Minoru Miyazato

Therapeutic receptor targets for lower urinary tract dysfunction

The functions of the lower urinary tract, to store and periodically release urine, are dependent on the activity of smooth and striated muscles in the bladder, urethra, and external urethral sphincter. During urine storage, the outlet is closed, and the bladder smooth muscle is quiescent. When bladder volume reaches the micturition threshold, activation of a micturition center in the dorsolateral pons (the pontine micturition center) induces a bladder contraction and a reciprocal relaxation of the urethra, leading to bladder emptying. During voiding, sacral parasympathetic (pelvic) nerves provide an excitatory input (cholinergic and purinergic) to the bladder and inhibitory input (nitrergic) to the urethra. These peripheral systems are integrated by excitatory and inhibitory regulation at the levels of the spinal cord and the brain. Injury or diseases of the nervous system, as well as drugs and disorders of the peripheral organs, can produce lower urinary tract dysfunction. In the overactive bladder (OAB) condition, therapeutic targets for facilitation of urine storage can be found at the levels of the urothelium, detrusor muscles, autonomic and afferent pathways, spinal cord, and brain. There is increasing evidence showing that the urothelium has specialized sensory and signaling properties including: (1) expression of nicotinic, muscarinic, tachykinin, adrenergic, bradykinin, and transient receptor potential (TRP) receptors, (2) close physical association with afferent nerves, and (3) ability to release chemical molecules such as adenosine triphosphate (ATP), acetylcholine, and nitric oxide. Increased expression and/or sensitivity of these urothelial-sensory molecules that lead to afferent sensitization have been documented as possible pathogenesis of OAB. Targeting afferent pathways and/or bladder smooth muscles by modulating activity of ligand receptors (e.g., neurokinin, ATP, or β3-adrenergic receptors) and ion channels (e.g., TRPV1 or K) could be effective to suppress OAB. In the stress urinary incontinence condition, pharmacotherapies targeting the neurally mediated urethral continence reflex during stress conditions such as sneezing or coughing could be effective for increasing the outlet resistance. Therapeutic targets include adrenergic and serotonergic receptors in the spinal cord as well as adrenergic receptors at the urethral sphincter, which can enhance urethral reflex activity during stress conditions and increase baseline urethral pressure, respectively.

GABA Receptor Activation in the Lumbosacral Spinal Cord Decreases Detrusor Overactivity in Spinal Cord Injured Rats

PURPOSEnWe investigated the effects of intrathecal application of gamma-aminobutyric acid A and B receptor agonists on detrusor overactivity in spinal cord injured rats.nnnMATERIALS AND METHODSnAdult female Sprague-Dawley rats were used. At 4 weeks after Th9-10 spinal cord transection awake cystometry and recordings of external urethral sphincter electromyogram were performed to examine the effect of intrathecal application of the gamma-aminobutyric acid A and B agonists muscimol and baclofen or the gamma-aminobutyric acid A and B antagonists bicuculline and saclofen (Tocris Cookson, Ellisville, Missouri), respectively, at the level of the L6-S1 spinal cord. The expression of glutamate decarboxylase 67 mRNA in the L6-S1 spinal cord and dorsal root ganglia was also assessed.nnnRESULTSnMuscimol and baclofen produced a dose dependent inhibition of the number (51% to 73% decrease) and amplitude (35% to 93% decrease) of nonvoiding bladder contractions and a decrease in micturition pressure. The effects of muscimol and baclofen were antagonized by bicuculline and saclofen, respectively. Bursting activity of external urethral sphincter electromyogram was inhibited, corresponding to the inhibition of bladder activity by muscimol and baclofen. Glutamate decarboxylase 67 mRNA levels in the spinal cord and dorsal root ganglia were decreased after spinal cord transection (55% and 84%, respectively).nnnCONCLUSIONSnThese results indicate that gamma-aminobutyric acid A and B receptor activation in the spinal cord inhibits detrusor overactivity. The decrease in glutamate decarboxylase 67 mRNA suggests hypofunction of GABAergic inhibitory mechanisms in the spinal cord. Therefore, stimulation of spinal GABAergic mechanisms could be effective for the treatment of detrusor overactivity after spinal cord injury.

Differential Roles of M2 and M3 Muscarinic Receptor Subtypes in Modulation of Bladder Afferent Activity in Rats

OBJECTIVESnTo investigate the effects of various muscarinic acetylcholine receptor (mAChR) antagonists, including selective M2 and M3 mAChR antagonists, on bladder overactivity. It has been proposed that the urothelium modulates the activity of bladder afferent pathways. However, the differential roles of mAChR subtypes in local bladder afferent activation remain unclear.nnnMETHODSnCystometry was performed in urethane-anesthetized female rats. We examined the effects of intravesical administration of antimuscarinic agents (nonselective mAChR antagonists: atropine sulfate, tolterodine tartrate, and propiverine hydrochloride; M2-selective antagonists: dimethindene maleate and methoctramine hemihydrate; M3-selective antagonists: darifenacin hydrobromide and 4-DAMP) on bladder overactivity induced by oxotremorine-M (oxo-M; nonselective mAChR agonist).nnnRESULTSnIntravesical administration of oxo-M (200 microM) elicited bladder overactivity as evidenced by decreased intercontraction interval, bladder capacity, and pressure threshold. These effects were blocked by intravesical administration of nonselective or M2-selective antagonists (30-60 microM), whereas M3-selective antagonists (150 microM) did not suppress the overactivity. When instilled intravesically by itself, none of the antimuscarinic agents (nonselective, M2-selective or M3-selective antagonists) affected any cystometric parameters.nnnCONCLUSIONSnThe M2 mAChR subtype plays an important role in the local cholinergic modulation of bladder afferent activity that contributes to bladder overactivity in normal rats. Therefore, it is expected that antimuscarinic agents that have antagonistic activity against M2 mAChR can be more beneficial for the treatment of patients with overactive bladder if enhanced acetylcholine mechanisms are involved in pathogenesis of overactive bladder.

Herpes simplex virus vector-mediated gene delivery of glutamic acid decarboxylase reduces detrusor overactivity in spinal cord injured rats

We examined whether replication-defective herpes simplex virus (HSV) vectors encoding the 67u2009kDa form of the glutamic acid decarboxylase (GAD67) gene product, the γ-aminobutyric acid (GABA) synthesis enzyme, can suppress detrusor overactivity (DO) in rats with spinal cord injury (SCI). One week after spinalization, HSV vectors expressing GAD and green fluorescent protein (GFP) (HSV-GAD) were injected into the bladder wall. Rats with SCI without HSV injection (HSV-untreated) and those injected with lacZ-encoding reporter gene HSV vectors (HSV-LacZ) were used as controls. Three weeks after viral injection, continuous cystometry was performed under awake conditions in all three groups. In the HSV-GAD group, the number and amplitude of non-voiding contractions (NVCs) were significantly decreased (40–45% and 38–40%, respectively) along with an increase in voiding efficiency, compared with HSV-untreated and HSV-LacZ groups, but micturition pressure was not different among the three groups. Intrathecal application of bicuculline partly reversed the decreased number and amplitude of NVCs, and decreased voiding efficiency in the HSV-GAD group. In the HSV-GAD group, GAD67 mRNA and protein levels were significantly increased in the L6-S1 dorsal root ganglia (DRG) compared with the HSV-LacZ group, while 57% of DRG cells were GFP-positive, and these neurons showed increased GAD67-like immunoreactivity compared with the HSV-LacZ group. These results indicate that GAD gene therapy effectively suppresses DO after SCI predominantly through the activation of spinal GABAA receptors. Thus, HSV-based GAD gene transfer to bladder afferent pathways may represent a novel approach for treatment of neurogenic DO.

SUPPRESSION OF DETRUSOR-SPHINCTER DYSYNERGIA BY GABA-RECEPTOR ACTIVATION IN THE LUMBOSACRAL SPINAL CORD IN SPINAL CORD INJURED RATS

We investigated the effects of intrathecal application of GABAA- or GABAB-receptor agonists on detrusor-sphincter dyssynergia (DSD) in spinal cord transection (SCT) rats. Adult female Sprague-Dawley rats were used. At 4 wk after Th9-10 SCT, simultaneous recordings of intravesical pressure and urethral pressure were performed under an awake condition to examine the effect of intrathecal application of GABAA and GABAB agonists (muscimol and baclofen, respectively) or GABAA and GABAB antagonists (bicuculline and saclofen, respectively) at the level of L6-S1 spinal cord. In spinal-intact rats, the effects of bicuculline and saclofen on bladder and urethral activity were also examined. During urethral pressure measurements, DSD characterized by urethral pressure increases during isovolumetric bladder contractions were observed in 95% of SCT rats. However, after intrathecal application of muscimol or baclofen, urethral pressure showed urethral relaxation during isovolumetric bladder contractions. The effective dose to induce inhibition of urethral activity was lower compared with the dose that inhibited bladder contractions. The effect of muscimol and baclofen was antagonized by intrathecal bicuculline and saclofen, respectively. In spinal-intact rats, intrathecal application of bicuculline induced DSD-like changes. These results indicate that GABAA- and GABAB-receptor activation in the spinal cord exerts the inhibitory effects on DSD after SCT. Decreased activation of GABAA receptors due to hypofunction of GABAergic mechanisms in the spinal cord might be responsible, at least in part, for the development of DSD after SCT.

Nitric Oxide–Mediated Suppression of Detrusor Overactivity by Arginase Inhibitor in Rats with Chronic Spinal Cord Injury

OBJECTIVESnWe investigated the effects of an arginase inhibitor on bladder overactivity and measured bladder arginase I and II mRNA levels in rats with chronic spinal cord injury (SCI).nnnMETHODSnWe performed awake cystometrograms 3 to 4 weeks after spinal cord transection in female rats. Cystometric parameters such as mean amplitudes and number of non-voiding contractions (NVCs), voided volume, voiding efficiency, and micturition pressure were evaluated before and after intravenous (i.v.) injection of an arginase inhibitor (nor-NOHA: N(omega)-hydroxy-nor-L-arginine) in SCI rats. We also examined the effects of an NOS inhibitor (L-NAME: N(omega)-nitro-L-arginine methyl ester hydrochloride) to determine whether suppression of bladder overactivity by arginase inhibition is mediated by increased production of NO. In addition, we measured mRNA levels of arginase I and II in SCI bladders using quantitative real-time polymerase chain reaction (qRT-PCR).nnnRESULTSnWe found that nor-NOHA (10 mg/kg, i.v.) significantly decreased the amplitude and number of NVCs. There were no significant changes in other parameters before and after administration of vehicle or nor-NOHA at any dose. When we administered L-NAME (20 mg/kg, i.v.) before nor-NOHA injection (10 mg/kg, i.v.), nor-NOHA-induced inhibition of NVCs was prevented. The relative levels of both arginase I and II mRNA in the bladder were significantly higher in SCI rats compared with spinal cord-intact rats.nnnCONCLUSIONSnThese results suggest that arginase inhibition can suppress SCI-induced bladder overactivity as indicated by a reduction in NVCs. Thus, arginase inhibition could be an effective treatment for neurogenic bladder overactivity in pathological conditions such as SCI.

Suppression of Detrusor-Sphincter Dyssynergia by Herpes Simplex Virus Vector Mediated Gene Delivery of Glutamic Acid Decarboxylase in Spinal Cord Injured Rats

PURPOSEnWe investigated whether replication defective herpes simplex virus vectors encoding genes of glutamic acid decarboxylase, the gamma-aminobutyric acid synthesis enzyme, could suppress detrusor-sphincter dyssynergia in rats with spinal cord injury.nnnMATERIALS AND METHODSnOne week after spinalization herpes simplex virus vectors expressing glutamic acid decarboxylase and green fluorescent protein were injected into the bladder wall. Spinal cord injured rats without herpes simplex virus injection (sham treated) and those injected with LacZ encoding herpes simplex virus vectors served as controls. Three weeks after viral injection we simultaneously recorded urethral and intravesical pressure in awake rats.nnnRESULTSnIn the glutamic acid decarboxylase group the urethral pressure increase during bladder contraction was significantly decreased by 77% to 79% compared with that in the sham treated and LacZ groups. Bladder activity and urethral baseline pressure did not differ among the 3 groups. Intrathecal application of the gamma-aminobutyric acid-A receptor antagonist bicuculline almost completely reversed the decrease in the urethral pressure increase during bladder contractions while intrathecal saclofen (Tocris Cookson, Ellisville, Missouri), a gamma-aminobutyric acid-B receptor antagonist, partially reversed it. In the glutamic acid decarboxylase group the mRNA of glutamic acid decarboxylase 67 was significantly increased in L6-S1 dorsal root ganglia, which is where bladder afferents originate, compared with that in the LacZ group.nnnCONCLUSIONSnHerpes simplex virus based glutamic acid decarboxylase gene transfer to bladder afferent pathway may represent a novel approach to detrusor-sphincter dyssynergia in cases of spinal cord injury.

Neurophysiology and therapeutic receptor targets for stress urinary incontinence

Stress urinary incontinence is the most common type of urinary incontinence in women. Stress urinary incontinence involves involuntary leakage of urine in response to abdominal pressure caused by activities, such as sneezing and coughing. The condition affects millions of women worldwide, causing physical discomfort as well as social distress and even social isolation. This type of incontinence is often seen in women after middle age and it can be caused by impaired closure mechanisms of the urethra as a result of a weak pelvic floor or poorly supported urethral sphincter (urethral hypermobility) and/or a damaged urethral sphincter system (intrinsic sphincter deficiency). Until recently, stress urinary incontinence has been approached by clinicians as a purely anatomic problem as a result of urethral hypermobility requiring behavioral or surgical therapy. However, intrinsic sphincter deficiency has been reported to be more significantly associated with stress urinary incontinence than urethral hypermobility. Extensive basic and clinical research has enhanced our understanding of the complex neural circuitry regulating normal function of the lower urinary tract, as well as the pathophysiological mechanisms that might underlie the development of stress urinary incontinence and lead to the development of potential novel strategies for pharmacotherapy of stress urinary incontinence. Therapeutic targets include adrenergic and serotonergic receptors in the spinal cord, and adrenergic receptors at the urethral sphincter, which can enhance urethral reflex activity during stress conditions and increase baseline urethral pressure, respectively. This article therefore reviews the recent advances in stress urinary incontinence research and discusses the neurophysiology of urethral continence reflexes, the etiology of stress urinary incontinence and potential targets for pharmacotherapy of stress urinary incontinence.

Central nervous targets for the treatment of bladder dysfunction

The functions of the lower urinary tract, to store and periodically release urine, are dependent on the activity of smooth and striated muscles in the urinary bladder, urethra, and external urethral sphincter. This activity is in turn controlled by neural circuits in the brain, spinal cord, and peripheral ganglia.

Surgical outcomes of laparoscopic adrenalectomy for patients with Cushing's and subclinical Cushing's syndrome: a single center experience

ObjectiveWe retrospectively examined the outcome of patients who underwent laparoscopic adrenalectomy for Cushing’s/subclinical Cushing’s syndrome in our single institute.Patients and methodsBetween 1994 and 2008, a total of 114 patients (29 males and 85 females, median age 54xa0years) with adrenal Cushing’s/subclinical Cushing’s syndrome were studied. We compared the outcome of patients who underwent laparoscopic adrenalectomy between intraperitoneal and retroperitoneal approaches. Surgical complications were graded according to the Clavien grading system. We also examined the long-term results of subclinical Cushing’s syndrome after laparoscopic adrenalectomy.ResultsLaparoscopic surgical outcome did not differ significantly between patients with Cushing’s syndrome and those with subclinical Cushing’s syndrome. Patients who underwent laparoscopic intraperitoneal adrenalectomy had longer operative time than those who received retroperitoneal adrenalectomy (188.2xa0min vs. 160.9xa0min). However, operative blood loss and surgical complications were similar between both approaches. There were no complications of Clavien grade III or higher in either intraperitoneal or retroperitoneal approach. We confirmed the improvement of hypertension and glucose tolerance in patients with subclinical Cushing’s syndrome after laparoscopic adrenalectomy.ConclusionsLaparoscopic adrenalectomy for adrenal Cushing’s/subclinical Cushing’s syndrome is safe and feasible in either intraperitoneal or retroperitoneal approach. The use of the Clavien grading system for reporting complications in the laparoscopic adrenalectomy is encouraged for a valuable quality assessment.