Involvement of protein tyrosine kinase in the InsP3-induced activation of Ca2+-dependent Cl- currents in cultured cells of the rat retinal pigment epithelium.
This combined study of patch-clamp and intracellular Ca2+ ([Ca2+]i) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca2+-dependent Cl- channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 microM) led to an increase in [Ca2+]i and activation of Cl- currents. In contrast, intracellular application of Ca2+ (10 microM) only induced transient activation of Cl- currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca2+ and to the blocker of ICRAC, La3+ (10 microM), despite the fact that both maneuvers led to a decline in [Ca2+]i. The InsP3-induced rise in Cl- conductance could be prevented either by thapsigargin-induced (1 microM) depletion of intracellular Ca2+ stores or by removal of Ca2+ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca2+-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 microM) had no effect. Inhibition of Ca2+/calmodulin kinase (KN-63, KN-92; 5 microM) reduced Cl--conductance in 50% of the cells investigated without affecting [Ca2+]i. Inhibition of protein tyrosine kinase (50 microM tyrphostin 51, 5 microM genistein, 5 microM lavendustin) reduced an increase in [Ca2+]i and Cl- conductance. In summary, elevation of [Ca]i by InsP3 leads to activation of Cl- channels involving cytosolic Ca2+ stores and Ca2+ influx from extracellular space. Tyrosine kinases are essential for the Ca2+-independent maintenance of this conductance.
Strauss
O
O
Institut für Klinische Physiologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany.
Steinhausen
K
K
Mergler
S
S
Stumpff
F
F
Wiederholt
M
M
D000818:Animals; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D018118:Chloride Channels / Q000502:physiology*; D007295:Inositol Phosphates / Q000502:physiology*; D015640:Ion Channel Gating / Q000502:physiology*; D018408:Patch-Clamp Techniques; D010857:Pigment Epithelium of Eye / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D051381:Rats; D015398:Signal Transduction
D018118:Chloride Channels; D007295:Inositol Phosphates; D011505:Protein-Tyrosine Kinases; D002118:Calcium