Chronic Dopamine D2 Receptor Activation Does Not Affect Survival and Differentiation of Cultured Dopaminergic Neurons: Morphological and Neurochemical Observations

Abstract: Primary cultures of rat ventral mesencephalon were used to elucidate the role of chronic stimulation of dopamine (DA) D₂ autoreceptors in the development of fetal dopaminergic neurons in vitro. Cultured dopaminergic neurons, as visualized by tyrosine hydroxylase immunocytochemistry, became more differentiated in the course of cultivation time and exhibited specific high-affinity uptake for [³H]DA. In rat striatal tissue, activation of D₂ receptors has been shown to inhibit the release of DA. Previously accumulated [³H]DA was released from the cultures upon depolarization in a Ca²⁺-dependent manner. K⁺-evoked [³H]DA release could be inhibited by the selective D₂ receptor agonists LY 171555 and N0437 in a concentration-dependent manner. The inhibitory effects of LY 171555 and N0437 were antagonized by the selective DA D₂ receptor antagonist sulpiride. These observations are indicative for the expression of functional D₂ receptors in the cultures. Daily treatment of these cultures for 7 days with LY 171555 or sulpiride did not lead to any change in protein content, the number of tyrosine hydroxylase-immunoreactive neurons, or the uptake capacity for [³_H]DA. Our data demonstrate that chronic stimulation of DA D₂ receptors does not impair survival or differentiation of cultured fetal dopaminergic neurons.     

Effects of Amphetamine on Carrier-Mediated and Electrically Stimulated Dopamine Release in Slices of Rat Caudate Putamen and Nucleus Accumbens

Abstract: The effects of (+)-amphetamine on carrier-mediated and electrically stimulated dopamine release were investigated using fast cyclic voltammetry in rat brain slices incorporating the nucleus accumbens, and in the caudate putamen. In the caudate putamen, dopamine release either increased with increasing frequency of local electrical stimulation (hot spots) or did not increase significantly (cold spots); dopamine release increased with increasing frequency of electrical stimulation in the nucleus accumbens. Local pressure application of (+)-amphetamine from a micropipette caused dopamine efflux at all sites examined, and this was not affected by sulpiride, indicating that efflux of dopamine caused by (+)-amphetamine is not regulated by dopamine D₂ autoreceptors. (+)-Amphetamine reduced single-pulse electrically stimulated dopamine release at all sites; sulpiride reversed this decrease, indicating that endogenous dopamine released by (+)-amphetamine activates dopamine D₂ autoreceptors. In nucleus accumbens and hot spots, (+)-amphetamine did not affect 20-pulse 50-Hz-stimulated dopamine release, whereas in cold spots it potentiated 20-pulse 50-Hz-stimulated dopamine release. We conclude that (+)-amphetamine modifies electrically stimulated dopamine release by uptake inhibition or by indirect activation of D₂ autoreceptors; the precise mechanism is determined by site and duration of electrical stimulation.     

Effects of the Dopamine D3 Antagonist PD 58491 and Its Interaction with the Dopamine D3 Agonist PD 128907 on Brain Dopamine Synthesis in Rat

Abstract: The dopamine (DA) D₃ receptor antagonist PD 58491 {3-[4-[1-[4-[2-[4-(3-diethylaminopropoxy)phenyl]-benzoimidazol-1-yl-butyl]-1 H -benzoimidazol-2-yl]-phenoxy]propyl]diethylamine} bound with high affinity and selectivity to recombinant human DA D₃ versus D_2L and D_4.2 receptors transfected into Chinese hamster ovary cells: K _i values of 19.5 n M versus 2,362 and >3,000 n M , respectively. In contrast, the putative DA D₃ receptor antagonist (+)-AJ76 displayed low affinity and selectivity for D₃ versus D_2L and D_4.2 receptors (91 n M vs. 253 and 193 n M , respectively). In vitro, PD 58491 (1 n M −1µ M ) exhibited D₃ receptor antagonist activity, reversing the quinpirole (10 n M )-induced stimulation of [³H]thymidine uptake in D₃ CHOpro-5 cells, but did not have any significant intrinsic activity by itself in this assay. PD 58491 did not decrease the γ-butyrolactone-induced increase in DA synthesis ( l -3,4-dihydroxyphenylalanine accumulation) in rat striatum, indicating that the compound possessed no in vivo DA D₂/D₃ receptor agonist action at DA autoreceptors. PD 58491 (3–30 mg/kg, i.p.) generally did not alter DA or serotonin synthesis in either the striatum or mesolimbic region of rat brain. The D₃-preferring agonist PD 128907 decreased DA synthesis in striatum and mesolimbic regions, and this effect was attenuated by pretreatment with PD 58491. These findings support the hypothesis that DA D₃ autoreceptors may in part modulate the synthesis and release of DA in striatum and mesolimbic regions.     

D1 Dopamine Receptor Binding and mRNA Levels Are Not Altered After Neonatal 6-Hydroxydopamine Treatment: Evidence Against Dopamine-Mediated Induction of D1 Dopamine Receptors During Postnatal Development

Abstract: The role of dopaminergic innervation on the postnatal developmental expression of D₁ dopamine receptors was investigated. Bilateral destruction of dopa-mine-containing neurons was achieved by treating rats intracisternally with 6-hydroxydopamine (6-OHDA) on postnatal day 3, and rats were killed on day 21. To ensure effective reduction of D₁ receptor activation by residual dopamine, a group of 6-OHDA-lesioned rats was given twice daily injections of the D₁ receptor antagonist SCH-23390, from day 4 to 20. D₁ dopamine receptor binding was assessed in the caudate—putamen, nucleus accumbens, and olfactory tubercle by quantitative autoradiographic analysis of [³H]SCH-23390 binding. In addition, the relative amount of D_1A receptor mRNA was assessed by in situ hybridization of a ³⁵S-labeled riboprobe. In the developing rats, neither the amount of [³H]SCH-23390 binding nor the amount of D_1A receptor mRNA was altered by 6-OHDA lesioning followed by chronic treatment with SCH-23390. Thus, bilateral destruction of dopamine-containing neurons and treatment with SCH-23390 in neonatal rats did not interfere with the developmental expression of D₁ receptors or alter the levels of mRNA that code for this receptor protein. Treatment of intact rats with SCH-23390 from postnatal day 4 to 20 also did not alter [³H]SCH-23390 binding or levels of D₁ receptor mRNA. However, adult rats treated chronically with SCH-23390 exhibited increased [³H]SCH-23390 binding but did not show a significant change in D₁ receptor mRNA levels.     

Alpha-synuclein aggregation and cell death triggered by energy deprivation and dopamine overload are counteracted by D2/D3 receptor activation

Progressive degeneration and intraneuronal Lewy bodies made of filamentous α-synuclein (α-syn) in dopaminergic cells of the nigrostriatal system are characteristics of Parkinson's disease (PD). Glucose uptake is reduced in some of the brain regions affected by PD neurodegenerative changes. Defects in mitochondrial activity in the substantia nigra have been observed in the brain of patients affected by PD and substantia nigra lesions can induce the onset of a secondary parkinsonism. Thus, energy starvation and consequently metabolic impairment to dopaminergic neurons may be related to the onset of PD. On this line, we evaluated the effect of nutrient starvation, reproduced ' in vitro ' by glucose deprivation (GD), in primary mesecephalic neuronal cultures and dopaminergic-differentiated SH-SY5Y cells, to evaluate if decreased glucose support to dopaminergic cells can lead to mitochondrial damage, neurodegeneration and α-syn misfolding. Furthermore, we investigated the effect of dopamine (DA) treatment in the presence of a DA-uptake inhibitor or of the D₂/D₃ receptor (D₂R/D₃R) agonist quinpirole on GD-treated cells, to evaluate the efficacy of these therapeutic compounds. We found that GD induced the formation of fibrillary aggregated α-syn inclusions containing the DA transporter in dopaminergic cells. These alterations were accompanied by dopaminergic cell death and were exacerbated by DA overload. Conversely, the block of DA uptake and D₂R/D₃R agonist treatment exerted neuroprotective effects. These data indicate that glucose starvation is likely involved in the induction of PD-related pathological changes in dopaminergic neurons. These changes may be counteracted by the block of DA uptake and by dopaminergic agonist treatment.     

Zinc Allosterically Modulates Antagonist Binding to Cloned D1 and D2 Dopamine Receptors

Abstract: Cations of various size and charge were used as atomic scale probes of D₁ and D₂ dopamine receptors. Those cations that perturbed the binding of D₁- and D₂-selective dopamine receptor antagonists were identified by screening at 5 m M cation. Pseudo-noble-gas-configuration d-transition metals, such as zinc, exerted a complete inhibition of specific binding, whereas most other cations had little or no effect. The nature of zinc's actions was characterized by measuring the radioligand binding properties of [³H]SCH-23390 and [³H]methylspiperone to cloned D_1A and D_2L dopamine receptors in either the presence or absence of Zn²⁺. Zinc exerts a low-affinity, dose-dependent, EDTA-reversible inhibition of the binding of subtype-specific antagonists primarily by decreasing the ligands' affinity for their receptors. The mechanism of zinc inhibition appears to be allosteric modulation of the dopamine receptor proteins because zinc increases the dissociation constant ( K _D) of ligand binding, Schild-type plots of zinc inhibition reach a plateau, and zinc accelerates antagonist dissociation rates. Here we demonstrate the effect of zinc on the binding of D₁- and D₂-selective antagonists to cloned dopamine receptors and show that the inhibition by zinc is through a dose-dependent, reversible, allosteric, two-state modulation of dopamine receptors.     

Dopaminergic Regulation of Septohippocampal Cholinergic Neurons

Abstract: The extent to which acetylcholine (ACh) release in the hippocampus is regulated by dopaminergic mechanisms was assessed using in vivo microdialysis in freely moving rats. Systemic administration of the dopamine (DA) receptor agonist apomorphine (1.0 mg/kg) or the specific D₁ agonist CY 208–243 (1.0 mg/kg) increased microdialysate concentrations of ACh in the hippocampus. The D₂ receptor agonist quinpirole (0.5 mg/kg) produced a small but statistically significant decrease in hippocampal ACh release. d -Amphetamine (2.0 mg/kg) increased ACh release, an effect that was blocked by the D₁ receptor antagonist SCH 23390 (0.3 mg/kg) but not by the D₂ antagonist raclopride (1.0 mg/kg). These findings suggest that endogenous DA stimulates septo-hippocampal cholinergic neurons primarily via actions at D₁ receptors. In addition, these results are similar to previous findings regarding the dopaminergic regulation of cortical ACh release, and suggest that the anatomical continuum formed by basal forebrain cholinergic neurons that project to the cortex and hippocampus acts as a functional unit, at least with respect to its regulation by DA.     

Inhibition of Adenylyl Cyclase Activity by a Homogeneous Population of Dopamine Receptors: Selective Blockade by Antisera Directed Against Gi1 and/or Gi2

Abstract: The 7315c pituitary tumor cell expresses a homogeneous population of dopamine receptors that are functionally similar to brain dopamine D₂ receptors. [³H]-Sulpiride binding to 7315c cell homogenates was specific and saturable, and K _i values for compounds to compete for these sites were highly correlated with values for the same compounds at D₂ receptors in brain. Dopamine maximally inhibited ∼65% of forskolin-stimulated cyclase activity in cell membranes. Some D₂ agonists had lower efficacies, suggesting that some compounds are partial agonists at this receptor. Removal of GTP from the assay buffer or pretreatment of the tissue with pertussis toxin abolished the inhibition of adenylyl cyclase by dopamine. Immunodetection of most of the known Gα subunits revealed that G_i1, G_i2, G_i3, G_o, G_q, and G_s are present in the 7315c membrane. Pretreatment with the AS antibody (which recognizes the C-terminal regions of Gα_i1 and Gα_i2) significantly attenuated the inhibition of adenylyl cyclase activity by dopamine, whereas antibodies to C-terminal regions of the other Gα subunits had no effect. These findings suggest that the dopamine D₂ receptor regulates cyclase inhibition predominantly via G_i1 and/or G_i2 and that the 7315c tumor cells provide a useful model for studying naturally expressed dopamine D₂ receptors in the absence of other dopamine receptor subtypes.     

[3H]Nemonapride and [3H]Spiperone Label Equivalent Numbers of D2 and D3 Dopamine Receptors in a Range of Tissues and Under Different Conditions

Abstract: [³H]Nemonapride and [³H]spiperone are very widely used to study dopaminergic systems in vitro and in vivo, but it has been reported that [³H]nemonapride and [³H]spiperone give markedly different B _max values for preparations of D₂ dopamine receptors from recombinant cell lines or animal tissues. We have used the two radioligands in parallel to study a range of dopamine receptors [D_2(short), D_2(long), and D₃] in different buffers. B _max values derived using either radioligand differ by an average of <20%, independent of receptor type or buffer conditions. All competition experiments show that the two ligands compete at a single site. It seems that [³H]spiperone and [³H]nemonapride do not differentiate between different forms or populations of D₂-like receptors.     

Ascorbic Acid Inhibits 125I-SCH 23982 Binding but Increases the Affinity of Dopamine for D1 Dopamine Receptors

Abstract: Effects of ascorbic acid (AA) on ¹²⁵I-SCH 23982 binding to D₁ dopaminergic receptors in membrane preparations from rat striatum were investigated. AA in the range of 0.03 µ M –0.33 m M inhibited 75% of specific binding of ¹²⁵I-SCH 23982 in a dose-dependent manner. At higher concentrations, this inhibition of binding activity by AA was less potent, and 3.3 m M AA inhibited only 30% of specific binding. Reduced glutathione did not alter the inhibition of binding by 0.33 m M AA, but reduced the inhibition by 3.3 m M AA to 8% of specific binding. The loss of specific binding by AA was rescued by 1 m M EDTA, an inhibitor of lipid peroxidation. In the absence of AA, competition experiments with the agonist, dopamine, revealed the presence of high-affinity ( K _h = 224.9 ± 48.9 n M ) and low-affinity ( K _l = 21,100 ± 2,400 n M ) binding sites. Although the maximum binding of ¹²⁵I-SCH 23982 decreased to 40% without affecting the K _D value in the presence of 1.67 m M AA, the value of the high-affinity site for dopamine was increased ( K _h = 23.3 ± 9.4 n M ) and that of the low-affinity site was decreased ( K _l = 136,800 ± 40,900 n M ). These results suggest that AA may affect D₁ dopamine receptor function by lipid peroxidation, competition with dopamine for low-affinity sites, and reduced oxidation of dopamine.     

Inhibition of Dopamine Agonist-Induced Phosphoinositide Hydrolysis by Concomitant Stimulation of Cyclic AMP Formation in Brain Slices

Abstract: We examined the effects of cyclic AMP on dopamine receptor-coupled activation of phosphoinositide hydrolysis in rat striatal slices. Forskolin, dibutyryl cyclic AMP, and the protein kinase A activator Sp -cyclic adenosine monophosphothioate ( Sp -cAMPS) significantly inhibited inositol phosphate formation stimulated by the dopamine D₁ receptor agonist SKF 38393. Conversely, the protein kinase A antagonist Rp -cyclic adenosine monophosphothioate ( Rp -cAMPS) dose-dependently potentiated the SKF 38393 effect. In the presence of 200 µ M Rp -cAMPS, the dose-response curves of the dopamine D₁ receptor agonists SKF 38393 and fenoldopam were shifted to the left and maximal agonist responses were markedly increased. The agonist EC₅₀ values, however, were not significantly altered by protein kinase A inhibition. Neither Sp -cAMPS nor Rp -cAMPS significantly affected basal inositol phosphate accumulation. These findings demonstrate that dopaminergic stimulation of phosphoinositide hydrolysis is inhibited by elevations in intracellular cyclic AMP. Dopamine receptor agonists that stimulate adenylyl cyclase could suppress their activation of phosphoinositide hydrolysis by concomitantly stimulating the formation of cyclic AMP in striatal tissue. The interaction between dopamine D₁ receptor-stimulated elevations in cyclic AMP and dopaminergic stimulation of inositol phosphate formation suggests a cellular colocalization of these dopamine-coupled transduction pathways in at least some cells of the rat striatum.     

Two Intracellular Signaling Pathways for the Dopamine D3 Receptor: Opposite and Synergistic Interactions with Cyclic AMP

Abstract: As cerebral neurons express the dopamine D₁ receptor positively coupled with adenylyl cyclase, together with the D₃ receptor, we have investigated in a heterologous cell expression system the relationships of cyclic AMP with D₃ receptor signaling pathways. In NG108-15 cells transfected with the human D₃ receptor cDNA, dopamine, quinpirole, and other dopamine receptor agonists inhibited cyclic AMP accumulation induced by forskolin. Quinpirole also increased mitogenesis, assessed by measuring [³H]thymidine incorporation. This effect was blocked partially by genistein, a tyrosine kinase inhibitor. Forskolin enhanced by 50–75% the quinpirole-induced [³H]thymidine incorporation. This effect was maximal with 100 n M forskolin, occurred after 6–16 h, was reproduced by cyclic AMP-permeable analogues, and was blocked by a protein kinase A inhibitor. Forskolin increased D₃ receptor expression up to 135%, but only after 16 h and at concentrations of >1 µ M . Thus, in this cell line, the D₃ receptor uses two distinct signaling pathways: it efficiently inhibits adenylyl cyclase and induces mitogenesis, an effect possibly involving tyrosine phosphorylation. Activation of the cyclic AMP cascade potentiates the D₃ receptor-mediated mitogenic response, through phosphorylation by a cyclic AMP-dependent kinase of a yet unidentified component. Hence, transduction of the D₃ receptor can involve both opposite and synergistic interactions with cyclic AMP.     

Activation of D1 and D2 Dopamine Receptors Inhibits Protein Kinase C Activity in Striatal Synaptoneurosomes

Abstract: The effects of D₁ and D₂ dopamine ligands on protein kinase C (PKC) activity were examined in synaptoneurosomes. Incubation with D₁ agonists (SKF 38393, fenodopam), in the presence of calcium, decreased the soluble and increased the particulate PKC activity. These effects were reversed by SCH 23390, which by itself had the opposite effect of increasing the soluble and decreasing the particulate PKC activity. In contrast, incubation with the D₂ agonists [LY 171555, (+)-3-(3-hydroxyphenyl)- N - n -propylpiperidine, RU 24213] increased the soluble and decreased the particulate PKC activity. These effects were reversed by sulpiride. (−)-3-(3-Hydroxyphenyl)- N - n -propylpiperidine had a D₂ antagonist profile. Apomorphine showed a biphasic dose-response change; i.e., it decreased particulate PKC activity at the D₂ receptor at low concentrations (0.1 µ M ) and increased it at the D₁ receptor at higher concentrations (10 µ M ). Pretreatment with tetrodotoxin or omission of calcium in the incubation medium did not alter the responses of the D₂ agonists, but it reversed the changes in PKC activity induced by the D₁ agonists and converted the biphasic response of apomorphine to a monophasic inhibition. These results indicate that (1) D₁ and D₂ dopamine receptors are negatively coupled to PKC and (2) the increase in particulate PKC activity seen with the D₁ drugs in the presence of calcium is mediated indirectly via a transneuronal effect.     

Opposing Actions of D1 and D2-Dopamine Receptors on Arachidonic Acid Release and Cyclic AMP Production in Striatal Neurons

Abstract: D₁-and D₂-dopamine receptors exert important physiological actions on striatal neurons, but the intracellular second messenger pathways activated by these receptors are still incompletely understood. Using primary cultures of rat striatal cells, we have examined the effects of activating D₁ or D₂ receptors on arachidonic acid (AA) release and cyclic AMP accumulation. In striatal neurons labeled by incubation with [³H]AA, D₂-receptor stimulation enhanced release of [³H]AA produced by application of the Ca²⁺ ionophore A23187 or of the purinergic agonist ATP. By contrast, D₁-receptor stimulation inhibited [³H]AA release. This inhibitory effect of D₁ receptors was accompanied by stimulation of adenylyl cyclase activity, measured as accumulation of cyclic AMP, and was mimicked by application of the adenylyl cyclase activator forskolin. The results indicate the existence of a novel signaling pathway for D₂ and D₁ receptors in striatum, potentiation and inhibition, respectively, of Ca²⁺-evoked AA release.     

Dopamine Receptor Stimulation Decreases Cytosolic γ Protein Kinase C Immunoreactivity in Rat Hippocampal Slices: Evidence for Increased Ca2+-Dependent Proteolysis

Abstract: The effect of dopamine (DA) receptor stimulation on the distribution of γ protein kinase C (γPKC) in hippocampal slices was assessed. Nanomolar concentrations of DA decreased cytosolic γPKC (56%) without altering membrane γPKC levels, resulting in decreased total γPKC immunoreactivity. The maximal decrease in cytosolic γPKC occurred at 20 min of incubation and was significantly blocked by the D₁ DA antagonist SCH 23390 (10⁻⁶ M ) but not by the D₂ antagonist sulpiride (10⁻⁵ M ). The D₁ agonists SKF 38393 and A 77636 mimicked the effect of DA with similar responses produced at 10 µ M and 1 n M , respectively. The D₂ agonist quinpirole had no effect on γPKC immunoreactivity, thus indicating that this dopaminergic response is mediated through a D₁-like receptor. DA had no effect on α, δ, or ζPKC isozyme immunoreactivity in the same hippocampal preparations. The DA-induced decrease in cytosolic γPKC immunoreactivity was blocked by the Ca²⁺-dependent protease inhibitor N -acetyl-Leu-Leu-norleucinal (100 µ M ) and by the inorganic Ca²⁺ channel blocker Co²⁺. The data suggest that DA stimulates a D₁-like DA receptor, which increases the influx of Ca²⁺ and activates the Ca²⁺-dependent proteolysis of γPKC.     

Inhibitory effects of aripiprazole on interferon-gamma-induced microglial activation via intracellular Ca2+ regulation in vitro

The activation of the inflammatory/immunological response system is suggested to be related to the pathophysiology of schizophrenia. Aripiprazole is a novel atypical antipsychotic, which is a high-affinity dopamine D₂ receptor partial agonist. Atypical antipsychotics, all of which have dopamine D₂ receptor antagonism, have recently reported to have significantly inhibitory effects on interferon (IFN)-γ-induced microglial activation in vitro . In the present study, we investigated whether or not aripiprazole also has anti-inflammatory effect on IFN-γ-induced microglial activation. Not quinpirole, dopamine D₂ full agonist, but aripiprazole significantly inhibited the generation of nitric oxide (NO) and tumor necrosis factor (TNF)-α from IFN-γ-activated microglia and suppressed the IFN-γ-induced elevation of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in murine microglial cells. Increased [Ca²⁺]_i has been reported to be required, but by itself not sufficient, for the release of NO and certain cytokines. As a result, we can speculate that aripiprazole may inhibit IFN-γ-induced microglial activation through the suppression of IFN-γ-induced elevation of [Ca²⁺]_i in microglia. Our results demonstrated that not only antipsychotics which have dopamine D₂ receptor antagonism but also aripiprazole have anti-inflammatory effects via the inhibition of microglial activation. Antipsychotics may therefore have a potentially useful therapeutic effect on patients with schizophrenia by reducing the microglial inflammatory reactions.     

Blockade of Striatal 5-Hydroxytryptmine2 Receptors Reduces the Increase in Extracellullar Concentrations of Dopamine Produced by the Amphhetamine analogue 3,4-Methylenedioxymethamphetamine

Abstract: 5-Hydroxytryptamine₂ (5-HT₂) receptor antagonists have been shown to interfere with the stimulation of striatal dopamine synthesis and release produced by the amphetamine analogue 3,4-methylenedioxymethamphetamine (MDMA). To localize the receptors responsible for the attenuation of MDMA-induced release, 5-HT₂ receptor antagonists were infused via the microdialysis probe directly into the brains of awake, freely moving rats before the systemic administration of MDMA. Intrastriatal infusions of the selective 5-HT₂ antagonist MDL 100, 907 produced a concentration-dependent inhibition of MDMA-induced dopamine release. Similar results were observed with intrastriatal infusions of the 5-HT₂ antagonist amperozide. In contrast, infusion of MDL 100, 907 into the midbrain region near the dopaminergic cell bodies was with out effect on the MDMA-induced elevation of extracellular dopamine in the ipsilateral striatum. Neither antagonist attenuated basal transmitter efflux nor the MDMA-stimulated release of [³H]dopamine from striatal slices in vitro indicating that the in vivo effect of the antagonists was not due to inhibition of the dopamine uptake carrier. Intrastriatal infusion of tetrodotoxin reduced both basal and MDMA-stimulated dopamine efflux and eliminated the effect of intrastriatal MDL 100, 907. The results indicate that 5-HT₂ receptors located in the striatum augment the release of dopamine produced by high doses of MDMA. Furthermore, these 5-HT₂ receptors appear to be located on nondopaminergic elements of the striatum.     

GABABR1a/R1b-Type Receptor Antisense Deoxynucleotide Treatment of Melanotropes Blocks Chronic GABAB Receptor Inhibition of High Voltage-Activated Ca2+ Channels

Abstract: GABA_B and dopamine D₂ receptors, both of which acutely inhibit adenylyl cyclase and high voltage-activated Ca²⁺ channels (HVA-CCs), are found in high levels in the melanotrope cells of the pituitary intermediate lobe. Chronic D₂ receptor agonist application in vitro has been reported to result in inhibition of HVA-CC activity by down-regulation. Here we report that chronic GABA_B, but not GABA_A, agonist treatment also resulted in HVA-CC inhibition. Two GABA_B receptor variants have been cloned and shown to inhibit adenylyl cyclase in HEK-293 cells. We have constructed an antisense deoxynucleotide knockdown-type probe that is complementary to 18 bp from the point at which the two sequences first become homologous. Chronic coincubation with baclofen and GABA_B antisense nucleotide completely eliminated the inhibition of the channels by baclofen alone but had no reversing effect on HVA-CC inhibition by the D₂ agonist quinpirole. A scrambled, missense nucleotide also had no reversing effect. Incubation with a D₂ antisense knockdown probe eliminated the ability of a D₂ agonist to inhibit the channels but had no effect on baclofen blockade. These results show the existence an R1a/R1b type of GABA_B receptor, which, like the D₂ receptor, is coupled to chronic HVA-CC inhibition in melanotropes.