Pharmacology & Toxicology 1992, 71, 95-102.
Amiloride Derivatives as Blockers of Na+/Ca*+Exchange: Effects on Mechanical and Electrical Function of Guinea-Pig Myocardium Erich Wettwer, Herbert Himmel and Ursula Ravens” Department of Pharmacology, University of Essen, Hufelandstrasse 55, D-4300 Essen 1, Germany (Received September 6, 1991; Accepted January 6, 1992) Abstract: The amiloride derivatives, 2’,3’-benzobenzamil (BB), 3’,4‘-dichlorobenzamil (DCB), and 5-(N-4-chlorobenzyl)-
2’,4‘-dimethylbenzamil (CBDB) are known as inhibitors of the Na+/Ca2+ exchange. This kind of drug action was recently suggested to be a new inotropic mechanism. In guinea-pig myocardium, we have studied the inotropic and the accompanying electrophysiological effects of the three compounds in order to assess their selectivity of action. In left atria and in papillary muscle, force of contraction increased with DCB and CBDB (atria only) a t a high concentration (5 x lo-’mol/l) and after long exposure time, whereas BB produced a negative inotropic effect. In the isolated perfused Langendorff heart, the amiloride derivatives tested decreased spontaneous heart rate and force of contraction and prolonged the duration of contraction. In isolated cardiac myocytes, sodium current, calcium current and the delayed rectifier were reduced by concentrations of BB, DCB and CBDB similar to the IC, values reported for the inhibition of the Na+/Ca2+ exchange. Our results demonstrate that the amiloride derivatives have multiple sites of action. It is concluded that more specific modulators of the Na+/Ca2+exchange are required in order to define their contribution to the regulation of contractile activation of the heart.
In heart muscle, the sodium-calcium countertransport mechanism (Na+lCa2+exchange) is involved in the regulation of sodium (Na+) and calcium (Ca2+)homeostasis by utilizing the electrochemical energy of the transmembrane Naf concentration gradient to expel1 Ca2+ from the cell (Reuter & Seitz 1968; Reuter 1974). This links changes in the Na+ concentration gradient and changes in intracellular Ca2+ concentration. Since Ca2+ play a dominant role in excitation-contraction coupling, the Na+/Ca2+ exchange contributes to the regulation of force of contraction. When the intracellular Na+ concentration is elevated, the Na+/Ca2+exchange induces a rise in the intracellular Ca2+ concentration and consequently causes a positive inotropic effect (Reuter 1974; Borchard & Ravens 1986). Furthermore, pharmacological modification of the activity of the Na+/Ca2+ exchange should also influence intracellular Ca2+and therefore force of contraction (Luciani & Floreani 1985), although the direction of change is more difficult to predict. In cardiac sarcolemmal membrane preparations, several amiloride derivatives are potent inhibitors of the Na+ /Ca2+ exchange transporter with IC5,, values < 1-5 x lo-’ mol/l (Siegl er al. 1984; Slaughter et al. 1988; for review, see Ravens & Wettwer 1989). In multicellular cardiac preparations, both positive and negative inotropic effects of amiloride and its derivatives have been reported: positive inotropic effect of amiloride in guinea-pig right atria (Pousti & Khoyi 1979; Floreani & Luciani 1984), positive inotropic effect of 3’,4’-dichlorobenzamil in guinea-pig left atria and papillary muscle (Floreani et al. 1987), negative inotropic effect 3’,4-
* To whom correspondence should be directed.
dichlorobenzamil in guinea-pig papillary muscle (Siegl et al. 1984), positive inotropic effect of amiloride in guinea-pig left atria, no effect in rat left atria (Cargnelli et al. 1989). The reasons for these controversial results could include differenes in experimental procedure, species variations or lack of specificity of various compounds. For example, some amiloride derivatives were shown to block also myocardial Ca2+currents (Bielefeld et al. 1986; Wettwer 1988). In the present investigation, we have used three amiloride derivatives, namely 2’,3’-benzobenzamil (BB), 3’4’-dichlorobenzamil (DCB), and 5-(N-4-chlorobenzyl)-2’,4-dimethylbenzamil (CBDB), to study their effects on cardiac force of contraction under identical experimental conditions. Several heart muscle preparations were used because different parts of the heart may show particular responses. We have also investigated the selectivity of these agents in single myocytes, because additional effects on membrane conductances may have profound effects on contractile activation. Some of our results have been reported previously in preliminary form wettwer et al. 1991). Materials and Methods Left atria. Guinea pigs (250-300 g) of either sex were stunned by a sharp blow on the head and their hearts removed quickly. Left atria were isolated and mounted in a 20 ml muscle chamber filled with modified Tyrode solution aerated with carbogen at a temperature of 35”. They were stimulated at 1 set.-', isometric tension was recorded via a strain gauge transducer and registered on a chart recorder. Papillary muscles. Right ventricular papillary muscles were carefully dissected from the hearts of guinea pigs and mounted in a 5 ml muscle chamber through which modified Tyrode solution recircu-
96
ERICH WETTWER ET AL.
lated from a 100 ml reservoir kept at 35". A jet stream of carbogen (95% O,, 5% CO,) simultaneously served for circulation and oxygenation of the solution. The preparations were stimulated via platinum electrodes at a frequency of 1 set.-' (square wave pulses of 3 msec. in duration, 20% above threshold voltage). Action potentials were recorded with conventional glass microelectrodes filled with KCI solution (3 mol/l), force of contraction was measured by an isometric force transducer (Statham UC2 cell). Both signals were displayed on the screen of a dual beam storage oscilloscope. At regular intervals, digitized data were stored on floppy disc for later analysis. Drugs were added in a cumulative manner to the reservoir of the bathing solution. Langendorj'preparations. After isolation of the heart, the aorta was tied around the cannula of a Langendorff apparatus for constant flow perfusion of the coronary vascular bed with modified Tyrode solution aerated with carbogen and maintained at 35" (unless indicated otherwise). The hearts were allowed to beat spontaneously. The frequency of contractions (beatdmin.) was counted separately for atria and ventricles over a time period of 15 sec. In control experiments the force-frequency relation was determined in electrically paced hearts. The sinus-nodes of these hearts were mechanically destroyed. The hearts were stimulated between 60 and 240 beatdmin. via an extracellular electrode placed on the left ventricle. Force of contraction was measured isometrically by a strain gauge transducer attached to the apex of the hearts and registered on a chart recorder. Drugs were added at a constant rate into the perfusion system.
Isolated myocytes. Single myocytes were isolated from the hearts of guinea pigs by a modification of the method of Bendukidze et al. (1985). Dissociation of cells was achieved by a brief period (5 min.) of perfusion of the whole hearts with a nominally calcium-free solution followed by 5-8 min. of perfusion in pronase-containing solution (pronase E 0.1 mg/ml, Serva, Heidelberg, Germany) supplemented with 200 pmol/l CaCI, for optimum enzyme activation and with fatty acid free albumin (A-6003, Sigma, Miinchen, Germany). Enzymatic dissociation continued by incubating the chopped tissue pieces under gentle mechanical agitation in fresh pronase-containing buffer three successive times. Rod-shaped cells ( 6 7 0 % ) were suspended in the superfluent, they showed no spontaneous activity in calcium-containing solution (3.6 mmol/l). The cells were stored at room temperature and used within 12 hr. As described in detail previously (Cragoe et al. 1987), electrical activity from the myocytes was recorded using patch electrodes (Hamill et al. 1989). A List L/M-EPC7 patch clamp amplifier was used for single electrode voltage clamp technique in connection with a PClamp data acquisition and analysis program (Axon Instruments). Electrodes with tip resistances of 2 4 MQ were made from filamented borosilicate glass and filled as indicated below. Composition of solutions. The modified Tyrode solution had the following composition (in mmolll): NaCl 126.7, KCI 5.4, MgCl, 1.05, CaCI, 1.8, NaH,PO, 0.42, NaHCO, 22, glucose 5. After equilibration with carbogen, the pH was 7.4. The nominally Ca'+-free solution had the following composition (in mmol/l): NaCl 140, KCI 10, MgSO, 5, KH2P041.2, glucose 20, taurine 50, MOPS (morpholinopropane sulphonic acid) 10, adjusted to pH 7.0 with KOH. The solution for superfusion of the cells for measurements of Ca2+ current contained (in mmol/l): NaCl 150, KCI 5.4, MgCI, 2.1, CaCI, 1.8, HEPES (hydroxyethyl-piperazinylethane-sulphonic acid) 10, glucose 10, adjusted to pH 7.4 with NaOH. The superfusion solution for measurements of Na+ current contained (in mmol/l): NaCl 30, CsCl 107, CaCI, 0.5, CdCI, 0.05, MgCI, 2.5, HEPES 10 (pH 7.4). The following electrode filling solutions were used (in mmol/l): (a) measurements of CaZ+current; KCI 150, MgCI, 5, EGTA (ethylene-glycol-bis-(P-amhoethylether)N,N,N',N'-tetra-acetic acid) 0.02, HEPES 10, adjusted to pH 7.2 with KOH; (b) measurements of Na+ current; CsCl 140, MgCI, 4, EGTA 10, HEPES 10, Na,ATP 4, adjusted to pH 7.3 with CsOH.
Drugs. The amiloride derivatives were a gift from Merck, Sharp & Dohme, West Point PA, U.S.A. They were dissolved in DMSO (dimethylsulphoxide) and distilled water and used the same day. The final concentration of DMSO solution never exceeded 1% vol/ vol.
Statistical analysis. Results are given as means k S.E.M. of n experiments. The statistical significance of differences was estimated according to Student's ttest for grouped observations. Differences were considered significant for P
Amiloride Derivatives as Blockers of Na+/Ca*+Exchange: Effects on Mechanical and Electrical Function of Guinea-Pig Myocardium Erich Wettwer, Herbert Himmel and Ursula Ravens” Department of Pharmacology, University of Essen, Hufelandstrasse 55, D-4300 Essen 1, Germany (Received September 6, 1991; Accepted January 6, 1992) Abstract: The amiloride derivatives, 2’,3’-benzobenzamil (BB), 3’,4‘-dichlorobenzamil (DCB), and 5-(N-4-chlorobenzyl)-
2’,4‘-dimethylbenzamil (CBDB) are known as inhibitors of the Na+/Ca2+ exchange. This kind of drug action was recently suggested to be a new inotropic mechanism. In guinea-pig myocardium, we have studied the inotropic and the accompanying electrophysiological effects of the three compounds in order to assess their selectivity of action. In left atria and in papillary muscle, force of contraction increased with DCB and CBDB (atria only) a t a high concentration (5 x lo-’mol/l) and after long exposure time, whereas BB produced a negative inotropic effect. In the isolated perfused Langendorff heart, the amiloride derivatives tested decreased spontaneous heart rate and force of contraction and prolonged the duration of contraction. In isolated cardiac myocytes, sodium current, calcium current and the delayed rectifier were reduced by concentrations of BB, DCB and CBDB similar to the IC, values reported for the inhibition of the Na+/Ca2+ exchange. Our results demonstrate that the amiloride derivatives have multiple sites of action. It is concluded that more specific modulators of the Na+/Ca2+exchange are required in order to define their contribution to the regulation of contractile activation of the heart.
In heart muscle, the sodium-calcium countertransport mechanism (Na+lCa2+exchange) is involved in the regulation of sodium (Na+) and calcium (Ca2+)homeostasis by utilizing the electrochemical energy of the transmembrane Naf concentration gradient to expel1 Ca2+ from the cell (Reuter & Seitz 1968; Reuter 1974). This links changes in the Na+ concentration gradient and changes in intracellular Ca2+ concentration. Since Ca2+ play a dominant role in excitation-contraction coupling, the Na+/Ca2+ exchange contributes to the regulation of force of contraction. When the intracellular Na+ concentration is elevated, the Na+/Ca2+exchange induces a rise in the intracellular Ca2+ concentration and consequently causes a positive inotropic effect (Reuter 1974; Borchard & Ravens 1986). Furthermore, pharmacological modification of the activity of the Na+/Ca2+ exchange should also influence intracellular Ca2+and therefore force of contraction (Luciani & Floreani 1985), although the direction of change is more difficult to predict. In cardiac sarcolemmal membrane preparations, several amiloride derivatives are potent inhibitors of the Na+ /Ca2+ exchange transporter with IC5,, values < 1-5 x lo-’ mol/l (Siegl er al. 1984; Slaughter et al. 1988; for review, see Ravens & Wettwer 1989). In multicellular cardiac preparations, both positive and negative inotropic effects of amiloride and its derivatives have been reported: positive inotropic effect of amiloride in guinea-pig right atria (Pousti & Khoyi 1979; Floreani & Luciani 1984), positive inotropic effect of 3’,4’-dichlorobenzamil in guinea-pig left atria and papillary muscle (Floreani et al. 1987), negative inotropic effect 3’,4-
* To whom correspondence should be directed.
dichlorobenzamil in guinea-pig papillary muscle (Siegl et al. 1984), positive inotropic effect of amiloride in guinea-pig left atria, no effect in rat left atria (Cargnelli et al. 1989). The reasons for these controversial results could include differenes in experimental procedure, species variations or lack of specificity of various compounds. For example, some amiloride derivatives were shown to block also myocardial Ca2+currents (Bielefeld et al. 1986; Wettwer 1988). In the present investigation, we have used three amiloride derivatives, namely 2’,3’-benzobenzamil (BB), 3’4’-dichlorobenzamil (DCB), and 5-(N-4-chlorobenzyl)-2’,4-dimethylbenzamil (CBDB), to study their effects on cardiac force of contraction under identical experimental conditions. Several heart muscle preparations were used because different parts of the heart may show particular responses. We have also investigated the selectivity of these agents in single myocytes, because additional effects on membrane conductances may have profound effects on contractile activation. Some of our results have been reported previously in preliminary form wettwer et al. 1991). Materials and Methods Left atria. Guinea pigs (250-300 g) of either sex were stunned by a sharp blow on the head and their hearts removed quickly. Left atria were isolated and mounted in a 20 ml muscle chamber filled with modified Tyrode solution aerated with carbogen at a temperature of 35”. They were stimulated at 1 set.-', isometric tension was recorded via a strain gauge transducer and registered on a chart recorder. Papillary muscles. Right ventricular papillary muscles were carefully dissected from the hearts of guinea pigs and mounted in a 5 ml muscle chamber through which modified Tyrode solution recircu-
96
ERICH WETTWER ET AL.
lated from a 100 ml reservoir kept at 35". A jet stream of carbogen (95% O,, 5% CO,) simultaneously served for circulation and oxygenation of the solution. The preparations were stimulated via platinum electrodes at a frequency of 1 set.-' (square wave pulses of 3 msec. in duration, 20% above threshold voltage). Action potentials were recorded with conventional glass microelectrodes filled with KCI solution (3 mol/l), force of contraction was measured by an isometric force transducer (Statham UC2 cell). Both signals were displayed on the screen of a dual beam storage oscilloscope. At regular intervals, digitized data were stored on floppy disc for later analysis. Drugs were added in a cumulative manner to the reservoir of the bathing solution. Langendorj'preparations. After isolation of the heart, the aorta was tied around the cannula of a Langendorff apparatus for constant flow perfusion of the coronary vascular bed with modified Tyrode solution aerated with carbogen and maintained at 35" (unless indicated otherwise). The hearts were allowed to beat spontaneously. The frequency of contractions (beatdmin.) was counted separately for atria and ventricles over a time period of 15 sec. In control experiments the force-frequency relation was determined in electrically paced hearts. The sinus-nodes of these hearts were mechanically destroyed. The hearts were stimulated between 60 and 240 beatdmin. via an extracellular electrode placed on the left ventricle. Force of contraction was measured isometrically by a strain gauge transducer attached to the apex of the hearts and registered on a chart recorder. Drugs were added at a constant rate into the perfusion system.
Isolated myocytes. Single myocytes were isolated from the hearts of guinea pigs by a modification of the method of Bendukidze et al. (1985). Dissociation of cells was achieved by a brief period (5 min.) of perfusion of the whole hearts with a nominally calcium-free solution followed by 5-8 min. of perfusion in pronase-containing solution (pronase E 0.1 mg/ml, Serva, Heidelberg, Germany) supplemented with 200 pmol/l CaCI, for optimum enzyme activation and with fatty acid free albumin (A-6003, Sigma, Miinchen, Germany). Enzymatic dissociation continued by incubating the chopped tissue pieces under gentle mechanical agitation in fresh pronase-containing buffer three successive times. Rod-shaped cells ( 6 7 0 % ) were suspended in the superfluent, they showed no spontaneous activity in calcium-containing solution (3.6 mmol/l). The cells were stored at room temperature and used within 12 hr. As described in detail previously (Cragoe et al. 1987), electrical activity from the myocytes was recorded using patch electrodes (Hamill et al. 1989). A List L/M-EPC7 patch clamp amplifier was used for single electrode voltage clamp technique in connection with a PClamp data acquisition and analysis program (Axon Instruments). Electrodes with tip resistances of 2 4 MQ were made from filamented borosilicate glass and filled as indicated below. Composition of solutions. The modified Tyrode solution had the following composition (in mmolll): NaCl 126.7, KCI 5.4, MgCl, 1.05, CaCI, 1.8, NaH,PO, 0.42, NaHCO, 22, glucose 5. After equilibration with carbogen, the pH was 7.4. The nominally Ca'+-free solution had the following composition (in mmol/l): NaCl 140, KCI 10, MgSO, 5, KH2P041.2, glucose 20, taurine 50, MOPS (morpholinopropane sulphonic acid) 10, adjusted to pH 7.0 with KOH. The solution for superfusion of the cells for measurements of Ca2+ current contained (in mmol/l): NaCl 150, KCI 5.4, MgCI, 2.1, CaCI, 1.8, HEPES (hydroxyethyl-piperazinylethane-sulphonic acid) 10, glucose 10, adjusted to pH 7.4 with NaOH. The superfusion solution for measurements of Na+ current contained (in mmol/l): NaCl 30, CsCl 107, CaCI, 0.5, CdCI, 0.05, MgCI, 2.5, HEPES 10 (pH 7.4). The following electrode filling solutions were used (in mmol/l): (a) measurements of CaZ+current; KCI 150, MgCI, 5, EGTA (ethylene-glycol-bis-(P-amhoethylether)N,N,N',N'-tetra-acetic acid) 0.02, HEPES 10, adjusted to pH 7.2 with KOH; (b) measurements of Na+ current; CsCl 140, MgCI, 4, EGTA 10, HEPES 10, Na,ATP 4, adjusted to pH 7.3 with CsOH.
Drugs. The amiloride derivatives were a gift from Merck, Sharp & Dohme, West Point PA, U.S.A. They were dissolved in DMSO (dimethylsulphoxide) and distilled water and used the same day. The final concentration of DMSO solution never exceeded 1% vol/ vol.
Statistical analysis. Results are given as means k S.E.M. of n experiments. The statistical significance of differences was estimated according to Student's ttest for grouped observations. Differences were considered significant for P
