Figure 1

U73343 and U73122 block MTX-induced Ca²⁺ influx and ethidium uptake. Upper panel. Fura-2 loaded BAECs were suspended in HBS and the fluorescence ratio was recorded as a function of time. Five traces are shown superimposed. At time 100 sec, U73343 was added at the final concentration indicated to the right of each trace. At time 300 sec 0.3 nM MTX was added to stimulate Ca²⁺ influx. Concentration-response curves were determined for both U73122 (n = 3) and U73343 (n = 5). Inset shows the % inhibition relative to DMSO control. Lower panel. Ethidium bromide (EB) uptake in BAECs was determined from the increase in fluorescence as a function of time as described in Materials and Methods. U73122 (red trace), U73343 (blue trace), or DMSO (black trace) were added at time 0, EB was added at 50 sec, and MTX was added at 200 sec. Values shown are normalized to the maximum fluorescence obtained by addition of digitonin at the end of each trace. Results shown are representative of 3 independent experiments.