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1 Medical Research Service and 2 Geriatric Research, Education and Clinical Center, South Texas Veterans Health Care System, Audie L. Murphy Division; and Departments of 3 Dental Diagnostic Science and 4 Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284
The effects of epidermal growth factor
(EGF) on intracellular calcium ([Ca2+]i)
responses to the muscarinic agonist carbachol were studied in a human
salivary cell line (HSY). Carbachol (10
4 M)-stimulated
[Ca2+]i mobilization was inhibited by 40%
after 48-h treatment with 5 × 1010 M EGF. EGF also
reduced carbachol-induced [Ca2+]i in
Ca2+-free medium and Ca2+ influx following
repletion of extracellular Ca2+. Under
Ca2+-free conditions, thapsigargin, an inhibitor of
Ca2+ uptake to internal stores, induced similar
[Ca2+]i signals in control and EGF-treated
cells, indicating that internal Ca2+ stores were unaffected
by EGF; however, in cells exposed to thapsigargin, Ca2+
influx following Ca2+ repletion was reduced by EGF.
Muscarinic receptor density, assessed by binding of the muscarinic
receptor antagonist
L-[benzilic-4,4'-3HCN]quinuclidinyl benzilate
([3H]QNB), was decreased by 20% after EGF treatment.
Inhibition of the carbachol response by EGF was not altered by phorbol
ester-induced downregulation of protein kinase C (PKC) but was enhanced
upon PKC activation by a diacylglycerol analog. Phosphorylation of mitogen-activated protein kinase (MAP kinase) and inhibition of the
carbachol response by EGF were both blocked by the MAP kinase pathway
inhibitor PD-98059. The results suggest that EGF decreases carbachol-induced Ca2+ release from internal stores and
also exerts a direct inhibitory action on Ca2+ influx. A
decline in muscarinic receptor density may contribute to EGF inhibition
of carbachol responsiveness. The inhibitory effect of EGF is mediated
by the MAP kinase pathway and is potentiated by a distinct modulatory
cascade involving activation of PKC. EGF may play a physiological role
in regulating muscarinic receptor-stimulated salivary secretion.
carbachol; signal transduction; protein kinase C; mitogen-activated protein kinase; epidermal growth factor
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