Sustained calcium influx activated by basic fibroblast growth factor in Balb-c 3T3 fibroblasts.
1. We have investigated the ionic events elicited in Balb-c 3T3 fibroblasts by basic fibroblast growth factor (bFGF), a peptide that binds to membrane receptors with tyrosine kinase activity and has a mitogenic action on many cell types. The peptide (0.2-100 ng ml-1) caused the appearance of an inward current, as observed in whole-cell patch-clamp experiments at a holding potential of -50 mV, that could last for tens of minutes and had a peak density of 4.6 +/- 2.6 pA pF-1. The reversal potential was 18.8 +/- 16.7 mV. 2. The current was reversibly abolished by removal of bFGF from the external bath. Inhibition of low-affinity FGF receptors had no effect on the activation of the inward current; it was completely abolished when cells were pre-incubated with tyrphostin or 5'-methylthioadenosine (MTA), two inhibitors of the tyrosine kinase activity of the high-affinity FGF receptors. The inward current was not activated by the emptying of internal calcium stores, as tested with 200 nM thapsigargin. 3. Values of peak current density comparable to control ones were obtained when either all Na+ ions or all Ca2+ ions were removed from the external solution; when both ions were completely removed, no inward current could be observed. The inward current was not affected by 2 microM nifedipine, and was reversibly blocked by the imidazole derivative SK&F 96365-A. 4. Measurements of free intracellular calcium concentration ([Ca2+]i) with the dye fura-2 showed that bFGF elicited sustained increases in [Ca2+]i that were completely dependent on external calcium and on the presence of the agonist and could last more than 1 h. 5. Single channel currents (conductance 7.9 pS) in response to bFGF stimulation could be recorded in the cell-attached configuration with 100 mM CaCl2 in the pipette. When the resting potential was brought near to 0 mV by external perfusion in a high-K+ solution, Vrev was about 0 mV. 6. We conclude that in Balb-c 3T3 cells bFGF induces an inward current that is carried at least partially by Ca2+ ions; this current in turn causes a long-lasting increase in intracellular Ca2+ concentration. The amplitude and time course of these bFGF-activated ionic events are compatible with their involvement in the control of cell proliferation.
Munaron
L
L
Department of Animal Biology, University of Torino, Italy.
Distasi
C
C
Carabelli
V
V
Baccino
F M
FM
Bonelli
G
G
Lovisolo
D
D
D016475:3T3 Cells; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology; D004553:Electric Conductivity; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D007477:Ions; D051379:Mice; D008807:Mice, Inbred BALB C; D018408:Patch-Clamp Techniques; D015398:Signal Transduction
D015220:Calcium Channels; D007477:Ions; D016222:Fibroblast Growth Factor 2; D002118:Calcium