title: Cytosolic calcium in the ischemic rabbit heart: assessment by pH- and temperature-adjusted rhod-2 spectrofluorometry.
abstract: OBJECTIVE
  Cytosolic calcium ([Ca(2+)](i)) mediates ischemia-reperfusion (I/R) injury, but magnitude and time course of I/R-induced [Ca(2+)](i) overload remain unclear. Fluorescent indicators are commonly used to measure [Ca(2+)](i), and have a dissociation constant (K(d)) that depends on pH and temperature. We hypothesized that changes of K(d) during I/R lead to misleading interpretations of [Ca(2+)](i) recordings.
  
  
  METHODS
  (1) In isolated rabbit hearts (n=4 each), intracellular pH was measured during I/R at 37 degrees C, 20 degrees C, and 4 degrees C with and without cardioplegic arrest by (31)P-NMR-spectroscopy. (2) K(d) for rhod-2 and calcium was determined at varying pH and temperature in in vitro experiments. (3) Isolated rabbit hearts were subjected to I/R, and [Ca(2+)](i) was recorded by surface rhod-2 spectrofluorometry. Finally, [Ca(2+)](i) was computed using either the conventional K(d), or the pH- and temperature-adjusted K(d).
  
  
  RESULTS
  K(d)(Ca(2+)Rhod-2) remained stable between pH 7.1 and 6.8, but increased exponentially with lower pH and/or temperature. Calculations using a static K(d) indicated that [Ca(2+)](i) rose only mildly during warm ischemia and did not rise during cardioplegic arrest, while a large Ca(2+) influx appeared to occur during early reperfusion. When the pH and temperature-adjusted K(d) was used for calculation, [Ca(2+)](i) rose significantly during ischemia (431+/-37% during 20 min 37 degrees C ischemia, and 78+/-19% during 20 min cardioplegic arrest at 20 degrees C). During early reperfusion, [Ca(2+)](i) decreased rapidly, without significant further [Ca(2+)](i) elevation.
  
  
  CONCLUSIONS
  In contrast to previous reports, [Ca(2+)](i) accumulation occurs during unprotected ischemia as well as hypothermic ischemia with cardioplegic arrest, without further net Ca(2+) influx on reperfusion. This finding has important implications for timing of protective strategies during myocardial ischemia.
mesh_terms: D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D005456:Fluorescent Dyes; D006324:Heart Arrest, Induced; D006575:Heterocyclic Compounds, 3-Ring; D006863:Hydrogen-Ion Concentration; D017202:Myocardial Ischemia / Q000378:metabolism*; D010477:Perfusion; D011817:Rabbits; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D006575:Heterocyclic Compounds, 3-Ring; C068483:rhod-2; D002118:Calcium