Epidermal growth factor-induced depletion of the intracellular Ca2+ store fails to activate capacitative Ca2+ ntry in a human salivary cell line

Epidermal growth factor (EGF) is a multifunctional factor known to influence proliferation and function of a variety of cells. The actions of EGF are mediated by EGF receptor tyrosine kinase pathways, including stimulation of phospholipase Cgamma and mobilization of intracellular Ca2+ ([Ca2+](i)). Generally, agonist-mediated Ca2+ mobilization involves both Ca2+ release from internal stores and Ca2+ influx activated by store depletion (i.e. capacitative or store-operated Ca2+ influx). However, the role of capacitative Ca2+ entry in EGF-mediated Ca2+ mobilization is still largely unknown. In this study, we compared [Ca2+], signals elicited by EGF with those induced by agents (the muscarinic receptor agonist carbachol and thapsigargin (Tg)) known to activate capacitative Ca2+ entry. Unlike carbachol and Tg, EGF (5 nM) elicited a transient [Ca2+](i) signal without a plateau phase in the presence of extracellular Ca2+ and also failed to accelerate Mn2+ entry. Repletion of extracellular Ca2+ to cells stimulated with EGF in the absence of Ca2+ elicited an increase in [Ca2+](i), indicating that EGF indeed stimulates Ca2+ influx. However, the influx was activated at lower EGF concentrations than those required to stimulate Ca2+ release. Interestingly, the phospholipase C inhibitor U73122 completely inhibited Ca2+ release induced by both EGF and carbachol and also reduced Ca2+ influx responsive to carbachol but had no effect on Ca2+ influx induced by EGF. EGF-induced Ca2+ influx was potentiated by low concentrations (<5 ng/ml) of oligomycm, a mitochondrial inhibitor that blocks capacitative Ca2+ influx in other systems. Transient expression of the hTRPC3 protein enhanced Ca2+ influx responsive to carbachol but did not increase EGF-activated Ca2+ influx. Both EGF and carbachol depleted internal Ca2+ stores. Our results demonstrate that EGF-induced Ca2+ release from internal stores does not activate capacitative Ca2+ influx. Rather, EGF stimulates Ca2+ influx via a mechanism distinct from capacitative Ca2+ influx induced by carbachol and Tg.