Misoprostol and the Sildenafil analog (PHAR-0099048) Modulate Cellular Efflux of cAMP and cGMP Differently

Abstract

<span>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 K_m-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.</span><span></span

Inhibition of PDE5A1 guanosine cyclic monophosphate (cGMP) hydrolysing activity by sildenafil analogues that inhibit cellular cGMP efflux

“…The aim of our work was to identify and characterise sildenafil‐like inhibitors with a balanced effect on cGMP hydrolysis and cGMP efflux. In our experimental set‐ups, we found virtually identical values K m values for PDE5 cGMP hydrolysis (1.7 μ m ) in the present work and K m values for high‐affinity cGMP transport as reported in previous studies: 2.4, 2.2 and 2.6 μ m . On the other hand, the K i values of sildenafil inhibition of PDE5A1 cGMP hydrolysis and high‐affinity cGMP efflux are extremely different.…”

“…Sildenafil interacts with ABCB1 (P‐glycoprotein) and ABCG2 (breast cancer resistance protein), ABCC4 (MRP4), ABCC5 (MRP5) and ABCC10 (MRP7) . It was not surprising that the sildenafil analogues identified with VLS inhibited activity of ABCC5 and ABCC4 . These analogues were characterised in this study to decide whether they were able to distinguish between the various cnPDEs and to determine their affinities for PDE5.…”

The role of OAT2 (SLC22A7) in the cyclic nucleotide biokinetics of human erythrocytes

“…The low affinity component of both pumps was nonselective with similar affinities (Jedlitschky et al, ; Orvoll et al, ; Schultz, Vaskinn, Kildalsen, & Sager, ). The results obtained in the present study of intact hRBC cyclic nucleotide efflux were in were in close agreement to the data from IOVs reported earlier uptake (Orvoll et al, ; Sager et al, ).…”

“…In IOVs from hRBC, the K m ‐values of selective high affinity cGMP efflux (2–4 μM) is 10–20 times lower than that of selective high affinity cAMP efflux (35 μM) (Orvoll et al, ; Sager et al, ). The low affinity component of both pumps was nonselective with similar affinities (Jedlitschky et al, ; Orvoll et al, ; Schultz, Vaskinn, Kildalsen, & Sager, ). The results obtained in the present study of intact hRBC cyclic nucleotide efflux were in were in close agreement to the data from IOVs reported earlier uptake (Orvoll et al, ; Sager et al, ).…”

The Effect of MRP5-Expression on Human Erythroleukemia (HEL) Cell Growth and cGMP Levels

“…Efflux of cGMP from hRBC (human erythrocytes) was composed of high and low affinity transport [7] and the cGMP transporter was identified as MRP5 (the ATP-Binding-Cassette transporter ABCC5) [8]. Cyclic AMP was not able to compete with the high affinity ATP-dependent cGMP transport [9] [10] but inhibited the low affinity transport [10]. Based on the observations in hRBC and the effects of elevating cGMP levels in HEK293 cells [11] we employed a human hemopoietic cell line; human erythroleukemia cells (HEL) [12] for the present study.…”