Alpha-stat calibration of indo-1 fluorescence and measurement of intracellular free calcium in rat ventricular cells at different temperatures.
To explore how to manage pH when calibrating Ca2+ probes at different temperatures, the dissociation constant (Kd) of indo-1 was determined both in pH-stat (pH is fixed despite the temperature) and in alpha-stat (pH changes with temperature as in cells). The results showed that the Kd was much more sensitive to temperature in pH-stat than in alpha-stat, demonstrating that alpha-stat calibration should be preferred when using a Ca2+ probe to measure intracellular free calcium ([Ca2+]i) at different temperatures. Based on the calibration in situ and in alpha-stat, we showed a striking increase of [Ca2+]i from 141+/-8 nM at 30 degrees C to 218+/-22 nM at 10 degrees C in indo-1 loaded rat ventricular cells, which supports that intracellular calcium overload takes place in cardiac myocytes of non-hibernating mammals during hypothermia.
Wang
S Q
SQ
College of Life Sciences, Peking University, Beijing, China. wangsq@grc.nia.nih.gov
Zhou
Z Q
ZQ
D000704:Analysis of Variance; D000818:Animals; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D002138:Calibration; D005260:Female; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007211:Indoles / Q000378:metabolism*; D008297:Male; D018613:Microscopy, Confocal; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013696:Temperature; D013816:Thermodynamics
D002021:Buffers; D005456:Fluorescent Dyes; D007211:Indoles; C048960:indo-1; D002118:Calcium