CA2+ SIGNALING IN SF9 INSECT CELLS AND THE FUNCTIONAL EXPRESSION OF A RAT-BRAIN M(5) 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 mu M) 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 mu M), substance P (50 nM), histamine (50 mu M), ATP (50 mu M), acetylcholine (10 or 100 mu M), carbachol (10 or 100 mu M), serotonin (50 mu M), epinephrine (10 mu M), or bradykinin (50 nM). The Ca2+-adenosinetriphosphatase inhibitors thapsigargin (200 nM) and 2,5-di-tert-butylhydroquinone (BHQ; 10 mu M) 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 M(5)) was incorporated into the baculovirus by homologous recombination. Addition of carbachol(100 mu M) increased [Ca2+](i) from 92.7 +/- 6.4 to 480 +/- 26 nM in Sf9 cells infected with recombinant virus containing the Mg receptor cDNA. The effect of carbachol on [Ca2+](i) was concentration dependent with a 50% effective concentration of similar to 30 mu M and was blocked by atropine (10 mu M) The response to carbachol was not seen in Sf9 cells infected with baculovirus containing an unrelated cDNA. These results demonstrate that the Sf9 cell can be used for the functional expression of mammalian receptors Linked to changes in Ca2+ homeostasis.