We also studied the effect of 24 hours of SNAP treatment on Na
+-dependent and -independent uptake of
l-cystine from the apical and basolateral fluid of RCECs. These experiments were conducted in an NaCl or choline chloride buffer after treating the cells with 0.1, 0.25, 0.5, 1.0, and 2.0 mM SNAP for 24 hours. Apical uptake of
l-cystine was not significantly changed with 0.1 mM SNAP, whereas it increased significantly (>6 fold) with 1 mM SNAP in both apical and basolateral fluids. By contrast, the increase of apical
l-cystine uptake was only approximately twofold after treatment with 2 mM SNAP for 24 hours
(Fig. 5A) . The pattern of stimulation of basolateral
l-cystine uptake was similar to that of apical uptake
(Fig. 5B) . For the treatment with 2 mM SNAP for 24 hours, the increase in basolateral uptake remained higher than fivefold. Treatment of RCECs with either a protein synthesis inhibitor (CHX) or RNA synthesis inhibitor (AD) caused the uptake rate for
l-cystine after 1 mM SNAP
(Fig. 6A) to decline to that of the untreated control. Basolateral uptake of
l-cystine was also increased by SNAP, although the SNAP sensitivity was different from that observed for apical
l-cystine uptake, in that a significant increase in basolateral
l-cystine uptake was observed even at 0.1 mM SNAP (not shown), and a nearly fivefold increase occurred with 1 mM SNAP
(Fig. 6B) . SNAP-induced stimulation of basolateral
l-cystine uptake was blocked similarly by either CHX or AD. The pattern of SNAP-induced increase in the uptake rate of
l-cystine from apical fluid observed in the absence of Na
+ (Fig. 6C) was almost identical with that observed in the presence of Na
+. Approximately 53% of the SNAP-induced
l-cystine uptake from apical fluid was due to the Na
+-independent transport component. In contrast, the basolateral Na
+-independent
l-cystine uptake rate was not significantly affected by SNAP
(Fig. 6D) . Therefore, virtually all SNAP-induced
l-cystine uptake from basolateral fluid was due to the Na
+-dependent transport component.