Kazumasa Matsumoto-Miyai

Extracellular Ca2+ regulates the stimulus-elicited ATP release from urothelium

Accumulating evidence shows that the epithelial cells in urinary bladder (urothelium) serve as a sensory organ in micturition and/or in nociception pathway by releasing ATP in response to mechanical and/or chemical stimuli. Here, we compared the effects of capsaicin, acetylcholine, and prostaglandin E(2) receptor EP1 agonist (ONO-DI-004) on the urothelial ATP release in primary cultured mouse urothelial cells in low Ca(2+) medium. All of these chemicals induced a gradual ATP release from urothelium, implying that the downstream Ca(2+) release from endoplasmic reticulum could trigger the ATP release. Consistent with this suggestion, blockade of inositol 1,4,5-triphosphate receptor reduced the distention-induced ATP release from urothelial tissues. The distention-induced ATP release was not affected by tetrodotoxin. However, an increase in extracellular Ca(2+) diminished both chemical- and distention-induced ATP release from urothelium. Thus raising the extracellular Ca(2+) concentration was found to inhibit stimulation-evoked ATP urothelial release.

Role of supraspinal and spinal α1-adrenergic receptor subtypes in micturition reflex in conscious rats

α(1)-Adrenergic receptor subtypes are widely distributed in the central nervous system and are involved in autonomic functions such as micturition. We investigated the presence and the role of supraspinal and/or spinal α(1)-adrenergic receptors in modulating the micturition reflex in conscious female Wistar rats. The expression of α(1)-adrenergic receptor subtypes in rat brain and lumbosacral spinal cord was studied using RT-PCR. Continuous-infusion cystometrograms were obtained in conscious rats, and α(1)-adrenergic receptor antagonists were administered via intracerebroventricular or intrathecal routes. The mRNA expression of α(1A)-, α(1B)-, and α(1D)-adrenergic receptors was detected in rat brain (midbrain and pons) and lumbosacral spinal cord (dorsal and ventral parts of spinal cord). In addition, intracerebroventricular injection of the α(1)-adrenergic receptor antagonist tamsulosin (1-10 μg), the selective α(1A)-adrenergic receptor antagonist silodosin (1-10 μg), and the selective α(1D)-adrenergic receptor antagonist BMY 7378 (1-10 μg) significantly prolonged the intercontraction interval (ICI) but did not alter maximum voiding pressure (MVP). Although intrathecal injection of BMY 7378 (0.0001-10 μg) did not affect ICI, tamsulosin and silodosin prolonged ICI in a dose-dependent manner. MVP was significantly reduced by intrathecal injection of tamsulosin (10 μg) but not by silodosin or BMY 7378 (0.0001-10 μg). Supraspinal α(1A)- and α(1D)-adrenergic receptors are apparently important for the regulation of reflex-bladder activity in conscious rats. Noradrenergic projection from the brain stem to the lumbosacral spinal cord may promote the afferent limb rather than the efferent limb of the micturition reflex pathway via α(1A)-adrenergic receptors.

Store-operated Ca2+ entry suppresses distention-induced ATP release from the urothelium

Epithelial cells in the urinary bladder (urothelium) trigger sensory signals in micturition by releasing ATP in response to distention of the bladder wall. Our previous study revealed the distinct roles of extracellular Ca(2+) and the Ca(2+) stores in the endoplasmic reticulum (ER) in urothelial ATP release. In the present study, we investigated the regulation of urothelial ATP release by Ca(2+) influx from the extracellular space and Ca(2+) release from the ER using a distention assay of the mouse bladder wall in a small Ussing chamber. Stimulation of Ca(2+) release from the ER in the mucosal side of the bladder induced significant ATP release without distention. Blockade of the inositol 1,4,5-triphosphate receptor reduced distention-induced ATP release, suggesting that Ca(2+) release from the ER is essential for the induction of urothelial ATP release. On the other hand, blockade of store-operated Ca(2+) entry (SOCE) from the extracellular space significantly enhanced distention-induced ATP release. Thus Ca(2+) release from the ER causes urothelial ATP release and depletion of Ca(2+) stores in the ER, which in turn causes the depletion-inducing SOCE to suppress the amount of urothelial ATP released.

Authentic role of ATP signaling in micturition reflex.

Adenosine triphosphate (ATP) is a signaling molecule that regulates cellular processes. Based on previous studies of bladder function over the past decade, bladder ATP signaling was thought to have an essential role in the normal micturition reflex. In this study, we performed detailed analyses of bladder function in purinergic receptor-deficient mice using the automated voided stain on paper method and video-urodynamics. Unexpectedly, a lack of P2X2 or P2X3 receptors did not affect bladder function under normal physiological conditions, indicating that bladder ATP signaling is not essential for normal micturition reflex. In contrast, we found that lipopolysaccharide (LPS) induced markedly high levels of ATP release from the urothelium. In addition, LPS-induced rapid bladder hyperactivity was attenuated in P2X2−/− and P2X3−/− mice. Contrary to the previous interpretation, our present findings indicate that bladder ATP signaling has a fundamental role in the micturition reflex, especially in bladder dysfunction, under pathological conditions. Therefore, the bladder ATP signaling pathway might be a highly promising therapeutic target for functional bladder disorders. This study newly defines an authentic role for bladder ATP signaling in the micturition reflex.

Regulatory Effects of 5-Hydroxytryptamine Receptors on Voiding Function.

A growing body of evidence suggests that 5-hydroxytryptamine (5-HT; serotonin) has both physiological and pathological functions in the lower urinary tract. A wide variety of 5-HT receptor subtypes are variably expressed in different organs, both peripheral and central. On urinary bladder smooth muscle, 5-HT1A, 5-HT2, 5-HT3, and 5-HT7 subtypes could function as postjunctional receptors. Postjunctional 5-HT2 receptors induce detrusor contraction of the bladder body. 5-HT1A is suggested to have a similar effect to 5-HT2, while 5-HT3 might suppress detrusor contraction evoked by direct muscle stimulation. Postjunctional 5-HT7 is reported to induce relaxation of the bladder neck, which might be required for efficient voiding. 5-HT1A, 5-HT2A, 5-HT2C, 5-HT3, 5-HT4, and 5-HT7 subtypes also could act as prejunctional receptors in autonomic excitatory nerve terminals. 5-HT2A, 5-HT2C, 5-HT3, 5-HT4, and 5-HT7 subtypes facilitate the neurogenic contraction of the detrusor by enhancing cholinergic or purinergic transmission, whereas 5-HT1A receptors might inhibit the release of acetylcholine in the detrusor. Furthermore, 5-HT1D could be involved in the suppression of ATP release from the urothelium, aiding visceral sensation of the urinary bladder. In the central pathways controlling the micturition reflex, 5-HT1A, 5-HT2A, and 5-HT7 are involved in regulation of bladder and urethral sphincter activities. Their functions, especially that of 5-HT1A, vary in a species- and site (spinal or supraspinal)- dependent manner. In addition to urinary bladder, 5-HT could be involved in prostate contraction and cell proliferation. Evidence indicates that 5-HT receptor subtypes may be novel therapeutic targets for lower urinary tract symptoms.FundingGrants-in-Aid for Scientific Research (C) (KAKENHI 23590707, 24590722, and 26460694) from the Japan Society for the Promotion of Science.

Urothelial α1D Receptor is Predominantly Involved in the Adrenergic Facilitation of Micturition Reflex

Previous studies using specific antagonists have shown that α1‐adrenergic receptors (AR) in the urinary bladder epithelium (urothelium) serve as a regulator of micturition reflex. To confirm the urothelial adrenergic effect on the micturition reflex, we investigated the functional role of the α1D‐AR subtype in the urothelium on micturition reflex using α1D‐AR knockout (α1D‐KO) mice. An immunohistochemical analysis showed that the α1D‐AR was localized to the umbrella cells in the mouse urothelium. Cystometry using wild‐type mice demonstrated that the intravesical infusion of noradrenaline (NA) into the urinary bladder shortened the intercontraction interval (ICI) in a dose‐dependent manner, without changing the maximum voiding pressure (MVP). In contrast, α1D‐KO mice exhibited no change in the ICI in response to the intravesical infusion of NA. These results indicate a predominant involvement of the α1D‐AR in facilitation of micturition reflex by NA. Interestingly, the intravenous injection of NA did not alter the ICI and the MVP in wild‐type mice. Thus, locally releasing NA might play an important role in the facilitation of micturition reflex via the urothelial α1D−AR subtype.

Serotonergic regulation of distention-induced ATP release from the urothelium

Serotonin [5-hydroxytryptamine (5-HT)] is involved in both motor and sensory functions in hollow organs, especially in the gastrointestinal tract. However, the involvement of 5-HT in visceral sensation of the urinary bladder remains unknown. Because distention-induced ATP release from the urothelium plays an essential role in visceral sensation of the urinary bladder, we investigated the regulation of urothelial ATP release by the 5-HT signaling system. RT-PCR and immunohistochemical analyses of the urothelium revealed specific expression of 5-HT1D and 5-HT4 receptors. The addition of 5-HT did not affect urothelial ATP release without bladder distention, but it significantly reduced distention-induced ATP release by physiological pressure during urine storage (5 cmH2O). The inhibitory effect of 5-HT on distention-elicited ATP release was blocked by preincubation with the 5-HT1B/1D antagonist GR-127935 but not by the 5-HT4 antagonist SB-204070. mRNA encoding tryptophan hydroxylase 1 was detected in the urinary bladder by nested RT-PCR amplification, and l-tryptophan or the selective serotonin reuptake inhibitor citalopram also inhibited ATP release, indicating that 5-HT is endogenously synthesized and released in the urinary bladder. The addition of GR-127935 significantly enhanced the distention-elicited ATP release 40 min after distention, whereas SB-204070 reduced the amount of ATP release 20 min after distention. These data suggest that 5-HT4 facilitates the distention-induced ATP release at an earlier stage, whereas 5-HT1D inhibits ATP release at a later stage. The net inhibitory effect of 5-HT indicates that the action of 5-HT on the urothelium is mediated predominantly by 5-HT1D.

PD70-06 THE ROLE OF UROTHELIAL ATP SIGNALING IN MICTURITION REFLEX

nociceptive neurons (TRPV1-ChR2 and Nav 1.8-Arch). These opsins were activated be either blue (ChR2) or green (Arch) laser or LED illumination of the bladder. We then measured the influence of opsin activation on bladder function using continuous infusion cystometry. In order to translate this into a more clinically relevant gene delivery system, bladder wall injection of herpes simplex viral vector (HSV) with a pan-neuronal promotor controlling expression of ChR2 or Arch were used in rats. RESULTS: In TRPV1-ChR2 mice, ChR2 activation with blue light resulted in voiding events in a half full bladder that increased in magnitude with increase of light intensity. Activation of Arch with green light could delay regular cystometric contractions in Nav 1.8-Arch mice. In rats transduced with HSV-ChR2, we were able to evoke voiding events with ChR2 activation by blue light illumination of the bladder, after less filling than under normal conditions (non-stimulated). These effects were not seen in rats injected with the control viral vector, HSVeYFP. Finally, illumination of the bladder with green light delayed regular cystometric contractions in rats injected with HSV-Arch but not in animals injecting with HSV-eYFP. CONCLUSIONS: Here we demonstrate bidirectional modulation of bladder function using optogenetics.. This bidirectional control of bladder function can be induced by selective targeting of nociceptive afferents. Further restriction of opsin expression to specific populations of bladder sensory fibers could lead to a better understanding of their role in bladder function and disease. The refinement of virally delivery methods for optogenetic proteins, together with the development of medical devices for light delivery could lead to development of future therapies for bladder dysfunction.

Nerve Growth Factor and Estrogen Receptor mRNA Expression in Paravertebral Muscles of Patients With Adolescent Idiopathic Scoliosis: A Preliminary Study

STUDY DESIGN Comparison of nerve growth factor (NGF) and estrogen receptor (ER)α messenger ribonucleic acid (mRNA) expression in bilateral paravertebral muscles in adolescent idiopathic scoliosis (AIS). This expression in AIS was compared with that of normal control subjects. OBJECTIVES To investigate NGF and ERα mRNA expression in bilateral paravertebral muscles in AIS and control subjects to clarify its association with the development and progression of spinal curvature. SUMMARY OF BACKGROUND DATA Paravertebral muscle abnormalities in AIS patients have been investigated through various methods. Despite the roles of NGF and ER in human skeletal muscles, the association with idiopathic scoliosis is still unclear. METHODS A total of 14 AIS patients (average age, 15.9 ± 2.2 years; average Cobb angle, 48.2° ± 8.9°) and 8 controls (average age, 27.3 ± 9.3 years) were included. Muscle samples were harvested from bilateral paravertebral muscles at the apical vertebral level. Nerve growth factor and ERα mRNA expression was evaluated by the real-time polymerase chain reaction. The researchers compared expression levels in bilateral paravertebral muscles in each group. The expression ratio, the expression at the convex side relative to the concave side, was compared between groups and the correlation between Cobb angle and expression ratio was analyzed. RESULTS Nerve growth factor and ERα mRNA expression on the convex side was higher than on the concave side in the AIS group (p = .024 and .007, respectively) and the expression ratio of NGF and ERα in the AIS group was higher than that of control subjects (p = .004 and .017, respectively). The expression ratio of NGF and the Cobb angle were significantly correlated (r = -0.5728; p = .0323). CONCLUSIONS In the AIS group, both NGF and ERα mRNA expression was asymmetric. The AIS group had higher expression ratios than control group and the NGF expression ratio was positively correlated to the Cobb angle.

Pulse Diode Laser Irradiation (830 nm) of Lumbosacral Spinal Roots Diminished Hyperreflexia-Induced by Acetic Acid or Prostaglandin E2 Infusion in Rat Urinary Bladder.

Objectives: Low power diode Iaser (830 nm) irradiation is a useful analgesic tool in superficial pain. Pulse laser irradiation allows us to increase the laser power because the non‐irradiation time reduces heating effects and/or direct tissue damage at the irradiation area. This new irradiation device using pulse laser was applied to the dorsal skin to investigate the effects on the micturition reflex in the rat by targeting underlying sacral spinal roots.