Uptake of dopamine by rat hepatocytes in vitro

The present results showed that uptake of dopamine (DA) by rat isolated hep-atocytes was mediated, in addition to simple diffusion, mainly by a transporter-involved process, with Km of 66. 8 μmol and Vmax of 52. 3 pmol·min-1/105 cells. The process was pH- and temperature-dependent and required an activation energy of 4. 12 kcal ·mol-1(Q10 = 1.25) in the range of 2.0-12.7 C and 13.0 kcal·mol -1(Q10 = 2. 0) in the range of 12.7 -39.0C. Cysteine residue having free thiol group was. unrelated to the activity of the transporter. Catecholamines, serotonin, and cocaine inhibited the DA transport , but tyramine (TA) and tryptamine, as well as benztropine and imipramine (which are potent inhibitors for hepatic TA transporter and neuronal DA transporter), had no inhibitory effect on the transport of DA in these cells. These results indicated that DA was taken up into hepatocytes by a distinct carrier. NaF and mastoparan influenced the transport activity in these cells further, suggesting that signal transduci     

Effects of bilobalide on rat astrocytes in vitro

In the previous studies we found that the treatment with amyloid-β25 - 35 peptide (Aβ 25 - 35) resulted in an elevation of monoamine oxidase B (MAO-B) expression in rat astrocytes,suggesting that the upregulation of MAO-B in Aβstimulated astrocytes may play an important role in the pathogenesis of Alzheimer's disease.In present studies,we reported that bilobalide, a sesqulterpene isolated from Ginkgo biloba leaves attenuated the upregulation of MAO-B expression induced by Aβ in rat astrocytes.A significant increase in the expression of vascular endothelial growth factor was also found when the astrocytes was treated with bilobalide.The results suggest that bilobalide may play important roles in the beneficial effects of     

Epoxide-diol pathway of Δ1 -THC in the rat in vitro

1. 1α,2α-Epoxyhexahydrocannabinol was identified as a metabolite of Δ₁-tetrahydrocannabinol.

2. The two trans-1,2-dihydroxyhexahydrocannabinol isomers, 1α,2β-dihydroxy-hexahydrocannabinol and 1β,2α-dihydroxyhexahydrocannabinol (as their acetates) were tentatively identified as metabolites from incubation of 1α,2α-epoxyhexahydrocannabinol with rat hepatic microsomes in vitro.

3. The 1α,2α-epoxyhexahydrocannabinol acetate was found to be a good substrate for epoxide hydratase as compared to styrene oxide.

4. The synthesis of metabolites of 1α,2α-epoxyhexahydrocannabinol is described.     

Combining in vitro embryotoxicity data with physiologically based kinetic (PBK) modelling to define in vivo dose–response curves for developmental toxicity of phenol in rat and human

In vitro assays are often used for the hazard characterisation of compounds, but their application for quantitative risk assessment purposes is limited. This is because in vitro assays cannot provide a complete in vivo dose–response curve from which a point of departure (PoD) for risk assessment can be derived, like the no observed adverse effect level (NOAEL) or the 95 % lower confidence limit of the benchmark dose (BMDL). To overcome this constraint, the present study combined in vitro data with a physiologically based kinetic (PBK) model applying reverse dosimetry. To this end, embryotoxicity of phenol was evaluated in vitro using the embryonic stem cell test (EST), revealing a concentration-dependent inhibition of differentiation into beating cardiomyocytes. In addition, a PBK model was developed on the basis of in vitro and in silico data and data available from the literature only. After evaluating the PBK model performance, effective concentrations (EC_x) obtained with the EST served as an input for in vivo plasma concentrations in the PBK model. Applying PBK-based reverse dosimetry provided in vivo external effective dose levels (ED_x) from which an in vivo dose–response curve and a PoD for risk assessment were derived. The predicted PoD lies within the variation of the NOAELs obtained from in vivo developmental toxicity data from the literature. In conclusion, the present study showed that it was possible to accurately predict a PoD for the risk assessment of phenol using in vitro toxicity data combined with reverse PBK modelling.     

Expression of Rho GDIα in rat osteoblasts intermittently exposed to parathyroid hormone in vitro and in vivo

Aim： To investigate the mechanism of the bone-forming effects of intermittent parathyroid hormone （PTH） administration and to search for novel molecules of bone anabolism via the PTH signaling pathway.
Methods： Primary cultures of rat osteoblasts （ROBs） were divided into an intermittent PTH-treated group （Itm） and a control group （Ctr）. Imitating the pharmacokinetics of intermittent PTH administration in vivo, the ROBs in the Itm group were exposed to PTH for 6 h in a 24-h incubation cycle, and the ROBs in the Ctr group were exposed to vehicle for the entire incubation cycle. The cells were collected at 6 h and 24 h of the final cycle, and the proteins in the Itm and Ctr groups were analyzed by two-dimensional electrophoresis （2-DE） coupled with peptide mass fingerprinting and matrix assisted laser desorption/ionization time-of-flight mass spectrometry （MALDITOF-MS） to detect proteins that were differentially expressed. The proteins with the most significant changes in vitro were validated by immunohistochemistry （IHC） in a rat model.
Results： The proteomics analysis indicated that a total of 26 proteins were up- or down-regulated in the Itm group compared with the Ctr group at 6 h and 24 h; among these, 15 proteins were successfully identified. These proteins mainly belong to the cytoskeleton and molecular chaperone protein families, and most of these have anti-apoptotic effects in various cells. Rho GDP-dissociation inhibitor a （RhoGDIα） and vimentin were the most significantly changed proteins. Further studies by IHC showed that the expression of RhoGDla in ROBs was significantly higher in PTH-treated sham-operated rats than in vehicle-treated sham-operated rats, but the difference was not significant between PTH-treated and vehicle-treated OVX rats. Vimentin expression was not changed in either PTH-treated sham- operated rats or PTH-treated OVX rats.
Conclusion： Our research suggests that intermittent PTH treatment induces changes in expression of many proteins i     

Interactions between dopamine D1 receptors and γ-aminobutyric acid mechanisms in substantia nigra pars reticulata of the rat: neurochemical and behavioral studies

RATIONALE: Several studies have shown that dopamine D1 agonists act on forebrain dopamine terminal regions to exert many of their behavioral effects. Yet, there is also a large number of D1 receptors in the substantia nigra pars reticulata (SNr), and these receptors are located mainly on terminals of gamma-aminobutyric acid (GABA)-ergic striatonigral neurons. OBJECTIVE: The present studies were undertaken to determine the behavioral and neurochemical effects of local administration of the D1 agonist SKF 82958 and to study the interactions between D1 and GABA mechanisms in SNr. METHODS: Microdialysis methods were used to characterize the effect of SKF 82958 on extracellular GABA, and several experiments studied the effects of nigral D1 stimulation on motor activity and investigated the behavioral significance of D1/GABA interactions in SNr. RESULTS: Local infusion of 10(-6) M SKF 82958 increased extracellular levels of SNr GABA, and this effect was blocked by co-infusion of the D1 antagonist SCH 23390. Bilateral SNr injections of SKF 82958 increased locomotor activity, and this effect was blocked by the GABA-A antagonist bicuculline. Intranigral bicuculline reduced motor activity, while the GABA-A agonist muscimol increased various motor activities in a manner similar to SKF 82958. CONCLUSIONS: The present results suggest that the D1 agonist SKF 82958 acts on D1 receptors in SNr to increase extracellular levels of GABA, and the increase in motor activity produced by nigral D1 stimulation is dependent on stimulation of GABA-A receptors. D1/GABA interactions in SNr are important for the modulation of basal ganglia output, which may have important implications for Parkinson's disease.     

γ-Hydroxybutyric acid (GHBA) induces pacemaker activity and inhibition of substantia nigra dopamine neurons by activating GABAB-receptors

Summary In the present study the actions of -hydroxybutyric acid (GHBA) on dopaminergic neurons in the rat substantia nigra (SN) were pharmacologically analysed utilising extracellular single unit recording techniques. Intravenous administration of GHBA was associated with several effects on the neuronal activity of nigral dopamine (DA) neurons. Low doses (<200 mg/kg) of GHBA produced a slight excitation of the neurons, concomitant with a regularised firing rhythm and lack of burst activity. In higher doses GHBA produced an even higher degree of regularisation but, in contrast to low doses, an inhibition of firing rate. Administration of the GABA_B-receptor agonist baclofen, in all essential respects, mimicked the effect of GHBA on the firing of nigral DA neurons. Both the regularisation of the firing pattern and inhibition of firing rate produced by systemic administration of GHBA were antagonised by the GABA_B-receptor antagonist CGP 35348 (200 mg/kg, i.v.).Our observations show that GHBA affects the firing pattern of nigral DA neurons in doses considerably lower than those required to inhibit the firing rate of the neurons. This action, as well as the decreased firing rate observed after high doses of GHBA, are mediated via activation of GABA_B-receptors. Correspondence to: G. Engberg at the above address     

β(2)-Adrenergic activity of 6-methoxykaempferol-3-O-glucoside on rat uterus: in vitro and in silico studies

6-Methoxykaempferol-3-O-glucoside (6-MKG) was isolated from a Sudanese herb (El-hazha). The pharmacological effects of 6-MKG were tested on isolated non-pregnant or late-pregnant rat uteri in vitro, whilst docking studies were carried out modelling of the binding of 6-MKG to the rat β(2)-adrenoceptor in silico. In vitro studies revealed that 6-MKG was able to relax both the non-pregnant and the late-pregnant uterine contractility with 50% of the E(max) of terbutaline, whilst the EC(50) for 6-MKG was at least half than that of terbutaline. The β(2)-adrenoceptors antagonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol(ICI118,551) competitively antagonised the relaxing effect of 6-MKG. Radioligand binding and cAMP studies confirmed the β(2)-adrenoceptors agonistic property of the compound. In in silico docking studies, 6-MKG bound to rat β(2)-adrenoceptors with low ?G(bind) value (-11.53±0.06 kcal/mol) and it interacted with four residues of the active site (Asp(113), Asn(312), Cys(191)and Tyr(316)). It is concluded that 6-MKG exerts weak β(2)-adrenoceptor agonistic activity and can be considered a natural compound with potential therapeutic significance in the field of premature pregnant uterine contractions and asthmatic problems.     

Effects of β-adrenoceptor antagonists on the firing rate of noradrenergic neurones in the locus coeruleus of the rat

Summary Acute i.v. administration of the non-selective -adrenoceptor antagonist dl-propranolol given in incremental doses (<40 mg/kg) did not affect the firing rate of locus coeruleus (LC) neurones in the rat, as revealed by single cell recording techniques. Furthermore, no effect was seen 4 h after a single i.p. dose of this -blocker (10 mg/kg). However, repeated treatment with dl-propranolol (1, 5, 10 or 20 mg/kg i.p., twice daily for 4 days) produced a significant, dose-dependent decrease of the average LC neuronal firing rate in comparison to controls. The dextro isomer of propranolol, which has negligible -blocking activity but the same local anaesthetic potency as the racemate, had no corresponding effect. The non-selective -adrenoceptor antagonist sotalol, which is one of the most hydrophilic -blockers, had much less inhibitory effect on LC neurones than dl-propranolol. The ₁-selective antagonist metoprolol did not change the firing of noradrenergic neurones in the LC after similar treatment for 4 days. However, when the rats were subjected to oral treatment for 28 days, metoprolol was found to produce a slight inhibitory effect although much less than dl-propranolol.In view of these findings we propose a stimulatory and mainly ₂-adrenoceptor-mediated control mechanism for the noradrencrgic neurones in the LC. This mechanism seems to be characterized by a delayed responsiveness.     

Inhibitory effects of ginkgolides on nitric oxide production in neonatal rat microglia in vitro

目的：观察银杏内酯Ａ和Ｂ，ＰＡＦ拮抗剂阿帕泛（Ａｐａ）和ＮＯＳ抑制剂ＬＮＡ对新生大鼠小胶质细胞（Ｍｉ）产生ＮＯ的影响．方法：以Ｇｒｉｅｓ反应测定亚硝酸盐含量表示ＮＯ量．结果：在静息Ｍｉ，ＧＡ，ＧＢ和Ａｐａ在１－１００００ｎｍｏｌ·Ｌ－１范围对Ｍｉ产生ＮＯ没有影响，但ＬＮＡ可浓度依赖性地抑制ＮＯ产生，其ＩＣ５０（９５％可信限）值为３．４（０．８－１４９）μｍｏｌ·Ｌ－１．而在激活的Ｍｉ，ＧＡ，ＧＢ和ＬＮＡ可浓度依赖性地抑制ＮＯ产生，其ＩＣ５０（９５％可信限）值分别为５．７（１．８－１８１），１．１（０．３－４４）和０．５（０．１－２．８）μｍｏｌ·Ｌ－１，但Ａｐａ不能抑制ＮＯ产生．结论：ＧＡ和ＧＢ抑制ＬＰＳ诱导Ｍｉ产生ＮＯ．     

Dual effects of pentobarbital on rat sacral dorsal commissural neurons in vitro

Ｄｕａｌｅｆｅｃｔｓｏｆｐｅｎｔｏｂａｒｂｉｔａｌｏｎｒａｔｓａｃｒａｌｄｏｒｓａｌｃｏｍｍｉｓｓｕｒａｌｎｅｕｒｏｎｓｉｎｖｉｔｒｏ１ＰＡＮＧＺｈｉＰｉｎｇ，ＸＵＴｉａｎＬｅ２，ＨＵＧｕｏＹｕａｎ３，ＬＩＪｉＳｈｕｏ（Ｄｅｐａｒｔｍｅｎｔｏ...     

Cytotoxicity and apoptotic gene expression in an in vitro model of the blood–brain barrier following exposure to poly(butylcyanoacrylate) nanoparticles

Background Poly(butylcyanoacrylate) (PBCA) nanoparticles (NPs) loaded with doxorubicin (DOX) and coated with polysorbate 80 (PS80) have shown efficacy in the treatment of rat glioblastoma. However, cytotoxicity of this treatment remains unclear.

Purpose The purpose of this study was to investigate cytotoxicity and apoptotic gene expression using a proven in vitro co-culture model of the blood–brain barrier.

Methods The co-cultures were exposed to uncoated PBCA NPs, PBCA-PS80 NPs or PBCA-PS80-DOX NPs at varying concentrations and evaluated using a resazurin-based cytotoxicity assay and an 84-gene apoptosis RT-PCR array.

Results The cytotoxicity assays showed PBCA-PS80-DOX NPs exhibited a decrease in metabolic function at lower concentrations than uncoated PBCA NPs and PBCA-PS80 NPs. The apoptosis arrays showed differential expression of 18 genes in PBCA-PS80-DOX treated cells compared to the untreated control.

Discussion As expected, the cytotoxicity assays demonstrated enhanced dose-dependent toxicity in the DOX loaded NPs. The differentially expressed apoptotic genes participate in both the tumor necrosis factor receptor-1 and mitochondria-associated apoptotic pathways implicated in current DOX chemotherapeutic toxicity.

Conclusion The following data suggest that the cytotoxic effect may be attributed to DOX and not the NPs themselves, further supporting the use of PBCA-PS80 NPs as an effective drug delivery vehicle for treating central nervous system conditions.     

Bladder selectivity of the novel β3-agonist ritobegron (KUC-7483) explored by in vitro and in vivo studies in the rat

We performed in vitro and in vivo experiments to evaluate the pharmacological profile of ritobegron and its effects on the bladder in rats. β(3)-AR selectivity was assessed using CHO cells expressing various subtypes of the human β-adrenoceptor (AR). Effects on isolated organs were evaluated using the organ-bath method. Effects on intravesical pressure, heart rate, and mean blood pressure were evaluated in urethane-anesthetized rats. Ritobegron increased cAMP accumulation in a concentration-dependent manner in CHO cells expressing any one of three human β-AR, its selectivity for β(3)-AR being 301-fold and 32-fold higher versus β(1)-AR and β(2)-AR, respectively. Ritobegron decreased the resting tension of the isolated bladder in a concentration-dependent manner (EC(50), 7.7 × 10(-8) mol/L; maximal relaxation, 97.0 %), and the β(3)-AR antagonist SR58894A produced a parallel rightward-shift of this concentration-response curve without altering the maximal response [pK(B) value, 6.43]. Ritobegron concentration-dependently increased atrial rate and decreased myometrial contractions in vitro, and its selectivity for the bladder was 2,078-fold higher versus the atria and 14-fold higher versus the uterus. In vivo, ritobegron induced a dose-dependent decrease in intravesical pressure (ED(50) 0.4 mg/kg), without affecting heart rate and only slightly lowering mean blood pressure. Thus, ritobegron displayed potent and selective β(3)-AR agonistic activity toward transfected human β-AR and exhibited a high selectivity for the bladder versus other organs in rats. Moreover, it decreased intravesical pressure with minimal effects on the cardiovascular system in anesthetized rats. These results suggest that ritobegron shows promise as a potential agent for the treatment of overactive bladder.     

Comparison of binding affinities of ω-conotoxin and amlodipine to N-type Ca~(2 ) channels in rat brain

目的：用大鼠脑对ωｃｏｎｏｔｏｘｉｎ（ωＣＴＸ）及氨氯地平与Ｎ型钙通道的内在关系进行分析．方法：将大鼠全脑匀浆于ＨＥＰＥＳ５０ｍｍｏｌ·Ｌ－１（ｐＨ７４）缓冲液中，经４００００×ｇ离心后，收集膜区域．以１２５Ｉωｃｏｎｏｔｏｘｉｎ（ＣＴＸ）作为放射配体测定．结果：１２５ＩωＣＴＸ与冷冻标本及新鲜标本结合的Ｂｍａｘ无区别．Ｎ型钙通道的Ｋｄ和Ｂｍａｘ值分为００２±００１ｍｍｏｌ·Ｌ－１和１０２９±１０８ｐｍｏｌ／ｇ蛋白质．ωＣＴＸ及氨氯地平的ｐＫｉ值分别为９５７以及＜４，普萘洛尔、哌唑嗪、阿托品、组胺的ｐＫｉ值也非常低．结论：Ｌ型钙离子拮抗剂氨氯地平与Ｎ离子通道的亲和性很低．     

Correlation of the intrinsic clearance of donepezil (Aricept®) between in vivo and in vitro studies in rat,dog and human

1. Donepezil hydrochloride (Aricept®) is used for the treatment of Alzheimer's disease. Here the correlation of the intrinsic clearance (Cl_int) of donepezil between the in vivo and in vitro states was studied in rat, dog and human. 2. In an experiment with ¹⁴C-donepezil and human microsomes the routes of metabolism were identified as N-dealkylation and O-demethylation, and no unknown metabolites were detected. 3. The Cl_int of donepezil in the male rat, female rat, dog and human liver microsomes were 33.7, 13.4, 37.0 and 6.35 μl/min/mg microsomal protein respectively, and sex difference in rat and interspecies difference in the estimated Cl_int were found. 4. After a single intravenous administration to the male rat, female rat and dog, total plasma clearance (Cl^P_total) was 78.6, 29.5 and 88.3 ml/min/kg respectively, and a sex difference was observed in rat. 5. After a single oral administration to the male rat, dog and healthy volunteer, Cl^P_total/F was 140, 105 and 2.35 ml/min/kg respectively, and remarkable differences were observed between animals and man. 6. The contribution of renal clearance to blood clearance (Cl_r) was low in all species. The predicted in vitro hepatic clearance (Cl_h-pre) was in the rank order: male rat (15.91 ml/min/kg) &gt; dog (7.96) &gt; female rat (7.67) &gt; human (1.04). Although Cl_h-pre was underestimated, Cl_h-pre was significantly correlated with that of ClBtotal in the different animal species and in man, indicating that the in vitro-in vivo ranking order was conserved.     

The effect of C-terminal fragment of ANF-ANF(24–28)OH on the cardiovascular system in rat

1. The effect of C-terminal fragment of ANF-ANF(24–28)OH on the cardiovascular system was investigated in rats2. _{In vivo} this pentapeptide caused the fall of systolic and diastolic blood pressure.3. ANF(24–28)OH increased cardiac contraction amplitude and changed coronary outflow in vitro.4. These experiments showed that the shorter fragment of ANF containing five amino acids: Asn24-Ser25-Phe26-Arg27-Tyr28-COOH is a bioactive substance.     

A time scaling approach to develop an in vitro–in vivo correlation (IVIVC) model using a convolution-based technique

In vitro–in vivo correlation (IVIVC) models prove very useful during drug formulation development, the setting of dissolution specifications and bio-waiver applications following post approval changes. A convolution-based population approach for developing an IVIVC has recently been proposed as an alternative to traditional deconvolution based methods, which pose some statistical concerns. Our aim in this study was to use a time-scaling approach using a convolution-based technique to successfully develop an IVIVC model for a drug with quite different in vitro and in vivo time scales. The in vitro and the in vivo data were longitudinal in nature with considerable between subject variation in the in vivo data. The model was successfully developed and fitted to the data using the NONMEM package. Model utility was assessed by comparing model-predicted plasma concentration-time profiles with the observed in vivo profiles. This comparison met validation criteria for both internal and external predictability as set out by the regulatory authorities. This study demonstrates that a time-scaling approach may prove useful when attempting to develop an IVIVC for data with the aforementioned properties. It also demonstrates that the convolution-based population approach is quite versatile and that it is capable of producing an IVIVC model with a big difference between the in vitro and in vivo time scales.     

Stimulation of central cholinergic neurons by(-)clausenamide in vitro

Ｓｔｉｍｕｌａｔｉｏｎｏｆｃｅｎｔｒａｌｃｈｏｌｉｎｅｒｇｉｃｎｅｕｒｏｎｓｂｙ（－）ｃｌａｕｓｅｎａｍｉｄｅｉｎｖｉｔｒｏ１ＤＵＡＮＷｅｎＺｈｅｎ，ＺＨＡＮＧＪｕｎＴｉａｎ２（ＤｅｐａｒｔｍｅｎｔｏｆＰｈａｒｍａｃｏｌｏｇｙ，Ｉｎｓｔｉｔｕｔｅ...     

Inhibitory effect of(-),( )clausenamide on acetylcholinesterase in vitro

用Ｅｌｍａｎ比色法测定乙酰胆碱酯酶（ＡＣｈＥ）的活性，观察（－），（＋）黄皮酰胺对小鼠脑组织海马，前脑皮层和红细胞膜ＡＣｈＥ活性的影响，以探索药物作用机制并比较左右旋体黄皮酰胺作用的异同．结果表明：（－），（＋）黄皮酰胺对海马和皮层ＡＣｈＥ均有抑制作用，（－）黄皮酰胺对小鼠脑皮层和海马ＡＣｈＥ抑制的ＩＣ５０（９５％可信限）值分别为０．３１（０．２７－０．３６）和０．３３（０．２８－０．３９）ｍｍｏｌ·Ｌ－１；（＋）黄皮酰胺对小鼠脑皮层和海马ＡＣｈＥ抑制的ＩＣ５０（９５％可信限）值分别为０．７１（０．５３－０．９４）和０．７７（０．５５－１．０７）ｍｍｏｌ·Ｌ－１．（－），（＋）黄皮酰胺对红细胞膜ＡＣｈＥ的抑制作用是可逆性的，抑制特点属于竞争与非竞争混合型抑制，（－）黄皮酰胺的Ｋｉ值为０．２６ｍｍｏｌ·Ｌ－１，Ｋｉ′值为１．２０５ｍｍｏｌ·Ｌ－１；（＋）黄皮酰胺Ｋｉ值为０．７２ｍｍｏｌ·Ｌ－１，Ｋｉ′值为１．８５６ｍｍｏｌ·Ｌ－１．提示：（－）黄皮酰胺作用强于（＋）黄皮酰胺．黄皮酰胺的抑制ＡＣｈＥ作用存在手性选择性．