Characterization of a human plasma membrane heme transporter in intestinaland hepatocyte cell lines

Heme is the most bioavailable form of dietary iron and a component of many cellular proteins. Controversy exists as to whether heme uptake occurs via specific transport mechanisms or passive diffusion. The aims of this study were to quantify cellular heme uptake with a fluorescent heme analog and to determine whether heme uptake is mediated by a heme transporter in intesti nal and hepatic cell lines. A zinc-substituted porphyrin, zinc mesoporphyri n (ZnMP), was validated as a heme homolog in uptake studies of intestinal ( Caco-2, I-407) and hepatic (HepG2) cell lines. Uptake experiments to determ ine time dependence, heme inhibition, concentration dependence, temperature dependence, and response to the heme synthesis inhibitor succinylacetone w ere performed. Fluorescence microscope images were used to quantify uptake and determine the cellular localization of ZnMP; ZnMP uptake was seen in in testinal and hepatic cell lines, with cytoplasmic uptake and nuclear sparin g. Uptake was dose-and temperature dependent, inhibited by heme competition , and saturated over time. Preincubation with succinylacetone augmented upt ake, with an increased initial uptake rate. These findings establish a new method for quantifying heme uptake in individual cells and provide strong e vidence that this uptake is a regulated, carrier-mediated process.