Demonstration of a muscarinic receptor-mediated cyclic GMP-dependent hyperpolarization of the membrane potential of mouse neuroblastoma cells using [3H]tetraphenylphosphonium

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'-tetra-acetic 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.