Neonatal 6-Hydroxydopamine and Adult SKF 38393 Treatments Alter Dopamine D1 Receptor mRNA Levels: Absence of Other Neurochemical Associations with the Enhanced Behavioral Responses of Lesioned Rats

Abstract: To study potential biochemical correlates of dopamine (DA) and serotonin receptor supersensitivity, rats were lesioned at 3 days after birth with 6-hydroxydopamine (6-OHDA; 67 µg in each lateral ventricle; desipramine pretreatment, 20 mg/kg i.p., 1 h) and then sensitized with the DA D₁ agonist, SKF 38393 HCl (3.0 mg/kg i.p. per day) either ontogenetically (daily, for 28 consecutive days from birth) and/or in adulthood (four weekly injections, 6–9 weeks from birth). Controls received vehicle in place of 6-OHDA or SKF 38393. Enhanced locomotor responses were observed after SKF 38393 at 6 weeks, only in rats that received SKF 38393 + 6-OHDA in ontogeny. Locomotor responses were further enhanced in this group after the last of four weekly SKF 38393 injections at the 9th week. These weekly SKF 38393 treatments also produced enhanced responses in 6-OHDA rats that did not receive SKF 38393 in ontogeny. When striata were studied at 11 weeks, the percentages of high and low affinity DA D₁ binding sites were not altered. Basal as well as DA-, NaF-, and forskolin-stimulated adenylyl cyclase activities also were not changed. Dot blot analysis showed that there was a reduction of mRNA levels for DA D₁, but not serotonin_1C, receptors in the 6-OHDA groups. However, SKF 38393 at 6–9 weeks eliminated this alteration. Based on these findings it can be proposed that supersensitization may be a consequence of altered neuronal cross talk rather than an imbalance of receptor elements per se.     

Characterization of the Phosphoinositide-Linked Dopamine Receptor in a Mouse Hippocampal-Neuroblastoma Hybrid Cell Line

Abstract: Previous studies have established that dopamine (DA) can stimulate phosphoinositide (PI) metabolism in the CNS and in the periphery. The present study summarizes our attempt to find a cell line that expresses this dopaminergic system. We describe that the stable clonal HN33.11 cell line, established by fusion of mouse hippocampal cells with neuroblastoma cells (N18TG2) that originate from A/J mouse, natively expresses the D₁ DA receptor system that couples to PI hydrolysis. In this cell line, 500 µM DA or SKF38393 produced 43 and 75% increases in inositol phosphate (IP) accumulations, respectively. In contrast, noradrenaline or 5-hydroxytryptamine did not affect IP accumulations. The formation of IP that was stimulated by DA or SKF38393 was selectively blocked by the D₁ DA receptor antagonist SCH23390 with IC₅₀ values of 13 and 16 µM. This response was not mediated by the D_1A DA receptor and was cyclic AMP-independent, as HN33.11 cells did not express this receptor, and DA or SKF38393 was unable to stimulate the formation of cyclic AMP. In Ca²⁺-free/100 µM EGTA medium, basal IP level was reduced by 31.5%, but SKF38393-stimulated PI hydrolysis was not affected. SKF38393-stimulated IP accumulation was also not affected by pertussis toxin (PTX) treatment (200 ng/ml), suggesting that this dopaminergic response is mediated by PTX-insensitive G proteins. Co-immunoprecipitation studies indicated that in membranes of HN33.11 cells, D₁-like binding sites are coupled to Gα_q protein. Blockade of SKF38393-induced PI hydrolysis with antiserum against phospholipase C (PLC) isozymes, performed in permeabilized cells, as well as co-immunoprecipitation studies implicate PLCβ3 and PLCβ4 in this dopaminergically mediated PI hydrolysis cascade. The results indicate that HN33.11 cells express a D₁-like DA receptor that couples to PLCβ3/4 via Gα_q protein. These cells may therefore be a useful model system for investigating this receptor system.     

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.     

The dopamine D1 receptor is expressed and facilitates relaxation in airway smooth muscle

Background

Dopamine signaling is mediated by G_s protein-coupled “D₁-like” receptors (D₁ and D₅) and G_i-coupled “D₂-like” receptors (D_2-4). In asthmatic patients, inhaled dopamine induces bronchodilation. Although the G_i-coupled dopamine D₂ receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle (ASM) cells, the G_s-coupled dopamine D₁-like receptor subtypes have never been identified on these cells. Activation of G_s-coupled receptors stimulates cyclic AMP (cAMP) production through the stimulation of adenylyl cyclase, which promotes ASM relaxation. We questioned whether the dopamine D₁-like receptor is expressed on ASM, and modulates its function through G_s-coupling.

Methods

The mRNA and protein expression of dopamine D₁-like receptor subtypes in both native human and guinea pig ASM tissue and cultured human ASM (HASM) cells was measured. To characterize the stimulation of cAMP through the dopamine D₁ receptor, HASM cells were treated with dopamine or the dopamine D₁-like receptor agonists ({"type":"entrez-nucleotide","attrs":{"text":"A68930","term_id":"4759850","term_text":"A68930"}}A68930 or {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393) before cAMP measurements. To evaluate whether the activation of dopamine D₁ receptor induces ASM relaxation, guinea pig tracheal rings suspended under isometric tension in organ baths were treated with cumulatively increasing concentrations of dopamine or {"type":"entrez-nucleotide","attrs":{"text":"A68930","term_id":"4759850","term_text":"A68930"}}A68930, following an acetylcholine-induced contraction with or without the cAMP-dependent protein kinase (PKA) inhibitor Rp-cAMPS, the large-conductance calcium-activated potassium (BK_Ca) channel blocker iberiotoxin, or the exchange proteins directly activated by cAMP (Epac) antagonist NSC45576.

Results

Messenger RNA encoding the dopamine D₁ and D₅ receptors were detected in native human ASM tissue and cultured HASM cells. Immunoblots confirmed the protein expression of the dopamine D₁ receptor in both native human and guinea pig ASM tissue and cultured HASM cells. The dopamine D₁ receptor was also immunohistochemically localized to both human and guinea pig ASM. The dopamine D₁-like receptor agonists stimulated cAMP production in HASM cells, which was reversed by the selective dopamine D₁-like receptor antagonists {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 or {"type":"entrez-protein","attrs":{"text":"SCH39166","term_id":"1052842517","term_text":"SCH39166"}}SCH39166. {"type":"entrez-nucleotide","attrs":{"text":"A68930","term_id":"4759850","term_text":"A68930"}}A68930 relaxed acetylcholine-contracted guinea pig tracheal rings, which was attenuated by Rp-cAMPS but not by iberiotoxin or NSC45576.

Conclusions

These results demonstrate that the dopamine D₁ receptors are expressed on ASM and regulate smooth muscle force via cAMP activation of PKA, and offer a novel target for therapeutic relaxation of ASM.     

Physicochemical Modulation of Agonist-Induced [35S]GTPγS Binding: Implications for Coexistence of Multiple Functional Conformations of Dopamine D1-Like Receptors

Dopamine agonist-stimulated [³⁵S]GTPγS binding to membrane G proteins was studied in select brain regions under experimental conditions that permit the activation of receptor coupling to the G proteins G_i, G_s, or G_q. Agents studied were agonists known to be effective at various dopamine receptor/effector systems and included quinelorane (D₂-like/G_i), SKF38393 (D₁-like/G_q, D₁-like/G_s), SKF85174 (D₁-like/G_s), and SKF83959 (D₁-like/G_q). Dopamine and SKF38393 significantly stimulated [³⁵S]GTPγS binding to normal striatal membranes by 161% and 67% above controls. Deoxycholate, which enhances agonist-induced phospholipase C (PLC) stimulation, markedly enhanced the agonistic effects of dopamine and SKF38393 to 530% and 637% above controls, respectively. The enhancing effects of deoxycholate were reversed if it was washed off the membranes before agonist addition. The thiol-reducing agent, dithiothreitol, completely abolished the effects of SKF38393 and SKF83959, whereas SKF85174 effects were augmented. Agonist responses were concentration-related, and highest efficacies were obtained in the hippocampus, thus paralleling both the brain regional distribution and agonist efficacies previously observed in phosphoinositide hydrolysis assays. These findings suggest that D₁-like receptor conformations that mediate agonist stimulation of G_s/adenylylcyclase may be structurally different from those that mediate G_q/PLC activation. Although the exact mechanism of deoxycholate's effect awaits elucidation, the results are consistent with the emerging concept of functional selectivity whereby deoxycholate could create a membrane environment that facilitates the transformation of the receptor from a conformation that activates G_s/adenylylcyclase to one that favors G_q/PLC signaling.     

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.     

Modulation of In Vivo Dopamine Release by D2 but Not D1 Receptor Agonists and Antagonists

The capacity of D1 and D2 agonists and antagonists to regulate the in vivo release and metabolism of dopamine (DA) in mesolimbic and nigrostriatal DA neurons of the mouse was determined using gas chromatographic and mass fragmentographic (GC-MF) analysis. DA release was inferred from levels of 3-methoxytyramine (3-MT) and DA metabolism was inferred from levels of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). DA release was increased by the D2 antagonists haloperidol and metoclopramide but not by the D1 antagonists SCH 23390 and SKF 83566. DA metabolism was increased by each of the four antagonists but to a greater extent with the D2 antagonists. The D2 agonists CGS 15855A and LY 171555 decreased DA release whereas the D1 agonist SKF 38393, at relatively high doses, only slightly affected DA release. Each of the three agonists decreased DA metabolism but again metabolism was more affected by the D2-selective drugs. The in vivo release of DA from mesolimbic and neostriatal DA neurons appears to be modulated by D2 but not by D1 receptors, whereas both receptor types can modulate DA metabolism.     

Regional distribution of monoamines and dopamine D1-and D2-receptors in the striatum of the rat

Dopamine (DA) D₁-and D₂-receptor densities were determined in 18 discrete areas of the caudate-putamen-globus pallidus of male Wistar rats and compared to local DA concentrations. All three parameters were found to decrease caudally. The globus pallidus was distinguished by the low concentration of DA and its receptors and high noradrenaline, (NA) content. While there were no mediolateral differences in DA or DA D₁-receptors, a clear mediolateral gradient was observed for DA D₂-receptors which extended over several sections of the brain. The ratio of DA D₁-to D₂-receptors was significantly higher in the dorsal than in the ventral areas of the mediolateral and caudal striatum. This is the first report of clear dorsoventral differences in parameters relating to DA activity in the striatum. These findings may be of particular significance in understanding the functional dichotomy between the dorsal and ventral striatum.     

In the ventral tegmental area, progestins have actions at D1 receptors for lordosis of hamsters and rats that involve GABA A receptors

In the ventral tegmental area (VTA), progestins facilitate lordosis via actions at gamma-aminobutyric acid (GABA)(A)/benzodiazepine receptor complexes (GBRs) and dopamine type 1 receptors (D1). The relationship between progestins' actions at GBRs and D1 in the VTA for facilitating sexual behavior of hamsters and rats was examined. Ovariectomized (ovx), estradiol (E(2); 10 microg)+progesterone (P; 250 microg; SC)-primed hamsters, with bilateral guide cannulae to the VTA, were pre-tested for sexual and motor behavior and infused with the GBR antagonist bicuculline (100 ng/side) or vehicle. Thirty minutes later, hamsters were re-tested and then infused with the D1 agonist SKF38393 (100 ng/side) or vehicle. Hamsters were post-tested 30 min later. Ovx, E(2) (10 microg)-primed rats were pre-tested, infused first with bicuculline or vehicle, second with SKF38393 or vehicle, third with 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP; 0, 100, or 200 ng) and were post-tested 10 and 60 min after 3alpha,5alpha-THP infusions. VTA infusions of SKF38393 increased lordosis of hamsters or rats. Bicuculline pretreatment reduced SKF38393- and/or progesterone-mediated increases in lordosis of E2-primed hamsters. In E2-primed rats, bicuculline blocked SKF38393- and/or 3alpha,5alpha-THP-mediated increases in lordosis. There were no effects on motor behavior. Thus, in the VTA, GBR activity modulates D1-mediated actions for lordosis of hamsters and rats.     

Further evidence for the involvement of D2, but not D1 dopamine receptors in dopaminergic control of striatal cholinergic transmission

The relative involvement of D₁ (cyclase linked) and D₂ dopamine receptors in dopaminergic control of striatal cholinergic transmission has been investigated in the rat by comparing the effects of SKF 38393 and LY 141865 (which act as specific agonists at D₁ and D₂ dopamine receptors, respectively) on striatal acetylcholine and dopamine metabolite concentrations and on the potassium-evoked release of ³H-acetylcholine from rat striatal slices. LY 141865 given systemically produced a dose-dependent increase in acetylcholine concentrations and a concomitant reduction of homovanillic and dihydroxyphenylacetic acid levels in the striatum (ED₅₀ 0.1 mg/kg) whereas SKF 38393 (1–30 mg/kg) did not. SKF 38393 (30 mg/kg) also failed to modify the LY 141865 (1 mg/kg) induced alterations of striatal acetylcholine and dopamine metabolite levels when given concomitantly with the latter compound. In experiments $\text{in vitro}$, LY 141865 reduced (EC₅₀ 0.14 μM), whereas SKF 38393 (up to 100 μM) failed to affect, the potassium-evoked release of ³H-acetylcholine from striatal slices. When given concomitantly with LY 141865, SKF 38393 (10 μM) did not modify the ability of the former compound to diminish striatal ³H-acetylcholine release. Finally, SKF 38393 also failed to affect the release of striatal ³H-acetylcholine after chemical lesion of the nigro-striatal dopaminergic pathway. The present results provide evidence for the involvement of D₂ but not D₁ dopamine receptors in dopaminergic control of striatal cholinergic transmission and indicate that D₁ dopamine receptors do not exert any modulatory influence on D₂ dopamine receptor mediated dopaminergic transmission.     

D1-D2 dopamine receptor interaction within the nucleus accumbens mediates long-loop negative feedback to the ventral tegmental area (VTA)

The objective of the present study was to examine the effects of perfusion of dopamine (DA) D1- and D2-like receptor agonists in the nucleus accumbens (ACB) on the long-loop negative feedback regulation of mesolimbic somatodendritic DA release in the ventral tegmental area (VTA) of Wistar rats employing ipsilateral dual probe in vivo microdialysis. Perfusion of the ACB for 60 min with the D1-like receptor agonist SKF 38393 (SKF, 1-100 microM) dose-dependently reduced the extracellular levels of DA in the ACB, whereas the extracellular levels of DA in the VTA were not changed. Similarly, application of the D2-like receptor agonist quinpirole (Quin, 1-100 microM) through the microdialysis probe in the ACB reduced the extracellular levels of DA in the ACB in a concentration-dependent manner, whereas extracellular levels of DA in the VTA were not altered. Co-application of SKF (100 microM) and Quin (100 microM) produced concomitant reductions in the extracellular levels of DA in the ACB and VTA. The reduction in extracellular levels of DA in the ACB and VTA produced by co-infusion of SKF and Quin was reversed in the presence of either 100 microM SCH 23390 (D1-like antagonist) or 100 microM sulpiride (D2-like antagonist). Overall, the results suggest that (a) activation of dopamine D1- or D2-like receptors can independently regulate local terminal DA release in the ACB, whereas stimulation of both subtypes is required for activation of the negative feedback pathway to the VTA.     

In Vivo Evidence that Nonneuronal β-Adrenoceptors as Well as Dopamine Receptors Contribute to Cyclic AMP Efflux in Rat Striatum

Abstract: We applied in vivo microdialysis to assess the effects of dopaminergic and β-adrenergic receptor stimulation on cyclic AMP efflux in rat striatum under chloral hydrate anesthesia. Dopamine (up to 1 mM) infused for 20 min through the probe did not increase cyclic AMP, whereas both the selective dopamine D₁ agonist SKF 38393 and D₂ antagonist sulpiride produced modest increases. It is interesting that the β-adrenoceptor agonist isoproterenol produced a marked increase (204.7% of basal level at 1 mM) which was antagonized by the β-adreno-ceptor antagonist propranolol. Pretreatment with a glial selective metabolic inhibitor, fluorocitrate (1 mM), by a 5-h infusion through the probe attenuated basal cyclic AMP efflux by 30.3% and significantly blocked the response to isoproterenol. By contrast, striatal injection of a neuro-toxin, kainic acid (2.5 μg), 2 days before the dialysis experiment did not affect basal cyclic AMP or the response to isoproterenol, but blocked the response to SKF 38393. These data demonstrate that β-adrenoceptors as well as dopamine receptors contribute to cyclic AMP efflux in rat striatum in vivo. They also suggest that basal and β-adre-noceptor-stimulated cyclic AMP efflux are substantially dependent on intact glial cells.     

Evidence for a Distinct D1Like Dopamine Receptor that Couples to Activation of Phosphoinositide Metabolism in Brain

Abstract: Dopamine and the D₁, receptor agonist SKF 38393 activate the phospholipase C-rnediated hydrolysis of phosphoinositides in brain slices. This action is selectively inhibited by SCH-23390, thus suggesting its mediation through the dopamine D₁ receptor. To determine if the dopamine receptor that mediates Phosphoinositide hydrolysis is the adenylyl, cyclase-linked D₁ receptor or a different subtype of the dopamine D₁ receptor, 20 benzazepine compounds that were previously characterized as selective dopamine D₁ receptor agonists were tested for stimulation of Phosphoinositide hydrolysis in rat striatal slices and for activation of adenylyl cyclase in rat striatal membranes. The compounds displayed a range of potencies and efficacies in stimulating adenylyl cyclase or Phosphoinositide hydrolysis. Compounds such as SKF 81427 and SKF 38393 were as efficacious as dopamine in stimulating Phosphoinositide hydrolysis, whereas other compounds, including SKF 85174 and SKF 86284, although showing high efficacy in stimulating cyclic AMP, failed to stimulate inositol phosphate formation. There was no correlation between the potencies (r= 0.016; p < 0.95) or efficacies (r=?0.294; p < 0.24) of the tested compounds in stimulating cyclic AMP formation and phosphoinositide hydrolysis. These observations indicate that the D₁-like dopamine receptor that mediates phosphoinositide hydrolysis is pharmacologically distinct from the classic D₁ receptor that is coupled to stimulation of cyclic AMP formation.     

Dopamine D1-D2 Receptor Heteromer-mediated Calcium Release Is Desensitized by D1 Receptor Occupancy with or without Signal Activation: DUAL FUNCTIONAL REGULATION BY G PROTEIN-COUPLED RECEPTOR KINASE 2*

We identified that activation of the G_q-linked dopamine D1-D2 receptor hetero-oligomer generates a PLC-dependent intracellular calcium signal. Confocal FRET between endogenous dopamine D1 and D2 receptors in striatal neurons confirmed a physical interaction between them. Pretreatment with SKF 83959, which selectively activates the D1-D2 receptor heteromer, or SKF 83822, which only activates the D1 receptor homo-oligomer, led to rapid desensitization of the D1-D2 receptor heteromer-mediated calcium signal in both heterologous cells and striatal neurons. This desensitization response was mediated through selective occupancy of the D1 receptor binding pocket. Although SKF 83822 was unable to activate the D1-D2 receptor heteromer, it still permitted desensitization of the calcium signal. This suggested that occupancy of the D1 receptor binding pocket by SKF 83822 resulted in conformational changes sufficient for desensitization without heteromer activation. Bioluminescence resonance energy transfer and co-immunoprecipitation studies indicated an agonist-induced physical association between the D1-D2 receptor heteromeric complex and GRK2. Increased expression of GRK2 led to a decrease in the calcium signal with or without prior exposure to either SKF 83959 or SKF 83822. GRK2 knockdown by siRNA led to an increase in the signal after pretreatment with either agonist. Expression of the catalytically inactive and RGS (regulator of G protein signaling)-mutated GRK2 constructs each led to a partial recovery of the GRK2-attenuated calcium signal. These results indicated that desensitization of the dopamine D1-D2 receptor heteromer-mediated signal can occur by agonist occupancy even without activation and is dually regulated by both the catalytic and RGS domains of GRK2.     

Effect of chronic haloperidol treatment on dopamine-induced inositol phosphate formation in rat brain slices

The effects of chronic haloperidol administration on the accumulation of inositol phosphates were examined in rat brain slices pre-labeled with [³H]myo-inositol and incubated with various dopaminergic drugs. Rats were treated with haloperidol-decanoate or its vehicle (sesame oil) for two, four or six weeks. Dopamine and the selective D₁ agonist, SKF38393, induced a significant increase in lithium-dependent accumulation of [³H]inositol monophosphate (IP₁) in the frontal cortex, hippocampus and striatum of vehicle-treated animals, while the selective D₂ agonist quinpirole did not show any effect on IP₁ accumulation. The actions of dopamine and SKF38393 were blocked by the D₁ antagonist, SCH23390, but not by the D₂ antagonist, spiperone, in all three brain regions. Haloperidol treatment did not affect basal phosphoinositide turnover in the three brain regions. Four or six weeks of haloperidol treatment significantly decreased dopamine-induced IP₁ accumulation in the striatum (by 30% and 25%, respectively), but not in the frontal cortex and the hippocampus. Four weeks of treatment with haloperidol significantly decreased IP₁ levels in the striatal slices when measured in the presence of quinpirole. However, the accumulation of IP₁ measured in the presence of SKF38393 was not significantly altered after haloperidol treatment. The loss of dopamine-sensitive IP accumulation was not observed in the presence of spiperone after haloperidol treatment. The number, but not the affinity, of [³H]sulpiride binding sites in the striatum was significantly increased (by 34–46%) after chronic haloperidol treatment. A timecourse study suggests that the inhibition by chronic haloperidol treatment of dopamine-induced phosphoinositide hydrolysis may involve an effect secondary to an increase in the number of dopamine D₂ receptors in the striatum.     

Enhanced Dopamine D1 and D2 Receptor Gene Expression in the Hippocampus of Hypoglycaemic and Diabetic Rats

Hypoglycaemic coma and brain injury are potential complications of insulin therapy. Hippocampal neurons are particularly vulnerable to hypoglycaemic stress leading to memory impairment. In the present article, we have investigated the dopamine (DA) content, homovanillic acid (HVA)/DA turnover ratio, DA D₁ and DA D₂ receptors in the hippocampus of insulin-induced hypoglycaemic (IIH) and streptozotocin induced diabetic rats where brain functions are impaired. The DA content decreased significantly in hippocampus of diabetic, diabetic +IIH and control +IIH rats compared to control. The HVA/DA turnover ratio also increased significantly in diabetic, diabetic +IIH and control +IIH rats compared to control. Scatchard analysis using [³H] DA in the hippocampus showed a significant increase in DA receptors of diabetic, diabetic +IIH and control +IIH rats with decreased affinity. Gene expression studies using Real-time PCR showed an increased expression of DA D₁ and DA D₂ receptors in the hippocampus of hypoglycaemic and diabetic rats. Our results indicate that the dopaminergic system is impaired in the hippocampus of hypoglycaemic and hyperglycaemic rats impairing DA related functions of hippocampus. We observed a prominent dopaminergic functional disturbance in the hypoglycaemic condition than in hyperglycaemia compared to control. This dopaminergic dysfunction in hippocampus during hypoglycaemia and hyperglycaemia is suggested to contribute to cognitive and memory deficits. This will have clinical significance in the treatment of diabetes.     

Oxytocin receptors in the nucleus accumbens shell are involved in the consolidation of maternal memory in postpartum rats

Female rats with maternal experience display a shorter onset of maternal responsiveness compared to those with no prior experience. This phenomenon called ‘maternal memory’ is critically dependent on the nucleus accumbens (NA) shell. We hypothesized that activation of OT receptors in the NA shell facilitates maternal memory. In Experiment 1, postpartum female rats given 1 hour of maternal experience were infused following the experience with either a high or low dose of an OT antagonist into the NA shell and tested for maternal behavior after a 10-day pup isolation period. Females receiving a high dose of the antagonist showed a significantly longer latency to exhibit full maternal behavior after the pup isolation period compared to females that received vehicle or a high dose of antagonist in a control region. In Experiment 2, postpartum female rats were infused with either a high or low dose of OT into the NA shell after a 15-minute maternal experience and tested for maternal behavior after a 10-day pup isolation period. There were no significant differences between the females infused with OT and females treated with a vehicle infused into the NA shell or with OT infused into the control region. One possible reason for a lack of facilitation is a floor effect, since females in the control groups displayed a rapid maternal response after the pup isolation period. These findings suggest that OT receptors, likely in combination with other neurotransmitters, in the NA shell play a role in the consolidation of maternal memory.     

Interactions of novel dopaminergic ligands with D-1 and D-2 dopamine receptors

The interactions of three novel dopaminergic ligands, SKF38393, SKF82526 and SKF83742, with D-1 and D-2 dopamine (DA) receptors have been investigated using radioligand binding techniques and computer modeling procedures. Using the bovine anterior pituitary D-2 DA receptor system, SKF38393 and SKF82526 behave as agonists demonstrating biphasic agonist/3H-antagonist competition curves. For both drugs, the high affinity phase comprised 30% of the total displacement curve. Such findings are atypical as previously tested classical dopamine agonists demonstrated high and low affinity displacement phases in equal proportions. Such behavior exhibited by the SKF agonists may be related to their activity as partial agonists. In contrast, SKF83742 behaves as an antagonist exhibiting homogeneous monophasic competition curves. Similar results are obtained in the rat striatal membrane D-2 DA receptor system. Both SKF38393 and SKF82526 also demonstrate shallow biphasic displacement curves on rat striatal D-1 receptors labeled with 3H-flupentixol whereas SKF83742/3H-flupentixol curves are uniphasic. Of all the ligands, only SKF38393 clearly demonstrates higher affinity for 3H-flupentixol labeled D-1 receptors.     

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.