Inhibition of cardiac sarcolemmal Na lH antiporter by opioids +
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S. M. PERIYASAMY Department of Phamcology, mdical College of Ohio, Toledo, OH 43699, U.5-A. Received November 13, 1991 PERIYASAMY, S. M. 1992. Inhibition of cardiac sarcolemmal Na+/H antiporter by opioids. Can. J. Physiol. Pharrnacol. 761: 1048- 1056. In our routine screening of chemicals that would inhibit cardiac sarcolemmal Na+/H+ antiporter, we discovered that some of the spioids produced inhibition of cardiac sarcolemmal Na+/H+ antiporter in micromolar concentrations. Using U-50,48$H, a selective x-opioid agonist, we characterized the nature of interaction between opioids and the Naf/H+ antiporter. The inhibitory effect of U-50,488H on Na+/H+antiporter was immediate and reversible, and was not mediated through the interaction with the opioid receptors but due to the direct interaction of U-58,488H with the Na+/H9 antiporter. The kinetic data show that in the presence of U-50,488H the Km for Na+ was increased from 2.5 f 8.2 to 5.8 0.3 d, while the V,,, (52.0 f 5.0 nmol . mg-I min-') remained the same. These results suggest that U-50,488H and Na+ csrnPete for the same site on the antiporter. When testing the effect of U-58,488H on other transport systems of cardiac sarcolemma, we found that U-50,488H also inhibited Na'/Ca2+ antipsrter and Na+/K+ pump but at much higher concentrations suggesting that U-50,488H shows some degree of selectivity for cardiac sarcolemmal Na+/H+ antiporter. When we compared the inhibitory potency of U-50,488H with amiloride and its analog, namely 5-(N,N-hexamethylene)amiloride,we found that U-58,488H (HC,, = 100 f 15 pM) was threefold more potent than amilsride (IC,, = 300 f 20 pM) but it was threefold less potent than the amiloride andog (1C50 = 30 & BO pM) in inhibiting cardiac sarcolemmal Na+/H+ antiporter. These results show that although U-58,488H is more potent than arniloride, the inhibitory characteristics of U-50,488H on cardiac sarcolernmal Na+/H+ antiporter are similar to amiloride. Key words: Na+/H+ antiporter, Na+ uptake, cardiac sarcolemmal vesicles, opioid agonists, U-50,488M.
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PEWYAMMY, S. M. 1992. Inhibition of cardiac sarcolemd Na '/H+ antiporter by opioids. Can. 1. Physiol. Pharmcol. 70 : 1048 - 1056. Lsrs de notre dkpistage de routine des produits chimiques qui inhiberaient l'antiport de Na+/H+ dans le sarcolemme cardiaque, nous avons constat6 que certains opioides provoquaient son inhibition en concentrations micromolaires. Nous avons caracterisd la nature de l'interaction entre les opioides et l'antiport Naf /H+en utilisant 1'U-50,488H, un agoniste opisyde-x selectif. L'effet inhibiteur d9U-50,488I-Isur l'antiport Na+/H+ a CtC immCdiat, rdversible et n'a pas Ct6 mdiC par B'interaction avec Bes rCcepteurs opioides, mais a rCsult6 de l'interaction directe entre U-58,488l-I et l'antiport Na+/Hf. Les donnkes cinCtiques montrent qu'en prksence d'U-50,488H, Ia valeur de Km pour le Na+ a CtC augmentee de 2,5 i 0,2 h 5,0 f 0'3 mM, alors que celle de V,,, (52'0 5,0 nmol mgsl . min-I) est demeurCe la mCme. Ces rCsultats sugg5rent que U-50,488M entre en cornpitition avec Na+ pour le mCme site sur l'antiport. Lorsqu'on a examink 19effetde U-50,488M sur d'autres systbmes ce transport dans Be sarcolemme eardiaque, nous avons constate que U-50,488H avait aussi inhibe l'antiport Naf /Ca2+ et la pompe Na+/Kf9mais B des concentrations beaucoup plus ClevCes, suggCrant que U-50,488H montre un certain degrC de s6lectivitC pour l'antiport Na+/H+ dans cette membrane. Lorsque nous avons compare le pouvoir ilfhibiteur de U-58,488M avec I'amiloride et son analogue, 5-(N,N-hexamCthylbm)amiBoride,nous avsns constat6 que U-50,488H (fC,, = 1 0 0 & 15 pM) Ctait d'un facteur 3 plus puissant que l'amiloride (HC,, = 300 9 20 pM), mais qu'il etait d'un facteur 3 moins puissant que I'analogue de l'amiloride = 30 f 10 pM) pour inhiber I'antiport Na+/H+ sarcolemmal cardiaque. Ces rksultats montrent que, bien qu'il soit plus puissant que l'amiloride, les carct6ristiques inhibitrices de U-50,488H sur I'antiport Naf /H+ sarco%emmalcardiaque ssnt similaires B celles de l'amiloride. Mots cb&s : antiport de Na+/H+, capture de Na+ , vCsicules du sarcolem~ecardiaque, agonistes opioides, U-50,488H. [Traduit par la rCdaction]
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Introduction Na+/H+ antiporter, which catdyzes the transmembrane exchange of Na+ for H + , has been shown to be present in the plasma membrane of a variety of cells including cardiac cells (Aronson 1985; Mahnensmith and Aronson 1985a; Seiler et al. 1985; Lazdunaski et al. 198%; Periyasamy et al. 1990). This antiporter is activated by various agonists including vasoactive agents (Nord et al. 1987; LydH et al. 1988; Gesek et a%. 1989; Saecomani et al. 1990) and growth factors (Little et al. 19891, and is inhibited by chemical agents having diverse chemical structure such as amilsride and its analogs (Weyman and Cragse B988), harmdine (Aronson and Bounds 19801, cirnetidine (Ganapathy et al. 1986a), clonidine (Ganapathy et al. 1986&), Ioperamide (Balkovetz et al. 19871, and quinidine (Mahnensmith and Aronson 1985b) . Of these inhibitors,
amiloride is used extensively to characterize the properties sf the Na+/H+ antiporter in the plasma membrane of various cell types. In several studies, it has been shown that the concentrations sf amiloride needed to produce 50% inhibition of Na+/H+ antiporter were in the range of 0.3 - 1.0 rnM (Seiler et al. 1985; Kakn et d . 1986; Escobdes and Canessa 1986). Furthermore, in two studies, concentration sf amiloride as high as 18 mM was used to produce complete inhibition of the Na+/H+ antiporter (Moseley et al. 1986; EaBelle 1986). In other studies, it has been reported that amiloride in concentrations that were used to inhibit Na+/H+ antiporter d s o produced inhibition of other transport systems such as the Naf /K+ pump (Soltoff and Mandel 1983) and the Naf /Ca2+ antiporter (Kaczorowski et d. 1985). To circumvent the problems associated with the use of amiloride, many amiloride
A ~ s w ~ v ~ ~ ~ EGTA, a o n r s :[ethylene-bis-(oxyeklayIenenitrilo)]tetraacetic acid; HEPES, N-(2-hydroxyethyl)-piperazine-N'-~-ethanesulfonic acid]; MES, 2-(N-morpho~ino)ethanesulfonicacid; MGA, N-methylglucamine; MOPS, 3-(N-morpholino)propanesulfonic acid. Printed in C a n d a i HrnprirnC au Canada
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analogs have been synthesized and these analogs have been shown ts be severdfold more potent and selective than amiloride in inhibiting the Naf/H+ antiporter (Kleyman and Cragoe 1988). However, only a few of these amiloride analogs are available c o m e r c i d l y for investigators working in this area. Therefore, our laboratory was involved in searching for chemicals that would selectively inhibit the Naf/H+ antiporter but not other transport systems. In our routine screening of chemicals that would inhibit cardiac sarcolemmal Naf /Hf antiporter, we found that some of the agents interacting with the opioid receptors were able to inhibit cardiac sarcolemmal Naf/H' antiporter; one such compound was U-50,488H, which has been reported as a selective x-opioid agonist (Vonvoigtlander et al. 1983). Further, we carried out a series sf experiments to elucidate the mechanismfs) by which U-50,488H produced inhibition of cardiac sarcslemmal Naf /H4 antiporter and also investigated its effect on other ion transport systems present in the cardiac sarcolemma.
TABLE1. Effects of various opioids on H+gradient-dependent ',Na+ uptake (,%a+ uptake mediated by Na+/H9 antiporter)
Materials and methods
The sarcolemmal vesicles collected at the interfaces of the 0.250.6 M sucrose layers were recovered by aspiration. They were then diluted with 3-4 vol. of ice-cold H 2 0 and the vesicles were sedimentd at 170 000 x g for 30 min. The pellets were suspended in 0.25 M sucrose and 10 mM histidine (pH 7.5) medium and used immediately or stored at -50'C. The purity of the vesicles was determined as described by Jones and Besch (1984). It was found that cardiac sarcolemmal vesicles prepared routinely in our laboratory were contaminated with less than 2 % of sarcoplasmic reticulum and 18% of mitochondria (Periyasamy et al. 1990). The vesicles thus prepared are mixtures of leaky vesicles, sealed inside-out vesicles, and sealed right side out vesicles; however the relative proportions of these vesicles vary according to preparative procedures used. Therefore we determined the relative proportions of these vesicles, as described by Periyasamy et al. (1990), and we found that the sarcolemmal vesicles prepared in our laboratory contained 38.0 f 2.0% leaky vesicles, and 62 f 4.0 sealed vesicles; of the sealed vesicles 94.0 f 4.0 % were right-side out and 6.0 1.B) % were inside out. As over 90% s f the sealed vesicles were right-side out, the data presented here are generated from the interaction of the substrates or inhibitors with the Na+/H+ antiporter facing the ewtracellular side of the membrane. Measurement ~ j - ' ~ N a 'uptak~mediated by Na+/HjF+ antiporter '=Na' uptake by the vesicles was measured by modification of the procedure described by Seiler et al. (1985) as described by Periyasamy et al. (1990). Freshly thawed vesicles were centrifuged at 100 000 x g for 15 min in a B e c h a n Airfuge. The sedimented vesicles were washed once by suspension and centrifugation with a medium containing 100 mM mannitol, 40 mM MES, 20 mM MGA, and 1 mM EGTA (pH 6.0). The vesicles were loaded at 23°C for 2 h with the above medium. An aliquot of the vesicles (20 yL containing 30-50 pg membrane protein) was diluted 10-fold into a 180-pE uptake medium containing 100 mM mannitol. 40 mM glycylglycine, 20 mM MGA, 1.0 mM 22NaCl, and I lnaM EGTA (pH 8.0). '2Na9 uptake was measured at 23'C for 20 s and was stopped by the addition of 3.0 mL of ice-cold stop solution containing 100 mM rnanmitol, 100 mM MgCl,, 8 mM HEPES, and 4 mM THPBS (pH 7.3). The diluted medium was filtered through 0.45-pm Millipore filters (Millipore Corp., Bedford, MA). The filters were washed 2 times with 3.0 mL of ice-cold stop solution. The difference between the 22Na' uptake in the presence (pHi 6, pH,, 8) and in the absence (pHi 6, pH, 6) sf a pH gradient was considered as 22Na' uptake mediated by the Na'/H+ antiporter. pH, and pH, refer to intravesicular and extravesicular pH values, respectively.
Sources of chemicaks Some of the chemicals used in the present study were purchased from the following sources: carrier-free 22NaC1and 15CaC12from New England Nuclear, Boston, MA; amiloride, dynorphin A (1 139, leueine-ertkephalin, methionine-edephalin, naloxone, and loperamide from Sigma Chemical Co., St. Louis, MQ; 5-(N,Nhexamethylene)amiloride from Research Biochemicals Inc., Natick, MA; and U-50,488H {3,4-dichloro-N-methyl-N-[2-(1-pyrro1idinyl)cyclohexyl]-benzene acetamide) from Upjohn Co.. Kalamazoo, MI. The following chemicals were received as gifts: U-62,0665 {3,4dichloro-N-methyl-Wr-[7-(1-pyrrolidiny)- l -oxaspiro-(4,5)dec-$-yl]bemeneacetamide) from Upjohn Co., &damazoo, MI; MI32266 [2-(P-furylmethyl)-5,9-die%azyl-2 '-hydroxy-6,7-benzomorphan]from Bsehringer Ingelheim, Ridgefield, CT; and ketocyclozine from Sterling -Winthrop Research Institute, Rensselaer, NY. All other chemicals used in the present study were of analytical grade. Preparation of cardiac sa~cokcmmlvesicles Cardiac sarcolemmal vesicles were prepared from left ventricular muscle of fresh beef heart by modification of the method of Jones and Besch (1984) as described by Kakar et al. (1987). Briefly, the tissues obtained from left ventricles were homogenized in a medium of 0.75 M NaCl and 10 mM histidine (pH 7.5) for 5 s with a Polytron BT-20 (Brinkmaman Instruments) set at half-maximal speed. The hsrnogenate was centrifuged at 14 000 x g for 20 min. The pellet was resuspended in the above medium and the suspension was h o m g enized and centrifuged as described above. The pellet was again resuspended in the above medium, homogenized and centrifuged for a third time. The pellet obtained from the third centrihgation was resuspended in a medium containing 10 mM NaHCO, and 5 mM histidine (pH 7.5). The suspension was homogenized three times for 30 s with the polytron BT-20 set at half-maximal speed. The homogenate was centrifuged at 14 000 x ,g for 20 min. The supernatant containing partially purified sarcolemmal vesicles was collected and centrifuged for 30 min at 44 000 X g. The resulting pellet (microsomes) was suspended in a medium sf 1 M sucrose, 150 mM NaCl, 50 mM tetrasodium pyrophosphate, and 100 mM Tris (pH 7. 1). The microsomes containing partially purified sarcolemmal vesicles were further purified by sucrose flotation. An aliquot of the membrane suspension (microsomes) was placed in polycarbornate centrifuge tubes and discontinuous sucrose gradients were built on the top of the membrane suspension with 0.6 M sucrose, 3QU mM NaC1, 50 mhd tetrasodium pyrophosphate, and 100 mM Tris (pH 7.1) and 0.25 M sucrose and 10 mM histidine (pH 7.5). The sucrose gradients containing the membranes were centrifuged at 370000 k g for 54 min. During centrifugation, the sarcolemal vesicles in the crude membrane suspension float toward the tops of the sucrose gradients.
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H+ gradient-dependent Opioids
Concentration (PA%)
"Na uptake (7% of control) +
Control Eeu-enkephalin Met-edcephalin Loperamide U-50,488H U-62,066E Ketocyclozine Dynophin A (I NOTE:The data represent the mean of three separate experiments and each experiment was conducted in triplicate. H + gradient-depenkmt 22Na+uptake was 16.5 f 3.0 nmol mg-' of protein . min-'. *p < 0.05, significant difference compared with control. **p < 0.01, significant difference compared with control.
+
Measurement ofZ2Nai uptake mediated by Na'/Ki pwnp uptake by the vesicles was measured as described by Kakar et al. (1987). The vesicles were washed and loaded with a medium
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CAN. J. PHYSIOL. PHARMACOL. VOL. 70. 1992
Time (seconds) FIG. 1. Effect of U-50,488H on the time course of 22Na+uptake in the presence and in the absence of H+ gradient. Vesicles were loaded overnight with acidic buffer (pH 4.0). 22Na+ uptake was measured as described in the Methods at the indicated times with and without 180 f l U-563,88H and in the presence (pHi 6 , pH, 8) and in the absence (pHi 6, pHo 6 ) of a pH gradient. The concentration of "Na+ in the uptake medium was 1- 0 mM.
containing 160 wM KC1 9 20 mM MOPS (pH 7.2). To initiate 22Naf uptake, 20 p L of the KCl-loaded vesicles was added to 200 p E of a reaction medium that contained 5 mM 22NaCl, 140 mM choline chloride, 1 mM MgCl,, 0.2 mM EGTA, 2.0 mM ATB, and 20 mM MOPS (pH 7.2). 22Na+uptake was measured at 23OC for 40 s and was terminated by diluting the reaction medium with 3.0 mL of an ice-cold 160 mM KCI and 2&M MOPS (pH '7.2) solution. The diluted medium was filtered and the filters were washed 2 times with ice-cold KC1-MOP solution. "Na' uptake mediated by the Na+/Kf pump was taken as the difference between the "Na+ uptake in the presence and in the absence of ATB. Measurement of 45C~z2+ uptake mediated by Na"/Cn2+ antiporter Nai-dependent Ca2+ uptake by the vesicles was measured at 223C for 20 s by modification of the procedure described by Vemuri and Philipson (1987). The vesicles were washed and loaded with a buffer containing 140 mM NaCl and 10 mM MOPS (pH 7.4). An aliquot of the NaCl-loaded vesicles (263 pk) was diluted 10-fold into a Na+-free uptake medium containing 140 mM KC1, 10 pM "CaC12, and 10 mM MOPS (pH 7.4). "Ca2+ uptake was stopped by diluting the uptake medium with 3.0 mh, of ice-cold stop solution containing 140 mM KC1 and 1 mM LaCI,; the diluted medium was filtered and the filters were washed 2 times with the stop solution. Simuitaneously, nonspecific Ca2+ uptake and binding were determined by diluting the NaC1-loaded vesicles into an uptake medium containing 143 mM NaCl instead of 140 mM KC1. Ca2+ uptake through the Na+/Ca2+ antiporter was obtained by subtracting the nonspecific Ca2+ uptake from the total Ca2+ uptake. The 22Na+and "Ca2+ accumulated inside the vesicles were determined by counting the filters in a gamma counter and liquid scintillation counter, respectively. The data were expressed as means SE and were statistically ana-
l y z d with the Student's b-test; a p value < 0.05 was considered significant. The data presented in the figures represent the mean of three separate experiments, which varied less than 5 % .
Results E&cts ofopioids OPZ22Na$ uptake mediated by cardiac sarcokentml Wra /El+ antiporter +
It is well established that in membrane vesicles the initial rate of 22Na+ uptake mediated by the Na+/HS antiporter is severdfold greater in the presence of an outward IS+ gradient than the rate of uptake either in the absence of a gradient or in the presence of an inward H + gradient. Therefore, the effects s f several opioids ow the initial rates of 22Na+uptake by the cardiac sarcslemmal vesicles in the presence of an outward-directed H+ gradient (pHi 6, pH, 8) were examined. Vesicles were loaded overnight with acidic buffer (pH 6.0), and the initial rate sf uptake of 22Na+ was measured as described in the Methods for 20 s with and without the indicated concentrations of spioids, and in the presence (pHi 6, pH, 8) and in the absence (pHi 6 , pH, 6) of a pH gradient. The difference between the uptake in the presence and in the absence of a pH gradient was the H+ gradient-dependent uptake. The results are presented in Table 1. As shown in Table 1, of the opioids tested, leucine-enkephalin and methisnine-enkephdin did not produce a significant inhibition, while others produced a moderate to marked inhibition of 22Na+ uptake. It appears from the data of Table 1 that U-50,488H seems to be more potent than other spioids in
TABLE2. Effect of U-50,488H on amiloride-sensitive "Na+ uptake in the absence (pHi 8, pH, 8) and in the presence of H+ gradient (pHi 6, pH, 8) 22Na+uptake (nmol - mg-' of protein - min-l)
Treatment Control (pHi 6, pH, 8) Control + U-50,488H (pHi 6, pH, 8) Control (pH, 8, pH, 8) Control + U-50,488H (pHi 8, pH, 8) Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by McMaster University on 11/14/14 For personal use only.
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NOTE: The data are the mean of three experiments and each experiment was performed in triplicate. See Results for details. **p < 0.01, significant difference compared with "Nat uptake in the absence of U-50,488H.
inhibiting the antiporter, and therefore, U-50,488H was used as a tool to elucidate the mechanism of inhibition s f the Na+/H+ antiporter produced by opioids.
Inhibition of 22hrhl uptake mdiated by cardiac sarcokemmk Na+/H+ antiporter by U-50,488Has a finctisn of time Figure 1 illustrates the 22Na+ uptake mediated by the Na+/H+ antiporter and the inhibition of the antiporter by U-50,488H as a function of time. "Na+ uptake by cardiac s a r c o % e m dvesicles was measured in the presence and absence of H+ gradient. In the absence of H + gradient (pHi 6 , pH, 6) the 22Na+ uptake was slow and increased gradually with time. On the other hand, 22Na+uptake was greatly stimulated in the presence of an outward-directed H + gradient (pHi 6, pH, $), indicating that the uptake was mediated by the antiporter. This H+gradient-dependent 22Na+uptake was significantly inhibited by U-50,48$H, while the inhibition of 22Na+ uptake by U-50,488H in the absence of H+ gradient was very minimal ((Fig. 1). It is possible that U-50,488H might have produced inhibition of the Na+/H+ antiporter by dissipating the pH gradient that existed across the vesicular membrane. To test this possibility, the following experiments were performed. We emmined the effect of U-50,488H on the amiloridesensitive "Na+ uptake in the presence (pHi 6, pH, 8) and in the absence (pHi $, pH,, 8) of a pH gradient. Amiloridesensitive 22Na+uptake was considered as uptake mediated by Na+/H+ antiporter. Vesicles were loaded with 100 mM mannitol, 40 rnM glycylglycine, 20 mM N-methylglucamine, and 1 mM EGFA (pH 8.0). 22Na+uptake was measured with and without arniloride by diluting the loaded vesicles into the uptake medium. that contained the same buffer used for loading the vesicles. This uptake represents the uptake in the absence of pH gradient (pHi 8, pH, 8). In another set of experiments, vesicles were loaded with 100 mM mannitol, 40 mM MES, 28 mM MGA, and I mM EGTA (pH 6.0). 22Na+uptake was measured with and without amiloride by diluting the loaded vesicles into the uptake medium that contained 100 mM mannitol, 40 mM glycylglycine, 20 m.M N-methylglucamine, and 1.O mM EGTA (pH 8.0). This uptake represents the uptake in the presence of pH gradient (pHi 6, pH, 8). The concentrations of Na+ and U-50,488H in the uptake medium were 1.0 mM and 100 pM, respectively. Amiloride-sensitive 22Na+ uptake was the difference between the uptake measured in the presence and in the absence of 2.5 mM amiloride. The data of these experiments are sumarized in Table 2, which shows that U-50,488H at a concentration of 100 pM significantly inhibited the amiloride-sensitive 2Wa+ uptake occurring in the presence as well as in the absence of a pH gradient. These findings indicate that the inhibition of the anti-
TABLE3. Reversibility of inhibition s f H '
gradientdependent "Na+ uptake by U-50,488H -
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Treatment
+
Control Control Control
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22Na"uptake (% of control)
+ 2.5 mM arniloride + 2.5 m M U-50,488H
After three washings
Control Control + 2.5 mM amiloride Control + 2.5 mM U-50,488H After six washings Control Control + 2.5 mM U-50,488H NOTE:The data are the. mean of three separate experiments and each experiment was carried out in triplicate. H9 gradient9 mediated by Na+/H+ dependent 22Na9 uptake ( 2 2 ~ auptake antiporter) in control (without washing) and after three sand six washings were found to be 16.5 f 3.0, 15.9 1 2.5, and 15.5 f 2.8 nmel mg-' of protein . wain-', respectively. **p < 0.01,significant difference compared with control. Tg, < 0.001, significant difference compared with control.
porter produced by U-50,488H was not due to the dissipation of the H + gradient that existed across the vesicular membrane. To examine whether or not the inhibition produced by U-50,488H on the antiporter was reversible, we carried out the experiments shown in Table 3. Vesicles were loaded with the pH 6.0 buffer as described in the Methods. The loaded vesicles were incubated with or without 2.5 mM U-50,488H for 5 min at room temperature. An aliquot of these vesicles was used to determine the uptake. The remaining vesicles were washed with the pH 6.0 buffer (used for loading) by repeated suspension and centrifugation. The washed vesicles were used to measure the uptake. The concentration of %$a+in the uptake medium was 1.0 mM. The H + gradientdependent 22Na+ uptake was calculated by taking the difference between the 22Na+ uptake in the presence and in the absence of H+ gradient. A similar experiment was carried out using amiloride. As shown in Table 3, amiloride (2.5 mM) and U-50,488H (2.5 HnE&I) produced inhibition of 22Na+ uptake by 80 and 85 % , respectively. After three washings, the inhibitory effect of amiloride was completely reversed while the effect of U-50,488H was reversed partially (50% of control). However, after six washings, the antiporter activity was restored almost to its control value (before treatment with U-50,488H). These data indicate that U=-50,488Hinhibited the antiporter reversibly, however, its dissociation from the antiporter was slow compared with amiloride, and also
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CAN. I. PHB'SIOL. PHARM.4GOL. VOL. 70. 1992
FIG. 2 . Double reciprocal plot of the inhibition of the Na+/H+ antiporter by U-50,488H. Vesicles were loaded with the pH 6.8 buffer. 22Na+uptake by the vesicles was measured at various concentrations of NaC1 in the presence (pHi 6, pH, 8) and in the absence of a pH gradient (pH, 4 , pH, 6 ) , and with and without 100 yM U-50,488H. The concentrations of 22Na+in the uptake medium were 0.5, 1.0, 2.5, and 5.0 mM. TABLE4. Effects of naloxone and MR2266 on the inhibition of H9 gradient-dependent "'Na9 uptake by U-50,488H
H9 gradient-dependent 22Na uptake (% 'of control) +
Opioids Control
100
U-50,488H
50f 4**
Naloxone MR 2266
90k5
U-58,488J-I + naloxone U-50,488H + MR 2264
88$6 45f 3**
47f 4""
NOTE: The data are the mean of three separate experiments and each experiment was conducted in triplicate. # + gradient-dependent 2 2 ~ a uptake ' ( 2 2 ~ auptake + mediated by Na+/Ht antiporter) was found to be 18.3 d 2.2 nmol . mg-' of protein min-'. **p < 0.01, significant difference compared with control.
U-50,488H did not produce any physical damage to the vesicles. Taken together, these findings suggest that the inhibition produced by U-50,488H on the Na+/H+ antiporter was not due to a nonspecific effect on cardiac sarcolemma. Mechanism of inhibition of the cardiac sarcokemml Na + / H f antiporter by U-50,488H With U - 5 0 . 4 8 8 ~being an opioid agonist and as cardiac sarcslernma have been shown to possess opioid receptors, it is conceivable that U-50,488H might cause inhibition through interacting with the opioid receptors of the cardiac sarcolemma. To test this possibility we performed the experiments
shown in Table 4. Vesicles were loaded with the pH 6.0 buffer. 22Na+ uptake by the vesicles was measured in the presence (pHi 6 , pH, 8) and in the absence of a pH gradient (pHi 6, pH, 6). The final concentrations of 22Na+, U-50,488H, naloxone, and M E 2 6 4 in the uptake medium were 1.0 mM, 100 pM, 100 pM, and 10 pM, respectively. Table 4 shows that both nalsxsne (a nonselective opioid antagonist) and MR2264 (a selective x-antagonist) produced a sad1 inhibition of the antiporter. As expected, U-50,488H (100 pM) reduced the activity of the antiporter by 50% of the control. However, when the effect of U-50,488H was studied in the presence of naloxone or MR2266, both failed to block the effect of U-50,488H on the antiporter. These data clearly indicate that the opioid receptors were not involved in mediating the effect of U-50,488H on the antiporter. To understand the nature of inhibition of the antiporter by U-50,488H, 22Na+uptake was measured in the presence s f varying concentrations of NaCl and with and without 100 pM U-50,488H. The data are presented in the farm of doublereciprocal plot and are shown in Fig. 2. As shown in Fig. 2, in the presence of U-50,488H, the Km for Na+ was increased from 2.5 f 0.2 to 5.0 f 0.3 mM, but the $/',, was unchanged (52.0 f 5.0 m o l mg-I - rnin-l). These data suggest that U-50,488B has a direct effect on the antiporter where it binds to the cation binding site and thereby it the transport of Na+. Comparison ofthe inhibitory efect of U-50,488Hwith amiloride a ~ l dthe arniloride analog on cardiac sarcokemmk NQ+/H+ antiporter Figure 3 describes the effects sf U-50,48$H, amiloride, and the amiloride analog on the Na+/H+ antiporter. The initial
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FIG. 3. Inhibition of cardiac sarcolemmal Na'/H' antiporter by U-%0,488H,arniloride, and the arniloride amlog. Vesicles were loaded with the pH 6.0 buffer. The loaded vesicles were diluted into the pH 8.0 or pH 6.0 buffer solution containing 1.0 mM 22NaCLand the indicated concentrations of compounds. '2Na+ uptake was measured as described in the Methods. H9 gradient-dependent uptake of "Na+ in the absence of compounds was represented as 100%.
rates of 22Na+ uptake by cardiac sarcolemmal vesicles that were dependent can H' gradient were inhibited by W-50,488H d l o r i d e and its analog in a concentration-depewdent manner. The concentration that produced 50% inhibition of 22Naf uptake was calculated for each of these compounds and was found to be 30 f 18 pM for the arniloride analog, 188 f 15 pM for U-50,48$H, and 300 f 20 pM for amiloride. These results suggest that the rank order of potency in inhibiting the antiporter was amiloride analog > U-50,488I-l > amiloride. Efleca of U-50,488-8, arniioride, and amiloride analog on cardiac Na+/K+ pump aad cardiac Na+/CaS antiporter W-50,48$H, arniloride, and the amiloride analog also inhibited the Na+/K+ pump and Na+/Cas antiporter of cardiac sarcolemrna (Figs. 4 and 5). However, the magnitude of the inhibition of the Na+/K+ pump or the Na+/CGf antiprter produced by these compounds was not the same. As shown in Figs. 4 and 5 the arniloride analog produced a complete inhibition of the Na+/K+ pump as well as the Na+/Ca2+ antiporter. On the other hand, amiloride and U-50,488H produced partial inhibition of the Na+/Kt pump and Na+/Ca2+ antiporter even with a concentration as high as 5.0 mM. The amiloride analog consistently produced an initial stimulation (30 -50%) at lower concentrations ( .S 100 p M ) followed by an inhibition of Na+/Ca2+antiprter at higher concentrations ( > 100 pM) in all experiments (Fig. 5). This phenomenon of initial stimulation was not observed with either amiloride or U-50,488H.
Discussie~n The data presented in this paper demonstrate that some of the opioids inhibited the cardiac sarcolemmal Na+/H+ anti-
porter. The interaction between the opioids and the Na+/Hf antiporter was further characterized using U-50,488H, a selective x-agonist. U-50,488H produced a concentrationdependent inhibition of 22Na+uptake that was dependent on H+ gradient (pHi 6, pH, 81, while it had very little effect on 22Na+uptake in the absence of Hf gradient (pHi 6, pH, 6) (Fig. 1). When 22Na+uptake was measured in the absence of H+ gradient (pHi 8, pH, 8) U-50,488H also caused a significant inhibition of the "Na+ uptake (Table 2). The above findings may appear to be contradictory but they are not. Although there was no H + gradient in both situations (pHi 6, pH, 6 and pHi 8, pH, 8), Na+/H+ antiporter activity was very little when the intravesicular and extravesicular pH was 6 and 6, while its activity was very significant when the intravesicular and extravesicular pH was 8 and 8 (Periyasarny et al. 1990; Seiler et al. 1985). As a result, U-58,488H produced a significant inhibition when the intravesicular and extravesicular pH was 8 and 8 but not when the intravesicular and extravesicular pH was 6 and 6. The inhibition produced by U-58,488H was immediate and reversible. Taken together, these results suggest that the inhibition caused by U-50,488H was not due to nonspecific damage to the vesicles or nonspecific action on the membrane nor due to the dissipation of the Hf gradient that existed across the vesicular membrane. Having ruled out the nonspecific actions as the mechanisms for the inhibition of the antiporter, it was speculated that U-58,488H might cause inhibition either by directly interacting with the antiporter or indirectly via interacting with the opioid receptors present in the cardiac sarcolemrna. There is overwhelming evidence in the literature that compounds that interact with the cell surface receptors influence the activity of the antiporter (Nard et al. 1987; Lydl et d.
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CAN. J. PHYSIOL. PHAWMACOL. VOE. TO, 1992
FIG.4. Inhibition of cardiac sarcolemmal Na+/K+ pump by U-50,4$8H, amiloride, and the amiloride analog. Vesicles were loaded with the 160 rraM KC1 - 20 mM MOPS (pH 7.2) solution. The loaded vesicles were diluted into a medium containing 5 mM 22NaCl, 140 mM choline chloride, B mM MgCI,, 8.2 mM EG'FA, 20 mM MOPS (pH 7.21, 2 mM AT$, and the indicated concentrations of compounds. 2 2 ~ a + uptake mediated by the Na+/Kt pump was taken as the difference between the "Nat uptake in the presence and in the absence of ATP. 22Nai uptake in the absence of compounds was represented as 100%.
FIG. 5. Inhibition sf cardiac sarcolemmal Na'/Ca2+ antiporter by U-50.488H, amiloride, and the arniloride analogs. Vesicles were loaded with the 140 mM NaCl - 10 mM MOPS (pH 7.4) solution, The loaded vesicles were diluted into a Na+-free medium containing 148 mM KC], 10 pM 45CitC12,10 mPvl MOPS @H 7.4), and the indicated concentrations of compounds. C$+ uptake was measured as described in the Methods. Ca2+ uptake mediated by Na+/Ca2+ antiporter was calculated as fhe difference between the Ca2' uptake in the presence and in the absence of Na+ gradient. 45Ca+ uptake in the absence of compounds was represented as 100%.
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1988; Gesek et al. 1989). Further, the presence of opioid receptors in the heart has been suggested by the studies of Caffrey et al. (1985) and kechner et d. (1985) and recently the presence of x-opioid receptors has been demonstrated in cardiac sarcoIemma (Ventura et al. 1989). U-50,488H being a selective x-agonist, was thought to inhibit the cardiac sarcolemmal Na +/H+ antiporter through interaction with the x-opioid receptors of cardiac sarcolemma. Therefore, the possibility of involvement of opioid receptors in the inhibitory action of U-50,488H on the antiporter was investigated. The effect of U-50,488H on cardiac sarcolemmal Na+/H+ antiporter was studied in the presence of naloxone, a nonselective opioid antagonist, and MR2266, a selective x-antagonist. The results of Table 4 show that neither of them blocked the effect of U-50,488H on Na+/H+ antiporter, suggesting that the inhibitory effect was not mediated through the interaction with the x-opioid receptors. On the other hand, the kinetic data of Fig. 2 provide evidence that U-50,488H produced inhibition of the antiporter by directly interacting with the antiporter. The data also show that the Vm,, of 22Na+ uptake was unchanged, whereas the K, for 22Na+ was increased in the presence of U-50,488H. These results suggest that U-50,488H acts at the Na+ binding site and thereby prevents the uptake of 22Na+into sarcslemal vesicles. The competitive nature of interaction between U-50,488H and Na+ on the antiporter was further substantiated by the studies showing that increasing the concentration of Na+ attenuated the ability of U-50,488H to inhibit the vesicular 22Na+ uptake (data not shown). The inhibition of 22Na+ uptake by U-50,488H was also reversible because removal of U-50,488H from the sarcolemmd vesicles by repeated washing restored the 22Na+ uptake almost to the pretreatment level (Table 3). This finding supports the view that the interaction of U-50,488H with the antiporter is via noncovalent binding. Although U-50,488H and amiloride interact with the antiporter through noncovalent binding, the affinity of U-50,488H for the antiporter is greater than the affinity of amiloride for the antiporter. This is evident from the rate of recovery of the antiporter from the inhibitory effects of U-50,488H and amiloride after washings (Table 3). is The of the antiporter produced similar in many respects to the inhibition caused by amiloride, cirnetidine, and clonidine. For instance, similar to U-50,488H, these inhibitors do not damage the membrane vesicles; they do not dissipate the H+ gradient and their inhibitory effect is completely reversible. Further, the inhibition caused by these compounds is competitive with respect to Na+ (Kinsella and Aronson 1981; Ganapathy et al. 1 9 8 6 ~ ;Ganapathy et al. 19866). It has k e n shown that loperamide, an opioid agonist, inhibited the antiporter from brush-border membrane vesicles isolated from human placenta, rabbit renal cortex, and rabbit small intestine (Balkovetz et d. 1987). In the present study, we have shown that loperamide also inhibited the antiporter from cardiac sarcolemmal vesicles prepared from bovine heart. Although we have not explored the mechanism of inhibition of the cardiac sarcolemmal Na+/H+ antiporter by loperamide, Bdkovetz et al. (1987) have shown that opioid receptors were not involved in the inhibition of the antiporter produced by loperamide, and they have demonstrated that loperamide interacts directly with the antiporter. These findings are similar to what we observed with the U-50,488H in the present study. Further, Balkovetz et al. (1987) have reported that the inhibition of the antiporter by loperamide is of mixed type and it interacts with the antiporter at more than one site. On the other hand, we have shown that U-50,488H U-509488H
inhibition is of competitive type and it binds to the antiporter at a single site. These observations indicate that although loperamide and U-50,488H interact with the antiporter directly, they may not share a common binding site on the antiporter. Amiloride, a potassium sparing diuretic, has been used by many investigators to explore the physiological role of the Na+/H+ antiporter in different cells. We, therefore, compared the potency of U-50,488H with the amiloride and amiloride analog, namely 5-(N,N-hexamethylene)amiloride, which has been reported as 500-fold more potent than amiloride in inhibiting the Na+/H+ antiporter from human neutrophils (Simchowitz and Cragse 1986). It is evident from Fig. 3 that the amiloride analog (ICSo = 30 f 10 pM) is three times more potent than U-50,488H (ICSo = 100 & 15 pM) which, in turn, is three times more potent than amiloride (I&jS0 = 300 f 20 p M ) . To h o w whether these compounds have an effect on other transport systems, we tested these compounds on the Na+/K+ pump and the Na+/Ca2+amtiporter of cardiac sarcolemm. Results of our studies show that U-%09488H, amiloride, and the amiloride analog also inhibited the Na+/K+ pump and the Na+/Ca2+ antiporter, but the concentrations needed to inhibit the pump and the Na"/Ca2+ antiporter are much higher (see Figs. 3, 4, and 5), suggesting that these compounds show some degree of selectivity for the Na+/H antiporter. In summary, some opioids, in addition to interacting with the opioid receptors, also produce nonopioid actions as observed in this study as well as in other studies (Maeda et al. 1987; Periyasamy and Hoss 1991; Hayes et al. 1988). It is, therefore, important to use these compounds carefully and judiciously in studying their effects mediated through opioid receptors. Although U-50,488H is more potent than amiloride in inhibiting the antiporter and shows some degree of selectivity for the antiporter, in our view, it is not an ideal compound to probe the role of Na+/H+ antiporter in different cellular processes. +
The author thanks Debra LeBarr for her excellent secretarial assistance, This work was supported by a National Institutes of Health grant H~-36573awarded by the ~~~i~~~~Heart, Lung, and ~ l Institute, ~ ~ United d statesPublic service, Depafilnent of Health and Human Services. Aronson, P. S. 1985. Kinetic properties of the plasma membrane Na+/PI+ exchanger. Annu. Rev. Physiol. 45: 545 - 560. Aronson, P. S . , and Bounds, S. E. 1988. Hamaline inhibition of Na+-dependent transport in renal microvillus membrane vesicles. Am. 9. Physiol. 238: F210-F217. Balkovetz, D. F., Miyamoto, Y.: Timppathi, C . , et a1. 1987. Hnhibition of brush-border membrane Na+-HS exchanger by Ioperarnide. B. Pharrnacol. Exp. Ther. 243: 1 50 - 154. Caffrey, J. E., Gaugl, J. F., and Jones, C. E. 1985. Local endogenous opiate activity in dog myocardium: receptors' blockade with nalsxone. Am. J. Physisl. 243: H382-H388. Escobales, N . , and Canessa, ha. 1986. Amiloride sensitive Naf transport in human red cdls. Evidence for Na4/H+ exchange system. J. Membr. Biol. 90: 21-28. Ganapathy, M. E., Eeibach, F. PI., Mahesh, V. B., et al. 1 9 8 6 ~ . Interaction of clonidine with human placental Na+-H+ exchanger. Biochern. Phrmacol. 35: 3989-399-4. Ganapathy, V., Balkovetz, D. F., Miyamoto, Y., et &el.1986b. Inhibition of human placental Na+-H+ exchanger by cirnetidine. J. Pharmacol. Exp. Ther. 239: 192- 197.
CAN. J. PHYSHOL. PHARMACOL. V 8 L . 70, 1992
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Gesek, F. A., Cragoe, E. J., Jr., and Strandhoy, J. W. 1989. Synergistic alpha,- and alpha,-adrenergic stimulation of rat proximal nephrsn Nai/H+ exchange. J. Pharmacol. Exp. Ther. 249: 694 700. Hayes, A. G., Birch, P. P., and Cavicchini, E. 1988. Evidence that the kappa-agonist U-50,488H has non-opioid actions. J. Pharm. Pharrnacol. 40: 718 -728. Jones, L. R.,and Besch, M. R., Jr. 1984. Isolation of canine cardiac sarcolemml vesicles. Methods Pharmacol. 5: 1 - 12. Kaczorowski, G.J., Bavors, F., Dethmers, J. K., eta!. 1985. Inhibition of Na+/Ca2+ exchange in pituitary p l a s m vesicles by analogs of amiloride. Biochemistry, 24: 1394 - 1403. Kahn, A. M.: Shelat, H., and Allen, J. C . 1986. Na+-H+ exchange is present in sarcolemmal vesicles from dog superior mesenteric artery. Am. b. Physiol. 250: H3 13-H3 19. Kakar, S. S., Huang, W. H., and Askari, A. 1987. Control of cardiac sodium pump by long-chain acyl coenzymes A. J. Biol. Chem. 262: 42-45. Kinsella, J. L., and Aronson, P. S . 1981. Amiloride inhibition of the Na -M exchanger in renal microvillus membrane vesicles. Am. J. Physiol. 241: F374 -F379. Kleyman, T. W., and Cragoe, E. J., Jr. 1988. Arniloride and its analog as tools in the study of ion transport. J. Membr. Biol. 105: 1-21. LaBelle, E. H. 1986. Amiloride-inhibited Na+ -H+ exchange in human kidney medulla microsomes. Am. J. Physiol. 251: F232F237. Lazdunski, M., Frelin, C., and Vigne, P. 1985. The sodium1 hydrogen exchange system in cardiac cells: its biochemical and pharmacological properties and its role in regulating internal concentrations of sodium and internal pH. J. Mol. Cell. Cardiol. 17: 1029- 1042. Eechner, W. B., Gurll, N. b,, and Reynolds, D.6. 1985. Intracoronary naloxone in hemorrhagic shock: dose-dependent stereospecific effects. Am. J. Physiol. 249: H272 -H277. Little, P. J., Weissberg, P. E., Cragoe, E. J., Jr., and Bobik, A. 1987. Sodium -hydrogen exchange in vascular smooth muscle: regulation by growth factors. %.Hypertens. S(Suppl. 5): S277 S279. Lyall, F., Morton, J. J., Lever, A. F., and Cragoe, E. J, 1988. Angiotensin I% activates Na+-H+ exchange and stimulates growth in cultured vascular smooth muscle cells. J. Hypertens, 6(Suppl. 4): S438 -S442. +
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Maeda, S . , Nakamae, J., and Inoki, R. 1987. Inhibition of cardiac Na+,K+-ATPase activity by dynorphin-A and ethylketocyclazseine. Life Sci. 42: 461 -468. Mahnensrnith, R. L., and Aronson, P. S. 1985a. The plasma membrane sodium-hydrogen exchanger and its role in physiological and pathophysiological processes. Circ. Res. 57: 773 - 788. Mahnensmith, R. L., and Aronson, P. S . 1985b. Interrelationships among quinidine, amiloride, and lithium as inhibitors of the renal Na+-PS+ exchanger. 9. Biol. Chem. 260: 12 586 - 12 592. Moseley, R. H.,Meier, P. J , , Aronson, P. S., and Boyer, J. L. 1986. Na- H exchange in rat liver basolateral but not camlicular plasma membrane vesicles. Am. J. Physiol. 250: G35 -G43. Nord, E. P., Howard, M. J., Hafezi. A., ef a!. 1987. Alpha,adrenergic agonists stimulate Na+/Hi antiport activity in the rabbit renal proximal tubule. J. Clin. Invest. 80: 1755- 1742. Periyasamy, S., and Hoss, W. 1991. Inhibition of carbachol stirnulated PI turnover by U-50,488H in rat hippocampus - involvement of GTP-binding protein. Eur. J . Pharmacol. 207: 10l - 189. Periyasamy, S. M., Kakar, %. S . , Garlid, K. D., and Askari, A. 1990. Ion specificity of cardiac sarcolemmal Na+/H+ antiporter. J. Bi01. Chem. 265: 6035-6041. Saccsmani, G., Mitchell, K. D., and Navar, G. 2990. Angistensin I1 stimulation of Na+-H+ exchange in proximal tubule cells. Am. 9. Physiol. 258: F1188-FZ195. Seiler, S. M., Cragoe, E. J., Jr., and Jones, E. R. 1985. Demonstration of a Na+/Wt exchange activity in purified canine cardiac sarcolemrnal vesicles. J. Biol. Chem. 260: 4869 -4876. Soltoff, S. P., and Mandel, k.J. 1983. Amiloride directly inhibits the (Na+,K+)ATPase activity of rabbit kidney proximal tubules. Science (Washington, DC), 220: 957 - 959. Vernuri, R., and Philipson, K. B. 1987. Phospholipid composition modulates the Na+-Ca2+ exchange activity of cardiac sarcolemma in reconstituted vesicles. Biochim. Biophys. Acta, 937: 258 -268. Ventura, C.,Bastagli, L., Bernardi, P., ef RC. 1989. Opioid receptors in rat cardiac sarcolemma: effect of phenylephrine and isoproterenol. Biochim. Biophys. Acta, 987: 69 -74. Vonvoightlander, P. F., h h t i , R. A., and Eudens, J. H. 1983. U-58,488H: A selective and stmcturally novel nsn-mu (kappa) opioid agonist. J. Pharmacol. Exp. Ther. 224: 7 - 12.
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S. M. PERIYASAMY Department of Phamcology, mdical College of Ohio, Toledo, OH 43699, U.5-A. Received November 13, 1991 PERIYASAMY, S. M. 1992. Inhibition of cardiac sarcolemmal Na+/H antiporter by opioids. Can. J. Physiol. Pharrnacol. 761: 1048- 1056. In our routine screening of chemicals that would inhibit cardiac sarcolemmal Na+/H+ antiporter, we discovered that some of the spioids produced inhibition of cardiac sarcolemmal Na+/H+ antiporter in micromolar concentrations. Using U-50,48$H, a selective x-opioid agonist, we characterized the nature of interaction between opioids and the Naf/H+ antiporter. The inhibitory effect of U-50,488H on Na+/H+antiporter was immediate and reversible, and was not mediated through the interaction with the opioid receptors but due to the direct interaction of U-58,488H with the Na+/H9 antiporter. The kinetic data show that in the presence of U-50,488H the Km for Na+ was increased from 2.5 f 8.2 to 5.8 0.3 d, while the V,,, (52.0 f 5.0 nmol . mg-I min-') remained the same. These results suggest that U-50,488H and Na+ csrnPete for the same site on the antiporter. When testing the effect of U-58,488H on other transport systems of cardiac sarcolemma, we found that U-50,488H also inhibited Na'/Ca2+ antipsrter and Na+/K+ pump but at much higher concentrations suggesting that U-50,488H shows some degree of selectivity for cardiac sarcolemmal Na+/H+ antiporter. When we compared the inhibitory potency of U-50,488H with amiloride and its analog, namely 5-(N,N-hexamethylene)amiloride,we found that U-58,488H (HC,, = 100 f 15 pM) was threefold more potent than amilsride (IC,, = 300 f 20 pM) but it was threefold less potent than the amiloride andog (1C50 = 30 & BO pM) in inhibiting cardiac sarcolemmal Na+/H+ antiporter. These results show that although U-58,488H is more potent than arniloride, the inhibitory characteristics of U-50,488H on cardiac sarcolernmal Na+/H+ antiporter are similar to amiloride. Key words: Na+/H+ antiporter, Na+ uptake, cardiac sarcolemmal vesicles, opioid agonists, U-50,488M.
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PEWYAMMY, S. M. 1992. Inhibition of cardiac sarcolemd Na '/H+ antiporter by opioids. Can. 1. Physiol. Pharmcol. 70 : 1048 - 1056. Lsrs de notre dkpistage de routine des produits chimiques qui inhiberaient l'antiport de Na+/H+ dans le sarcolemme cardiaque, nous avons constat6 que certains opioides provoquaient son inhibition en concentrations micromolaires. Nous avons caracterisd la nature de l'interaction entre les opioides et l'antiport Naf /H+en utilisant 1'U-50,488H, un agoniste opisyde-x selectif. L'effet inhibiteur d9U-50,488I-Isur l'antiport Na+/H+ a CtC immCdiat, rdversible et n'a pas Ct6 mdiC par B'interaction avec Bes rCcepteurs opioides, mais a rCsult6 de l'interaction directe entre U-58,488l-I et l'antiport Na+/Hf. Les donnkes cinCtiques montrent qu'en prksence d'U-50,488H, Ia valeur de Km pour le Na+ a CtC augmentee de 2,5 i 0,2 h 5,0 f 0'3 mM, alors que celle de V,,, (52'0 5,0 nmol mgsl . min-I) est demeurCe la mCme. Ces rCsultats sugg5rent que U-50,488M entre en cornpitition avec Na+ pour le mCme site sur l'antiport. Lorsqu'on a examink 19effetde U-50,488M sur d'autres systbmes ce transport dans Be sarcolemme eardiaque, nous avons constate que U-50,488H avait aussi inhibe l'antiport Naf /Ca2+ et la pompe Na+/Kf9mais B des concentrations beaucoup plus ClevCes, suggCrant que U-50,488H montre un certain degrC de s6lectivitC pour l'antiport Na+/H+ dans cette membrane. Lorsque nous avons compare le pouvoir ilfhibiteur de U-58,488M avec I'amiloride et son analogue, 5-(N,N-hexamCthylbm)amiBoride,nous avsns constat6 que U-50,488H (fC,, = 1 0 0 & 15 pM) Ctait d'un facteur 3 plus puissant que l'amiloride (HC,, = 300 9 20 pM), mais qu'il etait d'un facteur 3 moins puissant que I'analogue de l'amiloride = 30 f 10 pM) pour inhiber I'antiport Na+/H+ sarcolemmal cardiaque. Ces rksultats montrent que, bien qu'il soit plus puissant que l'amiloride, les carct6ristiques inhibitrices de U-50,488H sur I'antiport Naf /H+ sarco%emmalcardiaque ssnt similaires B celles de l'amiloride. Mots cb&s : antiport de Na+/H+, capture de Na+ , vCsicules du sarcolem~ecardiaque, agonistes opioides, U-50,488H. [Traduit par la rCdaction]
+
Introduction Na+/H+ antiporter, which catdyzes the transmembrane exchange of Na+ for H + , has been shown to be present in the plasma membrane of a variety of cells including cardiac cells (Aronson 1985; Mahnensmith and Aronson 1985a; Seiler et al. 1985; Lazdunaski et al. 198%; Periyasamy et al. 1990). This antiporter is activated by various agonists including vasoactive agents (Nord et al. 1987; LydH et al. 1988; Gesek et a%. 1989; Saecomani et al. 1990) and growth factors (Little et al. 19891, and is inhibited by chemical agents having diverse chemical structure such as amilsride and its analogs (Weyman and Cragse B988), harmdine (Aronson and Bounds 19801, cirnetidine (Ganapathy et al. 1986a), clonidine (Ganapathy et al. 1986&), Ioperamide (Balkovetz et al. 19871, and quinidine (Mahnensmith and Aronson 1985b) . Of these inhibitors,
amiloride is used extensively to characterize the properties sf the Na+/H+ antiporter in the plasma membrane of various cell types. In several studies, it has been shown that the concentrations sf amiloride needed to produce 50% inhibition of Na+/H+ antiporter were in the range of 0.3 - 1.0 rnM (Seiler et al. 1985; Kakn et d . 1986; Escobdes and Canessa 1986). Furthermore, in two studies, concentration sf amiloride as high as 18 mM was used to produce complete inhibition of the Na+/H+ antiporter (Moseley et al. 1986; EaBelle 1986). In other studies, it has been reported that amiloride in concentrations that were used to inhibit Na+/H+ antiporter d s o produced inhibition of other transport systems such as the Naf /K+ pump (Soltoff and Mandel 1983) and the Naf /Ca2+ antiporter (Kaczorowski et d. 1985). To circumvent the problems associated with the use of amiloride, many amiloride
A ~ s w ~ v ~ ~ ~ EGTA, a o n r s :[ethylene-bis-(oxyeklayIenenitrilo)]tetraacetic acid; HEPES, N-(2-hydroxyethyl)-piperazine-N'-~-ethanesulfonic acid]; MES, 2-(N-morpho~ino)ethanesulfonicacid; MGA, N-methylglucamine; MOPS, 3-(N-morpholino)propanesulfonic acid. Printed in C a n d a i HrnprirnC au Canada
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analogs have been synthesized and these analogs have been shown ts be severdfold more potent and selective than amiloride in inhibiting the Naf/H+ antiporter (Kleyman and Cragoe 1988). However, only a few of these amiloride analogs are available c o m e r c i d l y for investigators working in this area. Therefore, our laboratory was involved in searching for chemicals that would selectively inhibit the Naf/H+ antiporter but not other transport systems. In our routine screening of chemicals that would inhibit cardiac sarcolemmal Naf /Hf antiporter, we found that some of the agents interacting with the opioid receptors were able to inhibit cardiac sarcolemmal Naf/H' antiporter; one such compound was U-50,488H, which has been reported as a selective x-opioid agonist (Vonvoigtlander et al. 1983). Further, we carried out a series sf experiments to elucidate the mechanismfs) by which U-50,488H produced inhibition of cardiac sarcslemmal Naf /H4 antiporter and also investigated its effect on other ion transport systems present in the cardiac sarcolemma.
TABLE1. Effects of various opioids on H+gradient-dependent ',Na+ uptake (,%a+ uptake mediated by Na+/H9 antiporter)
Materials and methods
The sarcolemmal vesicles collected at the interfaces of the 0.250.6 M sucrose layers were recovered by aspiration. They were then diluted with 3-4 vol. of ice-cold H 2 0 and the vesicles were sedimentd at 170 000 x g for 30 min. The pellets were suspended in 0.25 M sucrose and 10 mM histidine (pH 7.5) medium and used immediately or stored at -50'C. The purity of the vesicles was determined as described by Jones and Besch (1984). It was found that cardiac sarcolemmal vesicles prepared routinely in our laboratory were contaminated with less than 2 % of sarcoplasmic reticulum and 18% of mitochondria (Periyasamy et al. 1990). The vesicles thus prepared are mixtures of leaky vesicles, sealed inside-out vesicles, and sealed right side out vesicles; however the relative proportions of these vesicles vary according to preparative procedures used. Therefore we determined the relative proportions of these vesicles, as described by Periyasamy et al. (1990), and we found that the sarcolemmal vesicles prepared in our laboratory contained 38.0 f 2.0% leaky vesicles, and 62 f 4.0 sealed vesicles; of the sealed vesicles 94.0 f 4.0 % were right-side out and 6.0 1.B) % were inside out. As over 90% s f the sealed vesicles were right-side out, the data presented here are generated from the interaction of the substrates or inhibitors with the Na+/H+ antiporter facing the ewtracellular side of the membrane. Measurement ~ j - ' ~ N a 'uptak~mediated by Na+/HjF+ antiporter '=Na' uptake by the vesicles was measured by modification of the procedure described by Seiler et al. (1985) as described by Periyasamy et al. (1990). Freshly thawed vesicles were centrifuged at 100 000 x g for 15 min in a B e c h a n Airfuge. The sedimented vesicles were washed once by suspension and centrifugation with a medium containing 100 mM mannitol, 40 mM MES, 20 mM MGA, and 1 mM EGTA (pH 6.0). The vesicles were loaded at 23°C for 2 h with the above medium. An aliquot of the vesicles (20 yL containing 30-50 pg membrane protein) was diluted 10-fold into a 180-pE uptake medium containing 100 mM mannitol. 40 mM glycylglycine, 20 mM MGA, 1.0 mM 22NaCl, and I lnaM EGTA (pH 8.0). '2Na9 uptake was measured at 23'C for 20 s and was stopped by the addition of 3.0 mL of ice-cold stop solution containing 100 mM rnanmitol, 100 mM MgCl,, 8 mM HEPES, and 4 mM THPBS (pH 7.3). The diluted medium was filtered through 0.45-pm Millipore filters (Millipore Corp., Bedford, MA). The filters were washed 2 times with 3.0 mL of ice-cold stop solution. The difference between the 22Na' uptake in the presence (pHi 6, pH,, 8) and in the absence (pHi 6, pH, 6) sf a pH gradient was considered as 22Na' uptake mediated by the Na'/H+ antiporter. pH, and pH, refer to intravesicular and extravesicular pH values, respectively.
Sources of chemicaks Some of the chemicals used in the present study were purchased from the following sources: carrier-free 22NaC1and 15CaC12from New England Nuclear, Boston, MA; amiloride, dynorphin A (1 139, leueine-ertkephalin, methionine-edephalin, naloxone, and loperamide from Sigma Chemical Co., St. Louis, MQ; 5-(N,Nhexamethylene)amiloride from Research Biochemicals Inc., Natick, MA; and U-50,488H {3,4-dichloro-N-methyl-N-[2-(1-pyrro1idinyl)cyclohexyl]-benzene acetamide) from Upjohn Co.. Kalamazoo, MI. The following chemicals were received as gifts: U-62,0665 {3,4dichloro-N-methyl-Wr-[7-(1-pyrrolidiny)- l -oxaspiro-(4,5)dec-$-yl]bemeneacetamide) from Upjohn Co., &damazoo, MI; MI32266 [2-(P-furylmethyl)-5,9-die%azyl-2 '-hydroxy-6,7-benzomorphan]from Bsehringer Ingelheim, Ridgefield, CT; and ketocyclozine from Sterling -Winthrop Research Institute, Rensselaer, NY. All other chemicals used in the present study were of analytical grade. Preparation of cardiac sa~cokcmmlvesicles Cardiac sarcolemmal vesicles were prepared from left ventricular muscle of fresh beef heart by modification of the method of Jones and Besch (1984) as described by Kakar et al. (1987). Briefly, the tissues obtained from left ventricles were homogenized in a medium of 0.75 M NaCl and 10 mM histidine (pH 7.5) for 5 s with a Polytron BT-20 (Brinkmaman Instruments) set at half-maximal speed. The hsrnogenate was centrifuged at 14 000 x g for 20 min. The pellet was resuspended in the above medium and the suspension was h o m g enized and centrifuged as described above. The pellet was again resuspended in the above medium, homogenized and centrifuged for a third time. The pellet obtained from the third centrihgation was resuspended in a medium containing 10 mM NaHCO, and 5 mM histidine (pH 7.5). The suspension was homogenized three times for 30 s with the polytron BT-20 set at half-maximal speed. The homogenate was centrifuged at 14 000 x ,g for 20 min. The supernatant containing partially purified sarcolemmal vesicles was collected and centrifuged for 30 min at 44 000 X g. The resulting pellet (microsomes) was suspended in a medium sf 1 M sucrose, 150 mM NaCl, 50 mM tetrasodium pyrophosphate, and 100 mM Tris (pH 7. 1). The microsomes containing partially purified sarcolemmal vesicles were further purified by sucrose flotation. An aliquot of the membrane suspension (microsomes) was placed in polycarbornate centrifuge tubes and discontinuous sucrose gradients were built on the top of the membrane suspension with 0.6 M sucrose, 3QU mM NaC1, 50 mhd tetrasodium pyrophosphate, and 100 mM Tris (pH 7.1) and 0.25 M sucrose and 10 mM histidine (pH 7.5). The sucrose gradients containing the membranes were centrifuged at 370000 k g for 54 min. During centrifugation, the sarcolemal vesicles in the crude membrane suspension float toward the tops of the sucrose gradients.
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H+ gradient-dependent Opioids
Concentration (PA%)
"Na uptake (7% of control) +
Control Eeu-enkephalin Met-edcephalin Loperamide U-50,488H U-62,066E Ketocyclozine Dynophin A (I NOTE:The data represent the mean of three separate experiments and each experiment was conducted in triplicate. H + gradient-depenkmt 22Na+uptake was 16.5 f 3.0 nmol mg-' of protein . min-'. *p < 0.05, significant difference compared with control. **p < 0.01, significant difference compared with control.
+
Measurement ofZ2Nai uptake mediated by Na'/Ki pwnp uptake by the vesicles was measured as described by Kakar et al. (1987). The vesicles were washed and loaded with a medium
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CAN. J. PHYSIOL. PHARMACOL. VOL. 70. 1992
Time (seconds) FIG. 1. Effect of U-50,488H on the time course of 22Na+uptake in the presence and in the absence of H+ gradient. Vesicles were loaded overnight with acidic buffer (pH 4.0). 22Na+ uptake was measured as described in the Methods at the indicated times with and without 180 f l U-563,88H and in the presence (pHi 6 , pH, 8) and in the absence (pHi 6, pHo 6 ) of a pH gradient. The concentration of "Na+ in the uptake medium was 1- 0 mM.
containing 160 wM KC1 9 20 mM MOPS (pH 7.2). To initiate 22Naf uptake, 20 p L of the KCl-loaded vesicles was added to 200 p E of a reaction medium that contained 5 mM 22NaCl, 140 mM choline chloride, 1 mM MgCl,, 0.2 mM EGTA, 2.0 mM ATB, and 20 mM MOPS (pH 7.2). 22Na+uptake was measured at 23OC for 40 s and was terminated by diluting the reaction medium with 3.0 mL of an ice-cold 160 mM KCI and 2&M MOPS (pH '7.2) solution. The diluted medium was filtered and the filters were washed 2 times with ice-cold KC1-MOP solution. "Na' uptake mediated by the Na+/Kf pump was taken as the difference between the "Na+ uptake in the presence and in the absence of ATB. Measurement of 45C~z2+ uptake mediated by Na"/Cn2+ antiporter Nai-dependent Ca2+ uptake by the vesicles was measured at 223C for 20 s by modification of the procedure described by Vemuri and Philipson (1987). The vesicles were washed and loaded with a buffer containing 140 mM NaCl and 10 mM MOPS (pH 7.4). An aliquot of the NaCl-loaded vesicles (263 pk) was diluted 10-fold into a Na+-free uptake medium containing 140 mM KC1, 10 pM "CaC12, and 10 mM MOPS (pH 7.4). "Ca2+ uptake was stopped by diluting the uptake medium with 3.0 mh, of ice-cold stop solution containing 140 mM KC1 and 1 mM LaCI,; the diluted medium was filtered and the filters were washed 2 times with the stop solution. Simuitaneously, nonspecific Ca2+ uptake and binding were determined by diluting the NaC1-loaded vesicles into an uptake medium containing 143 mM NaCl instead of 140 mM KC1. Ca2+ uptake through the Na+/Ca2+ antiporter was obtained by subtracting the nonspecific Ca2+ uptake from the total Ca2+ uptake. The 22Na+and "Ca2+ accumulated inside the vesicles were determined by counting the filters in a gamma counter and liquid scintillation counter, respectively. The data were expressed as means SE and were statistically ana-
l y z d with the Student's b-test; a p value < 0.05 was considered significant. The data presented in the figures represent the mean of three separate experiments, which varied less than 5 % .
Results E&cts ofopioids OPZ22Na$ uptake mediated by cardiac sarcokentml Wra /El+ antiporter +
It is well established that in membrane vesicles the initial rate of 22Na+ uptake mediated by the Na+/HS antiporter is severdfold greater in the presence of an outward IS+ gradient than the rate of uptake either in the absence of a gradient or in the presence of an inward H + gradient. Therefore, the effects s f several opioids ow the initial rates of 22Na+uptake by the cardiac sarcslemmal vesicles in the presence of an outward-directed H+ gradient (pHi 6, pH, 8) were examined. Vesicles were loaded overnight with acidic buffer (pH 6.0), and the initial rate sf uptake of 22Na+ was measured as described in the Methods for 20 s with and without the indicated concentrations of spioids, and in the presence (pHi 6, pH, 8) and in the absence (pHi 6 , pH, 6) of a pH gradient. The difference between the uptake in the presence and in the absence of a pH gradient was the H+ gradient-dependent uptake. The results are presented in Table 1. As shown in Table 1, of the opioids tested, leucine-enkephalin and methisnine-enkephdin did not produce a significant inhibition, while others produced a moderate to marked inhibition of 22Na+ uptake. It appears from the data of Table 1 that U-50,488H seems to be more potent than other spioids in
TABLE2. Effect of U-50,488H on amiloride-sensitive "Na+ uptake in the absence (pHi 8, pH, 8) and in the presence of H+ gradient (pHi 6, pH, 8) 22Na+uptake (nmol - mg-' of protein - min-l)
Treatment Control (pHi 6, pH, 8) Control + U-50,488H (pHi 6, pH, 8) Control (pH, 8, pH, 8) Control + U-50,488H (pHi 8, pH, 8) Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by McMaster University on 11/14/14 For personal use only.
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NOTE: The data are the mean of three experiments and each experiment was performed in triplicate. See Results for details. **p < 0.01, significant difference compared with "Nat uptake in the absence of U-50,488H.
inhibiting the antiporter, and therefore, U-50,488H was used as a tool to elucidate the mechanism of inhibition s f the Na+/H+ antiporter produced by opioids.
Inhibition of 22hrhl uptake mdiated by cardiac sarcokemmk Na+/H+ antiporter by U-50,488Has a finctisn of time Figure 1 illustrates the 22Na+ uptake mediated by the Na+/H+ antiporter and the inhibition of the antiporter by U-50,488H as a function of time. "Na+ uptake by cardiac s a r c o % e m dvesicles was measured in the presence and absence of H+ gradient. In the absence of H + gradient (pHi 6 , pH, 6) the 22Na+ uptake was slow and increased gradually with time. On the other hand, 22Na+uptake was greatly stimulated in the presence of an outward-directed H + gradient (pHi 6, pH, $), indicating that the uptake was mediated by the antiporter. This H+gradient-dependent 22Na+uptake was significantly inhibited by U-50,48$H, while the inhibition of 22Na+ uptake by U-50,488H in the absence of H+ gradient was very minimal ((Fig. 1). It is possible that U-50,488H might have produced inhibition of the Na+/H+ antiporter by dissipating the pH gradient that existed across the vesicular membrane. To test this possibility, the following experiments were performed. We emmined the effect of U-50,488H on the amiloridesensitive "Na+ uptake in the presence (pHi 6, pH, 8) and in the absence (pHi $, pH,, 8) of a pH gradient. Amiloridesensitive 22Na+uptake was considered as uptake mediated by Na+/H+ antiporter. Vesicles were loaded with 100 mM mannitol, 40 rnM glycylglycine, 20 mM N-methylglucamine, and 1 mM EGFA (pH 8.0). 22Na+uptake was measured with and without arniloride by diluting the loaded vesicles into the uptake medium. that contained the same buffer used for loading the vesicles. This uptake represents the uptake in the absence of pH gradient (pHi 8, pH, 8). In another set of experiments, vesicles were loaded with 100 mM mannitol, 40 mM MES, 28 mM MGA, and I mM EGTA (pH 6.0). 22Na+uptake was measured with and without amiloride by diluting the loaded vesicles into the uptake medium that contained 100 mM mannitol, 40 mM glycylglycine, 20 m.M N-methylglucamine, and 1.O mM EGTA (pH 8.0). This uptake represents the uptake in the presence of pH gradient (pHi 6, pH, 8). The concentrations of Na+ and U-50,488H in the uptake medium were 1.0 mM and 100 pM, respectively. Amiloride-sensitive 22Na+ uptake was the difference between the uptake measured in the presence and in the absence of 2.5 mM amiloride. The data of these experiments are sumarized in Table 2, which shows that U-50,488H at a concentration of 100 pM significantly inhibited the amiloride-sensitive 2Wa+ uptake occurring in the presence as well as in the absence of a pH gradient. These findings indicate that the inhibition of the anti-
TABLE3. Reversibility of inhibition s f H '
gradientdependent "Na+ uptake by U-50,488H -
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Treatment
+
Control Control Control
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22Na"uptake (% of control)
+ 2.5 mM arniloride + 2.5 m M U-50,488H
After three washings
Control Control + 2.5 mM amiloride Control + 2.5 mM U-50,488H After six washings Control Control + 2.5 mM U-50,488H NOTE:The data are the. mean of three separate experiments and each experiment was carried out in triplicate. H9 gradient9 mediated by Na+/H+ dependent 22Na9 uptake ( 2 2 ~ auptake antiporter) in control (without washing) and after three sand six washings were found to be 16.5 f 3.0, 15.9 1 2.5, and 15.5 f 2.8 nmel mg-' of protein . wain-', respectively. **p < 0.01,significant difference compared with control. Tg, < 0.001, significant difference compared with control.
porter produced by U-50,488H was not due to the dissipation of the H + gradient that existed across the vesicular membrane. To examine whether or not the inhibition produced by U-50,488H on the antiporter was reversible, we carried out the experiments shown in Table 3. Vesicles were loaded with the pH 6.0 buffer as described in the Methods. The loaded vesicles were incubated with or without 2.5 mM U-50,488H for 5 min at room temperature. An aliquot of these vesicles was used to determine the uptake. The remaining vesicles were washed with the pH 6.0 buffer (used for loading) by repeated suspension and centrifugation. The washed vesicles were used to measure the uptake. The concentration of %$a+in the uptake medium was 1.0 mM. The H + gradientdependent 22Na+ uptake was calculated by taking the difference between the 22Na+ uptake in the presence and in the absence of H+ gradient. A similar experiment was carried out using amiloride. As shown in Table 3, amiloride (2.5 mM) and U-50,488H (2.5 HnE&I) produced inhibition of 22Na+ uptake by 80 and 85 % , respectively. After three washings, the inhibitory effect of amiloride was completely reversed while the effect of U-50,488H was reversed partially (50% of control). However, after six washings, the antiporter activity was restored almost to its control value (before treatment with U-50,488H). These data indicate that U=-50,488Hinhibited the antiporter reversibly, however, its dissociation from the antiporter was slow compared with amiloride, and also
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CAN. I. PHB'SIOL. PHARM.4GOL. VOL. 70. 1992
FIG. 2 . Double reciprocal plot of the inhibition of the Na+/H+ antiporter by U-50,488H. Vesicles were loaded with the pH 6.8 buffer. 22Na+uptake by the vesicles was measured at various concentrations of NaC1 in the presence (pHi 6, pH, 8) and in the absence of a pH gradient (pH, 4 , pH, 6 ) , and with and without 100 yM U-50,488H. The concentrations of 22Na+in the uptake medium were 0.5, 1.0, 2.5, and 5.0 mM. TABLE4. Effects of naloxone and MR2266 on the inhibition of H9 gradient-dependent "'Na9 uptake by U-50,488H
H9 gradient-dependent 22Na uptake (% 'of control) +
Opioids Control
100
U-50,488H
50f 4**
Naloxone MR 2266
90k5
U-58,488J-I + naloxone U-50,488H + MR 2264
88$6 45f 3**
47f 4""
NOTE: The data are the mean of three separate experiments and each experiment was conducted in triplicate. # + gradient-dependent 2 2 ~ a uptake ' ( 2 2 ~ auptake + mediated by Na+/Ht antiporter) was found to be 18.3 d 2.2 nmol . mg-' of protein min-'. **p < 0.01, significant difference compared with control.
U-50,488H did not produce any physical damage to the vesicles. Taken together, these findings suggest that the inhibition produced by U-50,488H on the Na+/H+ antiporter was not due to a nonspecific effect on cardiac sarcolemma. Mechanism of inhibition of the cardiac sarcokemml Na + / H f antiporter by U-50,488H With U - 5 0 . 4 8 8 ~being an opioid agonist and as cardiac sarcslernma have been shown to possess opioid receptors, it is conceivable that U-50,488H might cause inhibition through interacting with the opioid receptors of the cardiac sarcolemma. To test this possibility we performed the experiments
shown in Table 4. Vesicles were loaded with the pH 6.0 buffer. 22Na+ uptake by the vesicles was measured in the presence (pHi 6 , pH, 8) and in the absence of a pH gradient (pHi 6, pH, 6). The final concentrations of 22Na+, U-50,488H, naloxone, and M E 2 6 4 in the uptake medium were 1.0 mM, 100 pM, 100 pM, and 10 pM, respectively. Table 4 shows that both nalsxsne (a nonselective opioid antagonist) and MR2264 (a selective x-antagonist) produced a sad1 inhibition of the antiporter. As expected, U-50,488H (100 pM) reduced the activity of the antiporter by 50% of the control. However, when the effect of U-50,488H was studied in the presence of naloxone or MR2266, both failed to block the effect of U-50,488H on the antiporter. These data clearly indicate that the opioid receptors were not involved in mediating the effect of U-50,488H on the antiporter. To understand the nature of inhibition of the antiporter by U-50,488H, 22Na+uptake was measured in the presence s f varying concentrations of NaCl and with and without 100 pM U-50,488H. The data are presented in the farm of doublereciprocal plot and are shown in Fig. 2. As shown in Fig. 2, in the presence of U-50,488H, the Km for Na+ was increased from 2.5 f 0.2 to 5.0 f 0.3 mM, but the $/',, was unchanged (52.0 f 5.0 m o l mg-I - rnin-l). These data suggest that U-50,488B has a direct effect on the antiporter where it binds to the cation binding site and thereby it the transport of Na+. Comparison ofthe inhibitory efect of U-50,488Hwith amiloride a ~ l dthe arniloride analog on cardiac sarcokemmk NQ+/H+ antiporter Figure 3 describes the effects sf U-50,48$H, amiloride, and the amiloride analog on the Na+/H+ antiporter. The initial
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FIG. 3. Inhibition of cardiac sarcolemmal Na'/H' antiporter by U-%0,488H,arniloride, and the arniloride amlog. Vesicles were loaded with the pH 6.0 buffer. The loaded vesicles were diluted into the pH 8.0 or pH 6.0 buffer solution containing 1.0 mM 22NaCLand the indicated concentrations of compounds. '2Na+ uptake was measured as described in the Methods. H9 gradient-dependent uptake of "Na+ in the absence of compounds was represented as 100%.
rates of 22Na+ uptake by cardiac sarcolemmal vesicles that were dependent can H' gradient were inhibited by W-50,488H d l o r i d e and its analog in a concentration-depewdent manner. The concentration that produced 50% inhibition of 22Naf uptake was calculated for each of these compounds and was found to be 30 f 18 pM for the arniloride analog, 188 f 15 pM for U-50,48$H, and 300 f 20 pM for amiloride. These results suggest that the rank order of potency in inhibiting the antiporter was amiloride analog > U-50,488I-l > amiloride. Efleca of U-50,488-8, arniioride, and amiloride analog on cardiac Na+/K+ pump aad cardiac Na+/CaS antiporter W-50,48$H, arniloride, and the amiloride analog also inhibited the Na+/K+ pump and Na+/Cas antiporter of cardiac sarcolemrna (Figs. 4 and 5). However, the magnitude of the inhibition of the Na+/K+ pump or the Na+/CGf antiprter produced by these compounds was not the same. As shown in Figs. 4 and 5 the arniloride analog produced a complete inhibition of the Na+/K+ pump as well as the Na+/Ca2+ antiporter. On the other hand, amiloride and U-50,488H produced partial inhibition of the Na+/Kt pump and Na+/Ca2+ antiporter even with a concentration as high as 5.0 mM. The amiloride analog consistently produced an initial stimulation (30 -50%) at lower concentrations ( .S 100 p M ) followed by an inhibition of Na+/Ca2+antiprter at higher concentrations ( > 100 pM) in all experiments (Fig. 5). This phenomenon of initial stimulation was not observed with either amiloride or U-50,488H.
Discussie~n The data presented in this paper demonstrate that some of the opioids inhibited the cardiac sarcolemmal Na+/H+ anti-
porter. The interaction between the opioids and the Na+/Hf antiporter was further characterized using U-50,488H, a selective x-agonist. U-50,488H produced a concentrationdependent inhibition of 22Na+uptake that was dependent on H+ gradient (pHi 6, pH, 81, while it had very little effect on 22Na+uptake in the absence of Hf gradient (pHi 6, pH, 6) (Fig. 1). When 22Na+uptake was measured in the absence of H+ gradient (pHi 8, pH, 8) U-50,488H also caused a significant inhibition of the "Na+ uptake (Table 2). The above findings may appear to be contradictory but they are not. Although there was no H + gradient in both situations (pHi 6, pH, 6 and pHi 8, pH, 8), Na+/H+ antiporter activity was very little when the intravesicular and extravesicular pH was 6 and 6, while its activity was very significant when the intravesicular and extravesicular pH was 8 and 8 (Periyasarny et al. 1990; Seiler et al. 1985). As a result, U-58,488H produced a significant inhibition when the intravesicular and extravesicular pH was 8 and 8 but not when the intravesicular and extravesicular pH was 6 and 6. The inhibition produced by U-58,488H was immediate and reversible. Taken together, these results suggest that the inhibition caused by U-50,488H was not due to nonspecific damage to the vesicles or nonspecific action on the membrane nor due to the dissipation of the Hf gradient that existed across the vesicular membrane. Having ruled out the nonspecific actions as the mechanisms for the inhibition of the antiporter, it was speculated that U-58,488H might cause inhibition either by directly interacting with the antiporter or indirectly via interacting with the opioid receptors present in the cardiac sarcolemrna. There is overwhelming evidence in the literature that compounds that interact with the cell surface receptors influence the activity of the antiporter (Nard et al. 1987; Lydl et d.
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CAN. J. PHYSIOL. PHAWMACOL. VOE. TO, 1992
FIG.4. Inhibition of cardiac sarcolemmal Na+/K+ pump by U-50,4$8H, amiloride, and the amiloride analog. Vesicles were loaded with the 160 rraM KC1 - 20 mM MOPS (pH 7.2) solution. The loaded vesicles were diluted into a medium containing 5 mM 22NaCl, 140 mM choline chloride, B mM MgCI,, 8.2 mM EG'FA, 20 mM MOPS (pH 7.21, 2 mM AT$, and the indicated concentrations of compounds. 2 2 ~ a + uptake mediated by the Na+/Kt pump was taken as the difference between the "Nat uptake in the presence and in the absence of ATP. 22Nai uptake in the absence of compounds was represented as 100%.
FIG. 5. Inhibition sf cardiac sarcolemmal Na'/Ca2+ antiporter by U-50.488H, amiloride, and the arniloride analogs. Vesicles were loaded with the 140 mM NaCl - 10 mM MOPS (pH 7.4) solution, The loaded vesicles were diluted into a Na+-free medium containing 148 mM KC], 10 pM 45CitC12,10 mPvl MOPS @H 7.4), and the indicated concentrations of compounds. C$+ uptake was measured as described in the Methods. Ca2+ uptake mediated by Na+/Ca2+ antiporter was calculated as fhe difference between the Ca2' uptake in the presence and in the absence of Na+ gradient. 45Ca+ uptake in the absence of compounds was represented as 100%.
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1988; Gesek et al. 1989). Further, the presence of opioid receptors in the heart has been suggested by the studies of Caffrey et al. (1985) and kechner et d. (1985) and recently the presence of x-opioid receptors has been demonstrated in cardiac sarcoIemma (Ventura et al. 1989). U-50,488H being a selective x-agonist, was thought to inhibit the cardiac sarcolemmal Na +/H+ antiporter through interaction with the x-opioid receptors of cardiac sarcolemma. Therefore, the possibility of involvement of opioid receptors in the inhibitory action of U-50,488H on the antiporter was investigated. The effect of U-50,488H on cardiac sarcolemmal Na+/H+ antiporter was studied in the presence of naloxone, a nonselective opioid antagonist, and MR2266, a selective x-antagonist. The results of Table 4 show that neither of them blocked the effect of U-50,488H on Na+/H+ antiporter, suggesting that the inhibitory effect was not mediated through the interaction with the x-opioid receptors. On the other hand, the kinetic data of Fig. 2 provide evidence that U-50,488H produced inhibition of the antiporter by directly interacting with the antiporter. The data also show that the Vm,, of 22Na+ uptake was unchanged, whereas the K, for 22Na+ was increased in the presence of U-50,488H. These results suggest that U-50,488H acts at the Na+ binding site and thereby prevents the uptake of 22Na+into sarcslemal vesicles. The competitive nature of interaction between U-50,488H and Na+ on the antiporter was further substantiated by the studies showing that increasing the concentration of Na+ attenuated the ability of U-50,488H to inhibit the vesicular 22Na+ uptake (data not shown). The inhibition of 22Na+ uptake by U-50,488H was also reversible because removal of U-50,488H from the sarcolemmd vesicles by repeated washing restored the 22Na+ uptake almost to the pretreatment level (Table 3). This finding supports the view that the interaction of U-50,488H with the antiporter is via noncovalent binding. Although U-50,488H and amiloride interact with the antiporter through noncovalent binding, the affinity of U-50,488H for the antiporter is greater than the affinity of amiloride for the antiporter. This is evident from the rate of recovery of the antiporter from the inhibitory effects of U-50,488H and amiloride after washings (Table 3). is The of the antiporter produced similar in many respects to the inhibition caused by amiloride, cirnetidine, and clonidine. For instance, similar to U-50,488H, these inhibitors do not damage the membrane vesicles; they do not dissipate the H+ gradient and their inhibitory effect is completely reversible. Further, the inhibition caused by these compounds is competitive with respect to Na+ (Kinsella and Aronson 1981; Ganapathy et al. 1 9 8 6 ~ ;Ganapathy et al. 19866). It has k e n shown that loperamide, an opioid agonist, inhibited the antiporter from brush-border membrane vesicles isolated from human placenta, rabbit renal cortex, and rabbit small intestine (Balkovetz et d. 1987). In the present study, we have shown that loperamide also inhibited the antiporter from cardiac sarcolemmal vesicles prepared from bovine heart. Although we have not explored the mechanism of inhibition of the cardiac sarcolemmal Na+/H+ antiporter by loperamide, Bdkovetz et al. (1987) have shown that opioid receptors were not involved in the inhibition of the antiporter produced by loperamide, and they have demonstrated that loperamide interacts directly with the antiporter. These findings are similar to what we observed with the U-50,488H in the present study. Further, Balkovetz et al. (1987) have reported that the inhibition of the antiporter by loperamide is of mixed type and it interacts with the antiporter at more than one site. On the other hand, we have shown that U-50,488H U-509488H
inhibition is of competitive type and it binds to the antiporter at a single site. These observations indicate that although loperamide and U-50,488H interact with the antiporter directly, they may not share a common binding site on the antiporter. Amiloride, a potassium sparing diuretic, has been used by many investigators to explore the physiological role of the Na+/H+ antiporter in different cells. We, therefore, compared the potency of U-50,488H with the amiloride and amiloride analog, namely 5-(N,N-hexamethylene)amiloride, which has been reported as 500-fold more potent than amiloride in inhibiting the Na+/H+ antiporter from human neutrophils (Simchowitz and Cragse 1986). It is evident from Fig. 3 that the amiloride analog (ICSo = 30 f 10 pM) is three times more potent than U-50,488H (ICSo = 100 & 15 pM) which, in turn, is three times more potent than amiloride (I&jS0 = 300 f 20 p M ) . To h o w whether these compounds have an effect on other transport systems, we tested these compounds on the Na+/K+ pump and the Na+/Ca2+amtiporter of cardiac sarcolemm. Results of our studies show that U-%09488H, amiloride, and the amiloride analog also inhibited the Na+/K+ pump and the Na+/Ca2+ antiporter, but the concentrations needed to inhibit the pump and the Na"/Ca2+ antiporter are much higher (see Figs. 3, 4, and 5), suggesting that these compounds show some degree of selectivity for the Na+/H antiporter. In summary, some opioids, in addition to interacting with the opioid receptors, also produce nonopioid actions as observed in this study as well as in other studies (Maeda et al. 1987; Periyasamy and Hoss 1991; Hayes et al. 1988). It is, therefore, important to use these compounds carefully and judiciously in studying their effects mediated through opioid receptors. Although U-50,488H is more potent than amiloride in inhibiting the antiporter and shows some degree of selectivity for the antiporter, in our view, it is not an ideal compound to probe the role of Na+/H+ antiporter in different cellular processes. +
The author thanks Debra LeBarr for her excellent secretarial assistance, This work was supported by a National Institutes of Health grant H~-36573awarded by the ~~~i~~~~Heart, Lung, and ~ l Institute, ~ ~ United d statesPublic service, Depafilnent of Health and Human Services. Aronson, P. S. 1985. Kinetic properties of the plasma membrane Na+/PI+ exchanger. Annu. Rev. Physiol. 45: 545 - 560. Aronson, P. S . , and Bounds, S. E. 1988. Hamaline inhibition of Na+-dependent transport in renal microvillus membrane vesicles. Am. 9. Physiol. 238: F210-F217. Balkovetz, D. F., Miyamoto, Y.: Timppathi, C . , et a1. 1987. Hnhibition of brush-border membrane Na+-HS exchanger by Ioperarnide. B. Pharrnacol. Exp. Ther. 243: 1 50 - 154. Caffrey, J. E., Gaugl, J. F., and Jones, C. E. 1985. Local endogenous opiate activity in dog myocardium: receptors' blockade with nalsxone. Am. J. Physisl. 243: H382-H388. Escobales, N . , and Canessa, ha. 1986. Amiloride sensitive Naf transport in human red cdls. Evidence for Na4/H+ exchange system. J. Membr. Biol. 90: 21-28. Ganapathy, M. E., Eeibach, F. PI., Mahesh, V. B., et al. 1 9 8 6 ~ . Interaction of clonidine with human placental Na+-H+ exchanger. Biochern. Phrmacol. 35: 3989-399-4. Ganapathy, V., Balkovetz, D. F., Miyamoto, Y., et &el.1986b. Inhibition of human placental Na+-H+ exchanger by cirnetidine. J. Pharmacol. Exp. Ther. 239: 192- 197.
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