Valproic acid (VPA), a widely prescribed drug for seizures and bipolar disorder, has been shown to be an inhibitor of histone deacetylase (HDAC). Our previous study has demonstrated that VPA pretreatment reduces lipopolysaccharide (LPS)-induced dopaminergic (DA) neurotoxicity through the inhibition of microglia over-activation. The aim of this study was to determine the mechanism underlying VPA-induced attenuation of microglia over-activation. Other HDAC inhibitors (HDACIs) were compared with VPA for their effects on microglial activity. We found that VPA induced apoptosis of microglia cells in a time and concentration-dependent manner. VPA-treated microglial cells showed typical apoptotic hallmarks including phosphatidylserine externalization, chromatin condensation and DNA fragmentation. Further studies revealed that trichostatin A (TSA) and sodium butyrate (SB), two structurally dissimilar HDACIs, also induced microglial apoptosis. The apoptosis of microglia was accompanied by the disruption of mitochondrial membrane potential and the enhancement of acetylation levels of the histone H3 protein. Moreover, pretreatment with Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access

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.