Epidermal growth factor-induced depletion of the intracellular Ca²⁺ store fails to activate capacitative Ca²⁺ entry 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 Cγ and mobilization ofintracellular 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 storeoperated Ca²⁺ influx). However, the role of capacitative Ca²⁺ entry in EGF-mediated Ca²⁺ mobilization is still largely unknown. In this study, we compared [Ca²⁺]_i 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⁺ infux. 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 oligomycin, 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.