NMR methods provide a unique approach for the investigation of metabolic and physiological processes in intact systems, perfused organs, cell suspensions, as well as by examination of cell extracts. Cellular cations play fundamental roles in hormonal signaling, and are also involved in the mediation of cell injury. The development of intracellular indicators for cytosolic cations and other parameters of interest has had a major impact on the field of cell biology. We have continued our work on the development and application of NMR indicators for intracellular cations and other parameters of interest. We are currently working on other fluorinated quinolones to obtain more selective Mg2+ NMR indicators, and on a trifluoromethylated analog of the calcium chelator BAPTA. Indicators developed as part of this porject have been utilized to study cell injury in perfused heart model systems. We have recently been performed to determine the effect of NO on sarcoplasmic reticulum (SR) Ca2+ using the fluorinated indicator TFBAPTA which we have found to load preferentially into the SR compartment of rabbit hearts. The indicator was loaded into the Langendorff perfused heart preparation that we have used previously, and the NO was generated by the precursor S-nitroso-N-acetyl-penicillamine (SNAP). In one series of studies, addition of 100 uM SNAP was followed by a 10 minute washout period, 20 minutes of global ischemia, and 20 minutes of reperfusion. We found that addition of SNAP resulted in a significant decrease in SR Ca2+ concentration, from 1.11 mM to 0.78 mM (p<0.001 with paired t-test). Following 20 minutes of global ischemia, SR Ca2+ fell to 0.70 mM in the control group and recovered to 1.06 during the first 10 minutes of reflow (p<0.005). In the SNAP treated hearts, the SR Ca2+ fell to a similar level, but did not recover by the end of reflow (0.72 mM). The basis for the SNAP-induced decrease in SR Ca2+ is currently under study.
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