Kidney International, Vol. 32 (1987), pp. 785—793 EDITORIAL REVIEW Molecular properties of amiloride action and of its Na transporting targets Amiloride (Fig. 1) was originally synthesized at Merck, Sharp and Dohme during the search for a nonsteroidal saliuretic agent with antikaliuretic properties. Amiloride administered to dog, rat and human results in modest increases in the urinary excretion of sodium and bicarbonate, while causing a striking reduction in the excretion of potassium. It is used as a diuretic mostly in combination with hydrochiorothiazide in the treat- ment of hypertension and congestive heart failure. Its clinical advantage over more potent diuretics, such as furosemide and ethacrynic acid, is that it prevents hypokaliemia to occur. Amiloride acts at two distinct segments of the nephron. It inhibits the Na4/H4 exchange system of the proximal tubule and the epithelial Na4 channel of the distal tubule. Early studies on the action of amiloride have been reviewed by Benos [1]. This review summarizes recent work aimed at defining in molecular terms the action of amiloride on its two targets. Extrarenal effects of amiloride are also considered in relation to the cardiovascular system. Amioride and the Na/H4 exchange system The first description of the presence in rat renal, brush border membrane vesicles of a coupled exchange of Na4 for H4 is due to Murer, Hopfer and Kinne [2]. It was subsequently shown that this exchange is inhibited by amiloride [3]. The main function of the N&7H4 exchanger of the renal proximal tubule is to acidify the glomerular filtrate and hence to promote the reabsorption of most of the filtered bicarbonate [4]. Protons released into the lumen titrate HC03 to carbonic acid and CO2 which then diffuses across the apical membrane. HC03 is regenerated inside the cells by carbonic anhydrase, thus leading to the accumulation of HC03 against its electro- chemical gradient. HC03 is then secreted at the basolateral membrane by passive diffusion or using coupled transport mechanisms involving Na4 and Cl- [4]. On the other hand Na4 that has entered the cells is transported to the blood by the (Na,K4)ATPase. The asymmetric distribution of the different ion transport pathways in the proximal tubule epithelium is essential for vectorial transport of Na4 and bicarbonate (Fig. 2). Both physiological and biochemical experiments showed that the Na4/H4 exchange system is preferentially localized in the apical membrane of the proximal tubule epithelium, whereas the (Na4 ,K)ATPase is localized in the basolateral membrane [6—9]. Physiological experiments [5] do, however, indicate that the Na/H exchange system is also present in the basolateral membrane of salamander proximal tubular cells. The Na4/H exchange system is not only present in kidney proximal tubular cells. It has now been identified in most eukaryotic cells. It is involved in the regulation of the intracel- lular pH, of the intracellular concentration of Na and of the cell volume. It plays an important role during the progression of the cells from the GJG1 phase to the S phase of the cell cycle. These topics have been recently reviewed [4, 10—18]. The mechanistic properties of the renal Na/H4 exchange system have been reviewed recently [10]. They can be summa- rized as follows: 1. The system is electroneutral with a 1: 1 stoichiometry. 2. Only Li and Na4 and possibly NH44 can be exchanged for H4. Guanidinium ions are antagonists of the system [19]. 3. The antiport is reversible. However under physiological conditions, the inward Na4 gradient across the apical mem- brane imposes the system to function as an uptake pathway for Na4 and as an effiux pathway for H4. 4. The interaction of the Na4/H4 exchanger with external Na4 and Li follows simple Michaelis—Menten kinetics [20]. Li4 presents a better affinity (KLI4 = 1 to 2 mM) for the antiport than Na (KNa4 = 5 to 10 mM). However, the maximal rate of Li/H4 exchange is lower than the maximum rate of Na4/H exchange. 5. An increase of the external pH increases the activity of the Na4/H4 exchanger. The upper panel of Figure 3 shows that the external pH dependence of the Na4/H4 exchanger is identical in rabbit kidney, brush border membranes and in chick cardiac cells. In the kidney external H4 behaves as a competitive inhibitor of Na4 uptake. This property allows the kidney to adjust tubular HC03 reabsorption to the luminal HC03 concentration [4]. In non-renal cells external W behaves either as a non-competitive inhibitor of Na4 uptake (such as, chick skeletal muscle-cells [21] and rat brain synaptosomes [22]) or as a mixed type inhibitor (such as Chinese Hamster Fibroblasts [231 and peripheral blood lym- phocytes [24]). 6. An intracellular acidification activates the Na4/H4 exchange system. Internal H4 ions interact cooperatively with the Na4/H4 exchanger [25], suggesting the presence of more than one internal binding site for H4. This property has important physiological consequences. A slight change in the value of the intracellular pH produces a much larger change in activity of the transporter for a cooperative system than for a system that follows simple Michaelis-Menten kinetics. Two extreme cases have been encountered. In kidney brush border membranes the cooperativity in the Na4/H4 exchange system is low. Hill coefficients describing the interaction of internal H4 with the exchanger are be- tween 1.2 and 1.3 [8, 26—28]. A much higher degree of cooperativity is observed in cardiac cells [31], skeletal muscle cells [29, 30], and brain synaptosomes [22], with values of the Hill coefficient comprized between 2.5 and 3.0. The lower panel of Figure 3 compares the internal pH dependence of the Na4fH exchanger in rabbit renal, brush 785