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Modification of rat carotid body glomus cell O2 sensing by RNA interference method
Author(s) -
Kim Insook,
Yang DJ,
Carroll John L
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.lb113-d
Subject(s) - glomus cell , rna interference , gene silencing , carotid body , transfection , immunostaining , microbiology and biotechnology , small interfering rna , hypoxia (environmental) , biology , intracellular , chemistry , rna , gene , biochemistry , neuroscience , immunology , electrophysiology , immunohistochemistry , organic chemistry , oxygen
The carotid bodies (CB) are the major arterial O 2 chemoreceptors in mammals. The CB glomus cell response to hypoxia is mediated by multiple ion channels, including TASK‐like background K + channels, other K + channels and voltage‐gated calcium channels. In order to investigate the physiological role of TASK‐like K + channels in CB O 2 sensing, we applied RNA interference technology by knocking down gene/protein expression and observing its functional effect on O 2 sensing. Dissociated CB glomus cells from 14 day old rats were incubated overnight and treated with siRNA of TASK‐1, TASK‐3 or non‐silencing siRNA with optimized transfection reagent. After cells were incubated for 48 hrs, gene expression was measured with real time PCR and protein expression was tested with immunostaining. Functional effects of siRNA exposure were assessed using fura‐2 to measure the glomus cell intracellular calcium ([Ca 2+ ] i ) response to hypoxia. Results: TASK‐3 siRNA reduced gene expression ~70%, while non‐silencing siRNA had no effect. TASK‐1siRNA exposure reduced TASK‐1 protein expression (immunostaining) and reduced the glomus cell [Ca 2+ ] i response to hypoxia ~70%, while non‐silencing siRNA had no effect. These findings suggest that TASK‐like K + channels play an important role in the glomus cell response to hypoxia and RNA interference methods represent a feasible approach to exploring the role of specific ion channels in O 2 CB sensing. (funded by University of Arkansas for Medical Sciences Dean’s Research Development Fund).