Effect of temperature on receptor-activated changes in [Ca2+]i and their determination using fluorescent probes

Several recent studies of intracellular Ca2+ concentration ([Ca2+]i using fluorescent indicators have involved experiments performed at nonphysiological temperatures, frequently room temperature. In the studies reported here, a reduction in temperature was shown to influence the measurement of [Ca2+]i in two major ways. The first is an effect on the value for the Kd (apparent dissociation constant) used in the calculation of [Ca2+]i. The Kd values for indo-1 and fura-2 were found to change with temperature in a manner analogous to the related compounds [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid and bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid. Experiments showed that the use of previously published Kd values (measured at 37 degrees C) in experiments at room temperature results in errors of at least 25% in the calculated [Ca2+]i. The second arises from the differential temperature sensitivities of the physiological processes that make up any particular value of [Ca2+]i. While resting [Ca2+]i was relatively unaffected by reduced temperature (27 degrees C, 13 degrees C), changes in [Ca2+]i following stimulation were profoundly influenced and in a way that was not simply predictable. Analysis of the responses indicated that the observed effects resulted largely from a temperature-dependent reduction in the rate of Ca2+ removal from the cytosol together with an additional component, namely a marked reduction in Ca2+ entry, at the lower temperature (13 degrees C). The net result was observed as changes in the initial rise in [Ca2+]i on stimulation, together with more profound, and complex, effects on the sustained elevation in [Ca2+]i following stimulation. This latter component was markedly increased at 27 degrees C, but was decreased in cells at 13 degrees C.