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

G. J. Wastek, J. R. Lopez, E. Richelson

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)15-20
Number of pages6
JournalMolecular Pharmacology
Volume19
Issue number1
StatePublished - 1981
Externally publishedYes

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Cyclic GMP
Muscarinic Receptors
Neuroblastoma
Membrane Potentials
Phosphates
tetraphenylphosphonium
Pyrilamine
Tubocurarine
Ethylene Glycol
Carbachol
Microelectrodes
Atropine
Acetic Acid
Ether
Cations
Suspensions
Potassium
Buffers
Clone Cells
Cell Count

ASJC Scopus subject areas

  • Pharmacology

Cite this

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.

In: Molecular Pharmacology, Vol. 19, No. 1, 1981, p. 15-20.

Research output: Contribution to journalArticle

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abstract = "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.",
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N2 - 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.

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