Inhibitory mechanisms of YC-1 and PMC in the induction of iNOS expression by lipoteichoic acid in RAW 264.7 macrophages

In the present study, the signal pathways involved in NO formation and iNOS expression in RAW 264.7 macrophages stimulated by LTA were investigated. We also compared the relative inhibitory activities and mechanisms of PMC, a novel potent antioxidant of alpha-tocopherol derivatives, with those of YC-1, an sGC activator, on the induction of iNOS expression by LTA in cultured macrophages in vitro and LTA-induced hypotension in vivo. LTA induced concentration (0.1-50 microg/mL)- and time (4-24 hr)-dependent increases in nitrite (an indicator of NO biosynthesis) in macrophages. Both PMC (50 microM) and YC-1 (10 microM) inhibited NO production, iNOS protein, mRNA expression, and IkappaBalpha degradation upon stimulation by LTA (20 microg/mL) in macrophages. On the other hand, PMC (50 microM) almost completely suppressed JNK/SAPK activation, whereas YC-1 (10 microM) only partially inhibited its activation in LTA-stimulated macrophages. Moreover, PMC (10 mg/kg, i.v.) and YC-1 (5 mg/kg, i.v.) significantly inhibited the fall in MAP stimulated by LTA (10 mg/kg, i.v.) in rats. In conclusion, we demonstrate that YC-1 shows more-potent activity than PMC at abrogating the expression of iNOS in macrophages in vitro and reversing delayed hypotension in rats with endotoxic shock stimulated by LTA. The inhibitory mechanisms of PMC may be due to its antioxidative properties, with a resulting influence on JNK/SAPK and NF-kappaB activations. YC-1 may be mediated by increasing cyclic GMP, followed by, at least partly, inhibition of JNK/SAPK and NF-kappaB activations, thereby leading to inhibition of iNOS expression.     

The blockade of cyclopiazonic acid-induced store-operated Ca2+ entry pathway by YC-1 in neutrophils

In the presence of external Ca2+, pretreatment of neutrophils with 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) inhibited the cyclopiazonic acid (CPA)-induced [Ca2+](i) elevation in a concentration- but not a time-dependent manner, while YC-1 had no effect on the Ca2+ signals in a Ca2+-free medium. YC-1 failed to inhibit ATP- and interleukin-8 (IL-8)-induced [Ca2+](i) changes. Addition of YC-1 after cell activation strongly inhibited the CPA-induced [Ca2+](i) changes. In a classical Ca2+ readdition protocol, a similar extent inhibition of Ca2+ spike by YC-1 introduced either prior to or after CPA stimulation was obtained. In rat neutrophils, mRNA for endothelial differentiation gene (edg)1, edg5, edg6 and edg8, the putative targets for sphingosine 1-phosphate (S1P), could be detected. However, S1P was found to have little effect on Ca(2+) signals. YC-1 did not inhibit but enhanced the sphingosine-induced [Ca2+](i) changes. Inhibition by YC-1 of CPA-induced [Ca2+](i) changes was not prevented by 7-nitroindazole and N-(3-aminomethyl)benzylacetamidine (1400W), two nitric oxide synthase (NOS) inhibitors, by aristolochic acid, a phospholipase A(2) inhibitor, or by suspension in a Na(+)-deprived medium. YC-1 did not affect the mitochondrial membrane potential. Moreover, YC-1 did not alter [Ca2+](i) changes in response to ionomycin after CPA and formyl-Met-Leu-Phe (fMLP) stimulation in a Ca2+-free medium. YC-1 had no effect on the basal [Ca2+](i) level, the pharmacologically isolated plasma membrane Ca2+-ATPase activity, and Ba2+ entry into CPA-activated cells. YC-1 alone resulted in the accumulation of actin filaments in neutrophils, while significantly reduced the intensity of actin filament staining in the subsequent activation with CPA. These results indicate that YC-1 inhibited CPA-activated store-operated Ca2+ entry (SOCE) probably through the direct blockade of channel activation and/or the disruption of the integrity of the actin cytoskeleton necessary for supporting Ca2+ entry pathway in neutrophils.     

吲唑类化合物的合成方法及应用的研究进展

吲唑是重要的有机合成中间体，其许多衍生物具有药物活性。本文综述了吲唑类化合物的合成方
法及改进方向，主要介绍了邻取代苯甲腈、取代芳香醛、全氟苯二甲酸、腙等为原料合成吲唑的方法、研究现状及
其发展前景，并对其生物药理活性进行综述。