Leukotriene C(4) (LTC(4)) does not share a cellular efflux mechanism with cGMP: characterisation of cGMP transport by uptake to inside-out vesicles from human erythrocytes.
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.
Sundkvist
E
E
Department of Pharmacology, Institute of Medical Biology, Faculty of Medicine, University of Tromso, N-9037, Tromso, Norway.
Jaeger
R
R
Sager
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D000975:Antioxidants / Q000494:pharmacology; D001693:Biological Transport, Active / Q000187:drug effects; D002413:Cations, Divalent / Q000494:pharmacology; D006152:Cyclic GMP / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D019803:Glutathione Disulfide / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D020024:Leukotriene Antagonists / Q000494:pharmacology; D017997:Leukotriene C4 / Q000378:metabolism*; D011422:Propionates / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D013696:Temperature
D000975:Antioxidants; D002413:Cations, Divalent; D020024:Leukotriene Antagonists; D011422:Propionates; D011804:Quinolines; D017997:Leukotriene C4; D004533:Egtazic Acid; C059141:verlukast; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D005978:Glutathione; D006152:Cyclic GMP; D004229:Dithiothreitol; D019803:Glutathione Disulfide