Antagonism of discriminative stimulus effects of Δ9-THC and (R)-methanandamide in rats

Rationale In previous drug discrimination studies we observed surmountable antagonism by Δ⁹-tetrahydrocannabinol (THC) in the presence of constant doses of SR-141716 [N-(piperidin-1-yl)-5-(4-chloro-phenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (0.3 and 1 mg/kg), but there was only marginal evidence for surmountable antagonism with combinations of SR-141716 and (R)-methanandamide, a chiral analog of the endocannabioid anandamide. Objective Here we examine antagonism where the cannabinoid CB1 receptor agonist [Δ⁹-THC and (R)-methanandamide] dose is held constant (i.e., the training dose) and the antagonist {i.e., SR-141716 and AM-251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; 2 ml/kg]} dose varied. We also tested the cannabinoid CB2 receptor antagonist SR-144528 {N-[(1S)-endo-1,3,3-trimethylbicyclo(2.2.1)heptan-2-yl]5-(4-chloro-3-methyl-phenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide}. Methods Different groups of rats were trained to discriminate between vehicle and three different doses of Δ⁹-THC (1.8, 3, and 5.6 mg/kg, presumably reflecting different efficacy demands) as well as 10 mg/kg (R)-methanandamide. Dose-generalization tests involved different doses of the cannabinoid CB1 receptor agonists. Antagonist tests varied the dose of the antagonist (range: 0.1 and 3 mg/kg for SR-141716 and AM-251, and 1 to 10 mg/kg for SR-144528). Results SR-141716 and AM-251 doses dependently blocked the agonist-induced discriminative stimulus effects. SR-141716 tended to be slightly more potent than AM-251. The effective dose 50 (ED₅₀) of SR-141716 was higher in the 5.6 mg/kg Δ⁹-THC-trained group relative to the two other Δ⁹-THC-trained groups. The cannabinoid CB2 receptor antagonist SR-144528 combined with the training dose of 1.8 mg/kg Δ⁹-THC, as well as when combined with the training dose of 10 mg/kg (R)-methanandamide, did not markedly change drug-appropriate (agonist) responses. Conclusion Data support that the discriminative stimulus effects of (R)-methanandamide and its overlap with the Δ⁹-THC cue are, indeed, CB1 receptor mediated events as revealed in antagonism tests with the selective central CB1 receptor antagonists SR-141716 and AM-251. The activation of cannabinoid CB2 receptors appears to be insignificant for these discriminations.     

荧光试剂法测定小鼠受精卵胞质游离Ca~(2 )

目的通过测定小鼠卵细胞受精过程中胞质游离Ｃａ２＋浓度的变化，研究其变化规律及在胚胎早期发育中的作用。方法采用Ｃａ２＋特导荧光试剂Ｆｕｒａ－２／ＡＭ和ＡＲ－ＣＭ－ＭＩＣ型单细胞阳离子测定系统，测定了休止于第二次成熟分裂中期（ＭⅡ）的小鼠卵母细胞受精过程中的胞质游离Ｃａ２＋浓度。结果小鼠卵受精过程中胞质游离Ｃａ２＋浓度发生波动或振荡。结论这对胚胎发育的早期研究有一定意义     

蜂毒素对大鼠血小板内钙浓度的影响

应用Ｆｕｒａ－２作为荧光试剂，测定大鼠血小板细胞内Ｃａ~（２＋）浓度变化。蜂毒素１．５ｍｇ／Ｌ使血小板细胞内Ｃａ~（２＋）浓度增加；悬液中加入ＣａＣｌ＿２１ｍｍｏｌ／Ｌ，血小板细胞内Ｃａ~（２＋）继续增加。维拉帕米３．１２５ｍｇ／Ｌ作用５ｍｉｎ后，蜂毒素仍可使血小板内Ｃａ~（２＋）增加．但加入ＣａＣｌ２血小板Ｃａ~（２＋）的浓度不再增加。提示蜂毒素促进大鼠血小板细胞内Ｃａ~（２＋）的释放和细胞外Ｃａ~（２＋）进入细胞内。     

黄芪抗γ射线辐射对小鼠淋巴细胞增殖和IL-2的作用研究

目的 :从免疫学方面探讨中药黄芪对辐射损伤小鼠淋巴细胞增殖和 IL - 2的研究。方法 :用 10 0 %黄芪水浸出液定期给小鼠灌胃 ,行γ线照射后检测小鼠淋巴细胞增殖和 IL - 2的产生。结果 :用药组小鼠脾淋巴细胞增殖和 IL - 2产生有显著增强作用 ,与照射组相比均有非常显著性差异 (P<0 .0 1)。结论 :黄芪有明显抗 γ线辐射、促进小鼠淋巴细胞增殖和IL- 2产生的作用。     

CR1409拮抗缩胆囊素升高细胞内Ca~(2+)的作用

CR1409是一种新合成的缩胆囊素(CCK)受体拮抗剂。本文使用荧光指示剂Fura-2作为细胞内钙离子的探测物,在离体的大白鼠胰腺细胞研究OR1409对CCK引起细胞内钙离子浓度升高的拮抗作用。结果表明:10μmol/L CR1409完全抑制1nmol/L CCK的作用,半效抑制浓度为0.37μmol/L,比另一种拮抗剂双丁酰环鸟嘌呤核苷(db cGMP)强100倍。随着CR1409浓度的递增,使CCK升高细胞内钙离子浓度的量效曲线右移,根据Schild方法计算,pA_2值为6.93(r=0.992)。CR1409对氯氨甲酰胆碱和蛙皮素无拮抗作用。实验结果证明CR1409是一种效力较强的、特异性的和竞争性的CCK受体拮抗剂。     

青蒿素对体外培养人U251细胞凋亡和增殖的作用

目的:探讨青蒿素对体外培养的人U251胶质瘤细胞株是否具有诱导凋亡的作用。方法:(1)体外培养的U251细胞培养液中按10μg/mL终浓度加入青蒿素培养3d后进行实验;(2)倒置显微镜下观察U251细胞形态;(3)用台盼蓝拒染色法进行U251细胞的活细胞计数;(4)用MTT比色法检测青蒿素对U251细胞的抑制率;(5)透射电镜下观察U251细胞的形态。结果:(1)光镜下U251细胞部分出现碎裂崩解;(2)台盼蓝法U251细胞的活细胞计数青蒿素组少于对照组(7.6±1.273vs16.7±1.593,P<0.05);(3)MTT比色法检测青蒿素对U251细胞的抑制率为(50.0±3.2)%;(4)透射电镜下U251细胞的亚细胞结构发生凋亡变化。结论:青蒿素对体外培养的人U251胶质瘤细胞株具有诱导凋亡和抑制增殖的作用。     

Taurocholate-stimulated leukotriene C4 biosynthesis and leukotriene C4-stimulated choleresis in isolated rat liver

Cysteinyl-containing leukotrienes seem to exert a cholestatic effect. However, leukotriene inhibitors were found to reduce bile salt efflux in isolated rat hepatocytes, suggesting a role for leukotrienes in bile flow formation. In the isolated rat liver, the effects of two different concentrations of leukotriene C₄ on bile flow and bile salt excretion are analyzed, as well as the possible effect of taurocholate on the hepatic production of cysteinyl-containing leukotrienes. Leukotriene C₄ (0.25 fmol) increased bile salt excretion (+22.2%; P < 0.05), whereas a much higher dose (0.25 × 10⁶ fmol) showed the known cholestatic effect, reducing bile salt excretion (−25.9%; P < 0.01). These dose-dependent biphasic effects were specific because they could be prevented by the simultaneous administration of cysteinyl-containing leukotriene antagonists. On the other hand, taurocholate administration induced a dose-dependent increase in biliary excretion of cysteinyl-containing leukotrienes. Furthermore, taurocholate increased messenger RNA levels of 5-lipoxygenase, a key enzyme in leukotriene biosynthesis. Taurocholate increase of hepatocyte intracellular calcium was not significant, suggesting that taurocholate effects are not mediated by stimulation of calcium metabolism. These results constitute evidence for the existence of a positive feedback mechanism by which bile salts stimulate the synthesis of leukotrienes that, in turn, stimulate bile salt excretion.     

Participation of the endocannabinoid system in lipopolysaccharide-induced inhibition of salivary secretion

Objective

The aim of the present paper was to assess whether lipopolysaccharide (LPS)-induced inhibition of salivary secretion involves the activation of the endocannabinoid system and the participation of tumor necrosis factor (TNF)α in the submandibular gland.

Design

Pharmacological approaches were performed by using CB1 and/or CB2 cannabinoid receptor antagonists, AM251 and AM630, respectively, injected into the submandibular gland, to study the participation of the endocannabinoid system in LPS inhibitory effects on metacholine-induced salivary secretion. To assess the participation of TNFα on LPS inhibitory effects, salivary secretion was studied in LPS treated rats after the intraglandular injection of etanercept, a soluble form of TNF receptor which blocks TNFα action. Finally, to evaluate the possible interplay between endocannabinoids and TNFα on the submandibular gland function reduced during LPS challenge, the salivary secretion was studied after the intraglandular injection of this cytokine alone or concomitantly with AM251 and AM630.

Results

AM251 and AM630, injected separately or concomitantly, partially prevented LPS-induced inhibition of salivation. Also, anandamide synthase activity was increased in submandibular glands extracted from rats 3 h after LPS injection, suggesting that the endocannabinoid system was activated in response to this challenge. On the other hand, etanercept, prevented the inhibitory effect of LPS on salivary secretion and moreover, TNFα injected intraglandularly inhibited salivary secretion, being this effect prevented by AM251 and AM630 injected concomitantly.

Conclusion

The present results demonstrate the participation of the endocannabinoid system and TNFα on salivary responses during systemic inflammation induced by LPS.