Dysregulated NO/PDE5 signaling in the sickle cell mouse lower urinary tract: Reversal by oral nitrate therapy

Abstract

Aims

Nitric oxide (NO) has a critical, but not well understood, influence in the physiology of the lower urinary tract. We evaluated the effect of NO/phosphodiesterase (PDE)5 signaling in voiding dysfunction in the sickle cell disease (SCD) mouse, characterized by low NO bioavailability.

Main methods

Adult SCD (Sickle) and wild-type (WT) male mice were treated daily with sodium nitrate (10 mM) or vehicle. After 18 days, blood was obtained for nitrite measurement, urethra was collected for organ bath study, and bladder and urethra were collected for Western blot analysis of PDE5 phosphorylation (Ser-92) (activated form). Non-anesthetized mice underwent evaluation of urine volume by void spot assay. eNOS phosphorylation (Ser-1177) and nNOS phosphorylation (Ser-1412) (positive regulatory sites) were evaluated in the bladder and urethra of untreated mice.

Key findings

Sickle mice exhibited decreased eNOS, nNOS, and PDE5 phosphorylation in the bladder and urethra, decreased plasma nitrite levels, increased relaxation of phenylephrine-contracted urethral tissue to an NO donor sodium nitroprusside, and increased total urine volume, compared with WT mice. Nitrate treatment normalized plasma nitrite levels, relaxation of urethra to sodium nitroprusside, PDE5 phosphorylation in the urethra and bladder, and urine volume in Sickle mice.

Significance

Derangement in PDE5 activity associated with basally low NO bioavailability in the bladder and urethra contributes to the molecular basis for voiding abnormalities in Sickle mice. Inorganic nitrate supplementation normalized voiding in Sickle mice through mechanisms likely involving upregulation of PDE5 activity. These findings suggest that interventions targeting dysregulatory NO/PDE5 signaling may ameliorate overactive bladder in SCD.

Introduction

Micturition depends on the coordination between the urinary bladder and the urethra. Storage of urine in the bladder requires bladder relaxation during the filling phase, while bladder emptying requires coordinated contraction of the bladder and relaxation of the urethra [1]. Increased contractions of bladder detrusor muscle during the storage phase result in a hypercontractile voiding disorder, the most common cause of overactive bladder syndrome (OAB) [2]. OAB is clinically characterized by urinary urgency with or without urinary urge incontinence and is usually accompanied by urinary frequency and nocturia. The estimated prevalence of OAB is between 11.8 and 24.7% with similar rates in men and women [3].

Detrusor overactivity may result from impairment in detrusor smooth muscle tone, neuronal input, and/or sensory signals originating in the bladder [2]. A wide range of neurotransmitters control urine storage and voiding, including nitric oxide (NO). NO mediates relaxation of urethral and bladder neck smooth muscle [[4], [5], [6], [7]]. NO also contributes to regulation of detrusor tone. Systemic NO synthase (NOS) inhibition, intravesical NO scavenging, and knockout of neuronal NOS (nNOS) and cGMP-dependent protein kinase result in bladder overactivity in rats and mice [[8], [9], [10], [11], [12]], while intravesically or systemically supplied NO donors reduce bladder contraction frequency [13,14]. However, NO donors and cGMP analogues have also been shown to evoke a complex response (relaxant, contractile, or biphasic in isolated human detrusor muscle) [15] or have only a modest relaxing effect on isolated rat and mouse detrusor muscle [[16], [17], [18]]. Furthermore, both NO-mediated cGMP-dependent relaxation [19] and cGMP‐independent contraction of the detrusor [20,21] have been reported, further complicating the understanding of NO's role in bladder physiology and pathophysiology.

OAB is common in the sickle cell disease (SCD) population, with rates that exceed twice that of age-matched control subjects [22]. Symptoms of OAB in SCD include urinary frequency, urgency and nocturia [22,23]. SCD is caused by a single point mutation in the β-globin gene of hemoglobin leading to polymerization of hemoglobin under hypoxic conditions, red blood cell fragility, hemolysis, vaso‐occlusive crises, and inflammation [24]. SCD is also characterized by a chronic state of reduced NO bioavailability [25]. We have shown that homozygous SCD mice, a humanized mouse model of SCD, have decreased endothelial NO bioavailability in the penis associated with decreased phosphodiesterase (PDE)5 function, such that cGMP accumulation occurs upon neurostimulation, which results in excessive cavernosal vasorelaxation and priapism [[26], [27], [28]]. We have also demonstrated that SCD mice exhibit enhanced voiding and non‐voiding bladder contractions and produce greater volumes of urine [29]. These basic science findings correlate with clinical observations that both priapism and OAB are common disorders among the SCD population [22].

We hypothesized that chronically aberrant NO regulatory signaling in the bladder and urethra contributes to voiding dysfunction in SCD. We evaluated the effects of NO deficiency in lower urinary tract function using the SCD mouse at baseline and after long-term dietary inorganic nitrate supplementation.