In the present study we have characterized ATP-dependent transport of cAMP and cGMP in physiological, but also supraphysiological concentrations. The uptake into inside-out vesicles from human erythrocytes could be dissected into two components with high and low affinity. The respective Km-values were 30.8 ± 5.2 and 352 ± 26 μM for cAMP and 2.6 ± 0.4 and 260 ± 15 μM for cGMP. The two cyclic nucleotides were unable to mutually inhibit cellular efflux for concentrations up to about 100 μM. At higher concentrations the inhibition curve showed a steep fall. The IC50-value for cAMP reduction of high affinity [3H]-cGMP transport was 695 ± 9 μM. The respective value for cGMP inhibition of [3H]-cAMP efflux was 284 ± 20 μM. These observations are compatible with two selective high affinity transport systems. Other endogenous substances such as prostaglandins did not discriminate between cyclic nucleotide transport. The IC50 values for inhibition of [3H]-cAMP and [3H]-cGMP were 4.1 and 4.2 μM for PGE1, 2.7 and 4.4 μM for PGE2, respectively. However, the prostaglandin analog misoprostol discriminated distinctly between cAMP and cGMP transport with respective IC50-values of 4.5 and 24 μM. The assumption that the specific PDE5-inhibitor sildenafil could distinguish between the two cyclic nucleotides was disproved with respective IC50 values of 3.8 and 2.9 μM for inhibition of [3H]-cAMP and [3H]-cGMP, respectively. However, at least one sildenafil analog (PHAR0099048) showed a clear difference with respective IC50 values of 2.0 and 0.52 μM. The other tested sildenafil analogs showed no or minor ability to discriminate with IC50 values of 0.16 and 0.17 μM for IS-39213, and 0.35 and 0.16 μM for IS-60049, respectively. In agreement with previous reports, the present study shows that proteins responsible for cyclic nucleotide transport are multiorganic anion pumps. However, the observation that drug analogs may discriminate between these two efflux systems makes them potential drug targets.
Members of the ATP-Binding-Cassette family, ABCC4, ABCC5 and ABCC11, have been identified as transport proteins for cyclic nucleotides [1-5]. The cellular efflux of these signal molecules was characterized decades ago, including cAMP (for review see [
Specific inhibitors of phosphodiesterase 5 (PDE5) like sildenafil, have been identified as potent inhibitors of ABCC5-mediated cGMP cellular efflux [
In addition we employed sildenafil derivates, identified by molecular modeling and virtual ligand screening (VLS) [
The following substances were employed: [3H]-cGMP and [3H]-cAMP (Perkin Elmer, Boston, MA), cGMPcAMP, misoprostol, PGE1 and PGE2 (Sigma Aldrich, Schnelldorf, Germany), PHAR0099048, IS-39213 and IS-60049 (Ambinter, Greenpharma SAS, Orléans, France) and sildenafil (Pfizer Inc., NY). Other chemicals were of analytical grade.
In the present study, IOVs were prepared using a modification of the method described by Steck [
In the present study [3H]-cAMP and [3H]-cGMP uptake into IOVs was determined in absence or in the presence of various inhibitors. IOVs were incubated for 60 minutes with or without 2.0 mM ATP in a mixture containing 20 mM Tris-HCl, 10 mM MgCl2, 1 mM EGTA, 2 µM [3H]-cGMP or 2 µM [3H]-cAMP, 121 mM KCl (pH 8.0) at 37˚, and substrates or inhibitors in concentrations up to 1 mM. The transport process was terminated with addition of ice-cold 1.47 mM KH2PO4, 8.1 mM K2HPO4 and 140 mM KCl (pH 7.6) and rapid filtration through nitrocellulose membranes (0.22 µm GSWP, Millipore, Billerica, MA) in a refrigerated laboratory (4˚C). The radioactivity on the filters was quantified by liquid scintillation (Ultima Gold XR, Packard, Groningen, The Netherlands) in a Packard 1900 TR Liquid Scintillation analyzer. DMSO was needed to dissolve some of the inhibitors and a similar concentration was added to the control samples.
Hofstee-inhibition plot was used to decompose biphasic curves to obtain low and high Km-values for cAMP and cGMP transport [
ATP-dependent radiolabeled cyclic nucleotide uptake into IOVs was determined in the presence of the respective unlabeled compound in concentrations from micromolar to millimolar. The Hofstee inhibition plot [
In a previous study we found that physiologic concentrations of cAMP did not reduce high affinity cGMP transport and only with 10% - 15% at 100 µM [
different transporters are responsible for high affinity efflux of cyclic nucleotides from hRBC.
The prostaglandins belong to a subclass of eicosanoids, termed prostanoids and proposed as specific ABCC4- substrates [
Sildenafil is a potent PDE5 inhibitor [
The cyclic nucleotides cAMP and cGMP are extruded from cells by an ATP-dependent process. The efflux from human erythrocytes comprises at least two components. A low affinity component was reported for cAMP transport in ghosts (Km = 400 - 500 µM) [
Inhibition studies support these results. The present work showed that cAMP, below 100 µM, was almost unable to reduce high affinity transport of [3H]-cGMP (
*n = 4, **n = 2.
100 µM with an IC50 of ≈280 µM. A low affinity state of ABCC4 as well as ABCC5 may account for this since intact HEK293 cells overexpressing ABCC4 or ABCC5 showed linear transport rates for the two cyclic nucleotides up to intracellular concentrations of about 600 µM [
The first report on cAMP transport with ABCC4 showed moderate to high affinity (Km ≈ 45 µM) [
The question whether ABCC5 is involved in cGMP efflux at all, is relevant [
ABCC5 was identified as a high affinity cGMP transporter with a Km of 2.1 µM [
In contrast to the selectivity observed for cyclic nucleotides, PGE1 and PGE2 (
It is difficult to explain the present observations with a single high affinity efflux pump for cyclic nucleotides. Our hypothesis is that ABCC4 and ABCC5 represent a similar low affinity transport system with low or no selectivity and that ABCC4 and ABCC5 are responsible for high affinity transport of cAMP and cGMP, respectively. The observation that two exobiotics showed a limited but distinct discrimination between the two transport systems encourages further studies on these pumps as potential drug targets.
The financial support from the Norwegian Cancer Society and the kind gift of sildenafil from Pfizer Inc. is gratefully acknowledged. Thanks are due to Natalia Smaglyukova for excellent technical assistance.