Agonist action at D2(long) dopamine receptors: ligand binding and functional assays

Clinical pathways are being introduced by hospitals to reduce costs and control unnecessary variation in care. We studied 766 inpatients to measure the impact of a perioperative clinical pathway for patients undergoing knee replacement surgery on hospital costs. One hundred twenty patients underwent knee replacement surgery before the development of a perioperative clinical pathway, and 63 patients underwent knee replacement surgery after pathway implementation. As control groups, we contemporaneously studied 332 patients undergoing radical prostatectomy (no clinical pathway in place for these patients) and 251 patients undergoing hip replacement surgery without a clinical pathway (no clinical pathway and same surgeons as patients having knee replacement surgery). Total hospitalization costs (not charges), excluding professional fees, were computed for all patients. Mean (+/-SD) hospital costs for knee replacement surgery decreased from $21,709 +/- $5985 to $17,618 +/- $3152 after implementation of the clinical pathway. The percent decrease in hospitalization costs was 1.56-fold greater (95% confidence interval 1.02-2.28) in the knee replacement patients than in the radical prostatectomy patients and 2.02-fold greater (95% confidence interval 1.13-5.22) than in the hip replacement patients. If patient outcomes (e.g., patient satisfaction) remain constant with clinical pathways, clinical pathways may be a useful tool for incremental improvements in the cost of perioperative care. Implications: Doctors and nurses can proactively organize and record the elements of hospital care results in a clinical pathway, also known as "care pathways" or "critical pathways." We found that implementing a clinical pathway for patients undergoing knee replacement surgery reduced the hospitalization costs of this surgery.     

Adenosine A2A receptors modulate the binding characteristics of dopamine D2 receptors in stably cotransfected fibroblast cells

In membrane preparations from rat striatum, where adenosine A2A and dopamine D2 receptors are coexpressed, stimulation of adenosine A2A receptors was found to decrease the affinity of dopamine D2 receptors for dopamine agonists. We now demonstrate the existence of this antagonistic interaction in a fibroblast cell line (Ltk-) stably transfected with the human dopamine D2 (long-form) receptor and the dog adenosine A2A receptor cDNAs (A2A-D2 cells). In A2A-D2 cells, but not in control cells only containing dopamine D2 receptors (D2 cells), the selective adenosine A2A agonist 2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethyl-carboxamido adenosine (CGS 21680) induced a 2-3-fold decrease in the affinity of dopamine D2 receptors for dopamine, as shown in competition experiments with dopamine versus the selective dopamine D2 antagonist [3H]raclopride. By contrast, activation of the constitutively expressed adenosine A2B receptors with 5'-N-ethyl-carboxamidoadenosine (NECA) did not modify dopamine D2 receptor binding. In A2A-D2 cells CGS 21680 failed to induce or induced only a small increase in adenosine-3',5'-cyclic-monophosphate (cAMP) accumulation. In D2 cells NECA- or forskolin-induced adenylyl cyclase activation was not associated with any change in dopamine D2 receptor binding. These results indicate that adenylyl cyclase activation is not involved in the adenosine A2A receptor-mediated modulation of the binding characteristics of the dopamine D2 (long-form) receptor.     

Left-right asymmetry of striatal dopamine D2 receptors

Brain functions may be lateralized to the right or the left hemisphere. However, the biochemical characteristics accompanying these functions are largely unknown. To test possible lateralization of striatal dopamine D2 receptors, we examined 18 volunteers using 123I-iodobenzamide and single photon emission tomography. The striatum-to-cerebellum D2 binding ratio was 1.93 +/- 0.22 (mean +/- S.D.) on the right side and 1.85 +/- 0.19 on the left side. In 14 subjects, D2 binding was higher in the right compared to the left striatum (P < 0.05). These results are supported by a meta-analysis performed on 15 studies reported in the literature. We conclude that side differences of striatal dopamine D2 receptors exist. We propose that motor activity could be responsible for our findings.     

Clozapine occupies high levels of dopamine D2 receptors

An important discrepancy has been noted concerning the number of dopamine D2 receptors which must be occupied in patients by clozapine, in contrast to other antipsychotic drugs, in order to achieve an antipsychotic effect. For example, when D2 receptors are labelled by radioactive raclopride or spiperone congeners in patients, psychosis-controlling doses of all antipsychotic drugs occupy about 70% or more of the D2 receptors in patients. However, equi-effective psychosis-controlling doses of clozapine (approximately 400 mg/day; approximately 70-130 nM in spinal fluid) only occupy between 20% and 50% of the D2 receptors in patients. This discrepancy of a consistently lower occupancy of D2 by psychosis-controlling doses of clozapine may be resolved when one considers that the neuroleptic concentration for half-occupancy of D2 receptors in vitro (i.e. the inhibition constant) depends on the radioligand used to label the receptor. Radioligands with higher tissue/buffer partition coefficients are less displaced by clozapine. This principle applies both in vitro and in vivo. Thus, allowing for this principle, psychosis-controlling doses of clozapine can be shown to occupy over 70% of the brain dopamine D2 receptors in patients, as found with other neuroleptics.     

Effects of chronic neuroleptic treatments on dopamine D1 and D2 receptors: homogenate binding and autoradiographic studies

The antipsychotic effects of neuroleptics are believed to be mediated via dopamine D2 receptor blockade; however, the anatomical and pharmacological targets of these drugs remain somewhat controversial. The purpose of this study was to examine the effects of chronic clozapine (CLZ) and haloperidol (HAL) treatments on the densities of DA D1 and D2 receptors. Adult male Sprague-Dawley rats (300-350 g) were treated for 21 days with either HAL (1 mg/kg/day, i.p.), CLZ (20 mg/kg/day, i.p.) or saline. Three days after ending the treatments, the brains were removed and used for biochemical assays of tissue DA and metabolites as well as for receptor studies. DA D1 and D2 receptors were labelled with [3H]SCH23390 and [3H]raclopride, respectively, and measured in the neostriatum by binding studies, and in autoradiograms of forebrain sections by quantitative densitometry. The autoradiographic measurements revealed significant increases in the densities of D2 receptors in nucleus accumbens, in the medio-ventral, latero-dorsal and latero-ventral quadrants of the rostral neostriatum, in caudal neostriatum and in globus pallidus of both HAL-(28-44%) and CLZ-treated (15-85%) animals. The HAL-induced up-regulation of D2 receptors in rostral and caudal neostriatum was homogenous, but CLZ produced a more uneven increase, with the highest absolute densities measured in latero-dorsal neostriatum, as well as with changes in the medio-dorsal rostral neostriatum. For D1 receptors, only CLZ and not HAL, produced significant increases in five regions, namely nucleus accumbens (43%) latero-dorsal rostral neostriatum (16%), caudal neostriatum (30%), globus pallidus (67%) and substantia nigra (12%). The observation that CLZ, contrary to HAL, also has an effect on D1 receptor densities may explain the greater therapeutic and selective efficacy with fewer side-effects of this agent, in comparison to other neuroleptics.     

D2 dopamine receptor antisense oligodeoxynucleotide inhibits the synthesis of a functional pool of D2 dopamine receptors

In vivo administration of an antisense oligonucleotide targeted toward the D2 dopamine (DA) receptor mRNA (D2 AS) markedly inhibited D2 receptor-mediated behaviors but produced only a relatively small reduction in the levels of D2 DA receptors in mouse striatum. This apparent dissociation between DA receptor-mediated behaviors and the levels of D2 DA receptors was addressed by inhibiting the total number of D2 DA receptors by intraperitoneal administration of the selective, irreversibly acting D2 DA receptor antagonist fluphenazine-N-mustard (FNM) and then determining the effects of D2 AS, administered intracerebroventricularly, on the rate of synthesis of D2 DA receptors and on the recovery of D2 receptor-mediated behaviors. FNM inactivated approximately 90% of D2 DA receptors within 4 hr of treatment, after which the receptors returned to normal levels by approximately 8 days. D2 AS treatment significantly inhibited the rate of recovery of D2 DA receptors in striatum of FNM-treated mice. FNM treatment also produced a number of behavioral alterations, including catalepsy, and the inhibition of stereotypic behavior induced by the D2/D3 DA receptor agonist quinpirole. Both of these behaviors returned to normal within 8 days after FNM treatment. D2 AS treatment delayed the restoration of these FNM-induced behaviors. Thus, it reduced the rate of disappearance of the cataleptic behavior induced by FNM and significantly delayed the restoration of the stereotypic behavior induced by quinpirole. The changes induced by D2 AS on D2 receptor-mediated behaviors were reversed on cessation of D2 AS treatment. A random oligomer given in the same amount and for the same length of time as that of the D2 AS had no significant effects on either D2 DA receptor synthesis or DA receptor-mediated behaviors. These studies demonstrate that in vivo administration of D2 AS decreased the rate of recovery of D2 DA receptors and inhibited the recovery of D2 DA receptor-mediated behaviors after irreversible receptor inactivation and suggest that D2 AS treatment inhibits the synthesis of a functional pool of D2 DA receptors.     

Multiple coupling of human D5 dopamine receptors to guanine nucleotide binding proteins Gs and Gz

We have demonstrated previously that D1 dopamine receptors are coupled to both Gs alpha and Go alpha. We examine here the coupling between human D5 dopamine receptors and G proteins in transfected rat pituitary GH4C1 cells. Similar to D1 receptors, cholera toxin treatment of cells reduced, but did not abolish, D5 agonist high-affinity binding sites, indicating D5 receptors couple to both Gs alpha and cholera toxin-insensitive G proteins. The interaction between D5 receptors and Gs alpha was confirmed by immunoprecipitation studies and by the ability of D5 receptors to stimulate adenylyl cyclase. Unlike D1 receptors, D5 receptors did not display any pertussis toxin-sensitive G-protein coupling to Go alpha or Gi alpha. D5 receptors were also not coupled to Gq alpha and were unable to mediate phosphatidylinositol metabolism. Instead, D5 sites appeared to be coupled to an AIF(-)4-sensitive, N-ethylmaleimide-resistant G protein. Anti-Gz alpha caused immunoprecipitation of 24.2 +/- 5.2% of G protein-associated D5 receptors, indicating coupling between D5 and Gz alpha. The coupling to Gz alpha was specific for D5 receptors, because similar associations were not detected between D1 receptors and Gz alpha.     

Opposing roles for dopamine D1 and D2 receptors in the regulation of hypothalamic tuberoinfundibular dopamine neurons

The purpose of the present study was to characterize pharmacologically dopamine D1 receptor-mediated inhibition of tuberoinfundibular dopamine neurons in males rats, and to determine if inhibitory dopamine D1 receptors oppose stimulatory dopamine D2 receptors and account for the inability of mixed dopamine receptor agonists to alter the activity of these neurons. Tuberoinfundibular dopamine neuronal activity was estimated by measuring the concentrations of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the median eminence, the region of the hypothalamus containing terminals of these neurons. Administration of the dopamine D1 receptor agonist (+/-)-1 phenyl-2,3,4,5-tetrahydro-(1 H)-3-benzazepine-7,8-diol (SKF38393) decreased median eminence DOPAC and increased plasma prolactin concentrations, whereas administration of the dopamine D1 receptor antagonist ((-)-trans,6,7,7a,8,9,13b-hexahydro-3-chloro-2-hydroxy-N-methyl-5H -benzo[d]naphtho-[2,1 b]azepine (SCH39166) increased median eminence DOPAC concentrations but had not effect on plasma prolactin. The inhibitory effect of SKF38393 on median eminence DOPAC concentrations was blocked by SCH39166. These results demonstrate that acute activation of dopamine D1 receptors inhibits the activity of tuberoinfundibular dopamine neurons and thereby increases prolactin secretion, and that under basal conditions dopamine D1 receptor-mediated inhibition of tuberoinfundibular dopamine neurons is tonically active. Administration of the dopamine D2 receptor agonist (5aR-trans)-5,5a,6,7,8,9,9a,10-octahydro-6-propyl-pyridol[2, 3-g]quinazolin-2-amine (quinelorane) increased median eminence DOPAC concentrations, and SKF38393 caused a dose-dependent reversal of this effect. Administration of the mixed dopamine D1/D2 receptor agonist R(-)-10,11-dihydroxy-apomorphine (apomorphine) had no effect per se, but blocked quinelorane-induced increases in DOPAC concentrations in the median eminence. These results reveal that concurrent activation of dopamine D1 and D2 receptors nullifies the actions of each of these receptors on tuberoinfundibular dopamine neurons, which likely accounts for the lack of an acute effect of mixed dopamine D1/D2 receptor agonists on these hypothalamic dopamine neurons.     

Visualization of D1 dopamine receptors on living nucleus accumbens neurons and their colocalization with D2 receptors

To examine the substrate for dopamine (DA) synaptic action in the nucleus accumbens (nAcc), we visualized the cellular and subcellular distribution of DA receptors on postnatal nAcc neurons in culture using fluoroprobe derivatives of DA receptor ligands. Previously, we have shown that rhodamine-N-(p-aminophenethyl)-spiperone (NAPS) (10 nM), a derivative of the D2 antagonist spiperone, labels D2-like receptors on living nAcc neurons. We now show that rhodamine-Sch-23390 (30 nM), a derivative of the D1 antagonist, labels D1-like receptors. Putative specific membrane labeling reached a plateau after about 20 min. Labeling was stereospecific, as it was unaffected by competition with (-)-butaclamol, but blocked with (+)-butaclamol. We found that 52 +/- 7% of nAcc medium-sized neurons showed D1 labeling, which extended onto the dendrites. Labeling was also seen on presynaptic terminals, often abutting D1-positive and D1-negative cell bodies, consistent with a presynaptic modulatory role for D1 receptors. Larger neurons, which may be GABAergic or cholinergic interneurons, were also labeled. By sequential labeling first with rhodamine-Sch-23390 and then rhodamine-NAPS, we found that 38 +/- 6% of medium-sized neurons express both D1- and D2-like receptors, indicating that D1-D2 interactions may occur at the level of single postsynaptic neurons.     

Behavioural sensitization of mesolimbic dopamine D2 receptors in chronic fluoxetine-treated rats

A common action of chronic antidepressant treatments is the potentiation of dopaminergic transmission in the limbic system. We now report that chronic, but not acute, treatment with fluoxetine (2.5 mg/kg by intragastric gavage once a day for 21 days) potentiates the locomotor stimulant effect of quinpirole, a selective dopamine D2/D3 receptor agonist. However, neither quinpirole-induced stereotypies nor the sedative effects elicited by low doses of this dopamine receptor agonist are influenced by chronic fluoxetine. These results suggest that fluoxetine, as well as classical antidepressants, sensitize postsynaptic dopamine D2/D3 receptors in the mesolimbic system.     

Dopamine-induced recruitment of dopamine D1 receptors to the plasma membrane

The recruitment of G protein-coupled receptors from the cytoplasm to the plasma membrane generally is believed to be a constitutive process. We show here by the use of both confocal microscopy and subcellular fractionation that, for at least one such receptor, this recruitment is regulated and not constitutive. Cells from a proximal tubular-like cell line, LLCPK1 cells, were incubated with either a D1 agonist, a dopamine precursor, or an inhibitor of dopamine metabolism to increase dopamine availability in the cell. Each of the three procedures led to a rapid translocation of dopamine D1 receptors from the cytosol to the plasma membrane.     

Photoaffinity labeling of D1 and D5 dopamine receptors

Although human D1 and D5 dopamine receptors are encoded by distinct genes and share only 50% sequence homology at the amino acid level, their pharmacological properties are identical. Using a selective D1 receptor photoaffinity radioligand, (+/-)-7-[125I]iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)-2,3,4,5-tetrahyd ro-1H-3-benzazepine ([125I]MAB), we have further probed the molecular properties of these receptors in transfected GH4C1 rat pituitary cells. Under reversible, non-covalent binding conditions, [125I]MAB bound to both the D1 and the D5 receptors with identical affinities, dopaminergic selectivity and stereospecificity. Upon photoactivation of the bound [125I]MAB, the label was incorporated into a approximately 64,000 mol. wt protein corresponding to the D1 dopamine receptor. However, there was no specific photoincorporation of the ligand observed in D5 receptors. The lack of [125I]MAB photolabeling of D5 receptors was independent of the cell line chosen, since similar results were obtained using other transfected cells. The data suggest that although both D1 and D5 receptors share structurally similar binding sites, the protein domains around the sites are different. Thus, although there are currently no specific compounds which bind preferentially to D1 or D5 receptors, these receptors can be distinguished from one another by the inability of [125I]MAB to photolabel D5, but not D1, receptors. Such selective targeting of a specific receptor may be useful in understanding the functional importance and/or interaction between closely related members of the same receptor family when co-expressed in the same cell.     

Imidazole binding to horse metmyoglobin: dependence upon pH and ionic strength

A series of substituted chalcone oxides (1,3-diphenyl-2-oxiranyl propanones) and structural analogs was synthesized to investigate the mechanism by which they inhibit soluble epoxide hydrolases (sEH). The inhibitor potency and inhibition kinetics were evaluated using both murine and human recombinant sEH. Inhibition kinetics were well described by the kinetic models of A. R. Main (1982, in Introduction to Biochemical Toxicology, pp. 193-223, Elsevier, New York) supporting the formation of a covalent enzyme-inhibitor intermediate with a half-life inversely proportional to inhibitor potency. Structure-activity relationships describe active-site steric constraints and support a mechanism of inhibition consistent with the electronic stabilization of the covalent enzyme-inhibitor intermediate. The electronic effects induced by altering the ketone functionality and the para-substitution of the phenyl attached to the epoxy C1 (i.e., the alpha-carbon) had the greatest influence on inhibitor potency. The direction of the observed influence was reversed for the inhibitory potency of glycidol (1-phenyl-2-oxiranylpropanol) derivatives. Recent insights into the mechanism of epoxide hydrolase activity are combined with these experimental results to support a proposed mechanism of sEH inhibition by chalcone oxides.     

Progressive supranuclear paralysis. Quantification of dopamine D2 receptors using radionuclide tomography

Progressive supranuclear palsy (PSP) may sometimes be misdiagnosed as Parkinson's disease in its early stages, hence an early positive diagnosis of PSP based on dopamine D2 receptor density could be extremely valuable. In the present case report, the absence of dopamine D2 receptors was clearly demonstrated in the striatum using 123I-iodobenzamide (IBZM) tomoscintigraphy. This illustrates the potential use of IBZM tomoscintigraphy to identify Parkinson-like's disease presenting with decreased dopamine D2 receptor density; and hence to predict L-Dopa effectiveness. Further studies are needed to evaluate the vaue of IBZM tomoscintigraphy in the different Parkinson's like diseases.     

Role of dopamine D1 and D2 receptors in the nucleus accumbens in mediating reward

The objectives of this study were to examine the involvement of D1 and D2 receptors within the nucleus accumbens (ACB) in mediating reinforcement. The intracranial self-administration (ICSA) of D1 and D2 agonists was used to determine whether activating D1 and/or D2 receptors within the ACB of Wistar rats is reinforcing. At concentrations of 0.25, 0.50, and 1.0 mM (25, 50, and 100 pmol/100 nl of infusion), neither the D1 agonist R(+)-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8-diol [SKF 38393 (SKF)] hydrochloride nor the D2 agonist (-)-quinpirole (Quin) hydrochloride was self-administered into the shell region of the ACB. On the other hand, equimolar mixtures of SKF and Quin (SKF+Quin), at concentrations of 0.25, 0.50, and 1.0 mM each, were significantly self-infused into the ACB shell. The core region of the ACB did not support the ICSA of SKF+Quin at any of these concentrations. Rats increased lever pressing when the response requirement was increased from a fixed ratio 1 (FR1) to FR3, and they responded significantly more on the infusion lever than they did on the control lever. Coadministration of either 0.50 mM R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4, 5-tetrahydro-1H-3-benzazepine (SCH 23390) hydrochloride, a D1 antagonist, or 0.50 mM S(-)-sulpiride, a D2 antagonist, completely abolished the ICSA of the mixture of SKF+Quin (each at 0.50 mM) into the ACB shell. The present results suggest that concurrent activation of D1- and D2-type receptors in the shell of the ACB had a cooperative effect on DA-mediated reward processes.     

Heterogeneous distributions of histamine H3, dopamine D1 and D2 receptors in rat brain

The changes of the histamine H3 and dopamine D1 or D2 receptor binding sites induced by quinolinic acid treatment were studied in order to discriminate the comparative distribution. This treatment resulted in similar decreases in histamine H3 and dopamine D1 receptor binding sites in the striatum and ipsilateral substantia nigra. Dopamine D2 receptor binding sites were relatively well conserved, whereas H3 receptors decreased considerably. These results suggest that histamine H3 and dopamine D1 receptor binding sites are localized on the striatonigral projection neurones which are together sensitive to quinolinic acid, and that the distributional compartment of dopamine D2 receptor binding sites is quite different from those of histamine H3 and dopamine D1 receptors.     

The roles of accumbal dopamine D1 and D2 receptors in maternal memory in rats.

Female rats show enhanced maternal responsiveness toward their young if they have had maternal experiences before. This kind of maternal experience-based memory is critically dependent on the mesolimbic dopamine (DA) system, especially the nucleus accumbens (NA) shell. However, the relative contributions of the two main DA receptor systems (D? and D?) within the shell have not been delineated. This study investigates the roles of dopamine D? and D? receptors in maternal memory by infusing a selective D? antagonist, SCH-23390; a selective D? antagonist, sulpiride; or a combination D?/D? antagonist, cis-Z-flupenthixol, into the NA shell of postpartum female rats. Sulpiride-infused rats showed a significantly longer latency to exhibit full maternal behavior following a 10-day pup isolation period in comparison to the controls that received a vehicle. Cis-Z-flupenthixol disrupted maternal memory to a greater extent, as rats receiving this showed the longest latencies to express maternal behavior. SCH-23390 infusions had only marginal effects. These findings suggest that both the D? and the D? receptor subtypes play a role in the consolidation of maternal memory and they might do so by mediating the motivational salience of pup stimulation. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Zn2+ modulation of ATP-responses at recombinant P2X2 receptors and its dependence on extracellular pH

1. Using recombinant P2X2 receptors expressed in Xenopus oocytes, the modulatory effects of zinc (Zn2+) on ATP-responses were studied under voltage-clamp conditions and at different levels of extracellular pH. 2. Zn2+ (0.3-300 microM) added to the bathing medium potentiated ATP-activated membrane currents, increasing ATP-responses by up to 20 fold. This potentiating effect was reversed on washout. Zn2+-potentiation was reduced in an exponential manner (decaying 1/e in 42 s) as the interval was lengthened between adding Zn2+ then ATP to the superfusate. 3. The potentiating effect of Zn2+ was progressively diminished by acidic shifts in extracellular pH (pHe) which, of itself, also potentiated ATP-responses at P2X2 receptors. The maximal potentiating effects of Zn2+ and H+ were not additive. 4. Neither Zn2+ nor H+ potentiation of ATP-responses was abolished by diethylpyrocarbonate (DEPC, 0.3-3 mM), which irreversibly denatures histidyl residues. Nine histidyl residues are present in the extracellular loop of P2X2 receptors. 5. Zn2+ also enhanced the blocking activity of the P2 receptor antagonist suramin at P2X2 receptors. Therefore, Zn2+ also mimics H+ in increasing suramin-activity at P2X2 receptors. 6. In summary, Zn2+ and H+ potentiate agonist and antagonist activity at P2X2 receptors but their effects are not wholly alike for receptor agonism. There, the potentiating effects of Zn2+ are time-dependent and gradually convert to inhibition while those of H+ are time-independent, persistent and more potent, suggesting that either these modulators interact in a different way with a single allosteric site or with different allosteric sites.