Introduction: Urine of patients with overactive bladder disease (OAB) shows low levels of neurotrophin NGF and stable amounts of its precursor proNGF. Higher levels of nitric oxide (NO) were shown in the same samples and may be a contributing factor of OAB.¹ Here, we investigated how NO may control the synthesis and secretion of NGF in bladder cells in vitro.

Methods: Primary cultures of urothelial (UROs) and smooth muscle cells (SMCs) from rat bladders were used. NO was measured using the Griess reaction. Expression of NGF, proNGF, and MMP-9 mRNA was determined by RT-qPCR. Intracellular MMP-9 was assessed by immunoblotting. ELISA kits were used to measure NGF, proNGF, and cyclic GMP (cGMP). The activity of major proteases involved in NGF metabolism (plasmin, MMP-7, MMP-9) was measured by specific enzymatic kits. Crisp-Cas9 was done to delete the genomic MMP-9 sequence.

Results: NO secretion increased in cells incubated in a hyperglycemic medium (27 mM). Incubation with the NO generator, sodium nitroprusside (SNP) (300 µM), led to decreased NGF and stable proNGF levels. SNP decreased cGMP levels in UROs but increased in SMCs. 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 UROs. SMCs had stable levels of NGF and MMP-9 mRNA, while UROs showed an increase. In Crispr-Cas9 transfected SMCs, NGF decrease was independent of MMP-9 but may be linked to decreased MMP-7 activity. Crispr-cas9 transfection in UROs partially inhibited the effect of SNP on NGF. MMP-7 levels were also enhanced by SNP. Plasmin activity was unaffected in both cell types.

Conclusions: High glucose levels increase the release of NO leading to decreased NGF. The signaling mechanism involves cGMP, MMP-7, and MMP-9 activity. These results are consistent with those obtained in urine of OAB patients,¹ suggesting that increased glycemia, by enhanced synthesis of NO, could be part of the pathological process.