Development of concepts, techniques and applications related to new stationary phases for high performance liquid chromatography (HPLC)

Abstract

The calcium ion (Ca) plays important roles in the living cell, from genetic and regulatory levels to a direct participation in the generation of electrical activity and contraction, as it is the case in muscle cells. In the heart, several Ca transporters are involved in the regulation of intracellular Ca concentration, so that Ca can be provided to and removed from the myofilaments in a proper amount and time-course for contraction and relaxation, respectively. The heart cells are still immature in newborn mammalians and they undergo marked structural and functional development in the first weeks after birth. There are indications that Ca regulation (and Ca fluxes through the several transporters) may be different in the hearts of adult and newborn animals, but quantitative information about Ca fluxes in intact, living cells is still lacking in the literature. The main goal of the present project is to determine, from a quantitative point of view, in cardiac myocytes from adult and newborn/young rats : a) sarcoplasmic reticulum (SR) Ca content; b) Ca influx into the cell via transmembrane currents; c) ability of inflowing Ca to trigger SR Ca release; d) relative participations of the several Ca transporters in cell relaxation; e) Ca uptake by isolated cardiac mitochondria; f) passive Ca binding characteristics; and g) in vivo calibration of the fluorescent Ca indicator indo-1. The methodology to be used has been previously tested and/or developed by the research team. Basic procedures involve patch-clamp tecniques, Ca concentration measurement with fluorescent indicators, cell shortening measurement by edge detection and estimations of the Ca fluxes and Ca binding. (AU)