Role of inositol (1,4,5)trisphosphate in epidermal growth factor-induced Ca2+ signaling in A431 cells.
The effects of epidermal growth factor on Ca2+ signaling in A431 cells were investigated. Epidermal growth factor induced a transient Ca2+ signal in the absence of external Ca2+ and a sustained response in the presence of extracellular Ca2+, indicating an ability to mobilize intracellular Ca2+ as well as the ability to increase Ca2+ entry from the extracellular space. The Ca(2+)-ATPase inhibitor thapsigargin also activated Ca2+ entry, and neither epidermal growth factor nor the guanine nucleotide-dependent protein-linked receptor agonist bradykinin activated additional Ca2+ entry over that due to thapsigargin. In nominally Ca(2+)-free medium, the addition of bradykinin to A431 cells rapidly but transiently increased inositol 1,4,5-trisphosphate and, in parallel fashion, transiently increased cytosolic Ca2+. Unexpectedly, under these experimental conditions, epidermal growth factor elicited a small but significant Ca2+ signal after the addition of bradykinin. Experiments were designed to determine whether the Ca2+ response to epidermal growth factor after bradykinin results from mobilization of Ca2+ by an inositol 1,4,5-trisphosphate-independent mechanism. Epidermal growth factor stimulated additional inositol 1,4,5-trisphosphate formation in bradykinin-treated cells. Furthermore, the Ca2+ signals elicited by both bradykinin and epidermal growth factor were blocked in cells microinjected with the inositol 1,4,5-trisphosphate receptor antagonist heparin, whereas the intracellular Ca(2+)-ATPase inhibitor thapsigargin still mobilized Ca2+. Finally, histamine, a less efficacious guanine nucleotide-dependent protein-linked receptor agonist, as well as photolyzed, microinjected, caged inositol 1,4,5-trisphosphate, also mobilized Ca2+ after bradykinin. The results of this study show (i) that epidermal growth factor activates intracellular Ca2+ release as well as Ca2+ entry, the latter most likely resulting from an indirect effect due to the depletion of intracellular Ca2+ pools, (ii) that the actions of epidermal growth factor on Ca2+ homeostasis can be fully accounted for by inositol 1,4,5-trisphosphate formation, and (iii) that the ability of A431 cells to produce Ca2+ signals when epidermal growth factor is applied after bradykinin can be explained by the rapid and complete desensitization of the bradykinin stimulated phospholipase C activity.
Hughes
A R
AR
Laboratory of Cellular and Molecular Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
Bird
G S
GS
Obie
J F
JF
Thastrup
O
O
Putney
J W
JW
D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000502:physiology; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000502:physiology*; D014407:Tumor Cells, Cultured
D004815:Epidermal Growth Factor; D015544:Inositol 1,4,5-Trisphosphate; D066246:ErbB Receptors; D001920:Bradykinin; D002118:Calcium