Dynamic changes in NADPH-diaphorase staining reflect activity of nitric oxide synthase: Evidence for a dopaminergic regulation of striatal nitric oxide release

In fixed tissue, neuronal NADPH-diaphorase staining results from nitric oxide synthase (NOS) activity. Neuronal NOS only synthesizes nitric oxide once activated by the binding of Ca²⁺/calmodulin. We show here that neuronal NADPH-diaphorase staining is also dependent on Ca²⁺/calmodulin, implying that only activated NOS is detected. In addition, in bovine pulmonary endothelial cells, carbachol and bradykinin dramatically and rapidly increase the intensity of NADPH-diaphorase staining. Furthermore, administration of MK801, an NMDA antagonist, decreases neuronal NADPH-diaphorase staining. This suggests that the intensity of the NADPH-diaphorase staining is related to the level of enzyme activation at the moment of tissue fixation. The potential of exploiting this observation to detect cellular activation of NOS is illustrated by the observations that the intensity of NADPH-diaphorase staining in rat striatal neurones is decreased following systemic treatment with the D1-like dopamine receptor antagonist SCH23390, and increased by the D2-like antagonist eticlopride. These results therefore provide strong evidence that the NADPH-diaphorase reaction can be used to monitor NOS activity at a cellular level of resolution, and reveal a dopaminergic regulation of NOS activity in the striatum mediated by D1-like and D2-like dopamine receptors.     

Characterisation of dopamine receptors in insect (Apis mellifera) brain

In vitro binding experiments using the vertebrate D₁ dopamine receptor ligand [³H]SCH23390 and the vertebrate D₂ dopamine receptor ligand [³H]spiperone were conducted on membrane preparations of honey bee (Apis mellifera) brain. Specific binding of [³H]SCH23390 was saturable and reversible. Analysis of saturation data gave an apparent K_d of 6.3 ± 1.0 nM and B_max of 1.9 ± 0.2 pmol/mg protein for a single class of binding sites. The specificity of high affinity [³H]SCH23390 binding was confirmed in displacement experiments using a range of dopaminergic antagonists and agonists. The rank order of potency for antagonists was: R(+)-SCH23390 > cis-(Z)-flupentixol ≥ chlorpromazine > fluphenazine> S(+)-butaclamol > spiperone. R(±)-SKF38393 and dopamine were the most effective agonists tested. [³H]SCH23390 labels a site in bee brain that is similar, but not identical to the vertebrate D₁ dopamine receptor subtype. [³H]Spiperone also bound with high affinity to bee brain homogenates. Scatchard analysis of [³H]spiperone saturation data revealed a curvilinear plot suggesting binding site heterogeneity. The high affinity site had a apparent K_d of 0.11 ± 0.02 nM and B_max of 9.2 ± 0.5 fmol/mg protein. The calculated values for the low affinity site were a K_d of 19.9 nM and B_max of 862 fmol/mg protein. Kinetic analyses also indicated that [³H]spiperone recognises a heterogeneous population of sites in bee brain. Furthermore, agonist competition studies revealed a phenolaminergic as well as a dopaminergic component to [³H]spiperone binding in bee brain. The rank order of potency of dopaminergic antagonists in competing for [³H]spiperone binding was: spiperone > fluphenazine> S(+)-butaclamol > domperidone> R(+)-SCH23390 > S(−)-sulpiride.     

Dopamine receptor blockade and synthesis inhibition during exaggerated natriuresis in spontaneously hypertensive rats

Hansell , P. & Sjöquist , M. 1992. Dopamine receptor blockade and synthesis inhibition during exaggerated natriuresis in spontaneously hypertensive rats. Acta Physiol Scand 144 , 269–276. Received 21 September 1990, accepted 11 October 1991. ISSN 0001–6772. Department of Physiology and Medical Biophysics, Biomedical Centre, University of Uppsala, Sweden. The influence of dopamine receptor blockade and synthesis inhibition on natriuresis induced by isotonic saline volume expansion was investigated in anaesthetized spontaneously hypertensive rats and normotensive Wistar-Kyoto rats. The aim of the study was to elucidate the mechanisms underlying the phenomenon of exaggerated natriuresis during volume expansion that has been observed in spontaneously hypertensive rats. Volume expansion, at 5 % of body weight, resulted in a larger and faster natriuretic response in spontaneously hypertensive rats than in Wistar-Kyoto rats. Sixty minutes after commencement of volume expansion the natriuretic response (accumulated sodium excretion) in Wistar-Kyoto rats (n = 8) was only 24% of that in spontaneously hypertensive rats (n = 17). When spontaneously hypertensive rats were pretreated with the dopamine receptor blockers haloperidol (n= 14, 1 mg kg^-1), SCH23390 (n = 8, 30 μg h^-1 kg^-1) or the dopamine synthesis inhibitor benserazide (n = 8, 50 mg kg^-1; n = 5, 100 mg kg^-1), the natriuretic response to volume expansion was only 16, 35, 59 and 42%, respectively, of that in untreated SHR. The corresponding proportion in the haloperidol-treated (n= 8) compared with untreated Wistar-Kyoto rats was 22%. In conclusion, isotonic volume loading results in more pronounced natriuresis in spontaneously hypertensive than in Wistar-Kyoto rats. Dopamine receptor blockade and synthesis inhibition attenuate the expansion of exaggerated natriuresis in spontaneously hypertensive rats and reduces the volume expansion natriuresis in Wistar-Kyoto rats, indicating that the dopamine system plays an important role.     

Power spectral analysis of electroencephalographic desynchronization induced by cocaine in rats: Correlation with microdialysis evaluation of dopaminergic neurotransmission at the medial prefrontal cortex

We evaluated the effect and action mechanisms of cocaine on electroencephalographic (EEG) activity in adult male Sprague-Dawley rats anesthetized with chloral hydrate (400 mg/kg, i.p., with 80 mg/kg/h supplements). On-line and real-time power spectral analysis of the EEG activity continuously quantified its root mean square (RMS) and mean power frequency (MPF) values, and the power of its spectral frequency components (low frequency: 0–4 Hz; high frequency: 4–20 Hz). Administration of cocaine (1.5 or 3.0 mg/kg, i.v.) dose-dependently induced EEG desynchronization, as manifested by a reduction in RMS and an elevation in MPF values, coupled with a differential decrease in both high and low frequency components. Samples collected by in vivo microdialysis at the medial prefrontal cortex (mPFC) and analyzed by HPLC showed that the elevation of cocaine and dopamine (DA) level in the dialysate reached its peak during the time interval when maximal activation of EEG occurred. This EEG activation was antagonized by microinfusion into the mPFC via reverse microdialysis of R(+)-SCH 23390, a selective antagonist for D-1 receptors; sulpiride, a selective antagonist for D-2 receptors; or haloperidol, a nonspecific dopamine antagonist. These results suggest that dopaminergic neurotran:3mission at the mPFC may be intimately related to the specific spectral pattern of alteration in EEG activity elicited by cocaine in the rat and that both D-1 and D-2 receptors may be involved in the process. © 1994 Wiley-Liss, Inc.     

D1 receptor activation enhances sciatic nerve stimulation-induced inhibition of nigrostriatal dopamine neurons

Nigrostriatal dopamine (NSDA) neurons have been hypothesized to play an important regulatory role in neostriatal sensorimotor integration. In order to provide further information on the nature of sensory modulation of NSDA cells, we have examined the pharmacology of the responsiveness of these neurons to peripheral nerve stimulation. The selective D1 dopamine receptor agonist SKF 38393 enhanced the normal inhibition of NSDA neurons produced by electrical stimulation of the sciatic nerve. The SKF 38393-induced enhancement, but not the basal stimulation-induced inhibition itself, was blocked by prior hemitransection of the forebrain and was reversed by the selective D1 antagonist SCH 23390 but not by the selective D2 antagonist 1-sulpiride. SCH 23390 alone, however, exerted no effect on this inhibition. The selective D1 receptor agonist fenoldopam, which does not cross the blood-brain barrier, also failed to alter the response to sciatic nerve stimulation (i.v. administration). Thus, central D1 receptors (rostral to the midbrain) appear to be involved in a system which mediates phasic control over sensory modulation of NSDA neuronal activity.     

多巴胺对小鼠神经母细胞瘤和大鼠神经胶质瘤的杂交(NG108)细胞在体外的毒性(英文)

目的:研究多巴胺(DA)对小鼠神经母细胞瘤和大鼠神经胶质瘤的杂交细胞NG108的毒性.方法:用MTT法测定NG108细胞的活性.结果:当DA浓度在100 μmol L~(-1)时对NG108细胞有毒性作用.这时的细胞活性仅为对照的1/4左右.DA受体拮抗剂舒必利和Sch-23390不能阻断DA毒性,表明DA对NG108细胞的毒性作用不是由DA受体介导的.DA(125 μmol L~(-1))的毒性作用能被过氧化氢酶(50 kU L~(-1))、超氧化物歧化酶(50kU L~(-1))和维生素C(200 μmol L~(-1))抑制.它们分别能使NG108细胞的活性由DA单独作用时的25.9±11.0％上升到74.8±4.4％、72.3±4.5％和71.4±2.3％.结论:DA对NG108细胞的毒性作用是由DA氧化代谢产生的有毒产物如过氧化氢引起的.     

Cooperative activation of D1-like and D2-like dopamine receptors in the nucleus accumbens shell is required for the reinstatement of cocaine-seeking behavior in the rat

Activation of D1-like (D1, D5) or D2-like (D1, D3, D4) dopamine receptors in the nucleus accumbens shell is sufficient to reinstate cocaine-seeking behavior in rats. The goal of these experiments was to assess whether cooperative activation of D1-like and D2-like dopamine receptors in the accumbens shell is required to promote cocaine reinstatement. Rats were initially trained to self-administer cocaine (0.25 mg, i.v.) using a fixed-ratio schedule of reinforcement for approximately 21 days. Animals subsequently underwent an extinction phase during which saline was substituted for cocaine. Once cocaine self-administration behavior was extinguished (defined as <15% of the total responses maintained during self-administration), dopamine receptor agonist-induced reinstatement of cocaine seeking was assessed. Administration of the selective D1/5 agonist R-(+)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF-81297) (1.0 microg) or the D2/3 receptor agonist trans-(-)-(4aR)-4,4a,5,6,7,8,8a,9-octahydro-5-propyl-1H-pyrazolo[3,4-g]quinoline hydrochloride (quinpirole) (3.0 microg) directly into the nucleus accumbens shell promoted reinstatement of cocaine seeking. In order to determine if endogenous dopamine tone in the accumbens shell is required for dopamine receptor agonist-induced reinstatement of cocaine seeking, D1/5 or D2/3 dopamine receptor antagonists were administered into the nucleus accumbens shell prior to a selective dopamine receptor agonist. Microinfusion of the D2/3 dopamine receptor antagonist sulpiride ((S)-5-aminosulfonyl-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-methoxybenzamide) (1.0 microg) into the nucleus accumbens shell 10 minutes prior to SKF-81297 (1.0 microg) blocked the ability of this D1-like dopamine receptor agonist to reinstate cocaine seeking. Similarly, administration of the selective D1/5 dopamine receptor antagonist R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH-23390) (1.0 microg) into the nucleus accumbens shell prior to quinpirole (3.0 microg) blocked reinstatement of drug-seeking behavior elicited by this D2/3 dopamine receptor agonist. Moreover, intra-accumbal shell co-administration of subthreshold doses of quinpirole (1.5 microg) and SKF-81297 (0.1 microg) promoted cocaine-seeking behavior. Collectively, these results indicate that cooperative activation of D1-like and D2-like dopamine receptors in the nucleus accumbens shell is necessary to reinstate cocaine seeking in rats.     

Prefrontal cortex D1 modulation of the reinforcing properties of cocaine

The involvement of the dopaminergic pathway from the ventral tegmental area (VTA) to the nucleus accumbens (NAcc) in the reinforcing properties of many drugs of abuse is well established. Though the prefrontal cortex (PFC) exhibits significant influence over activity in this pathway, its role in drug abuse is less defined. The present experiment investigated the impact of PFC D1 activity on cocaine self-administration (0.25, 0.75 mg/kg/inj) under progressive (PR) and fixed ratio (FR) schedules of reinforcement by assessing immediate and delayed effects of bilateral intra-PFC infusions of a D1 agonist (SKF 38393; 0.23 microg/side) and antagonist (SCH 23390; 0.25 microg/side). Immediately following infusion of dopaminergic agents or vehicle, no significant changes in self-administration occurred under any tested condition. However, 24 h after intra-PFC antagonist treatment, significantly lower PR breakpoints were observed for low (0.25 mg/kg), but not moderate (0.75 mg/kg) unit doses of self-administered cocaine. Locomotor activity levels during these assessments were unaffected by intra-PFC treatments. On an FR-3 schedule of reinforcement, the 0.25 cocaine unit dose elicited higher total cocaine intake and hyperlocomotor activation during a shorter session, but intra-PFC treatment had no significant effects on the number of reinforced responses or behavioral activity. The observation of decreased cocaine breakpoints after intra-PFC DA antagonist treatment reflects decrements in cocaine reinforcement efficacy. This finding corresponds temporally with previous work showing increased NAcc DA levels after similar treatment. Current findings demonstrate that transient changes in PFC DA neurotransmission can specifically influence reinforced behaviors without affecting overall behavioral activation.     

Effects of dopamine antagonists on methamphetamine-induced dopamine release in high and low alcohol preference rats

The authors have previously shown that high alcohol preference rats (HAP) have a significantly higher sensitivity than low alcohol preference rats (LAP) for methamphetamine (MAP). In this study, changes in dopamine and serotonin release induced by MAP (1?mg/kg, intraperitoneally) after pre-treatment with D1 and D2 receptor antagonists were examined in the striatum of rats with different alcohol preferences to elucidate differences in receptor levels between the two rat strains. D1 receptor antagonist SCH23390 or D2 receptor antagonist haloperidol were administrated intracerebroventricularly 10?min before MAP stimulation. This study investigated the effect of methamphetamine-induced dopamine and serotonin release in striatum using microdialysis of freely moving rats coupled to ECD-HPLC. With haloperidol treatment both strains of rats showed a significantly greater maximum increase on MAP-induced dopamine release compared with respective control rats. However, after SCH23390 treatment only HAP rats showed a significantly greater increase in dopamine release compared with controls. SCH23390 blocks mainly D1 receptors only in the post-synaptic membrane, whereas haloperidol blocks D2 receptors in both the pre-synaptic and post-synaptic membranes. The MAP-induced increase in dopamine release following haloperidol pre-treatment was greater than SCH23390 pre-treatment in both strains. This result indicates that D2 receptors (autoreceptors) in the pre-synaptic membrane were blocked, leading to the elimination of the feedback function that regulates dopamine release. These data suggested that alcohol preference is associated with the action of MAP, and the dopaminergic mechanism, specifically the D1 system in the striatum, might have a different pathway dependent on alcohol preference.     

Recent developments in reversing glycopeptide-resistant pathogens

Recent studies addressing vancomycin-resistance phenomena are surveyed. Besides leading semi-synthetic glycopeptides: BI-397 and LY 333328, other new synthetic derivatives are also presented. Rational design based on the known mode of action is seen in recent research from both industrial and academic groups. Thus, Biosearch Italia SpA worked on new synthetic glycopeptides with a modified binding pocket in order to find drugs active against both vancomycin-sensitive and -resistant strains. Eli Lilly & Co. and research groups at Stanford, Cambridge and Harvard synthesised covalently linked dimers in order to copy the dimerisation and membrane anchoring effects. Both directions proved to be rewarding and some interesting compounds with potent in vitro activity against vancomycin-resistant enterococci (VRE) were found from these investigations. A new mechanistic proposal that can account for the bioactivity of LY 333328 against VRE was summarised. Research on new agents with modes of action different to that of glycopeptide antibiotics have also been successful and recent investigations related to RP-59500, SCH-27899 and U-100766 are presented.