Preferential Vulnerability of Nucleus Accumbens Dopamine Binding Sites to Low-Level Lead Exposure: Time Course of Effects and Interactions with Chronic Dopamine Agonist Treatments

Abstract: This study examined the hypotheses that low-level lead (Pb) exposure would increase dopamine (DA) binding sites, would do so preferentially in nucleus accumbens, and that such effects would be modified by concurrent DA agonist treatment. D₁-like and D₂-like binding sites and the dopamine transporter (DT) were measured autoradiographically in caudate-putamen and nucleus accumbens of rats exposed from weaning to 0, 50, or 150 ppm Pb acetate drinking solutions with or without concurrent chronic intermittent intraperitoneal injections of the D₁-like agonist SKF 82958 or the DA agonist apomorphine after 2 weeks (no injections), 8 months, or 12 months of Pb exposure. Pb selectively decreased DA binding in nucleus accumbens. Decreases in D₂-like and DT sites were sustained across the 12-month exposure, whereas D₁-like sites evidenced recovery at 12 months. Chronic intermittent DA agonist treatments reversed these effects of Pb in nucleus accumbens, restoring receptor and DT binding levels to normal, despite decreasing binding sites of non-Pb-treated rats. These studies implicate increased DA availability as a mechanism of Pb-induced DA system changes. They also raise the possibility that Pb exposure could serve as a predisposing factor in neurodegenerative diseases associated with DA system dysfunction or could alter the course of DA-based therapeutic treatments.     

MIF-1 fails to modify agonist-induced oral activity in neonatal 6-OHDA-treated rats

-Prolyl- -leucyl-glycinamide (MIF-1) is known to attenuate apomorphine-induced stereotypies in adult rats that are lesioned as neonates with 6-hydroxydopamine (6-OHDA). To test whether MIF-1 would affect dopamine (DA) agonist-induced and serotonin (5-HT) agonist-induced oral activity, both intact and neonatal 6-OHDA-treated rats were studied. Rats at 3 days from birth were injected with desipramine (20 mg/kg, IP), 1 h before 6-OHDA HBr (100 μg, salt form, in each lateral ventricle) or its vehicle, saline-ascorbic acid (0.1%). At approximately 6 months rats were treated with MIF-1 (0.1, 1.0, or 10.0 mg/kg, IP), 10 min before SKF 39393 HCl (1.0 mg/kg, IP) or m-chlorophenylpiperazine 2HCl (m-CPP 2HCl; 0.5 mg/kg, IP), DA D₁ and 5-HT_1C,2 receptor agonists, respectively. Although both agonists increased oral activity in control and neonatal 6-OHDA-treated rats, MIF-1 did not modify the response. In rats that received either of the three doses of MIF-1 for 21 consecutive days, there was still no observed effect of MIF-1 on the oral response of control and 6-OHDA-lesioned rats to SKF 38393 and m-CPP. These findings indicate that MIF-1 does not modify the oral activity response of supersensitized D₁ and 5-HT_1C receptors in adult rats that are lesioned neonatally with 6-OHDA.     

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.     

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.     

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.     

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.     

Dopamine D1 and D2 Receptors and Their Signal System Present in Coated Vesicles Prepared from Bovine Striatal Tissue

Abstract: Coated vesicles (CVs) isolated from bovine striatal tissue were examined to determine whether they are associated with dopamine signal systems consisting of dopamine D₁ and D₂ receptors, G proteins, and adenylate cyclase. Dopamine receptors in CVs were characterized by a dopamine D₁ receptor antagonist, [³H]SCH 23390, and a dopamine D₂ receptor antagonist, [³H]-spiroperidol. The bindings of both ligands were specifically saturable and reversible with a dissociation constant ( K _D) of 0.65 and 0.5 n M , respectively. Dopaminergic antagonists and agonists inhibited the specific bindings of [³H]SCH 23390 and [³H]spiroperidol in a stereoselective and concentration-dependent manner with an appropriate rank order potency for dopamine D₁ or D₂ receptors. The regulations of the agonist binding by guanyl-5-ylimidodiphosphate were observed. ADP ribosylation of the CVs with [³²P]NAD demonstrated predominant labeling of bands of M_r 47,000–52,000, 42,000–45,000, and 40,000-39,000, which corresponded to the known molecular weights of the α subunits of G_s and G_i proteins. The presence of α and β subunits of G proteins in the CVs was also confirmed by immunoblotting assay. Adenylate cyclase activity, which was stimulated by SKF 38393 and inhibited by dopamine D₂ receptor agonists, was present in the CVs. These findings suggest that the dopamine D₁ and D₂ receptors in the CVs couple with adenylate cyclase via G_s/G_i protein.     

Repeated Reserpine Increases Striatal Dopamine Receptor and Guanine Nucleotide Binding Protein RNA

Abstract: In the present study the effects of repeated administration of reserpine on striatal dopamine receptor and guanine nucleotide binding protein mRNAs were determined. Twenty-four hours after seven consecutive daily injections of reserpine—a treatment that is known to produce functional sensitization of D₁ and D₂ dopamine receptors—the level of striatal D₁ dopamine receptor mRNA was unchanged. However, the level of mRNA for the G protein G_sα was increased by 127%. After extended reserpine treatment for 14 days, levels of both striatal D₁ DA receptor and G_sα mRNAs were elevated by 99 and 78%, respectively. Seven days of reserpine treatment also increased levels of mRNA of the striatal D₂ dopamine receptor and of G proteins G_i2α and G_oα by 200, 79, and 32%, respectively. After 14 days of reserpine treatment the level of striatal D₂ dopamine receptor mRNA was increased by twofold. In contrast, levels of the mRNAs coding for the G proteins G_i2α and G_oα were unchanged. These data suggest that dopamine receptors and their respective G proteins play important roles in the development of sensitization of striatal dopamine receptors during repeated reserpine treatment. Furthermore, the persistent increase in level of striatal G_sα mRNA suggests that this G protein is necessary to maintain supersensitivity of the striatal D₁ dopamine receptor system following long-term dopamine depletion.     

Activation of post-synaptic dopamine D₁ receptors promotes the release of tissue plasminogen activator in the nucleus accumbens via PKA signaling

We have previously demonstrated that tissue plasminogen activator (tPA) plays an important role through the conversion of plasminogen to plasmin in the release of dopamine in the nucleus accumbens (NAc) evoked by depolarization or the systemic administration of drugs of abuse such as morphine and nicotine. In the present study, we examined the mechanisms by which drugs of abuse increase extracellular tPA activity in the NAc in vivo using in situ zymography. The dopamine D₁ receptor (D₁R) agonist SKF38393, but not D₂ receptor agonist quinpirole, significantly increased extracellular tPA activity in the NAc. The effect of SKF38393 was blocked by pre-treatment with the dopamine D₁R antagonist SCH23390. Microinjection of Rp-cAMPs, a protein kinase A inhibitor, into the NAc completely blocked the effect of SKF38393. Systemic administration of morphine and methamphetamine increased extracellular tPA activity in the NAc, and these effects were completely blocked by pre-treatment with SCH23390 and raclopride. The results suggest that activation of post-synaptic dopamine D₁Rs in the NAc leads to an increase in extracellular tPA activity via protein kinase A signaling. Furthermore, dopamine D₂ receptors are also involved in the release of tPA induced by morphine and methamphetamine.     

In Vivo Microdialysis Evidence for Transient Dopamine Release by Benzazepines in Rat Striatum

Abstract: The effects of benzazepine derivatives on extracellular levels of dopamine (DA) and l -3,4-dihydroxyphenylacetic acid (DOPAC) in the dorsal striatum of freely moving rats were studied using in vivo microdialysis. Direct injection of SKF-38393 (0.5 or 1.5 µg/0.5 µl), a selective D₁ receptor agonist, into the striatum through a cannula secured alongside a microdialysis probe produced a rapid dose-dependent transient increase in striatal DA efflux and a more gradual reduction in efflux of DOPAC. The rapid increase in DA efflux was not affected by infusion of tetrodotoxin (TTX; 2 µ M ) or Ca²⁺-free Ringer's solution and occurred after either enantiomer of SKF-38393. A TTX-insensitive increase in DA level similar to that induced by SKF-38393 was also seen after other benzazepines acting as agonists (SKF-75670 and SKF-82958, each 1.5 µg in 0.5 µl) and antagonists (SCH-23390, 1.5 µg in 0.5 µl) at the D₁ receptor and after (+)-amphetamine. These effects were inhibited by infusion of nomifensine (100 µ M ). It is concluded that the transient increases in striatal DA efflux seen after intrastriatal injection of SKF-38393 and other benzazepines are not mediated by presynaptic D₁ receptors but by an amphetamine-like action on the dopamine transporter.     

Methamphetamine-Induced Dopamine Overflow and Injury to Striatal Dopamine Terminals: Attenuation by Dopamine D1 or D2 Antagonists

Abstract: Pharmacological blockade of either D₁ or D₂ dopamine (DA) receptors prevents damage of striatal DA terminals by repeated doses of methamphetamine (m-AMPH). Because the substantial DA overflow produced by multiple m-AMPH treatments appears to contribute to the subsequent injury, we have investigated the effects of blockade of D₁ or D₂ receptors on m-AMPH-induced DA efflux using in vivo microdialysis. Four treatments with m-AMPH (4 mg/kg, s.c., 2-h intervals) produced large increases in striatal DA overflow, with particularly marked overflow (10 times the basal values) following the fourth injection. Administered by themselves, four injections of the D₁ antagonist SCH 23390 or the D₂ antagonist eticlopride (0.5 mg/kg, i.p., 2-h intervals) significantly increased striatal DA overflow. However, treatment with either SCH 23390 or eticlopride 15 min before each of four m-AMPH injections attenuated the marked DA peak otherwise seen after the fourth m-AMPH injection. These effects on DA overflow were related to subsequent DA depletions. Although our m-AMPH regimen produced a 54% reduction in striatal DA tissue content 1 week later, pretreatments with either the D₁ or the D₂ antagonist completely prevented subsequent DA content depletions. Furthermore, the DA content of striatal tissue remaining 1 week after m-AMPH treatment was significantly correlated with the magnitude of the cumulative DA overflow during the m-AMPH treatment ( r = -0.69). Thus, the extensive DA overflow seen during neurotoxic regimens of m-AMPH appears critical to the subsequent neurotoxicity, and the neuroprotective action of DA receptor antagonists seems to result from their attenuation of stimulant-induced DA overflow.     

Dopamine D1 Agonist SKF 38393 Increases the State of Phosphorylation of ARPP-21 in Substantia Nigra

Abstract: ARPP-21 is a cyclic AMP-regulated phosphoprotein (M_r= 21,000) that has a distribution in brain similar to that of DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, M_r= 32,000). It is enriched in the medium-sized spiny neurons in the striatum and in the striatonigral nerve terminals in the pars reticulata of the substantia nigra. The present study shows that dopamine D₁ agonist SKF 38393 increases the state of phosphorylation of ARPP-21 by 26% in nigral slices and that pretreatment of the slices with D₁ antagonist SCH 23390 blocks this effect. These results demonstrate that ARPP-21 is a dopamine-regulated phosphoprotein. Because D₁ receptors are localized on nerve terminals of striatonigral pathway, the phosphorylation of ARPP-21 is likely to mediate some of the intracellular effects of dopamine on these terminals.     

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.     

Effects of Cerebral Ischemia on Regional Dopamine Release and D1 and D2 Receptors

Abstract: To expand on the nature of regional cerebral vulnerability to ischemia, the release of dopamine (DA) and dopaminergic (D₁ and D₂) receptors were investigated in Mongolian gerbils subjected to bilateral carotid artery occlusion (15 min) alone or with reflow (1–2 h). Extracellular cortical and striatal content of DA and its metabolites was measured by microdialysis using HPLC with electrochemical detection. The kinetic properties of D₁ and/or D₂ receptor binding sites were determined in cortical and striatal membranes with the use of radiolabeled ligands (¹²⁵I-SCH23982 and [³H]YM-09151-2, respectively). The ischemic release of DA from the striatum was greater (400-fold over preischemic level) than that from the cortex (12-fold over preischemic content). The affinity for the D₁-receptor ligand was lower ( K _D= 1.248 ± 0.047 n M ) after ischemia than that for sham controls ( K _D= 0.928 ± 0.032 n M, p < 0.001). The number of binding sites for D₂ receptors decreased in striatum ( B _max= 428 ± 18.4 fmol/mg of protein) after ischemia compared with sham controls ( B _max= 510 ± 25.2 fmol/mg of protein, p < 0.05). D₁ or D₂ binding sites were not changed either in the ischemic cortex or postischemic striatum and cortex. The findings strongly suggest that the ischemic release of DA from striatum is associated with early transient changes in D₁- and D₂-mediated DA neurotransmission.     

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.     

Temperature Sensitivity of Agonist High-Affinity Binding Sites of Solubilized and Reconstituted D1 Dopamine Receptors

Abstract: Solubilization of rat striatal membranes with sodium cholate, followed by reconstitution into phospholipid vesicles, leads to a 6.5-fold increase in the agonist high-affinity binding sites of the D₁ dopamine receptor. These high-affinity binding sites display differential sensitivity toward temperature. When reconstituted receptors were preincubated for 1 h at 0–4°C (on ice) or at 22°C (room temperature) followed by radioligand binding assays with dopamine, neither the high-affinity values of the receptor for dopamine nor the percent receptors in the high-affinity state (31–39%) were changed from control reconstituted receptors, which were not subject to any preincubations. At 30°C, there was a partial loss in the number of high-affinity D₁ receptors with only 25% of the total receptor population in the high-affinity state; there was no change in the affinity values of the high-affinity binding sites. At 37°C, there was a 40% loss in total number of D₁ receptor binding sites. All the high-affinity binding sites were lost and the remaining 60% of binding activity represented the low-affinity binding state of the receptor. These results indicate that the high-affinity binding sites of the reconstituted D₁ dopamine receptors are uniquely sensitive to higher temperatures.     

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.     

Chimeric D2/D3 Dopamine Receptors Efficiently Inhibit Adenylyl Cyclase in HEK 293 Cells

Abstract: Despite a high degree of sequence homology, the dopamine D₂ and D₃ receptors have substantially different second messenger coupling properties. We have used chimeric D₂/D₃ receptors to investigate the contribution of the intracellular loops to the signaling properties of these receptors. In HEK 293 cells, D₂ receptors inhibit prostaglandin E₁-stimulated cyclic AMP levels by >90%, whereas D₃ receptors inhibit cyclic AMP accumulation by only 20%. In chimeras that have the second or third intracellular loop, or both loops simultaneously, switched between the D₂ and D₃ receptors, the maximal inhibition of adenylyl cyclase is 60–90%. In addition, the potency of quinpirole to inhibit adenylyl cyclase activity at some of the chimeras is altered compared with the wild-type receptors. It appears that the intracellular loops of the D₃ receptor are capable of interacting with G proteins, as when these loops are expressed in the D₂ receptor, the chimeras inhibit adenylyl cyclase similarly to the wild-type D₂ receptor. Our data suggest that the overall conformation of the D₃ receptor may be such that it interacts with G proteins only weakly, but when the intracellular loops are expressed in another context or the D₃ receptor structure is altered by the introduction of D₂ receptor sequence, this constraint may be lifted.     

Pharmacological Characterization of Tachykinin Receptors Controlling Acetylcholine Release from Rat Striatum: An In Vivo Microdialysis Study

Abstract: The regulation of striatal cholinergic function by tachykinins was examined in urethane-anesthetized rats by using microdialysis. Substance P (0.01–1 µ M ), [Sar⁹,Met(O₂)¹¹]substance P (1–10 µ M ), septide (0.1–3 µ M ), neurokinin (NK) A (0.1–10 µ M ), and senktide (0.1–10 µ M ) produced concentration-dependent increases in striatal acetylcholine (ACh) release. Septide was the most potent agonist for inducing release of ACh, whereas the stimulating effect of senktide was less pronounced and more progressive in onset. The response to septide was prevented by intraperitoneal administration of the nonpeptide NK₁ antagonist SR 140333 (1–3 mg/kg) but not by the nonpeptide NK₂ receptor antagonist SR 48968, indicating that the effect was mediated specifically by NK₁ receptors. ACh release caused by NKA was reduced by SR 48968 (1–3 mg/kg) and slightly affected by SR 140333, indicating a principal role for NK₂ receptors in the peptide response. The similar efficacy of SR 140333 and SR 48968 in blocking substance P-induced ACh release suggested that the effect of this peptide involves the stimulation of both NK₁ and NK₂ receptors. Finally, our results indicate that the increase in striatal ACh release induced by the D₁ agonist (+)-SKF-38393 (3 µ M ) may be mediated indirectly through local release of NKA or substance P acting at NK₂ receptors.     

Dopamine D2 Receptor-Deficient Mice Exhibit Decreased Dopamine Transporter Function but No Changes in Dopamine Release in Dorsal Striatum

Abstract : Presynaptic D₂ dopamine (DA) autoreceptors, which are well known to modulate DA release, have recently been shown to regulate DA transporter (DAT) activity. To examine the effects of D₂ DA receptor deficiency on DA release and DAT activity in dorsal striatum, we used mice genetically engineered to have two (D₂^+/+), one (D₂^+/-), or no (D₂^-/-) functional copies of the gene coding for the D₂ DA receptor. In vivo microdialysis studies demonstrated that basal and K⁺-evoked extracellular DA concentrations were similar in all three genotypes. However, using in vivo electrochemistry, the D₂^-/- mice were found to have decreased DAT function, i.e., clearance of locally applied DA was decreased by 50% relative to that in D₂^+/+ mice. In D₂^+/+ mice, but not D₂^-/- mice, local application of the D₂-like receptor antagonist raclopride increased DA signal amplitude, indicating decreased DA clearance. Binding assays with the cocaine analogue [³H]WIN 35,428 showed no genotypic differences in either density or affinity of DAT binding sites in striatum or substantia nigra, indicating that the differences seen in DAT activity were not a result of decreased DAT expression. These results further strengthen the idea that the D₂ DA receptor subtype modulates activity of the striatal DAT.