Muscarinic receptor-mediated cyclic GMP formation by cultured nerve cells-. Ionic dependence and effects of local anesthetics

Elliott Richelson, Franklyn G. Prendergast, Silvia Divinetz-Romero

Research output: Contribution to journalArticle

65 Citations (Scopus)

Abstract

A new assay technique for measuring receptor-mediated cyclic GMP formation by cultured mouse neutroblastoma cells was used to study the role of ions in, and the effects of local anesthetics on, the function of the muscarinic receptor. The technique involved radioactively labeling intracellular stores of GTP by incubating cells with [3H]guanine and isolating [3H]cyclic GMP with a cation exchange resin (Dowex-50+) column. High-pressure liquid chromatography of cell extracts and of eluates from the Dowex column showed that after 45 min the majority of the radioactivity in the cell extracts was [3H]GTP and that, for carbamylcholine-stimulated cells, greater than 90 per cent of the radioactivity in the eluates was [3H]cyclic GMP. In the absence of external Na+ ([Na+]e, with cesium chloride as osmotic filler) or external ([Ca2+]e), the carbamylcholine-stimulated formation of [3H]cyclic GMP was about 60 and 10 per cent of control, respectively, while removal of other ions had no significant effect. There was little difference in the responses at 10 mM vs 110 mM-[Na+]e, whereas the optimal [Ca2+], was around 5 mM. Ca2+ increased [3H]cyclic GMP formation in response to carbamylcholine without affecting the apparent affinity of this agonist for the receptor. Local anesthetics were apparently competitive inhibitors of carbamylcholine with equilibrium dissociation constants (KB) in the range of 6-250 μM. The rank order for the apparent affinity of local anesthetics for the muscarinic receptor was tetracaine = butacaine = procaine > dibucaine = lidocaine > ethyl aminobenzoate.

Original languageEnglish (US)
Pages (from-to)2039-2048
Number of pages10
JournalBiochemical Pharmacology
Volume27
Issue number16
DOIs
StatePublished - 1978

Fingerprint

Cyclic GMP
Muscarinic Receptors
Local Anesthetics
Neurons
Carbachol
Cultured Cells
Radioactivity
Guanosine Triphosphate
Cell Extracts
Dibucaine
Benzocaine
High pressure liquid chromatography
Cation Exchange Resins
Ions
Tetracaine
Procaine
Guanine
Lidocaine
Labeling
Fillers

ASJC Scopus subject areas

  • Pharmacology

Cite this

Muscarinic receptor-mediated cyclic GMP formation by cultured nerve cells-. Ionic dependence and effects of local anesthetics. / Richelson, Elliott; Prendergast, Franklyn G.; Divinetz-Romero, Silvia.

In: Biochemical Pharmacology, Vol. 27, No. 16, 1978, p. 2039-2048.

Research output: Contribution to journalArticle

Richelson, Elliott ; Prendergast, Franklyn G. ; Divinetz-Romero, Silvia. / Muscarinic receptor-mediated cyclic GMP formation by cultured nerve cells-. Ionic dependence and effects of local anesthetics. In: Biochemical Pharmacology. 1978 ; Vol. 27, No. 16. pp. 2039-2048.
@article{8c464cdaa926437aa975143eeea4ca5b,
title = "Muscarinic receptor-mediated cyclic GMP formation by cultured nerve cells-. Ionic dependence and effects of local anesthetics",
abstract = "A new assay technique for measuring receptor-mediated cyclic GMP formation by cultured mouse neutroblastoma cells was used to study the role of ions in, and the effects of local anesthetics on, the function of the muscarinic receptor. The technique involved radioactively labeling intracellular stores of GTP by incubating cells with [3H]guanine and isolating [3H]cyclic GMP with a cation exchange resin (Dowex-50+) column. High-pressure liquid chromatography of cell extracts and of eluates from the Dowex column showed that after 45 min the majority of the radioactivity in the cell extracts was [3H]GTP and that, for carbamylcholine-stimulated cells, greater than 90 per cent of the radioactivity in the eluates was [3H]cyclic GMP. In the absence of external Na+ ([Na+]e, with cesium chloride as osmotic filler) or external ([Ca2+]e), the carbamylcholine-stimulated formation of [3H]cyclic GMP was about 60 and 10 per cent of control, respectively, while removal of other ions had no significant effect. There was little difference in the responses at 10 mM vs 110 mM-[Na+]e, whereas the optimal [Ca2+], was around 5 mM. Ca2+ increased [3H]cyclic GMP formation in response to carbamylcholine without affecting the apparent affinity of this agonist for the receptor. Local anesthetics were apparently competitive inhibitors of carbamylcholine with equilibrium dissociation constants (KB) in the range of 6-250 μM. The rank order for the apparent affinity of local anesthetics for the muscarinic receptor was tetracaine = butacaine = procaine > dibucaine = lidocaine > ethyl aminobenzoate.",
author = "Elliott Richelson and Prendergast, {Franklyn G.} and Silvia Divinetz-Romero",
year = "1978",
doi = "10.1016/0006-2952(78)90064-3",
language = "English (US)",
volume = "27",
pages = "2039--2048",
journal = "Biochemical Pharmacology",
issn = "0006-2952",
publisher = "Elsevier Inc.",
number = "16",

}

TY - JOUR

T1 - Muscarinic receptor-mediated cyclic GMP formation by cultured nerve cells-. Ionic dependence and effects of local anesthetics

AU - Richelson, Elliott

AU - Prendergast, Franklyn G.

AU - Divinetz-Romero, Silvia

PY - 1978

Y1 - 1978

N2 - A new assay technique for measuring receptor-mediated cyclic GMP formation by cultured mouse neutroblastoma cells was used to study the role of ions in, and the effects of local anesthetics on, the function of the muscarinic receptor. The technique involved radioactively labeling intracellular stores of GTP by incubating cells with [3H]guanine and isolating [3H]cyclic GMP with a cation exchange resin (Dowex-50+) column. High-pressure liquid chromatography of cell extracts and of eluates from the Dowex column showed that after 45 min the majority of the radioactivity in the cell extracts was [3H]GTP and that, for carbamylcholine-stimulated cells, greater than 90 per cent of the radioactivity in the eluates was [3H]cyclic GMP. In the absence of external Na+ ([Na+]e, with cesium chloride as osmotic filler) or external ([Ca2+]e), the carbamylcholine-stimulated formation of [3H]cyclic GMP was about 60 and 10 per cent of control, respectively, while removal of other ions had no significant effect. There was little difference in the responses at 10 mM vs 110 mM-[Na+]e, whereas the optimal [Ca2+], was around 5 mM. Ca2+ increased [3H]cyclic GMP formation in response to carbamylcholine without affecting the apparent affinity of this agonist for the receptor. Local anesthetics were apparently competitive inhibitors of carbamylcholine with equilibrium dissociation constants (KB) in the range of 6-250 μM. The rank order for the apparent affinity of local anesthetics for the muscarinic receptor was tetracaine = butacaine = procaine > dibucaine = lidocaine > ethyl aminobenzoate.

AB - A new assay technique for measuring receptor-mediated cyclic GMP formation by cultured mouse neutroblastoma cells was used to study the role of ions in, and the effects of local anesthetics on, the function of the muscarinic receptor. The technique involved radioactively labeling intracellular stores of GTP by incubating cells with [3H]guanine and isolating [3H]cyclic GMP with a cation exchange resin (Dowex-50+) column. High-pressure liquid chromatography of cell extracts and of eluates from the Dowex column showed that after 45 min the majority of the radioactivity in the cell extracts was [3H]GTP and that, for carbamylcholine-stimulated cells, greater than 90 per cent of the radioactivity in the eluates was [3H]cyclic GMP. In the absence of external Na+ ([Na+]e, with cesium chloride as osmotic filler) or external ([Ca2+]e), the carbamylcholine-stimulated formation of [3H]cyclic GMP was about 60 and 10 per cent of control, respectively, while removal of other ions had no significant effect. There was little difference in the responses at 10 mM vs 110 mM-[Na+]e, whereas the optimal [Ca2+], was around 5 mM. Ca2+ increased [3H]cyclic GMP formation in response to carbamylcholine without affecting the apparent affinity of this agonist for the receptor. Local anesthetics were apparently competitive inhibitors of carbamylcholine with equilibrium dissociation constants (KB) in the range of 6-250 μM. The rank order for the apparent affinity of local anesthetics for the muscarinic receptor was tetracaine = butacaine = procaine > dibucaine = lidocaine > ethyl aminobenzoate.

UR - http://www.scopus.com/inward/record.url?scp=0018073481&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0018073481&partnerID=8YFLogxK

U2 - 10.1016/0006-2952(78)90064-3

DO - 10.1016/0006-2952(78)90064-3

M3 - Article

C2 - 214089

AN - SCOPUS:0018073481

VL - 27

SP - 2039

EP - 2048

JO - Biochemical Pharmacology

JF - Biochemical Pharmacology

SN - 0006-2952

IS - 16

ER -