Total Cell Associated | ei + es-Mediated Uptake | es-Mediated Uptake | ||||
---|---|---|---|---|---|---|
Max | Vi | Max | Vi | Max | Vi | |
μM | pmol/μl/s | μM | pmol/μl/s | μM | pmol/μl/s | |
HN-5a | ||||||
−Na/−ATP | 8.9 ± 0.6 | 0.48 ± 0.03 | 6.4 ± 0.4 | 0.29 ± 0.02 | 5.4 ± 0.4 | 0.34 ± 0.05 |
−Na/+ATP | 5.4 ± 0.32-a | 0.49 ± 0.07 | 2.4 ± 0.82-a | 0.12 ± 0.03 | 1.8 ± 0.42-a | 0.16 ± 0.01 |
+Na/+ATP | 5.7 ± 0.3 | 0.48 ± 0.07 | 2.6 ± 0.3 | 0.08 ± 0.02 | 2.2 ± 0.2 | 0.13 ± 0.04 |
GEM-8e | ||||||
−Na/−ATP | 9.0 ± 0.5 | 0.57 ± 0.06 | 6.9 ± 0.5 | 0.25 ± 0.04 | 5.8 ± 0.5 | 0.22 ± 0.02 |
−Na/+ATP | 5.7 ± 0.62-a | 0.67 ± 0.17 | 3.6 ± 0.62-a | 0.20 ± 0.05 | 2.4 ± 0.42-a | 0.30 ± 0.06 |
+Na/+ATP | 6.8 ± 0.8 | 0.58 ± 0.19 | 3.1 ± 0.6 | 0.13 ± 0.03 | 2.8 ± 0.3 | 0.12 ± 0.04 |
ATP-depleted (−ATP) and ATP-replete (+ATP) cells were equilibrated in either normal Dulbecco’s PBS (+Na) or in an Na+-free PBS buffer (−Na; iso-osmotic replacement with Li+) and then incubated with 10 μM [3H]gemcitabine in absence (total cell associated) and presence of 100 nM NBMPR or 10 μM NBMPR/dipyridamole for various times as shown in Fig. 4. Equilibrative transporter (ei + es)-mediated uptake was calculated as total cell associated [3H]gemcitabine minus that observed in presence of 10 μM NBMPR/dipyridamole. NBMPR-sensitive (es) transporter-mediated uptake was calculated as difference in cell accumulation of [3H]gemcitabine in presence and absence of 100 nM NBMPR. Hyperbolic curves were fitted to resulting time course data and extrapolated to obtain estimates of steady-state intracellular concentrations of [3H]gemcitabine (Max) and initial rate of influx (Vi). Each value represents mean ± S.E. of four independent experiments.
↵2-a Significantly different from corresponding data obtained using ATP-depleted cells (one-way ANOVA with Newman-Keuls post test, p < .05).