Interacting Presynaptic k-Opioid and GABAA Receptors Modulate Dopamine Release from Rat Striatal Synaptosomes

Abstract— The presynaptic regulation of stimulated dopa-mine release from superfused rat striatal synaptosomes by opioids and γ-aminobutyric acid (GABA) was studied. It was found that in addition to dopamine D₂ autoreceptors, calcium-dependent K⁺-stimulated [³H]dopamine release was inhibited through activation of a homogeneous population of k -opioid receptors in view of the potent inhibitory effect of the k -selective agonist U69.593 (EC₅₀ 0.2 nM) and its antagonism by norbinaltorphimine. Neither μ-nor δ-selective receptor agonists affected release of [³H]-dopamine. In addition, GABA potently inhibited the evoked [³H]dopamine release (EC₅₀ 0.4 nM) through activation of GABA_A receptors in view of the GABA-mimicking effect of muscimol, the sensitivity of its inhibitory effect to picro-toxin and bicuculline, and the absence of an effect of the GABA_B receptor agonist baclofen. In the presence of a maximally effective concentration of GABA, U69,593 did not induce an additional release-inhibitory effect, indicating that these receptors and the presynaptic D₂ receptor are colocalized on the striatal dopaminergic nerve terminals. The excitatory amino acid agonists N-methyl-d -aspartate and kainate, as well as the cholinergic agonist carbachol, stimulated [³H]dopamine release, which was subject to k -opioid receptor-mediated inhibition. In conclusion, striatal dopamine release is under regulatory control of multiple excitatory and inhibitory neurotransmitter by activation of colocalized presynaptic receptors for excitatory amino acids, acetylcholine, dopamine, dynorphins, and GABA within the dopaminergic nerve terminals. Together, these receptors locally control ongoing dopamine neurotransmission.     

8-[3H]Hydroxy-2-(di-n-propylamino) tetralin binding sites in goldfish retina

The binding sites of 8-[³H]hydroxy-2-(di-n-propylamino)tetralin ([³H]DPAT) were characterized in the retina of goldfish in order to evaluate the selectivity of the ligand for serotonin_1A (5HT_1A) receptors. Specificity of the binding was performed in the presence of serotonergic and dopaminergic agonists and antagonists. Buspirone, spriroxatrine and 5-methoxy-N,N-dimethyltryptamine were potent inhibitors, followed by propranolol, citalopram, imipramine and desipramine. Serotonin was not a potent inhibitor, and its interaction with the binding sites of [³H]DPAT was complex. Nomifensine displayed an important inhibition, however, other dopamine uptake blockers, such as bupropion and GBR-12909, were less potent. Haloperidol was also a good inhibitor, but the D₁ receptor agonist, SKF-38393, the D₂ receptor antagonist, sulpiride, and dopamine did not inhibit the binding. GppNHp inhibited the binding in the micromolar range. The analysis of saturation experiments by isotopic dilution, using buspirone to determine nonspecific binding, revealed two sites. The number of binding sites defined by buspirone were higher than the ones defined by nomifesine. The specific binding, using buspirone for definition, was reduced by the intraocular injection of 6-hydroxydopamine. This investigation demonstrates that [³H]DPAT labels 5HT_1A receptors in goldfish retina, but also interacts with a non-5HT receptor site. These receptors seem to be localized in dopaminergic neurons.     

Neuroleptics Up-Regulate Adenosine A2a Receptors in Rat Striatum: Implications for the Mechanism and the Treatment of Tardive Dyskinesia

Abstract: Neuroleptics, which are potent dopamine receptor antagonists, are used to treat psychosis. In the striatum, dopamine subtype-2 (D₂) receptors interact with high-affinity adenosine subtype-2 (A_2a) receptors. To examine the effect of various neuroleptics on the major subtypes of striatal dopamine and adenosine receptors, rats received 28 daily intraperitoneal injections of these drugs. Haloperidol (1.5 mg/kg/day) increased the density of striatal D₂ receptors by 24% without changing their affinity for [³H]sulpiride. Haloperidol increased the density of striatal A_2a receptors by 33% (control, 522.4 ± 20.7 fmol/mg of protein; haloperidol, 694.6 ± 23.6 fmol/mg of protein; p < 0.001) without changing their affinity for [³H]CGS-21680 (control, 19.2 ± 2.2 nM; haloperidol, 21.4 ± 2.3 nM). In contrast, haloperidol had no such effect on striatal dopamine subtype-1 (D₁) and adenosine subtype-1 (A₁) receptors. Binding characteristics and the pharmacological displacement profile of the increased [³H]CGS-21680 binding sites confirmed them as A_2a receptors. Comparing different classes of neuroleptics showed that the typical neuroleptics haloperidol and fluphenazine (1.5 mg/kg/day) increased D₂ receptor densities, whereas the atypical neuroleptics sulpiride (100 mg/kg/day) and clozapine (20 mg/kg/day) did not (control, 290.3 ± 8.7 fmol/mg of protein; haloperidol, 358.1 ± 6.9 fmol/mg of protein; fluphenazine, 381.3 ± 13.6 fmol/mg of protein; sulpiride, 319.8 ± 18.9 fmol/mg of protein; clozapine, 309.2 ± 13.7 fmol/mg of protein). Similarly, the typical neuroleptics increased A_2a receptor densities, whereas the atypical neuroleptics did not (control, 536.9 ± 8.7 fmol/mg of protein; haloperidol, 687.9 ± 28.0 fmol/mg of protein; fluphenazine, 701.1 ± 31.6 fmol/mg of protein; sulpiride, 563.3 ± 27.2 fmol/mg of protein; clozapine, 550.9 ± 40.9 fmol/mg of protein). There were no differences in affinities for [³H]CGS-21680 or [³H]sulpiride among the various treatment groups. This study demonstrates that typical neuroleptics induce comparable up-regulation in both striatal D₂ and A_2a receptors. Thus, A_2a receptors might be a pharmacologic target for the development of novel therapeutic strategies to minimize the adverse effects of antipsychotic treatment.     

The D3 dopamine receptor inhibits dopamine release in PC-12/hD3 cells by autoreceptor signaling via PP-2B, CK1, and Cdk-5

The function of the D₃ dopamine (DA) receptor remains ambiguous largely because of the lack of selective D₃ receptor ligands. To investigate the function and intracellular signaling of D₃ receptors, we established a PC‐12/hD3 clone, which expresses the human D₃ DA receptor in a DA producing cell line. In this model, we find that the D₃ receptor functions as an autoreceptor controlling neurotransmitter secretion. Pre‐treatment with 3,6a,11, 14‐tetrahydro‐9‐methoxy‐2 methyl‐(12H)‐isoquino[1,2‐b] pyrrolo[3,2‐f][1,3] benzoxanzine‐1‐carboxylic acid, a D₃ receptor preferring agonist, dose‐dependently suppressed K⁺‐evoked [³H]DA release in PC‐12/hD3 cells but not in the control cell line. This effect was prevented by D₃ receptor preferring antagonists GR103691 and SB277011‐A. Furthermore, activation of D₃ receptors significantly inhibits forskolin‐induced cAMP accumulation and leads to transient increases in phosphorylation of cyclin‐dependent kinase 5 (Cdk5), dopamine and cAMP‐regulated phosphoprotein of M_r 32 000 and Akt. Because we observed differences in Cdk5 phosphorylation as well as Akt phosphorylation after DA stimulation, we probed the ability of Cdk5 and phosphatidylinositol‐3 kinase (PI3K) to influence DA release. Cdk5 inhibitors, roscovitine, or olomoucine, but not the PI3K inhibitor wortmannin, blocked the D₃ receptor inhibition of DA release. In a complimentary experiment, over‐expression of Cdk5 potentiated D₃ receptor suppression of DA release. Pertussis toxin, 3‐[(2,4,6‐trimethoxyphenyl)methylidenyl]‐indolin‐2‐one and cyclosporine A also attenuated D₃ receptor‐mediated inhibition of DA release indicating that this phenomenon acts through Gi/oα and casein kinase 1, and phosphatase protein phosphatase 2B (calcineurin), respectively. In support of previous data that D₃ DA receptors reduce transmitter release from nerve terminals, the current results demonstrate that D₃ DA receptors function as autoreceptors to inhibit DA release and that a signaling pathway involving Cdk5 is essential to this regulation.     

Receptor-mediated inhibition of octopamine-stimulated adenylate cyclase in the optic lobe of Octopus vulgaris

1. Inhibition of octopamine-stimulated adenylate cyclase was studied in the optic lobe of Octopus vulgaris.2. The octopamine antagonist, mianserin, and the dopamine D₂ agonists, PPHT and metergoline, induced dose-dependent inhibition of octopamine-stimulated adenylate cyclase activity.3. The binding of the tritiated benzazepine neuroleptic YM-09151-2 to octopus membranes and the displacement of [³H]YM-09151-2 by PPHT, metergoline and spiperone were consistent with the presence of a D₂-like dopamine receptor in the octopus optic lobe.4. The conclusion is drawn that octopamine-stimulated adenylate cyclase in the octopus is negatively regulated by a dopamine D₂-like receptor.     

Dopaminergic Receptors in Bovine Retina and Their Interaction with Thyrotropin-Releasing Hormone

A superfusion technique was employed to study the release of [3H]dopamine from isolated bovine retina. Only K+-stimulated release was observed from both light- and dark-adapted retina; release by other stimuli was from dark-adapted retina only. Light-evoked release of [3H]dopamine from dark-adapted retina was blocked by thyrotropin-releasing hormone (TRH), which has previously been identified as a retinal neuropeptide. TRH itself released small amounts of [3H]dopamine from dark-adapted retina. These results are interpreted as indicating that TRH acts as a modulator of dopaminergic activity in retina through the agency of presynaptic autoreceptors. Evidence of the existence of a feedback inhibition system, probably mediated by dopaminergic autoreceptors, was found by the inclusion of sulpiride, a dopaminergic D2 receptor antagonist in the perfusate, which, in a stereoselective manner, enhanced spontaneous and light-evoked release of [3H]dopamine. On the other hand, dopamine (1 microM) reduced these effects. TRH did not affect the high-affinity uptake system for dopamine in retina; this, then, could not account for the effects on release. Radioligand binding showed a specific, saturable high-affinity binding system for [3H]TRH, with an apparent KD of 2.2 nM and a Bmax of 23 fmol/mg protein in bovine retinal membranes. Displacement experiments showed that specific [3H]TRH binding was displaced in the nanomolar range by spiperone and in the micromolar range by dopamine, whereas L-(--)-sulpiride was virtually inactive in displacing [3H]TRH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of temperature and ionic environment on the specific binding of 3H(—)sulpiride to membranes from different rat brain regions

Sulpiride is an antipsychotic drug endowed with the properties of a dopamine antagonist. The failure of sulpiride to inhibit neostriatal dopamine stimulated adenylate cyclase activity indicated that this drug is a selective D₂ receptor antagonist. In this study we used a novel synthesized ²H(—)sulpiride with very high specific activity (72 Ci/mol) and characterized the temperature sensitivity of the binding sites labeled by this compound. Kinetic analysis of ³H(—)sulpiride binding in rat striatum showed unstable behavior when incubation was performed at 37 or 30°C. However when experiments were carried out at 15 or 10°C, binding reached a stable steady-state within 10 min. Scatchard analysis of binding isotherms obtained at 10°C showed a 5-fold increase in the maximum number of binding sites and a decrease in K_d values to one-third those obtained at 37°C. Pharmacological characterization of the binding sites labeled by ³H(—)sulpiride at 10°C showed a greater affinity for antagonists but not for agonists than 37°C. Under both experimental condition, ³H(—)sulpiride binding sites were Na⁺ and GTP-sensitive. The temperature sensitive binding phenomenon appeared to be area specific. ³H(—)sulpiride binding sites in tissues other than from striatum were influenced less or not at all by changes in incubation temperature.     

[3H]Dopamine Labeling of D3 Dopaminergic Sites in Human, Rat, and Calf Brain

Abstract: The binding of [³H]dopamine to brain regions of calf, rat, and human was investigated. The calf caudate contained the highest density of [³H]dopamine binding sites, with a B_max value of 185 fmol/mg protein, whereas rat and human striatum contained one-third this number of sites. The K_D values for [³H]dopamine in all tissues were 2–3 nM. Dopaminergic catecholamines (dopamine, apomorphine, 6,7-dihydroxy-2-aminotetralin, and N-propylnorapomorphine) inhibited the binding of [³H]dopamine in all three species, at low concentrations, with IC₅₀ values of 1.5 to 6 nM. Neuroleptics, in contrast, inhibited the binding at high concentrations (with IC₅₀ values of 200 to 40,000 nM). The [³H]dopamine binding sites were saturable, heat-labile, and detectable only in dopamine-rich brain regions; these sites differed from D₂ dopamine sites (labeled by [³H]butyrophenone neuroleptics), and from D_l dopamine sites (labeled by [³H]thioxanthene neuroleptics) associated with the dopamine-stimulated adenylate cyclase. We have, therefore, called these high-affinity [³H]dopamine binding sites D₃ sites. [³H]Apomorphine and [³H]ADTN also appeared to label D₃ sites. These ligands however, were less selective than [³H]dopamine, and labeled sites other than D₃ as well. Assay conditions were important in determining the parameters of [³H]dopamine binding. The optimum conditions for selective labeling of the D₃ dopaminergic sites, using [³H]dopamine, required the presence of EDTA and ascorbate.     

Dopamine receptors in the rat brain: quantitative autoradiographic localization using [3H]sulpiride

In vitro labeling of tissue sections with [³H]sulpiride has been utilized in the present study to autoradiographically localize D₂-dopamine receptors in the rat brain. Preliminary biochemical studies, using slide-mounted tissue sections, were performed to define the optimal labeling conditions for this binding. Autoradiograms were generated by apposition of the labeled tissue sections to tritium-sensitive film. Specific binding sites for [³H]sulpiride were localized to the caudate-putamen, nucleus accumbens, olfactory tubercle, glomerular layer of the olfactory bulb, pituitary, laminae I and III of the entorhinal cortex, substantia nigra, lateral mammillary nucleus and the stratum-lacunosum moleculare of the hippocampus. The high selectivity of [³H]sulpiride for the D₂-dopamine receptor indicates that it is a valuable tool for the autoradiographic localization and quantitation of neuroleptic receptors.     

Pre- and Postnatal Ontogeny and Characterization of Dopaminergic D2, Serotonergic S2, and Spirodecanone Binding Sites in Rat Forebrain

Abstract: The ontogeny of binding sites for [³H] spiperone was studied in time-pregnant rats. Binding of [³H]spiperone to fresh homogenates of pre- and postnatal rat forebrain was characterized by Scatchard analysis and competition experiments with a number of dopaminergic and serotonergic agonists and antagonists and additional substances. A convenient discrimination of three high-affinity sites, i.e., the dopaminergic D₂, serotonergic S₂, and spirodecanone (Sd) sites, was obtained with l-(–)sulpiride and cis-flupenthixol. The analgesic R5573 was found not to be specific for the Sd site but to interact with all three sites. The three binding sites became detectable in sequential order. S₂ and D₂ binding sites were first found at embryonic days 15.75 and 17.75, respectively. The Sd site did not appear before postnatal day 8. All three binding sites reached adult values at approximately postnatal day 30. During the prenatal period, the increase in the number of D₂ binding sites paralleled the rise in forebrain dopamine concentrations. The kinetics of D₂ and S₂ sites were the same at embryonic day 19.75 and postnatal day 30. These observations provide evidence for the presence of the receptor substrate for actions of neuroleptics on dopaminergic and serotonergic systems during fetal life.     

Dopamine receptors in canine caudate nucleus

Summary Physiological, pharmacological, histochemical and biochemical studies indicate that dopamine receptors are heterogenous in the, central nervous system with each individual functions. This review describes pharmacological and biochemical characteristics of dopamine receptors, particularly in canine caudate nucleus, which have been studied in our laboratory with a brief comparison to the current studies by other workers in similar research fields.Two distinct dopamine receptors have been characterized by means of [³H]dopamine binding to the synaptic membranes from canine caudate nucleus. One of the receptors with a Kd of about 3 M for dopamine may be associated with adenylate cyclase and referred to as D, receptor. The other receptor with a Kd of about 10 nM for dopamine is independent of adenylate cyclase and referred to as D₂. A photochemical irreversible association of [³H]dopamine with the membraneous receptors makes it possible to separate D₁ and D₂ receptors from one another by gel filtration on a Sephadex G-200 column after solubilization with Lubrol PX. On the basis of selective inhibition of [³H]dopamine binding to D₁ and D₂ receptors, dopamine antagonists can be classified into three classes: D₁-selective (YM-09151-2), D₂-selective (sulpiride) and nonselective (haloperidol, chlorpromazine). Effects of these typical antagonists on the metabolism of rat brain dopamine suggest that D₁ receptor is more closely associated with the neuroleptic-induced increase in dopamine turnover. Studies with 28 benzamide derivatives and some classical neuroleptics reveal that apomorphine-induced stereotypy displays a greater association with D₁ than with D₂ receptors.Dopamine-sensitive adenylate cyclase in canine caudate nucleus can be solubilized with Lubrol PX in a sensitive form to either dopamine, Gpp(NH)p or fluoride. Sephadex G-200 gel filtration separates adenylate cyclase from D₁ receptors with a concomitant loss of dopamine sensitivity. Addition of the D₁ receptor fraction to the adenylate cyclase restores the responsiveness to dopamine. The solubilized dopamine-unresponsive adenylate cyclase can be further separated into two distinct fractions by a batch-wise treatment with GTP-sepharose: a catalytic unit which does not respond to fluoride, and a guanine nucleotide regulatory protein. The regulatory protein confers distinct responsiveness to Gpp(NH)p and fluoride upon adenylate cyclase. These results indicate that dopamine-sensitive adenylate cyclase is composed of at least three distinct units; D₁ receptor, guanine nucleotide regulatory protein and adenylate cyclase.     

D2 Receptor Antagonists Do Not Modify Guanine Nucleotide-Sensitive Interactions Between Dopamine and D1 Dopamine Receptors Under In Vitro Conditions

Abstract: This study investigated possible D₁/D₂ interactions in rat and bovine striatal tissue by examining the effects of D₂ antagonists on the action of dopamine at D₁ dopamine receptors. In addition, the extent to which D₂ antagonists may induce an agonist low-affinity state of the D₁ receptor was evaluated in comparison with the effects of the guanine nucleotide analogue 5′-guanylylimidodiphosphate [Gpp(NH)p]. In saturation experiments dopamine caused a dose-dependent decrease in rat striatal and bovine caudate D₁ receptor density. This effect of dopamine, which has been shown to be sensitive to Gpp(NH)p, was not altered by pretreatment with either of the selective D₂ antagonists eticlopride (200 nM) or domperidone (200 nM). Results from displacement experiments show that the affinity of dopamine for D₁ receptors and the proportion of receptors in an agonist high-affinity state, are reduced by Gpp(NH)p (100 µM) but not by eticlopride. A molar excess of dopamine (100 µM) promotes the dissociation of (±)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-ol ([³H]SCH 23390) from rat striatal D₁ receptors at a rate that is significantly slower than when dissociation is initiated using 1 µM piflutixol. After pretreatment with Gpp(NH)p, [³H]SCH 23390 dissociation induced by dopamine occurred at an even slower rate. Pretreatment with eticlopride had no effect on the dopamine-induced rate of [³H]SCH 23390 dissociation. These results indicate that all experimental approaches detected dopamine effects at D₁ receptors that are Gpp(NH)p sensitive and D₂ antagonist insensitive and provide no evidence to support a D₁/D₂ link operating at the receptor level.     

Dopamine receptors in the goldfish retina: 3H-spiroperidol and 3H-domperidone binding; and dopamine-stimulated adenylate cyclase activity

Dopamine receptors in the goldfish retina have been examined by binding studies using ³H-spiroperidol and ³H-domperidone as specific ligands, and by measuring retinal adenylate cyclase activities in the presence and absence of dopamine. Our results indicate that washed membranes from goldfish retinal homogenate bind a variety of dopamine agonists and antagonists with high affinities and with characteristics similar to those reported for the brain, with the exception that in this retina there is virtually no binding of the specific D₂ receptor antagonist, ³H-domperidone. In addition, there is a very low basal activity of adenylate cyclase which can be greatly stimulated by dopamine, possibly reflecting a high degree of coupling between this enzyme and the dopamine receptor. Taken together, our findings indicate that the goldfish retina contains a high density of D₁ type dopamine receptors and few, if any, D₂ type receptors.     

Dopamine inhibition of anterior pituitary adenylate cyclase is mediated through the high-affinity state of the D2 receptor

The diterpinoid forskolin stimulated adenylate cyclase activity (measured by conversion of [³H]-ATP to [³H]-cAMP) in anterior pituitary from male and female rats. Inhibition of stimulated adenylate cyclase activity by potent dopaminergic agonists was demonstrable only in female anterior pituitary. The inhibition of adenylate cyclase activity displayed a typically dopaminergic rank order of agonist potencies and could be completely reversed by a specific dopamine receptor antagonist. The IC₅₀ values of dopamine agonist inhibition of adenylate cyclase activity correlated with equal molarity with the dissociation constant of the high-affinity dopamine agonist-detected receptor binding site and with the IC₅₀ values for inhibition of prolactin secretion. These findings support the hypothesis that it is the high-affinity form of the D₂ dopamine receptor in anterior pituitary which is responsible for mediating the dopaminergic function of attenuating adenylate cyclase activity.     

Reconstitution of affinity-purified dopamine D2 receptor binding activities by specific lipids

The role of lipids in maintaining ligand binding properties of affinity-purified bovine striatal dopamine D₂ receptor was investigated in detail. The receptor, purified on a haloperidol-linked Sepharose CL6B affinity column, exhibited low [³H]spiroperidol binding unless reconstituted with soybean phospholipids. In order to understand the role of individual phospholipids in maintaining the receptor binding activity, the purified preparation was reconstituted separately with individual phospholipids and assayed for [³H]spiroperidol binding. Except for phosphatidylcholine and phosphatidylethanolamine, that respectively restored 30 and 20% binding as compared to that obtained with soybean lipids, reconstitution with other lipids had very little effect. When various combinations of phospholipids were used for reconstitution, a phosphatidylcholine and phosphatidylserine mixture seemed to almost fully restore the receptor binding. A mixture of phosphatidylcholine and phosphatidylethanolamine was as effective as phosphatidylcholine alone in reconstituting ligand binding; however, when phosphatidylserine was also included in the mixture, there was a pronounced increase in binding (about 2-fold compared to the soybean lipids and about 6-fold compared to the phosphatidylcholine-phosphatidylethanolamine mixture). Substitution of other phospholipids or cholesterol for phosphatidylserine in phosphatidylcholine and phosphatidylethanolamine mixture had little effect. Maximal reconstitution of [³H]spiroperidol binding was obtained with phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine mixture (2:2:1, w/w) at a concentration of 0.5 mg/ml. The reconstituted receptor exhibited high affinity binding for [³H]spiroperidol which was comparable to that obtained with membrane or solubilized preparations. Various dopaminergic antagonists and agonists showed appropriate order of potency for the reconstituted receptor. The presently described reconstitution data suggest a role of specific phospholipids in preserving the binding properties of dopamine D₂ receptor and should prove useful in studies on functional reconstitution of the receptor.     

THE UPTAKE AND RELEASE OF [3H]DOPAMINE IN THE GOLDFISH RETINA

Abstract— The uptake and release of [³H]dopamine was studied in the goldfish retina with the following results: (1) when goldfish retinas were incubated with 2 ± 10^-7m -[³H]dopamine for less than 20min and processed for autoradiography. most of the label was associated with dopaminergic terminals that contact certain horizontal cells. Biochemical analysis showed that > 93% of this label was [³H]-dopamine. (2) [³H]dopamine uptake saturated with increasing dopamine concentration and followed Michaelis-Menten kinetics. This uptake could be explained by a single ‘high-affinity’ mechanism with a K_m of 2.61 ± 0.41 ± 10^-7m and a V_max of 66 ± 12 ± 10^-12 mol/min/mg protein. (3) [³H]dopamine uptake was temperature-dependent with a temperature coefficient of 1.7 and an energy of activation of 11.4 kcal/mol. (4) The initial rate of uptake was unaffected by the absence of Ca²⁺ or the presence of Co²⁺; however, more than 85, uptake was blocked in the absence of external Na⁺. (5) Neither 1 mm -cyanide nor 5 mm -iodoacetate blocked more than 30% of uptake individually; however, in combination > 70% of uptake was blocked. (6) Centrally acting drugs benztropine and diphenylpyraline inhibited at least 60–70% of [³H]dopamine uptake. (7) [³H]dopamine in the retina could be released by increasing the external K⁺ concentration. This release was Ca²⁺ -dependent and was blocked by 10mm -Co²⁺ or 2Omm -Mg²⁺. The amount of [³H]dopamine released was not affected by the presence of benztropine, diphenylpyraline or fluphenazine in the incubation medium. These studies add further support for dopamine as a neurotransmitter used by interplexiform cells of the goldfish retina.     

[3H]Bicuculline Methochloride Binding to Low-Affinity γ-Aminobutyric Acid Receptor Sites

Abstract: The binding of [³H]bicuculline methochloride (BMC) to mammalian brain membranes was characterized and compared with that of [³H]γ-aminobutyric acid ([³H]GABA). The radiolabeled GABA receptor antagonist showed significant displaceable binding in Tris-citrate buffer that was improved by high concentrations of chloride, iodide, or thiocyanate, reaching >50% displacement in the presence of 0.1 M SCN^?. An apparent single class of binding sites for [³H]BMC (K_D= 30 nM) was observed in 0.1 M SCN^? for fresh or frozen rat cortex or several regions of frozen and thawed bovine brain. The B_max was about 2 pmol bound/mg of crude mitochondrial plus microsomal membranes from unfrozen washed and osmotically shocked rat cortex, similar to that for [³H]GABA. Frozen membranes, however, showed decreased levels of [³H]BMC binding with no decrease or an actual increase in [³H]GABA binding sites. [³H]BMC binding was inhibited by GABA receptor specific ligands, but showed a higher affinity for antagonists and lower affinity for agonists than did [³H]GABA binding. Kinetics experiments with [³H]GABA binding revealed that low- and high-affinity sites showed a similar pharmacological specificity for a series of GABA receptor ligands, but that whereas all agonists had a higher affinity for slowly dissociating high-affinity [³H]GABA sites, bicuculline had a higher affinity for rapidly dissociating low-affinity [³H]GABA sites. This reverse potency between agonists and antagonists during assay of radioactive antagonists or agonists supports the existence of agonist- and antagonist-preferring conformational states or subpopulations of GABA receptors. The differential affinities, as well as opposite effects on agonist and antagonist binding by anions, membrane freezing, and other treatments, suggest that [³H]BMC may relatively selectively label low-affinity GABA receptor agonist sites. This study, using a new commercially available preparation of [³H]bicuculline methochloride, confirms the report of bicuculline methiodide binding by Mohler and Okada (1978), and suggests that this radioactive GABA antagonist will be a valuable probe in analyzing various aspects of GABA receptors.     

Effect of ibogaine on serotonergic and dopaminergic interactions in striatum from mice and rats

The effect of ibogaine (Endabuse, NIH 10567) on serotonin uptake and release, and on serotonergic modulation of dopamine release, was measured in striatal tissue from rats and mice. Two hours after treatment in vivo with ibogaine (40 mg/kg i.p.), the uptake of labeled [³H]serotonin and [³H]dopamine uptake in striatal tissue was similar in the ibogaine-treated animal to that in the control. The 5HT_1B agonist CGS-12066A (10^–5 M) had no effect on stimulation-evoked tritium release from mouse or rat striatal tissue preloaded with [³H]serotonin; however, it elevated tritium efflux from striatal tissue preloaded with [³H]dopamine. This increase was not seen in mice treated with ibogaine 2 or 18 hours previously, or in rats treated 2 hours before. Dopamine autoreceptor responses were not affected by ibogaine pretreatment in either mouse or rat striatal tissue; sulpiride increased stimulation-evoked release of tritium from tissue preloaded with [³H]dopamine. The long-lasting effect of ibogaine on serotonergic functioning, in particular, its blocking of the 5HT_1B agonist-mediated increase in dopamine efflux, may have significance in the mediation of its anti-addictive properties.Special issue dedicated to Dr. Sidney Ochs.     

Solubilization and Characterization of Striatal Dopamine Receptors

Abstract: Dopamine receptor binding proteins were sol-ubilized with the detergent 3–(3–cholamidopropyl) dimethylammonio - 2 - hydroxy - 1– propanesulfonate (CHAPSO) from bovine and rat striatal membranes. The binding of the dopamine antagonist [³H]spiroperidol ([³H]Spi) to the solubilized dopamine receptors was determined by the polyethyleneglycol method. The CHAPSO-solubilized dopamine receptor binding proteins remain in the supernatant fraction following centrifuga-tion at 100,000 ×g for 2 h. The CHAPSO-solubilized dopamine receptor proteins, as well as the prelabeled [³H]Spi-receptor protein complex, bind specifically to wheat germ agglutinin (WGA)-agarose columns, which is consistent with an identification as glycoproteins. HPLC analysis of the CHAPSO-solubilized, prelabeled [³H]Spi-receptor protein complex (CHAPSO preparation) reveals association with a high molecular weight form, indicating the formation of aggregates and/or micelles. Treatment of the WGA-agarose-bound [³H]Spi-receptor protein complex with digitonin (CHAPSO-digitonin preparation) results in dissociation of the high molecular weight form into lower molecular weight forms. The HPLC profile of the prelabeled [³H]Spi-receptor complex in the CHAPSO-digitonin preparation reveals two radioactive peaks. The major peak had a retention time of 16 min, corresponding to an apparent MW of 175,000, whereas the minor peak had a retention time of 21 min, corresponding to an apparent MW of 49,000. The CHAPSO-solubilized dopamine receptor binding proteins are sensitive to modulation by GTP, indicating that the association with the GTP binding component is preserved in the “soluble” state. The potencies of dopamine antagonists and agonists for inhibiting the binding of [³H]Spi to CHAPSO-solubilized dopamine receptor proteins are similar to those for membrane-bound proteins. Chronic treatment with haloperidol increases the B_max, and does not change the K_D for [³H]Spi in the CHAPSO-solubilized and in the membrane-bound preparations. Thus, the CHAPSO-solubilized dopamine receptor proteins retain the binding characteristics of the supersensitive membrane-bound dopamine receptors.     

Effect of melatonin agonists and antagonists on horizontal cell spinule formation and dopamine release in a fish retina

The crucian carp retina was used to study the effects of the melatonin antagonist p697 (N-pentanoyl 2-benzyltryptamine) and the melatonin agonists [+]- and [-]-AMMTC (N-acetyl-4-aminomethyl-6-methoxy-9-methyl-1,2,3,4-tetrahydrocarbazol e) on horizontal cell spinule formation, an indicator of the state of retinal adaptation. DH97 was capable of both counteracting dark-adaptive spinule degradation and inducing light-adaptive spinule formation at the beginning of the dark phase. Addition of dopamine receptor blockers opposed the action of DH97 on spinules, with SCH 23930, a D1 dopamine receptor antagonist, being more effective than the D2 receptor antagonist sulpiride. DH97 induced a twofold increase in dopamine release. We conclude that melatonin acts as a dark signal within the teleost retina by inhibiting the dopaminergic system. In accordance with this, both enantiomers of AMMTC prevented light-induced spinule formation, and reduced dopamine release to below dark-adaptive baseline levels. We suggest that the suppression of spinule formation by AMMTC may be due to either a direct inhibitory interaction between the melatonin agonist and horizontal cell dopamine D1 receptors, or an inhibitory effect on the activity of the dopamine-releasing interplexiform cells.