cerebral cortical microsomal membranes using [ 3H]IP 3 as the ligand. The binding was displaceable (IC 50 for IP 3 = 28 nM), high affinity ( K d = 20 nM) and saturable ( B max = 7.75 pmol/mg protein). Kinetic studies showed an extremely rapid time-course of [ 3H]IP 3 binding with half-maximal binding achieved in less than 1 min and equilibrium binding was attained by around 5 min. These results are in accord with the proposed function of IP 3 in mobilizing intracellular calcium from endoplasmic reticulum.

Differential regulation of M 2 muscarinic receptors by guanine nucleotides in rat cerebral cortical and cardiac membranes

Differential regulation of M 2 muscarinic receptors by guanine nucleotides in rat cerebral cortical and cardiac membranes

Guanine nucleotide and NaF stimulation of phospholipase C activity in rat cerebral-cortical membranes. Studies on substrate specificity.

Guanyl-5'-yl imidodiphosphate (p[NH]ppG) stimulated a rapid phospholipase C-mediated breakdown of exogenously added phosphatidylinositol 4,5-bisphosphate (PIP2) in rat cerebral-cortical membranes, with half-maximal activation at approx. 33 microM. NaF stimulated phospholipase C activity, with half-maximal activation at 0.5 mM. Stimulation of phospholipase C activity by NaF exhibited pH optima at approx. 5.5 and 7.0, with the stimulatory activity at pH 7.0 greater than that at pH 5.5. With p[NH]ppG, only stimulation at pH 7.0 was observed. Neither p[NH]ppG nor NaF stimulated hydrolysis of added phosphatidylinositol (PI) or phosphatidylinositol 4-phosphate (PIP). Mg2+ (0.5 mM) potentiated p[NH]ppG-stimulated breakdown of PIP2. Ca2+ increased basal and p[NH]ppG-stimulated breakdown of PIP2. PI breakdown was stimulated only by high Ca2+ concentrations and was unaffected by p[NH]ppG at any Ca2+ concentration examined. These results indicate that, in cerebral-cortical membranes, activation of phospholipase C by guanine nucleotides or fluoride directly increases a phospholipase C activity which specifically hydrolyses PIP2.

Molecular probes for extracellular adenosine receptors

Derivatives of adenosine receptor agonists ( N 6-phenyladenosines) and antagonists (1,3-dialkyl-8-phenylxanthines) bearing functionalized chains suitable for attachment to other molecules have been reported [Jacobson et al., J. med. Chem. 28, 1334 and 1341 (1985)]. The “functionalized congener” approach has been extended to the synthesis of spectroscopic and other probes for adenosine receptors that retain high affinity ( K i ∼ 10 −9−10 −8 M) in A 1-receptor binding. The probes have been synthesized from an antagonist xanthine amine congener (XAC) and an adenosine amine congener (ADAC). [ 3H]ADAC has been synthesized and found to bind highly specifically to A 1-adenosine receptors of rat and calf cerebral cortical membranes with K D values of 1.4 and 0.34 nM respectively. The higher affinity in the bovine brain, seen also with many of the probes derived from ADAC and XAC, is associated with phenyl substituents. The spectroscopic probes contain a reporter group attached at a distal site of the functionalized chain. These bifunctional ligands may contain a spin label (e.g. the nitroxyl radical TEMPO) for electron spin resonance spectroscopy, or a fluorescent dye, including fluorescein and 4-nitrobenz-2-oxa-l,3-diazole (NBD), or labels for 19F nuclear magnetic resonance spectroscopy. Potential applications of the spectroscopic probes in characterization of adenosine receptors are discussed.

Effects of slow cooling in situ and freeze-thaw procedures on [ 3H]hemicholinium-3 binding in rat cerebral cortical plasma membranes

Effects of slow cooling in situ and freeze-thaw procedures on [ 3H]hemicholinium-3 binding in rat cerebral cortical plasma membranes

Biphasic binding of 125I-iodocyanopindolol to beta-adrenergic receptors in rat cerebral cortical membranes. I. Assessment by the use of agonists.

When saturation binding studies of β-adrenoceptors in rat brain with 125I-iodocyanopindolol (125I-ICYP) were carried out by means of radioligand binding assay, the Scatchard plots showed a biphasic character. The addition of guanosine triphosphate (GTP, 3× 10-4 M) had no effect on the biphasic character. The inhibition constants of various ligands for the high-and low-affinity sites of 125I-ICYP binding were determined from the biphasic Scatchard plots. Competition by the agonists at the high-affinity sites was in the order of l-isoproterenol> l-epinephrine > l-norepinephrine, whereas at the low-affinity sites, the order was l-isoproterenol > l-epinephrine = l-norepinephrine. The specificities of the agonists for the high-and low-affinity sites were high = low (l-isoproterenol), high >low (l-epinephrine), or high < low (l-norepinephrine). These results suggest that two distinct types of β-adrenoceptor sites can be differentiated in rat cerebral cortical membranes.

Effect of 5-(3-[4-(4-fluorophenyl)-1-piperazinyl]-propoxy)indan (BP-528) on benzodiazepine receptor bindings and gamma-aminobutyric acid release.

The action of a new type of anti-anxiety compound, 5-(3-[4-(4-fluorophenyl)-1-piperazinyl]-propoxy)indan (BP-528), was tested on benzodiazepine receptor bindings and on [3H]-GABA release. BP-528 did not alter [3H]-diazepam binding to rat cerebral cortical and hippocampal membranes either in the presence or absence of GABA; and the binding of [3H]-propyl-beta-carboline-3-carboxylate at low concentration (0.04 nM), which labels only the type I benzodiazepine receptor, was not changed by BP-528. BP-528 did not interact with the GABA-benzodiazepine receptor complex, which is related to the anti-anxiety activity of benzodiazepines. This compound affected neither GABA binding nor GABA uptake. Ten micromolar BP-528 depressed high K+-induced [3H]-GABA release from preloaded rat hippocampal slices. However, the same concentration of BP-528 also inhibited high K+-induced calcium uptake by rat cerebral cortical synaptosomes.

Effect of 5-{3-[4-(4-Fluorophenyl)-1-Piperazinyl]-Propoxy}indan (BP-528) on Benzodiazepine Receptor Bindings and γ-Aminobutyric Acid Release

The action of a new type of anti-anxiety compound, 5-{3-[4-(4-fluorophenyl)-1-piperazinyl]-propoxy}indan (BP-528), was tested on benzodiazepine receptor bindings and on [3H]-GABA release. BP-528 did not alter [3H]-diazepam binding to rat cerebral cortical and hippocampal membranes either in the presence or absence of GABA; and the binding of [3H]-propyl-β-carboline-3-carboxylate at low concentration (0.04 nM). which labels only the type I benzodiazepine receptor, was not changed by BP-528. BP-528 did not interact with the GABA-benzodiazepine receptor complex, which is related to the antianxiety activity of benzodiazepines. This compound affected neither GABA binding nor GABA uptake. Ten micromolar BP-528 depressed high K+-induced [3H]-GABA release from preloaded rat hippocampal slices. However, the same concentration of BP-528 also inhibited high K+-induced calcium uptake by rat cerebral cortical synaptosomes.

Beta-adrenergic receptors and their mode of coupling to adenylate cyclase

articleBeta-adrenergic receptors and their mode of coupling to adenylate cyclaseA. LevitzkiA. LevitzkiPublished Online:01 Jul 1986https://doi.org/10.1152/physrev.1986.66.3.819MoreSectionsPDF (5 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat Previous Back to Top Download PDF FiguresReferencesRelatedInformationCited ByCombining resonance energy transfer methods reveals a complex between the α 2A -adrenergic receptor, Gα i1 β 1 γ 2 , and GRK2The FASEB Journal, Vol. 24, No. 12Genesis and Regulation of the Heart AutomaticityMatteo E. Mangoni, and Joël Nargeot1 July 2008 | Physiological Reviews, Vol. 88, No. 3Role of β-Adrenoceptor Signaling in Skeletal Muscle: Implications for Muscle Wasting and DiseaseGordon S. Lynch, and James G. Ryall1 April 2008 | Physiological Reviews, Vol. 88, No. 2Cholesterol as a determinant of cooperativity in the M2 muscarinic cholinergic receptorBiochemical Pharmacology, Vol. 74, No. 2Effects of β2-agonist administration and exercise on contractile activation of skeletal muscle fibersGordon S. Lynch, Alan Hayes, Siun P. Campbell, and David A. Williams1 October 1996 | Journal of Applied Physiology, Vol. 81, No. 4Unoccupied β-adrenoceptor-induced adenylyl cyclase stimulation in turkey erythrocyte membranesEuropean Journal of Pharmacology: Molecular Pharmacology, Vol. 268, No. 2Acetylcholine reversal of isoproterenol-stimulated sodium currents in rabbit ventricular myocytes.Circulation Research, Vol. 72, No. 3The signal transduction between β-receptors and adenylyl cyclaseLife Sciences, Vol. 52, No. 26Positive inotropic effects of CGRP and isoprenaline: Analogies and differencesNeuropeptides, Vol. 23, No. 3Effect of albumin on adenylate cyclase receptor-related signal transduction of human peripheral blood mononuclear cellsBiochemical Pharmacology, Vol. 44, No. 2Alpha-1 adrenergic receptor number and receptor density in isolated hepatocytes from foetal, juvenile and adult ratsCellular Signalling, Vol. 4, No. 3Critique2 July 2016 | Journal of Psychopharmacology, Vol. 6, No. 1Interaction betweenα2- and β-adrenergic receptors in rat cerebral cortical membranes: clonidine-induced reduction in agonist and antagonist affinity for β-adrenergic receptorsBrain Research, Vol. 542, No. 2The regulation of adenylyl cyclase by receptor-operated G proteinsPharmacology & Therapeutics, Vol. 50, No. 3Rhythms in second messenger mechanismsPharmacology & Therapeutics, Vol. 51, No. 2Effect of oxygen tension on catecholamine-induced formation of cAMP and on swelling of carp red blood cellsA. Salama, and M. Nikinmaa1 November 1990 | American Journal of Physiology-Cell Physiology, Vol. 259, No. 5The effect of aluminum on markers for synaptic neurotransmission, cyclic AMP, and neurofilaments in a neuroblastoma × glioma hybridoma (NG108-15)Brain Research, Vol. 528, No. 1Biochemical and autoradiographic analysis of β-adrenoceptors in rat nasal mucosaEuropean Journal of Pharmacology, Vol. 182, No. 3Sites and mechanisms of Ca2+ movement in non-excitable cellsCell Calcium, Vol. 10, No. 5Optimal features of a new β-blockerAmerican Heart Journal, Vol. 116, No. 5Functional ß-adrenergic receptors in a human mammary cell line (HBL-100)Biochemical Pharmacology, Vol. 37, No. 15Transmembrane signalling to adenylate cyclase in mammalian cells and in Saccharomyces cerevisiaeTrends in Biochemical Sciences, Vol. 13, No. 8A potassium conductance contributes to the action of somatostatin-14 to suppress ACTH secretionBrain Research, Vol. 444, No. 2Regulation of cardiac ion channels by catecholamines, acetylcholine and second messenger systemsProgress in Biophysics and Molecular Biology, Vol. 52, No. 3Central adrenergic receptors2 July 2016 | Journal of Psychopharmacology, Vol. 1, No. 2 More from this issue > Volume 66Issue 3July 1986Pages 819-854 Copyright & PermissionsCopyright © 1986 the American Physiological Societyhttps://doi.org/10.1152/physrev.1986.66.3.819PubMed3016770History Published online 1 July 1986 Published in print 1 July 1986 Metrics

Nature of the N6 region of the adenosine receptor in guinea-pig ileum and rat vas deferens.

This study explored the nature of the purine domain N6 regions of the presynaptic adenosine receptors of guinea-pig ileum and of rat vas deferens. The experimental design tested a model of these receptors which is complementary to the structure of the N6 substituent of the classical A1 adenosine receptor agonist N6-1-phenyl-2R-propyladenosine, (R-PIA). Assays of activity employed ileal segments or the midportions of vasa deferentia under continuous electrical stimulation at 0.2 Hz. Structure activity correlations compared the EC-50s for twitch inhibition. As shown previously, R-PIA was 60-80 times more potent than its S diastereomer, the resultant of the positive contribution of propyl C-3 to activity as well as the negative influence of steric hindrance exerted by propyl C-3 of the S diastereomer. Other pairs of diastereomers having a chiral center adjacent to N6 showed that the stereoselectivity of the PIAs was generalizable. Biological activity appears to reside wholly in the N6 alkyl moiety; the phenyl or aryl groups of similar size actually diminished potency. The receptor subregions interacting with propyl C-1 and C-3 of R-PIA are each large enough to accommodate two - but not three - methylene residues, each methylene contributing additively to activity. Hydrophobicity is a prominent attribute of the propyl C-1 and C-3 subregions. The potencies of these analogs as inhibitors of presynaptic transmission in ileum or vas deferens are covariant with inhibition of [3H]N6-cyclohexyl-adenosine binding to rat cerebral cortical membranes. Singular exceptions to this generalization may represent organ- or species-dependent differences in receptor fine structure.

3H]xanthine amine congener of 1,3-dipropyl-8-phenylxanthine: an antagonist radioligand for adenosine receptors.

An amine-functionalized derivative of 1,3-dipropyl-8-phenylxanthine has been prepared in tritiated form as a xanthine amine congener ([3H]XAC) for use as an antagonist radioligand for adenosine receptors. [3H]XAC has higher receptor affinity, higher specific activity, lower nonspecific membrane binding, and more favorable hydrophilicity than 1,3-diethyl-8-[3H]phenylxanthine, the xanthine commonly used for adenosine receptor binding. In rat cerebral cortical membranes, [3H]XAC exhibits saturable, specific binding with a Kd of 1.23 nM and a Bmax of 580 fmol/mg of protein at 37 degrees C. N6-(R-Phenylisopropyl)adenosine is a more potent inhibitor of [3H]XAC binding than is 5'-N-ethylcarboxamidoadenosine, indicating that binding is to an A1-adenosine receptor. In the absence of GTP, the inhibition curves for adenosine agonists versus [3H]XAC binding are biphasic, indicating that [3H]XAC is binding to low- and high-affinity agonist states of the A1 receptor. In the presence of GTP, adenosine analogs exhibit monophasic, low-affinity inhibition of binding of [3H]XAC. Inhibition of [3H]XAC binding by theophylline or by various 8-phenylxanthines is monophasic, and the potencies are commensurate with the potencies of these xanthines as adenosine receptor antagonists. The receptor sites in calf brain membranes exhibit a higher affinity (Kd = 0.17 nM) for [3H]XAC, whereas sites in guinea pig exhibit a slightly lower affinity (Kd = 3.0 nM). Densities of [3H]XAC binding sites are similar in brain membranes from all species.

Target size of the adenosine Ri receptor.

The adenosine receptor of rat cerebral-cortical membranes was examined by radiation inactivation. In control membranes the receptor is distributed between high- and low-affinity states, that can be preferentially expressed by Mg2+ ions and guanine nucleotides respectively. Upon exposure to increasing doses of radiation, the high-affinity receptor decayed linearly as a function of radiation dose. This decay rate corresponded to a target size of 63,000 Da, when compared with the decay of the muscarinic cholinergic receptor that was also measured in these membranes.

Solubilization and characterization of the 5-hydroxytryptamine transporter complex from rat cerebral cortical membranes

The 5-hydroxytryptamine transporter complex from rat cerebral cortical membranes was solubilized with digitonin. The affinity of the solubilized transporter complex for [ 3H]paroxetine, a very selective and potent inhibitor of 5-hydroxytryptamine uptake, was not affected and remained unchanged when compared with the parent membrane preparation. The solubilization yield of membrane-bound [ 3H]paroxetine binding sites was 42%. The pharmacological profile of the solubilized transporter complex was similar to that of the intact transporter in membranes of the cerebral cortex, with the exception of tryptamine, which exhibited a 10-fold loss in potency to inhibit [ 3H]paroxetine binding to the solubilized transporter when compared to membranes. The Stokes radius determined by gel filtration was 7.6 nm. This successful solubilization of the neuronal 5-hydroxytryptamine transporter complex is the starting point for purification of this macromolecular moiety.

Enhancement of [3H]Clonidine Binding to Rat Cerebral Synaptic Membranes by Treatment with Arachidonic Acid, Prostaglandin (PG) D2, PGE2 and PGF2α

Pretreatment of cerebral synaptic membranes with arachidonic acid (10 nM—1 μM) at 37°C for 40 min caused a significant increase in [3H]clonidine binding but pretreatment at 4°C did not. Scatchard analysis showed that 100 nM arachidonic acid pretreatment resulted in a significant elevation of the Bmax value of the low affinity site. PGD2, PGE2 and PGF2α (10-100 μM) pretreatment also enhanced binding. These polyunsaturated fatty acids could be involved in regulation of the α2-adrenoceptor binding in rat cerebral cortical membranes.