OBJECTIVETo set the measuring method of tyrosine hydroxylase activity in the brain of conscious rats.

METHODBy using microdialysis and High Performance Liquid Chromatography-Electrochemical Detector system, the 3, 4-dihydrioxphenylalanine (DOPA) formation in the striatum of 6-hydroxdopamine-pretreated rats during infusion of an L-aromatic amino-acid decarboxylase inhibitor (NSD1015) was monitored.

RESULTThe absence of DOPA in dialysates of 6-hydroxdopamine-pretreated rats, the measurable DOPA and the steady decreasing of 3,4-dihydroxyphenylacetic acid(DOPAC) during infusion of NSD1015 and the disappearance of DOPA after administration of alpha-methyl-rho-tyrosine indicated that the dialyzed DOPA was derived from dopaminergic nerve terminals. After intraperitoneal administration of dopamine receptor agonist apomorphine the DOPA output was deseased. After intraperitoneal administration of dopamine recepter antagonist haloperidol, the DOPA output was increased. The study showed that twenty-four hours ofter implantation of the probe with infusion of 0.01 mmol.L-1 NSD1015, the DOPA level in the striatum of 6-hydroxdopamine-pretreated rats was 0.39 +/- 0.12 pmol/min (X +/- S, n = 5).

CONCLUSIONThe DOPA concentration in striatal dialysates could be considered as an index of tyrosion hydroxlase activity during infusion of 0.01 mM NSD1015. The method in vivo to monitor tyrosine hydroxlase activity in the brain is reliable.

Animals ; Apomorphine ; pharmacology ; Brain ; enzymology ; Dihydroxyphenylalanine ; metabolism ; Dopamine Agonists ; pharmacology ; Dopamine Antagonists ; pharmacology ; Female ; Haloperidol ; pharmacology ; Male ; Microdialysis ; Rats ; Rats, Sprague-Dawley ; Tyrosine 3-Monooxygenase ; metabolism

A series of nitrogen-containing benzoheterocyclic derivatives were synthesized and tested for their antipsychotic activities. Their structures were confirmed by 1H NMR and HR-MS. Preliminary in vitro pharmacological trials showed that most of the target compounds have high affinity with D2 and 5-HT(2A) receptors. Among the tested compounds, 20 exhibited the highest affinity and D2 partial agonist activity. In vivo studies showed 20 has potent antipsychotic activities on apomorphine mice model, which is a chance to find a better precursor of D2 partial agonist for further optimization.
Animals ; Antipsychotic Agents ; chemical synthesis ; pharmacology ; Dopamine Agonists ; chemical synthesis ; pharmacology ; Drug Design ; Isoquinolines ; chemical synthesis ; pharmacology ; Mice ; Receptors, Dopamine D2 ; Structure-Activity Relationship

OBJECTIVETo research the mechanism of dopamine (DA) controlled memory in mice.

METHODSMice received i.p. injection of scopolamine (0.3 mg/kg, SCOP 0.3, and 3.0mg/kg, SCOP 3.0, respectively, n = 10) and saline (NS, n = 10) for 60 days in experiment 1. Memory of mice was detected by dark avoidance behavior in the 53" d and the 60"' d. Animals were sacrificed after the memory test; brain tissues were processed for Fos-ir and TH-ir by immunohistochemistry. Mice were divided into four groups according results of expri-ment 1, they received i.p. injection of apomorphine (0.1 mg/kg, APO 0.1, 0.5 mg/kg, APO 0.5, and 2.0 mg/kg, APO2.0 respectively, n = 10).

RESULTSMemory was inhibited in mice injected scopolamine 3.0 mg/kg. Latency was significantly less than in NS group, only 1/ 4 that of NS group (P > 0.05). The number of mistake of SCOP 3.0 group increased about four times than that of NS group (P > 0.05). But there was no difference of latency and number of mistake between SCOP 0.3 and NS group in expriment 1. Scopolamine-induced memory deficit was associated with decreased cellular activation, indicated by Fos immunoreactive (ir) staining, in NAcc CA1 and CA3 (P < 0.05), and also associated with decreases in the number of cells labeled for tyrosine hydroxylase (TH-ir), the rate limiting enzyme for dopamine conversion (P < 0.01) and the number of cells co-labeled for TH-ir/Fos-ir (P <0.01) in the ventral tegmental area(VTA), apomorphine lessened scopolamine-induced memory deficit in experiment 2. The number of cells co-labeled for TH-ir/Fos-ir (P <, 0.05) was increased in VTA after apomorphine treatment.

CONCLUSIONApomorphine lessened scopolamine-induced memory deficit in mice by increasing DA activities in VTA.

Animals ; Apomorphine ; pharmacology ; Disease Models, Animal ; Dopamine Agonists ; pharmacology ; Male ; Memory Disorders ; chemically induced ; drug therapy ; Mice ; Scopolamine Hydrobromide ; toxicity

OBJECTIVETo investigate the role of dopamine-1 receptor in the modulation of basic respiration rhythm.

METHODSNewborn SD rat (0-3 days, n=20) brain stem slices containing the medial region of the nucleus retrofacialis (mNRF) were prepared with the hypoglossal nerve roots retained. The respiratory rhythmical discharge activity (RRDA) of the hypoglossal nerve was recorded using suction electrodes on these preparations, and the effects of dopamine-1 receptor on RRDA were investigated by application of the specific agonist of dopamine-1 receptor A68930 at different concentrations (0, 1, 2, and 5 micromol/L) in the perfusion solution.

RESULTSThe respiratory cycles (RC) and the expiratory time (TE) decreased progressively with gradual increment of the integrated amplitude (IA) after A68930 administration, and their changes were the most conspicuous at 5 min after the administration. A68930 at the concentrations of 2 and 5 micromol/L resulted in the most obvious changes in RC, TE, and IA (P<0.05), but IA exhibited no significant variation at 1 min after perfusion with 2 micromol/L A68930 (P>0.05). RC and TE were gradually shortened after treatment with increasing concentrations of A68930, which also caused gradual increment of IA, and at the concentration of 5 micromol/L, RC, TE, and IA all showed the most obvious changes (P<0.01).

CONCLUSIONSDopamine-1 receptor plays a role in the modulation of RRDA in isolated neonatal rat brainstem slice. A68930 may increase the frequency of respiration by shortening TE and enhance the respiratory activity by increasing the amplitude of inspiratory discharge of the respiratory neurons.

Animals ; Animals, Newborn ; Cell Separation ; Chromans ; pharmacology ; Dopamine Agonists ; pharmacology ; In Vitro Techniques ; Medulla Oblongata ; cytology ; physiology ; Neurons ; cytology ; Rats ; Rats, Sprague-Dawley ; Receptors, Dopamine ; physiology ; Respiration ; drug effects

D5 receptor is a subtype of dopamine D1-like receptor, and it plays a functional role in many neurological disorders. Some natural compounds from Chinese herbs, which were shown to have the property as that of receptor agonist, might provide a rich source in search of new candidates for therapeutic use. For exploring this possibility, we developed a cell-based high throughput (HTS) D5 receptor assay to screen the herb-based natural compound library established in our centre. The D5 receptor plasmid (hD5R/pcDNA3.1) and reporter gene plasmid (4 x CRE/TK/Luci/pGL3) were co-transfected into HEK293 cell line. After G418 being selected, the monoclonal cell lines bearing hD5R and the reporter gene were established and used for agonist screening. To optimize the assay condition, the effects of some factors such as cell number per well, incubation time, and the doses of SKF38393 (a potent selective partial D1-like agonist) were examined by using forskolin, a positive compound for cAMP response element. The best condition for this HTS assay included: the cell number at 5 x 10(4)/mL, the dose of forskolin at 5-20 micromol/L, the dose of SKF38393 at 100 nmol/L-100 micromol/L, and the agonist incubation time at 6 -8 h. Thereafter, water extracts from more than 200 Chinese herbs in our library were screened and three of these water extracts showed positive activity, with higher or similar activity as SKF38393. In conclusion, we have established a cell-based HTS assay for D5 receptor agonist screening, and by use of this HTS assay, 3 Chinese herbs maybe contain components exhibiting D5 receptor agonist property. This work provides an alternative vision of how to use herb medicines and a way to develop potential drugs for treatment of neurological disorders.
Cell Line ; Dopamine Agonists ; isolation & purification ; Drug Evaluation, Preclinical ; methods ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; High-Throughput Screening Assays ; methods ; Humans ; Plants, Medicinal ; chemistry ; Receptors, Dopamine D5 ; agonists

Intermittent administrations of dopaminergic agents in hemiparkinsonian rat enhances the behavioral response to subsequent administration of the drugs. This phenomenon is known as "priming" and thought as comparable to drug-induced dyskinesia in patients with Parkinson's disease. We investigated the behavioral and electrophysiological changes in 6-hydroxydopamine (6-OHDA)-lesioned hemiparkinsonian rats after repeated administrations of apomorphine. Administration of apomorphine (0.32 mg/kg, intraperitoneal, i.p.) twice daily for 6 days enhanced the rotation induced by apomorphine from 341 turns/hour at the beginning to 755 turns/hr at the end. At the same time, the response to selective D2 agonist quinpirole (0.26 mg/kg, i.p.) was also enhanced from 203 to 555 turns/hr. Extracellular single unit recording revealed no significant difference in the basal firing rates of substantia nigra pars reticulata (SNr) neurons between the ipsilateral and contralateral side of the 6-OHDA lesion regardless of the repeated administrations of apomorphine. In SNr of the lesion side, the units with burst firing pattern were found more frequently after repeated administrations of apomorphine and the suppressive effect of quinpirole on the firing rate was enhanced. These findings suggest that the increased percentage of the burst units is the important electrophysiological change in the development of enhanced response to selective D2 agonist.
Animal ; Apomorphine/*pharmacology ; Dopamine Agonists/*pharmacology ; MPTP Poisoning/physiopathology ; Male ; Oxidopamine/toxicity ; Parkinsonian Disorders/*physiopathology ; Quinpirole/pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, Dopamine D2/*drug effects ; Substantia Nigra/*drug effects/physiology

OBJECTIVETo explore the possible differential trafficking properties of the dopamine D1-like receptor subtypes, D1 receptor and D5 receptor.

METHODSTo visualize distributions of dopamine D1-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.

RESULTSIn resting conditions, D1 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 D1 receptors appeared in the cytosol, indicating that D1 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.

CONCLUSIONOur 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.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine ; pharmacology ; Acetylcholine ; pharmacology ; Animals ; Cell Line ; Dopamine Agonists ; pharmacology ; HeLa Cells ; Humans ; Luminescent Proteins ; genetics ; Mice ; Microscopy, Confocal ; methods ; Neuroblastoma ; Protein Transport ; drug effects ; Rats ; Receptors, Dopamine D1 ; metabolism ; Receptors, Dopamine D5 ; metabolism ; Subcellular Fractions ; metabolism ; ultrastructure ; Transfection ; methods

Gerbil forebrain ischemia/reperfusion(I/R) injury model was used to study the effects of D(1) and D(2) receptor agonists and antagonists on neuronal apoptosis of hippocampal CA1 area. All animals were tested for habituation deficits in an open field test on the 1st, 3rd and 7th days after reperfusion. The animals were then killed, and brains underwent paraffin embedding for hematoxylin-eosin staining, in situ terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling (TUNEL) staining and immunohistochemistry (bax, bcl-2). The result of open field test showed that the I/R group was significantly impaired (higher activity scores) when compared with the control group. Pretreatment with pergolide significantly reduced this habituation impairment. Forebrain ischemia for 5 min resulted in extensive CA1 apoptosis on the 3rd and 7th days after I/R injury. About 95% neurons in hippocampal CA1 area entered apoptosis and only 2%-7% pyramidal neurons stayed alive due to an inhibition of bcl-2 expression and an increase in bax expression. Pretreatment of pergolide attenuated neuronal damage caused by transient ischemia. Infusion of pergolide could induce the expression of bcl-2 and reduce the expression of bax. Pretreatment with SKF38393, SCH23390 and spiperone had no effects on these changes in this transient I/R injury model. All these results indicate that pergolide plays an important role in the protection of hippocampal neurons from apotosis through upregulating the expression of bcl-2 protein and reducing the expression of bax protein.
2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine ; pharmacology ; Animals ; Apoptosis ; Brain ; physiopathology ; Brain Ischemia ; physiopathology ; Dopamine Agonists ; pharmacology ; Dopamine Antagonists ; pharmacology ; Gerbillinae ; Hippocampus ; physiopathology ; Ischemic Attack, Transient ; physiopathology ; Male ; Neurons ; physiology ; Neuroprotective Agents ; pharmacology ; Pergolide ; pharmacology ; Prosencephalon ; physiopathology ; Proto-Oncogene Proteins ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Receptors, Dopamine D1 ; Receptors, Dopamine D2 ; Reperfusion Injury ; physiopathology ; bcl-2-Associated X Protein

OBJECTIVETo explore the expressions of HO-2 and CO in the corpus cavernosum of castrated rats in order to further study the pathogenesis of erectile dysfunction (ED).

METHODSWe randomly divided 72 male SD rats into four groups: normal control, sham operation, castration, and castration + ZnPP. We detected intracavernous pressure (ICP) and penile erection in the basic condition and after apomorphine (APO) induction, determined the expression of the HO-2 protein in the corpus cavernosum by laser scanning confocal microscopy, and measured the level of CO by spectrophotometry during different periods of penile erection.

RESULTSThe ICP in the basic condition and that after APO induction and the rate of penile erection were decreased significantly in the castration group ([11.68 ± 0.69] mmHg, [54.81 ± 3.86] mmHg, and 33.3%) and the castration + ZnPP group ([11.20 ± 0.71] mmHg, [41.17 ± 5.41] mmHg, and 22.2%) as compared with the normal control ([22.83 ± 2.66] mmHg, [66.92 ± 7.77] mm-Hg, and 100%) and the sham operation group ([23.35 ±2.22] mmHg, [70.43 ?7. 22] mmHg, and 100%) (all P <0. 01). After APO induction, ICP in the castration + ZnPP group was remarkably reduced in comparison with that in the castration group (P < 0.01), and so was the expression of the HO-2 protein before and during penile erection in the castration (445.4 ± 23.7 and 847.4 ± 35.0) and the castration + ZnPP group (390.1 ± 29.7 and 526.0 ± 52.5) compared with the normal control (512.7 ±57.4 and 1145.2 ± 89.8) and the sham operation group (583.7 ± 8.0 and 1016.3 ± 79.8), the expression of the HO-2 protein significantly decreased in the castration group (445.4 ± 23.7 and 847.4 ± 35.0) (P < 0.05 or 0.01), markedly lower in the castration + ZnPP than in the castration group during penile erection (P < 0.01) but with no significant differences among the four groups after it. Before, during and after penile erection, the levels of CO were remarkably decreased in the castration ([20.59 ± 1.01], [32.53 ± 1.26], and [18.71 ± 1.22] x 10(-7) nmol/L) and the castration +ZnPP group ([12.52 ± 1.05], [21.90 ± 1.02], and [16.56 ± 0.55] x 10(-7) nmol/L) as compared with the normal control ([26.76 ± 1.41], [48.25 ± 1.01], and [27.10 ± 1.58 ] x 10(-7) nmol/L) and the sham operation group ([25.41 ± 2.09], [ 47.90 ± 1.22], and [25.67 ± 1.20] x 10(-7) nmol/L) (P < 0.05 or 0.01), significantly lower in the castration + ZnPP than in the castration group during penile erection (P < 0.01).

CONCLUSIONDecreased expressions of HO-2 and CO may correlate with erectile dysfunction in castrated rats.

Animals ; Apomorphine ; pharmacology ; Carbon Monoxide ; metabolism ; Dopamine Agonists ; pharmacology ; Erectile Dysfunction ; etiology ; Humans ; Male ; Molecular Chaperones ; metabolism ; Orchiectomy ; Penile Erection ; drug effects ; Penis ; drug effects ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley

The present study was to investigate the role of dopamine D1 receptors and its relationship with glutamate, N-methyl-D-aspartic acid (NMDA) receptor and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor in depression induced by chronic unpredictable mild stress (CUMS). CUMS-induced depression model was established in Sprague-Dawley rats, and intrahippocampal microinjections of D1 dopamine receptor agonist SKF38393, non-competitive NMDA receptor antagonist MK-801 and AMPA receptor antagonist NBQX were respectively adopted by rat brain stereotaxic coordinates. The behavioral observations were conducted by measurement of weight changes, sucrose preference test, open-field test and tail suspension test. The concentration of glutamic acid and the expression of its receptors' subunits were detected by HPLC and Western blot, respectively. The results showed that, compared with control group, CUMS rats showed depression-like behavioral changes, higher concentration of glutamic acid, lower expressions of NMDA receptor (NR1) and AMPA receptor (GluR2/3) in hippocampus. Pretreatment with injection of SKF38393 could rescue such depression effect of CUMS, decrease the concentration of glutamic acid, and increase the expressions of NMDA receptor (NR1), AMPA receptor (GluR2/3) in hippocampus. Pretreatment with MK-801 could enhance the antidepressant effect of SKF38393, while NBQX weakened. These results suggest that agonists of D1 dopamine receptor could reduce the concentration of glutamic acid in hippocampus, and its antidepressant effect may be mediated by AMPA receptor partially.
2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine ; pharmacology ; Animals ; Depression ; etiology ; physiopathology ; Dizocilpine Maleate ; pharmacology ; Excitatory Amino Acid Antagonists ; Glutamates ; metabolism ; Hippocampus ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, AMPA ; metabolism ; Receptors, Dopamine D1 ; agonists ; physiology ; Stress, Physiological ; physiology