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1.
Abstract: Coated vesicles (CVs) isolated from bovine striatal tissue were examined to determine whether they are associated with dopamine signal systems consisting of dopamine D1 and D2 receptors, G proteins, and adenylate cyclase. Dopamine receptors in CVs were characterized by a dopamine D1 receptor antagonist, [3H]SCH 23390, and a dopamine D2 receptor antagonist, [3H]-spiroperidol. The bindings of both ligands were specifically saturable and reversible with a dissociation constant ( K D) of 0.65 and 0.5 n M , respectively. Dopaminergic antagonists and agonists inhibited the specific bindings of [3H]SCH 23390 and [3H]spiroperidol in a stereoselective and concentration-dependent manner with an appropriate rank order potency for dopamine D1 or D2 receptors. The regulations of the agonist binding by guanyl-5-ylimidodiphosphate were observed. ADP ribosylation of the CVs with [32P]NAD demonstrated predominant labeling of bands of Mr 47,000–52,000, 42,000–45,000, and 40,000-39,000, which corresponded to the known molecular weights of the α subunits of Gs and Gi proteins. The presence of α and β subunits of G proteins in the CVs was also confirmed by immunoblotting assay. Adenylate cyclase activity, which was stimulated by SKF 38393 and inhibited by dopamine D2 receptor agonists, was present in the CVs. These findings suggest that the dopamine D1 and D2 receptors in the CVs couple with adenylate cyclase via Gs/Gi protein.  相似文献   

2.
Abstract: D1-and D2-dopamine receptors exert important physiological actions on striatal neurons, but the intracellular second messenger pathways activated by these receptors are still incompletely understood. Using primary cultures of rat striatal cells, we have examined the effects of activating D1 or D2 receptors on arachidonic acid (AA) release and cyclic AMP accumulation. In striatal neurons labeled by incubation with [3H]AA, D2-receptor stimulation enhanced release of [3H]AA produced by application of the Ca2+ ionophore A23187 or of the purinergic agonist ATP. By contrast, D1-receptor stimulation inhibited [3H]AA release. This inhibitory effect of D1 receptors was accompanied by stimulation of adenylyl cyclase activity, measured as accumulation of cyclic AMP, and was mimicked by application of the adenylyl cyclase activator forskolin. The results indicate the existence of a novel signaling pathway for D2 and D1 receptors in striatum, potentiation and inhibition, respectively, of Ca2+-evoked AA release.  相似文献   

3.
The existence of pre-synaptic auto- and hetero receptors which modulate neurotransmitter release is well documented. Emerging evidence show that in some cases these pre-synaptic receptors may also cross-talk with each other. The aim of the present work was to investigate whether acetylcholine receptors (nAChRs) and dopamine (DA) autoreceptors, which are both able to modulate DA release, functionally interact on the same nerve endings. We used rat and mouse nucleus accumbens synaptosomes pre-labeled with [3H]DA and exposed to nicotinic and dopaminergic receptor ligands. Both nicotinic agonists and 4-aminopyridine (4-AP) provoked [3H]DA release which was inhibited by quinpirole and blocked by sulpiride and raclopride. Both the inhibitory effect of quinpirole and the stimulatory effect of (−)nicotine did not change when the nAChRs or the DA receptors were desensitized. (−)Nicotine and 4-AP were able to stimulate [3H]DA overflow also in mouse synaptosomes and this overflow was partially inhibited by quinpirole. In the β2 knockout mice quinpirole was still able to inhibit the [3H]DA overflow elicited by 4-AP. To conclude: in rat and mouse the (−)nicotine evoked-release can be modulated by D2/D3 autoreceptors present on the DA terminals and nAChRs function is independent from D2/D3 autoreceptors which themselves may function independently from the activation of nAChRs.  相似文献   

4.
Abstract: The role of dopaminergic innervation on the postnatal developmental expression of D1 dopamine receptors was investigated. Bilateral destruction of dopa-mine-containing neurons was achieved by treating rats intracisternally with 6-hydroxydopamine (6-OHDA) on postnatal day 3, and rats were killed on day 21. To ensure effective reduction of D1 receptor activation by residual dopamine, a group of 6-OHDA-lesioned rats was given twice daily injections of the D1 receptor antagonist SCH-23390, from day 4 to 20. D1 dopamine receptor binding was assessed in the caudate—putamen, nucleus accumbens, and olfactory tubercle by quantitative autoradiographic analysis of [3H]SCH-23390 binding. In addition, the relative amount of D1A receptor mRNA was assessed by in situ hybridization of a 35S-labeled riboprobe. In the developing rats, neither the amount of [3H]SCH-23390 binding nor the amount of D1A receptor mRNA was altered by 6-OHDA lesioning followed by chronic treatment with SCH-23390. Thus, bilateral destruction of dopamine-containing neurons and treatment with SCH-23390 in neonatal rats did not interfere with the developmental expression of D1 receptors or alter the levels of mRNA that code for this receptor protein. Treatment of intact rats with SCH-23390 from postnatal day 4 to 20 also did not alter [3H]SCH-23390 binding or levels of D1 receptor mRNA. However, adult rats treated chronically with SCH-23390 exhibited increased [3H]SCH-23390 binding but did not show a significant change in D1 receptor mRNA levels.  相似文献   

5.
1. Production of heterotrophic bacterioplankton was estimated monthly by the tritiated thymidine and leucine incorporation methods during the draining and filling of the mesotrophic Lake Pareloup (over a 2.5-years sampling program).
2. Rates of 3H-leucine (leu) and 3H-thymidine (thy DNA) incorporation generally paralleled each other but the ratio of leu/thy DNA incorporation rates was higher for the draining period (34.5 mean) than during and after filling (11.5 mean).
3. After draining, the highest ratios were observed during periods of low temperature and low bacterial specific activity, while DNA labeling by 3H-thymidine was reduced. However, bacterial production estimates obtained by 3H-leucine (BPL) and 3H-thymidine (BPT) incorporation methods were generally well correlated and the average BPL/BPT ratio was equal to 0.78.
4. In addition, both methods were applied during a diel cycle in three lakes of different trophic status. An increase of leu/thy DNA incorporation rates was noted from the oligotrophic to the eutrophic system. In the absence of Cyanobacteria, BPL and BPT values were quite concordant on average.
5. In situations of unbalanced growth, BPL and BPT values can diverge but when considered over a sufficient period of time they were found to be in agreement.  相似文献   

6.
Abstract: [3H]Nemonapride and [3H]spiperone are very widely used to study dopaminergic systems in vitro and in vivo, but it has been reported that [3H]nemonapride and [3H]spiperone give markedly different B max values for preparations of D2 dopamine receptors from recombinant cell lines or animal tissues. We have used the two radioligands in parallel to study a range of dopamine receptors [D2(short), D2(long), and D3] in different buffers. B max values derived using either radioligand differ by an average of <20%, independent of receptor type or buffer conditions. All competition experiments show that the two ligands compete at a single site. It seems that [3H]spiperone and [3H]nemonapride do not differentiate between different forms or populations of D2-like receptors.  相似文献   

7.
Abstract: Cations of various size and charge were used as atomic scale probes of D1 and D2 dopamine receptors. Those cations that perturbed the binding of D1- and D2-selective dopamine receptor antagonists were identified by screening at 5 m M cation. Pseudo-noble-gas-configuration d-transition metals, such as zinc, exerted a complete inhibition of specific binding, whereas most other cations had little or no effect. The nature of zinc's actions was characterized by measuring the radioligand binding properties of [3H]SCH-23390 and [3H]methylspiperone to cloned D1A and D2L dopamine receptors in either the presence or absence of Zn2+. Zinc exerts a low-affinity, dose-dependent, EDTA-reversible inhibition of the binding of subtype-specific antagonists primarily by decreasing the ligands' affinity for their receptors. The mechanism of zinc inhibition appears to be allosteric modulation of the dopamine receptor proteins because zinc increases the dissociation constant ( K D) of ligand binding, Schild-type plots of zinc inhibition reach a plateau, and zinc accelerates antagonist dissociation rates. Here we demonstrate the effect of zinc on the binding of D1- and D2-selective antagonists to cloned dopamine receptors and show that the inhibition by zinc is through a dose-dependent, reversible, allosteric, two-state modulation of dopamine receptors.  相似文献   

8.
Wang Y  He Q  Qin H  Xu J  Tong J  Gao L  Xu J 《Life sciences》2006,79(2):182-192
Thy-1 nephritis (Thy-1 N), namely, anti-Thy-1 or anti-thymocyte serum (ATS) induced nephritis (ATSN), is a typical model of human mesangioproliferative glomerulonephritis. The pathologic changes of glomerular mesangial cells (GMCs) in Thy-1 N are complement-dependent, especially C5b-9 complexes, but the role of C5b-9 in the mechanism of Thy-1 N has not been defined. Because previous studies have demonstrated that sublytic C5b-9 can increase production of several inflammatory mediators from resident glomerular cells, we utilized the isolated human membrane-bound C5b-9 complexes to stimulate the cultured rat GMCs and examined whether the GMCs can also induce the synthesis of nitric oxide (NO) in vitro. Simultaneously, the effects of antiserum against rat C5b-9 and NG-monomethyl-L-arginine (L-NMMA, NO inhibitor), including interfering with the formation of C5b-9, reducing NO production and GMCs injury were observed. The results showed that sublytic C5b-9 can increase synthesis of inducible NO from the stimulated GMCs, and that the anti-C5b-9 antiserum can obviously inhibit the pathologic changes in Thy-1 N, while L-NMMA can decrease the GMCs damage although the effect is not so significant as that of the anti-C5b-9 antiserum. These findings indicate that the synthesis of NO by GMCs can be promoted by sublytic C5b-9, and that lesions of GMCs in rats with Thy-1 N are prevented by either inhibiting C5b-9 formation or NO elevation in advance. The pathologic changes of GMCs in Thy-1 N are indeed complement C5b-9-dependent, and the glomerular injury can be mediated in part through elevation of NO from the GMCs after the sublytic C5b-9 stimulation.  相似文献   

9.
SUMMARY. 1. The literature contains a number of curves relating time (days) required for median hatch (=D2) to water temperature (T,°C) for the eggs of several salmonid fishes. There are relatively few data on the relationships between time to median eyed (= D1), days) or time to median swim-up (= D3, days) and temperature.
2. From published data, over most of the range 0-9.5°C, approximate relationships are D,=0.5D2 for Atlantic salmon (Salmo salar L.) and D3 = 1.7D2 for eight species of Salmo and Oncorhynchus.
3. Field and hatchery tests suggest that these are useful empirical models for approximate prediction of D1 and D3 from D2 for most salmonids.  相似文献   

10.
Abstract: Primary cultures of rat ventral mesencephalon were used to elucidate the role of chronic stimulation of dopamine (DA) D2 autoreceptors in the development of fetal dopaminergic neurons in vitro. Cultured dopaminergic neurons, as visualized by tyrosine hydroxylase immunocytochemistry, became more differentiated in the course of cultivation time and exhibited specific high-affinity uptake for [3H]DA. In rat striatal tissue, activation of D2 receptors has been shown to inhibit the release of DA. Previously accumulated [3H]DA was released from the cultures upon depolarization in a Ca2+-dependent manner. K+-evoked [3H]DA release could be inhibited by the selective D2 receptor agonists LY 171555 and N0437 in a concentration-dependent manner. The inhibitory effects of LY 171555 and N0437 were antagonized by the selective DA D2 receptor antagonist sulpiride. These observations are indicative for the expression of functional D2 receptors in the cultures. Daily treatment of these cultures for 7 days with LY 171555 or sulpiride did not lead to any change in protein content, the number of tyrosine hydroxylase-immunoreactive neurons, or the uptake capacity for [3H]DA. Our data demonstrate that chronic stimulation of DA D2 receptors does not impair survival or differentiation of cultured fetal dopaminergic neurons.  相似文献   

11.
Abstract: The ability of human and rat D2(short) and D2(long) dopamine receptors to activate microtubule-associated protein (MAP) kinase (Erk1/2) and p70 S6 kinase has been investigated in recombinant cells expressing these receptors. In cells expressing the D2(short) receptor, dopamine activated both enzymes in a transient manner but with very different time courses, with activation of Erk being much quicker. Activation of both enzymes by dopamine was dose-dependent and could be prevented by a range of selective dopamine antagonists. Excellent correlations were observed between the potencies of the antagonists for blocking enzyme activation and their affinities for the D2 dopamine receptor. Activation of Erk and of p70 S6 kinase via the D2 dopamine receptors was prevented by pretreatment of the cells with pertussis toxin, indicating the involvement of G proteins of the Gi or Go family. Inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase) were found to block substantially, but not completely, activation of p70 S6 kinase by dopamine, suggesting the involvement of PI 3-kinase-dependent and -independent signalling pathways in its control by dopamine. p70 S6 kinase activation was completely blocked by rapamycin. In the case of Erk, activation was partially blocked by wortmannin or LY294002, indicating a possible link with PI 3-kinase.  相似文献   

12.
Abstract: To expand on the nature of regional cerebral vulnerability to ischemia, the release of dopamine (DA) and dopaminergic (D1 and D2) receptors were investigated in Mongolian gerbils subjected to bilateral carotid artery occlusion (15 min) alone or with reflow (1–2 h). Extracellular cortical and striatal content of DA and its metabolites was measured by microdialysis using HPLC with electrochemical detection. The kinetic properties of D1 and/or D2 receptor binding sites were determined in cortical and striatal membranes with the use of radiolabeled ligands (125I-SCH23982 and [3H]YM-09151-2, respectively). The ischemic release of DA from the striatum was greater (400-fold over preischemic level) than that from the cortex (12-fold over preischemic content). The affinity for the D1-receptor ligand was lower ( K D= 1.248 ± 0.047 n M ) after ischemia than that for sham controls ( K D= 0.928 ± 0.032 n M, p < 0.001). The number of binding sites for D2 receptors decreased in striatum ( B max= 428 ± 18.4 fmol/mg of protein) after ischemia compared with sham controls ( B max= 510 ± 25.2 fmol/mg of protein, p < 0.05). D1 or D2 binding sites were not changed either in the ischemic cortex or postischemic striatum and cortex. The findings strongly suggest that the ischemic release of DA from striatum is associated with early transient changes in D1- and D2-mediated DA neurotransmission.  相似文献   

13.
Abstract: We found that the binding of [3H]prazosin, a selective ligand for α1-adrenergic recognition sites, is significantly lower in the frontal cortex of the genetically epilepsy-prone rats (GEPRs), as compared with normal Sprague-Dawley rats. Scatchard analysis reveals a decrease in the B max of [3H]prazosin binding with no change in the apparent K D, suggesting that there are fewer α1-adrenergic recognition sites in the frontal cortex of the GEPR. This abnormality is associated with a reduced capacity of norepinephrine (NE) to stimulate [3H]inositol monophosphate ([3H]IP1) formation in frontal cortex slices prelabeled with [3H]inositol. No significant differences in [3H]prazosin binding as well as NE-stimulated [3H]IP1 formation have been observed in other brain regions including hippocampus, corpus striatum, and inferior colliculus. These results indicate that a deficit in the α1-adrenergic receptor system in the frontal cortex may play a role in the seizure process in the GEPR.  相似文献   

14.
Vitamin D3 at low concentration (10−9 M) inhibited the growth of Phaseolus vulgaris L. (cv. Contrancha) roots in vitro as measured by elongation (14 h) and [3H]-leucine incorporation into protein (2 h), and increased their labelling with 45Ca2+ (2 h). Cycloheximide and puromycin (50 u.M) blocked vitamin D3 stimulation of root 45Ca2+ labelling, indicating that it is mediated by de novo protein synthesis. The calcium ionophore X-537A (10−5JW) induced similar changes both in root elongation and 45Ca2+ uptake (14 h). This may indicate that the inhibitory effects of the sterol on root growth are mediated by changes in Ca2+ fluxes. However, this interpretation should be further strengthened by additional studies as the ionophore may have acted on root growth, affecting physiological processes other than Ca2+ transport.  相似文献   

15.
Aims:  Sheep are important carriers of Shiga toxin-producing Escherichia coli (STEC) in several countries. However, there are a few reports about ovine STEC in American continent.
Methods and Results:  About 86 E. coli strains previously isolated from 172 healthy sheep from different farms were studied. PCR was used for detection of stx 1, stx 2, eae, ehxA and saa genes and for the identification of intimin subtypes. Restriction fragment length polymorphism (RFLP)–PCR was performed to investigate the variants of stx 1 and stx 2, and the flagellar antigen ( fli C) genes in nonmotile isolates. Five isolates were eae + and stx , and belonged to serotypes O128:H2/β-intimin (2), O145:H2/γ, O153:H7/β and O178:H7/ε. Eighty-one STEC isolates were recovered, and the stx genotypes identified were stx 1c stx 2d-O118 (46·9%), stx 1c (27·2%), stx 2d-O118 (23·4%), and stx 1c stx 2dOX3a (2·5%). Pulsed-field gel electrophoresis (PFGE) revealed 27 profiles among 53 STEC and atypical enteropathogenic Escherichia coli (EPEC) isolates.
Conclusions:  This study demonstrated that healthy sheep in São Paulo, Brazil, can be carriers of potential human pathogenic STEC and atypical EPEC.
Significance and Impact of the Study:  As some of the STEC serotypes presently found have been involved with haemolytic uraemic syndrome (HUS) in other countries, the important role of sheep as sources of STEC infection in our settings should not be disregarded.  相似文献   

16.
Abstract: The human D4 dopamine receptor has been expressed in Sf9 insect cells where it appears to couple to endogenous G proteins. Increased guanine nucleotide exchange to G proteins is a reflection of receptor activation and can be followed using a [35S]GTPγS binding assay. By measuring D4 receptor stimulation of [35S]-GTPγS binding we have been able to characterize several dopaminergic compounds for their functional activity at this receptor. In Sf9 cells expressing the D4 receptor, dopamine, quinpirole, and dp -2-aminodihydroxy-1,2,3,4-tetrahydronaphthalene were all full agonists, whereas (−)-apomorphine appeared to be a partial agonist. No increase in [35S]GTPγS binding was observed for noninfected cells or cells infected with an unrelated sequence. The quinpirole-stimulated [35S]GTPγS binding could be inhibited by the antagonists clozapine, eticlopride, and haloperidol, and a Schild analysis of these data showed that all three compounds were acting as competitive antagonists of D4 receptors. The rank order of affinities derived from the Schild analysis correlated with that obtained from [3H]spiperone competition binding assays. In conclusion, we have shown that, using this assay system, it is possible to investigate functionally the pharmacology of a recombinant G protein-coupled receptor in the absence of any information regarding the eventual second messenger pathways involved.  相似文献   

17.
18.
Cloning and Characterization of a Mouse σ1 Receptor   总被引:1,自引:1,他引:0  
Abstract: A cDNA clone (S2-1a) isolated from a mouse brain cDNA library, using a guinea pig σ1 cDNA as probe, has high homology to the predicted protein sequence of the guinea pig (88%) and human (90%) σ1 receptors. Northern analysis revealed a major mRNA of ∼1.8 kb in a wide range of mouse tissues, with highest levels in brain, liver, kidney, and thymus. Southern analysis and chromosomal mapping in the mouse suggested a single-copy gene in region A5-B2 of chromosome 4. Expression of the clone in MCF-7 and CHO cells led to a pronounced increase in (+)-[3H]pentazocine binding with a selectivity profile consistent with σ1 receptors. In vitro translation yielded a protein of ∼28 kDa, as did transfection of a probe containing the hemagglutinin (HA) epitope (S2-1a.HA) into CHO cells, as determined by western analysis using an antibody directed against HA. (+)-[3H]-Pentazocine binding to immunopurified HA-tagged receptor demonstrated conclusively that S2-1a.HA encodes a high-affinity (+)-[3H]pentazocine binding site with characteristics of a murine σ1 receptor. An antisense oligodeoxynucleotide designed from S2-1a potentiated opioid analgesia in vivo.  相似文献   

19.
Abstract: K+-evoked acetyl[3H]choline ([3H]ACh) release was inhibited in a concentration-dependent manner by apomorphine and the D2 agonist quinpirole in striatal slices prepared from euthyroid and hypothyroid rats. However, there was a significant increase in the maximum inhibition observed with both agonists in the hypothyroid compared with the euthyroid group, which paralleled the increased D2 agonist sensitivity reported for stereotyped behavior. The D2 antagonist raclopride decreased, and the D, antagonist SCH 23390 increased, the inhibition of [3H]ACh release by apomorphine, confirming an inhibitory role for D2 receptors and an opposing role for D1 receptors. Because there is no difference in D1 or D2 receptor concentration between the euthyroid and hypothyroid groups, it is suggested that thyroid hormone modulation of D2 receptor sensitivity affects a receptor-mediated event. Following intrastriatal injection of pertussis toxin (PTX), apomorphine no longer inhibited [3H]ACh release. In fact, increased [3H]- ACh release was observed, an effect reduced by SCH 23390, providing evidence that D1 receptors enhance [3H]- ACh release, and confirming that a PTX-sensitive G protein mediates the D2 response. As it has been reported that thyroid hormones modulate G protein expression, this mechanism may underlie their effect on dopamine agonist- mediated inhibition of ACh.  相似文献   

20.
Abstract: The dopamine (DA) D3 receptor antagonist PD 58491 {3-[4-[1-[4-[2-[4-(3-diethylaminopropoxy)phenyl]-benzoimidazol-1-yl-butyl]-1 H -benzoimidazol-2-yl]-phenoxy]propyl]diethylamine} bound with high affinity and selectivity to recombinant human DA D3 versus D2L and D4.2 receptors transfected into Chinese hamster ovary cells: K i values of 19.5 n M versus 2,362 and >3,000 n M , respectively. In contrast, the putative DA D3 receptor antagonist (+)-AJ76 displayed low affinity and selectivity for D3 versus D2L and D4.2 receptors (91 n M vs. 253 and 193 n M , respectively). In vitro, PD 58491 (1 n M −1µ M ) exhibited D3 receptor antagonist activity, reversing the quinpirole (10 n M )-induced stimulation of [3H]thymidine uptake in D3 CHOpro-5 cells, but did not have any significant intrinsic activity by itself in this assay. PD 58491 did not decrease the γ-butyrolactone-induced increase in DA synthesis ( l -3,4-dihydroxyphenylalanine accumulation) in rat striatum, indicating that the compound possessed no in vivo DA D2/D3 receptor agonist action at DA autoreceptors. PD 58491 (3–30 mg/kg, i.p.) generally did not alter DA or serotonin synthesis in either the striatum or mesolimbic region of rat brain. The D3-preferring agonist PD 128907 decreased DA synthesis in striatum and mesolimbic regions, and this effect was attenuated by pretreatment with PD 58491. These findings support the hypothesis that DA D3 autoreceptors may in part modulate the synthesis and release of DA in striatum and mesolimbic regions.  相似文献   

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