J Pharmacol Exp Ther. 2014 Apr;349(1):2-9.

Soluble guanylyl cyclase (sGC) degradation and impairment of nitric oxide-mediated responses in urethra from obese mice: reversal by the sGC activator BAY 60-2770.

Alexandre EC, Leiria LO, Silva FH, Mendes-Silvério CB, Calmasini FB, Davel AP, Mónica FZ, De Nucci G, Antunes E.

Departments of Pharmacology (E.C.A., L.O.L., F.H.S., C.B.M.S., F.B.C., F.Z.M., G.D.N., E.A.) and Anatomy, Cellular Biology, Physiology, and Biophysics (A.P.C.D.), University of Campinas (UNICAMP), Campinas, São Paulo, Brazil.

 

Abstract

Obesity has emerged as a major contributing risk factor for overactive bladder (OAB), but no study examined urethral smooth muscle (USM) dysfunction as a predisposing factor to obesity-induced OAB. This study investigated the USM relaxant machinery in obese mice and whether soluble guanylyl cyclase (sGC) activation with BAY 60-2770 [acid 4-({(4-carboxybutyl) [2-(5-fluoro-2-{[4-(trifluoromethyl) biphenyl-4-yl] methoxy} phenyl) ethyl] amino} methyl) benzoic] rescues the urethral reactivity through improvement of sGC-cGMP (cyclic guanosine monophosphate) signaling. Male C57BL/6 mice were fed for 12 weeks with a high-fat diet to induce obesity. Separate groups of animals were treated with BAY 60-2770 (1 mg/kg per day for 2 weeks). Functional assays and measurements of cGMP, reactive-oxygen species (ROS), and sGC protein expression in USM were determined. USM relaxations induced by NO (acidified sodium nitrite), NO donors (S-nitrosoglutathione and glyceryl trinitrate), and BAY 41-2272 [5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine] (sGC stimulator) were markedly reduced in obese compared with lean mice. In contrast, USM relaxations induced by BAY 60-2770 (sGC activator) were 43% greater in obese mice (P < 0.05), which was accompanied by increases in cGMP levels. Oxidation of sGC with ODQ [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one] (10 μM) potentiated BAY 60-2770-induced USM responses in the lean group. Long-term oral BAY 60-2770 administration fully prevented the impairment of USM relaxations in obese mice. Reactive-oxygen species (ROS) production was enhanced, but protein expression of β1 second guanylate cyclase subunit was reduced in USM from obese mice, both of which were restored by BAY 60-2770 treatment. In conclusion, impaired USM relaxation in obese mice is associated with ROS generation and down-regulation of sGC-cGMP signaling. Prevention of sGC degradation by BAY 60-2770 ameliorates the impairment of urethral relaxations in obese mice.

PMID: 24421320

 

SUPPLEMENT:

Obesity has been frequently reported as a major contributing factor for the lower urinary tract symptoms (LUTS) and overactive bladder (OAB) [1-3]. The prevalence of OAB and metabolic syndrome in the U.S. adult population is 10.7% and 24%, respectively [4,5]. A recent study estimated that 2.6 billion individuals (61.9%) worldwide experience at least one symptom (nocturia, frequency or urgency), with a projected increase to 3.1 billion (62.7%) in 2018 [6]. Anatomically, the lower urinary tract (LUT) consists of the urinary bladder and urethra, the former of which act to promote to promote urine storage and emptying. Urethra contributes to urinary continence by relaxing during the voiding phase and by contracting during the urine storage phase [7].

The nitric oxide (NO) – soluble guanylate cyclase (sGC) – cyclic GMP signaling pathway governs many aspects of cellular function via interaction with specific kinases, ion channels and phosphodiesterases (PDE). Soluble guanylyl cyclase (sGC) is a heterodimeric complex consisting of two subunits, α and β. Each subunit contains three common domains, namely: (1) N-terminal haem-binding domain that mediates the NO sensitivity of the enzyme, (2) dimerization domain, and (3) C-terminal catalytic domain, which is responsible for the conversion of GTP to cGMP [8]. The so-called stimulators and activators of sGC developed over the past two decades are of potential interest as treatment for cardiovascular and urological diseases. These compounds comprise two novel groups of small molecule compounds that increase the enzymatic activity of sGC through NO-independent mechanisms. The efficacy of these compounds differs depending on the oxidation state of sGC enzyme. Like the endogenous ligand NO, the sGC stimulators (BAY 41-2272 as prototype) increase sGC activity only when the heme iron is in its reduced state (Fe2+). On the contrary, the sGC activators (BAY 58-2667 and BAY 60-2770 as prototypes) preferentially activate the enzyme when the haem iron is in its oxidized state (Fe3+ instead of Fe2+) or even when the heme group is missing [9,10]. Figure 1 illustrates the putative mechanism of action of sGC stimulators and activators.

Nitric oxide (NO) produced by nitrergic nerves supplying the urethra smooth muscle is the predominant neurotransmitter that mediates urethral relaxations during the voiding phase [8]. Once released, NO binds to the prosthetic group containing the reduced Fe2+ haem moiety in sGC, leading to intracellular accumulation of the second messenger molecule cGMP that ultimately reduces cytosolic Ca2+ levels, causing smooth muscle relaxation [11]. Removal or oxidation (Fe3+) of the haem moiety leads to the NO-insensitive form of the enzyme [12]. Therefore, targeting sGC – cGMP pathway may be of therapeutical benefit to treat LUTS and OAB, as smooth muscle relaxations at the level of urethra, prostate and trigone may reduce bladder outlet obstruction.

A recent study in mice has implicated obesity as a major contributing factor for voiding dysfunction and overactive bladder [13], which is greatly ameliorated by chronic treatment with the sGC activator BAY 60-2770 [14]. Therefore, we have decided to use this same murine model of obesity-associated voiding dysfunction to evaluate the effects of BAY 60-2270 in the urethral dysfunction. Our results showed that obese mice display urethral dysfunction associated with sGC oxidation and reduction of β1 subunit expression [15]. Two-week therapy with BAY 60-2770 in obese mice normalized the levels of reactive-oxygen species (ROS) and fully rescued the expression of β1 sGC subunit in the urethral tissues, thus preventing the urethral dysfunction [15]. Our present work highlights the possibility of targeting the urethral smooth muscle with sGC activators to treat oxidative stress-associated OAB or outlet obstruction.

Acknowledgements

Eduardo Costa Alexandre and Edson Antunes are grateful to CNPq (National Council for Scientific and Technological Development) for financial support.

Contacts

Edson Antunes, Ph.D.
Department of Pharmacology
Faculty of Medical Sciences,
State University of Campinas (UNICAMP)
13084-971
Campinas (SP), Brazil
E-mail: edson.antunes@uol.com.br ; antunes@fcm.unicamp.br

Eduardo Costa Alexandre, MSc.
Department of Pharmacology
Faculty of Medical Sciences,
State University of Campinas (UNICAMP)
13084-971
Campinas (SP), Brazil
E-mail: edu86alex@gmail.com

 

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Figure1
Fig 1. Nitric oxide (NO) activates soluble guanylate cyclase (sGC), a heterodimer enzyme composed by α and β subunits and a prosthetic heme (Fe2+) group, which converts guanosine triphosphate (GTP) into cyclic guanosine monophosphate (cGMP). Nitric oxide and stimulators (BAY 41-2272) activate sGC in its reduced form (Fe2+) whereas activators (BAY 60-2770) act preferentially when sGC is in a oxidized (Fe3+) state. Adapted from Mónica FZ et al (2014) [16].

 

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