30 min and then the bladder was allowed to drain with a catheter in place for 60 min as the studies continued. After killing the rats the function of the bladder was assessed, with carbachol and 100 m M KCl, and the levels of malondialdehyde (MDA, a marker of lipid peroxidation), 8-hydroxydeoxyguanosine (8-OHdG; a marker of oxidative DNA damage), heat-shock protein 70 (HSP 70) and its mRNA were measured. RESULTSAUR increased the intravesical pressure and decreased blood flow, and subsequent catheterization decreased the intravesical pressure and increased blood flow. Edaravone induced a decrease in blood flow in the bladder during the urinary retention and subsequent catheterization compared to the blood flow in the AUR group. Edaravone resulted in protection of the contractile responses to both carbachol and KCl in a dose-dependent manner. The MDA concentration, 8-OHdG content and expressions of HSP-70 and its mRNA in the AUR group were significantly larger than those of the control group. Edaravone markedly suppressed the accumulations of MDA and 8-OHdG in the bladder, and reduced the expressions of HSP 70 and its mRNA. CONCLUSIONThese results indicate that edaravone reduces the oxidative stress and prevents the bladder dysfunction caused by AUR and subsequent catheterization.

Our data indicate that noninsulin dependent diabetes induces alterations in the muscarinic receptor system, which may contribute to the development of diabetic cystopathy.

As there are increasing evidences that human diabetes induces cardiovascular dysfunction, we investigated the type-2 diabetes-induced endothelial dysfunction in the early and late-stage Goto-Kakizaki (GK) rat aorta. We performed organ bath studies, and examined the changes in expression levels of muscarinic M(3) receptor, endothelial, inducible, and neuronal nitric oxide synthase (eNOS, iNOS, and nNOS, respectively) mRNAs in the rat aorta utilizing real-time polymerase chain reaction in 12-week-old and 70-week-old GK rats as well as in age-matched Wistar rats. In the 12-week-old GK rat aorta, a significant increase in norepinephrine-induced contraction and a significant decrease in acetylcholine-induced relaxation as well as significant increases in expression levels of muscarinic M(3) receptor and eNOS and a significant decrease in nNOS mRNAs were observed compared to age-matched controls. In the older GK rat aorta, significant decreases in acetylcholine- and nitroglycerine-induced relaxations as well as significant decreases in the expression levels of muscarinic M(3) receptor, eNOS, iNOS, and nNOS mRNAs were observed compared to those in the younger GK rats. In contrast, although significant decreases in acetylcholine and nitroglycerine-induced relaxations were observed, the expression levels of muscarinic M(3) receptor, eNOS, iNOS, and nNOS mRNAs in the older Wistar rats aorta were unchanged, increased, increased and decreased, respectively, compared to the younger Wistar rat aorta. These results indicate that endothelial dysfunction in the rat aorta progresses with age and development of diabetes condition, and that decreased relaxations in the late-stage rat aorta may be due to these alterations.

We attempted to clarify the effects of cyclohexenonic long-chain fatty alcohol (N-hexacosanol) on nitric oxide synthase (NOS) in streptozotocin-induced diabetic nephropathy. After induction of experimental diabetes with streptozotocin, rats were maintained for 8 weeks with or without treatment by N-hexacosanol (8 mg/kg i.p. every day). Urinary albumin excretion, blood chemistry, immunoblot analysis, and real-time polymerase chain reactions (real-time PCR) of endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) were investigated. Although N-hexacosanol had no effects on serum glucose or insulin level, it normalized serum creatinine and urinary albumin excretion. N-hexacosanol was found to improve the diabetes-induced alterations in the eNOS, iNOS, and nNOS protein and their mRNA levels. Histologically, N-hexacosanol inhibited the progression to glomerular sclerosis. Our data suggest that N-hexacosanol improves diabetes-induced NOS alterations in the kidney, resulting in the amelioration of diabetic nephropathy.

We evaluated the effects of N-hexacosanol, a cyclohexenonic long-chain fatty alcohol, on muscarinic receptors in diabetic rat ileal dysfunction. Eight-week-old male SD rats were divided into four groups. After induction of diabetes (streptozotocin 50 mg/kg, i.p.), three groups were maintained for eight weeks with treatment by N-hexacosanol (0, 2 or 8 mg/kg, s.c. every day). Ileum function was investigated by organ bath studies using carbachol and KCl, and the expression levels of muscarinic M 2 and M 3 receptors were investigated by real-time polymerase chain reaction. Various concentrations of subtype-selective muscarinic antagonists, i.e., atropine (non-selective), pirenzepine (M 1 selective), methoctramine (M 2 selective), and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, M 1 /M 3 selective), were used in this study. In the presence and absence of these antagonists, contractile response curves to increasing concentrations of carbachol were investigated. Treatment with N-hexacosanol did not alter the diabetic status of the rats, but did significantly prevent the carbachol-induced hypercontractility in diabetic rat ileum. Estimation of the pA 2 values for atropine, pirenzepine, methoctramine, and 4-DAMP indicated that the carbacholinduced contractile response in the ileum is mainly mediated through the muscarinic M 3 receptor subtype in all groups. Furthermore, N-hexacosanol significantly prevented the diabetes-induced up-regulation of intestinal muscarinic M 2 and M 3 receptor mRNAs in streptozotocin-diabetic rats. Our data indicated that N-hexacosanol exerts preventive effects with respect to carbachol-induced hypercontractility in the diabetic rat ileum without qualitative alteration of the muscarinic receptor system.Gastrointestinal smooth muscles receive a variety of excitatory and inhibitory inputs from the enteric nervous system. As cholinergic nerves are activated, the neurotransmitter acetylcholine (ACh) is released from their terminals. Subsequently, ACh acts on smooth muscle cells to activate cell-surface muscarinic receptors, thus activating various intracellular signaling pathways, and in turn inducing smooth muscle contraction (6, 7). To date, five muscarinic receptor subtypes (M 1 -M 5 ) have been identified (5,20). A recent reverse transcriptase-polymerase chain reaction (RT-PCR) study reported the possible expression of all five subtypes in gastrointestinal smooth muscle (5). The muscarinic M 2 and M 3 receptor subtypes are preferentially expressed, with a preponderance of the former subtype. In general, the muscarinic M 3 receptor subtype in smooth muscle is coupled to the G protein Gq/G11 and mediates the