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Evaluation of the effect of the inhibitor of cAMP and cGMP efflux transporters, mK 571 in the bladder and prostate of healthy and obese mice and tissues from patients with Overactive Bladder and Benign Prostatic Hyperplasia

Grant number: 18/07364-1
Support type:Scholarships in Brazil - Doctorate (Direct)
Effective date (Start): October 01, 2018
Effective date (End): February 28, 2021
Field of knowledge:Biological Sciences - Pharmacology - Autonomic Pharmacology
Principal Investigator:Fabíola Taufic Monica Iglesias
Grantee:Gabriela Maria Bertollotto
Home Institution: Faculdade de Ciências Médicas (FCM). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
Associated research grant:17/15175-1 - Modulation of soluble guanylate cyclase and the intracellular levels of cyclic nucleotides in the lower urinary tract and prostate, AP.TEM

Abstract

The intracellular concentration of the second cGMP and cAMP messengers is controlled by the rate of production through the activity of the enzymes soluble guanylate cyclase (GCs) and particulate or adenylate cyclase (AC), respectively and by clearance either by the action of phosphodiesterases or by the activity of the transporters of efflux. Transporters of the subtype MRP4 and MRP5 are part of a large family of transmembrane proteins that utilize the energy of ATP hydrolysis to pump endogenous and xenobiotic compounds out of the cell, and among these endogenous compounds are cAMP and cGMP. The expression of MRP4 and MPR5 in the human lower urinary tract, such as the bladder, cavernous body, urethra and prostate, has been demonstrated. Our group has been showing in Obesity and middle age models that GCs are degraded, resulting in low intracellular levels of cGMP, which seems to favor hypercontractility of the bladder, urethral and prostatic tissues in these pathological conditions. Therefore, we postulate that MRP4 and MRP5 play a relevant role in regulating the tone of this smooth muscle.To test these possibilities of MRP4 and MRP5 by controlling the intracellular levels of cyclic nucleotides in lower urinary tract tissues in healthy and pathological animals (Obesity) we outlined functional protocols in vivo (cystometry) and in vitro (concentration curves and frequency-response) (western blotting, RT-PCR, immunofluorescence and ELISA) obtained from obese mice and respective control groups. In most of our studies we will employ the MRP4 inhibitor, MK-571. In terms of molecular studies, we are proposing (i) to evaluate the localization, gene expression and protein expression of MRP4 and MRP5; (ii) quantify cyclic nucleotide levels (cGMP and cAMP) and evaluate their downstream signaling (PKG, p-VASP 239 and p-VASP 157) in the absence and presence of MK-571; and (iii) gene silencing of MRP4, MRP5 and MRP8 in bladder and prostate smooth muscle primary culture, and quantification of nucleotides produced intra- and extracellularly, as well as VASP-239 and VAPS-157 expression. n terms of functional studies, we will (i) investigate the in vivo (cystometry) and in vitro effect of MK-571 on the bladder, urethra and prostate in the cAMP and cGMP elevating agent-induced relaxations. Molecular and/or functional studies (as proposed above) will be used in human bladder, prostate and urethra obtained from patients with neurogenic bladder and/or BPH (and cadaveric donor tissues as a control), in the presence and absence of MK- 571. Data from this project may indicate that MRP4 and MRP5 are important in the physiological control of cyclic nucleotides in lower urinary tract tissues and whether these transporters could be relevant targets in the treatment of urologic complications associated with Obesity. (AU)