Rat Striatal Membranes Research Articles

Synthesis and dopamine receptor binding of bridged tricyclic dopamine analogues

The bridged tricyclic dopamine analogues exo-2-( N-n-propylamino)-6,7-dihydroxybenzonorbornene, exo-2-( N,N-di- n-propylamino)-6,7-dihydroxybenzonorbornene, and exo-2-amino-6,7-dihydroxybenzobicyclo[2.2.2]octene have been synthesized and assayed for their effects on the binding of [ 3H]spiperone and [ 3H]apomorphine to rat striatal membranes. The inactivity of these compounds is rationalized on the basis of proposed topographical models of dopamine receptors.

2-Chloro-N6-cyclopentyladenosine: a highly selective agonist at A1 adenosine receptors.

2-Chloro-N6-cyclopentyladenosine (CCPA) was synthesized as a potential high affinity ligand for A1 adenosine receptors. Binding of [3H]PIA to A1 receptors of rat brain membranes was inhibited by CCPA with a Ki-value of 0.4 nM, compared to a Ki-value of 0.8 nM for the parent compound N6-cyclopentyladenosine (CPA). Binding of [3H]NECA to A2 receptors of rat striatal membranes was inhibited with a Ki-value of 3900 nM, demonstrating an almost 10,000-fold A1-selectivity of CCPA. CCPA inhibited the activity of rat fat cell membrane adenylate cyclase, a model for the A1 receptor, with an IC50-value of 33 nM, and it stimulated the adenylate cyclase activity of human platelet membranes with an EC50-value of 3500 nM. The more than 100-fold A1-selectivity compares favourably with a 38-fold selectivity of CPA. Thus, CCPA is an agonist at A1 adenosine receptors with a 4-fold higher selectivity and 2-fold higher affinity than CPA, and a considerably higher selectivity than the standard A1 receptor agonist R-N6-phenylisopropyladenosine (R-PIA). CCPA represents the agonist with the highest selectivity for A1 receptors reported so far.

Effects of toluene treatment in vivo and in vitro on the binding characteristics of [ 3H]neurotensin in rat striatal membranes

The effects of treatment with toluene in vivo (80 ppm, 3 days, 6 h/day) and in vitro (19 μmol/ml) were analyzed on the binding characteristics of [ 3H]neurotensin in rat striatal membranes. Exposure to toluene in vivo did not produce any significant effects on the binding characteristics of [ 3H]neurotensin. However, the addition of toluene in vitro caused a trend for a decreased B max value and produced a significantly reduced K D value of [ 3H]neurotensin binding. The absence of effects at 80 ppm indicates that the neurotensin receptor is relatively insensitive to toluene exposure, in contrast to, e.g. the dopamine agonist binding sites. Furthermore, the toluene response of the neurotensin receptor, as seen after treatment in vitro, is different from the responses seen in many monoamine receptors, which show decreased affinities following toluene exposure. It is possible that toluene is mediating its effects on the neurotensin receptor by changing the lipid micro-environment in which the receptor is situated. Another explanation would be that toluene selectively acts on the monoamine receptors, e.g. the more sensitive dopamine receptors, which through receptor-receptor interactions would cause the response seen in the neurotensin receptor. However, it cannot be excluded that the suggested receptor-receptor interaction itself is affected by toluene.

3H]N-[1-(2-Benzo(b)thiophenyl) cycohexyl]piperidine ([ 3H]BTCP): a new phencyclidine analog selective for the dopamine uptake complex

A benzothiopenyl group instead of a phenyl ring on phencyclidine (PCP) yields a molecule N-[1-(2-benzo( (b)thiophenyl)cyclohexyl]piperidine (BTCP), which is one of the more potent known dopamine (DA) uptake inhibitors (IC 50=7 nM). This compound also has low affinity for the PCP receptor (K 0.5=6 μM). The sodium-dependent [ 3H]BTCP binding to rat striatal membranes was investigated. [ 3H]BTCP bound to two different sites: one with very high affinity (K d1=0.9 nM, B max1=3.5 pmol/mg protein) which paralleled the distribution of dopaminergic nerve endings and a second with lower affinity (K d2=20 nM, B max2=7.5 pmol/mg protein). There was a good correlation between the abilities of drugs specific for the DA uptake complex and of PCP analogs to inhibit high affinity [ 3H]BTCP binding and [ 3H]DA synaptosomal uptake. This study also demonstrated that PCP interacts with the DA uptake site since it is a competitive inhibitor of high affinity [ 3H]BTCP binding. This site, however, is not the PCP receptor, which has a different pharmacological selectivity.

Ionic requirements for the specific binding of [3H]GBR 12783 to a site associated with the dopamine uptake carrier.

At 0 degrees C, when Na+ was the only cation present in the incubation medium, increasing the Na+ concentration from 3 to 10 mM enhanced the affinity of [3H]1-[2-(++di-phenylmethoxy)ethyl]-4-(3-phenyl-2-propenyl)piperaz ine [( 3H]GBR 12783) for the specific binding site present in rat striatal membranes without affecting the Bmax. For higher Na+ concentrations, specific binding values plateaued and then slightly decreased at 130 mM Na+. In a 10 mM Na+ medium, the KD and the Bmax were, respectively, 0.23 nM and 12.9 pmol/mg of protein. In the presence of 0.4 nM [3H]GBR 12783, the half-maximal specific binding occurred at 5 mM Na+. A similar Na+ dependence was observed at 20 degrees C. Scatchard plots indicated that K+, Ca2+, Mg2+, and Tris+ acted like competitive inhibitors of the specific binding of [3H]GBR 12783. The inhibitory potency of various cations (K+, Ca2+, Mg2+, Tris+, Li+, and choline) was enhanced when the Na+ concentration was decreased from 130 to 10 mM. In a 10 mM Na+ medium, the rank order of inhibitory potency was Ca2+ (0.13 mM) greater than Mg2+ greater than Tris+ greater than K+ (15 mM). The requirement for Na+ was rather specific, because none of the other cations acted as a substitute for Na+. No anionic requirement was found: Cl-, Br-, and F- were equipotent. These results suggest that low Na+ concentrations are required for maximal binding; higher Na+ concentrations protect the specific binding site against the inhibitory effect of other cations.

O-176 - L-DOPA modulates the characteristics of 3H-dihydroxyalprenolol binding sites in rat striatal membrane

O-176 - L-DOPA modulates the characteristics of 3H-dihydroxyalprenolol binding sites in rat striatal membrane

L-DOPA stereoselectively increases the number of β-adrenergic binding sites in rat striatal membrane

L-DOPA stereoselectively increases the number of β-adrenergic binding sites in rat striatal membrane

Characterization of the radioiodinated analogue of SCH 23390: In vitro and in vivo D-1 dopamine receptor binding studies

A new radioiodinated molecule, 125I-SCH 38840 (previously referred to as 125I-SCH 23982), has been recently reported to be a D-1 dopamine receptor ligand. The current study confirms and expands the characterization of both the radiolabeled and unlabeled forms of this compound, as well as describing the development of an in vivo D-1 receptor binding assay utilizing the 125I-SCH 38840. The binding of 125I-SCH 38840 to rat striatal membranes, in vitro , was saturable and exhibited a K D of 1.47 nM. Competition studies using 125I-SCH 38840 exhibited a pharmacological profile consistent with the proposal that 125I-SCH 38840 was binding to the D-1 receptor. Further studies with the unlabeled SCH 38840 demonstrated that it inhibited dopamine-stimulated adenylate cyclase with a K I of 66.1 nM, indicating that SCH 38840 was acting as a D-1 antagonist. Behavioral studies demonstrated that SCH 38840 (MED = 1.0 mg/kg, s.c.) blocked conditioned avoidance responding in rats, a measurement considered predictive of anti-psychotic activity in man. In vivo binding of 125I-SCH 38840 to rat striatum following s.c. administration was specific. Peak striatal levels were observed 1 h after injection, with measurable binding observed out to 8 h post-treatment. The displacement of the in vivo binding by unlabeled standards again suggested a D-1 selective interaction. The half-life of the in vivo binding of 125I-SCH 38840 was approximately 1.25 h, and was nearly equivalent to the half-life of the anti-CAR activity of unlabeled SCH 38840. These results clearly demonstrate the D-1 nature of SCH 38840's behavioral activity and strengthen the anti-psychotic potential of a D-1 antagonist.

Neurotensin reduces the affinity of D-2 dopamine receptors in rat striatal membranes.

Acta Physiologica ScandinavicaVolume 131, Issue 4 p. 625-626 Neurotensin reduces the affinity of D-2 dopamine receptors in rat striatal membranes G. VON EULER, Corresponding Author G. VON EULER Department of Histology and Neurobiology, Karolinska Institute, Stockholm, SwedenDepartment of Histology and Neurobiology, Karolinska Institute, Box 60400, S-104 01 Stockholm, Sweden.Search for more papers by this authorK. FUXE, K. FUXE Department of Histology and Neurobiology, Karolinska Institute, Stockholm, SwedenSearch for more papers by this author G. VON EULER, Corresponding Author G. VON EULER Department of Histology and Neurobiology, Karolinska Institute, Stockholm, SwedenDepartment of Histology and Neurobiology, Karolinska Institute, Box 60400, S-104 01 Stockholm, Sweden.Search for more papers by this authorK. FUXE, K. FUXE Department of Histology and Neurobiology, Karolinska Institute, Stockholm, SwedenSearch for more papers by this author First published: December 1987 https://doi.org/10.1111/j.1748-1716.1987.tb08285.xCitations: 45AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume131, Issue4December 1987Pages 625-626 RelatedInformation

Ganglioside GM1 treatment prevents the effects of subacute exposure to toluene on binding characteristics in rat striatal membranes

The effects of ganglioside GM1 treatment (10 mg/kg, i.p., 3 days, once daily) were analyzed on N-[3H]propylnorapomorphine ([3H]NPA) binding characteristics in striatal membrane preparations from air or toluene (80 ppm, 3 days, 6 h/day) exposed adult male rats. It was found that toluene exposure induced increased Kd and Bmax values for [3H]NPA binding. These changes could be blocked by GM1 treatment, which by itself had no significant effects on the binding characteristics of [3H]NPA. The addition of toluene (9.3 mumol/ml) in vitro had similar effects on [3H]NPA binding characteristics as toluene exposure in vivo. These results indicate that the effects of toluene exposure on dopamine receptors are due to an alteration of membrane fluidity which is prevented by GM1 treatment.

Correlation of adenosine receptor affinities and cardiovascular activity

Binding affinities of 28 adenosine analogs at A1 adenosine receptors (rat whole brain membranes, [ 3H]N6-cyclohexyladenosine, CHA), and at A2 adenosine receptors (rat striatal membranes, [ 3H]NECA) were compared to their EC 25 values for decreasing heart rate and increasing coronary flow in the isolated rat heart. Heart rate (an A1 response) correlated with A1 binding affinity (r 2=0.71, p<0.0001) but not with A2 binding affinity (r 2=0.007, n.s.); conversely, coronary flow (an A2 response) correlated with A2 binding affinity (r 2=0.83, p<0.0001) but not with A1 binding affinity (r 2=0.05, n.s.). These results confirm that the brain A1 and A2 receptors, studied by binding methods, bear close similarities to their respective counterparts in the heart, studied by means of functional responses.

Further in vitro and in vivo studies with the putative presynaptic dopamine agonist N,N-dipropyl-7-hydroxy-2-aminotetralin.

The in vitro binding of the putative dopamine autoreceptor agonist [3H]DP-7-ATN to rat striatal membrane homogenates was investigated. The maximum number of binding sites Bmax was 497.5 +/- 50.2 fmol/mg protein and the affinity constant KD was 8.3 +/- 1.5 nM using 10 microM (+) butaclamol to define non-specific binding. Lesion of the left medium forebrain bundle by 6-hydroxydopamine resulted in an almost complete loss of dopamine in the striatum but did not affect the binding of [3H]DP-7-ATN. The binding of [3H]DP-7-ATN to the homogenates of the dopaminergic cell bodies in the substantia nigra revealed a Bmax of 542.4 +/- 40.1 fmol/mg protein and a KD of 11.1 +/- 1.3 nM. The pharmacological profile of the binding was characterized as being to D-2 receptors. No direct in vitro evidence could be found for a selective binding to DA autoreceptors. The dopamine uptake inhibitor GBR 12909 interacted in a noncompetitive manner with the in vitro binding of [3H]DP-7-ATN and the latter compounds uptake into isolated synaptosomes was not through the specific dopamine uptake system but rather through diffusion. GBR 12909 failed to reveal any agonistic or antagonistic activity in the GBL model but was able to antagonize the hypomotility in rats induced by 0.25 mg/kg DP-7-ATN. The inhibitory effect of DP-7-ATN on DA release was also demonstrated using in vivo brain dialysis in conscious rats. Based on the above results, the possibility is discussed that the release regulating DA autoreceptors, which might be coupled to the reuptake complex, and the DA biosynthesis regulating autoreceptors, are different entities.

Antisera against the 3–17 sequence of rat G αi recognize only a 40 kDa G-protein in brain

To obtain antisera specific for the GTP-binding protein G iα we immunized rabbits against a synthetic peptide derived from the N-terminal (3–17) sequence predicted from the rat G iα cDNA clone published by Itoh et al. (1986) (Proc. Natl. Acad. Sci. USA 83, 3776–3780). Western-blot analysis of bovine brain G-proteins purified and resolved by hydrophobic chromatography and of rat striatal membranes, indicate that this antiserum does not recognize 41 kDa α i or 39 kDa α o. However, it reacts with a 40 kDa α-subunit. The data suggest that the sequence deduced from the rat G αi cDNA corresponds to a G 40α protein and that N-terminus directed antisera are useful tools to discriminate between two different G αi-like types of G-proteins present in mammalian brain.

Effects of subacute toluene exposure on protein phosphorylation levels in rat frontoparietal and striatal membranes.

Effects of subacute toluene exposure (80 p.p.m. toluene in air, 5 + 4 days, 6 h day-1) were analysed on calcium (Ca2+)- and cyclic adenosine monophosphate (cAMP)-induced protein phosphorylation levels in membrane preparations from the frontoparietal cortex and the striatum of the adult male rat. After protein separation by gel electrophoresis, the amount of radioactive phosphate incorporated from adenosine 5'-[gamma-32P] triphosphate, tetra-(ethylammonium) salt ([32P]ATP) was measured indirectly by autoradiography. The 21 most phosphorylated protein bands were then analysed by computerized image analysis. In the frontoparietal cortex no protein bands were significantly affected after cAMP-induced back phosphorylation, while after Ca2+ stimulation there was a decreased incorporation of [32P]ATP in a 22,000 protein band. In the striatum there was a reduced incorporation of [32P]ATP in a 26,000 protein band after cAMP-induced back phosphorylation, and in four bands of 20,000, 21,000, 52,000 and 134,000, respectively, after Ca2+ stimulation. The reduced incorporation of [32P]ATP in these proteins indicated increased original phosphorylation levels after toluene exposure. A comparison between the frontoparietal cortex and the striatum showed a selective vulnerability of phosphorylation processes in striatal membrane protein bands. In conclusion, toluene exposure at low doses augments membrane protein phosphorylation levels in the rat forebrain and especially in the striatum, probably leading to changes in information handling and/or metabolic changes.

Interactions of amineptine with the neuronal dopamine uptake system: neurochemical in vitro and in vivo studies.

The effects of amineptine on 3H-dopamine uptake and 14C-dopamine release have been studied simultaneously in double labelling test performed on rat striatal synaptosomes. 3H-dopamine uptake was completely inhibited at 10 microM amineptine, a concentration which produced only a weak 14C-DA release (13% of the 14C-radioactivity stored). The IC 50 for the inhibition of 3H-DA uptake was not modified by a previous treatment with reserpine whereas the IC 50 of (+) amphetamine and the IC 50 of clomipramine were decreased 9 fold and increased two fold, respectively. In binding studies on rat striatal membranes amineptine displaces in vitro the 3H-GBR 12783, bound specifically to a component of the neuronal DA uptake complex. The apparent affinity of amineptine for this binding site was more than 150 times higher than its affinity for the binding site of 3H-desipramine on rat cortical membranes. In mice, increasing doses of amineptine injected i.p. reduced in a dose dependent manner the specific retention of radioactivity in the striatum after an i.v. injection of a tracer dose of 3H-GBR 12783. These data indicate that amineptine inhibits DA uptake and is virtually devoid of DA releasing effects. It displays a relatively low affinity for the NE uptake system. Its neurochemical profile in the double labelling test clearly differs from that of (+) amphetamine and from that of classical tricyclic anti-depressants.

The effect of ethanol and haloperidol on dopamine receptor (D2) density

The study was performed to investigate the influence of ethanol on haloperidol-induced changes of the dopamine (D 2) receptors in rat striatal membrane preparations. Subchronic administration of the neuroleptic in the drinking water resulted in an increase of the number of binding sites in a dose-dependent manner. Simultaneous treatment with both haloperidol and ethanol prevented the rise of D 2 receptors.

Effect of repeated administration of manganese on the striatal cholinergic and dopaminergic receptors in the rat

Previous studies suggest that chronic administration of manganese produces symptoms akin to Parkinson's disease, which is believed to be due to derangement in the central cholinergic and dopaminergic system. In the present study the effect of repeated administration of manganese chloride to male Sprague-Dawley rats on the binding of [ 3H]spiroperidol and [ 3H]quinuclidinyl benzilate to striatal dopaminergic and cholinergic receptors, respectively, was determined. Daily intraperitoneal injections of manganese chloride (15 mg/kg) for 15 days followed by a 24 h drug-free period did not alter the receptor density ( B max) or the apparent dissociation constant ( K d) of either [ 3H]spiroperidol or [ 3H]quinuclidinyl benzilate to the rat striatal membranes. It is concluded that chronic treatment with manganese does not change the plasticity of post-synaptic dopaminergic or cholinergic receptors in the rat striatum.

Sodium independence of the binding of [ 3H]GBR 12783 and other dopamine uptake inhibitors to the dopamine uptake complex

When Na + was the only cation present in the incubation medium used for the determination of the specific binding of [ 3H]GBR 12783 in rat striatal membranes, the Na +-dependence between 10 and 210 mM Na + was not observed. In media with low (10 mM) or high (130 mM) Na + concentration, mazindol and nomifensine competed with [ 3H]GBR 12783 for its specific binding site with the same affinities. With the exception of amineptine, all the tested catecholamine uptake inhibitors were equally potent to compete with [ 3H]GBR 12783 when Na + concentration was decreased from 130 to 10 mM. These data suggest that the media previously used for the binding studies of tritiated inhibitors of dopamine uptake (Tris-ions buffer and Krebs-Ringer medium) contain ions which could exert inhibitory effects on the specific binding at low Na + concentration.

Biochemical and pharmacological characterization of [3H]GBR 12935 binding in vitro to rat striatal membranes: labeling of the dopamine uptake complex.

Binding of the selective dopamine (DA) uptake inhibitor [3H]GBR 12935 to rat striatal membranes was characterized biochemically and pharmacologically. [3H]GBR 12935 binding at 0 degree C was reversible and saturable and Scatchard analysis indicated a single binding site with a KD of 5.5 nM and a Bmax of 760 pmol/mg tissue. [3H]GBR 12935 labeled two binding sites. One binding site was identified as the classic DA uptake site, since methylphenidate, cocaine, diclofensine, and Lu 19-005 potently inhibited [3H]GBR 12935 binding to it. Binding to the second site was inhibited by high concentrations of the above compounds. IC50 values for inhibition of [3H]GBR 12935 binding to the DA uptake site were proportional to IC50 values for inhibition of DA uptake. However, substrates of DA uptake, e.g., DA and 1-methyl-4-phenylpyridine, and DA releasers, e.g., the amphetamines, inhibited [3H]GBR 12935 binding less than DA uptake. Rate experiments excluded the possibility that these "weak" inhibitors affected the binding by allosteric coupled binding sites. The second binding site was not a noradrenergic, serotonergic, or GABAergic uptake site. Neither was it a dopaminergic, acetylcholinergic, histaminic, serotonergic, or adrenergic receptor. However, [3H]GBR 12935 was potently displaced from it by disubstituted piperazine derivatives, i.e., flupentixol and piflutixol. DA uptake and the DA uptake binding site of [3H]GBR 12935 were located primarily in the striatum, but the piperazine acceptor site was distributed uniformly throughout the brain. Also only the DA uptake binding site was destroyed by 6-OH-DA. Thus, [3H]GBR 12935 labels the classic DA uptake site in rat striatum and also a piperazine acceptor site. Substrates for DA uptake and releasers of DA inhibited [3H]GBR 12935 binding with low potency, but did not alter the rate constants for [3H]GBR 12935 binding. Therefore inhibitors of DA uptake label the carrier site and prevent the carrier process.

The characterization of [ 3H]sulpiride binding sites in rat striatal membranes

The characteristics of [ 3H]sulpiride binding to the D 2 dopamine receptor in rat striatal membranes were examined under several conditions. In the absence of sodium ions, the specific binding of [ 3H]sulpiride could not be detected. In the direct binding experiment, at 25 °C, the affinity of [ 3H]sulpiride for D 2 receptors was increased in a dose-dependent manner of sodium ions whereas magnesium ions have opposite effects on an affinity of [ 3H]sulpiride binding. The lowering incubation temperature (4 °C) also produced a further increase in affinity of the ligand. Under all conditions, [ 3H]sulpiride labeled a single homogenous site of the receptor. On the other hand, the result from quantitative analysis of agonist/[ 3H]sulpiride competition curves indicated an existence of high (RH) and low (RL) affinity states for agonists and the proportion of two-affinity states was modulated by guanosine triphosphate (GTP), magnesium ions and lowering temperatures. GTP, together with sodium ions, caused a full conversion of RH to RL, while an increase in the affinity for agonists with a partial conversion of RL to RH could be induced by magnesium ions at 25 °C. At a lower (4 °C) temperature, the agonist competition curve indicated an existence of a single agonist low-affinity state (RL) and then, the effects of GTP and magnesium ions in the agonist affinity observed at 25 °C were abolished. These results can be incorporated into a two-step, ternary complex model involving an inhibitory guanine nucleotide binding protein for the agonist and antagonist interaction with D 2 receptors. The regulation of [ 3H]sulpiride binding by sodium ions and guanine nucleotides is analogous to that of several receptors which are inversely coupled to adenylate cyclase systems. Accordingly, the specific binding of [ 3H]sulpiride appears to represent the agonist low-affinity state of D 2 receptors.