Peter Y Zhao1, Geliang Gan1, Shaomin Peng1, Shao-Bin Wang2, Bo Chen3, Ron A Adelman3, Lawrence J Rizzolo2. 1. Department of Surgery, Yale University, New Haven, Connecticut, United States. 2. Department of Surgery, Yale University, New Haven, Connecticut, United States Department of Ophthalmology & Visual Science, Yale University, New Haven, Connecticut, United States. 3. Department of Ophthalmology & Visual Science, Yale University, New Haven, Connecticut, United States.
Abstract
PURPOSE: Calcium regulates many functions of the RPE. Its concentration in the subretinal space and RPE cytoplasm is closely regulated. Transient receptor potential (TRP) channels are a superfamily of ion channels that are moderately calcium-selective. This study investigates the subcellular localization and potential functions of TRP channels in a first-passage culture model of human fetal RPE (hfRPE). METHODS: The RPE isolated from 15- to 16-week gestation fetuses were maintained in serum-free media. Cultures were treated with barium chloride (BaCl2) in the absence and presence of TRP channel inhibitors and monitored by the transepithelial electrical resistance (TER). The expression of TRP channels was determined using quantitative RT-PCR, immunoblotting, and immunofluorescence confocal microscopy. RESULTS: Barium chloride substantially decreased TER and disrupted cell-cell contacts when added to the apical surface of RPE, but not when added to the basolateral surface. The effect could be partially blocked by the general TRP inhibitor, lanthanum chloride (LaCl3, ~75%), or an inhibitor of calpain (~25%). Family member-specific inhibitors, ML204 (TRPC4) and HC-067047 (TRPV4), had no effect on basal channel activity. Expression of TRPC4, TRPM1, TRPM3, TRPM7, and TRPV4 was detected by RT-PCR and immunoblotting. The TRPM3 localized to the base of the primary cilium, and TRPC4 and TRPM3 localized to apical tight junctions. The TRPV4 localized to apical microvilli in a small subset of cells. CONCLUSIONS: The TRP channels localized to subdomains of the apical membrane, and BaCl2 was only able to dissociate tight junctions when presented to the apical membrane. The data suggest a potential role for TRP channels as sensors of [Ca(2+)] in the subretinal space. Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.
PURPOSE:Calcium regulates many functions of the RPE. Its concentration in the subretinal space and RPE cytoplasm is closely regulated. Transient receptor potential (TRP) channels are a superfamily of ion channels that are moderately calcium-selective. This study investigates the subcellular localization and potential functions of TRP channels in a first-passage culture model of human fetal RPE (hfRPE). METHODS: The RPE isolated from 15- to 16-week gestation fetuses were maintained in serum-free media. Cultures were treated with barium chloride (BaCl2) in the absence and presence of TRP channel inhibitors and monitored by the transepithelial electrical resistance (TER). The expression of TRP channels was determined using quantitative RT-PCR, immunoblotting, and immunofluorescence confocal microscopy. RESULTS:Barium chloride substantially decreased TER and disrupted cell-cell contacts when added to the apical surface of RPE, but not when added to the basolateral surface. The effect could be partially blocked by the general TRP inhibitor, lanthanum chloride (LaCl3, ~75%), or an inhibitor of calpain (~25%). Family member-specific inhibitors, ML204 (TRPC4) and HC-067047 (TRPV4), had no effect on basal channel activity. Expression of TRPC4, TRPM1, TRPM3, TRPM7, and TRPV4 was detected by RT-PCR and immunoblotting. The TRPM3 localized to the base of the primary cilium, and TRPC4 and TRPM3 localized to apical tight junctions. The TRPV4 localized to apical microvilli in a small subset of cells. CONCLUSIONS: The TRP channels localized to subdomains of the apical membrane, and BaCl2 was only able to dissociate tight junctions when presented to the apical membrane. The data suggest a potential role for TRP channels as sensors of [Ca(2+)] in the subretinal space. Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.
Authors: Li-Ting Su; Maria A Agapito; Mingjiang Li; William T N Simonson; Anna Huttenlocher; Raymond Habas; Lixia Yue; Loren W Runnels Journal: J Biol Chem Date: 2006-01-25 Impact factor: 5.157
Authors: Arvydas Maminishkis; Shan Chen; Stephen Jalickee; Tina Banzon; Guangpu Shi; Fei E Wang; Todd Ehalt; Jeffrey A Hammer; Sheldon S Miller Journal: Invest Ophthalmol Vis Sci Date: 2006-08 Impact factor: 4.799
Authors: David M Gamm; J Nicholas Melvan; Rebecca L Shearer; Isabel Pinilla; Grzegorz Sabat; Clive N Svendsen; Lynda S Wright Journal: Invest Ophthalmol Vis Sci Date: 2008-02 Impact factor: 4.799
Authors: Tony Sourisseau; Anastasios Georgiadis; Anna Tsapara; Robin R Ali; Richard Pestell; Karl Matter; Maria S Balda Journal: Mol Cell Biol Date: 2006-03 Impact factor: 4.272
Authors: Elena Oancea; Joris Vriens; Sebastian Brauchi; Janice Jun; Igor Splawski; David E Clapham Journal: Sci Signal Date: 2009-05-12 Impact factor: 8.192
Authors: Brian J Siroky; Nancy K Kleene; Steven J Kleene; Charles D Varnell; Raven G Comer; Jialiu Liu; Lu Lu; Nolan W Pachciarz; John J Bissler; Bradley P Dixon Journal: Am J Physiol Renal Physiol Date: 2017-01-25
Authors: Mauricio A Retamal; Edison P Reyes; Isaac E García; Bernardo Pinto; Agustín D Martínez; Carlos González Journal: Front Cell Neurosci Date: 2015-07-27 Impact factor: 5.505