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Norketamine, the Main Metabolite of Ketamine, Induces Mitochondria-Dependent and ER Stress-Triggered Apoptotic Death in Urothelial Cells via a Ca(2+)-Regulated ERK1/2-Activating Pathway

Ketamine-associated cystitis is characterized by suburothelial inflammation and urothelial cell death. Norketamine (NK), the main metabolite of ketamine, is abundant in urine following ketamine exposure. NK has been speculated to exert toxic effects in urothelial cells, similarly to ketamine. Howeve...

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Autores principales: Lin, Jhe-Wei, Lin, Yi-Chun, Liu, Jui-Ming, Liu, Shing-Hwa, Fang, Kai-Min, Hsu, Ren-Jun, Huang, Chun-Fa, Chang, Kai-Yao, Lee, Kuan-I, Chang, Kai-Chih, Su, Chin-Chuan, Chen, Ya-Wen
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9102902/
https://www.ncbi.nlm.nih.gov/pubmed/35563057
http://dx.doi.org/10.3390/ijms23094666
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author Lin, Jhe-Wei
Lin, Yi-Chun
Liu, Jui-Ming
Liu, Shing-Hwa
Fang, Kai-Min
Hsu, Ren-Jun
Huang, Chun-Fa
Chang, Kai-Yao
Lee, Kuan-I
Chang, Kai-Chih
Su, Chin-Chuan
Chen, Ya-Wen
author_facet Lin, Jhe-Wei
Lin, Yi-Chun
Liu, Jui-Ming
Liu, Shing-Hwa
Fang, Kai-Min
Hsu, Ren-Jun
Huang, Chun-Fa
Chang, Kai-Yao
Lee, Kuan-I
Chang, Kai-Chih
Su, Chin-Chuan
Chen, Ya-Wen
author_sort Lin, Jhe-Wei
collection PubMed
description Ketamine-associated cystitis is characterized by suburothelial inflammation and urothelial cell death. Norketamine (NK), the main metabolite of ketamine, is abundant in urine following ketamine exposure. NK has been speculated to exert toxic effects in urothelial cells, similarly to ketamine. However, the molecular mechanisms contributing to NK-induced urothelial cytotoxicity are almost unclear. Here, we aimed to investigate the toxic effects of NK and the potential mechanisms underlying NK-induced urothelial cell injury. In this study, NK exposure significantly reduced cell viability and induced apoptosis in human urinary bladder epithelial-derived RT4 cells that NK (0.01–0.5 mM) exhibited greater cytotoxicity than ketamine (0.1–3 mM). Signals of mitochondrial dysfunction, including mitochondrial membrane potential (MMP) loss and cytosolic cytochrome c release, were found to be involved in NK-induced cell apoptosis and death. NK exposure of cells also triggered the expression of endoplasmic reticulum (ER) stress-related proteins including GRP78, CHOP, XBP-1, ATF-4 and -6, caspase-12, PERK, eIF-2α, and IRE-1. Pretreatment with 4-phenylbutyric acid (an ER stress inhibitor) markedly prevented the expression of ER stress-related proteins and apoptotic events in NK-exposed cells. Additionally, NK exposure significantly activated JNK, ERK1/2, and p38 signaling and increased intracellular calcium concentrations ([Ca(2+)](i)). Pretreatment of cells with both PD98059 (an ERK1/2 inhibitor) and BAPTA/AM (a cell-permeable Ca(2+) chelator), but not SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor), effectively suppressed NK-induced mitochondrial dysfunction, ER stress-related signals, and apoptotic events. The elevation of [Ca(2+)](i) in NK-exposed cells could be obviously inhibited by BAPTA/AM, but not PD98059. Taken together, these findings suggest that NK exposure exerts urothelial cytotoxicity via a [Ca(2+)](i)-regulated ERK1/2 activation, which is involved in downstream mediation of the mitochondria-dependent and ER stress-triggered apoptotic pathway, consequently resulting in urothelial cell death. Our findings suggest that regulating [Ca(2+)](i)/ERK signaling pathways may be a promising strategy for treatment of NK-induced urothelial cystitis.
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spelling pubmed-91029022022-05-14 Norketamine, the Main Metabolite of Ketamine, Induces Mitochondria-Dependent and ER Stress-Triggered Apoptotic Death in Urothelial Cells via a Ca(2+)-Regulated ERK1/2-Activating Pathway Lin, Jhe-Wei Lin, Yi-Chun Liu, Jui-Ming Liu, Shing-Hwa Fang, Kai-Min Hsu, Ren-Jun Huang, Chun-Fa Chang, Kai-Yao Lee, Kuan-I Chang, Kai-Chih Su, Chin-Chuan Chen, Ya-Wen Int J Mol Sci Article Ketamine-associated cystitis is characterized by suburothelial inflammation and urothelial cell death. Norketamine (NK), the main metabolite of ketamine, is abundant in urine following ketamine exposure. NK has been speculated to exert toxic effects in urothelial cells, similarly to ketamine. However, the molecular mechanisms contributing to NK-induced urothelial cytotoxicity are almost unclear. Here, we aimed to investigate the toxic effects of NK and the potential mechanisms underlying NK-induced urothelial cell injury. In this study, NK exposure significantly reduced cell viability and induced apoptosis in human urinary bladder epithelial-derived RT4 cells that NK (0.01–0.5 mM) exhibited greater cytotoxicity than ketamine (0.1–3 mM). Signals of mitochondrial dysfunction, including mitochondrial membrane potential (MMP) loss and cytosolic cytochrome c release, were found to be involved in NK-induced cell apoptosis and death. NK exposure of cells also triggered the expression of endoplasmic reticulum (ER) stress-related proteins including GRP78, CHOP, XBP-1, ATF-4 and -6, caspase-12, PERK, eIF-2α, and IRE-1. Pretreatment with 4-phenylbutyric acid (an ER stress inhibitor) markedly prevented the expression of ER stress-related proteins and apoptotic events in NK-exposed cells. Additionally, NK exposure significantly activated JNK, ERK1/2, and p38 signaling and increased intracellular calcium concentrations ([Ca(2+)](i)). Pretreatment of cells with both PD98059 (an ERK1/2 inhibitor) and BAPTA/AM (a cell-permeable Ca(2+) chelator), but not SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor), effectively suppressed NK-induced mitochondrial dysfunction, ER stress-related signals, and apoptotic events. The elevation of [Ca(2+)](i) in NK-exposed cells could be obviously inhibited by BAPTA/AM, but not PD98059. Taken together, these findings suggest that NK exposure exerts urothelial cytotoxicity via a [Ca(2+)](i)-regulated ERK1/2 activation, which is involved in downstream mediation of the mitochondria-dependent and ER stress-triggered apoptotic pathway, consequently resulting in urothelial cell death. Our findings suggest that regulating [Ca(2+)](i)/ERK signaling pathways may be a promising strategy for treatment of NK-induced urothelial cystitis. MDPI 2022-04-23 /pmc/articles/PMC9102902/ /pubmed/35563057 http://dx.doi.org/10.3390/ijms23094666 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Lin, Jhe-Wei
Lin, Yi-Chun
Liu, Jui-Ming
Liu, Shing-Hwa
Fang, Kai-Min
Hsu, Ren-Jun
Huang, Chun-Fa
Chang, Kai-Yao
Lee, Kuan-I
Chang, Kai-Chih
Su, Chin-Chuan
Chen, Ya-Wen
Norketamine, the Main Metabolite of Ketamine, Induces Mitochondria-Dependent and ER Stress-Triggered Apoptotic Death in Urothelial Cells via a Ca(2+)-Regulated ERK1/2-Activating Pathway
title Norketamine, the Main Metabolite of Ketamine, Induces Mitochondria-Dependent and ER Stress-Triggered Apoptotic Death in Urothelial Cells via a Ca(2+)-Regulated ERK1/2-Activating Pathway
title_full Norketamine, the Main Metabolite of Ketamine, Induces Mitochondria-Dependent and ER Stress-Triggered Apoptotic Death in Urothelial Cells via a Ca(2+)-Regulated ERK1/2-Activating Pathway
title_fullStr Norketamine, the Main Metabolite of Ketamine, Induces Mitochondria-Dependent and ER Stress-Triggered Apoptotic Death in Urothelial Cells via a Ca(2+)-Regulated ERK1/2-Activating Pathway
title_full_unstemmed Norketamine, the Main Metabolite of Ketamine, Induces Mitochondria-Dependent and ER Stress-Triggered Apoptotic Death in Urothelial Cells via a Ca(2+)-Regulated ERK1/2-Activating Pathway
title_short Norketamine, the Main Metabolite of Ketamine, Induces Mitochondria-Dependent and ER Stress-Triggered Apoptotic Death in Urothelial Cells via a Ca(2+)-Regulated ERK1/2-Activating Pathway
title_sort norketamine, the main metabolite of ketamine, induces mitochondria-dependent and er stress-triggered apoptotic death in urothelial cells via a ca(2+)-regulated erk1/2-activating pathway
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9102902/
https://www.ncbi.nlm.nih.gov/pubmed/35563057
http://dx.doi.org/10.3390/ijms23094666
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