Demonstration of a muscarinic receptor-mediated cyclic GMP-dependent hyperpolarization of the membrane potential of mouse neuroblastoma cells using [3H]tetraphenylphosphonium
Wastek, G.J.; Lopez, J.R.; Richelson, E.
Molecular Pharmacology 19(1): 15-20
1981
ISSN/ISBN: 0026-895X PMID: 6259508 Document Number: 169966
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 K+ 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 .mu.M. Under equilibrium conditions at 37.degree. C, the addition of 1 mM carbachol significantly increased net [3H]TPP accumulation from 519 .+-. 30 pmol/106 cells to 1160 .+-. 33 pmol/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 .mu.M), but not d-tubocurarine (10 .mu.M) or pyrilamine (10 .mu.M) prevented the increase in [3H]TPP accumulation. This agonist-mediated hyperpolarization was abolished either by adding ethylene glycol bis(.beta.-aminoethyl ether)-N,N'-tetraacetic acid to the cells or by using a Ca2+-free buffer. Under similar conditions, cGMP increased net [3H]TPP accumulation to 1050 .+-. 31 pmol/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 cGMP.