Measurement of intracellular calcium using bioluminescent aequorin expressed in human cells.
Changes in intracellular free calcium concentration ([Ca2+]i) are involved in many important physiological responses. Detecting changes in [Ca2+]i is crucial to understanding the physiologic roles of intracellular free calcium. We have characterized changes of [Ca2+]i in human cells transfected with apoaequorin cDNA. When reconstituted in vivo by incubating transfected cells with coelenterazine, aequorin emits light upon binding free calcium and acts as a bioluminescent indicator for calcium. We have used this system to determine the concentration response relationship of serotonin for its receptor. Cells cotransfected with serotonin receptor cDNA and apoaequorin cDNA emitted light upon treatment with serotonin. The light emission responses were saturable and serotonin concentration-dependent, and they were inhibited by serotonin antagonists. Human 293 cells that stably express apoaequorin have been created. This system should facilitate the investigation of [Ca2+]i involvement in physiological and pathophysiological responses.
Sheu
Y A
YA
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia.
Kricka
L J
LJ
Pritchett
D B
DB
D000331:Aequorin / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D001059:Apoproteins / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D008163:Luminescent Measurements; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics; D037401:Scyphozoa; D014162:Transfection
D001059:Apoproteins; D011994:Recombinant Proteins; C060014:apoaequorin; D000331:Aequorin; D004247:DNA; D002118:Calcium