C. Elsing et al., EFFECT OF SURFACE AND INTRACELLULAR PH ON HEPATOCELLULAR FATTY-ACID UPTAKE, American journal of physiology: Gastrointestinal and liver physiology, 34(6), 1996, pp. 1067-1073
Fatty acids enter hepatocytes, at least in part, by a carrier-mediated
uptake mechanism. The importance of driving forces for fatty acid upt
ake is still controversial. To evaluate possible driving mechanisms fo
r fatty acid transport across plasma membranes, we examined the role o
f transmembrane proton gradients on fatty acid influx in primary cultu
red rat hepatocytes. After hepatocytes were loaded with SNARF-1 acetox
ymethyl ester, changes in intracellular pH (pH(i)) under different exp
erimental conditions were measured and recorded by confocal laser scan
ning microscopy. Fatty acid transport was increased by 45% during cell
ular alkalosis, achieved by adding 20 mM NH4Cl to the medium, and a co
ncomitant paracellular acidification was observed. Fatty acid uptake w
as decreased by 30% during cellular acidosis after withdrawal of NH4Cl
from the medium. Cellular acidosis activates the Na+/H+ antiporter to
export excessive protons to the outer cell surface. Inhibition of Na/H+ antiporter activity by amiloride diminishes pH(i) recovery and the
reby accumulation of protons at the outer surface of the plasma membra
ne. Under these conditions, fatty acid uptake was further inhibited by
57% of control conditions. This suggests stimulation of fatty acid in
flux by an inwardly directed proton gradient. The accelerating effect
of protons at the outer surface of the plasma membrane was confirmed b
y studies in which pH of the medium was varied at constant pH(i). Sign
ificantly higher fatty acid influx rates were observed at low buffer p
H. Recorded differences in fatty acid uptake appeared to be independen
t of changes in membrane potential, because BaCl2 did not influence in
itial uptake velocity during cellular alkalosis and paracellular acido
sis. Moreover, addition of oleate-albumin mixtures to the NH4Cl incuba
tion buffer did not change the observed intracellular alkalinization.
In contrast, after cells were acid loaded, addition of oleate-albumin
solutions to the recovery buffer increased pi-Ii recovery rates from 0
.21 +/- 0.02 to 0.36 +/- 0.05 pH units/min (P < 0.05), indicating that
fatty acids further stimulate Na+/H+ antiporter activity during pH(i)
recovery from an acid load. It is concluded that carrier-mediated upt
ake of fatty acids in hepatocytes follows an inwardly directed transme
mbrane proton gradient and is stimulated by the presence of H+ at the
outer surface of the plasma membrane.