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Z. Yuan, H. Yang, P. Reinach; Interdependence between EGF Receptor Elicited Calcium and pH Transients in Rabbit Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3207.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: EGF receptor linked signaling involves transient increases in intracellular calcium, pH and stimulation of protein kinase C activity. We report here on some of the interrelationships among these pathways in SV40-immortalized rabbit corneal epithelial cells(rRCEC). Methods: rRCEC were loaded with either 2 µM fura-2/AM or BCECF-AM to monitor with fluorescence digital imaging cytosolic Ca2+ concentration ([Ca2+]i) and pH (pHi),respectively. At about 20% confluence, they were dye loaded for 30 min in an incubator at 37 °C containing 95% air and 5% CO2. Each experiment was performed 6- 8 times and in all cases employed 8-10 cells. Results: Irrespective of the presence or absence of extracellular calcium, EGF ( 5 ng/ml) had a biphasic effect on pHi. It initially fell from 7.09 to 6.85, but then rose to 7.22 after 20 min and stabilized at this value for the next 30 min. Such alkalinization was inhibited by either: 1) an isosmotic substitution of NaCl with choline chloride; 2) 100 µM EIPA; 3) 10 µM calphostin C. In contrast, 10 µM PDBu caused pHi to rise after 50 min from 7.09 to 7.87. Neither the acidifying nor the alkalinizing effects of EGF were altered by either calcium removal (2 mM EGTA) and/or clamping intracellular calcium with 10 µM BAPTA . In a calcium containing medium, EGF (5 ng/ml) induced a Ca2+ transient that reached 3-fold above its baseline value. EIPA induced nearly a 2-fold transient increase in [Ca 2+ ]i. Following exposure to PDBu, EIPA elicited a transient that was 2-fold greater than the one elicited by EIPA alone. In contrast, following 10 µM calphostin C, EIPA instead caused [Ca 2+ ]I to fall below the baseline. EIPA induced a transient in Ca-free medium following depletion of endoplasmic reticulum calcium stores with 10 µM cyclopiazonic acid (CPA). Conclusions: EGF receptor stimulation of Na:H exchange activity is not dependent on mobilization of intracellular calcium by this growth factor. However, EGF appears to induce increases in Na:H exchange activity that are dependent on EGF activation of protein kinase C. EIPA, in addition to inhibiting Na:H exchange activity, mediates calcium release from a calcium store other than the endoplasmic reticulum. Such release is enhanced following PKC activation.
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