CALCIUM WAVES INDUCED BY LARGE VOLTAGE PULSES IN FISH KERATOCYTES

Intracellular calcium waves in fish keratocytes are induced by the app lication of electric field purses with amplitudes between 55 and 120 V /cm and full width at half-maximum of 65-100 ms. Calcium concentration s were imaged using two-photon excited fluorescence microscopy (Denk e t al., 1990. Science. 248:73-76; Williams et al. 1994. FASEB J. 8:804- 813) and the ratiometric calcium indicator indo-1. The applied electri c field pulses induced waves with fast calcium rise times and slow dec ays, which nucleated in the lamellipodium at the hyperpolarized side o f the cells and, less frequently, at the depolarized side. The effecti veness of wave generation was determined by the change induced in the membrane potential, which is about half the field trength times the ce ll width in the direction of the field. Stimulation of waves began at voltage drops across the cell above 150 mV and saturated at voltage dr ops above 300 mV, where almost all cells exhibited a wave. Waves were not induced in low-calcium media and were blocked by the nonselective calcium channel blockers cobalt chloride and verapamil, but not by spe cific organic antagonists of voltage-sensitive calcium channel conduct ance. Thapsigargin stopped wave propagation in the cell body, indicati ng that calcium release from intracellular stores is necessary. Thus a voltage pulse stimulates Ca2+ influx through calcium channels in the plasma membrane, and if the intracellular calcium concentration reache s a threshold, release from intracellular stores is induced, creating a propagating wave. These observations and the measured parameters (av erage velocity similar to 66 mu m/s and average rise time similar to 6 8 ms) are consistent with a wave amplification model in which v = root D/tau determines the effective diffusivity of the propagating molecul es, D approximate to 300 mu m(2)/s.