N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, amiloride analogues, and renal Na+/H+ antiporter

Abstract

N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) is a carboxyl-activating agent and has been shown to inhibit the renal Na+/H+ antiporter. The purposes of the present studies were to characterize the kinetics of inhibition of the Na+/H+ antiporter by EEDQ and to determine whether amiloride analogues affect the ability of EEDQ to inhibit the rate of Na+/H+ exchange. Brush-border membrane vesicles (BBMV) were prepared from rabbit kidneys; Na+/H+ exchange rate was assessed by the fluorescence quenching of acridine orange. EEDQ produced a concentration-dependent inhibition of Na+/H+ exchange; the effect was to decrease the maximum activity (Vmax) from 5.51 to 2.03 fluorescence units X mg protein-1 X S-1) and Km (from 14.1 to 8.7 mM) compared with control BBMV. Pretreatment of BBMV with amiloride before the addition of EEDQ maintained both Vmax and Km at values that were not significantly different from those for control BBMV. Compared with a series of analogues, amiloride was only the third most potent inhibitor of the rabbit renal Na+/H+ antiporter; amiloride, however, provided the greatest protection against inhibition of the antiporter by the subsequent addition of EEDQ. These findings suggest that the 2-carbonylguanidininum moiety and 6-chloro atom are important for binding of amiloride to sites at or near the antiporter; the group at position 5 is important in determining the ability of amiloride to protect against inhibition of the Na+/H+ antiporter by EEDQ. Finally, the ability of amiloride to protect against inactivation of the renal Na+/H+ antiporter by EEDQ is reversible.

Tonoplast Na+/H+ Antiport Activity and Its Energization by the Vacuolar H+-ATPase in the Halophytic Plant Mesembryanthemum crystallinum L

“…This result indicates that Na+/H+ exchange is an electroneutral process with a 1:l stoichiometry, and it also precludes the possibility that the H + efflux from the vesicles occurred simply as a response to an inside-positive Nat diffusion potential generated on addition of Na+ to the externa1 medium. Finally, Na+/H+ exchange in M. crystallinum was inhibited by the diuretic drug amiloride, a known inhibitor of the Na+/H+ antiporter in animals and plants (Blumwald and Poole, 1985;Rocco et al, 1987), with a sensitivity similar to that observed for the Na+/H+ antiporter in B. vulgaris (Blumwald and Poole, 1985).…”

“…These results suggest that the dissipation of the preset pH gradient by Na+ is not due to electrically driven Ht movement through conductive pathways but is consistent with an electroneutral Na+/H+ antiport. The diuretic drug amiloride has been shown to inhibit Na+ uptake via the N a f / H + exchange system in animals rapidly, reversibly, and competitively by binding at or near the Na+-binding site(s) (Rocco et al, 1987). In plants, amiloride has been shown to inhibit competitively the Na+/H+ antiport in Beta vulgaris (Blumwald and Poole, 1985), with a Ki for Na'-dependent H+ fluxes in tonoplast vesicles of 140 WM.…”

Is there a Cl−−OH− exchange (Cl−−H+ cotransport) mechanism in the brush-border membrane of the intestine of the fresh water trout (Salmo gairdneri, R.)?

“…This was obtained when CCCP was added to the incubation medium at two different concentrations. Furthermore, in both presence and absence of CCCP, C1-uptake shows nonsaturable properties, whereas a C1--OH-exchange (CI--H + cotransport) is a saturable mechanism as a Na+-H + exchange (Aronson, Nee & Suhm, 1982;Rocco, Cragoe & Warnock, 1987). The regression lines obtained in Fig.…”