Journal of Receptor Research
ISSN: 0197-5110 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/irst19
Receptor Binding Profiles of Amiloride Analogues Provide no Evidence for a Link Between Receptors + + and the Na /H Exchange, But Indicate a Common Structure on Receptor Proteins Anja Garritsen, Adriaan P. Ijzerman, Martin M. Th. Tulp, Edward J. Cragoe & Willem Soudijn To cite this article: Anja Garritsen, Adriaan P. Ijzerman, Martin M. Th. Tulp, Edward J. Cragoe & Willem Soudijn (1991) Receptor Binding Profiles of Amiloride Analogues Provide +
+
no Evidence for a Link Between Receptors and the Na /H Exchange, But Indicate a Common Structure on Receptor Proteins, Journal of Receptor Research, 11:6, 891-907, DOI: 10.3109/10799899109064686 To link to this article: http://dx.doi.org/10.3109/10799899109064686
Published online: 26 Sep 2008.
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Date: 11 May 2016, At: 22:59
JOURNAL OF RECEPTOR RESEARCH, 11(6), 891-907 (1991)
RECEPTOR BINDING PROFILES OF AMILORIDE ANALOGUES PROVIDE NO EVIDENCE FOR A LINK BETWEEN RECEPTORS AND THE Na+/H+EXCHANGER, BUT INDICATE A COMMON STRUCTURE ON RECEPTOR PROTEINS
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Anja Ganitsen', Adriaan P. IJzerman", Martin Th.M. Tulp*, Edward J. Cragoe, Jr.3 and Willem Soudijn' Center for Bio-Pharmaceutical Sciences, Div. Med. Chem., P.O. Box 9502, 2300 RA Leiden, the Netherlands, * Duphar BV, C.J. van Houtenlaan 36, 1381 CP Weesp and P.O. Box 631548, Nacogdoches, TX 75963, USA ABSTRACT Amiloride and its analogues affect radioligand binding to the adenosine-A, receptor. In this paper, the specificity of this effect is investigated by generating receptor binding profiles for amiloride and two of its analogues. A limited structure-activity relationships study is performed to probe the relationship between inhibition of receptor binding by amiloride analogues and the effects of these compounds on Na' transport, in particular Na+/H+exchange. The receptor binding profiles of amiloride, benzamil and 5'-(N,Nhexamethy1ene)amilonde (HMA) indicate that the compounds affect a variety of receptors and that none of the compounds is highly selective for any of these. The SAR study indicates that it is very unlikely that a direct coupling between receptors and Na'/H+ exchange or another amiloride-sensitive ion transport system is responsible for the inhibition of receptor binding. A correlation between the signal transduction systems coupled to the receptors involved and the potency of the amiloride analogues is also absent. The varying nature of the receptors, affected by amiloride or its analogues, suggests a wide-spread presence of an amiloride binding site on receptors and other membrane proteins. INTRODUCTION Amiloride, a potassium sparing diuretic, has been used as a pharmacological probe for Na' transport systems (1-3). The drug inhibits various systems with different potencies: in concentrations < 1 pM,epithelial sodium channels are 891 Copyright 0 1991 by Marcel Dekker, Inc.
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TABLE 1. The affinity of amiloride and its analogues for Na+/H+exchange, the epithelial sodium channel and Na'lCa'' exchange. System amiloride
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N a + W exchange (3,4) '%a+ efflux 84 human neutrophils, [NaCl] 140 mM N a + W exchange ( 5 ) =Na+ uptake 7 chick muscle, [NaCl] 3 mM Na'W exchange (6) 2 binding of [3H]EPAd rabbit kidney, [NaCl] 0 mM Na+ channel (3) electrophysiology frog skin, [NaCl] 110 mM Na+ channel (7) binding of [3H]phenamil pig kidney, INaCI] 0 mM
Affinity (JLM) HMA'/EIPAb
benzamil
0.1610.3 8
> lo00
n.d.'IO.050
100
n.d.10.017
> 10
0.35
> lo/> 10
0.038
4.0
n.d.ll.0
0.300
Na+lCa2+exchange (38) "%a2+ uptake 1100 pituitary plasma membrane vesicles [NaCl],, 0 mM
100/130
100
Affinity in pM refers to all four compounds. ' HMA, 5-(N,NN-hexamethylene)amiloride; The closely related analogue E P A (5-(N-ethyl-N-isopropyl)amiloride)is included in the tab., since the affinity of HMA has not alsways been determined. ' n.d., not determined, EPA, 5-(N-ethylN-propy1)amiloride
blocked, between 1 and 100 pM, Na+/H' exchange is inhibited and in the millimolar range Na+/Ca'+ exchange is affected (tab. 1). Besides the parent compound, amiloride analogues are available that affect mainly N a + W exchange (the 5-amino-substituted analogues such as HMA) or the epithelial sodium channel (the analogues bearing a substituent at a terminal guanidino nitrogen atom such as benzamil) (tab. 1) (1-3). As the affinities of HMA and benzamil for both processes differ by several orders of magnitude, these two compounds can be used to define which particular Na+ transporter is involved. Benzamil and HMA are both more potent inhibitors of Na+/Ca*+exchange than amiloride, but have very low affinities for this exchanger of about 100 pM.
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RECEPTOR BINDING PROFILES OF AMILORIDE ANALOGUES
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The mere fact that a physiological or pharmacological process is modulated by amiloride or its analogues is often regarded as evidence for the involvement of one of the above-mentioned transport systems. It has become clear, however, that amiloride is not only an ion transport inhibitor, but has a variety of other effects. Thus, several papers have been published on effects of amiloride on radioligand binding to the adrenergic and muscarinic receptors (9-17). Amiloride-sensitive Na' transport appears to be influenced by agonists for these receptors, an effect possibly directly related to the effects of amiloride on radioligand-receptor binding. The most likely candidate is Na+/H+exchange, as amilorid:: usually inhibits receptor binding with Ki values between 10 and 100 @A. This resulted in the hypothesis of Limbird and coworkers, that G,coupled receptors are somehow directly linked to the plasma membrane N a W exchanger (13,16,18). We showed recently that amiloride displaces radioligand binding to the, Gi coupled, adenosine-A, receptor in a calf brain membrane preparation with a Ki value of 2 pM (19), a concentration well below the Ki value for the adrenergic receptors (10-13,16,17). However, the structure-activity relationships (SAR) of 8 amiloride analogues for inhibition of adenosine-A, receptor binding are not at all consistent with the involvement of Na+/H+exchange or another Na' transporter, and a direct link seems very unlikely. The present study was undertaken to determine the specificity of amiloride and its analogues for the adenosine-A, receptor and, in addition, to establish whether amiloride-sensitivity is a general phenomenon among receptors that are coupled to certain signal transduction systems. A possible relationship between the inhibition of radioligand-receptor binding and the ion transport blocking properties of the amiloride analogues is addressed by means of a limited S A R study.
MATERIALS Amiloride was kindly donated by Merck Sharp and Dohme (Haarlem, the Netherlands, USP grade). 5-(N,N-Hexamethylene)amilorideand benzamil were synthesized as decribed previously (20). All other chemicals were obtained from standard commercial sources and were of analytical grade.
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GARRITSEN ET AL.
TABLE 2. Radioreceptor methodologies used in assessing the receptor binding profile of amiloride, HMA and benzamil. Unless otherwise indicated rat tissue was used. receptor (sub)type adenosine-A,
forebrain (calf) forebrain (rat) adenosine-A, c. striatum total brain a,-adrenergic G-adrenergic total brain~cerebellum p ,2-adrenergic cerebral cortex dopamine-D, n. caudate dopamine-D, corpus striatum 5-HT1, frontal cortex 5-HT,, frontal cortex 5-HTlC choroid plexus (pig) 5-HT1, n. caudate (bovine) 5-HT, frontal cortex 5-HT3 nb x g cells (mouse) frontal cortex 5-HT,,, histamine-HI total brain muscarine-M, hippocampus muscarine-M, atrium heart muscarine-M, submandibular gland p-opiate total brainarebellurn K-opiate total brain-rebellum Gopiate total brain-rebellum Ca2+channel (DHP) cerebral cortex Caz+channel (VER) forebrain benzodiazepine total brain GABA, cerebellum glycine medulla + pons LTD, lung (guinea pig) TRH total brain-rebellum CCK, cerebral cortex CCK, pancreas Substance P tot. br.-cerebral cortex
,
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tissue
[,H]liganb
ref
DPCPX DPCPX NECAb prazosine clonidine DHA' dopamine spipero 8-OH-DPAT serotonin* serotonin serotonind spiperone GR 38032F" paroxetine mepyramine pirenzepine N-Me-scopolamine N-Me-scopolamine naloxone EKC DADLE nitrendipine D-888 diazepam dihydromuscimol strychnine LTD, MeTRH CCK-8 CCK-8 Substance P
19 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 36 36 31 38 39 40 41 42 43 44 45 46 47 47 48
'Abbreviations used: CCK, cholecystokinin; DADLE, D-Ala-D-Leu-enkephalin; DHA, dihydroalprenolol; DHP, dihydropyridine; DPCPX, 8-cyclopentyl-1,3dipropylxanthine; EKC, ethylketocyclazocine; GABA, y-aminobutyric acid; 5HT, serotonin; LTD,, leukotriene-D,; MeTRH, methyl-thyrotropin releasing hormone; NECA, N-ethylcarboxamidoadenosine;8-OH-DPAT, 8-hydroxy-2-(din-propy1)tetraline; VER, verapamil b50nM N6-cyclopentyladenosinewas added to prevent binding of [,H]NECA to adenosine A, receptors. '1 pM serotonin was added to prevent binding of [,HIDHA to 5-HT, receptors. '30 nM unlabelled 8-OH-DPAT and 30 nM 4-iodo-2,5dimethoxyphenylisopropylamine were added in order to block 5-HT1, and 5HT,, receptors. "Method identical to this reference except [,H]GR 38032F was used as ligand, rather than [,H]ICS 205,930.
RECEPTOR BINDING PROFILES OF AMILORIDE ANALOGUES
895
METHODS
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Binding methodology The three drugs were evaluated in binding assays for activity at 31 neurotransmitter receptors. Most binding assays were performed by Duphar BV according to well documented methods summarized in tab. 2. In general, drug solutions were pipetted manually in displacement experiments, whilst the [3H]ligand solutions and tissue suspensions were pipetted automatically by a Filterprep 101 (Ismatec, Ziirich, Switzerland), which further performed the assays up to and including the addition of scintillation cocktail to the glass fiber filters (Whatman GFB). The adenosine receptor assays were performed manually according to the methods of Ganitsen et al. (19) and Lohse et al. (21) for the A, receptor and Bruns et al. (22) for the A2 receptor, using a Millipore sampling manifold. Data analysis The concentrations of unlabelled drug causing 50 % displacement of the specific binding of a radioligand (IC, values) were obtained by computerized log-probit linear regression analysis of the data obtained in the experiments in which 4-6 different concentrations of the test compound were used. Inhibition constants (K, values) were calculated with the Cheng-Prusoff equation (49): K, = ICd(l+[L]/K.J in which [L] stands for the concentration radioligand and K, for the equilibrium dissociation constant of the radioligand. The average Ki values were calculated from at least three values obtained in independent experiments. All incubations were done in duplicate or triplicate. RESULTS The inhibition constants of amiloride, HMA and benzamil for 31 neurotransmitter recognition sites are presented in tab. 3. In general, the drugs are not very potent at any of the receptors compared to standard receptor agonists and antagonists, but in relation to the ‘specific’ actions of amiloride and its derivatives (tab. 1). some values appear relevant.
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GARRITSEN E T AL.
TABLE 3. Receptor binding profile of amiloride, HMA and benzamil. Assays were performed according to the methods summarized in tab. 2. The Ki values, in pM, are expressed as means k SE for 3 experiments. Ki values presented as > 10 pM are, in general, the result of two experiments.
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Receptor (sub)type Amiloride adenosine-A,(rat) 11 f 0.3 adenosine-A, (calf) 2.0 f 0.2 adenosine-A, 17 + 3 a,-adrenergic > 10 &-adrenergic > 10 p,,-adrenergic > 10 dopamine-D, > 10 dopamine-D2 > 10 5-HTtA > 10 5-HT1, > 10 5-HT1, > 10 5-HT1, > 10 5-HT2 > 10 5-HT, > 10 5-HT,pmk, > 10 histamine-HI > 10 muscarine-M, > 10 muscarine-M2 > 10 muscarine-M, > 10 p-opiate > 10 K-opiate > 10 &opiate > 10 Ca2+channel (DHP) > 10 Ca2+channel (VER) > 10 benzodiazepine > 10 GABA, > 10 glycine > 10 LTD, > 10 > 10 TRH CCK, > 10 CCK, > 10 Subskce P > 10
(%).
HMA
Benzamil
1.2 k0.05 0.41 +_ 0.03 4.6 +0.7 > 10 5.2 k0.8 > 10 > 10 > 10 > 10 > 10 6.7 k 1.2 > 10 0.40 k 0.06 > 10 7.9 +_ 3.3 5.6 +_ 1.2 3.6 k 1.0 2.9 f 0.5 4.7 f O . 8 0.061 +_0.021 3.9 f0.6 1.0 fO.2 > 10 0.054 kO.019 > 10 > 10 > 10 > 10 > 10 > 10 > 10 > 10
' average percentage inhibition at 10 pM amiloride; was enhanced by amiloride.
0.98k 0.05 0.65k 0.04 6.4 f 0.7 2.0 f 0.3 2.2 k 0.5 > 10 > 10 7.6 f 0.8 1.9 +_ 0.3 > 10 > 10 > 10 1.4 +_ 0.1 2.2 k 0.7 1.9 f 0.3 3.2 k 0.2 2.9 f 0.7 5.8 f 1.1 2.8 -I: 0.5 1.1 k 0.40 > 10 > 10 > 10 2.7 f 1.0 > 10 > 10 > 10 > 10 > 10 > 10 > 10 > 10
Note that CCK, binding
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RECEPTOR BINDING PROFILES OF AMILORIDE ANALOGUES
897
As reported previously, amiloride has a K, value of 2 p M for the adenosine-A, receptor in calf brain (19). Among the receptors tested, the adenosine-A, receptor in calf brain appears to be most sensitive to amiloride. Both HMA and benzamil are more potent than amiloride. Amiloride and HMA are ca. 5 fold less potent in rat brain than in calf brain, whereas benzamil is equally effective. At all other receptors amiloride has K, values above 10 pM. H M A displays affinities in the submicromolar range for the adenosine A, (410 nM), the p-opiate (61 nM), the 5-HT2 (400 nM) and the verapamil receptor (54 nM) and in the 1-10 pM range for a multitude of other receptors. Benzamil has an affinity in the submicromolar range for the A, receptor, and between 1 and 10 ph4 for about half of the other receptors tested. CCK binding in the pancreas is enhanced by amiloride, reminiscent of the allosteric enhancement of atrial namuretic factor binding (50). This effect deserves further investigation. One of the aims of this study was to provide a broader basis for testing the hypothesis that Gi coupled receptors might be linked to the Na+/H+ exchanger (18). In tab. 4 the signal transduction mechanisms for the receptors tested are presented. G, coupled receptors (group 3) are not particularly sensitive to amiloride analogues. Moreover, they are differentially affected by the drugs. Conversely, receptors coupled to different signal transduction systems can be influenced by amiloride, benzamil or HMA in a similar way. For instance, radioligand binding to the a,-adrenergic (group 2), the 5-HT,, (group 3) and the 5HT, receptor (group 4) is displaced by the analogues with similar K, values. The muscarinic receptor subtypes are affected in an identical manner, although the second messenger systems of the MI and M2receptor differ. Coupling of a receptor to a G protein, in general, and G,, in particular, is neither a guarantee nor a prerequisite for inhibition by amiloride or its analogues. The, G protein coupled, P-adrenergic, dopamine-D,, 5-HT,, and 5HT,,,and several peptide receptors have Ki values > 10 ph4. In contrast, the verapamil and 5-HT3 receptor (51) are not coupled to G proteins, but are affected by benzamil with K, values of 2.7 and 2.2 pM, respectively. In addition, Velly et al. (52) reported K, values of several 5-amino-substituted
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GARRITSEN ET AL.
TABLE 4. Classification of receptors according to signal transduction mechanism.
GROUP
effect
G protein?
receptors
1: stimulatory receptors adenylate cyclase
activation
Yes
adenosine-A, PI,-adrenoceptor dopamine-D,
2: stimulatory receptors PIP,-PLC
activation
Yes
a,-adrenergic histamine-H, 5-HT,, muscarine-M,, LTD., TRH CCK,
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3: inhibitory receptors
adenylate cyclase PIP,-PLC calcium channel potassium channel
inhibition inhibition inhibition activation
adenosine-A, &-adrenoceptor dopamine-D, 5-HT1,, muscanne-M, p-opiate &opiate K-opiate
4: (ligand gated) ion channels voltage dependent calcium channel
activation ?
no
DHP VER
cation channel
activation
no
5-HT3
chloride channel
activation
n0
facilitation of activation
no
GABA, glycine benzodiazepine
no
5-HTV,
Yes
substance P CCK,
5: others transport protein potassium channel ?
inhibition
Information was obtained from refs 52-55. T h e inhibitory receptors (group 3) are often coupled to multiple mechanisms (18). It is, however, not yet clear which one will be the most important mechanism for each receptor. Therefore all the possibilities are listed.
RECEPTOR BINDING PROFILES OF AMILORIDE ANALOGUES
a99
analogues for inhibition of [3H]batrachotoxinbinding to the voltage-sensitive sodium channel around 0.35 pM. Other receptors with an intrinsic ion channel (group 4: benzodiazepine, GABA,, glycine and DHP receptors) are insensitive to amiloride and its analogues. DISCUSSION
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Specificity of amiloride and its analogues In the present study, a multitude of receptors has been screened for affinity for amiloride and its analogues. The overall view emerging from the results is that amiloride and its analogues are largely non-selective compounds, that interact with many membrane receptors, ion transporters and other membrane proteins. Comparing the values in tab. 3 with each other and with data from the literature must be done with a certain caution. Species, tissue, cell type or Na' concentrations in the assays vary and this may account for differences in the Ki values observed. In the literature no general tendencies have been reported. Thus, the affinity of amiloride for g-adrenoceptors is independent of the NaCl concentration in porcine brain (16), but in kidney membranes NaCi enhances the affinity of amiloride, whereas the effect of NaCl in platelets varies for unknown reasons (12). In contrast, binding to the adenosine-A, receptor is markedly attenuated by NaCl (19). Species differences may occur as evident from the Ki values for the adenosine-A, receptor in rat and calf brain, respectively (tab. 3). Nonetheless, the receptor binding assays presented here are employed in the screening of new drugs and together generate a profile representative for the potency of drugs at different receptors. The rank order of potency of amiloride and its analogues in a particular binding assay is a sound parameter in this study, as this is, obviously, not affected by differences between the binding assays. A few limitations of the comparison between the activity at different receptors and ion transport proteins should also be pointed out. First of all, binding data are compared with a physiological response. In this context, it is important to realize, that the effects on Na+/H+exchange are well correlated
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GARRITSEN ET AL.
with the inhibition of binding of tritiated analogues to the antiporter by these substances (636). Such a correlation has not been established for the epithelial sodium channel. [3H]Phenamilbinds to pig kidney membranes and rat brain homogenates but amiloride has a relatively low affinity (4 pM) for this site compared to its affinity for the epithelial sodium channel, and EIPA is more potent than amiloride (757). This suggests that the ['Hlphenamil binding site is not the epithelial sodium channel (2). Accordingly, a binding correlate is not available for the epithelial sodium channel and we could compare the effects on receptor binding only with effects on Na+ transport. Comparing the values for inhibition of receptor binding in tab. 3 with each other and with the data available from the literature, presented in tab. 1, amiloride nor its analogues is selective for the A, receptor, the subject of our previous study (19), although it displays a relatively high affinity for this receptor. Overall, amiloride itself is a slightly selective blocker of the epithelial sodium channel, as is benzamil. Side effects due to the interaction with neurotransmitter receptors will only occur at concentrations 10-20 fold higher than those needed for blockade of Na+ transport through the epithelial sodium channel. HMA is not selective for any of the processes, known to be affected by this compound. Its relatively high potency for the p-opiate receptor suggests that this analogue might be used as a lead compound for a novel type of opiate receptor ligands. The interaction with the L-type Caz+ channel was recently also reported by Garcia et al. (58). The Ki values in our study, however. are much lower.
Are G; coupled receDtors linked to Na+/f4+exchange? It has been hypothesized that a link might exist between Gi coupled receptors and the Na+/H+exchanger (18). Evidence for such a coupling should meet several criteria, some of which can well be addressed by radioligand binding studies. (1) The affinity of amiloride and its analogues should be in the proper concentration range, keeping in mind that the actions of the drugs can be counteracted by Na+ (1-3). (2) The structure-activity profile obtained must be consistent with the generally accepted profile for one of the transport systems. A S A R study should
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RECEPTOR BINDING PROFILES OF AMILORIDE ANALOGUES
901
include at least one 5-amino-substitutedanalogue and one analogue bearing a substituent on a terminal guanidino nitrogen atom. If we apply these criteria to the results in tab. 3, it can be concluded that (1) the epithelial sodium channel is not involved in any of the amiloride-receptor interactions, as the Ki values of amiloride are too high; (2) at some receptor types both analogues are more potent than amiloride, excluding the involvement of the epithelial sodium channel or Na+W exchange, but resembling the SAR profile of Na+/Ca2+exchange inhibition. The Ki values of HMA for inhibition of muscarinic, adenosine-A,, 5-HT2 and F-opiate receptor binding are, however, 25, 250, 250 and 1600 times lower than for inhibition of Na+/Ca2+ exchange; (3) the involvement of Na+/H+exchange can be rejected in 15 assays, where benzamil is the most active analogue or benzamil and HMA are both more active than amiloride and in another 13 assays where H M A has a Ki value > 10 J.LM, which is not in agreement with its high affinity for the Na+/H' antiporter. For the remaining receptors (5-HT,,, K-opiate and &opiate), Na+ transport experiments and more detailed SAR studies may give a final answer. In these cases, the order of potency, HMA > amiloride or benzamil, is, in our opinion, more likely to be a coincidence than an indication for the involvement of the Na'W exchanger. A direct relation between the signal transduction system coupled to the receptors tested and their sensitivity to amiloride and its analogues is not evident. Receptors linked to identical mechanisms react differently, and vice versa, indicating that not the second messenger system, but a domain of the receptor molecule involved in ligand binding determines its sensitivity to amiloride and its analogues. Interestingly, mainly the receptors for the classical neurotransmitters are affected. Considering the recently demonstrated homology between many receptors molecules ( 5 9 , a conserved amiloride binding region may indeed exist. It is conceivable that amiloride and its analogues interact with the Na+ binding site responsible for the reciprocal regulation of agonist and antagonist binding, a feature common to many G protein coupled receptors (see among others ref. 37). However, in previous studies on the interaction between the adenosine-A, receptor and amiloride and its analogues, we demonstrated that these compounds affect agonist as well as antagonist binding to this receptor
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GARRITSEN ET AL.
in identical concentrations. Na' largely reduces the affinity of amiloride, independent of the fact whether an agonist or antagonist radioligand was used (19). Recently, evidence against an interaction of amiloride with the Na' binding site was provided by Horstman et al. (59), based on mutagenesis of the porcine q-adrenergic receptor. They identified Asp79, an amino acid residue in the second transmembrane domain of the porcine q-adrenergic receptor, as the residue responsible for the modulation of radioligand binding to this receptor by Na' ions. Mutation of this residue that is highly conserved among G protein coupled receptors, to Asn abolishes Na+ sensitivity of receptor binding, without changing other binding properties of agonists and antagonists. The mutant form of the q-adrenergic receptor is still sensitive to amiloride and its analogues, suggesting that, at least for this receptor, the effects of Na' and amiloride are mediated by different domains of the receptor. In conclusion, amiloride, HMA and benzamil are nonselective inhibitors of binding to a variety of receptors. H M A is a fairly potent p-opiate receptor and verapamil receptor ligand. The Ki values of amiloride and benzamil for receptors are 10-20 fold higher than their reported affinity for the epithelial sodium channel. The additional effects of these compounds on receptor binding may conmbute to their pharmacological profile. The interaction of amiloride and its analogues with neurotransmitter receptors is clearly not related to the Na' transport-inhibiting properties of the compounds. The wide-spread amiloride-sensitivity found among receptors points to a common smcture in many neurotransmitter receptors and other membrane proteins, at or close to the ligand binding site, which recognizes amiloride and its analogues, thereby excluding the binding of the standard ligands. REFERENCES 1. Benos, D.J. Amiloride: a molecular probe of sodium transport in tissues and cells, Am. J. Physiol. 242, C131-145, 1982. 2. Garty, H.; and Benos, D.J. Characteristics and regulatory mechanisms of the amiloride-blockable Na' channel, Physiol. Rev. 68, 309-373,1988.
3. Kleyman, T.R.; and Cragoe, Jr, E.J. Adoride and its analogs as tools in the study of ion transport, J. Membrane Biol. 105, 1-21, 1988.
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4. Simchowitz, L.; and Cragoe, Jr, E.J. Inhibition of chemotactic factor-activated Na+/H+exchange in human neutrophils by analogues of amiloride: structureactivity relationships in the amiloride series, Mol. Pharmacol. 30, 112-120, 1986. 5. Vigne, P.; Frelin C.; Cragoe, Jr, E.J.; and Lazdunski M. Structure-activity relationships of amiloride and certain of its analogues in relation to the blockade of the Na+/H+exchange system, Mol. Pharmacol. 25, 131-136, 1984.
6. Vigne, P.; Frelin, C.; Audinot, M.; Borsotto, M.; Cragoe Jr, E.J.; and Lazdunski, M. [3H]Ethylisopropylamiloride, a radio-labelled diuretic for the analysis of the Na+/H+exchange system. Its use with kidney cell membranes, EMBO J. 3, 2647-2651, 1984.
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7. Barbry, P.; Frelin, C.; Vigne, P.; Cragoe, Jr E.J.; and Lazdunski M. [3H]Phenamil, a radiolabelled diuretic for the analysis of the miloride-sensitive Na+ channels in kidney membranes, Biochem. Biophys. Res. Comm. 135, 25-32, 1986. 8. Kaczorowski, G.J.; Barros, F.; Dethmers, J.K.; Trumble, M.J.; and Cragoe, Jr E.J. Inhibition of Na+/Ca2+exchange in pituitary plasma membrane vesicles by analogues of amiloride, Biochemistry 24, 1394-1403, 1985. 9. Friedrich, T.; and Burckhardt, G. Inhibition and labeling of the rat renal Na+/H+ exchanger by an antagonist of muscarinic acetylcholine receptors, Biochem. Biophys. Res. Comm. 157, 921-929, 1988. 10. Haussinger, D.; Brodde, 0.-E.; and Starke, K. Alpha-adrenoceptor antagonistic action of amiloride, Biochem. Pharmacol. 36, 3509-3515, 1987. 11. Howard, M.J.; Mullen, M.D.; and Insel, P.A. Amiloride interacts with renal
a- and P-adrenergic receptors, Am. J. Physiol. 253, F21-25, 1987. 12. Howard, M.J.; Hughes, R.J.; Motulsky, H.J.; Mullen, M.D.; and Insel, P.A. Interactions of amiloride with a-and P-adrenergic receptors: amiloride reveals an allosteric site on G-adrenergic receptors, Mol. Pharmacol. 32, 53-58, 1987.
13. Isom, L.L.; Cragoe, Jr E.J.; and Limbird, L.E. Multiple receptors linked to inhibition of adenylate cyclase accelerate Na+/H+exchange in neuroblastoma x glioma cells via a mechanism other than decreased CAMP accumulation, J. Biol. Chem. 262, 17504-17509, 1987 and (correction) J. Biol. Chem. 263, 16513, 1988. 14. Kuijpers, G.A.J.; De Pont, J.J.H.H.M.; van Nooy, I.G.P.; Fleuren-Jakobs, A.M.M.; Bonting, S.L.; and Rodrigues De Miranda, J. Amiloride is a cholinergic antagonist in the rabbit pancreas, Biochim. Biophys. Acta. 804, 237-244, 1984. 15. Martinez, J.R.; Barker, S.; and Camden, J. Amiloride inhibits *'Na+ uutake and [3H]QNB binding in rat submandibular gland cells, Eur. J. Pharmacol. i64, 335339, 1989.
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16.Nunnari, J.M.; Repaske, M.G.; Brandon, S.; Cragoe, Jr E.J.; and Limbird, L.E. Regulation of porcine brain q adrenergic receptors by Na+, H' and inhibitors of Na+/H+exchange, J. Biol. Chem. 262, 12387-12392,1987. 17. Yablonsky, F.; and Dausse, J.P. Amiloride interacts with [3H]idazoxan and [3H]rauwolscine binding sites in rabbit urethra, Eur. J. Pharmacol. 164, 167-170, 1989. 18. Limbird, L.E. Receptors linked to inhibition of adenylate cyclase: additional signaling mechanisms, FASEB J. 2, 2686-2695,1988.
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19. Garritsen, A.; IJzerman, A.P.; Beukers, M.W.; Cragoe, Jr E.J.; and Soudijn, W. Interaction of amiloride and its analogues with adenosine A, receptors in calf brain, Biochem. Pharmacol. 40, 827-834,1990. 20. Cragoe, E.J., Jr.; Woltersdorf, Jr O.W.; Bicking, J.B.; Kwong, S.F.; and Jones, J.H. Pyrazine diuretics II. N-Amidino-3-mino-5-substituted 6-halopyrazinecarboxamides, J. Med. Chem. 10, 66-75,1967. 21. Lohse, M.J.; Klotz, K.-N. ; Lindenborn-Fotinos, J.; Reddington, M.; Schwabe U., and Olsson, R.A. 8-cyclopentyl-1,3-dipropylxanthine(DPCPX) - a selective high affinity antagonist radioligand for A, adenosine receptors, NaunynSchmiedeberg's Arch. Pharmacol. 336, 204-210,1987. 22.Bruns, R.F.; Lu, G.H.; and Pugsley, T.A. Characterization of the A, adenosine receptor labeled by [%]NECA in rat smatal membranes, Mol. Pharmacol. 29, 331-346,1986. 23. Timmermans, P.B.M.W.M.; Karamat Ali, F.; Kwa, H.Y.; Schoop, A.M.C.; Slothorst-Grisdijk, F.P.; and Van Zwieten, P.A. Identical antagonist selectivity of central and peripheral a,-adrenoceptors, Mol. Pharmacol. 20, 295-301,1981. 24. U'Prichard, D.; Greenberg, D.A.; and Snyder, S.H. Binding characteristics of a radiolabeled agonist and antagonist at central nervous system alpha noradrenergic receptors, Mol. Pharmacol. 13, 454-473,1977.
25. Nahorski, S.R. Heterogeneity of cerebral p-adrenoceptor binding sites in various vertebrate species, Eur. J. Pharmacol. 51, 199-209,1978. 26. Burt, D.R.; Creese, I.; and Snyder, S.H.Properties of ['Hlhaloperidol and [Wldopamine binding associated with dopamine receptors in calf brain membranes, Mol. Pharmacol. 12, 800-812,1976.
27. Creese, I.; Schneider, R.; and Snyder, S.H. 'H-Spiroperidol labels dopamine receptors in pituitary and brain, Eur. J. Pharmacol. 46, 377-381,1977. 28. Gozlan, H.; El Mestikawy, S.; Pichat, L.; Glowinsky, J.; and Hamon, M. Identification of presynaptic serotonin autoreceptors using a new ligand r3H]PAT, Nature 305, 140-142,1983.
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32. Creese, I.; and Snyder, S.H. 'H-Spiroperidol labels serotonin receptors in rat cerebral cortex and hippocampus, Eur. J. Pharmacol 49, 201-204, 1978. 33. Hoyer, D.; and Neijt, H.C. Identification of serotonin 5-HT3 recognition sites by radioligand binding in NG108-15 neuroblastoma-glioma cells, Eur. J. Pharmacol. 143, 291-292, 1987. 34. Habert, E.; Graham, D.; Tahraoui, L.; Claustre, Y.; and Langer, S.Z. Characterization of ['Hlparoxetine binding to rat cortical membranes, Eur. J. Pharmacol. 118, 107-114, 1985. 35. Tran, V.T., Chang, R.S.L.; and Snyder, S.H. Histamine HI receptors identified in mammalian brain membranes with [3H]mepyramine, Proc. Natl. Acad. Sci. U.S.A. 75, 6290-6294, 1978. 36. Doods, H.N. Affinity and selectivity of muscarinic antagonists for the M,, M, and M, binding sites, In: Characterization and classification of muscarinic receptors and their subtypes (dissertation) p. 40-53. University of Amsterdam, Amsterdam, 1987. 37. Pert, C.B.; and Snyder, S.H. Opiate receptor binding of agonists and antagonists affected differentially by sodium, Mol. Pharmacol. 10, 868-879, 1974. 38. Hiller, J.M.; and Simon, E.J. Specific, high affinity [3H]ethyLketocyclazocine binding in rat central nervous system: lack of evidence for k receptors, J. Pharmacol. Exp. Ther. 214, 516-519, 1980. 39. Leslie, F.M.; Chavkin, C.; and Cox, B.M. Opioid binding properties of brain and peripheral tissues, evidence for heterogeneity in opioid ligand binding sites, J. Pharmacol. Exp. Ther. 214, 395-402, 1980. 40. Ehlert, F.J.; Roeske, W.R.; Itoga, E.; and Yamamura, H.I. The binding of ['HI-Ntrendipine to receptors for calcium channel antagonists in the heart, cerebral cortex and ileum of rats, Life Sci. 30, 2191-2202, 1982.
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41. Reynolds, I.J.; Snowman, A.M.; and Snyder, S.H. ['H]Desmethoxyverapami1 labels multiple calcium channel modulator receptors in brain and skeletal muscle membranes: differentiation by temperature and dihydropyridines, J. Pharmacol. Exp. Ther. 237, 731-738, 1986. 42. Braesmp, C.; and Squires, R.F. Pharmacological characterization of benzodiazepine receptors in the brain, Eur. I. Pharmacol. 48, 263-270, 1978. 43. Herschel, M.; and Baldessarini, R.J. Evidence for two types of binding of 'HGABA and 'H-muscimol in rat cerebral cortex and cerebellum, Life Sci. 24, 1849-1854, 1979.
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44. Muller, W. E.; and Snyder, S.H. Strychnine binding associated with synaptic glycine receptors in rat spinal cord membranes: ionic influences, Brain Res. 147, 107-116, 1978. 45. Mong, S.; Wu, H.-L.; Hogaboom, G.K.;Clark, M.A.; and Crooke, S.T. Characterization of the leukomene D, receptor in guinea-pig lung, Eur. J. Pharmacol. 102, 1-11, 1984. 46. Taylor, R.L.; and Burt, D.R. Preparation of 'H-[3-Me-Hisz]TRH as an improved ligand for TRH receptors, Neuroendocrinol. 32, 310-316, 1981. 47. Van Dijk, A.; Richards, J.G.; Trzeciak, A.; Gillessen, D.; and Mohler, H. Cholecystokinin receptors: biochemical demonstration and autoradiographical localization in rat brain and pancreas using ['H]cholecystokinin-8 as radioligand, J. Neurosci. 4, 1021-1033, 1984. 48. Perrone, M.H.; Diehl, R.E.; and Haubrich, D.R. Binding of [3H]Substance P to putative Substance P receptors in rat brain membranes, Eur. J. Pharmacol. 95, 131-133, 1983. 49. Cheng, Y.C.; and Prusoff, W.H. Relationship between the inhibition constant (K,)and the concentration of inhibitor which causes 50 percent inhibition (I,) of an enzymatic reaction, Biochem. Pharmacol. 22, 3099-3108, 1973. 50. DeLean, A. Amilonde potentiates amal namuretic factor inhibitory action by increasing receptor binding in bovine adrenal zona glomerulosa, Life Sci. 39, 1109-1116, 1986. 51. Derkach, V.; Surprenant, A.; and North, R.A. 5-HT3receptors are membrane ion channels, Nature 339. 706, 1989. 52. Velly, J.; Grima, M.; Decker, N.; Cragoe, Jr E.J.; and Schwartz, J. Effects of amiloride and its analogues on ['H]batrachotoxinin-A 20-01 benzoate binding, FHItetracaine binding and "Na' influx, Eur. J. Pharmacol. 149, 97-105, 1988. 53. Linden, J.; and Delahunty, T.M. Receptors that inhibit phosphoinositide breakdown, Trends Pharmacol. Sci. 10, 114-120, 1989.
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54. Michell, R. Transmembrane signalling, Trend Pharmacol. Sci. 9, centerfold april, 1988. 55. Strange, P.G. The structure and mechanism of neurotransmitter receptors, Biochem. J. 249, 309-318, 1988. 56. Jean, T.; Frelin, C.; Vigne, P.; Barbry, P.; and Lazdunski, M. Biochemical properties of the Na+/H+exchange system in rat brain synaptosomes, J. Biol. Chem. 260, %78-9684, 1985.
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57. Vigne, P.; Champigny, G.; Marsault, R.; Barbry, P.; Frelin, C.; and Lazdunski, M. A new type of amiloride-sensitive cationic channel in endothelial cells of brain microvessels, J. Biol. Chem. 264, 7663-7668, 1989. 58. Garcia, M.L.; King, V.F.; Shevell, J.L.; Slaughter, 3.; Suarez-Kurtz, G.; Winquist, R.J.; and Kaczorowski, G.J., Amiloride analogues inhibit L-type calcium channels and display calcium entry blocker activity, J. Biol Chem. 265, 37633771, 1990. 59. Horstman, D.A.; Brandon, S.; Wilson, A.L.; Guyer, C.A.; Cragoe, Jr E.J.; and Lmbird, L.E. An aspartate conserved among G-protein receptors confers allosteric regulation of cq-adrenergic receptors by sodium,-J. Biol. Chem. 265,21590-21595, 1990.
ISSN: 0197-5110 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/irst19
Receptor Binding Profiles of Amiloride Analogues Provide no Evidence for a Link Between Receptors + + and the Na /H Exchange, But Indicate a Common Structure on Receptor Proteins Anja Garritsen, Adriaan P. Ijzerman, Martin M. Th. Tulp, Edward J. Cragoe & Willem Soudijn To cite this article: Anja Garritsen, Adriaan P. Ijzerman, Martin M. Th. Tulp, Edward J. Cragoe & Willem Soudijn (1991) Receptor Binding Profiles of Amiloride Analogues Provide +
+
no Evidence for a Link Between Receptors and the Na /H Exchange, But Indicate a Common Structure on Receptor Proteins, Journal of Receptor Research, 11:6, 891-907, DOI: 10.3109/10799899109064686 To link to this article: http://dx.doi.org/10.3109/10799899109064686
Published online: 26 Sep 2008.
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Date: 11 May 2016, At: 22:59
JOURNAL OF RECEPTOR RESEARCH, 11(6), 891-907 (1991)
RECEPTOR BINDING PROFILES OF AMILORIDE ANALOGUES PROVIDE NO EVIDENCE FOR A LINK BETWEEN RECEPTORS AND THE Na+/H+EXCHANGER, BUT INDICATE A COMMON STRUCTURE ON RECEPTOR PROTEINS
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Anja Ganitsen', Adriaan P. IJzerman", Martin Th.M. Tulp*, Edward J. Cragoe, Jr.3 and Willem Soudijn' Center for Bio-Pharmaceutical Sciences, Div. Med. Chem., P.O. Box 9502, 2300 RA Leiden, the Netherlands, * Duphar BV, C.J. van Houtenlaan 36, 1381 CP Weesp and P.O. Box 631548, Nacogdoches, TX 75963, USA ABSTRACT Amiloride and its analogues affect radioligand binding to the adenosine-A, receptor. In this paper, the specificity of this effect is investigated by generating receptor binding profiles for amiloride and two of its analogues. A limited structure-activity relationships study is performed to probe the relationship between inhibition of receptor binding by amiloride analogues and the effects of these compounds on Na' transport, in particular Na+/H+exchange. The receptor binding profiles of amiloride, benzamil and 5'-(N,Nhexamethy1ene)amilonde (HMA) indicate that the compounds affect a variety of receptors and that none of the compounds is highly selective for any of these. The SAR study indicates that it is very unlikely that a direct coupling between receptors and Na'/H+ exchange or another amiloride-sensitive ion transport system is responsible for the inhibition of receptor binding. A correlation between the signal transduction systems coupled to the receptors involved and the potency of the amiloride analogues is also absent. The varying nature of the receptors, affected by amiloride or its analogues, suggests a wide-spread presence of an amiloride binding site on receptors and other membrane proteins. INTRODUCTION Amiloride, a potassium sparing diuretic, has been used as a pharmacological probe for Na' transport systems (1-3). The drug inhibits various systems with different potencies: in concentrations < 1 pM,epithelial sodium channels are 891 Copyright 0 1991 by Marcel Dekker, Inc.
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GARRITSEN ET AL.
TABLE 1. The affinity of amiloride and its analogues for Na+/H+exchange, the epithelial sodium channel and Na'lCa'' exchange. System amiloride
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N a + W exchange (3,4) '%a+ efflux 84 human neutrophils, [NaCl] 140 mM N a + W exchange ( 5 ) =Na+ uptake 7 chick muscle, [NaCl] 3 mM Na'W exchange (6) 2 binding of [3H]EPAd rabbit kidney, [NaCl] 0 mM Na+ channel (3) electrophysiology frog skin, [NaCl] 110 mM Na+ channel (7) binding of [3H]phenamil pig kidney, INaCI] 0 mM
Affinity (JLM) HMA'/EIPAb
benzamil
0.1610.3 8
> lo00
n.d.'IO.050
100
n.d.10.017
> 10
0.35
> lo/> 10
0.038
4.0
n.d.ll.0
0.300
Na+lCa2+exchange (38) "%a2+ uptake 1100 pituitary plasma membrane vesicles [NaCl],, 0 mM
100/130
100
Affinity in pM refers to all four compounds. ' HMA, 5-(N,NN-hexamethylene)amiloride; The closely related analogue E P A (5-(N-ethyl-N-isopropyl)amiloride)is included in the tab., since the affinity of HMA has not alsways been determined. ' n.d., not determined, EPA, 5-(N-ethylN-propy1)amiloride
blocked, between 1 and 100 pM, Na+/H' exchange is inhibited and in the millimolar range Na+/Ca'+ exchange is affected (tab. 1). Besides the parent compound, amiloride analogues are available that affect mainly N a + W exchange (the 5-amino-substituted analogues such as HMA) or the epithelial sodium channel (the analogues bearing a substituent at a terminal guanidino nitrogen atom such as benzamil) (tab. 1) (1-3). As the affinities of HMA and benzamil for both processes differ by several orders of magnitude, these two compounds can be used to define which particular Na+ transporter is involved. Benzamil and HMA are both more potent inhibitors of Na+/Ca*+exchange than amiloride, but have very low affinities for this exchanger of about 100 pM.
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RECEPTOR BINDING PROFILES OF AMILORIDE ANALOGUES
893
The mere fact that a physiological or pharmacological process is modulated by amiloride or its analogues is often regarded as evidence for the involvement of one of the above-mentioned transport systems. It has become clear, however, that amiloride is not only an ion transport inhibitor, but has a variety of other effects. Thus, several papers have been published on effects of amiloride on radioligand binding to the adrenergic and muscarinic receptors (9-17). Amiloride-sensitive Na' transport appears to be influenced by agonists for these receptors, an effect possibly directly related to the effects of amiloride on radioligand-receptor binding. The most likely candidate is Na+/H+exchange, as amilorid:: usually inhibits receptor binding with Ki values between 10 and 100 @A. This resulted in the hypothesis of Limbird and coworkers, that G,coupled receptors are somehow directly linked to the plasma membrane N a W exchanger (13,16,18). We showed recently that amiloride displaces radioligand binding to the, Gi coupled, adenosine-A, receptor in a calf brain membrane preparation with a Ki value of 2 pM (19), a concentration well below the Ki value for the adrenergic receptors (10-13,16,17). However, the structure-activity relationships (SAR) of 8 amiloride analogues for inhibition of adenosine-A, receptor binding are not at all consistent with the involvement of Na+/H+exchange or another Na' transporter, and a direct link seems very unlikely. The present study was undertaken to determine the specificity of amiloride and its analogues for the adenosine-A, receptor and, in addition, to establish whether amiloride-sensitivity is a general phenomenon among receptors that are coupled to certain signal transduction systems. A possible relationship between the inhibition of radioligand-receptor binding and the ion transport blocking properties of the amiloride analogues is addressed by means of a limited S A R study.
MATERIALS Amiloride was kindly donated by Merck Sharp and Dohme (Haarlem, the Netherlands, USP grade). 5-(N,N-Hexamethylene)amilorideand benzamil were synthesized as decribed previously (20). All other chemicals were obtained from standard commercial sources and were of analytical grade.
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GARRITSEN ET AL.
TABLE 2. Radioreceptor methodologies used in assessing the receptor binding profile of amiloride, HMA and benzamil. Unless otherwise indicated rat tissue was used. receptor (sub)type adenosine-A,
forebrain (calf) forebrain (rat) adenosine-A, c. striatum total brain a,-adrenergic G-adrenergic total brain~cerebellum p ,2-adrenergic cerebral cortex dopamine-D, n. caudate dopamine-D, corpus striatum 5-HT1, frontal cortex 5-HT,, frontal cortex 5-HTlC choroid plexus (pig) 5-HT1, n. caudate (bovine) 5-HT, frontal cortex 5-HT3 nb x g cells (mouse) frontal cortex 5-HT,,, histamine-HI total brain muscarine-M, hippocampus muscarine-M, atrium heart muscarine-M, submandibular gland p-opiate total brainarebellurn K-opiate total brain-rebellum Gopiate total brain-rebellum Ca2+channel (DHP) cerebral cortex Caz+channel (VER) forebrain benzodiazepine total brain GABA, cerebellum glycine medulla + pons LTD, lung (guinea pig) TRH total brain-rebellum CCK, cerebral cortex CCK, pancreas Substance P tot. br.-cerebral cortex
,
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tissue
[,H]liganb
ref
DPCPX DPCPX NECAb prazosine clonidine DHA' dopamine spipero 8-OH-DPAT serotonin* serotonin serotonind spiperone GR 38032F" paroxetine mepyramine pirenzepine N-Me-scopolamine N-Me-scopolamine naloxone EKC DADLE nitrendipine D-888 diazepam dihydromuscimol strychnine LTD, MeTRH CCK-8 CCK-8 Substance P
19 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 36 36 31 38 39 40 41 42 43 44 45 46 47 47 48
'Abbreviations used: CCK, cholecystokinin; DADLE, D-Ala-D-Leu-enkephalin; DHA, dihydroalprenolol; DHP, dihydropyridine; DPCPX, 8-cyclopentyl-1,3dipropylxanthine; EKC, ethylketocyclazocine; GABA, y-aminobutyric acid; 5HT, serotonin; LTD,, leukotriene-D,; MeTRH, methyl-thyrotropin releasing hormone; NECA, N-ethylcarboxamidoadenosine;8-OH-DPAT, 8-hydroxy-2-(din-propy1)tetraline; VER, verapamil b50nM N6-cyclopentyladenosinewas added to prevent binding of [,H]NECA to adenosine A, receptors. '1 pM serotonin was added to prevent binding of [,HIDHA to 5-HT, receptors. '30 nM unlabelled 8-OH-DPAT and 30 nM 4-iodo-2,5dimethoxyphenylisopropylamine were added in order to block 5-HT1, and 5HT,, receptors. "Method identical to this reference except [,H]GR 38032F was used as ligand, rather than [,H]ICS 205,930.
RECEPTOR BINDING PROFILES OF AMILORIDE ANALOGUES
895
METHODS
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Binding methodology The three drugs were evaluated in binding assays for activity at 31 neurotransmitter receptors. Most binding assays were performed by Duphar BV according to well documented methods summarized in tab. 2. In general, drug solutions were pipetted manually in displacement experiments, whilst the [3H]ligand solutions and tissue suspensions were pipetted automatically by a Filterprep 101 (Ismatec, Ziirich, Switzerland), which further performed the assays up to and including the addition of scintillation cocktail to the glass fiber filters (Whatman GFB). The adenosine receptor assays were performed manually according to the methods of Ganitsen et al. (19) and Lohse et al. (21) for the A, receptor and Bruns et al. (22) for the A2 receptor, using a Millipore sampling manifold. Data analysis The concentrations of unlabelled drug causing 50 % displacement of the specific binding of a radioligand (IC, values) were obtained by computerized log-probit linear regression analysis of the data obtained in the experiments in which 4-6 different concentrations of the test compound were used. Inhibition constants (K, values) were calculated with the Cheng-Prusoff equation (49): K, = ICd(l+[L]/K.J in which [L] stands for the concentration radioligand and K, for the equilibrium dissociation constant of the radioligand. The average Ki values were calculated from at least three values obtained in independent experiments. All incubations were done in duplicate or triplicate. RESULTS The inhibition constants of amiloride, HMA and benzamil for 31 neurotransmitter recognition sites are presented in tab. 3. In general, the drugs are not very potent at any of the receptors compared to standard receptor agonists and antagonists, but in relation to the ‘specific’ actions of amiloride and its derivatives (tab. 1). some values appear relevant.
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GARRITSEN E T AL.
TABLE 3. Receptor binding profile of amiloride, HMA and benzamil. Assays were performed according to the methods summarized in tab. 2. The Ki values, in pM, are expressed as means k SE for 3 experiments. Ki values presented as > 10 pM are, in general, the result of two experiments.
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Receptor (sub)type Amiloride adenosine-A,(rat) 11 f 0.3 adenosine-A, (calf) 2.0 f 0.2 adenosine-A, 17 + 3 a,-adrenergic > 10 &-adrenergic > 10 p,,-adrenergic > 10 dopamine-D, > 10 dopamine-D2 > 10 5-HTtA > 10 5-HT1, > 10 5-HT1, > 10 5-HT1, > 10 5-HT2 > 10 5-HT, > 10 5-HT,pmk, > 10 histamine-HI > 10 muscarine-M, > 10 muscarine-M2 > 10 muscarine-M, > 10 p-opiate > 10 K-opiate > 10 &opiate > 10 Ca2+channel (DHP) > 10 Ca2+channel (VER) > 10 benzodiazepine > 10 GABA, > 10 glycine > 10 LTD, > 10 > 10 TRH CCK, > 10 CCK, > 10 Subskce P > 10
(%).
HMA
Benzamil
1.2 k0.05 0.41 +_ 0.03 4.6 +0.7 > 10 5.2 k0.8 > 10 > 10 > 10 > 10 > 10 6.7 k 1.2 > 10 0.40 k 0.06 > 10 7.9 +_ 3.3 5.6 +_ 1.2 3.6 k 1.0 2.9 f 0.5 4.7 f O . 8 0.061 +_0.021 3.9 f0.6 1.0 fO.2 > 10 0.054 kO.019 > 10 > 10 > 10 > 10 > 10 > 10 > 10 > 10
' average percentage inhibition at 10 pM amiloride; was enhanced by amiloride.
0.98k 0.05 0.65k 0.04 6.4 f 0.7 2.0 f 0.3 2.2 k 0.5 > 10 > 10 7.6 f 0.8 1.9 +_ 0.3 > 10 > 10 > 10 1.4 +_ 0.1 2.2 k 0.7 1.9 f 0.3 3.2 k 0.2 2.9 f 0.7 5.8 f 1.1 2.8 -I: 0.5 1.1 k 0.40 > 10 > 10 > 10 2.7 f 1.0 > 10 > 10 > 10 > 10 > 10 > 10 > 10 > 10
Note that CCK, binding
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RECEPTOR BINDING PROFILES OF AMILORIDE ANALOGUES
897
As reported previously, amiloride has a K, value of 2 p M for the adenosine-A, receptor in calf brain (19). Among the receptors tested, the adenosine-A, receptor in calf brain appears to be most sensitive to amiloride. Both HMA and benzamil are more potent than amiloride. Amiloride and HMA are ca. 5 fold less potent in rat brain than in calf brain, whereas benzamil is equally effective. At all other receptors amiloride has K, values above 10 pM. H M A displays affinities in the submicromolar range for the adenosine A, (410 nM), the p-opiate (61 nM), the 5-HT2 (400 nM) and the verapamil receptor (54 nM) and in the 1-10 pM range for a multitude of other receptors. Benzamil has an affinity in the submicromolar range for the A, receptor, and between 1 and 10 ph4 for about half of the other receptors tested. CCK binding in the pancreas is enhanced by amiloride, reminiscent of the allosteric enhancement of atrial namuretic factor binding (50). This effect deserves further investigation. One of the aims of this study was to provide a broader basis for testing the hypothesis that Gi coupled receptors might be linked to the Na+/H+ exchanger (18). In tab. 4 the signal transduction mechanisms for the receptors tested are presented. G, coupled receptors (group 3) are not particularly sensitive to amiloride analogues. Moreover, they are differentially affected by the drugs. Conversely, receptors coupled to different signal transduction systems can be influenced by amiloride, benzamil or HMA in a similar way. For instance, radioligand binding to the a,-adrenergic (group 2), the 5-HT,, (group 3) and the 5HT, receptor (group 4) is displaced by the analogues with similar K, values. The muscarinic receptor subtypes are affected in an identical manner, although the second messenger systems of the MI and M2receptor differ. Coupling of a receptor to a G protein, in general, and G,, in particular, is neither a guarantee nor a prerequisite for inhibition by amiloride or its analogues. The, G protein coupled, P-adrenergic, dopamine-D,, 5-HT,, and 5HT,,,and several peptide receptors have Ki values > 10 ph4. In contrast, the verapamil and 5-HT3 receptor (51) are not coupled to G proteins, but are affected by benzamil with K, values of 2.7 and 2.2 pM, respectively. In addition, Velly et al. (52) reported K, values of several 5-amino-substituted
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GARRITSEN ET AL.
TABLE 4. Classification of receptors according to signal transduction mechanism.
GROUP
effect
G protein?
receptors
1: stimulatory receptors adenylate cyclase
activation
Yes
adenosine-A, PI,-adrenoceptor dopamine-D,
2: stimulatory receptors PIP,-PLC
activation
Yes
a,-adrenergic histamine-H, 5-HT,, muscarine-M,, LTD., TRH CCK,
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3: inhibitory receptors
adenylate cyclase PIP,-PLC calcium channel potassium channel
inhibition inhibition inhibition activation
adenosine-A, &-adrenoceptor dopamine-D, 5-HT1,, muscanne-M, p-opiate &opiate K-opiate
4: (ligand gated) ion channels voltage dependent calcium channel
activation ?
no
DHP VER
cation channel
activation
no
5-HT3
chloride channel
activation
n0
facilitation of activation
no
GABA, glycine benzodiazepine
no
5-HTV,
Yes
substance P CCK,
5: others transport protein potassium channel ?
inhibition
Information was obtained from refs 52-55. T h e inhibitory receptors (group 3) are often coupled to multiple mechanisms (18). It is, however, not yet clear which one will be the most important mechanism for each receptor. Therefore all the possibilities are listed.
RECEPTOR BINDING PROFILES OF AMILORIDE ANALOGUES
a99
analogues for inhibition of [3H]batrachotoxinbinding to the voltage-sensitive sodium channel around 0.35 pM. Other receptors with an intrinsic ion channel (group 4: benzodiazepine, GABA,, glycine and DHP receptors) are insensitive to amiloride and its analogues. DISCUSSION
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Specificity of amiloride and its analogues In the present study, a multitude of receptors has been screened for affinity for amiloride and its analogues. The overall view emerging from the results is that amiloride and its analogues are largely non-selective compounds, that interact with many membrane receptors, ion transporters and other membrane proteins. Comparing the values in tab. 3 with each other and with data from the literature must be done with a certain caution. Species, tissue, cell type or Na' concentrations in the assays vary and this may account for differences in the Ki values observed. In the literature no general tendencies have been reported. Thus, the affinity of amiloride for g-adrenoceptors is independent of the NaCl concentration in porcine brain (16), but in kidney membranes NaCi enhances the affinity of amiloride, whereas the effect of NaCl in platelets varies for unknown reasons (12). In contrast, binding to the adenosine-A, receptor is markedly attenuated by NaCl (19). Species differences may occur as evident from the Ki values for the adenosine-A, receptor in rat and calf brain, respectively (tab. 3). Nonetheless, the receptor binding assays presented here are employed in the screening of new drugs and together generate a profile representative for the potency of drugs at different receptors. The rank order of potency of amiloride and its analogues in a particular binding assay is a sound parameter in this study, as this is, obviously, not affected by differences between the binding assays. A few limitations of the comparison between the activity at different receptors and ion transport proteins should also be pointed out. First of all, binding data are compared with a physiological response. In this context, it is important to realize, that the effects on Na+/H+exchange are well correlated
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with the inhibition of binding of tritiated analogues to the antiporter by these substances (636). Such a correlation has not been established for the epithelial sodium channel. [3H]Phenamilbinds to pig kidney membranes and rat brain homogenates but amiloride has a relatively low affinity (4 pM) for this site compared to its affinity for the epithelial sodium channel, and EIPA is more potent than amiloride (757). This suggests that the ['Hlphenamil binding site is not the epithelial sodium channel (2). Accordingly, a binding correlate is not available for the epithelial sodium channel and we could compare the effects on receptor binding only with effects on Na+ transport. Comparing the values for inhibition of receptor binding in tab. 3 with each other and with the data available from the literature, presented in tab. 1, amiloride nor its analogues is selective for the A, receptor, the subject of our previous study (19), although it displays a relatively high affinity for this receptor. Overall, amiloride itself is a slightly selective blocker of the epithelial sodium channel, as is benzamil. Side effects due to the interaction with neurotransmitter receptors will only occur at concentrations 10-20 fold higher than those needed for blockade of Na+ transport through the epithelial sodium channel. HMA is not selective for any of the processes, known to be affected by this compound. Its relatively high potency for the p-opiate receptor suggests that this analogue might be used as a lead compound for a novel type of opiate receptor ligands. The interaction with the L-type Caz+ channel was recently also reported by Garcia et al. (58). The Ki values in our study, however. are much lower.
Are G; coupled receDtors linked to Na+/f4+exchange? It has been hypothesized that a link might exist between Gi coupled receptors and the Na+/H+exchanger (18). Evidence for such a coupling should meet several criteria, some of which can well be addressed by radioligand binding studies. (1) The affinity of amiloride and its analogues should be in the proper concentration range, keeping in mind that the actions of the drugs can be counteracted by Na+ (1-3). (2) The structure-activity profile obtained must be consistent with the generally accepted profile for one of the transport systems. A S A R study should
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include at least one 5-amino-substitutedanalogue and one analogue bearing a substituent on a terminal guanidino nitrogen atom. If we apply these criteria to the results in tab. 3, it can be concluded that (1) the epithelial sodium channel is not involved in any of the amiloride-receptor interactions, as the Ki values of amiloride are too high; (2) at some receptor types both analogues are more potent than amiloride, excluding the involvement of the epithelial sodium channel or Na+W exchange, but resembling the SAR profile of Na+/Ca2+exchange inhibition. The Ki values of HMA for inhibition of muscarinic, adenosine-A,, 5-HT2 and F-opiate receptor binding are, however, 25, 250, 250 and 1600 times lower than for inhibition of Na+/Ca2+ exchange; (3) the involvement of Na+/H+exchange can be rejected in 15 assays, where benzamil is the most active analogue or benzamil and HMA are both more active than amiloride and in another 13 assays where H M A has a Ki value > 10 J.LM, which is not in agreement with its high affinity for the Na+/H' antiporter. For the remaining receptors (5-HT,,, K-opiate and &opiate), Na+ transport experiments and more detailed SAR studies may give a final answer. In these cases, the order of potency, HMA > amiloride or benzamil, is, in our opinion, more likely to be a coincidence than an indication for the involvement of the Na'W exchanger. A direct relation between the signal transduction system coupled to the receptors tested and their sensitivity to amiloride and its analogues is not evident. Receptors linked to identical mechanisms react differently, and vice versa, indicating that not the second messenger system, but a domain of the receptor molecule involved in ligand binding determines its sensitivity to amiloride and its analogues. Interestingly, mainly the receptors for the classical neurotransmitters are affected. Considering the recently demonstrated homology between many receptors molecules ( 5 9 , a conserved amiloride binding region may indeed exist. It is conceivable that amiloride and its analogues interact with the Na+ binding site responsible for the reciprocal regulation of agonist and antagonist binding, a feature common to many G protein coupled receptors (see among others ref. 37). However, in previous studies on the interaction between the adenosine-A, receptor and amiloride and its analogues, we demonstrated that these compounds affect agonist as well as antagonist binding to this receptor
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in identical concentrations. Na' largely reduces the affinity of amiloride, independent of the fact whether an agonist or antagonist radioligand was used (19). Recently, evidence against an interaction of amiloride with the Na' binding site was provided by Horstman et al. (59), based on mutagenesis of the porcine q-adrenergic receptor. They identified Asp79, an amino acid residue in the second transmembrane domain of the porcine q-adrenergic receptor, as the residue responsible for the modulation of radioligand binding to this receptor by Na' ions. Mutation of this residue that is highly conserved among G protein coupled receptors, to Asn abolishes Na+ sensitivity of receptor binding, without changing other binding properties of agonists and antagonists. The mutant form of the q-adrenergic receptor is still sensitive to amiloride and its analogues, suggesting that, at least for this receptor, the effects of Na' and amiloride are mediated by different domains of the receptor. In conclusion, amiloride, HMA and benzamil are nonselective inhibitors of binding to a variety of receptors. H M A is a fairly potent p-opiate receptor and verapamil receptor ligand. The Ki values of amiloride and benzamil for receptors are 10-20 fold higher than their reported affinity for the epithelial sodium channel. The additional effects of these compounds on receptor binding may conmbute to their pharmacological profile. The interaction of amiloride and its analogues with neurotransmitter receptors is clearly not related to the Na' transport-inhibiting properties of the compounds. The wide-spread amiloride-sensitivity found among receptors points to a common smcture in many neurotransmitter receptors and other membrane proteins, at or close to the ligand binding site, which recognizes amiloride and its analogues, thereby excluding the binding of the standard ligands. REFERENCES 1. Benos, D.J. Amiloride: a molecular probe of sodium transport in tissues and cells, Am. J. Physiol. 242, C131-145, 1982. 2. Garty, H.; and Benos, D.J. Characteristics and regulatory mechanisms of the amiloride-blockable Na' channel, Physiol. Rev. 68, 309-373,1988.
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