Pingle Sandeep C, Sanchez Joseph F, Hallam Daniel M, Williamson Andrea L, Maggirwar Sanjay B, Ramkumar Vickram
Department of Pharmacology, Southern Illinois University School of Medicine, Box 19230, Springfield, IL 62974-1222, USA.
Mol Pharmacol. 2003 Jun;63(6):1238-47. doi: 10.1124/mol.63.6.1238.
The expression of inducible nitric-oxide synthase (iNOS) in vascular smooth muscle cells leads to prolonged vasorelaxation in vivo and contributes to the profound vasodilation induced by bacterial lipopolysaccharide (LPS) in septic shock. This induction of iNOS depends, in large part, on activation of nuclear factor (NF)-kappaB. Hypertonicity regulates the activity of NF-kappaB in different cell lines; as such, we propose that it should also regulate the expression of iNOS. Thus, the goal of this study was to determine whether hypertonicity regulates iNOS expression and function in smooth muscle cells and to elucidate the mechanism(s) underlying this process. Treatment of hamster ductus deferens (DDT1MF-2) cells and porcine aortic smooth muscle cells with either mannitol (50 mM) or NaCl (50 mM) reduced LPS-stimulated iNOS expression and nitric oxide release. Both of these agents also reduced the activation of NF-kappaB induced by LPS, tumor necrosis factor-alpha and interleukin-1beta in smooth muscle cells. This inhibitory action was caused by suppression of IkappaB-alpha phosphorylation, a prerequisite for ubiquitination and degradation of this protein, and showed additivity with N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG-132), an inhibitor of proteasomal degradation of IkappaB-alpha. Furthermore, exposure to mannitol inhibited the activity of IkappaB kinase, an enzyme involved in phosphorylation of IkappaB-alpha. Mannitol was unable to affect the induction of iNOS produced by overexpression of RelA in DDT1MF-2 cells, suggesting that this agent does not have additional downstream inhibitory actions on this activated NF-kappaB subunit. Taken together, these data suggest that these hypertonic solutions may prove useful as anti-inflammatory agents, especially against conditions associated with increased NF-kappaB activity.
诱导型一氧化氮合酶（iNOS）在血管平滑肌细胞中的表达可导致体内血管舒张延长，并促成脓毒性休克中细菌脂多糖（LPS）诱导的显著血管扩张。iNOS的这种诱导在很大程度上依赖于核因子（NF）-κB的激活。高渗性调节不同细胞系中NF-κB的活性；因此，我们推测它也应调节iNOS的表达。因此，本研究的目的是确定高渗性是否调节平滑肌细胞中iNOS的表达和功能，并阐明这一过程的潜在机制。用甘露醇（50 mM）或氯化钠（50 mM）处理仓鼠输精管（DDT1MF-2）细胞和猪主动脉平滑肌细胞，可降低LPS刺激的iNOS表达和一氧化氮释放。这两种试剂还降低了LPS、肿瘤坏死因子-α和白细胞介素-1β在平滑肌细胞中诱导的NF-κB激活。这种抑制作用是由抑制IκB-α磷酸化引起的，IκB-α磷酸化是该蛋白泛素化和降解的前提条件，并且与N-苯甲酰氧基羰基（Z）-亮氨酰-亮氨酰-亮氨酸（MG-132）具有相加性，MG-132是IκB-α蛋白酶体降解的抑制剂。此外，暴露于甘露醇可抑制IκB激酶的活性，IκB激酶是一种参与IκB-α磷酸化的酶。甘露醇无法影响DDT1MF-2细胞中RelA过表达产生的iNOS诱导，这表明该试剂对这种活化的NF-κB亚基没有额外的下游抑制作用。综上所述，这些数据表明这些高渗溶液可能被证明是有用的抗炎剂，尤其是针对与NF-κB活性增加相关的病症。
