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.     

A dopamine- and octopamine-sensitive adenylate cyclase in the nervous system of Octopus vulgaris

• 1.1. Adenylate cyclase activity was assayed in the optic lobe of Octopus vulgaris.
• 2.2. Both octopamine and dopamine stimulate the octopus adenylate cyclase, apparently by competing with the same receptor site.
• 3.3. (±)-2-Amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene-HBr (6,7-ADTN) and a number of phenylethanolamine derivatives stimulate the octopus adenylate cyclase activity.
• 4.4. The dopamine D-1 antagonists R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-HCl (SCH-23390) and (±)-7-bromo-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-HCl (SKF-83566) are unable to antagonize the effects of dopamine and octopamine, and similarly ineffective is the agonist (±)-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8-diol-HCl (SKF-38393).
• 5.5. No detectable binding of labelled SCH-23390 occurs on membrane preparations from octopus optic lobe.

     

Adenylate cyclase from sea urchin eggs is positively and negatively regulated by D-1 and D-2 dopamine receptors

The adenylate cyclase present in membranes prepared from sea urchin eggs is sensitive to dopamine stimulation. The receptor sites coupled to sea urchin adenylate cyclase were characterized by means of specific agonists and antagonists. The D-1 dopamine agonist SKF-38393 was able to stimulate enzyme activity, while the two D-1 dopamine antagonists, SCH-23390 and SKF-83566, suppressed the stimulatory effect of dopamine. In addition, the D-2 dopamine agonists, PPHT and metergoline, brought about a dose-dependent inhibition of dopamine-stimulated adenylate cyclase activity. These data show that: (i) in sea urchin eggs adenylate cyclase is regulated by dopamine receptors; (ii) these receptors share characteristics with D-1 and D-2 dopamine receptors present in the mammalian brain.     

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.     

Pharmacological characterization of dopamine-sensitive adenylate cyclase in the salivary glands of Locusta migratoria L.

The locust (Locusta migratoria) salivary gland adenylate cyclase was stimulated by both dopamine and serotonin (6 and 5-fold stimulation respectively). Since their effects were additive, it is concluded that these neurotransmitters are acting on two discrete receptor types.The agonist and antagonist specificity profiles were very similar to those of the D₁ receptor coupled with an adenylate cyclase in vertebrates. They were clearly different from those of the D₂ receptor of vertebrates. No evidence for octopamine sensitive adenylate cyclase was found in the salivary glands of L. migratoria.In addition to previous electrophysiological and histological studies (House and Ginsborg, 1979) these results suggest that dopamine has a neurotransmitter function in the salivary glands of locusts and that its function is mediated by cyclic AMP.     

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.     

Evidence for two types of β-adrenergic-sensitive adenylate cyclase activities in bovine cerebellum

A latent, as well as an expressed form of adenylate cyclase coupled to β-adrenergic receptors is present in intact crude synaptosomal preparations from bovine cerebellum. The latent adenylate cyclase activity was assayed in Krebs-Ringer buffer by [³H]adenine labeling and was found to be coupled to a β₁-like adrenergic receptor. The externally accessible adenylate cyclase assayed in the same with [³H]ATP was stimulated via β₂-adrenergic receptors.     

Dopamine-induced functional activation of Gαq mediated by dopamine D1-like receptor in rat cerebral cortical membranes

Although multiple roles of dopamine through D₁-like (D₁ and D₅) and D₂-like (D₂, D₃, and D₄) receptors are initiated primarily through stimulation or inhibition of adenylyl cyclase via G_s/olf or G_i/o, respectively, there have been many reports indicating diverse signaling mechanisms that involve alternative G protein coupling. In this study, dopamine-induced Gα_q activation in rat brain membranes was investigated. Agonist-induced Gα_q activation was assessed by increase in guanosine-5′-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding to Gα_q determined by [³⁵S]GTPγS binding/immunoprecipitation assay in rat brain membranes. Dopamine-stimulated Gα_q functionality was highest in cortex as compared to hippocampus or striatum. In cerebral cortical membranes, this effect was mimicked by benzazepine derivatives with agonist properties at dopamine D₁-like receptors, that is, SKF83959, SKF83822, R(+)-SKF81297, R(+)-SKF38393, and SKF82958, but not by the compounds with dopamine D₂-like receptor agonist properties except for aripiprazole. Against expectation, stimulatory effects were also induced by SKF83566, R(+)-SCH23390, and pergolide. The pharmacological profiling by using a series of antagonists indicated that dopamine-induced response was mediated through dopamine D₁-like receptor, which was distinct from the receptor involved in 5-HT-induced response (5-HT_2A receptor). Conversely, the responses induced by SKF83566, R(+)-SCH23390, and pergolide were most likely mediated by 5-HT_2A receptor, but not by dopamine D₁-like receptor. Caution should be paid when interpreting the experimental data, especially in behavioral pharmacological research, in which SKF83566 or R(+)-SCH23390 is used as a standard selective dopamine D₁-like receptor antagonist. Also, possible clinical implications of the agonistic effects of pergolide on 5-HT_2A receptor has been mentioned.     

6,7-Dihydroxy-2-dimethylaminotetralin (TL-99) stimulates postjunctional D-1 and D-2 dopamine receptors

6,7-Dihydroxy-2-dimethylaminotetralin (TL-99) mimicks the ability of dopamine either to enhance adenylate cyclase activity in homogenates of goldfish retina or to inhibit adenylate cyclase activity in homogenates of the intermediate lobe (IL) of the rat pituitary gland previously treated with cholera toxin. Both the dopamine stimulated adenylate cyclase activity in the fish retina and the dopamine inhibited adenylate cyclase activity in the rat IL are associated with cells postjunctional to the dopaminergic neurons innervating these tissues. Therefore, the present data do not support the contention that TL-99 is a selective presynaptic dopamine receptor agonist.     

Adrenocorticotropin/α-Melanocyte-Stimulating Hormone (ACTH/MSH)-Like Peptides Modulate Adenylate Cyclase Activity in Rat Brain Slices: Evidence for an ACTH/MSH Receptor-Coupled Mechanism

Abstract: The regulation of adenylate cyclase activity by adrenocorticotropin/α-melanocyte–stimulating hormone (ACTH/MSH)-like peptides was investigated in rat brain slices using a superfusion method. Adenylate cyclase activity was concentration-dependently increased by ACTH-(1–24), α-MSH (EC₅₀ values 16 and 6 nM, respectively), and [Nle⁴,D-Phe⁷]α-MSH (EC₅₀ value 1.6 nM), in the presence of forskolin (1 μM, optimal concentration). 1-9-Dideoxy-forskolin did not augment the response of adenylate cyclase to ACTH-(1–24). Various peptide fragments were tested for their ability to enhance [³H]cyclic AMP production. [Nle⁴,D-Phe⁷]α-MSH increased [³H]cyclic AMP formation with a maximal effect of 30% and was more potent than ACTH-(1–24), ACTH-(1–16)-NH₂, α-MSH, ACTH-(1–13)-NH₂, [MetO⁴]α-MSH, [MetO₂⁴,D-Lys⁸,Phe⁹]ACTH-(4–9), ACTH-(7–16)-NH₂, ACTH-(1–10), and ACTH-(11–24), in order of potency. This structure–activity relationship resembles that found for the previously described peptide-induced display of excessive grooming. ACTH-(1–24) stimulated adenylate cyclase activity in both striatal (maximal effect, ?20%) and septal slices (maximal effect, ?40%), but not in hippocampal or cortical slices. Lesioning of the dopaminergic projections to the striatum did not result in a diminished effect of [Nle⁴,D-Phe⁷]α-MSH on [³H]cyclic AMP accumulation, which indicates that the ACTH/MSH receptor–stimulated adenylate cyclase is not located on striatal dopaminergic terminals. ACTH-(1–24) did not affect the dopamine D₁ or D₂ receptor–mediated modulation of adenylate cyclase activity. Based on the present data, we suggest that the binding of endogenous ACTH or α-MSH to a putative ACTH/MSH receptor in certain brain regions leads to the activation of a signal transduction pathway using cyclic AMP as a second messenger.     

Chemical lesion and drug induced supersentivity and subsensitivity of caudate dopamine receptors

In order to elucidate the mechanism of denervation supersensitivity, the effects of 6-hydroxydopamine lesion, placed in the substantia nigra, were examined on rat brain caudate adenylate cyclase and ³H-haloperidol binding to membrane dopamine receptors. In addition, the effects of chronic administration of L-DOPA, bromocriptine and piribedil were also investigated on ³H-haloperidol binding and dopamine, K⁺ isoproterenol (IPNE) and 2-Cl-adenosine stimulated formation of cyclic AMP in caudate slices. 6-Hydroxydopamine lesions resulted in significantly greater stimulation of adenylate cyclase by dopamine at various concentrations tested. The haloperidol binding sites were increased by 28% on lesioned side caudate without changes in dissociation constants (K_D). Three weeks after treatment with L-DOPA, bromocriptine or piribedil, the ³H-haloperidol binding sites were decreased by 40% with no change in K_D. The stimulatory effect of dopamine on cyclic AMP formation was also abolished, although there was no change in IPNE, K⁺, or 2-Cl-adenosine stimulated cyclic AMP formation in caudate slices, suggesting a specific effect of dopamine agonists on dopamine receptors. The results of these studies suggest a close relationship between at least some populations of dopamine receptors and adenylate cyclase in the caudate nucleus.     

Octopamine- and dopamine-sensitive adenylate cyclase in the brain ofLocusta migratoria during its development

Octopamine- and dopamine-sensitive adenylate cyclases were studied in the brain of Locusta migratoria during its metamorphosis. In the adult brain the effects of octopamine and dopamine on adenylate cyclase were additive, suggesting the presence of separate populations of adenylate cyclase-linked receptors for octopamine and dopamine. There are no separate receptors for noradrenaline. Octopamine stimulates adenylate cyclase in both adult and larval brain; however, in adult brain octopamine is more potent than in larval brain. Dopamine stimulates adenylate cyclase activity only in adult brain. The sensitivity of adenylate cyclase to octopamine changes during the development of the animal. Phentolamine and cyproheptadine are potent antagonists of octopamine-stimulated adenylate cyclase, while propranolol has a weak effect. No cytosol factor which would modulate either basal or octopamine-stimulated adenylate cyclase was found. The effect of GTP and octopamine on adenylate cyclase was synergistic in adult brain but not in larval brain, while the effect of GppNHp and octopamine was synergistic in both adult and larval brains.     

Interaction of metergoline with striatal dopamine system

The intragastric of intraperitoneal administration of metergoline (2.5–10 mg/kg) stimulates dopamine (DA) synthesis in the rat and mouse brain. In vitro, metergoline blocks DA-sensitive adenylate cyclase and displaces H³-haloperidol from specific binding sites in striatal homogenates.     

Effect of neuropeptide-EI on the binding of [3H]SCH 23390 to the dopamine D1 receptor in rat striatal membranes

We have previously demonstrated that neuropeptide-EI, at high doses, stimulates the production of cAMP, in caudate putamen, through the activation of adenylate cyclase coupled to specific D₁ receptors. The aim of the present work was to find evidences for a probable interaction between this neuropeptide and the dopamine D₁ receptor in the mammalian central nervous system. The present data show that neuropeptide-EI, at high concentrations, affected both the maximum binding and the apparent affinity of [n-methyl-³H] (R)-(+)-8 chloro-2,3,4,5- tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hemimaleate to the dopamine D₁ receptor in a concentration-dependent manner.     

Agonist-induced desensitization of the D-2 dopamine receptor in the intermediate lobe of the rat pituitary gland

The mechanism of agonist-induced desensitization of the D-2 dopamine receptor in the intermediate lobe (IL) of the rat pituitary gland was investigated. Exposure of neurointermediate lobe to 60 microM (-)apomorphine (APO) for 60 min altered the binding of [125I]-N-(p-aminophenethyl)spiperone (NAPS), a D-2 receptor-specific ligand. The capacity of the tissue to bind the ligand (Bmax) was not significantly altered by the exposure to (-)APO but the affinity for [125I]NAPS was decreased 3.6-fold in (-)APO-exposed tissue. The molar potency of YM-09151-2, a D-2 receptor-specific antagonist, showed a minimal difference between in control and (-)-APO-exposed tissue. However, the molar potency of (-)APO towards the D-2 receptor was diminished. The loss of [125I]NAPS binding in (-)APO-exposed tissue was reversed by the addition of guanyl nucleotide. These data suggest that exposure to agonist causes a persistent occupancy of the high affinity state of the receptor. Exposure to (-)APO had no effect on either basal or forskolin-activated adenylate cyclase activity of the intermediate lobe. However, the inhibitory effect of (-)APO upon adenylate cyclase activity of IL homogenates was diminished when the tissue was exposed to (-)APO before homogenization. Furthermore, the ability of GTP but not 5'-guanylyl imidodiphosphate [Gpp(NH)p] to inhibit enzyme activity diminished in the (-)APO-exposed tissue. These data suggest that an agonist-induced desensitization of D-2 receptor in rat IL is thought to occur by uncoupling the receptor from the inhibitory guanyl nucleotide binding protein (Gi) or potentiating the hydrolysis of GTP by Gi.     

Effects of neuroleptics on 3H-haloperidol and 3H-cis(Z)-flupenthixol binding and on adenylate cyclase activity in vitro.

The subcellular localization of dopamine-sensitive adenylate cyclase was studied in rat brain striatum and compared to the distribution of dopamine binding sites. The highest specific activity of adenylate cyclase activities sensitive to dopamine was associated almost exclusively with synaptic membranes (mithchondrial fraction; P₂). Using [³H] haloperidol and [³H] apomorphine as markers for the dopamine receptor, specific binding was observed in both the mitochondrial (P₂) and microsomal (P₃) fractions. Data for the mitochondrial fraction revealed a heterogeneity of binding sites. Two saturable sites for [³H] haloperidol were observed with K_d values of 2.5nM and 12.5nM respectively. Overall, the localization of multiple binding sites in the crude synaptosomal fraction correlates well with the localization of dopamine-sensitive adenylate cyclase in this fraction.     

α<Subscript>2A</Subscript>- and α<Subscript>2C</Subscript>-Adrenoceptors as Potential Targets for Dopamine and Dopamine Receptor Ligands

The poor norepinephrine innervation and high density of Gi/o-coupled α_2A- and α_2C-adrenoceptors in the striatum and the dense striatal dopamine innervation have prompted the possibility that dopamine could be an effective adrenoceptor ligand. Nevertheless, the reported adrenoceptor agonistic properties of dopamine are still inconclusive. In this study, we analyzed the binding of norepinephrine, dopamine, and several compounds reported as selective dopamine D₂-like receptor ligands, such as the D₃ receptor agonist 7-OH-PIPAT and the D₄ receptor agonist RO-105824, to α₂-adrenoceptors in cortical and striatal tissue, which express α_2A-adrenoceptors and both α_2A- and α_2C-adrenoceptors, respectively. The affinity of dopamine for α₂-adrenoceptors was found to be similar to that for D₁-like and D₂-like receptors. Moreover, the exogenous dopamine receptor ligands also showed high affinity for α_2A- and α_2C-adrenoceptors. Their ability to activate Gi/o proteins through α_2A- and α_2C-adrenoceptors was also analyzed in transfected cells with bioluminescent resonance energy transfer techniques. The relative ligand potencies and efficacies were dependent on the Gi/o protein subtype. Furthermore, dopamine binding to α₂-adrenoceptors was functional, inducing changes in dynamic mass redistribution, adenylyl cyclase activity, and ERK1/2 phosphorylation. Binding events were further studied with computer modeling of ligand docking. Docking of dopamine at α_2A- and α_2C-adrenoceptors was nearly identical to its binding to the crystallized D₃ receptor. Therefore, we provide conclusive evidence that α_2A- and α_2C-adrenoceptors are functional receptors for norepinephrine, dopamine, and other previously assumed selective D₂-like receptor ligands, which calls for revisiting previous studies with those ligands.     

Independent variation of β-adrenergic receptor binding and catecholamine-stimulated adenylate cyclase activity in rat erythrocytes

Isoproterenol-stimulated adenylate cyclase activity in membrane preparations of reticulocyte-rich (90%) erythrocytes from phenylhydrazine-treated rats is 9 times greater than in untreated animals (1% reticulocytes); basal and fluoride-stimulated activities are also enhanced 2 and 4-fold respectively. In contrast, the number of β-adrenergic receptor sites detected by the binding of ¹²⁵I-hydroxybenzylpindolol (¹²⁵I-HYP) is increased only 40% in these same preparations. The dissociation constant (K_D) of ¹²⁵I-HYP and the IC₅₀ of (-)-isoproterenol for receptor binding sites are unchanged, as is the EC₅₀ of (-)-isoproterenol for activation of adenylate cyclase. The disproportionately large increase in the activity of the isoproterenol-sensitive adenylate cyclase, compared with the small increase in the number of ¹²⁵I-HYP binding sites indicates that the functions of catecholamine recognition and consequent adenylate cyclase response can vary independently and suggest that the receptor and the cyclase may be autonomous molecular entities.     

Effect of a novel benzamide, YM-09151-2, on rat serum prolactin levels

YM-09151-2(cis-N-(1-benzyl-2-methylpyrrolidin-3-yl) -5-chloro-2-methoxy-4-methylaminobenzamide) is a new benzamide which has been reported to be an antagonist of dopamine (DA) at D1-type DA receptors. In the present study, we have examined the extent of the interaction of YM-09151-2 with D2-type DA receptors in the anterior pituitary gland of the rat. YM-09151-2 was found to be a potent antagonist of these receptors as extremely low doses of this compound produced a marked, dose-dependent elevation of serum prolactin concentrations. This benzamide was also a potent blocker of DA-induced inhibition of prolactin release in vitro and was very effective in displacing 3H-spiperone from bovine pituitary membranes: IC50, 1.04nM. These results are consistent with recent evidence that YM-09151-2 is also a potent antagonist of the D2-type receptors in the intermediate lobe of the rat pituitary gland.