DEVELOPMENT OF INTRACELLULAR INDICATORS AND ION TRANSPORT STUDIES

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It is well recognized that ions can function as intracellular messengers. It is not surprising, therefore, that alterations in ion transport frequently cause cellular dysfunction. For example, many oncogene products alter ion transport. The goal of this project is twofold: (1) development of new indicators for the measurement of intracellular ion concentrations, and (2) investigation of ion transport and its regulation, and the perturbations which accompany pathogenic states. Previously we have developed new NMR-active indicators for cytosolic calcium, magnesium, and chloride, and for cell volume. These NMR indicators are generally (although not necessarily) fluorinated molecules since fluorine-19 is 100% abundant, highly sensitive for NMR detection, and since cells are essentially free of any background fluorine NMR signals. Two major projects over the past year were: (1) development of a new, high dissociation constant (KD) intracellular calcium ion indicator, "TFBAPTA", and (2) synthesis and evaluation of several derivatives of "NEAP", our recently developed pH sensitive indicator. One of the principal limitations on the use of cytosolic calcium ion indicators in the perfused heart has been the significant perturbation of contractility which results from the introduction of intracellular calcium ion chelators. This was the primary motivation for our development of a new indicator, TFBAPTA, which has a considerably higher calcium ion dissociation constant and is therefore essentially non-perturbing. Although not suited for the determination of basal calcium ion concentrations, this indicator is useful for measuring the increases which accompany perturbations such as ischemia or the administration of certain chemical agents. We have also made several further modifications of the pH indicator NEAP in order to reduce leakage and to add an internal reference signal in order to facilitate quantitation. We are also continuing studies on the transport mechanisms that regulate cytosolic free magnesium well below its electrochemical equilibrium.