NS-AUA 2024 Annual Meeting Abstracts – Basic Science

Introduction: Urine storage and voiding are controlled by the peripheral and central nervous systems.Neurotrophins are hormones essential for the maintenance and activity of nerves irrigating the bladder.A dysregulation in the ratio of brainderived neurotrophic factor (BDNF) and its precursor (proBDNF) was observed in the urine of a female cohort with overactive bladder (OAB).Atrial natriuretic peptide (ANP) and nitric oxide (NO) levels in the same samples were found to be elevated.We herein examine the natriuretic peptide receptors (NPRs), NO, and cell pathways involved in the release of BDNF by bladder cells.Methods: Primary cultures of rat bladder smooth muscle cells (SMCs) and urothelial cells (UROs) were incubated for 24 hours with natriuretic peptide A (ANP) (100 nM).ELISA kits were used to measure cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP), BDNF, and proBDNF.Matrix metalloproteinase-9 (MMP-9) activity was measured by enzymatic kinetics.NO was measured using the Griess method.Natriuretic receptor expression was measured by transcription polymerase chain reaction (RTqPCR) after Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 genomic edition.Results: SMCs are a major source of BDNF and proBDNF while urothelial cells (UROs) secrete most of the MMP-9, the enzyme converting proBDNF into mature BDNF, as confirmed by deletion of MMP-9 using CRISPR-Cas9.Incubation of SMCs with ANP (100 nM) for 24 hours decreased secretion of proBDNF and increased that of BDNF.ANP only increased the secretion of MMP-9 in UROs.Knockdown of the natriuretic peptide receptor 1 (NPR-1) gene by CRISPR-Cas9 plasmid completely abolished the increase in BDNF secretion elicited by ANP.On the other hand, an increase in cGMP was triggered by ANP and followed by an increase in cAMP levels.Dibutyryl cAMP (500 M) by itself mimicked the effect of ANP by increasing BDNF secretion and MMP-9 proteolytic activity.In parallel, ANP incubation led to a decrease in NO content, and L-NAME, a nitric oxide synthase (NOS) enzyme inhibitor, increased BDNF secretion but decreased MMP-9.Conclusions: These data suggest that in SMCs, ANP controls the secretion of BNDF and its precursor by binding NPR-1 involving at least two different pathways; one requiring an increase in cGMP, and the other a decrease in NO synthesis.ANP also increases the secretion of MMP-9 from UROs, enhancing the conversion of proBDNF to mature BDNF.