银杏叶提取物中银杏内酯A、B、C和白果内酯的提取、分离和结构鉴定

用柱层析结合溶剂法,从银杏叶提取物(EGb)中一次分得银杏内酯A、B、C和白果内酯BB.其结构经IR、1HNMR、13CNMR、MS等波谱确证,纯度经HPLC检测均为单一峰.     

HPLC-ELSD法测定白果中银杏内酯A、银杏内酯B和银杏内酯C

目的建立HPLC-ELSD法同时测定白果药材中银杏内酯A、银杏内酯B和银杏内酯C 3个萜杏内酯成分。方法采用Agilent 5TC C18色谱柱(250 mm×4.6 mm,5μm),以甲醇–四氢呋喃–水(25∶10∶65)为流动相,体积流量1 m L/min,柱温30℃,蒸发光散射检测器检测,对照品进样量为10、20μL,供试品溶液进样量为5~20μL。结果银杏内酯A在0.37~5.92μg、银杏内酯B在2.8~44.8μg、银杏内酯C在0.65~10.4μg时线性关系良好。银杏内酯A、银杏内酯B、银杏内酯C平均回收率分别为92.40%、95.03%、91.29%,RSD值分别为2.75%、2.06%、2.88%。结论本法操作简单,重复性好,为白果药材的质量控制提供了依据。     

HPLC法测定乌药中乌药内酯、乌药醚内酯、异乌药内酯的含量

目的采用HPLC法同时测定乌药中乌药内酯、乌药醚内酯、异乌药内酯含量的方法。方法色谱柱为KromasilC18柱（250mm×4.6mm,5μm）,流动相：甲醇-乙腈-水（35：25：40）;检测波长：235nm;柱温：40℃;流速：1ml/min。结果乌药内酯、乌药醚内酯、异乌药内酯的进样量分别在0.06～0.56μg（r=0.9992,n=6）、0.05～0.53μg（r=0.9992,n=6）、0.05～0.51μg（r=0.9993,n=6）的范围内与峰面积呈良好的线性关系。乌药内酯的平均回收率为99.12%,RSD为2.49%;乌药醚内酯的平均回收率为103.34%,RSD为1.59%;异乌药内酯的平均回收率为99.27%,RSD为3.58%。结论该法简单、可靠,可作为控制乌药药材质量的参考标准。     

HPLC法测定化橘红中水合橘皮内酯、橘皮内酯、马尔敏和葡萄内酯的含量

目的:建立HPLC法同时测定化橘红药材中水合橘皮内酯、橘皮内酯、马尔敏和葡萄内酯的含量。方法:采用Diamonsil C18色谱柱(250 mm×4.6 mm,5μm),以甲醇(A)-水(B)为流动相,二元梯度洗脱(0~6 min,49%A;6~15 min,49%A→75%A;15~26 min,75%A→100%A;26~30 min,100%A;30~32 min,100%A→49%A;32~35 min,49%A),流速1.0 m L·min-1,检测波长324 nm,柱温30℃。结果:水合橘皮内酯、橘皮内酯、马尔敏和葡萄内酯的进样量分别在0.378 0~2.268μg(r=0.999 7)、0.088 56~0.531 4μg(r=0.999 7)、0.089 58~0.537 5μg(r=0.999 8)和0.053 38~0.320 3μg(r=0.999 9)范围内呈良好的线性关系;平均回收率(n=6)分别为100.6%、101.5%、99.68%、98.93%,RSD为2.9%、2.7%、2.2%、2.3%。12批化橘红样品中水合橘皮内酯、橘皮内酯、马尔敏和葡萄内酯含量测定结果分别为0.123%~0.452%、0.036%~0.094%、0~0.090%、0~0.035%。结论:经方法学验证,本法可用于化橘红药材中水合橘皮内酯、橘皮内酯、马尔敏和葡萄内酯的含量测定。     

HPLC-MS法测定银杏叶片中银杏内酯A、B、C和白果内酯

目的建立测定银杏叶片中银杏内酯A、B、C及白果内酯的HPLC-MS法。方法采用HPLC-MS法,Zorbax SB-C_(18)色谱柱(250 mm×4.6 mm,5μm);流动相:甲醇–10 mmol乙酸铵水溶液,梯度洗脱;柱温:30℃;体积流量:1 m L/min;进样量:10μL。采用离子阱(ESI)负离子模式进行检测,检测模式为母离子全扫描方式(Scan),并进行二次离子化扫描。雾化器压力310.28 k Pa,毛细管电压3.5 k V,碰撞诱导解离电压150 V,干燥气为N_2,干燥器温度350℃,干燥气流量8.0 L/min。结果银杏内酯A、B、C和白果内酯在0.100 6~2.012 0、0.102 0~2.040 0、0.050 0~1.000 0、0.101 6~2.032 0 mg/m L显示良好的线性关系;平均加样回收率分别为97.1%、97.0%、98.2%、97.8%,RSD值分别为1.73%、1.68%、1.76%、1.83%(n=6)。结论该法灵敏度高、选择性高、检测限低,为银杏叶片的质量控制提供依据。     

雷公藤硫氰酸基内酯醇的半合成研究

目的合成雷公藤硫氰酸基内酯醇。方法以雷公藤甲素为起始原料,通过亲核取代反应生成雷公藤硫氰酸基内酯醇。结果总收率为79．5％,目标化合物的结构经IR、^1H—NMR、MS等方法确证。结论利用半合成技术开发高效低毒的新药是一条重要途径。     

HPLC-ELSD法测定注射用银杏二萜内酯中银杏内酯A、B、C、K

目的建立测定注射用银杏二萜内酯中银杏内酯A、B、C、K的HPLC-ELSD法。方法采用HPLC-ELSD法,Phenomenex Luna C18色谱柱(250 mm×4.6 mm,5μm),流动相为四氢呋喃–甲醇–水,梯度洗脱;体积流量为1.0 mL/min;柱温为30℃;漂移管温度为105℃,氮气体积流量为3.0 L/min。结果注射用银杏二萜内酯中银杏内酯A、B、C、K分别在1.70~8.50μg(r=0.999 7)、2.45~12.5μg(r=0.999 8)、0.30~1.50μg(r=0.999 7)、0.25~1.25μg(r=0.999 7)呈良好的线性关系;平均回收率分别为101.03%、100.86%、100.78%、101.02%,RSD值分别为0.87%、0.29%、1.30%、1.34%。结论该方法简便准确,重复性好,可用于注射用银杏二萜内酯中银杏内酯A、B、C、K的测定。     

高速逆流色谱法分离木香中的木香烃内酯和去氢木香内酯

目的 应用高速逆流色谱法分离制备木香中的木香烃内酯和去氢木香内酯.方法 应用正己烷-乙酸乙酯-甲醇-水(2:0.5:2:1)为两相溶剂系统,卡机转速为850 r·min-1,流速为2.0 ml·min-1,检测波长为254 nm.结果 从100 mg木香粗提物中分离得到34.6 mg木香烃内酯、44.3 mg去氧木香内酯,所得产物经HPLC检测纯度均大于98%.结论 所用方法分离制备木香烃内酯和去氢木香内酯具有简便、快速的优点.     

高速逆流色谱法分离制备化橘红中的柚皮苷、橙皮内酯水合物和异橙皮内酯

目的:建立高速逆流色谱法高效、快速分离制备化橘红中3个主要成分柚皮苷、橙皮内酯水合物、异橙皮内酯的新方法。方法:化橘红用9 5%乙醇超声提取,经石油醚(6 0~9 0℃)、二氯甲烷、正丁醇分级萃取初步分离,再进行高速逆流色谱分离。石油醚层所用的溶剂体系为正己烷-乙酸乙酯-甲醇-水(1∶1∶1∶1),二氯甲烷层所用的溶剂体系为正己烷-乙酸乙酯-甲醇-水(1∶2∶1∶2.5),正丁醇层所用两相溶剂体系为乙酸乙酯-正丁醇-水(4∶1∶5),高速逆流色谱仪转速8 5 7 r.min-1,所得产物的纯度经HPLC检测,结构经NMR和M S鉴定。结果:从化橘红粗提物中分离得到纯度高于9 8.0%的柚皮苷、橙皮内酯水合物,纯度高于9 5.0%的异橙皮内酯,得率均高于2 5%。结论:由该法从化橘红中制备柚皮苷、橙皮内酯水合物、异橙皮内酯简便、快速,所得产物纯度高,适合于其对照品的大规模制备。     

银杏萜内酯的药理作用与银杏天宝所含萜内酯的评价

银杏萜内酯的药理作用与银杏天宝所含萜内酯的评价吕玉涛,李遐松,任晖（贵州信邦制药公司贵阳市５５０００３）银杏萜内酯含量的多少,对银杏叶制剂质量具有关键性作用［１］。为提高银杏天宝总内酯含量,经开发研究,总内酯已达８．８７％～１０．０４％。现将有关情况...     

Mechanisms of accumulation of tyramine,metaraminol, and isoproterenol in isolated chromaffin granules and ghosts

The effects of the transmembrane pH gradient (ΔpH) and the transmembrane potential gradient (ΔΨ) on the uptake of several sympathomimetic amines were investigated, using bovine adrenal chromaffin granules isolated in isotonic sucrose. As previously described [R. Johnson and A. Scarpa, J. biol. Chem.254, 3750 (1979)], freshly isolated chromaffin granules maintain an intragranular pH of 5.5 as measured by [¹⁴C]methylamine distribution and, in the presence of ATP, generate a ΔΨ of 80 mV, positive inside, as measured by [¹⁴C]thiocyanate distribution. When tryamine, metaraminol, and isoproterenol (1–50 mM) were added to well-buffered suspensions of granules at pH 7.0, a dose-related alkalinization of the granule interior was observed. Study of the time-resolved influx of the same amines labeled radiochemically (5–21 μM) revealed that all the amines were accumulated against an apparent concentration gradient. However, while accumulation of [¹⁴C]serotonin and [³H]isoproterenol was totally inhibited by reserpine, [¹⁴C]tryramine accumulation was inhibited by only 60% and [¹⁴C]metaraminol uptake was unaffected. The ATP-dependent generation of a ΔΨ produced a stimulation of amine uptake in the order: serotonin > isoproterenol > tyramine; metaraminol accumulation was not enhanced by ATP addition. The relationship between the electrochemical proton gradient $\text{(Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext><mtext>+</mtext>}}\text{)}$ and the electrochemical gradient for each of the sympathomimetic amines $\text{(Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>A</mtext>}}\text{)}$ was investigated utilizing chromaffin ghosts devoid of endogenous matrix gradients or components. All amines were accumulated in the presence of ΔpH alone. In the presence of ΔΨ alone, [¹⁴serotonin, [¹⁴C]tyramine, and [³H]isoproterenol were accumulated, but no [³H]metaraminol uptake was demonstrable. The results indicate that serotonin and isoproterenol accumulated in isolated chromaffin granules and ghosts via a reserpine-sensitive mechanism, driven by the magnitude of the electrochemical proton gradient. Conversely, metaraminol permeated the membrane of the chromaffin granule through the apolar lipid phase and distributed according to the ΔpH alone. Tyramine uptake proceeded by both mechanisms. The implications of the mechanism of accumulation of these potent physiologic and pharmacologie agents for their in vivo action are discussed.     

Effects of bradykinin on signal transduction,cell proliferation,and cytokine,prostaglandin E2 and collagenase-1 release from human corneal epithelial cells

1. We recently demonstrated the presence of phospholipase C-coupled bradykinin (BK) B₂-receptors in human primary and SV40 virus-immortalized corneal epithelial (CEPI) cells.
2. The aims of the present studies were to demonstrate the specific binding of [³H]-BK to CEPI cell membranes and to study its pharmacological characteristics. In addition, we wished to study the functional coupling of the BK receptors to various physiological and pathological mechanisms in the CEPI cells, including phosphoinositide (PI) turnover, intracellular Ca²⁺-mobilization ([Ca²⁺]_i), cell proliferation (via [³H]-thymidine incorporation), and the release of various cytokines, collagenase-1 (matrix metalloproteinase-1) and prostaglandin E₂ (PGE₂).
3. Specific [³H]-BK binding comprised 83±2% of the total binding, and was of high affinity (K_d=1.66±0.52 nM, n=5), saturable (B_max=640±154 fmol g⁻¹ wet weight) and reversible. Competition studies yielded the following affinity values for BK and a number of BK-related peptides: Hoe-140 (D-Arg-[Hyp³,Thi⁵,D-Tic⁷,Oic⁸]BK; icatibant): K_i=0.17±0.07 nM; BK: K_i=1.0±0.11 nM; [Tyr⁸]-BK: K_i=12.9±2.3 nM; [des-Arg⁹]-BK: K_i>9,200 nM (all n=3–5)).
4. BK potently stimulated PI turnover (EC₅₀=2.3±0.3 nM; n=7) and [Ca²⁺]_i mobilization (EC₅₀=8–20 nM) in CEPI cells and both responses were inhibited in a concentration-dependent manner by 100 nM–10 μM Hoe-140, a selective B₂-receptor antagonist, and also inhibited by the selective phospholipase C (PLC) inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) (IC₅₀=3.0±1.6 μM). BK-induced [Ca²⁺]_i mobilization was reduced by about 30% in the presence of 4 mM EGTA, but was not significantly affected by 100 nM nifedipine.
5. BK (0.1 nM–10 μM) significantly (P<0.05–0.001) stimulated [³H]-thymidine incorporation into CEPI cellular DNA. However, while interleukin-1α (IL-1α; 10 ng ml⁻¹) potently stimulated the release of IL-6, IL-8 and granulocyte macrophage colony-stimulating factor from CEPI cells, BK (0.1 nM–10 μM) was without effect.
6. Whilst phorbol-12-myristate-13-acetate (PMA; 3 μg ml⁻¹) and 10% foetal bovine serum (positive control agents) significantly stimulated the release of both MMP-1 and PGE₂ from CEPI cells, BK (0.1 nM–10 μM) was without any significant effect under these conditions.
7. In conclusion, these data indicate that the CEPI cells express high-affinity [³H]-BK binding sites representing B₂-subtype BK receptors coupled to PI turnover and [Ca²⁺]_i mobilization which appear to stimulate [³H]-thymidine incorporation into cellular DNA. In contrast, BK failed to elicit the release of PGE₂, various cytokines and MMP-1 from CEPI cells. These results suggest that BK may have a potential role in corneal epithelium wound healing by stimulating cell proliferation.

     

Carrier-dependent and Ca2+-dependent 5-HT and dopamine release induced by (+)-amphetamine, 3,4-methylendioxy-methamphetamine,p-chloroamphetamine and (+)-fenfluramine

1. The mechanism underlying 5-hydroxytryptamine (5-HT) and/or dopamine release induced by (+)-amphetamine ((+)-Amph), 3,4-methylendioxymethamphetamine (MDMA), p-chloroamphetamine (pCA) and (+)-fenfluramine ((+)-Fen) was investigated in rat brain superfused synaptosomes preloaded with the ³H neurotransmitters.
2. Their rank order of potency for [³H]-5-HT-releasing activity was the same as for inhibition of 5-HT uptake (pCA⩾MDMA⩾(+)-Fen>>(+)-Amph). Similarly, their rank order as [³H]-dopamine releasers and dopamine uptake inhibitors was the same ((+)-Amph>>pCA=MDMA>>(+)-Fen). We also confirmed that the release induced by these compounds was prevented by selective transporter inhibitors (indalpine or nomifensine).
3. [³H]-5-HT and/or [³H]-dopamine release induced by all these compounds was partially (31–80%), but significantly Ca²⁺-dependent. Lack of extracellular Ca²⁺ did not alter uptake mechanisms nor did it modify the carrier-dependent dopamine-induced [³H]-dopamine release. (+)-Amph-induced [³H]-dopamine release and pCA- and MDMA-induced [³H]-5-HT release were significantly inhibited by ω-agatoxin-IVA, a specific blocker of P-type voltage-operated Ca²⁺-channels, similar to the previous results on (+)-Fen-induced [³H]-5-HT release.
4. Methiothepin inhibited the Ca²⁺-dependent component of (+)-Amph-induced [³H]-dopamine release with high potency (70 nM), as previously found with (+)-Fen-induced [³H]-5-HT release. The inhibitory effect of methiothepin was not due to its effects as a transporter inhibitor or Ca²⁺-channel blocker and is unlikely to be due to its antagonist properties on 5-HT_1/2, dopamine or any other extracellular receptor.
5. These results indicate that the release induced by these compounds is both ‘carrier-mediated'' and Ca²⁺-dependent (possibly exocytotic-like), with the specific carrier allowing the amphetamines to enter the synaptosome. The Ca²⁺-dependent release is mediated by Ca²⁺-influx (mainly through P-type Ca²⁺-channels), possibly triggered by the drug interacting with an unknown intracellular target, affected by methiothepin, common to both 5-HT and dopamine synaptosomes.

     

Biochemical and behavioural characterization of EMPA,a novel high-affinity,selective antagonist for the OX2 receptor

Background and purpose:

The OX₂ receptor is a G-protein-coupled receptor that is abundantly found in the tuberomammillary nucleus, an important site for the regulation of the sleep-wake state. Herein, we describe the in vitro and in vivo properties of a selective OX₂ receptor antagonist, N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulphonyl)-amino]-N-pyridin-3-ylmethyl-acetamide (EMPA).

Experimental approach:

The affinity of [³H]EMPA was assessed in membranes from HEK293-hOX₂-cells using saturation and binding kinetics. The antagonist properties of EMPA were determined by Schild analysis using the orexin-A-or orexin-B-induced accumulation of [³H]inositol phosphates (IP). Quantitative autoradiography was used to determine the distribution and abundance of OX₂ receptors in rat brain. The in vivo activity of EMPA was assessed by reversal of [Ala¹¹,D-Leu¹⁵]orexin-B-induced hyperlocomotion during the resting phase in mice and the reduction of spontaneous locomotor activity (LMA) during the active phase in rats.

Key results:

[³H]EMPA bound to human and rat OX₂-HEK293 membranes with K_D values of 1.1 and 1.4 nmol·L⁻¹ respectively. EMPA competitively antagonized orexin-A-and orexin-B-evoked accumulation of [³H]IP at hOX₂ receptors with pA₂ values of 8.6 and 8.8 respectively. Autoradiography of rat brain confirmed the selectivity of [³H]EMPA for OX₂ receptors. EMPA significantly reversed [Ala¹¹,D-Leu¹⁵]orexin-B-induced hyperlocomotion dose-dependently during the resting phase in mice. EMPA, injected i.p. in rats during the active phase, reduced LMA dose-dependently. EMPA did not impair performance of rats in the rotarod procedure.

Conclusions and implications:

EMPA is a high-affinity, reversible and selective OX₂ receptor antagonist, active in vivo, which should prove useful for analysis of OX₂ receptor function.     

Inhibition by mepacrine and p-bromophenacylbromide of phosphoinositide hydrolysis,glucose oxidation,calcium uptake and insulin release in rat pancreatic islets

Mepacrine and p-bromophenacylbromide were both found to impair ³H-inositol phosphate production in response to both nutrient and hormone-neurotransmitter stimuli in islets prelabelled with ³H-inositol. Both drugs also inhibited net ⁴⁵Ca uptake in response to glucose or glibenclamide and considerably modified the patterns of ⁴⁵Ca and ⁸⁶Rb efflux from perifused islets under both basal and glucose-stimulated conditions. In addition, the oxidation of [U-¹⁴C] glucose in islets was impaired by either mepacrine or p-bromophenacylbromide. These inhibitory effects were found to be concentration-related for both mepacrine (0.01–1.0 mM) and p-bromophenacylbromide (0.03–0.3 mM) and were accompanied, in general, by a similar degree of inhibition of insulin secretion. These results suggest that both mepacrine and p-bromophenacylbromide can inhibit phospholipase C activity in intact islets, but also impair ⁴⁵Ca and ⁸⁶Rb fluxes and oxidation of nutrients. The diversity of these drugs' inhibitory actions makes them unsuitable tools for examining the role of specific cellular processes in the regulation of islet function.     

Synthesis,characterization, and antimicrobial evaluation of carbostyril derivatives of 1H-pyrazole

A new series of 12 derivatives of 4-pyrazolyl-N-arylquinoline-2,5-dione (4a–l) were synthesized by one pot base catalyzed cyclocondensation reaction of 1-aryl-5-chloro-3-methyl-1H-pyrazole-4-carbaldehyde (1a–c), Meldrum’s acid (2) and 3-arylamino-5,5-disubstitutedcyclohex-2-enone (3a–d). All the compounds were characterized by elemental analysis, FT-IR, ¹H NMR and ¹³C NMR spectral data and were screened, against six bacterial pathogens, namely Bacillus subtilis, Clostridium tetani, Streptococcus pneumoniae, Salmonella typhi, Vibrio cholerae, Escherichia coli and antifungal activity, against two fungal pathogens Aspergillus fumigatus and Candida albicans, using broth microdilution MIC (minimum inhibitory concentration) method. Some of the compounds were found to be equipotent or more potent than commercial drugs, against most of the employed strains, as evident from the screening data.     

Rat liver kininase,a serine peptidase

A serine peptidase (RLK₁) was partially purified from rat liver homogenates. Its molecular weight was 80,000, and its optimum pH was 7.5. Bz-Tyr-O-Et was hydrolyzed by the enzyme, which was inhibited by Ip₂PF, PMSF and by Tos-Phe-CH₂Cl. The bonds cleaved by the enzyme were Phe⁵-Ser⁶ and Phe⁸-Arg⁹, when bradykinin was used as substrate.     

FAAH?/? Mice Display Differential Tolerance,Dependence, and Cannabinoid Receptor Adaptation After Δ9-Tetrahydrocannabinol and Anandamide Administration

Repeated administration of Δ⁹-tetrahydrocannabinol (THC), the primary psychoactive constituent of Cannabis sativa, induces profound tolerance that correlates with desensitization and downregulation of CB₁ cannabinoid receptors in the CNS. However, the consequences of repeated administration of the endocannabinoid N-arachidonoyl ethanolamine (anandamide, AEA) on cannabinoid receptor regulation are unclear because of its rapid metabolism by fatty acid amide hydrolase (FAAH). FAAH^−/− mice dosed subchronically with equi-active maximally effective doses of AEA or THC displayed greater rightward shifts in THC dose–effect curves for antinociception, catalepsy, and hypothermia than in AEA dose–effect curves. Subchronic THC significantly attenuated agonist-stimulated [³⁵S]GTPγS binding in brain and spinal cord, and reduced [³H]WIN55,212-2 binding in brain. Interestingly, AEA-treated FAAH^−/− mice showed less CB₁ receptor downregulation and desensitization than THC-treated mice. Experiments examining tolerance and cross-tolerance indicated that the behavioral effects of THC, a low efficacy CB₁ receptor agonist, were more sensitive to receptor loss than those of AEA, a higher efficacy agonist, suggesting that the expression of tolerance was more affected by the intrinsic activity of the ligand at testing than during subchronic treatment. In addition, the CB₁ receptor antagonist, rimonabant, precipitated a markedly reduced magnitude of withdrawal in FAAH^−/− mice treated subchronically with AEA compared with mice treated repeatedly with THC. The findings that repeated AEA administration produces lesser adaptive changes at the CB₁ receptor and has reduced dependence liability compared with THC suggest that pharmacotherapies targeting endocannabinoid catabolic enzymes are less likely to promote tolerance and dependence than direct acting CB₁ receptor agonists.     

Characterization of enzymatic acetylcholine synthesis by mouse brain,rat sperm,and purified carnitine acetyltransferase

Enzymatic acetylcholine synthesis by mouse brain, rat sperm, and purified pigeon breast muscle carnitine acetyltransferase was monitored. Optimal assay procedure, choline substrate requirements, the extent of acetylcholine synthesis, and the effects of acetyltransferase inhibitors were investigated to determine if acetylcholine in rat sperm is synthesized by choline acetyltransferase or by another enzyme that utilizes choline as a substrate. Of two assay procedures tested, the liquid cation-exchange procedure utilizing a butyronitrile-tetraphenylboron extraction was judged superior to an anion-exchange resin procedure. The latter procedure gave falsely high acetylcholine synthesis readings due to another acetylated contaminant (probably acetylcarnitine). The K_m for choline substrate in acetylcholine syntheses by mouse brain, which is a source of choline acetyltransferase, was 0.623 mM choline; this was 300 times less than the choline substrate K_m with rat sperm (207 mM choline) and 80 times less than the K_m with purified carnitine acetyltransferase (50.6 mM choline). Rat sperm had a V_max[3718 pmoles AcCh · min^?1 · (mg protein)^?1] that was > 2-fold that of mouse brain [1603 pmoles AcCh · min^?1·(mg protein)^?1]. A specific inhibitor of choline acetyltransferase, 4-(1-naphthylvinyl)pyridine (500 μM), abolished acetylcholine synthesis by mouse brain, but it caused only a 52 per cent inhibition of acetylcholine synthesis by rat sperm and only a 12 per cent inhibition of acetylcholine synthesis by purified carnitine acetyltransferase. These data indicate that an enzyme other than classical or “true” choline acetyltransferase is responsible for acetylcholine synthesis by rat sperm. Because of the high content of carnitine acetyltransferase in rat sperm and the ability of carnitine acetyltransferase from pigeon breast muscle to synthesize acetylcholine, carnitine acetyltransferase is the most probable enzyme responsible for acetylcholine synthesis in rat sperm.     

Assessment of genotoxicity of Lidocaine,Prilonest and Septanest in the drosophila wing-spot test

The scope of this study was to characterize the likely interaction Lidocaine^®, Prilonest^® and Septanest^® have with DNA, with a view to quantitatively and qualitatively establishing mutagenic, clastogenic, and/or recombinagenic activity of those compounds. The wing somatic mutation and recombination test in Drosophila melanogaster, which detects simultaneously point and chromosomal mutations as well as recombination induced by the activity of genotoxins of direct and indirect action, was used. Each of the anesthetics was tested at different concentrations, administered orally for 48 h to 3rd-stage larvae, in two independent experiments, with concurrent negative controls. The results obtained revealed that only Prilonest^® exhibits genotoxic activity in somatic cells, being able to induce exclusively homologous recombination. Additionally, it was possible to conclude that the genotoxic effect attributed to Prilonest^® is not related to metabolites produced via the P450-type enzymes. However, both Lidocaine^® and Septanest^® are unable to induce either events related to gene and chromosomal mutation, or reciprocal recombination.