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Am J Physiol. 1994 Jun;266(6 Pt 1):C1736-43.

Ca2+ signaling in Sf9 insect cells and the functional expression of a rat brain M5 muscarinic receptor.

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1
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030.

Abstract

The purpose of the present study was to examine Ca2+ signaling mechanisms in Sf9 cells and to demonstrate expression and functional linkage of a mammalian receptor to changes in cytosolic free Ca2+ concentration ([Ca2+]i). Addition of p-octopamine (50 microM to fura 2-loaded Sf9 cells produced a small transient increase in [Ca2+]i from a basal level of 58 +/- 10 to 194 +/- 7.6 (SD) nM. The response to octopamine was inhibited by both cyproheptadine and chlorpromazine and was mimicked by clonidine. In contrast, [Ca2+]i did not change in response to dopamine (50 microM), substance P (50 nM), histamine (50 microM), ATP (50 microM), acetylcholine (10 or 100 microM), carbachol (10 or 100 microM), serotonin (50 microM), epinephrine (10 microM), or bradykinin (50 nM). The Ca(2+)-adenosinetriphosphatase inhibitors thapsigargin (200 nM) and 2,5-di-tert-butylhydroquinone (BHQ; 10 microM) increased [Ca2+]i to 307 +/- 13 and 137 +/- 20 nM, respectively. In contrast to BHQ, the response to thapsigargin was attenuated by La3+ or removal of extracellular Ca2+ and increased by elevation of extracellular Ca2+. These results suggest that thapsigargin but not BHQ stimulates Ca2+ influx. The rat brain muscarinic receptor (subtype M5) was incorporated into the baculovirus by homologous recombination. Addition of carbachol (100 microM) increased [Ca2+]i from 92.7 +/- 6.4 to 480 +/- 26 nM in Sf9 cells infected with recombinant virus containing the M5 receptor cDNA. The effect of carbachol on [Ca2+]i was concentration dependent with a 50% effective concentration of approximately 30 microM and was blocked by atropine (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS).

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