Abstract
The present study was conducted to characterise the transporter(s) responsible for the uptake of cyclic nucleotides to human erythrocytes. Western blotting showed that hRBC expressed OAT2 (SLC22A7), but detection of OAT1 (SLC22A6), or OAT3 (SLC22A8) was not possible. Intact hRBC were employed to clarify the simultaneous cyclic nucleotide egression and uptake. Both these opposing processes were studied. The K(m) -values for high affinity efflux was 3.5 ± 0.1 and 39.4 ± 5.7 μM for cGMP and cAMP, respectively. The respective values for low affinity efflux were 212 ± 11 and 339 ± 42 μM. The uptake was characterised with apparently low affinity and similar K(m) -values for cGMP (2.2 mM) and cAMP (0.89 mM). Using an iterative approach in order to balance uptake with efflux, the predicted real K(m) -values for uptake were 100-200 μM for cGMP and 50-150 μM for cAMP. The established OAT2-substrate indomethacin showed a competitive interaction with cyclic nucleotide uptake. Creatinine, also an OAT2 substrate, showed saturable uptake with a K(m) of 854 ± 98 μM. Unexpectedly, co-incubation with cyclic nucleotides showed an uncompetitive inhibition. The observed K(m) -values were 399 ± 44 and 259 ± 30 μM for creatinine, in the presence of cGMP and cAMP, respectively. Finally, the OAT1-substrate para-aminohippurate (PAH) showed some uptake (K(m) -value of 2.0 ± 0.4 mM) but did not interact with cyclic nucleotide or indomethacin transport.