The effect of pH and temperature on the dissociation constant for fura-2 and their effects on [Ca(2+)](i) 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 Ca(2+) concentrations ([Ca(2+)](i)), at physiological temperatures, in poikilothermic animals living at low temperatures. The K(d) for fura-2 was found to decrease with increasing temperature (5-37 degrees C) and DeltaH, 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 K(d) are corrected for, fura-2 is a suitable tool for measurements of [Ca(2+)](i) at temperatures of 5-37 degrees C. The present study demonstrates that Atlantic cod enterocytes, acclimated to 37 degrees C, show a lower basal [Ca(2+)](i) (65 +/- 8 nM) compared to enterocytes acclimated to 10 degrees C (161 +/- 6 nM). Furthermore, addition of 10 mM Ca(2+) increases the [Ca(2+)](i) by 526%, when compared to basal [Ca(2+)](i), in cells 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.
Larsson
D
D
Department of Zoology/Zoophysiology, Göteborg University, Sweden. d.larsson@zool.gu.se
Larsson
B
B
Lundgren
T
T
Sundell
K
K
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D005399:Fishes / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D016257:Fura-2 / Q000031:analogs & derivatives* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000166:cytology; D008954:Models, Biological; D013696:Temperature
D005456:Fluorescent Dyes; C049925:fura-2-am; D002118:Calcium; D016257:Fura-2