The peripheral and central nervous systems control urine storage and voiding by inducing relaxation and contraction of the bladder wall. Neurotrophins are hormones released by cells of the urinary tract and essential in the maintenance and activity of nerve endings irrigating the bladder. Among them, Brain-derived neurotrophic factor (BDNF) promotes neuroregeneration by binding Trk receptor B while its precursor proBDNF triggers inflammation and apoptosis through receptor p75NTR. Imbalance in the urinary ratio of BDNF to proBDNF was found to be a viable biomarker for overactive bladder syndrome (OAB). On the other hand, nitric oxide (NO) and natriuretic papetide A (ANP) urine levels are found increased in the urine of female patients with OAB. We here examined the relation between ANP and NO, whose intracellular signalings involve cGMP, with BDNF and proBDNF secretion in bladder smooth muscle cells.

Smooth muscle (SMCs) and urothelial (UROs) cells were grown from female rat bladder. BDNF, proBDNF, cyclic nucleotides (cGMP and cAMP) levels were measured using specific ELISA kits. matrix metalloproteinase-9 (MMP-9) activity was assessed using an enzymatic kit. Nitric oxide was measured by the Griess method. Intracellular pathways were examined by semi-quantitative immunoblotting. CrisprCas9 plasmids were designed to target genomic MMP-9 or NPR-1.

Receptors NPR1, NPR2 and NPR3 were found expressed in rat urothelial and smooth muscle cells in vitro. SMCs are a main source of BDNF and proBDNF in the bladder while UROs secrete most of matrix metalloproteinase-9 (MMP-9), the enzyme converting proBDNF into BDNF, as confirmed by the accumulation of proBDNF after genomic deletion of MMP-9 by CriscprCas9. Incubation of SMCs with ANP (100 nM) for 24 hours decreased the secretion of proBDNF and increased BDNF one. On the other hand, ANP increased the secretion of MMP-9. Co-culture increased the ratio BDNF/proBDNF from SMC suggesting that MMP-9 released by URO targets SMCs. Regarding intracellular pathways regulating BDNF synthesis and secretion in SMCs, knockdown of NPR-1 gene completely abolished the increase in BDNF secretion elicited by ANP while sham cells receiving an empty plasmid were unaffected. On the other hand, we observed an increase in cGMP that in turn increases cAMP after ANP incubation. Dibutyryl cAMP added to the medium (500 µM) yielded the same results than ANP including increases in BDNF secretion and enhanced MMP-9 proteolytic activity. In parallel, ANP triggered a decrease in nitric oxide (NO) synthesis from SMCs. L-NAME, a general NOS enzyme inhibitor, increased BDNF secretion but decreased MMP-9. In accordance, addition of the nitric oxide generator sodium nitroprusside (300 microM) to the culture medium suppresses BDNF secretion elicited by ANP.

ANP and nitric oxide levels are both upregulated in OAB while proBDNF ones are decreased and BDNF stable. Our data suggest that the imbalance in mature to proneurotrophins observed in urine samples from aging female patients with OAB could results from ANP increasing the conversion of proBDNF into BDNF, leading to a decrease in proBDNF, and from nitric oxide preventing the increase in BDNF.

These results demonstrate that ANP controls the secretion of BDNF and its precursor in bladder tissue by acting on both URO and SMC cells. In SMCs, at least two distinct pathways, one involving cyclic GMP and one nitric oxide, are stimulated by ANP.

Continence 12S (2024) 101369
DOI: 10.1016/j.cont.2024.101369