Nitric oxide donor trans-[RuCl([15]aneN4)NO]2+ as a possible therapeutic approach for Chagas' disease

Background and purpose:

Benznidazole (Bz) is the therapy currently available for clinical treatment of Chagas'' disease. However, many strains of Trypanosoma cruzi parasites are naturally resistant. Nitric oxide (NO) produced by activated macrophages is crucial to the intracellular killing of parasites. Here, we investigate the in vitro and in vivo activities against T. cruzi, of the NO donor, trans-[RuCl([15]aneN₄)NO]²⁺.

Experimental approach:

Trans-[RuCl([15]aneN₄)NO]²⁺was incubated with a partially drug-resistant T. cruzi Y strain and the anti-proliferative (epimastigote form) and trypanocidal activities (trypomastigote and amastigote) evaluated. Mice were treated during the acute phase of Chagas'' disease. The anti-T. cruzi activity was evaluated by parasitaemia, survival rate, cardiac parasitism, myocarditis and the curative rate.

Key results:

Trans-[RuCl([15]aneN₄)NO]²⁺ was 10- and 100-fold more active than Bz against amastigotes and trypomastigotes respectively. Further, trans-[RuCl([15]aneN₄)NO]²⁺ (0.1 mM) induced 100% of trypanocidal activity (trypomastigotes forms) in vitro. Trans-[RuCl([15]aneN₄)NO]²⁺ induced permanent suppression of parasitaemia and 100% survival in a murine model of acute Chagas'' disease. When the drugs were given alone, parasitological cures were confirmed in only 30 and 40% of the animals treated with the NO donor (3.33 µmol·kg⁻¹·day⁻¹) and Bz (385 µmol·kg⁻¹·day⁻¹), respectively, but when given together, 80% of the animals were parasitologically cured. The cured animals showed an absence of myocarditis and a normalisation of cytokine production in the sera. In addition, no in vitro toxicity was observed at the tested doses.

Conclusions and implications:

These findings indicate that trans-[RuCl([15]aneN₄)NO]²⁺is a promising lead compound for the treatment of human Chagas'' disease.This article is commented on by Machado et al., pp. 258–259 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00662.x and to view a related paper in this issue by Silva et al. visit http://dx.doi.org/10.1111/j.1476-5381.2010.00524.x     

神经肽DGAVP和Org2766对神经细胞内游离Ca~(2+)的影响

运用Ca~(2+)指示剂Fura-2作为细胞内钙离子的荧光探针,利用AR—CM—MIC阳离子测定系统,检测了分离的神经细胞内游离钙及其变化,并观测了DGAVP和Org2766对蛋白质合成抑制剂茴香霉素(ANI)引起细胞内钙离子浓度([Ca~(2+)]_i)变化的影响。结果表明茴香霉素可使[Ca~(2+)]_i显著升高,且有量效关系;DGAVP本身并不引起[Ca~(2+)]_i发生显著变化,但适当剂量的DGAVP可显著对抗一定剂量范围内ANI升高[Ca~(2+)]_i的作用,提示DGAVP对抗ANI的蛋白质合成抑制效应可能是通过拮抗ANI升高[Ca~(2+)]_i这一途径实现的,另一神经肽Org2766则可能不是通过这一机制发生作用。从细胞内Ca~(2+)的角度看,这两种肽的作用机理显然是不同的。     

五味子酚对氧自由基损伤小鼠脾淋巴细胞的保护作用

研究了五味子酚(Sal)对氧自由基损伤小鼠脾淋巴细胞的影响。体外实验结果表明,Sal在5×10^-6mol·L^-1时对Fe²⁺-VitC引起的脾淋巴细胞GSH含量降低有明显的抑制作用,且能阻抑Fe²⁺-Cys引起的MDA生成增加,改善细胞膜的流动性。用扫描电镜观察到Sal在5×10^-4mol·L^-1时可逆转Fe²⁺-VitC引起的脾淋巴细胞表面微绒毛皱折减少、细胞变形等病理改变。体内高氧分压应激损伤小鼠实验表明,igSal20mg·kg^-1×8d可逆转脾淋巴细胞SOD活性代偿性增高,并提高脾淋巴细胞内GSH含量。以上结果提示Sal对氧自由基损伤脾淋巴细胞有保护作用。     

Neuropeptide Y receptor in pig spleen: binding characteristics, reduction of cyclic AMP formation and calcium antagonist inhibition of vasoconstriction

Specific, high-affinity binding sites for 125I-porcine neuropeptide Y (NPY) were demonstrated in membranes from the pig spleen. The equilibrium dissociation constant (KD) of the receptor 125I-NPY complex was 532 +/- 87 pM and the maximal number of specific binding sites (Bmax) 23 +/- 3 fmol/mg protein. The Scatchard plot for 125I-NPY binding under equilibrium conditions showed a best-fit to a straight line, whereas the dissociation appeared biphasic. 125I-NPY binding was unaffected by adrenoceptor antagonists and was inhibited by the guanosine triphosphate (GTP) analogue guanylylimidodiphosphate, suggesting regulation by a GTP binding protein. A series of NPY analogues showed a good correlation between binding, inhibition of forskolin-induced cyclic adenosine monophosphate (cAMP) formation and vasoconstrictor activity in vivo. A large carboxyl terminal portion of NPY and the carboxyl terminal amide were essential for binding, inhibition of cAMP formation and vasoconstrictor effects. The NPY fragment 13-36, which has been reported to act only on prejunctional NPY receptors, showed only a 10-fold lower potency than NPY-(1-36) both in binding to splenic membranes and vasoconstrictor activity in vivo. Phenylephrine increased phosphatidyl inositol turnover whereas NPY-(1-36) or -(13-36) did not induce formation of inositol phosphates. The calcium antagonists felodipine and nifedipine attenuated the splenic vasoconstrictor response to NPY in vivo but not the NPY-evoked inhibition of cAMP accumulation or the specific binding of 125I-NPY.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of substrate interactions on in vitro fatty acid synthesis in adipose tissue of normal and genetically obese mice

Summary Fatty acid synthesis was measured in vitro in pieces of adipose tissue from lean and obese-hyperglycaemic (ob/ob) mice, using ¹⁴C-glucose or ¹⁴C-lactate and ³H₂O to obtain absolute rates of total fatty acid synthesis. In the presence of lipoproteintriglyceride (2.5 mol/l) metabolic interaction occurred which decreased glucose incorporation into fatty acids by 30% in lean mouse tissue, but not in obese mouse tissue. In the absence of added insulin, the contribution of glucose to total fatty acid synthesis was 69% in obese mouse tissue, significantly lower than the value of 87% obtained in lean mouse tissue. Insulin increased the contribution of glucose to total synthesis in both lean and obese mouse tissues, although the value in obese mouse tissue (83%) remained lower than the value in lean mouse tissue (100%). Lactate was not a major precursor for fatty acid synthesis. When both lactate (2 mmol/l) and glucose (15 mmol/l) were present, the contribution of lactate to total fatty acid synthesis was not increased in obese mouse tissue, suggesting that even in the presence of insulin, about 30% of the carbon was provided by intracellular precursors.