Sirmakesyan Stephanie, Hajj Aya, Hamouda Aalya, Cammisotto Philippe, Campeau Lysanne
Lady Davis Institute for Medical Research, Montreal, Quebec, Canada.
Lady Davis Institute for Medical Research, Montreal, Quebec, Canada; Urology Department, Jewish General Hospital, Montreal, Quebec, Canada.
Nitric Oxide. 2023 Nov 1;140-141:30-40. doi: 10.1016/j.niox.2023.09.002. Epub 2023 Sep 10.
Urine samples of female patients with overactive bladder (OAB) are characterized by low levels of nerve growth factor (NGF) and elevated concentrations of nitric oxide (NO) compared to healthy controls. We therefore examined how NO might regulate NGF synthesis using rat bladder smooth muscle (SMCs) and urothelial (UROs) cells in culture. In UROs, incubation in hyperglycemic conditions to mimic insulin insensitivity present in the OAB cohort increased secretion of NO and concomitantly decreased NGF, except when the NO synthase inhibitor, l-NAME (1 mM) was present. Sodium nitroprusside (SNP) (300 μM, 24 h), a NO generator, decreased NGF levels and decreased cyclic GMP (cGMP) content, a process validated by the cGMP synthase inhibitor ODQ (100 μM). Alternatively, SNP increased mRNA of both NGF and matrix metalloproteinase-9 (MMP-9). MMP-9 knockout of UROs by Crispr-Cas9 potently decreased the effect of SNP on NGF, implying a dependent role of NO on MMP-9. On the other hand, matrix metalloproteinase-7 (MMP-7) activity was increased by SNP, which taken together with increase in NGF mRNA, suggests a compensatory mechanism. In SMCs, hyperglycemic conditions had the same effect on extracellular content of NO and NGF than in UROs. SNP also decreased NGF secretion but increased cGMP content. Stable permeable analogs of cGMP 8-(4-Chlorophenylthio)-cGMP (1 mM) and N2,2'-O-Dibutyryl-cGMP (3 mM) inhibited NGF release. NGF and MMP-9 mRNA expression was unchanged by SNP. Deletion of MMP-9 in SMCs by Crispr-Cas9 did not alter the effect of SNP. Finally, SNP decreased MMP-7 activity, diminishing the conversion of proNGF to NGF. These results demonstrate that enhanced NO secretion triggered by high glucose decreases NGF secretion through pathways unique for each cell type that involve cGMP and proteases MMP-7 and MMP-9. These results might help to explain our observations from the urine from patients with OAB associated with metabolic syndrome.
与健康对照相比，膀胱过度活动症（OAB）女性患者的尿液样本具有神经生长因子（NGF）水平低和一氧化氮（NO）浓度升高的特征。因此，我们使用培养的大鼠膀胱平滑肌（SMC）和尿路上皮（URO）细胞研究了NO可能如何调节NGF合成。在URO中，在高血糖条件下孵育以模拟OAB队列中存在的胰岛素不敏感，会增加NO的分泌并同时降低NGF，除非存在NO合酶抑制剂L-NAME（1 mM）。一氧化氮供体硝普钠（SNP）（300 μM，24小时）降低了NGF水平并降低了环磷酸鸟苷（cGMP）含量，这一过程通过cGMP合酶抑制剂ODQ（100 μM）得到验证。另外，SNP增加了NGF和基质金属蛋白酶-9（MMP-9）的mRNA。通过Crispr-Cas9敲除URO中的MMP-9可有效降低SNP对NGF的作用，这意味着NO对MMP-9具有依赖性作用。另一方面，SNP增加了基质金属蛋白酶-7（MMP-7）的活性，这与NGF mRNA的增加一起表明了一种补偿机制。在SMC中，高血糖条件对NO和NGF细胞外含量的影响与URO中的相同。SNP也降低了NGF分泌，但增加了cGMP含量。cGMP的稳定可渗透类似物8-（4-氯苯硫基）-cGMP（1 mM）和N2,2'-O-二丁酰-cGMP（3 mM）抑制了NGF的释放。SNP对NGF和MMP-9 mRNA表达没有影响。通过Crispr-Cas9在SMC中删除MMP-9并没有改变SNP的作用。最后，SNP降低了MMP-7活性，减少了前体NGF向NGF的转化。这些结果表明，高血糖引发的NO分泌增加通过涉及cGMP以及蛋白酶MMP-7和MMP-9的每种细胞类型特有的途径降低了NGF分泌。这些结果可能有助于解释我们从患有代谢综合征的OAB患者尿液中观察到的现象。
