Measurement of changes in functional muscarinic acetylcholine receptor density in single neuroblastoma cells using calcium release kinetics

Samuel Sheng-Hung Wang, S. H. Thompson

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

Calcium release in response to the activation of muscarinic M1 and histamine H1 receptors was studied in single N1E-115 cells using Fura-2 imaging. The objective was to relate changes in the kinetics of Ca release with reductions in functional receptor density resulting from receptor desensitization. Calcium release increased and its time course accelerated with increasing carbachol concentration with an EC50 = 96 ± 8 μM. This value is similar to the binding KD (100 μM) and the similarity shows that the activation of calcium release is limited by the number of muscarinic receptors. In contrast, the EC50 for Ca release in response to histamine is 4.0 ± 0.7 μM while the binding KD is 8.3 μM and, therefore, H1 receptors appear to be in approximately 2-fold excess over the minimum number necessary to fully engage the Ca release mechanism. Functional surface receptor number was assayed in the population of cells by counting the total number of cells responding to agonist. A 5 min exposure to 1 mM carbachol caused 12% of cells to lose their ability to respond to carbachol, with no change in their response to histamine. Interpolating from the dose-response curve taken before desensitization, this is equivalent to an average 23% reduction in the number of muscarinic receptors. In individual cells the latency to Ca release is dose-dependent in the absence of excess receptors. The loss of functional receptors was therefore estimated from the increase in latency after desensitization, and varied from 5-48% of receptors (22 ± 18%). Muscarinic desensitization did not depend on IP3-evoked Ca release, Ca entry, protein kinase C, NO, or cGMP. We conclude that in a population, the number of cells responding and in single cells, the latency to Ca release can serve as measures of functional receptor density.

Original languageEnglish (US)
Pages (from-to)483-496
Number of pages14
JournalCell Calcium
Volume15
Issue number6
DOIs
StatePublished - Jan 1 1994
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Physiology
  • Molecular Biology
  • Cell Biology

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