Calcium entry in rabbit corneal epithelial cells: evidence for a nonvoltage dependent pathway.
We performed experiments to elucidate the calcium influx pathways in freshly dispersed rabbit corneal epithelial cells. Three possible pathways were considered: voltage-gated Ca++ channels, Na+/Ca++ exchange, and nonvoltage-dependent Ca(++)-permeable channels. Whole cell inward currents carrying either Ca++ or Ba++ were not detected using voltage clamp techniques. We also used imaging technology and the Ca(++)-sensitive ratiometric dye fura 2 to measure changes in intracellular Ca++ concentration ([Ca]i). Bath perfusion with NaCl Ringer's solution containing the calcium channel agonist Bay-K-8644 (1 microM), or Ni++ (40 microM), a blocker of many voltage-dependent calcium channels, did not affect [Ca++]i. Membrane depolarization with a KCl Ringer's bath solution resulted in a decrease in [Ca++]i. These results are inconsistent with the presence of voltage gated Ca++ channels. Nonvoltage gated Ca++ entry, on the other hand, would be reduced by membrane depolarization and enhanced by membrane hyperpolarization. Agents which hyperpolarize via stimulation of K+ current, such as flufenamic acid, resulted in an increase in ratio intensity. The cells were found to be permeable to Mn++ and bath perfusion with 5 mM Ni++ decreased [Ca++]i suggesting that the Ca++ conductance was blocked. These results are most consistent with a nonvoltage gated Ca++ influx pathway. Finally, replacing extracellular Na+ with Li+ resulted in an increase in [Ca++]i if the cells were first Na(+)-loaded using the Na+ ionophore monensin and ouabain, a Na(+)-K(+)-ATPase inhibitor. These results suggest that Na+/Ca++ exchange may also regulate [Ca++]i in this cell type.
Rich
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Department of Physiology, Mayo Foundation, Rochester, Minnesota 55905, USA.
Rae
J L
JL
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D003315:Cornea / Q000166:cytology*; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D005439:Flufenamic Acid / Q000494:pharmacology; D016257:Fura-2; D015640:Ion Channel Gating / Q000502:physiology*; D017136:Ion Transport / Q000502:physiology; D008274:Magnesium / Q000494:pharmacology; D008564:Membrane Potentials / Q000502:physiology; D009532:Nickel / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011817:Rabbits; D012964:Sodium / Q000378:metabolism
D015220:Calcium Channels; D010042:Ouabain; D005439:Flufenamic Acid; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D009532:Nickel; D012964:Sodium; D008274:Magnesium; D002118:Calcium; D016257:Fura-2