Anoxia/reoxygenation enhances spontaneous contractile activity via TRPA1 channel and COX2 activation in isolated rat whole bladder.

PURPOSE: Bladder ischemia/reperfusion is an important etiologic factor for overactive bladder disease. The occurrence of this disease is closely associated with enhanced spontaneous contractile activity of the bladder. However, the relationship between bladder ischemia/reperfusion and altered spontaneous bladder contractions (SBC) remains poorly studied. Therefore, the present study investigated whether ischemia/reperfusion affects SBC ex vivo. METHODS: SBC was measured using isolated whole bladder preparations from rats. The preparations were exposed to anoxia (95% N2 ) for 0.5-6 h, followed by reoxygenation (95% O2 ) in Krebs medium. RESULTS: Anoxia followed by reoxygenation significantly enhanced the amplitude of SBC without affecting its frequency in an anoxic duration-dependent manner. The 5 h anoxia/reoxygenation-induced enhancement of SBC amplitude was completely suppressed by an antioxidant combination of L(+)-ascorbate/D, L-α-tocopherol, or N-acetyl cysteine. Additionally, the enhanced SBC amplitude was inhibited in a concentration-dependent manner by the nonselective TRP antagonist ruthenium red, or selective TRPA1 antagonists HC-030031 or AP-18. A similar inhibitory effect was obtained after repeated treatment with the TRPA1 agonist allyl isothiocyanate, as it induced acute desensitization of TRPA1 channels. Further, the enhanced SBC amplitude was significantly diminished by the nonselective cyclooxygenase (COX) inhibitor indomethacin or selective COX2 inhibitor NS-398, but not by the selective COX1 inhibitor SC-560 and 5-lipoxygenase inhibitor MK-886. CONCLUSIONS: The study findings reveal that the spontaneous contractile activity of the bladder is significantly enhanced in response to anoxia/reoxygenation, and that oxidative stress and activation of TRPA1 and COX2 (the resulting production of prostaglandins) are involved in the enhanced SBC activity.

In vivo bladder selectivity of imidafenacin, a novel antimuscarinic agent, assessed by using an effectiveness index for bladder capacity in rats.

Imidafenacin (KRP-197) is a novel antimuscarinic agent for overactive bladder treatment. The inhibitory effect of imidafenacin on detrusor contraction has been adopted for assessing their bladder selectivity, but this is becoming less convincing as an effectiveness index. We, therefore, reevaluated the bladder selectivity of imidafenacin and other antimuscarinics using their effects on the bladder capacity as an effectiveness index. Bladder capacity was measured by intermittent cystometry in urethane-anesthetized rats. In the tissues related to antimuscarinic side effects, the inhibitory actions were measured each on salivary secretion by electrical stimulation of chorda tympani, on rhythmical contractions in colon, and on carbamylcholine-induced bradycardia. Imidafenacin, solifenacin succinate, tolterodine tartrate, and propiverine hydrochloride significantly increased the bladder capacity, with minimum effective doses of 0.003, 1, 0.03, and 3 mg/kg (i.v.), respectively. The antimuscarinics tested, except for propiverine hydrochloride, shared a common property of increasing bladder capacity at a dose which did not affect micturition pressure. The relative bladder selectivity of imidafenacin, solifenacin succinate, and tolterodine tartrate was 15-, 1.7-, and 2.5-fold higher over salivary gland; 150-, 1.9-, and 9.2-fold higher over colon; and 50-, 12-, and 4.6-fold higher over heart, respectively, than that of propiverine hydrochloride. Thus, imidafenacin shows the most highly selective for bladder over the tissues related to major antimuscarinic side effects, compared to the other three well-known antimuscarinics tested in the rat.

Ibudilast, a mixed PDE3/4 inhibitor, causes a selective and nitric oxide/cGMP-independent relaxation of the intracranial vertebrobasilar artery.

Ibudilast, a mixed phosphodiesterase (PDE) 3/4 inhibitor, is a cerebral vasodilator widely used in Japan for treating post-stroke dizziness. However, little studies have been conducted on the vasorelaxant effects of PDE inhibitors in the vertebrobasilar artery associated with dizziness onset. The in vitro vasorelaxant properties of ibudilast were, therefore, investigated by comparing with known selective PDE inhibitors, using vertebrobasilar arteries. Vasorelaxant activities of PDE3, PDE4, PDE5 inhibitors, and ibudilast were assessed in 5-hydroxytryptamine precontracted ring preparations from rabbit intracranial and extracranial vertebrobasilar arteries. Ibudilast more selectively relaxed the intracranial than extracranial artery. Similarly, selective PDE3 and PDE4 inhibitors showed higher selectivity for intracranial arteries. Furthermore, like selective PDE4 inhibitor, the vasorelaxation by ibudilast accompanied by increase in cAMP levels was inhibited by the adenylyl cyclase inhibitor SQ22536 in intracranial arteries. Next, it was examined whether nitric oxide (NO)/cGMP signaling is involved in this vasorelaxation in intracranial arteries. The suppression of NO/cGMP signaling by an NO synthase inhibitor or a guanylyl cyclase inhibitor potentiated the vasorelaxion by a PDE3 inhibitor and reduced that by a PDE4 inhibitor, while either suppression of the signaling had little influence on that by ibudilast. These results suggest that ibudilast has the high vasoselectivity for intracranial artery based on a mixed PDE3 and PDE4-inhibition, and effectively relaxes intracranial arteries independently of NO/cGMP signaling because of its vasorelaxation compensated by either PDE3- or PDE4-inhibition depending on the state of NO/cGMP signaling change.

Design and synthesis of novel 7-heterocycle-6-trifluoromethyl-3-oxoquinoxaline-2-carboxylic acids bearing a substituted phenyl group as superior AMPA receptor antagonists with good physicochemical properties.

We describe the design, synthesis, and physicochemical and biological properties of a novel series of 7-heterocycle-6-trifluoromethyl-3-oxoquinoxaline-2-carboxylic acids bearing a substituted phenyl group joined through a urethane or urea linkage to the heterocycle at the 7 position. Introduction of the trifluoromethyl group at the 6 position conferred good biological activity, including neuroprotective effects, as well as good physicochemical properties. In terms of alpha-amino-3-hydroxy-5-methylisoxazole propionate receptor (AMPA-R) affinity, a urea linkage was equivalent to a urethane linkage and a pyrrole ring at the 7 position reduced affinity in comparison with an imidazole ring. Among this series, compound 14h (KRP-199), which has a 4-carboxyphenyl group joined through a urethane linkage to a 7-imidazolyl heterocycle, was found to possess high potency and selectivity for the AMPA-R in vitro and to exhibit good neuroprotective effects in vivo. Furthermore, the compound showed good physicochemical properties, including stability to light and good solubility in aqueous solutions.

Neuroprotective effects of KCL-440, a new poly(ADP-ribose) polymerase inhibitor, in the rat middle cerebral artery occlusion model.

It is reported that ischemic brain injury is mediated by the activation of poly(ADP-ribose) polymerase (PARP). In this study, we examined the pharmacological profile of KCL-440, a new PARP inhibitor, and its neuroprotective effects in the rat acute cerebral infarction model induced by photothrombotic middle cerebral artery (MCA) occlusion. In an in vitro study, KCL-440 exhibited potency with regard to inhibition of PARP activity, with an IC50 value of 68 nM. An in vivo pharmacokinetic study showed that the brain concentration of KCL-440 was sufficient to inhibit PARP activity during the intravenous infusion of KCL-440 at the rate of 1 mg/kg/h. KCL-440 at various doses or saline was administered for 24 h immediately after the MCA occlusion. Administration of KCL-440 led to a dose-dependent reduction in the infarct size at 24 h after MCA occlusion. Infarct sizes were 44.8% +/- 3.0% (n = 8), 40.5% +/- 1.1% (n = 8), 38.2% +/- 1.4% (n = 8), 35.1% +/- 2.1% (n = 8), 34.2% +/- 2.3% (n = 7), 32.6% +/- 1.9% (n = 8), and 31.0% +/- 2.1% (n = 5) at doses of 0, 0.01, 0.03, 0.1, 0.3, 1.0, and 3.0 mg/kg/h. When compared to the control group, a statistically significant difference was observed in the doses that were higher than 0.03 mg/kg/h. When the infusion of KCL-440 (1 mg/kg/h, n = 8) was started at 1 h after the MCA occlusion, a significant reduction in infarct size was observed; this was not observed when KCL-440 infusion was started 2 or 3 h after the MCA occlusion. Furthermore, increased poly(ADP-ribose) immunostaining was confirmed at the ischemic border zone 2 h after the MCA occlusion, and it was reduced by KCL-440 treatment. These results suggest that KCL-440 is a possible neuroprotective agent with high blood-brain barrier permeability and high PARP inhibitory activity.