Inhibition of Nicotinic Responses by Cotinine in Bovine Adrenal Chromaffin Cells

Abstract: We studied the effects of cotinine, the major metabolite of nicotine, on nicotine-induced increase in [³H]phorbol dibutyrate binding, activation of protein kinase C and [³H]noradrenaline release in primary cultured bovine adrenal chromaffin cells. Cotinine (1 mM, 15 min.) and nicotine (10 μM, 5 min.) increased the [³H]phorbol binding by 100% and 150%, respectively. Both a short-term (10 min.) and a long-term (24 hr) pretreatment with cotinine inhibited the effect of nicotine. A 24 hr pretreatment with cotinine (1 mM) also reduced the nicotine-induced increase in membrane-bound protein kinase C activity. Cotinine pretreatment (10 min.) dose-dependently inhibited the release of [³H]noradrenaline induced by nicotine and dimethylphenylpiperazinium. Cotinine pretreatment did not reduce the [³H]noradrenaline release induced by high extracellular potassium (56 mM) or veratrine (10 mg l^–1)- The results indicate that cotinine inhibits activation of protein kinase C and noradrenaline release induced by nicotinic agonists in primary cultures of bovine adrenal chromaffin cells. The results suggest that pre-existing cotinine could modify responses to acute exposure to nicotine in neural systems.     

微囊化牛嗜铬细胞移植对大鼠镇痛作用的研究

目的探讨海藻酸钠-多聚赖氨酸-海藻酸钠(APA)微囊化牛嗜铬细胞(BCC)蛛网膜下腔移植镇痛的作用机制和APA微囊的免疫隔离效果。方法取SD大鼠96只，随机分为空囊组(APA组)、微囊化细胞组(APA—BCC组)和裸细胞组(BCC组)，每组又分为4个亚组(2周组、4周组、6周组和8周组)．在三组大鼠的脊髓蛛网膜下腔分别植入含空囊的DMEM、微囊化BCC和单纯BCC。测定移植术后各组大鼠脑脊液灌流液中儿茶酚胺含量的变化、8周组大鼠对福尔马林刺激的行为学变化及脊髓后角c—fos基因表达的变化。结果微囊化细胞组和裸细胞组大鼠脑脊液中儿茶酚胺含量高于空囊组；微囊化细胞组大鼠的缩腿舔爪时间短于空囊组和裸细胞组，脊髓后角c—fos表达阳性神经元数目少于空囊组和裸细胞组。结论微囊化牛嗜铬细胞移植对福尔马林致痛大鼠有镇痛作用，可以提高脑脊液中儿茶酚胺的含量，可以抑制福尔马林致痛大鼠脊髓后角c—fos基因的表达。     

Comparative Studies of Chromaffin Cell Proliferation in the Adrenal Medulla of Rats and Mice

Spontaneous and drug-induced pheochromocytomas are common inrats and rare in mice. The antihypertensive drug reserpine hasbeen shown to both induce pheochromocytomas and stimulate chromaffincell proliferation in rats, leading to the hypothesis that reserpinecauses pheochromocytomas indirectly by providing a proliferativesetting in which DNA damage may occur. The present investigationwas undertaken to obtain baseline information on the relationshipacross species between chromaffin cell proliferation and pheochromocytomas.Basal chromaffin cell proliferation was compared in age-matchedyoung adult mice and rats. In addition, mice were studied foradrenal medullary responses to reserpine, and mouse chromaffincells in vitro were studied for responses to agents that aremitogenic for cultured rat chromaffin cells. Concurrently maintainedF-344 rats and several strains of mice showed no significantdifference in basal BrdU incorporation over a 1-week period.Mice also showed an adrenal medullary proliferative responseto reserpine that was comparable to the response previouslyreported for rats. However, there was a marked disparity betweenrat and mouse chromaffin cells in vitro, and cultured mousechromaffin cells did not respond to any mitogens. The in vivodata indicate that interspecies differences in basal-or reserpine-stimulatedchromaffin cell proliferation sufficient to account for differentfrequencies of pheochromocytomas are not detectable at a singletime point in young adult animals. However, the possibilitythat such differences might emerge with aging has not been ruledout. These data further suggest either that stimulation of chromaffincell proliferation might be necessary but not sufficient fordevelopment of pheochromocytomas or that stimulated proliferationin mice might not be sustained. The inability of cultured mousechromaffin cells to respond to mitogens raises the speculationof whether mechanisms that prevent proliferation of normal chromaffincells in vitro might also help to protect mice from developingpheochromocytomas.     

Pentazocine Inhibits Norepinephrine Transporter Function by Reducing its Surface Expression in Bovine Adrenal Medullary Cells

(±)-Pentazocine (PTZ), a non-narcotic analgesic, is used for the clinical management of moderate to severe pain. To study the effect of PTZ on the descending noradrenergic inhibitory system, in the present study we examined the effect of [³H]norepinephrine (NE) uptake by cultured bovine adrenal medullary cells and human neuroblastoma SK-N-SH cells. (?)-PTZ and (+)-PTZ inhibited [³H]NE uptake by adrenal medullary cells in a concentration-dependent (3 – 100 μM) manner. Eadie-Hofstee analysis of [³H]NE uptake showed that both PTZs caused a significant decrease in the V_max with little change in the apparent K_m, suggesting non-competitive inhibition. Nor-Binaltorphimine and BD-1047, κ-opioid and σ-receptor antagonists, respectively, did not affect the inhibition of [³H]NE uptake induced by (?)-PTZ and (+)-PTZ, respectively. PTZs suppressed specific [³H]nisoxetine binding to intact SK-N-SH cells, but not directly to the plasma membranes isolated from the bovine adrenal medulla. Scatchard analysis of [³H]nisoxetine binding to SK-N-SH cells revealed that PTZs reduced the B_max without changing the apparent K_d. Western blot analysis showed a decrease in biotinylated cell-surface NE transporter (NET) expression after the treatment with (?)-PTZ. These findings suggest that PTZ inhibits the NET function by reducing the amount of NET in the cell surface membranes through an opioid and σ-receptor–independent pathway.     

Effects of Four Local Anaesthetics on the Membrane Stability of Isolated Rat Liver Lysosomes

Abstract The effects of the local anaesthetics bupivacaine, lidocaine, mepivacaine and prilocaine on lysosomal membrane stability have been studied on isolated rat liver lysosomes. The lysosomal membranes were stabilized by the local anaesthetics only within a very narrow range of concentration (around 0.001 M). In lower concentrations there was no effect of the substances while in higher concentrations there was a marked labilizing effect on the lysosomal membranes. The phlogistic effect of an injected suspension of lysosomes in the rat paw was increased by mepivacaine in concentrations of 1 % (0.04 M) and higher. The labilizing effects of local anaesthetics on lysosomes were probably due to precipitation of lysosomal membrane proteins by the local anaesthetics.     

Interaction of Local Anaesthetics with Histamine H1 Receptors in Guinea-pig Ileum

The interaction of amine local anaesthetics and related compounds with histamine H₁ receptors was investigated in guinea-pig ileal longitudinal muscle. Quinacrine, chloroquine, tetracaine and procaine inhibited [³H]mepyramine binding to solubilized membrane from ileal muscle with pK_i values of 5.27 ± 0.11, 5.66 ± 0.01, 4.28 ± 0.08 and 3.97 ± 0.11, respectively. The pK_B values obtained from the initial parallel shift of the dose-response curves for histamine in the presence of these drugs were 549 ± 0.11, 6.14 ± 0.09, 4.86 ± 0.06 and 4.58 ± 0·06, respectively, in reasonable agreement with the pK_i values. The combined dose-ratio test with both local anaesthetics and antagonist (mepyramine) present showed that tetracaine and procaine were competitive and chloroquine was partially competitive, but that quinacrine was not competitive at histamine H₁ receptors. These local anaesthetics inhibited histamine-induced desensitization in guinea-pig ileum. Receptor occupancy (%) by agonist decreased from 95.2 (without inhibitor) to 73.9, 42.8, 35.9 and 33.9 in the presence of quinacrine, chloroquine, tetracaine or procaine, respectively, under the conditions where each inhibitor drug induced half maximum inhibition of desensitization. The results suggested that most of these local anaesthetics interacted competitively at histamine H₁ receptors and inhibited desensitization through their antagonizing actions, whereas quinacrine interacted allo-sterically and inhibited desensitization through a separate action.     

3种麻醉剂对兔Oddi括约肌肌电运动的影响

目的:研究麻醉剂速眠新、氯胺酮、戊巴比妥钠对兔Oddi括约肌(SO)肌电运动的影响。方法:取家兔24只,采用改良的Thomas法间置空肠建立SO肌电运动检测通道,术后10d时将存活的16只随机分为3组,分别静脉注射速眠新(n=6)、氯胺酮(n=5)、戊巴比妥钠(n=5),在清醒状态及麻醉起效后5min各组耳缘静脉注射胆囊收缩素(CCK)20、100ng.kg-1(2次给药至少间隔3min),期间连续记录3种麻醉剂给药前、后及CCK给予后的SO肌电运动,以运动指数及其增幅、兴奋效应持续时间等指标表示,同时以未加麻醉剂时给予CCK前、后的肌电运动为对照。结果:与给予麻醉剂前比较,速眠新和氯胺酮组给药后SO肌电运动指数明显增加(P<0.05),而戊巴比妥钠组变化不明显;与对照组比较,CCK使速眠新和氯胺酮组SO肌电运动指数增幅降低,戊巴比妥钠组运动指数增幅增加、兴奋效应持续时间延长(P<0.05)。结论:3种麻醉剂对兔SO肌电运动均有影响,以速眠新和氯胺酮的影响较大,戊巴比妥钠影响相对较小。     

微囊肾上腺髓质嗜铬细胞蛛网膜下隙移植治疗晚期癌痛病人的疗效观察

目的:观察微囊肾上腺髓质嗜铬细胞移植到晚期癌痛病人蛛网膜下隙后的镇痛效果,并检测血儿茶酚胺浓度变化。方法:选择15例晚期癌症伴剧烈疼痛的病人,将经过提取、分离、纯化并以微囊包裹的牛肾上腺髓质嗜铬细胞移植到病人的蛛网膜下隙,移植后采用VAS法评价镇痛效果,并观察移植前后血中儿茶酚胺浓度变化、哌替啶用量改变及副作用。结果;移植后病人疼痛明显缓解,移植前后VAS有显著性差异(P<0.01)　显效率为26.7％,有效率为60％,总有效率为86.67％) 移植后血儿茶酚胺浓度与移植前相比具有显著性意义(P<0.01)移植后哌替啶用量明显低于移植前(P<0.01).结论:采用微囊肾上腺髓质嗜铬细胞蛛网膜下隙移植可明显缓解晚期癌痛病人疼痛,且副作用少。     

Paraesthesia after Local Anaesthetics: An Analysis of Reports to the FDA Adverse Event Reporting System

This study was aimed to evaluate the possible alert signals of paraesthesia by local anaesthetics, focusing on those used in dentistry. A case/non‐case study of spontaneous adverse events recorded in FAERS (FDA Adverse Event Reporting System) between 2004 and 2011 was performed. Cases were represented by the reports of reactions grouped under the term ‘Paraesthesias and dysaesthesias’ involving local anaesthetics (ATC: N01B*); non‐cases were all other reports of the same drugs. Reporting odds ratios (ROR) with the relevant 95% confidence intervals (95CI) were calculated. Alert signal was considered when number of cases >3 and lower limit of ROR 95CI > 1. To estimate the specificity of signals for dentistry, the analysis was restricted to the specific term “Oral Paraesthesia” and to reports concerning dental practice. Overall, 528 reports of ‘Paraesthesias and dysaesthesias’ were retrieved, corresponding to 573 drug–reaction pairs (247 lidocaine, 99 bupivacaine, 85 articaine, 30 prilocaine, 112 others). The signal was significant only for articaine (ROR=18.38; 95CI = 13.95–24.21) and prilocaine (2.66; 1.82–3.90). The analysis of the specific term “Oral Paraesthesia” retrieved 82 reports corresponding to 90 drug–reaction pairs (37 articaine, 19 lidocaine, 14 prilocaine, 7 bupivacaine, 13 others) and confirmed the signal for articaine (58.77; 37.82–91.31) and prilocaine (8.73; 4.89–15.57). The analysis of reports concerning dental procedures retrieved a signal for articaine, both for any procedures (8.84; 2.79–27.97) and for non‐surgical ones (15.79; 1.87–133.46). In conclusion, among local anaesthetics, only articaine and prilocaine generated a signal of paraesthesia, especially when used in dentistry.     

Effects of Cadmium on Bovine Corneal Endothelial Cells In Vitro

Abstract

Cadmium at a concentration of 0.5 µg/ml has been shown to be toxic to bovine corneal endothelial cells in vitro. The toxicity could be observed within 24 hr and exhibited a dose-response relationship. After exposure to 0.1 µg/ml cadmium for 24 hr, the cells were morphologically normal. Prolonged treatment, however, caused a decrease in the density of nuclei at 168 hr. At higher dosages (0.5 and 1.0 µg/ml cadmium), a similar decrease in the density of nuclei was observed at 48 hr. The effect was aggravated by extending the exposure time to 168 hr. We propose that cadmium may interfere with the energy metabolism in bovine corneal endothelial cells in vitro, leading to the disruption of the monolayer structure and cell shape.     

癌症患者使用麻醉药品存在的问题及对策

随着癌症患病率呈上升趋势,越来越多的癌症患者形成了一个不容忽视的群体,他们所承受的痛苦来自癌症及其引发的疼痛.因此,控制疼痛也是癌症治疗的一个重要组成部分.     

Effects of Tetrandrine on Cytosolic Free Calcium in Cultured Bovine Aortic Smooth Muscle Cells

利用Ｆｕｒａ２技术和ＡＲＣＭＭＩＣ阳离子系统研究了粉防己碱对传代培养牛主动脉血管平滑肌细胞内游离钙的影响。粉防己碱（１～１００μｍｏｌ·Ｌ－１）不影响血管平滑肌细胞的静息钙，但在有胞外钙存在时，对高钾，５羟色胺，ＡＴＰ，血管紧张素Ｉ，去甲肾上腺素引起的胞内钙升高有抑制作用；在无胞外钙存在时，粉防己碱对苯肾上腺素引起的胞内钙升高也有一定的抑制作用。粉防己碱对高钾升高内钙的抑制作用呈剂量依赖性，ＩＣ５０值为９．２（９５％可信限：５．７～１４．９）μｍｏｌ·Ｌ－１。上述结果提示粉防己碱对血管平滑肌细胞的电压依赖性钙通道和受体调控性钙通道均有阻滞作用，对受体调控性钙通道阻滞作用主要表现为阻滞钙离子内流。     

cAMP/PKA信号通路在肾上腺肿瘤中的作用

目的：探讨cAMP/PKA信号通路在肾上腺肿瘤中的作用。方法将人肾上腺肿瘤H295R细胞随机分为对照组、实验组A及实验组B,对照组不进行处理,实验组A加入cAMP衍生物db-cAMP,实验组B加入db-cAMP及cAMP/PKA信号通路抑制剂H-89,分别检测细胞PKA活性;应用Western Blot方法检测cAMP/PKA信号通路下游CREB蛋白的磷酸化水平;CCK8实验检测细胞72 h的增殖水平;糖皮质激素分泌实验检测3组H295R的激素分泌能力。结果实验组A的PKA活性水平、CREB蛋白的磷酸化水平、细胞增殖水平、糖皮质激素分泌水平均较对照组和实验组B显著升高(P<0．05);而实验组B与对照组比较差异无统计学意义(P<0．05)。结论 cAMP/PKA信号通路的异常活化能够促进肾上腺肿瘤的形成与功能的发挥。     

牛视网膜毛细血管内皮细胞的选择性分离培养方法

利用胰蛋白酶对牛视网膜内皮细胞和周皮细胞消化存在时间差的特点,进行细胞的选择性分离培养。经过3代分离、传代培养,经采用Ⅷ因子抗体,SP法进行染色并在显微镜下观察鉴定,证明可以获得纯度>98%、并能连续传代的牛视网膜毛细血管内皮细胞。该方法是较简便易行的牛视网膜内皮细胞选择性分离培养方法。     

Effect of Aggregation on the Hydrodynamic Properties of Bovine Serum Albumin

Purpose

To systematically analyze shape and size of soluble irreversible aggregates and the effect of aggregate formation on viscosity.

Methods

Online light scattering, refractive index and viscosity detectors attached to HPLC (Viscotek®) were used to study aggregation, molecular weight and intrinsic viscosity of bovine serum albumin (BSA). Irreversible aggregates were generated by heat stress. Bulk viscosity was measured by an oscillating piston viscometer.

Results

As BSA was heated at a higher concentration or for a longer time, the relative contribution, molecular weight and intrinsic viscosity of aggregate species increased. Molecular shape was evaluated from intrinsic viscosity values, and aggregates were estimated to be more asymmetric than monomer species. The presence of aggregates resulted in an increase in bulk viscosity when relative contribution of very high molecular weight species exceeded 10%.

Conclusions

For model system and conditions studied, generation of higher order aggregate species was concluded to be associated with an increase in molecular asymmetry. Elevated viscosity in the presence of aggregated species points to molecular asymmetry being a critical parameter affecting solution viscosity of BSA.

     

Effect of General Anaesthetics and Organic Solvents on α1–Adrenoceptors in the Myometrium

Abstract: The α₁ selective radioligand ³H–prazosin was used to assay α₁ receptors in membranes prepared from the rabbit myometrium. ³H–Prazosin was found to bind to a single high affinity site in these membranes which was the presumed α₁ receptor. A series of general anaesthetics and organic solvents were tested for their ability to inhibit ³H–prazosin binding. The order of potency of the tested agents to inhibit the binding was: chloroform = halothane = trichloroethylene > carbon tetrachloride > dichloromethane. The depression of ³H–prazosin binding seemed to be induced on the α₁ receptor since non–specific radioligand binding was not affected as revealed by a saturation experiment with ³H–prazosin where halothane was used as inhibiting agent. Computer analysis of the latter experiment also showed that halothane depressed mainly the affinity of ³H–prazosin for the α₁ receptor. The ability of the general anaesthetics and organic solvents to inhibit contractions elicited by α₁ stimulation with phenylephrine in the rabbit uterus was also investigated. In these tests the order of potency for the inhibition of the contractile response was: carbon tetrachloride ≥ halothane = chloroform > trichloroethylene > dichloromethane. The mechanism of action for α₁ receptor and myometrial depression is discussed