omega-agatoxin-iva and indo-1

Intracellular calcium waves in fish keratocytes are induced by the application of electric field pulses with amplitudes between 55 and 120 V/cm and full width at half-maximum of 65-100 ms. Calcium concentrations were imaged using two-photon excited fluorescence microscopy (Denk et al., 1990 Science. 248:73-76; Williams et al. 1994 FASEB J. 8:804-813) and the ratiometric calcium indicator indo-1. The applied electric field pulses induced waves with fast calcium rise times and slow decays, which nucleated in the lamellipodium at the hyperpolarized side of the cells and, less frequently, at the depolarized side. The effectiveness of wave generation was determined by the change induced in the membrane potential, which is about half the field strength times the cell width in the direction of the field. Stimulation of waves began at voltage drops across the cell above 150 mV and saturated at voltage drops 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 specific organic antagonists of voltage-sensitive calcium channel conductance. Thapsigargin stopped wave propagation in the cell body, indicating 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 reaches a threshold, release from intracellular stores is induced, creating a propagating wave. These observations and the measured parameters (average velocity approximately 66 micron/s and average rise time approximately 68 ms) are consistent with a wave amplification model in which[equation, see text] determines the effective diffusivity of the propagating molecules, D approximately 300 micron2/s (Meyer, 1991. Cell. 64:675-678).

Topics: Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Chelating Agents; Electric Stimulation; Fluorescent Dyes; Goldfish; Indoles; Keratinocytes; Microscopy, Fluorescence; Models, Biological; omega-Agatoxin IVA; omega-Conotoxin GVIA; omega-Conotoxins; Peptides; Second Messenger Systems; Spider Venoms; Thapsigargin