Ca2+ signaling in Sf9 insect cells and the functional expression of a rat brain M5 muscarinic receptor.
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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Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030.
Rajan
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Schilling
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D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D002461:Cell Line, Transformed; D018076:DNA, Complementary; D005260:Female; D016257:Fura-2; D007313:Insecta; D007425:Intracellular Membranes / Q000378:metabolism; D009655:Octopamine / Q000494:pharmacology; D051381:Rats; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D015398:Signal Transduction
D018076:DNA, Complementary; D011976:Receptors, Muscarinic; D009655:Octopamine; D002118:Calcium; D016257:Fura-2