Specific interaction of 5-(N-methyl-N-isobutyl)amiloride with the organic cation-proton antiporter in human placental brush-border membrane vesicles. Transport and binding.
The interaction of 5-(N-methyl-N-isobutyl)amiloride (MIBA) with brush-border membrane vesicles isolated from normal human term placentas was investigated using two parameters: binding and transport. The binding of MIBA to placental membranes was specific and temperature- and pH-dependent, and the apparent dissociation constant (Kd) for the process was 58 +/- 2 microM. The binding was inhibited by other amiloride analogs and also by clonidine and cimetidine with a rank order potency: MIBA > benzamil > dimethylamiloride > amiloride > clonidine > cimetidine. These compounds also inhibited Na(+)-H+ exchanger activity in these membrane vesicles, but with a different order of potency: dimethylamiloride > MIBA > amiloride > benzamil > cimetidine > clonidine. The membrane vesicles were also able to transport MIBA into the intravesicular space, and the transport was stimulated many-fold by the presence of an outwardly directed H+ gradient across the membrane. The H+ gradient was the driving force for uphill accumulation of MIBA inside the vesicles. The transport process was electrically silent. The transport of MIBA was inhibited by other amiloride analogs and by clonidine and cimetidine, and the order of potency was the same as the order with which these compounds inhibited the binding of MIBA. The Michaelis-Menten constant (Kt) for the transport process was 46 +/- 2 microM. The binding as well as the transport were also inhibited by Na+ and Li+. Interestingly, tetraethylammonium and N1-methylnicotinamide, two of the commonly used substrates in organic cation transport studies, failed to inhibit the binding and transport of MIBA. Furthermore, although the outwardly directed H+ gradient-dependent uphill transport of tetraethylammonium could be demonstrated in renal brush-border membrane vesicles, there was no evidence for the presence of a transport system for this prototypical organic cation in placental brush-border membrane vesicles. It is concluded that the human placental brush-border membranes possess an organic cation-proton antiporter which accepts MIBA as a substrate, the low affinity binding site for MIBA observed in these membranes represents this antiporter, and that the placental organic cation-proton antiporter is distinct from the widely studied renal organic cation-proton antiporter.
Prasad
P D
PD
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100.
Leibach
F H
FH
Mahesh
V B
VB
Ganapathy
V
V
D000584:Amiloride / Q000031:analogs & derivatives* / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008871:Microvilli / Q000378:metabolism*; D009536:Niacinamide / Q000031:analogs & derivatives / Q000494:pharmacology; D010920:Placenta / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers; D013654:Taurine / Q000378:metabolism; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000378:metabolism / Q000494:pharmacology
D002352:Carrier Proteins; D006146:Guanidines; D017923:Sodium-Hydrogen Exchangers; D013757:Tetraethylammonium Compounds; D013654:Taurine; D009536:Niacinamide; D019789:Tetraethylammonium; C024785:5-methylnicotinamide; D000584:Amiloride; C056369:5-(N-methyl-N-isobutyl)amiloride; D012964:Sodium; D019791:Guanidine