FURA-2 FLUORESCENT TECHNIQUE FOR THE ASSESSMENT OF CA2+ HOMEOSTASIS IN CARDIOMYOCYTES

Ca2+ homeostasis plays a pivotal role in maintaining cell growth and f unction. Many heart diseases are related to the abnormalities in Ca2mobilization and extrusion. Ca2+-sensitive fluorescent dyes have been used successfully to estimate intracellular free Ca2+ ([Ca2+](i)) leve l and the mechanisms of Ca2+ movements in living cells. This article i s focused on the methodology involving the use of Fura-2/AM or free Fu ra-2 to measure agonist-induced Ca2+ mobilization as well as the mecha nisms of changes in [Ca2+](i) in cardiomyocytes. Methods involving Fur a-2 technique for the measurement of Ca2+ extrusion from the cells and Ca2+ reuptake by sarcoplasmic reticulum (SR) are also described. The prevention of KCl-induced increase in the intracellular Ca2+ is shown by chelating the extracellular Ca2+ with EGTA or by the presence of Ca 2+-channel inhibitors such as verapamil and diltiazem. The involvement of SR in the ATP-induced increase in intracellular Ca2+ is illustrate d by the use of Ca2+-pump inhibitors, thapsigargin and cyclopiazonic a cid as well as ryanodine which deplete the SR Ca2+ storage. The use of 2-nitro-4-carboxyphenyl N,N-diphenyl carbamate (NCDC), an inhibitor o f inositol 1,4,5-trisphosphate (IP3) production, is described for the attenuation of phosphatidic acid (PA) induced increase in Ca2+-mobiliz ation. The increase in intracellular Ca2+ in cardiomyocytes by PA, unl ike that by KCl or ATP, was observed in diabetic myocardium. Thus, it appears that the Fura-2 method for the measurement of Ca2+ homeostasis in cardiomyocytes is useful in studying the pathophysiology and pharm acology of Ca2+ movements.