DEVELOPMENT OF INTRACELLULAR INDICATORS AND ION TRANSPORT STUDIES

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Intracellular indicators for cytosolic ions have greatly extended our understanding of the role of these ions under normal and pathological conditions. Recent efforts have focused on several calcium ion indicators with relatively high (tens of micromolar) dissociation constants. Although in general measurements are most accurate if the KD is matched to the ion concentration under study, high KD indicators have several advantages and potential applications. These include: (1) reduced buffering of ion transients; (2) application to situations where the ion level may increase well beyond the basal value; (3) application to studies of organelles in which the calcium ion levels may be considerably higher than the cytosolic level. For example, the buffering of cytosolic calcium transients which results from loading a perfused rat heart with 5FBAPTA, the current NMR indicator of choice, significantly alters contractility. The indicator TFBAPTA which we have synthesized and evaluated provides an attractive solution to the above problems. This indicator has two fluorine resonances, one of which does not shift upon calcium complexation, and the second of which shifts by 10 ppm. At typical field strengths used, the calcium sensitive resonance is in fast exchange on the chemical shift scale. One important advantage of a fast exchange indicator such as TFBAPTA is that if the indicator loads into different cellular compartments with different calcium ion concentrations, separate resonances will be observed. An analogous high KD fluorescent indicator, fura-F has also been synthesized. Fura-F has a calcium ion KD of 20fM, and exhibits an excitation shift upon calcium ion complexation similar to fura-2. In addition to the indicator development work, we have also initiated studies to investigate the effect of ~-agonists on Mgi and magnesium efflux from perfused rat heart and S49 lymphoma cells using magnesium ion indicators previously developed as part of this program. Preliminary studies show no changes in Mgi, but a significant magnesium efflux upon addition of isoproterenol to perfused rat heart.