Masahito Kawatani

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.

Effects of Pituitary Adenylate Cyclase—Activating Polypeptide on Facial Nerve Recovery in the Guinea Pig

Objectives/Hypothesis Pituitary adenylate cyclase–activating polypeptide (PACAP) has neurotrophic effects of neural regeneration and gives protection to the nervous system. We investigated whether PACAP had a neurotrophic effect on peripheral motoneurons and whether PACAP could facilitate glial cell line–derived neurotrophic factor (GDNF), a neurotrophin, in nerve regeneration. The presence and distribution of PACAP receptors following facial nerve transection were also investigated.

Nitric oxide raises cytosolic concentrations of Ca2+ in cultured nodose ganglion neurons from rabbits.

The effects of donors of nitric oxide (NO), namely, S-nitroso-N-acetylpenicillamine-(SNAP) and E-methyl-2-E-hydroxyimino-5-nitro-6-methoxy-3-hexeneamide (NOR1), on cytosolic concentrations of Ca2+ ([Ca2+]i) were studied in cultured nodose ganglion neurons from newborn and young rabbits by loading with fura-2AM and microfluorometry. Application of SNAP (5 microM-1 mM) increased [Ca2+]i in 46% of neurons tested. The threshold dose of the response was 10 microM and the response increased in a dose-dependent fashion. The increase in [Ca2+]i at 50 microM averaged 74 +/- 8% above the control value. [Ca2+]i rose immediately after injection of SNAP and the plateau level was maintained in the presence of SNAP.NOR1, another donor of NO, increased [Ca2+]i with an average increase of 82 +/- 7% at 50 microM. Quantitation of NO gas in the solution of NOR1 by a redox chemiluminescence method revealed the constant release of 0.06 ppm NO from 5 ml of a 20 microM solution of NOR1. These data suggest that NO released from donors of NO induced an increase in [Ca2+]i in nodose ganglion neurons and, therefore, that NO might play a role as a transmitter or a modulator in autonomic primary sensory systems.

Effectiveness of an anti‐inflammatory drug, loxoprofen, for patients with nocturia

Abstract

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.

Effects of noradrenaline on cytosolic concentrations of Ca2+ in cultured corpus cavernosum smooth muscle cells of the rabbit

An immunohistochemical examination of cultured corpus cavernosum cells of the rabbit showed that they were composed of many smooth muscle cells retaining -smooth muscle actin and a few of endothelial cells. Application of noradrenaline (NA, 0.5-100 microM) to the culture dose-dependently increased cytosolic concentrations of Ca2+ ([Ca2+]i) in smooth muscle cells. The NA (10 microM)-induced [Ca2+]i increase was dose-dependently inhibited by phenoxybenzamine (1 nM-1 microM). The inhibition was 71% at 0.1 microM and was completely made at 1 microM. An alpha(1)-adrenoceptor antagonist, prazosin (0.1 microM), inhibited the [Ca2+]i response by 42%, and propranolol (1 microM) enhanced the response by 118%. These adrenoceptor antagonists showed almost the same effects on a contractile response of corporal tissue strips to NA (10 microM) as the effects on the [Ca2+]i response. Our results suggest that NA-induced [Ca2+]i increase was mixed with a large increase through 1-adrenoceptors and a small decrease through beta-adrenoceptors. The increased [Ca2+]i could play a role of intracellular messenger for leading to contraction of cavernosal smooth muscle cells.

PACAP facilitate the nerve regeneration factors in the facial nerve injury

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported as a strong neurotrophic factor in the various sites of nervous system. The facial nerve injury is one of the common problems in patients at the Otolaryngology since the nerve damage could occur easily due to the anatomical characteristics. Once it happens, the regeneration is little observed and functional recovery is poor. Thus, we investigated that PACAP might have some influence for regeneration after the facial nerve transaction in the guinea pig. PACAP treatment accelerated time for the appearance of compound muscle action potentials (CMAP) after the nerve transaction (first appeared at 1 versus 2 weeks in control) and shortened the latency at 4 weeks. The number of myelinated fibers increased at 4 weeks. Histochemical demonstration of GAP-43, a growth cone protein, was observed at the injury area at 2-4 days. PACAP increased the level of glial cell line-derived neurotrophic factor (GDNF), a neurotrophin, in facial target muscles at 1 day-4 weeks. These data indicated that PACAP promotes the regeneration factors and increases the possibility of functional recovery following the facial nerve injury.

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.

MK-801 inhibits the micturition reflex in chronic bladder irritation caused by crystalluria in the rat

Urodynamic and pharmacological studies were performed to investigate the effect of crystalluria on the micturition reflex and the involvement of glutamatergic transmission. The rats, which were given LP-805 (100 mg/kg/day) orally for 12 days, voided crystalluria. The pH of these crystalluria (LP-805 urine) was the same as normal urine. The amount of crystals was 70-100/division magnified 400 x. The end of the crystals was sharp. Intravesical administration of LP-805 urine induced hyperreflexia of the micturition reflex in normal rats. When the infusion solution was changed to LP-805 urine from saline, the latency was reduced to 57.6+/-2.1% of control in single cystometrogram (CMG) or was reduced to 51.4+/-0.9% of control in continuous CMG. The voiding volume was reduced to 52.1+/-3.6% of control in single CMG or was reduced to 62.5+/-0.8% of control in continuous CMG. These parameters were recovered after LP-805 urine was removed. Intravesical administration of acetic acid did not induce hyperreflexia of the micturition reflex in LP-805-treated rats. These data suggest that the chronic irritation by aculeate crystals might induce hyperreflexia of the micturition reflex, which increase afferent neuronal activity. Intravenous administration of MK-801 (0.001 to 1 mg/kg) inhibited the micturition reflex in a dose-dependent manner. The ID50 in LP-805-treated rats (0.03 mg/kg i.v.) was lower than that in normal rats (0.56 mg/kg i.v.). After chronic irritation of the bladder epithelium, MK-801 sensitivity was enhanced for the micturition reflex. These data suggested that crystalluria elicit hyperreflexia in the micturition reflex that mediated with NMDA glutamatergic receptors.

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.