PT  - JOURNAL ARTICLE
AU  - WASTEK, GREGORY J.
AU  - LOPEZ, J. RAFAEL
AU  - RICHELSON, ELLIOTT
TI  - Demonstration of a Muscarinic Receptor-Mediated Cyclic GMP-Dependent Hyperpolarization of the Membrane Potential of Mouse Neuroblastoma Cells Using [<sup>3</sup>H]Tetraphenylphosphonium
DP  - 1981 Jan 01
TA  - Molecular Pharmacology
PG  - 15--20
VI  - 19
IP  - 1
4099  - http://molpharm.aspetjournals.org/content/19/1/15.short
4100  - http://molpharm.aspetjournals.org/content/19/1/15.full
SO  - Mol Pharmacol1981 Jan 01; 19
AB  - The lipophilic cation [3H]tetraphenylphosphonium ([3H]TPP) was used to measure the transmembrane potential (Vm) 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’ Vm 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’ Vm 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(β-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’ Vm 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’ Vm 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’ Vm subsequent to an increase in intracellular cyclic GMP. 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.