Leukotriene C4 (LTC4) does not share a cellular efflux mechanism with cGMP: characterisation of cGMP transport by uptake to inside-out vesicles from human erythrocytes

Abstract

The transport of cGMP out of cells is energy requiring and has characteristics compatible with an ATP-energised anion pump. In the present study a model with inside-out vesicles from human erythrocytes was employed for further characterisation of the cGMP transporter. The uptake of leukotriene C(4) (LTC(4)), a substrate for multidrug resistance protein (MRP), was concentration-dependently inhibited by the leukotriene antagonist MK571 (IC(50)=110+/-20 nM), but cGMP was unable to inhibit LTC(4) uptake. Oxidised glutathione (GSSG) and glutathione S-conjugates caused a concentration-dependent inhibition of [(3)H]cGMP uptake with IC(50) of 2200+/-700 microM for GSSG, 410+/-210 microM for S-(p-nitrobenzyl)glutathione and 37+/-16 microM for S-decylglutathione, respectively. Antioxidants such as reduced glutathione and dithiothreitol did not influence transport for concentrations up to 100 microM, but both inhibited cGMP uptake with approx. 25% at 1 mM. The cGMP pump was sensitive to temperature without activity below 20 degrees C. The transport of cGMP was dependent on pH with maximal activity between pH 8.0 and 8.5. Calcium caused a concentration-dependent inhibition with IC(50) of 43+/-12 microM. Magnesium gave a marked activation in the range between 1 and 20 mM with maximum effect at 10 mM. The other divalent cations, Mn(2+) and Co(2+), were unable to substitute Mg(2+), but caused some activation at 1 mM. EDTA and EGTA stimulated cGMP transport concentration-dependently with 50% and 100% above control at 100 microM, respectively. The present study shows that the cGMP pump has properties compatible with an organic anion transport ATPase, without affinity for the MRP substrate LTC(4). However, the blockade of the cGMP transporter by glutathione S-conjugates suggests it is one of several GS-X pumps.

The Multidrug Resistance Protein 5 Functions as an ATP-dependent Export Pump for Cyclic Nucleotides

“…The knowledge about the structure and function of the protein families involved in cGMP synthesis and degradation has grown vastly the last years, whereas little was known so far about the molecular identity of the proteins that mediate the cellular export. Studies in membrane vesicles from human erythrocytes suggested that cGMP is transported by an organic anion transport ATPase (12)(13)(14). The reported characteristics of this transport system in erythrocytes, including the K m value of 2.4 M for cGMP (13) and the lack of affinity for the MRP1 substrate leukotriene C 4 (14), are similar to our findings on MRP5-mediated cGMP transport (see Fig.…”

“…Studies in membrane vesicles from human erythrocytes suggested that cGMP is transported by an organic anion transport ATPase (12)(13)(14). The reported characteristics of this transport system in erythrocytes, including the K m value of 2.4 M for cGMP (13) and the lack of affinity for the MRP1 substrate leukotriene C 4 (14), are similar to our findings on MRP5-mediated cGMP transport (see Fig. 3, Table I, and "Inhibition of MRP5-mediated cGMP Transport").…”

“…Both cAMP, as well as cGMP, secretion has been shown to be unidirectional and energy-dependent (4 -8, 10). Furthermore, a primary active transport of cGMP has been demonstrated directly in inside-out membrane vesicles from human erythrocytes (12)(13)(14). The transport of both cyclic nucleotides has been shown to be inhibited by probenecid, suggesting that this export is mediated by a transporter for amphiphilic anions (10,13,15).…”

Transepithelial urate transport by avian renal proximal tubule epithelium in primary culture

“…Luminal application of MK-571, a leukotriene C 4 receptor antagonist, dramatically reduced both urate secretory and reabsorptive flux. This compound is a known substrate for MRP2, MRP4 and the organic anion transporter oatp1 (Sundkvist et al, 2000), also known to be present in mammalian BBM; however, urate is not transported by MRP2 (Van Aubel et al, 2005). Interestingly, amino acid alignment of the chicken MRP4-like sequence against human MRP4 indicates 86% sequence similarity.…”

“…; Russel et al, 2002) and MK571 (Sundkvist et al, 2000) should compete for urate transport sites. Addition of MTX (500·mol·l -1 ) to the interstitium alone strongly inhibited net urate secretion by effectively reducing the secretory flux and stimulating the reabsorptive flux (Fig.·4).…”