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NEUROPHARMACOLOGY

Activation of Muscarinic Cholinergic Receptors on Human SH-SY5Y Neuroblastoma Cells Enhances Both the Influx and Efflux of K⁺ under Conditions of Hypo-Osmolarity

Departments of Pharmacology (D.J.F., S.K.F.) and Neurosurgery and Molecular and Integrative Physiology (R.F.K.), and Molecular and Behavioral Neuroscience Institute (A.M.H., S.K.F.), University of Michigan, Ann Arbor, Michigan

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.

Address correspondence to: Dr. Stephen K. Fisher, Molecular and Behavioral Neuroscience Institute, University of Michigan, 5039 Biomedical Science Research Building, Ann Arbor, MI 48109-2200. E-mail: skfisher{at}umich.edu