Ketamine abusers develop severe lower urinary tract symptoms. The major aims of the present study were to elucidate ketamine-induced ulcerative cystitis and bladder apoptosis in association with oxidative stress mediated by mitochondria and the endoplasmic reticulum (ER). Sprague-Dawley rats were distributed into three different groups, which received normal saline or ketamine for a period of 14 or 28 days, respectively. Double-labeled immunofluorescence experiments were performed to investigate tight junction proteins for urothelial barrier functions. A TUNEL assay was performed to evaluate the distribution of apoptotic cells. Western blot analysis was carried out to examine the expressions of urothelial tight junction proteins, ER stress markers, and apoptosis-associated proteins. Antioxidant enzymes, including SOD and catalase, were investigated by real-time PCR and immunofluorescence experiments. Ketamine-treated rats were found to display bladder hyperactivity. This bladder dysfunction was accompanied by disruptions of epithelial cadherin- and tight junction-associated proteins as well as increases in the expressions of apoptosis-associated proteins, which displayed features of mitochondria-dependent apoptotic signals and ER stress markers. Meanwhile, expressions of mitochondria respiratory subunit enzymes were significantly increased in ketamine-treated bladders. Conversely, mRNA expressions of the antioxidant enzymes Mn-SOD (SOD2), Cu/Zn-SOD (SOD1), and catalase were decreased after 28 days of ketamine treatment. These results demonstrate that ketamine enhanced the generation of oxidative stress mediated by mitochondria- and ER-dependent pathways and consequently contributed to bladder apoptosis and urothelial lining defects. Such oxidative stress-enhanced bladder cell apoptosis and urothelial barrier defects are potential factors that may play a crucial role in bladder overactivity and ulceration.

1 7-[2-[4-(2-chlorophenyl)piperazinyl]ethyl]-1,3-dimethylxanthine (KMUP-1) produces tracheal relaxation, intracellular accumulation of cyclic nucleotides, inhibition of phosphodiesterases (PDEs) and activation of K þ channels. 2 KMUP-1 (0.01-100 mM) induced concentration-dependent relaxation responses in guinea-pig epithelium-intact trachea precontracted with carbachol. Relaxation responses were also elicited by the PDE inhibitors theophylline, 3-isobutyl-1-methylxanthine (IBMX), milrinone, rolipram and zaprinast (100 mM), and a K ATP channel opener, levcromakalim. 3 Tracheal relaxation induced by KMUP-1 was attenuated by epithelium removal and by pretreatment with inhibitors of soluble guanylate cyclase (sGC) (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 1 mM), nitric oxide synthase (N o -nitro-L-arginine methyl ester, 100 mM), K þ channels (tetraethylammonium, 10 mM), K ATP channels (glibenclamide, 1 mM), voltage-dependent K þ channels (4-aminopyridine, 100 mM) and Ca 2 þ -dependent K þ channels (charybdotoxin, 0.1 mM or apamin, 1 mM). 4 Both KMUP-1 (10 mM) and theophylline nonselectively and slightly inhibited the enzyme activity of PDE3, 4 and 5, suggesting that they are able to inhibit the metabolism of adenosine 3 0 ,5 0 -cyclic monophosphate (cyclic AMP) and guanosine 3 0 ,5 0 -cyclic monophosphate (cyclic GMP). Likewise, the effects of IBMX were also measured and its IC 50 values for PDE3, 4 and 5 were 6.571.2, 26.373.9 and 31.775.3 mM, respectively. 5 KMUP-1 (0.01-10 mM) augmented intracellular cyclic AMP and cyclic GMP levels in guinea-pig cultured tracheal smooth muscle cells. These increases in cyclic AMP and cyclic GMP were abolished in the presence of an adenylate cyclase inhibitor SQ 22536 (100 mM) and an sGC inhibitor ODQ (10 mM), respectively. 6 KMUP-1 (10 mM) increased the expression of protein kinase A (PKA RI ) and protein kinase G (PKG 1a1b ) in a time-dependent manner, but this was only significant for PKG after 9 h. 7 Intratracheal administration of tumour necrosis factor-a (TNF-a, 0.01 mg kg À1 ) induced bronchoconstriction and exhibited a time-dependent increase in lung resistance (R L ) and decrease in dynamic lung compliance (C dyn ). KMUP-1 (1.0 mg kg À1 ), injected intravenously for 10 min before the intratracheal TNF-a, reversed these changes in R L and C dyn . 8 These data indicate that KMUP-1 activates sGC, produces relaxation that was partly dependent on an intact epithelium, inhibits PDEs and increases intracellular cyclic AMP and cyclic GMP, which then increases PKA and PKG, leading to the opening of K þ channels and resulting tracheal relaxation.

1 KMUP-1 (1, 3, 5 mg kg 71 , i.v.), a xanthine derivative, produced dose-dependent sustained hypotensive and short-acting bradycardiac eects in anaesthetized rats. This hypotensive eect was inhibited by pretreatment with glibenclamide (5 mg kg 71 , i.v.). 2 In endothelium-intact or denuded aortic rings preconstricted with phenylephrine, KMUP-1 caused a concentration-dependent relaxation. This relaxation was reduced by endothelium removal, the presence of NOS inhibitor L-NAME (100 mM) and sGC inhibitors methylene blue (10 mM) and ODQ (1 mM). 3 The vasorelaxant eects of KMUP-1 was attenuated by pretreatment with various K + channel blockers TEA (10 mM), glibenclamide (1 mM), 4-AP (100 mM), apamin (1 mM) and charybdotoxin (ChTX, 0.1 mM). 4 Increased extracellular potassium levels (30 ± 80 mM) caused a concentration-related reduction of KMUP-1-induced vasorelaxations. Preincubation with KMUP-1 (1, 10, 100 nM) increased the AChinduced maximal vasorelaxations mediated by endogenous NO release, and enhanced the potency of exogenous NO-donor SNP. 5 The vasorelaxant responses of KMUP-1 (0.01, 0.05, 0.1 mM) together with a PDE inhibitor IBMX (0.5 mM) had an additive action. Additionally, KMUP-1 (100 mM) aected cyclic GMP metabolism since it inhibited the activity of PDE in human platelets. 6 KMUP-1 induced a dose-related increase in intracellular cyclic GMP levels in rat A10 vascular smooth muscle (VSM) cells, but not cyclic AMP. The increase in cyclic GMP content of KMUP-1 (0.1 ± 100 mM) was almost completely abolished in the presence of methylene blue (10 mM), ODQ (10 mM), and L-NAME (100 mM). 7 In conclusion, these results indicate that KMUP-1 possesses the following merits: (1) stimulation of NO/sGC/cyclic GMP pathway and subsequent elevation of cyclic GMP, (2) K + channels opening, and (3) inhibition of PDE or cyclic GMP breakdown. Increased cyclic GMP display a prominent role in KMUP-1-induced VSM relaxations.

1 In isolated endothelium-intact or denuded rabbit corpus cavernosum preconstricted with phenylephrine, KMUP-1 (0.001 ± 10 mM) caused a concentration-dependent relaxation. 2 This relaxation of KMUP-1 was attenuated by endothelium removed, high K + and pretreatments with a soluble guanylyl cyclase (sGC) inhibitor ODQ (1 mM), a NOS inhibitor L-NAME (100 mM), a K + channel blocker TEA (10 mM), a K ATP channel blocker glibenclamide (1 mM), a voltage-dependent K + channel blocker 4-AP (100 mM) and Ca 2+-dependent K + channel blockers apamin (1 mM) and charybdotoxin (ChTX, 0.1 mM). 3 The relaxant responses of KMUP-1 (0.01, 0.05, 0.1 mM) together with a PDE inhibitor IBMX (0.5 mM) had additive actions on rabbit corpus cavernosum smooth muscle (CCSM). 4 KMUP-1 (0.01 ± 10 mM) induced increase of intracellular cyclic GMP level in the primary cell culture of rabbit CCSM. This increase in cyclic GMP content was abolished in the presence of ODQ (10 mM). 5 Both KMUP-1 and sildena®l at 0.2, 0.4, 0.6 mg kg 71 caused increases of intracavernous pressure (ICP) and duration of tumescene (DT) in a dose-dependent manner. These in vivo activities of ICP for sildena®l and KMUP-1 are consistent with those of in vitro eects of cyclic GMP. 6 KMUP-1 has the following merits: (1) inhibition of PDE or cyclic GMP breakdown, (2) stimulation of NO/sGC/cyclic GMP pathway, and (3) subsequent stimulation of K + channels, in rabbit CCSM. We suggest that these merits play prominent roles in KMUP-1-induced CCSM relaxation-associated increases of ICP and penile erection.

Three new butanolides, tenuifolide A (1), isotenuifolide A (2), and tenuifolide B (3), a new secobutanolide, secotenuifolide A (4), and one new sesquiterpenoid, tenuifolin (5), along with 16 known compounds were isolated from the stems of Cinnamomum tenuifolium. Their structures were determined by spectroscopic analyses. Compound 4 was found to induce apoptotic-related DNA damage, increase sub-G1 cells, and inhibit the growth of human prostate cancer cells, DU145. In addition, treatment with 4 significantly increased intracellular H2O2 and/or peroxide. The results show that 4 induced (a) noticeable reduction of mitochondrial transmembrane potential (DeltaPsim); (b) significant increase in the ratio of cytochrome c concentration (cytosol/mitochondria); and (c) subsequent activation of caspase-9/caspase-3. Antiproliferation caused by 4 was found to markedly decrease when pretreated with caspase-9/caspase-3 inhibitor. In ROS scavenging, antioxidant, NADPH oxidase, and NO inhibitor studies, pretreatment of DU145 cells with either DPI, dexamethasone, L-NAME, or mannitol decreased 4-induced intracellular DCF fluorescence of ROS. These results suggest that an increase of H2O2 and/or peroxide by 4 is the initial apoptotic event and 4 has anticancer effects on DU145 cells.