Abstract
The ability of receptor activation to regulate osmosensitive K+ fluxes (monitored as 86Rb+) in SH-SY5Y neuroblastoma has been examined. Incubation of SH-SY5Y cells in buffers rendered increasingly hypotonic by a reduction in NaCl concentration resulted in an enhanced basal efflux of Rb+ (threshold of release, 200 mOsM) but had no effect on Rb+ influx. Addition of the muscarinic cholinergic agonist, oxotremorine-M (Oxo-M), potently enhanced Rb+ efflux (EC50 = 0.45 μM) and increased the threshold of release to 280 mOsM. Oxo-M elicited a similarly potent, but osmolarity-independent, enhancement of Rb+ influx (EC50 = 1.35 μM). However, when incubated under hypotonic conditions in which osmolarity was varied by the addition of sucrose to a fixed concentration of NaCl, basal- and Oxo-M-stimulated Rb+ influx and efflux were demonstrated to be dependent upon osmolarity. Basal- and Oxo-M-stimulated Rb+ influx (but not Rb+ efflux) were inhibited by inclusion of ouabain or furosemide. Both Rb+ influx and efflux were inhibited by removal of intracellular Ca2+ and inhibition of protein kinase C activity. In addition to Oxo-M, agonists acting at other cell surface receptors previously implicated in organic osmolyte release enhanced both Rb+ efflux and influx under hypotonic conditions. Oxo-M had no effect on cellular K+ concentration in SH-SY5Y cells under physiologically relevant reductions in osmolarity (0–15%) unless K+ influx was blocked. Thus, although receptor activation enhances the osmosensitive efflux of K+, it also stimulates K+ influx, and the latter permits retention of K+ by the cells.
Footnotes
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This study was supported by National Institutes of Health Grants NS23831 (to S.K.F.), NS 034709 (to R.F.K.), and GM007767 (to D.J.F.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.135475.
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ABBREVIATIONS: CNS, central nervous system; GPCR, G-protein-coupled receptor; mAChR, muscarinic cholinergic receptor; DIOA, R-(+)-[(2-n-butyl-6,7-dichlooro-2-cyclopentenyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid; DMEM, Dulbecco's modified Eagle's medium; ANOVA, analysis of variance; Oxo-M, oxotremorine-M; NKCC, Na+-K+-2Cl– cotransporter; KCC, K+-Cl– cotransporter; PKC, protein kinase C; PAR, protease-activated receptor; S1P, sphingosine-1-phosphate; LPA, lysophosphatidic acid.
- Received December 19, 2007.
- Accepted February 14, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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