Dopamine D4 receptor protected against hyperglycemia-induced endothelial dysfunction via PI3K /eNOS pathway.

Hyperglycemia-induced endothelial dysfunction is generally believed to be the basis of diabetic vascular complications. Dopamine receptors is known to play an important protective role in diabetes. However, the protective effect of dopamine receptors against hyperglycemia-induced endothelial damage in diabetic rats is still unknown. In the present study, we established a cell model of hyperglycemia-induced endothelial dysfunction by treating human umbilical vein endothelial cells (HUVEC) with high glucose. MTT and lactate dehydrogenase assays results showed that high glucose treatment significantly reduced the cell viability and down-regulated dopamine D4 receptor. Pre-treatment with PD168077, a specific D4 receptor agonist, greatly improved endothelial cell viability and decreased apoptosis. Furthermore, pharmacological inhibition of phosphoinositide 3-kinase (PI3K) and endothelial nitric oxide synthase (eNOS) eliminated the protective effect of D4 receptor against endothelial injury. More importantly, the expression level of D4 receptor was also dramatically down-regulated in the arterial endothelium of rats with streptozotocin-(STZ)-induced diabetes, and the STZ-induced impairment of acetylcholine-induced vasodilation was reversed by activation of D4 receptor. In conclusion, our results indicated that dopamine D4 receptor protected against hyperglycemia-induced endothelial dysfunction via the PI3K/eNOS pathway, which may provide a novel strategy in the treatment of diabetes.

Activation of angiotensin II type 1 receptors increases D4 dopamine receptor expression in rat renal proximal tubule cells.

Both the dopaminergic and renin-angiotensin systems play important roles in the regulation of blood pressure. Our previous study showed that the stimulation of dopaminergic D4 receptors reduced angiotensin II type 1 (AT1) receptor expression in renal proximal tubule (RPT) cells. In this study, we tested whether AT1 receptors, in return, would regulate D4 receptor expression and function in RPT cells. Expression of the D4 receptor from Wistar-Kyoto (WKY) or spontaneously hypertensive rats (SHRs) RPT cells and renal cortex tissues were determined by western blot, and Na+-K+ ATPase activity was determined using an enzyme assay. Urine volume and urine sodium of WKY rats and SHRs treated with or without D4 receptor stimulation were measured. Thus, activation of AT1 receptors with angiotensin II (Ang II) increased D4 receptor protein expression in RPT cells, and this increase was blocked by nicardipine, a calcium influx blocker. The D4 receptor agonist PD168077 inhibited Na+-K+ ATPase activity in WKY RPT cells but not in SHR RPT cells. Ang II pre-treatment promoted D4 receptor-mediated inhibition of Na+-K+ ATPase in RPT cells in WKY rats but not in SHRs. Meanwhile, Ang II pre-treatment augmented the natriuretic effect of PD168077 in WKY rats but not in SHRs. In conclusion, AT1 stimulation can regulate the expression and natriuretic function of dopaminergic D4 receptors in RPT cells and might be involved in the pathogenesis of essential hypertension.

Effect of D3 dopamine receptor on dopamine D4 receptor expression and function in renal proximal tubule cells from Wistar-Kyoto rats and spontaneously hypertensive rats.

BACKGROUND: Dopamine receptors induce natriuresis in kidney. Previous studies have shown interactions between different subtypes of dopamine receptors in renal proximal tubule (RPT) cells. We hypothesize that D3 receptors have an interaction with D4 receptors in RPT cells from normotensive rats (Wistar-Kyoto, WKY) and spontaneously hypertensive rats (SHRs). METHODS: Immunoblotting and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to examine the expression of D3 and D4 receptors. Na-K-ATPase activity was used to measure the function of receptors. The distribution and colocalization of D3 and D4 receptors were detected by confocal microscopy and co-immunoprecipitation. RESULTS: D3 receptor agonist PD128907 increased the mRNA and protein expression of D4 receptors in RPT cells from WKY rats, but decreased that from SHRs. In the presence of PLC blocker (U73122, 10-mol/l) or PKC inhibitor 19 -31 (10-mol/l), the up-regulation of D3 receptor on D4 receptor was lost in WKY cells. Moreover, stimulation with PD128907 for 30 minutes decreased D4 receptor degradation in WKY cells, not in SHR cells. D3 and D4 receptors colocalized and co-immunoprecipitated in RPT cells. PD128907 increased co-immunoprecipitation of D3 and D4 receptors in WKY RPT cells, but not in SHR RPT cells. Pre-treatment with D3 receptor agonist also increases D4 receptor mediated inhibitory effect on Na-K-ATPase activity in WKY cells, but not in SHR cells. CONCLUSION: Renal D3 receptor regulates the expression and function of D4 receptor in RPT cells via PLC /PKC signaling pathway, the loss of this interaction might be involved in the pathogenesis of hypertension.

Pancreatic Endocrine Effects of Dopamine Receptors in Human Islet Cells.

OBJECTIVE: To date, there are no reports on the cellular localization of dopamine receptors in the human pancreas. In our study, we determined the localization and expression of 5 dopamine receptors (D(1), D(2), D(3), D(4), and D(5)) in normal human pancreas tissue. METHODS: Human nonpathological pancreas tissues were fixed with 4% paraformaldehyde, paraffin-embedded, and processed for immunohistochemical analysis to detect dopamine receptors in the human pancreas tissue by using double immunofluorescent labeling and confocal microscopy. RESULTS: We found that the D(1) receptor is present in ß cells; the D(2) receptor is expressed by α, δ, and pancreatic polypeptide cells; the D(4) receptor is expressed by ß and polypeptide cells; whereas the D(5) receptor is expressed only by δ cells. CONCLUSIONS: Our results identify the dopamine receptors (D(1)-D(5)) in normal pancreas tissue and provide a morphological basis for studying the pancreatic endocrine effects of dopamine and suggest a new target for the clinical treatment of diabetes.

Labeling of benzodioxin piperazines with fluorine-18 as prospective radioligands for selective imaging of dopamine D4 receptors.

The D(4) receptor is of high interest for research and clinical application but puts high demands on appropriate radioligands to be useful tools for investigation. Search for adequate radioligands suitable for in vivo imaging is therefore still in progress. The potential neuroleptic drug 6-(4-[4-fluorobenzyl]piperazin-1-yl)benzodioxin shows high affinity and selectivity to the D(4) receptor. Derivatization of this lead structure by adding hydrophilic moieties was carried out in order to lower its lipophilicity what led to three new putative dopamine receptor D(4) ligands. A comprehensive description of the syntheses of standard compounds and corresponding labeling precursors is given which were obtained in satisfactory yields. Furthermore, the radiosyntheses by direct (18) F-labeling and build-up synthesis were compared. All derivatives of 6-(4-[4-fluorobenzyl]-piperazin-1-yl)benzodioxin were successfully synthesized in (18) F-labeled form with radiochemical yields of 9-35% and molar activities of 30-60 GBq/µmol using one-pot procedures.

The dopamine D4/D2 receptor antagonist affinity ratio as a predictor of anti-aggression medication efficacy.

Aggression is a major clinical problem that spans multiple medical and psychiatric diagnoses. The etiology of aggression is complex, comprising social, psychological, and biological factors. Treatment of aggression is equally heterogeneous with structural (environmental), behavioral, and pharmacologic approaches. Among medications, mood stabilizers and antipsychotics are usually considered first line. Antidepressants are frequently recommended but their anti-aggression effect is limited. Within the available limited data, clozapine stands out as more effective than other antipsychotic medications. One of the prominent differences between clozapine and less effective medications is that the affinity of the compound to the dopamine D4 receptor is quite high and significantly greater than to the D2 receptor (defined as a ratio that is >1). Medications that inhibit both receptors equally, e.g., haloperidol, have anti-aggression properties that are less than seen with clozapine. A specific variant of the D4 receptor with an expansion of the third cytoplasmic loop, which interacts with the G protein second messenger system, has been implicated in the etiology of aggression. The proposal is put forward that blockade of the D4 receptor, without concomitant significant blockade of D2, increases the anti-aggression effect of the medication. This hypothesis can be tested using a new antipsychotic, asenapine, which is the only other antipsychotic with antagonistic affinity ratio of D4/D2>1.

Dopamine receptor subtypes in the native human heart.

The present study first time detected D1-D5 dopamine receptor subtypes in the native human heart simultaneously, found presence of D1, D2, D4, and D5 in cardiac tissues, and revealed distribution features of dopamine receptor subtypes in the epicardium, myocardium, and endocardium. Samples from four native hearts coming from young brain-dead donors, which for technical reason were not used for transplants, were studied. Dopamine receptors were revealed by immunochemistry technique and immunoblot analysis. Morphometrical quantification of the density of each receptor subtype was performed by an image analyzer. Our results demonstrate that only four subtypes of dopamine receptors can be found in cardiac tissues: D1, D2, D4, and D5. These dopamine receptors have been detected in endocardium, myocardium, and epicardium. D1 receptors were stored primarily in the epicardial layer. Dopamine receptors are distributed in the wall of both atria and ventricles, and its transmural gradient can be described in the wall of the human heart. Sections of atria and ventricles exposed to antidopamine receptor antibodies showed fluorescent-positive reaction in the epicardium, myocardium and endocardium. D4 receptor immune reactivity was remarkably less intense than D2 receptor immune reactivity. All the subtypes of dopamine receptors are in close relationships with all cardiac structures. Our findings provide a favorable basis for researching the role of dopamine receptors in controlling functions of the human heart and in the pathogenesis of cardiovascular diseases.

Should we be cautious on the use of commercially available antibodies to dopamine receptors?

Evidence indicate that it is difficult to obtain specific antibodies to G protein-coupled receptors and different technical difficulties may allow the generation of antibodies that lack specificity. We conducted experiments to validate the specificity of commercially available antibodies raised against dopamine (DA) receptors hD(1), hD(4), and hD(5) using a transfection approach: we studied whether, in HEK 293 cells selectively transfected with the various cloned subtypes, each antibody generates bands only in cells expressing its cognate receptor but not in those expressing the other DA receptors. Our results demonstrated that hD(1) and hD(4) receptor antibodies recognize not only their respective epitope, but also other DA receptor subtypes, while for the hD(5) receptor detection, we observed a signal only in the lane loaded with hD(5)-transfected HEK 293 cells, although with a lack of purity. Therefore, we recommend caution on the use of commercially available DA receptor antibodies.

Presence of D4 dopamine receptors in human prefrontal cortex: a postmortem study.

OBJECTIVE: The aim of our study was to explore the presence and the distribution of D4 dopamine receptors in postmortem human prefrontal cortex, by means of the binding of [3H]YM-09151-2, an antagonist that has equal affinity for D2, D3 and D4 receptors. It was therefore necessary to devise a unique assay method in order to distinguish and detect the D4 component. METHOD: Frontal cortex samples were harvested postmortem, during autopsy sessions, from 5 subjects. In the first assay, tissue homogenates were incubated with increasing concentrations of [3H]YM-09151-2, whereas L-745870, which has a high affinity for D4 and a low affinity for D2/D3 receptors, was used as the displacer. In the second assay, raclopride, which has a high affinity for D2/D3 receptors and a low affinity for D4 receptors, was used to block D2/D3. The L-745870 (500 nM) was added to both assays in order to determine the nonspecific binding. RESULTS: Our experiments revealed the presence of specific and saturable binding of [3H]YM-09151-2. The blockade of D2 and D3 receptors with raclopride ensured that the D4 receptors were labeled. The mean maximum binding capacity was 88 +/- 25 fmol/mg protein, and the dissociation constant was 0.8 +/- 0.4 nM. DISCUSSION AND CONCLUSIONS: Our findings, although not conclusive, suggest that the density of D4 receptors is low in the human prefrontal cortex.