The effect of pH and temperature on the dissociation constant for fura-2 and their effects on [Ca2+], in enterocytes from a poikilothermic animal, Atlantic cod (Gadus morhua)

In this study, we investigated the validity of the fluorescent probe fura-2 in determinations of intracellular Ca2+ concentrations ([Ca2+](i)), at phy siological temperatures, in poikilothermic animals living at low temperatur es. The K-d for fura-2 was found to decrease with increasing temperature (5 -37 degrees C) and Delta H, in the Van't Hoff isochore equation, was determ ined to be 11.03 kJ/mol, when pH was corrected to 7.2 for all temperatures tested. The absorption maxima (340 nm) and isobestic point (360 nm) for the UV spectra of fura-2 were not affected by temperature. Thus, if pH-and tem perature-dependent changes in and are corrected for, fura-2 is a suitable t ool for measurements of [Ca2+](i) at temperatures of 537 degrees C. The pre sent study demonstrates that Atlantic cod enterocytes, acclimated to 37 deg rees C, show a lower basal [Ca2+](i) (65 +/- 8 nM) compared to enterocytes acclimated to 10 degrees C (161 +/- 6 nM). Furthermore, addition of 10 mM C a2+ increases the [Ca2+](i) by 526%, when compared to basal [Ca2+](i), in c ells at 37 degrees C but only by 36%, in cells kept at 10 degrees C. Thus, performing experiments at unphysiological temperatures results in cellular responses that would not be observed under physiological conditions. (C) 19 99 Academic Press.