Characterization of ascorbic acid transport by adrenomedullary chromaffin cells. Evidence for Na+-dependent co-transport

Abstract

Ascorbic acid transport by bovine adrenomedullary chromaffin cells in primary cultures has been characterized. Ascorbic acid uptake can be measured by either high performance liquid chromatography with electrochemical detection or radiometric techniques with L-[1-14C]ascorbic acid. The transport system is temperature- and energy-dependent and exhibits Michaelis-Menten kinetics with an apparent Km of 29 microM when the external Na+ concentration is 150 mM. Uptake of ascorbate by chromaffin cells is ouabain-sensitive and dependent on the presence of external Na+. Ascorbate transport by chromaffin cells is, thus, an active process driven by the Na+ electrochemical gradient. The kinetics of this co-transport system fits an "affinity type" model where binding of Na+ to the carrier increases the affinity to ascorbate and vice versa. Thus, the data suggest that binding of either Na+ or ascorbate induces a conformational change in the transporter, which results in a change in the association constant for the second ligand while the mobility of the carrier remains unchanged. Cellular uptake of ascorbate into adrenomedullary chromaffin cells appears to be followed by its distribution into several subcellular compartments. One subcellular compartment for concentration of ascorbate is the chromaffin vesicle where it accumulates at a relatively slow rate. The interrelationships between ascorbate transport and other aspects of ascorbate metabolism and chromaffin vesicle function and dopamine beta-hydroxylation are also considered.