Molecular Pharmacology, Vol 19, 15-20, Copyright © 1981 by the American Society for Pharmacology and Experimental Therapeutics

Demonstration of a Muscarinic Receptor-Mediated Cyclic GMP-Dependent Hyperpolarization of the Membrane Potential of Mouse Neuroblastoma Cells Using [³H]Tetraphenylphosphonium

¹ Departments of Psychiatry and Pharmacology, Mayo Foundation, Rochester, Minnesota 55901

The lipophilic cation [³H]tetraphenylphosphonium ([³H]TPP) was used to measure the transmembrane potential (V_m) of cultured mouse neuroblastoma cells (clone N1E-115) in suspension. These cells accumulated approximately twice as much [³H]TPP in low-K⁺ phosphate-buffered saline (PBS) as they did in high-K⁺ PBS. Accumulation in the presence of either low or high potassium was both time- and temperature-dependent. At equilibrium, [³H]TPP accumulation in low-K⁺ and high-K⁺ PBS increased with increasing cell number, and net accumulation increased linearly with the external [³H]TPP concentration between 0.1 and 50 µM. Under equilibrium conditions at 37°, the addition of 1 mM carbachol significantly increased net [³H]TPP accumulation from 519 ± 30 pmoles/10⁶ cells to 1160 ± 33 pmoles/10⁶ cells within 1 min. This increase was equivalent to a hyperpolarization of the cells’ V_m from approximately -66 ± 5 mV to -79 ± 5 mV. Direct measurements with microelectrodes under these same conditions showed that there was an immediate and significant hyperpolarization of the cells’ V_m from -62.3 ± 0.5 mV to -72.0 ± 1.3 mV. Atropine (1 µM), but not d-tubocurarine (10 µM) or pyrilamine (10 µM) prevented the increase in [³H]TPP accumulation. This agonist-mediated hyperpolarization was abolished either by adding ethylene glycol bis(-aminoethyl ether)-N,N'-tetraacetic acid to the cells or by using a Ca²⁺-free buffer. Under similar conditions, cyclic GMP increased net [³H]TPP accumulation to 1050 ± 31 pmoles/10⁶ cells within 10 min (i.e., an increase equivalent to a hyperpolarization of the cells’ V_m from -66 ± 5 mV to -76 ± 6 mV). Direct electrophysiological measurements under these same conditions showed that there was a significant hyperpolarization of the cells’ V_m from -62.3 ± 0.5 mV to -71.2 ± 1.5 mV after a period of 3.8 ± 0.6 min. These data suggest that muscarinic receptor responses in these cells may be mediated by a hyperpolarization of the cells’ V_m subsequent to an increase in intracellular cyclic GMP.

Note:
ACKNOWLEDGMENTS We thank Drs. H. R. Kaback and A. J. Blume (Roche Institute of Molecular Biology) for supplying our initial batch of [³H]TPP. We also thank Drs. J. R. Blinks and S. R. Taylor for their helpful discussion and criticism of the manuscript.

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