M_(4) muscarinic receptors regulates dopamine/DARPP-32 signaling and glutamate transmis⁃sion to balance dopaminergic D1 function in mouse dorsal striatum

OBJECTIVE Abnormal striatal dopaminergic and glutamatergic neurotransmis⁃sion is central to the pathophysiology of schizo⁃phrenia.In this study,we investigated the roles of M4 receptor interplay with D1 signaling in stria⁃tal neurotransmission that affect glutamatergic transmission to control the etiology of neuropsy⁃chiatric disorders.METHODS To study dorsal striatum(DS)region-specific neuronal and behav⁃ioral responses modulated by M4 receptors,we used clustered regularly interspaced short palin⁃dromic repeats-associated protein 9 technology to generate mice lacking M4 in the dorsal stria⁃tum(DS-M4-KD).The M4 positive allosteric modu⁃lator,VU0467154,were used to study the phar⁃macologically profiles with M4 receptor stimula⁃tion in WT mice.Oxotremorine M(Oxo-M),a no subtype-selective muscarinic agonist,was used to show that mAchRs activation,in order to dissect the particular function of M4,in DS-M4-KD mice.Open filed test and forced swim test were used to assess the change of psychiatric-like behav⁃iors.Western blotting and immunohistochemistry were used to detect protein levels of phosphory⁃lation site of dopamine-and cAMP-regulated phosphoprotein of 32 ku(DARPP-32).Whole-cell patch-clamp recording was used to assess M4-mediated cholinergic inhibition of glutamater⁃gic synaptic input transmission.RESULTS West⁃ern blotting and immunohistochemistry assay showed VU0467154(5 mg·kg-1,ip)promoted phosphorylation of DARPP-32 at Thr75,and atten⁃uated D1-dependent phosphorylation of DARPP-32 at Thr34 within the mouse DS.Consistently,the Oxo-M(4μg,icv)also increased DARPP-32 phosphorylation at site Thr75 to reversed phos⁃phorylation at site Thr34 in WT mice,but not in DS-M4-KD mice.In parallel with altered DARPP-32 responses,VU0467154 or Oxo-M evoked a psychological stress response and reversed D1-induced hyperlocomotion in mice in open field test and force swim tests.However,Oxo-M sup⁃pression of D1-depengdeng behavioral respons⁃es was impaired in DS-M4-KD mice.Whole-cell patch recording showed that VU0467154 or Oxo-M mediated endogenous cholinergic inhibition of miniature excitatory postsynaptic currents through M4 receptors,which in turn suppressed D1-depen⁃dent glutamatergic synaptic transmission in the DS.CONCLUSION This study provides evidence for the role of M4 receptors in regulation of dopa⁃mine/DARPP-32 signaling and glutamate respons⁃es in the DS,and therefore modulation of psychi⁃atric behaviors associated with D1 signaling.This results indicate the mechanisms of treatments targeting M4 in psychiatric disorders.

-94 G/A polymorphism in the dopamine D1 receptor gene is associated with schizophrenia in a Chinese Han population from Shandong province

The correlation between -94 G/A polymorphism in the dopamine D1 receptor gone and schizophrenia remains poorly understood despite extensive research. This study sought to evaluate the genotypes and allele frequencies of the -94 G/A polymorphism in the dopamine D1 receptor gone by real-time PCR using TaqMan fluorescent probes. One hundred and sixty-two patients with schizophrenia and 101 healthy controls living in Shandong province of China were evaluated. Experimental results showed that the G/A genotype distribution was significantly higher in the schizophrenia patients than in healthy controls. The frequencies of G allele and A allele were not significantly different between the schizophrenia patients and the controls. Thus, the -94 G/A polymorphism in the dopamine D1 receptor gone was found to be associated with schizophrenia in a Chinese Han population from Shandong province.

Effects of endogenous dopamine induced by low concentration atropine eye drops on choroidal neovascularization in high myopia mice

AIM:To evaluate effects of endogenous dopamine induced by low concentration atropine eye drops on choroidal neovascularization(CNV)in high myopia mice.METHODS:The C57BL/6J mice were deprived of the right eye for 4wk,and the high myopia was diagnosed by optometry,the diopter was less than-6.00 D,and CNV was induced by 532 nm laser.The changes of dopamine D1 receptor(DRD1),dopamine D2 receptor(DRD2),and vascular endothelial growth factor A(VEGFA)were detected by Western blot technology at 0.5,1,2h,and 7d after 0.01%,0.05%,and 0.1%atropine eye drops,respectively,the area of CNV was measured.RESULTS:Significant increases were observed on the expression of DRD2 in mouse high myopia model at 0.5,1,2h,7d with 0.05%and 0.1%atropine eye drops(P<0.05).Significant decreases were observed on the expression of DRD1 and VEGFA in mouse high myopia model at 0.5,1,2h,7d with 0.05%and 0.1%atropine eye drops(P<0.05).The area of CNV induced by laser in the drug-treated group was significantly smaller than that in the control group,and the higher the concentration,the more significant the inhibitory effect(P<0.05).CONCLUSION:The 0.01%,0.05%,0.1%atropine eye drops can decrease the level of VEGFA and inhibit high myopia CNV indirectly by up-regulating the level of DRD2 and down-regulating the level of DRD1,and the effect of 0.05%and 0.1%atropine eye drops is more significant.

Differential distributions and trafficking properties of dopamine D1 and D5 receptors in nerve cells

Objective To explore the possible differential trafficking properties of the dopamine D 1-like receptor subtypes, D 1 receptor and D5 receptor. Methods To visualize distributions of dopamine D 1-like receptor subtypes at subcellular level, the yellow and cyan variants of green fluorescent protein （GFP） were used to tag D1 and D5 receptors. After transfection with the tagged dopamine receptors, the neuroblastoma cells NG108-15 were treated with D1 agonist SKF38393 or acetylcholine （ACh）. Then we observed the subcellular distributions of the tagged receptors under the confocal microscopy and tried to determine trafficking properties by comparing their distribution patterns before and after the drug treatment. Results In resting conditions, D 1 receptors located in the plasma membrane of NG108-15 cells, while D5 receptors located in both plasma membrane and cytosol. With the pre-treatment of SKF38393, the subcellular distribution of D1 receptors was changed. The yellow particle-like fluorescence of tagged D 1 receptors appeared in the cytosol, indicating that D 1 receptors were internalized into cytosol from the cell surface. Same situation also occurred in ACh pre-treatment. In contrast, the subcellular distribution of D5 receptors was not changed after SKF38393 or ACh treatment, indicating that D5R was not translocated to cell surface. Interestingly, when D1 and D5 receptors were co-expressed in the same cell, both kept their distinct subcellular distribution patterns and the trafficking properties. Conclusion Our present study reveals that in NG108-15 nerve cells, dopamine D1 and D5 receptors exhibit differential subcellular distribution patterns, and only D1 receptor has a marked trafficking response to the drug stimulation. We further discuss the potential role of the differential trafficking properties of D1-like receptors in complex modulation of DA signaling.

Increased dopamine and its receptor dopamine receptor D1 promote tumor growth in human hepatocellular carcinoma

Background:Dopamine and dopamine receptor D1(DRD1),a member of the dopamine receptor family,have been indicated to play important roles in cancer progression,but dopamine secretion in hepatocellular carcinoma(HCC)and the effects of DRD1 on HCC remain unclear.This study was designed to explore the contribution of the dopaminergic system to HCC and determine the relationship between DRD1 and prognosis in HCC patients.Methods:The dopamine metabolic system was monitored using enzyme-linked immunosorbent assays(ELISAs).The expression of DRD1 was detected by microarray analysis,immunohistochemistry(IHC),and quantitative real-time PCR(qRT-PCR).Stable DRD1 knockout and overexpression cell lines were established for investigation.Transwell,colony formation,and Cell Counting Kit 8(CCK8)assays were performed to assess the malignant behaviors of cancer cells.The cAMP/PI3K/AKT/cAMP response element-binding(CREB)signaling pathway was evaluated by Western blot.This pathway,which is agitated by DRD1 in striatal neurons,had been proven to participate in tumor progression.Xenograft HCC tumors were generated for in vivo experiments.Results:Dopamine secretion increased locally in HCC due to an imbalance in dopamine metabolism,including the upregulation of dopa decarboxylase(DDC)and the downregulation of monoamine oxidase A(MAOA).Dopamine promoted the proliferation and metastasis of HCC.DRD1 was highly expressed in HCC tissues and positive DRD1 expression was related to a poor prognosis in HCC patients.The upregulation of DRD1 agitated malignant activities,including proliferation and metastasis in HCC by regulating the cAMP/PI3K/AKT/CREB pathway,and the downregulation of DRD1 had opposing effects.The effects of dopamine on HCC was reversed by depleting DRD1.SCH23390,a selective DRD1 antagonist,inhibited the proliferation and metastasis of HCC cells both in vitro and in vivo.Conclusion:Dopamine secretion was locally increased in HCC and promoted HCC cell proliferation and metastasis.DRD1 was found to exert positive effects on HCC progression and play a vital role in the dopamine system,and could be a potential therapeutic target and prognostic biomarker for HCC.

Pharmacodynamic model of dopamine D1 receptor agonists in the treatment of breast cancer lung metastasis

Previous study has shown that dopamine D1 receptor(D1DR)agonists,fenoldopam(FEN)and l-stepholidine(l-SPD),have inhibitory effects on breast cancer lung metastasis.To quantitatively describe and predict the pharmacodynamic(PD)properties of FEN and l-SPD and to explore the PD model structure of cancer metastasis treating drugs,we used the data of lung metastasis in 4T1 breast cancer mice under the treatment of either FEN or l-SPD,and established a PD model.The PD model assumed an exponential growth for both primary tumor and metastasis.The primary tumor emitted cells to form metastases,and the cell emitting rate was proportional to power form of the primary tumor weight.The total number of lung metastasis was set as the target value.D1DR agonists inhibited metastasis by inhibiting cell emitting rate instead of the growth rate of primary tumor or metastasis.The model results showed that the decrease in the number of lung metastases was roughly proportional to the square of the drug dose.The values of PD coefficient reflected the inhibitory ability of the drugs,and that of l-SPD(0.274 kg/mg)was greater than that of FEN(0.0393 kg/mg).This PD model can quantitatively describe the effects of FEN and l-SPD on the progression of lung metastasis in 4T1 primary breast cancer mice and can predict the time course of drug efficacy at multiple doses,providing a reference for PD model structure of other drugs for cancer metastasis indication.

Disruption of dopamine D1 receptor phosphorylation at serine 421 attenuates cocaine-induced behaviors in mice

Dopamine D1 receptors（D1Rs） play a key role in cocaine addiction, and multiple protein kinases such as GRKs, PKA, and PKC are involved in their phosphorylation. Recently, we reported that protein kinase D1 phosphorylates the D1 R at S421 and promotes its membrane localization. Moreover, this phosphorylation of S421 is required for cocaineinduced behaviors in rats. In the present study, we generated transgenic mice over-expressing S421A-D1 R in the forebrain. These transgenic mice showed reduced phospho-D1R（S421） and its membrane localization, and reduced downstream ERK1/2 activation in the striatum. Importantly, acute and chronic cocaine-induced locomotor hyperactivity and conditioned place preference were significantly attenuated in these mice. These findings provide in vivo evidence for the critical role of S421 phosphorylation of the D1 R in its membrane localization and in cocaine-induced behaviors. Thus, S421 on the D1 R represents a potential pharmacotherapeutic target for cocaine addiction and other drug-abuse disorders.

New therapeutic strategies targeting D1-type dopamine receptors for neuropsychiatric disease

The neurotransmitter dopamine acts via two major classes of receptors, Dl-type and D2-type. D1 receptors are highly expressed in the striatum and can also be found in the cerebral cortex. Here we review the role of D1 dopamine signaling in two major domains： L-DOPA-induced dyskinesias in Parkinson＇s disease and cognition in neuropsychiatric disorders. While there are many drugs targeting D2-type receptors, there are no drugs that specifically target D1 receptors. It has been difficult to use selective Dl-receptor agonists for clinical applications due to issues with bioavailability, binding affinity, pharmacological kinetics, and side effects. We propose potential therapies that selectively modulate D1 dopamine signaling by targeting second messengers downstream of D1 receptors, aUosteric modulators, or by making targeted modifications to Dl-receptor machinery. The development of therapies specific to Dl-receptor signaling could be a new frontier in the treatment of neurological and psychiatric disorders.

Gut-brain crosstalk regulates craving for fatty food

Patients undergoing Roux-en-Y gastric bypass(RYGB)surgery elicit striking loss of body weight. Anatomical restructuring of the gastrointestinal(GI) tract, leading to reduced caloric intake and changes in food preference, are thought to be the primary drivers of weight loss in bariatric surgery patients. However, the mechanisms by which RYGB surgery causes a reduced preference for fatty foods remain elusive. In a recent report, Hankir et al described how RYGB surgery modulated lipid nutrient signals in the intestine of rats to blunt their craving for fatty food. The authors reported that RYGB surgery restored an endogenous fat-satiety signaling pathway, mediated via oleoylethanolamide(OEA), that was greatly blunted in obese animals. In RYGB rats, high fat diet(HFD) led to increased production of OEA that activated the intestinal peroxisome proliferation activator receptors-α(PPARα). In RYGB rats, activation of PPARα by OEA was accompanied by enhanced dopamine neurotransmission in the dorsal striatum and reduced preference for HFD. The authors showed that OEA-mediated signals to the midbrain were transmitted via the vagus nerve. Interfering with either the production of OEA in enterocytes, or blocking of vagal and striatal D1 receptors signals eliminated the decreased craving for fat in RYGB rats. These studies demonstrated that bariatric surgery led to alterations in the reward circuitry of the brain in RYGB rats and reduced their preference for HFD.

Compound C17 inhibits the lung metastasis of breast cancer

Breast cancer is the most common cancer in females with extremely high lethality mainly due to the occurrence of metastasis,which is closely related to cancer stem-like cells(CSCs).It has been reported that CSC frequency in drug-resistant breast cancer and non-small cell lung cancer is reduced by activating dopamine D1 receptor(D1 DR).In the present study,we aimed to investigate the effect of a compound C17 that can be used orally for breast cancer metastasis as well as the underlying mechanism involving the activation of D1 DR.The confocal immunofluorescence analysis demonstrated that D1 DR was up-regulated by C17.The cell survival and colony formation were inhibited by C17 through the detection by Sulforhodamine B colorimetric(SRB)assay and colony formation assay,respectively.Results from both wound healing assay and transwell assay demonstrated that C17 inhibited the migration of 4T1 cells.Flow cytometry analysis indicated that C17 significantly reduced the CSC frequency.In addition,C17 could inhibit the lung metastasis in 4T1 orthotopic mouse model of breast cancer without obvious toxicity,and it could up-regulate the expression of intratumoral E-cadherin and down-regulate the expressions of Snail and N-cadherin in primary breast tumor,which might be related to the activation of D1 DR.Our findings provided a potential candidate compound for the treatment of metastatic breast cancer with good compliance and safety.

QAP21 reduces stemness and mobility of metastatic breast cancer cells involving D1DR activation

Breast cancer is the second leading cause of cancer death in women mainly due to metastasis,which is closely related to cancer stemness.Evidence has shown that cancer stem-like cells(CSCs),which are responsible for cancer stemness,can be decreased by activating dopamine D1 receptor(D1 DR).In the present study,we aimed to explore the pharmacological effects as well as the underlying mechanisms of QAP21,a newly synthesized compound that can be orally administered,in metastatic breast cancer cells.Our results showed that QAP21 dose-dependently inhibited the ability of colony formation in 4 T1 and MDA-MB-231 cells.Cell mobility,including cell migration and invasion,was also remarkably inhibited.Besides,QAP21 significantly inhibited mammosphere formation and decreased CSC proportion,indicating reduced cancer stemness.We further verified that the nuclear factor-kappa B(NF-κB)/Akt/epithelial-mesenchymal transition(EMT)pathway was markedly impacted by QAP21 treatment.Moreover,QAP21 up-regulated the expressions of D1 DR and its second messengers,including cAMP and cGMP,which can be increased when D1 DR is activated.SCH 23390,a specific D1 DR antagonist,partially or completely reversed the above-mentioned effects of QAP21,indicating that D1 DR activation might be involved in the underlying mechanism of QAP21.In summary,QAP21 effectively reduced breast cancer stemness and cell mobility,indicating its potential use for metastatic breast cancer therapy.

Dopaminergic system does not play a major role in the precipitated cannabinoid withdrawal syndrome

AIM:To determine the dopaminergic system involvement in precipitated cannabinoid withdrawal syndrome.METHODS:The dopamine D_(1)receptor antagonist SCH23390 or the dopamine D_(2)receptor antagonist sulphide was administered to rats chronically treated with either△^(9)-tetrahydrocannabinol(THC)or vehicle.Subjects were then injected with either SR141716A or vehicle and behavior was observed for 1 h.RESULTS:Administration of the cannabinoid receptor antagonist SR141716A to animals chronically treated with THC as described by Tsou et al(1995)produced a profound withdrawal syndrome.Treatment with dopamine antagonists did not attenuate cannabinoid precipitated withdrawal syndrome in THC tolerant animals while the agonists increased the syndrome.CONCLUSION:It is unlikely that the dopaminergic system plays a major role in mediating the behavioral aspects of the cannabinoid withdrawal syndrome.