Amiloride and its analogs as tools to inhibit Na+ transport via the Na+ channel, the Na+/H+ antiport and the Na+/Ca2+exchanger
Christian FRELIN1,Pascal BARBRYI,Paul VIGNE1,
Olivier CHASSANDEI,Edward J. CRAGOE Jr.2 and Michel LAZDUNSKI’
‘Centre de Biochimie, Centre National de la Recherche Scientifique, Parc Valrose,06034 Nice Cedex, France,and 2Merck,Sharp & Dohme Research Laboratories,West Point,PA 19486,U.S.A.
(Received 6-11-1987,accepted 5-1-1988)
Summary- Amiloride analogs inhibit a number of transmembrane Na+ transport systems: 1) the epithelium Na+ channel, 2) the Na+/H+ exchange system and 3) the Na+/Ca2+ exchange system. Structure-activity relationships using amiloride derivatives with selected modification of each of the functional groups of the molecule indicate that the 3 Na+ transporting systems have distinct pharmaco-logical profiles.5-N Disubstituted derivatives of amiloride, such as ethylisopropylamiloride are the most potent inhibitors of the Na+ /H+ exchange system. Conversely, amiloride derivatives that are substitut-ed on the guanidino moiety, such as phenamil, are potent inhibitors of the epithelium Na+ channel. It is thus possible, by using selected amiloride derivatives to inhibit selectively one or another of the Na+ transport systems.
amiloride/Na+ channel/Na+/H+ exchange/Na+/Ca2+exchange
High affinity inhibitors are essential tools for the identification of membrane structures involved in cation transport and for the analyses of their mechanism and their physiological function. Well-known examples are ouabain and other digitalis compounds for the (Na+, K+)-ATPase; tetrodotoxin, veratridine,batrachotoxin,scor-pion and sea anemone toxins for the voltage-dependent Na+ channel; dihydropyridines and phenylalkylamines for Ca2+ channels; apamin for the Ca2+-dependent K+ channel; furosemide and bumetanide for the Na+/K+/Cl-co-trans-port system,etc.
Amiloride (Fig. 1), a potent diuretic mole-cule,has recently been shown to inhibit a num-ber of important membrane structures involved in Na+ transport. These are the epithelial Na+ channel, the Na+/H+ antiport and the Na+/Ca2+ exchange system. Detailed reviews

on the action mechanism of amiloride and amilo-ride derivatives on Na+ transport in epithelial tissues have already appeared in which more detailed information than that presented here will be found [1,2]. The main purpose of the present article is to compare the respective sensi-tivities of Na+ channels,Na+/H+exchangers and Na+/Ca2+ exchangers to amiloride and derivatives in view of choosing the adequatc molecule to block one or another of these trans-port systems.
The Na+ channel of epithelia
Amiloride blocks Na+ transport by isolated amphibian epithelia. The site of action of amilo-ride is the apical Na+ channel that has been extensively studied by electrophysiological tech-niques [2]. The physiological function of this type of Na+ channel is to allow Na+ and osmoti-cally obliged water reabsorption.

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Fig.1.Structural formulae of amiloride and amiloride derivatives.
The analysis of structure-activity relation-ships for the inhibition of the epithelial Na+ channel of amphibia by various amiloride deri-vatives [3-5] has led to the discovery of high affinity inhibitors of this Na* transport system. Efficient blocking of the apical Na+ channel requires: 1) a positively charged carbony!guani-dinium side chain with a substitution of one of the hydrogens of the guanidino group by a phe-nyl or a benzyl substituent to increase the blocking potency, 2) an amino group in position 5 and 3) a medium-sized halogen atom (Ċl orBr) at position 6 of the pyrazine ring.
Less is known about the pharmacological pro-perties of the epithelial Na+channel of the mam-malian kidney. O’Neil and Boulpaep [6] have shown that amiloride inhibits Na+ transport with an ICso (50% inhibitory concentration) of 0.1 μM in isolated perfused rabbit cortical col-lecting tubules.
Fig.2B presents typical dose-response curves for inhibition of Na+ channels in pig kidney microsomes by phenamil, benzamil, amiloride and 5-N ethylisopropylamiloride. The best com-pound for the inhibition of the Na+ channel in this preparation is phenamil which has an affinity (Kos) for the apical Na+ channel of about 40 nM [7].The pharmacological efficacy of the different

molecules for the inhibition of the Na+ channel in pig kidney membranes is: phenamil(Ko.s= 40 nM) > benzamil (Kos =300 nM) > amilo-ride, 5-N ethylisopropylamiloride(Ko.s=3-10 μM). Exact affinities of the different amilo-ride derivatives may vary with animal species and tissue origin [1]. Phenamil also appears to be the best compound for inhibiting Na+ channels in frog epithelia [8]. In using amiloride and ami-loride derivatives, it should be kept in mind that the action of these molecules on the apical Na+ channel can be antagonized by external Na+[1, 2,9].
The Na+/H+antiport
The Na+/H+ antiport is a ubiquitous membrane structure that is also inhibited by amiloride. Its main properties have been reviewed recently [10-12].The Na+/H+ antiport is an important pathway for Na+ uptake which is used for the regulation of the intracellular pH in various cell types [13].Modulation of the activity of the Na+ / H+ antiport is involved in fertilization,cell growth and differentiation and in cell volume regulation[10-14].
Inhibition of Na+ transport



22Na* uptake (% of maximum)

Fig.2.The epithelial Na+ channel.A.Diagram showing the route of Na+ reabsorption by kidney distal tubule cells.Na+from the lumen enters cells via the amiloride-sensitive Na+ channel.Na+is then transported across the basolateral membrane via the (Na+,K+)-ATPase.B.Dose-response curves for the inhibition of the Na* channel of pig kidney microsomes by phenamil (Phena.,*), benzamil (Benz.,),amiloride (Ami.,V) and ethylisopropylamiloride (EIPA, A) from 22Na+ flux measurements [7].
The first analysis of structure-activity rela-tionships for several amiloride derivatives in relation with the blockade of the Na+ /H+ anti-port was performed with skeletal muscle cells in culture [15, 16]. It was followed by studies on fibroblasts [16, 17], cardiac cells [18], A431 car-cinoma celIs [19], kidney cells and brush-border membranes [20], etc. These studies have shown that the Na+/H+ antiport in the different cell types has the same pharmacological properties. Modification of the amiloride structure has the following effects on the inhibition of the Na+/H+ antiport: 1) substitution of the carbo-nylguanidinium moiety decreases the potency of the molecule, in contrast to what was observed for the epithelial Na+ channel; 2) substitution of H atoms on the amino group at position 5 of the pyrazine ring dramatically increases the potency of the inhibitor. 5-N Disubstituted derivatives of amiloride are more potent inhibitors than 5-N monosubstituted derivatives.
The best compound found so far to inhibit the Na+/H+ antiport is 5-N ethylisopropylamilo-ride[15](Fig. 1) which was shown to be very efficient in preventing the Na+-dependent recov-ery of the internal pH after cytoplasmic acidifica-

tion. Fig. 3C shows representative dose-re-sponse curves for the inhibition of the Na*/H antiport in chick cardiac cells by amiloride derivatives. The respective efficacies of the dif-ferent molecules are:5-N ethylisopropylamil-oride (Ko.s = 50 nM) >5-N dimethylamiloride (Ko.s = 500 ‘nM) > amiloride(Ko.s=8μM)> benzamii(Ko.s=100 μM).
The action of amiloride, and also of its more potent derivatives, on the Na+/H*antiport is antagonized by external Na+.As a consequence, under physiological conditions of external Na-(140 mM), apparent affinities for amiloride de-rivatives are shifted by a factor of 7-10 towards higher concentrations.
The Na+/Ca2+antiport
A membrane transport system catalyzing the electrogenic exchange of Na* for Ca2+ has been described in cardiac and neuronal cells. This sys-tem is believed to play an important role in exci-tation to contraction and in excitation to secre-tion coupling. It is also inhibited by amiloride derivatives(Fig.4).Structure-activity relation-

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ships in the amiloride series show that the phar-macological profile of the Na+/Ca2+ antiport is clearly distinct from that of the Na+/H+ anti-port[12,21-23].Amiloride derivatives that are substituted on the guanidino moiety and that have a low inhibitory activity on the Na+/H+ antiport are the most potent inhibitors of the Na+/Ca2- antiport. Fig. 4B shows typical

dose-response curves for the inhibition by amil-oride derivatives on the Na+/Ca2+ antiport of chick cardiac cells. The pharmacological profile of the Na+/Ca2+exchanger follows the sequence: dichlorobenzamil (Ko.s = 30 μM)> benzamil(Ko.s=200 μM)>> amiloride,5-N ethylisopropylamiloride. In this cardiac prepara-tion, 5-N ethylisopropylamiloride was found to

(日n日日22Na* uptake (%

Fig.3.The Na+/H’ antiport.The Na+/H* antiport is the major mechanism enabling cardiac cells to recover from intracellular acidosis.Chick cardiac cells, loaded with the fluorescent trapped pH, indicator biscarboxylethylcarboxyfluorescein were subject-ed to intracellular acidosis by the NHį, prepulse technique (11]’ A. Imposing an inward Na+ gradient promotes pH, recovery, which is due to the activity of the Na+ /H+ antiport. B. Ethylisopropylamiloride (EIPA), the most potent derivative of amiloride for inhibiting the Na+/H* antiport prevents Na+induced pH,recovery.C.Dose-response curves for the inhibition of cardiac Na+/H+ antiport by benzamil (Benz., =), amiloride (Ami., V), dimethylamiloride (DMA,·)and ethylisopropylamiloride (EIPA,A) from 22Na+ flux measurements [16].


Fig.4.The Na+/Ca2+ exchanger.A.Diagram showing how (Na+. K+)-ATPase and the Na+/Ca2+ exchanger work together in the plasma membrane of chick cardiac cells. After inhibition of the (Na+, K+)-ATPase with ouabain, the internal Na+ concen-tration increases and Na+ is released from the cell through the Na+/Ca2+ exchanger in exchange for Ca2+.B.Dose-response curves for the inhibition of the Na+/Ca2+ exchanger of chick cardiac cells by ethylisopropylamilbride (EIPA, A), amiloride (Ami.,V),benzamil (Benz.,)and dichlorobenzamil (D.C.Benz.,)fromCa2flux measurements [i1].
Inhibition of Na+ transport

be inactive in inhibiting the Na+/Ca2+ ex-changer.Other authors using other preparations have reported that 5-N disubstituted derivatives of amiloride are as potent as benzamil deriva-tives for inhibiting Ca2+ uptake mediated by the Na+ /Ca2+ exchanger [23]. However, the possi-bility of an indirect effect which would be pri-marily due to the blockade of the Na+/H+ exchanger as described for chick cardiac cells [12, 18]has not been completely eliminated.
A comparison of the Fig. 2B and 4B shows that the order of potency of the different amil-oride derivatives for inhibiting the Na+/Ca2+ exchanger is close to that found for the epithelial Na+ channel.However,inhibitor concentrations required to block the Na+/Ca2+ antiport are much higher than those required to inhibit the epithelial Na+ channel. For instance,the ICso for phenamil inhibition of the epithelial Na+chan-nel is 40 nM, whereas it is 0.4 mM for the Na+/Ca2+ exchanger.The derivatives of benza-mil that have been found to be active on the Na+/Ca2+exchange system also inhibit voltage-dependent Ca2+ channels in the heart [24,25]. For that reason, they cannot be used to deter-mine the physiological role of the Na+/Ca2+ exchange system in excitation-contraction coupling.
Other effects of amiloride
In addition to their now well-characterized actions on the epithelial Na+ channel,the Na+/H+antiport and the Na+/Ca2+ antiport, amiloride and its derivatives have been shown to inhibit a number of other cellular activities. Amiloride blocks protein synthesis in whole cells and in cell-free systems [7, 17]; it inhibits the (Na+, K+)-ATPase [19, 26] and several protein kinases [24, 27]. However, all these effects have been observed with very high concentrations of amiloride or of amiloride derivatives (0.1-1 mM) and are less sensitive to modifications of the amiloride structure. Conversely,both the epithelial Na+ channel and the Na+/H+ anti-port are inhibited by low concentrations of se lected amiloride derivatives and the inhibitory effect is highly sensitive to relatively small modifications of the chemical structure of the inhibitor. Detailed structure-activity relation-ships for the inhibition by amiloride derivatives of selected protein kinases have not yet been established.

Amiloride is a molecule with multiple and diverse pharmacological properties.However, chemical manipulation of the amiloride struc-ture led to selected amiloride derivatives which are able to block specifically and with a high affinity one or another of the amiloride-sensitive Na+ transport systems. Two groups of com-pounds are of particular interest: first, the 5-N disubstituted derivatives of amiloride which are highly potent and specific inhibitors of the Na+ /H+ antiport; secondly,the amiloride deri-vatives that are substituted on the guanidino moiety,such as phenamil and benzamil,which constitute highly specific inhibitors of the epithe-lial Na+ channel. It is now easily feasible to in-hibit specifically and independently the Na+/H+ antiport and/or the Na+ channel. A recent illustration of the use of phenamil and EIPA to determine the respective roles of the Na+/H+ exchange system and of the epithelium Na+ channel in transepithelium ion transport can be found in [28]. Inhibitors of the Na+/Ċa2* exchange system in the amiloride series are not yet potent enough to be used routinely and it will be necessary to devise new derivatives with an affinity increased by at least 2 orders of magni-tude. Also, it would be useful to have amiloride derivatives which inhibit the Na+/Ca2+ antiport without affecting the Na+channel.
One of the interests of specific and highly potent inhibitors is the possibility to prepare them in a radiolabeled form in order to biochem-ically characterize their receptor site. This is now possible using [3H]ethylpropylamiloride for the Na+ /H+ exchanger [20, 29]. For the epithelium Na+ channel [3H]bromobenzamil has been used as a photoaffinity labeling agent in bovine kid-ney microsomes [30].More recently [H]phen-amil has been used to monitor the purification of the epithelium Na+ channel of pig kidney micro-somes. The purified [3H]phenamil receptor has been identified as being composed of 2 90-100 kDa polypeptide chains cross-linked by disulfide bridges[31].
This work was supported by grants from the Centre National de la Recherche Scientifique,the Institut National de la Santé et de ia Recherche Médicale, the Fondation sur les Maladies Vasculaires and the

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Fondation pour la Recherche Médicale.We are grateful to Dr. T. Jean for assistance and Martine Valetti for secretarial help.
1 Benos D.J.(1982) Am.J. Physiol. 242, C131-C145
2 Lindemann B.(1984) Annu. Rev.Physiol.46, 497-515
3 Cuthbert A.W.&Fanelli G.M.(1978) Br.J. Pharmacol.63,139-149
4 Li J.H.Y.,Cragoe E.J. Jr.& Lindemann B. (1985)J.Membr.Biol.83,45-56
5 Benos D.J.. Simon S.A.,Mandel L.J.&Cala P.M.(1976)J. Gen.Physiol.68,43-63
6 O’Neil R.G.& Boulpaep E.L.(1979)J.Membr. Biol.50,365-387
7 Barbry P.,Frelin C.,Vigne P.,Cragoe E.J.Jr.& Lazdunski M.(1986) Biochem. Biophys. Res. Commun.135,25-32
8 Garvin J.L.,Simon S.A.,Cragoe E.J.Jr.&Man-del L.J. (1985) J. Membr.Biol.87,45-54
9 Aceves J. & Cuthbert A.W.(1979)J.Physiol. (London)295,491-504
10 Aronson P.S. (1985) Annu. Rev. Physiol.47, 545-560
11 Frelin C.,Vigne P. & Lazdunski M.(1985) in: Hormones and Cell Regulation, vol. 9 (Dumont J.E.,Hamprecht B.&Nunez J.,eds.),Elsevier, Amsterdam pp.259-268
12 Lazdunski M.,Frelin C.& Vigne P.(1985)J. Mol.Cell.Cardiol.17,1029-1042
13 Roos A.& Boron W.F.(1981) Physiol.Rev.61, 296-434
14 Jean T.,Frelin C.,Vigne P.& Lazdunski M. (1986) Eur.J.Biochem.160,211-219
15 Vigne P.,Frelin C.,Cragoe E.J. Jr.& Lazdunski M.(1983) Biochem. Biophys. Res. Commun. 116,86-90

16 Vigne P.,Frelin C.,Cragoe E.J.Jr.& Lazdunski M.(1984) Mol.Pharmacol.25,131-136
17 L’Allemain G.,Franchi A.,Cragoe E.J.Jr.& Pouysségur J. (1984) J. Biol. Chem. 259, 4313-4319
18 Frelin C., Vigne P. & Lazdunski M.(1984)J. Biol.Chem.259,8880-8885
19 Zhuang Y.X.,Cragoe E.J.,Shaikewitz T.,Gla-ser L. & Cassel D.(1984)Biochemistry 23, 4481-4488
20 Vigne P.,Frelin C., Audinot M.,Borsotto M., Cragoe E.J. Jr. & Lazdunski M.(1984) EMBO J.3,2647-2651
21 Siegl P.K.S.,Cragoe E.J. Jr.,Trumble M.J.& Kaczorowski G.J. (1984) Proc. Natl. Acad.Sci. USA 81,3238-3242
22 Schellenberg G.D.,Anderson L.,Cragoe E.J.& Swanson P.D.(1985) Mol. Pharmacol. 27, 537-543
23 Kaczorowski G.J.,Barros F.,Dethmers J.K., Trumble M.J.&Cragoe E.J.(1985)Biochemistry 24,1394-1403
24Kim D.&Smith T.W.(1986) Mol.Pharmacol. 30,164-170
25 Bielefeld D.R.,Hadley R.W.,Vassilev P.M.& Hume J.R.(1986) Circ.Res.59,381-389
26 Soltoff S.P.& Mandel L.J.(1983) Science 220, 957-959
27 Besterman J.F.,May W.S.,Levine H.,Cragoe E.J.& Cuatrecasas D. (1985)J.Biol.Chem.260, 1155-1159
28 Ehrenfeld J.,Cragoe E.J. Jr.&Harvey B.J. (1987) Pflügers Arch. Eur. J. Physiol.409, 200-207
29 Vigne P.,Jean T.,Barbry P.,Frelin C.,Fine L.G. & Lazdunski M. (1985) J.Biol.Chem.260, 14120-14125
30 Kleyman T.R.,Yulo T.,Ashbaugh C.,Landry D.,Cragoe E.J. Jr.,Karlin A. &Al-Awqati Q. (1986)J. Biol.Chem.261,2839-2843
31 Barbry P.,Chassande O.,Vigne P.,Frelin C., Ellory J.C.,Cragoe E.J. Jr.&Lazdunski M. (1987) Proc. Natl. Acad. Sci. USA 84,4836-4840