Introduction: Urine of female patients with overactive bladder syndrome (OAB) was found to contain low levels of nerve growth factor (NGF) compared to healthy controls, with stable concentrations of its precursor proNGF. The same samples displayed elevated levels of nitric oxide (NO) that were associated to insulin resistance, a potential causative factor of OAB related to metabolic syndrome. The present study aims to determine if NO is directly linked to the control of NGF secretion from bladder cells.
Methods: Primary cultures of urothelial and smooth muscle cells (SMCs) were grown from collagenase digestion of rat bladders. Expression of NGF, proNGF and MMP-9 mRNA were measured by RT-qPCR. Intracellular MMP-9 was assessed by immunoblotting. Levels of NGF, proNGF and cyclic GMP were measured by ELISA kits. Activities of plasmin and MMP-7 (enzymes converting proNGF into NGF) and MMP-9 (the major protease degrading NGF into peptides) were assessed
Results: Incubation of cells in a hyperglycemic medium (27 mM glucose) led to increased secretion of nitric oxide and in parallel decreased NGF in the culture medium. The NO generator sodium nitroprusside (SNP) (300 μM) incubated for 24 hours also decreased NGF secretion while proNGF levels were stable. SNP potently increased cyclic GMP levels in SMCs, but decreased it in urothelial cells. Stable permeable analogs of cGMP, 8-(4-Chlorophenylthio)-cGMP (3 mM) and N2,2'-O-Dibutyryl-cGMP (1 mM) respectively decreased and increased NGF secretion in SMCs and urothelial cells, mimicking the changes observed in cGMP by SNP. On the other hand, RT-qPCR revealed stable expression of NGF and MMP-9 mRNA in SMCs, while they were increased in urothelial cells. In Crispr-Cas9 transfected SMCs, MMP-9 expression was abolished and decrease in NGF secretion by SNP was unaffected demonstrating that SNP effect in SMCs is not mediated by MMP-9. On the other hand, SNP decreases MMP-7 activity and did not affect plasmin levels, showing a decrease of NGF synthesis from proNGF. In urothelial cells, Crispr-cas9 potently inhibited the effect of SNP on NGF secretion while MMP-7 levels were enhanced, suggesting increase in NGF proteolysis and a compensatory mechanism to increase NGF.
Conclusions: Nitric oxide decreases NGF secretion by cells of the bladder through cyclic GMP and matrix metalloproteinases 7 and 9. This control is regulated differently between urothelial and SMCs. Our findings are in concordance with our previous studies of proNGF and NGF in the urine of OAB patients, and link the secretion of NGF to hyperglycaemia/insulin resistance and nitric oxide levels.