The transport of FFA across the plasma membrane represents one of the earliest points at which FFA metabolism can be controlled by cardiac myocytes. Using novel methods to measure the intracellular unbound concentration of FFA ([FFA
i
]), the first direct measurements of FFA transport across cardiac plasma membranes have been performed in freshly isolated cardiac myoctyes. Measurements of the unbound concentrations of FFA (FFA
u
) in the aqueous phase were performed using the fluorescent ratio probe ADIFAB. Cardiac myocytes were microinjected with ADIFAB, and the transport of oleate and palmitate was determined by monitoring [FFA
i
] using fluorescence ratio microscopy. FFA influx was initiated by rapidly increasing the extracellular concentration of FFA
u
([FFA
o
]) using FFA-BSA complexes, which clamped [FFA
o
] at fixed values. The time course of influx was monitored from the change in [FFA
i
], which rose exponentially to a steady state level (k
influx
~ 0.01 s
−1
). Once steady state was achieved, efflux was initiated by changing the extracellular media back to zero [FFA
o
]. Efflux was monitored by the decrease in [FFA
i
] which, like influx, revealed exponential behavior (k
efflux
~ 0.02 s
−1
). At steady state [FFA
i
] was greater than [FFA
o
] by a factor of ~3.5, indicating that during influx FFA are pumped up a concentration gradient. Both the initial rate of transport and the gradient ([FFA
i
] > [FFA
o
]) revealed saturation with increasing [FFA
o
]. The initial rate of influx saturated at [FFA
o
] > 200 nM, while the [FFA
i
] > [FFA
o
] gradient was relatively constant (~ 3.5) but began to decrease and approached 1 at [FFA
o
] > 200 nM. The efflux rate constant decreased for [FFA
o
] > zero, suggesting that efflux may be regulated by a mechanism that senses the level of circulating FFA
u
. Our results indicate that the mechanism of FFA transport across cardiac myocytes is regulated by the plasma membrane and allows for the efficient storage and release of FFA from cardiac myocytes. We suggest that this mechanism involves an as yet unknown membrane protein pump which enables the cells to accumulate surprisingly high concentrations of FFA. The ability to measure [FFA
i
] and the demonstration of efflux are significant steps in understanding cardiac FFA metabolism.
This research has received full or partial funding support from the American Heart Association, AHA Western States Affiliate (California, Nevada & Utah).
Production of long-chain free fatty acids from metabolically engineered Rhodobacter sphaeroides heterologously producing periplasmic phospholipase A2 in dodecane-overlaid two-phase culture
