Spatiotemporal calcium signaling in a Drosophila melanogaster cell line stably expressing a Drosophila muscarinic acetylcholine receptor.
A muscarinic acetylcholine receptor (mAChR), DM1, expressed in the nervous system of Drosophila melanogaster, has been stably expressed in a Drosophila S2 cell line (S2-DM1) and used to investigate spatiotemporal calcium changes following agonist activation. Carbamylcholine (CCh) and oxotremorine are potent agonists, whereas application of the vertebrate M1 mAChR agonist, McN-A-343, results in a weak response. Activation of S2-DM1 receptors using CCh resulted in an increase in intracellular calcium ([Ca(2+)](i)) that was biphasic. Two distinct calcium sources were found to contribute to calcium signaling: (1) internal stores that are sensitive to both thapsigargin and 2-aminoethoxydiphenyl borate and (2) capacitative calcium entry. Spatiotemporal imaging of individual S2-DM1 cells showed that the CCh-induced [Ca(2+)](i) transient resulted from a homogeneous calcium increase throughout the cell, indicative of calcium release from internal stores. In contrast, ionomycin induced the formation of a "calcium ring" at the cell periphery, consistent with external calcium influx.
Cordova
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Dupont Crop Protection Products, Stine Haskell Research Center, Elkton Road, Newark, Delaware 19714, USA. daniel.cordova@usa.dupont.com
Delpech
V Raymond
VR
Sattelle
D B
DB
Rauh
J J
JJ
D000818:Animals; D001896:Boron Compounds / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002460:Cell Line; D018679:Cholinergic Agonists / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004331:Drosophila melanogaster; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors
D001896:Boron Compounds; D018679:Cholinergic Agonists; D004791:Enzyme Inhibitors; D007476:Ionophores; D011976:Receptors, Muscarinic; D015759:Ionomycin; D019284:Thapsigargin; C109986:2-aminoethoxydiphenyl borate; D002118:Calcium; D016257:Fura-2