The Effect of pH and Temperature on the Dissociation Constant for Fura-2 and Their Effects on [Ca2+]iin 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 physiological temperatures, in poikilothermic animals living at low temperatures. The Kdfor fura-2 was found to decrease with increasing temperature (5–37°C) and ΔH,in the Van't Hoff isochore equation, was determined 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 temperature-dependent changes in Kdare corrected for, fura-2 is a suitable tool for measurements of [Ca2+]iat temperatures of 5–37°C. The present study demonstrates that Atlantic cod enterocytes, acclimated to 37°C, show a lower basal [Ca2+]i(65 ± 8 nM) compared to enterocytes acclimated to 10°C (161 ± 6 nM). Furthermore, addition of 10 mM Ca2+increases the [Ca2+]iby 526%, when compared to basal [Ca2+]i, in cells at 37°C but only by 36%, in cells kept at 10°C. Thus, performing experiments at unphysiological temperatures results in cellular responses that would not be observed under physiological conditions.