Leukotriene C-4 (LTC4) does not share a cellular efflux mechanism with cGMP: characterisation of cGMP transport by uptake to inside-out vesicles fromhuman erythrocytes

The transport of cGMP out of cells is energy requiring and has characterist ics compatible with an ATP-energised anion pump. In the present study a mod el with inside-out vesicles from human erythrocytes was employed for furthe r characterisation of the cGMP transporter. The uptake of leukotriene C-4 ( LTC4), a substrate for multidrug resistance protein (MRP), was concentratio n-dependently inhibited by the leukotriene antagonist MK571 (IC50 = 110 +/- 20 nM), but cGMP was unable to inhibit LTC4 uptake. Oxidised glutathione ( GSSG) and glutathione S-conjugates caused a concentration-dependent inhibit ion of [H-3]cGMP uptake with IC50 of 2200 +/- 700 mu M for GSSG, 410+/-210 mu M for S-(p-nitrobenzyl)glutathione and 37 +/- 16 mu M for S-decylglutath ione, respectively. Antioxidants such as reduced glutathione and dithiothre itol did not influence transport for concentrations up to 100 mu M, but bot h inhibited cGMP uptake with approx, 25% at 1 mM. The cCMP pump was sensiti ve to temperature without activity below 20 degrees C. The transport of cGM P was dependent on pH with maximal activity between pH 8.0 and 8.5. Calcium caused a concentration-dependent inhibition with IC50 of 43 +/- 12 mu M. M agnesium gave a marked activation in the range between 1 and 20 mM with max imum effect at 10 mM. The other divalent cations, Mn2+ and Co2+, were unabl e to substitute Mg2+, but caused some activation at 1 mM. EDTA and EGTA sti mulated cGMP transport concentration-dependently with 50% and 100% above co ntrol at 100 mu M, respectively. The present study shows that the cGMP pump has properties compatible with an organic anion transport ATPase, without affinity for the MRP substrate LTC4. However, the blockade of the cGMP tran sporter by glutathione S-conjugates suggests it is one of several GS-X pump s. (C) 2000 Elsevier Science B.V. All rights reserved.