J Mol
Cell
Cardiol
24,
EScts
731-740
(1992)
of Na+/H+
W. Scholz,
U. Albus, Hoechst
(Received
AG,
Exchange W. Linz, Postfach
19 March
Inhibitors
P. Martorana, 800320,
1991,
6230
accepted
in Cardiac H. J. Lang,
Frankfurt/M.
in revisedform
80,
Ischemia
B.A.
Schiilkens
Germany
21 February
1992)
W. SCHOLZ, U. ALBUS, W. LINZ, P. MARTORANA, H. J. LANG AND B.A. SCH~LKENS. Effects of Na’/H’ Exchange Inhibitors in Cardiac Ischemia. Journal ofMolecular and Cellular Cardiology (1992) 24, 733-741. To investigate a possible protective role of Na ’ /H ’ exchange inhibition under ischemic conditions isolated rat hearts were subjected to regional ischemia and reperfusion. In these experiments all 6 untreated hearts suffered ventricmol/l amiloride or 3 x 10 ’ mol/l 5-(N-ethyl-N-isoular fibrillation on reperfusion. Addition of 1 x 10e5 propyl)amiloride (EIPA) markedly decreased the incidence and duration of ventricular fibrillation or even suppressed fibrillation completely as in the case of 1 x 10 - 6 mol/l EIPA. Both compounds diminished the activities of lactate dehydrogenase and creatine kinase in the venous effluent of the hearts during ischemia. At the end of the experiments tissue contents of glycogen, ATP and creatine phosphate were increased in the treated hearts as compared to control hearts. In an additional experiment the beneficial effects of Na’/H + exchange inhibition during ischemia was confirmed in viuo with anaesthetized rats undergoing coronary artery ligation. In these animals amiloride or EIPA pretreatment caused a marked reduction of ventricular premature beats and ventricular tachycardia as well as a complete suppression of ventricular fibrillation. The concentration dependent inhibition of Na ’ influx via Na ’ /H + exchange by amiloride and EIPA was investigated in erythrocytes from hypercholesterolemic rabbits with Na+/H+ exchange activated by exposure to hyperosmotic medium. Furthermore the inhibition of Na + influx by EIPA after intracellular acidification was studied in cardiac myocytes of neonatal rats. Both agents were effective in the same order of potency in the ischemic isolated working rat heart as in the erythrocyte model in which they inhibited Na+/H+ exchange. In addition the anti-ischemic effects of EIPA were seen over the same concentration range over which the compound inhibited Na+ /H + exchange in acidified cardiac myocytes. This indicates that the anti-ischemic actions and the effects observed in erythrocytes or cardiac cells might be caused by a common mechanism. We suggest that amiloride and EIPA show cardioprotective and antiarrhythmic effects in ischemia and reperfusion which could probably be attributed to Na’ /H + exchange inhibition. KI:Y
WORDS:
Na + /H + exchange;
Ischemia;
Amiloride;
Introduction All eukaryotic cells are equipped with an Na+ /H + exchanger which extrudes H + from the cytosol in exchange for Na+, which provides the driving force by its gradient across the plasma membrane [1, 21. Amiloride, a clinically useful diuretic, as well as the more specific 5-(N-ethyl-N-isopropyl)amiloride (EIPA) have been shown to inhibit Na+ influx caused by an activation of Na+/H + exchange in several types of cells [3-51. In cardiac cells where the exchanger is excessively activated during ischemia by a drop of intracellular pH [S, 7] Na+/H + exProofs should Frankfurt/M. OOZZ-2828/92/070733
be sent to: Wolfgang 80, Germany. + 09 $03.00/O
Scholz,
Hoechst
AG.
EIPA;
Arrhythmia;
Cardiac
protection
change inhibition could play a beneficial role since the deleterious Na+ influx in this condition is believed to originate mainly from Na + /H + exchange [ 8, 91. These ischemic cells seem to run into a vicious cycle, since an inincrease in cytosolic sodium in turn activates Na’ /K+-ATPase [ 9, 101 which in turn increases ATP consumption. During ischemia the anaerobic metabolism of glucose terminates in lactic acid. This leads to a further decrease of intracellular pH and to a further activation of Na+/H+ exchange, resulting in energy depletion, cellular Na+ overload and finally cellular Ca2 + overload [ 6, 9, II] due to a coupling of Na+- and Ca* + transport. SBU
Cardiovascular
Agents,
H821.
0 1992
P.O.B.
Academic
800320,
Press
6230
Limited
732
W. Scholz et al.
Especially in ischemic cardiac tissue where Na+ /H + exchange is the dominant pH regulating ion transport system [ 7, I,?] these pathophysiological events could lead to energy starvation, increased excitability and precipitation of cellular death. Based on this assumption inhibitors of Na+ /H + exchange like amiloride and EIPA should be able to interrupt this vicious cycle, to conserve cellular energy stores and to diminish excitability and necrosis in cardiac ischemia. In the present study the effect of the Na + /H + exchange inhibitors amiloride and EIPA was investigated in isolated working rat hearts subjected to ischemia-reperfusion injury. In addition, the action of both compounds on ischemia induced arrhythmias was examined in anaesthetized rats with ligation of the left coronary artery. Further experiments investigated the inhibition of Na+ influx via Na + /H + exchange by amiloride and EIPA in rabbit erythrocytes and the inhibitory effect of EIPA on intracellular acidification activated Na+/H + exchange, in cardiac myocytes. Methods Sodium [email protected] into erythrocytes New Zealand white rabbits (Ivanovas) were fed a rabbit standard chow with 2% cholesterol for six weeks to activate the Na + /H + exchange [ 131. This facilitates measurement of sodium influx via Na + /H + exchange by flame photometry in the erythrocytes. Blood was drawn from the ear artery of the rabbits and prevented from coagulating by the addition of 25 W/ml potassium heparin. Hematocrit was determined in duplicate by centrifugation. The initial sodium content of the erythrocytes was measured in 1001.11 aliquots. To determine the amiloride sensitive sodium influx into erythrocytes, 100~1 of blood were diluted into 5ml of buffer (in mmol/l: NaCl 140, KC1 3, sucrose 150, ouabain 0.1, Trishydroxy-methylaminomethane 20, pH 7.4) made hyperosmolar by the addition of sucrose and incubated for 60 min at 37’C. Subsequently, the erythrocytes were washed three centrifugation in icecold times by MgC$ouabain-solution (in mmol/l: MgC12 112, ouabain 0.1). For determination of intracellular Na+ content, the cells were hemolysed in distilled water
in a final volume of 2 .O ml, the cell membranes were centrifuged and the sodium concentration of the supernatant was measured by flame photometry with an Eppendorf flame photometer (Netheler + Hinz GmbH Hamburg) at a wavelength of 589nm. Net influx of sodium into the erythrocytes was calculated from the difference between the initial sodium content and the sodium content after the incubation. Amiloride sensitive Na+-influx was calculated from the difference between Na+ content of erythrocytes incubated with and without amiloride 3 x 10 - 4 mol/l.
Sodium-in+
into cardiac myocytes
Rat myocardial cells were isolated by trypsin digestion of hearts from neonatal rats. The cells were cultured on 35mm dishes and grown to confluency in Dulbeco’s Minimum Essential Medium (DMEM, GIBCOR) in an atmosphere with 10% CO1. After confluency the cells were used for measurement of 22Na+ influx. The cells were washed twice with Krebs-Ringer-solution buffered with Hepes/Tris (KRB) in which sodium chloride had been replaced by choline chloride (in mmoyl: choline chloride, 130, CaC12 1.5, KC1 5, MgCl, 1, Hepes 20, pH7.0 with Tris) and then incubated for twenty minutes at 37’C in the same buffer with added 0.1% bovine serum albumin (BSA) and lOmmol/l glucose. The culture dishes were then incubated for another 10min with Na+-propionate for cytosolic acidification and stimulation of Na+/H + exchange. The compounds were dissolved in 500pl/dish KRB in which 50% of the sodium chloride had been replaced by choline chloride containing additionally P@i/ml **Na+-bicarbonate, and 5-(N-ethyl-N-isopropyl)amiloride (EIPA). After the stimulation period sodium influx was terminated by washing the cells twice with an icecold stop solution (in mmol/l: MgCl, 0.1, Tris 10, pH 7.0). Subsequently, the cells were lyzed with 250~1 trichloric acid and scraped from the dishes. Radioactivity was determined in a Packard gamma counter. Measurement of Na + /H + exchange was performed with n = 6. Results are given in mean f S.D.
Na+/H+
Exchange
Isolated working rut heart preparation Isolated working rat heart preparation from Wistar rats of both sexes weighing 280-300 g were used in all experiments as described before [ 141. Animals were injected intraperitoneally with sodium heparin (500 U/kg) 1 h before killing. The hearts were excised and placed in icecold perfusion medium until contraction had ceased, approximately 5 s. The isolated hearts were then perfused via the aorta at a constant perfusion pressure equivalent to 65 mmHg (15) with a modified Krebs-Henseleit buffer of the following composition (mmol/l: NaCl 113.8, NaHC03 22, KC1 4.7, KH2P04 1.2, MgS04 1.1, CaC12 2.5, glucose 11.0, Napyruvate 2.0). The medium was gassed with 95% 02 plus 5% CO* at 37°C and pH7.4. The perfusate was not recirculated. A silicone balloon was then introduced into the left ventricle through the mitral valve. Preload was held at 7.4 mmHg and afterload as well as coronary perfusion were kept constant at 65 mmHg. Amiloride and EIPA were present in the respective experiments throughout the perfusion period. The compounds were dissolved in the medium without the use of any solvent. After the perfusion had commenced the hearts immediately began to pump and to perform pressure volume work (for detailed description see 14). The hearts were perfused for an initial period of 20min (control perfusion period). Acute regional myocardial ischemia was then produced by clamping the left anterior descending coronary artery close to its origin for 15 min (ischemic period). Thereafter, the clip was reopened and changes during reperfusion were monitored for 30min (reperfusion period). The following parameters were measured: left ventricular presure (LVP) via a Statham pressure transducer P23 Db, which on differentiation yielded LV dPldt,,, and heart rate (HR). Coronary flow (CF) was determined by an electromagnetic flow probe in the aortic cannula. An epicardial electrocardiogram recording was obtained during the reperfusion period via two silver electrodes attached to the heart. All parameters were recorded on a Brush 2600 recorder. Lactate release, lactate dehydrogenase (LDH) and creatine kinase (CK) activities
in Cardiac
Ischemia
733
were measured by spectrophotometrical measurements in the perfusate [II] collected from the coronary effluent. All heart weights are given in g of wet tissue. Wet weight of the hearts (approximately 1.2 g) was determined immediately after excision from the rats and before they were mounted in the perfusion chamber. Dry tissue weight (approximately 240 mg) was measured after drying the hearts for 12h at 110%. After the experiments, hearts were rapidly frozen in liquid nitrogen and stored at - 80%. For tissue analysis of lactate, glycogen and high energy phosphates (HEP) 500 mg of the left ventricle were put into 5 ml ice-cold HC104 (0.6 mol/l) and homogenized with an Ultra-Turrax (Junke & Kunkel, IkaWerk, Typ TP). Glycogen was hydrolized to glucose with amyloglycosidase (pH 4.8) [ 141. Lactate was assayed spectrophotometrically according to F. No11 [16]. ATP and creatine phosphate (CP) were measured according to Trauschold et al. and Heinz et al. published in Methods of Enzymatic Analysis [ 17, 181, Statistical analysis of the data obtained was performed with Student’s t-test for independent groups. Differences were considered significant if PcO.05. Results are given as mean values? S.E.M.
Anaesthetized rat Male Sprague-Dawley rats weighing 280444g were anaesthetized with sodium pentobarbital (60 mg/kg i.p.). The rats were intubated and respirated with room air lo-13 ml/kg at 54 breaths/min by a Rhema@ respirator (Frankfurt, Germany). A catheter was inserted into the left carotid artery and connected to a pressure transducer (Type P23Dd, Statham, Hato Ray, Puerto Rico) to record blood pressure with a Brush Mark 220 polygraph. The ECG was recorded from lead 2 and arrhythmias were evaluated according to the guidelines of the Lambeth conventions [19\ where: VPB’s (ventricular premature beats) are defined as discrete and identifiable premature QRS complexes (premature in relation to the P wave). All the VPB’s occurring in the ischemic period (30min) were counted. VT (ventricular tachycardia) was defined as a
734
w. Scholz et al.
run of four or more consecutive ventricular premature beats. The duration of VT, in seconds, was measured. VF (ventricular fibrillation) was defined as a signal for which individual QRS deflections could no longer be distinguished from one another. The duration of VF, in seconds, was measured. The heart rate was counted from the ECG. Thoracotomy for coronary artery ligation was performed at the fifth intercostal space. During an equilibration period of 15 min rats with spontaneous arrhythmias or a systolic blood pressure below 70 mmHg were rejected. The left coronary artery was then ligated for 30mins. For intravenous applications a catheter was placed into the right jugular vein. Amiloride (30 mg/kg) was given intraperitoneally 15 min before coronary artery occlusion. EIPA (1 mg/kg) was given intravenously as a bolus 5 min before coronary artery occlusion, followed by a constant infusion of 0.01 mg/kg/min. The results are presented as mean + S.E.M. Differences between mean values were analysed with the Student’s t-test and differences between numbers of animals with arrhythmia were analysed with the x2-test.
Results Sodium inJEux into erythrocytes As shown in Figure 1 both Na+/H+ exchange inhibitors decreased sodium influx into rabbit erythrocytes in a dose dependent manner. The IC,, of amiloride was 1.5 x 10e5 mol/l.
3-
(mmolll) FIGURE 1. Concentration dependent inhibition at Na’ influx via Na’ /H + exchange by amiloride and EIPA in rabbit erythrocytes (mean + s E.M., n = 6) (- - -: Amiloride; EIPA).
The derivative of amiloride, EIPA, was about 500 times more potent in this assay with an ICsO of 5 x lo-* mol/l.
Sodium influx into rat myocardial cells Rat cardiac myocytes acidified with sodium propionate showed a marked increase of Na* influx during the incubation period as compared with controls (Fig. 2). While there was hardly any inhibition of Na+ influx by 10 ’ mol/l EIPA under control conditions, the increase of Na + influx caused by propionate was totally inhibited by EIPA 10 m6 mol/l. Almost no inhibition of Na+ influx in propionate treated myocytes could be observed with 10-j mol/l EIPA.
Isolated working
rat heart preparation
To investigate a possible protective role of Na + /H + exchange inhibition under ischemic conditions isolated rat hearts were subjected to ischemia and reperfusion. In these experiments all six untreated hearts suffered ventricular fibrillation with an incidence of 100% and a mean duration of about 17min during the reperfusion period of 30 min (Fig. 3). Perfusion of the isolated hearts with lo-’ mol/l amiloride or lo-’ mol/l EIPA did not cause any change. However, when the concentration of amiloride or EIPA was increased in the perfusion medium to 10 - ’ or 3 x 10 ’ mol/l, respectively, the incidence of ventricular fibrillation as well as the duration of the fibrillation was markedly decreased. EIPA at 10 m6 mol/l totally suppressed ventricular fibrillation (Fig. 3). During ischemia LV dPldt was significantly increased by about 35 %, while coronary flow (CF) was slightly decreased with 10. ‘, mol/l amiloride in comparison with control hearts (Fig. 4). The release of LDH and CK from the hearts into the venous effluent was slightly diminished during ischemia (Fig. 4). Analysis of the energy stores of the treated hearts at the end of the experiments showed significant differences in the levels of glycogen, ATP and CP as compared to control hearts. Tissue contents of glycogen, ATP and CP were increased by 48%) 200% and 200% respectively from 88.4 + 3.4, 5.3 + 0.5 and
Na+/H+
Exchange
in
Cardiac
735
Ischemia
1000 900 600 aJ 700 'j h 600 2xX3 bM 300 200 100 o_.
_.
control
Proplonote
+EIPA 10-S
FIGURE 2. Inhibition ation by Na+ propionate.
2oi T
of “Na+ Relative
mflux 22 Na+
Propkmote +EIPA lo-’
(mol/l)
into rat cardiac myocytes by EIPA with and without intracellular acidificinflux given as counts per min per culture plate (mean + s D , n = 6).
T
( Cardiodynamics
1Venous
effhxnt
( -dial
tii
FIGURE 5. Effect of EIPA 10 -” mol/l on isolated working rat hearts during &hernia and rcperfusion. Cardiodynamics during ischemia, enzymes in the venous effluent at the beginning of reperfusion and HEP stores after &hernia and reperfusion (mean + s E M , R = 6 in each group, ‘PCO.05).
MolA FIGURE 3. Effect of am&ride duration of ventricular fibrillation hearts during reperfusion (mean group) (I: Control: a: Amiloride;
Proplonote +EIPA 10-s
and EIPA on the in isolated working rat f S.E M , R = 6 in each 0: EIPA).
/enwseffluent
Myoa7rdiol tissue I
*
FI(GURE 4. Effect of amiloride 10 - ’ mol/l on isolated working rat hearts during ischemia and reperfusion Cardiodynamirs during &hernia, enzymes in the venous effluent at the beginning of reperfusion and HEP stores after ix-hernia and reperfusion (meant s E M . n = 6 in each group, *P
Cell
Cardiol
24,
EScts
731-740
(1992)
of Na+/H+
W. Scholz,
U. Albus, Hoechst
(Received
AG,
Exchange W. Linz, Postfach
19 March
Inhibitors
P. Martorana, 800320,
1991,
6230
accepted
in Cardiac H. J. Lang,
Frankfurt/M.
in revisedform
80,
Ischemia
B.A.
Schiilkens
Germany
21 February
1992)
W. SCHOLZ, U. ALBUS, W. LINZ, P. MARTORANA, H. J. LANG AND B.A. SCH~LKENS. Effects of Na’/H’ Exchange Inhibitors in Cardiac Ischemia. Journal ofMolecular and Cellular Cardiology (1992) 24, 733-741. To investigate a possible protective role of Na ’ /H ’ exchange inhibition under ischemic conditions isolated rat hearts were subjected to regional ischemia and reperfusion. In these experiments all 6 untreated hearts suffered ventricmol/l amiloride or 3 x 10 ’ mol/l 5-(N-ethyl-N-isoular fibrillation on reperfusion. Addition of 1 x 10e5 propyl)amiloride (EIPA) markedly decreased the incidence and duration of ventricular fibrillation or even suppressed fibrillation completely as in the case of 1 x 10 - 6 mol/l EIPA. Both compounds diminished the activities of lactate dehydrogenase and creatine kinase in the venous effluent of the hearts during ischemia. At the end of the experiments tissue contents of glycogen, ATP and creatine phosphate were increased in the treated hearts as compared to control hearts. In an additional experiment the beneficial effects of Na’/H + exchange inhibition during ischemia was confirmed in viuo with anaesthetized rats undergoing coronary artery ligation. In these animals amiloride or EIPA pretreatment caused a marked reduction of ventricular premature beats and ventricular tachycardia as well as a complete suppression of ventricular fibrillation. The concentration dependent inhibition of Na ’ influx via Na ’ /H + exchange by amiloride and EIPA was investigated in erythrocytes from hypercholesterolemic rabbits with Na+/H+ exchange activated by exposure to hyperosmotic medium. Furthermore the inhibition of Na + influx by EIPA after intracellular acidification was studied in cardiac myocytes of neonatal rats. Both agents were effective in the same order of potency in the ischemic isolated working rat heart as in the erythrocyte model in which they inhibited Na+/H+ exchange. In addition the anti-ischemic effects of EIPA were seen over the same concentration range over which the compound inhibited Na+ /H + exchange in acidified cardiac myocytes. This indicates that the anti-ischemic actions and the effects observed in erythrocytes or cardiac cells might be caused by a common mechanism. We suggest that amiloride and EIPA show cardioprotective and antiarrhythmic effects in ischemia and reperfusion which could probably be attributed to Na’ /H + exchange inhibition. KI:Y
WORDS:
Na + /H + exchange;
Ischemia;
Amiloride;
Introduction All eukaryotic cells are equipped with an Na+ /H + exchanger which extrudes H + from the cytosol in exchange for Na+, which provides the driving force by its gradient across the plasma membrane [1, 21. Amiloride, a clinically useful diuretic, as well as the more specific 5-(N-ethyl-N-isopropyl)amiloride (EIPA) have been shown to inhibit Na+ influx caused by an activation of Na+/H + exchange in several types of cells [3-51. In cardiac cells where the exchanger is excessively activated during ischemia by a drop of intracellular pH [S, 7] Na+/H + exProofs should Frankfurt/M. OOZZ-2828/92/070733
be sent to: Wolfgang 80, Germany. + 09 $03.00/O
Scholz,
Hoechst
AG.
EIPA;
Arrhythmia;
Cardiac
protection
change inhibition could play a beneficial role since the deleterious Na+ influx in this condition is believed to originate mainly from Na + /H + exchange [ 8, 91. These ischemic cells seem to run into a vicious cycle, since an inincrease in cytosolic sodium in turn activates Na’ /K+-ATPase [ 9, 101 which in turn increases ATP consumption. During ischemia the anaerobic metabolism of glucose terminates in lactic acid. This leads to a further decrease of intracellular pH and to a further activation of Na+/H+ exchange, resulting in energy depletion, cellular Na+ overload and finally cellular Ca2 + overload [ 6, 9, II] due to a coupling of Na+- and Ca* + transport. SBU
Cardiovascular
Agents,
H821.
0 1992
P.O.B.
Academic
800320,
Press
6230
Limited
732
W. Scholz et al.
Especially in ischemic cardiac tissue where Na+ /H + exchange is the dominant pH regulating ion transport system [ 7, I,?] these pathophysiological events could lead to energy starvation, increased excitability and precipitation of cellular death. Based on this assumption inhibitors of Na+ /H + exchange like amiloride and EIPA should be able to interrupt this vicious cycle, to conserve cellular energy stores and to diminish excitability and necrosis in cardiac ischemia. In the present study the effect of the Na + /H + exchange inhibitors amiloride and EIPA was investigated in isolated working rat hearts subjected to ischemia-reperfusion injury. In addition, the action of both compounds on ischemia induced arrhythmias was examined in anaesthetized rats with ligation of the left coronary artery. Further experiments investigated the inhibition of Na+ influx via Na + /H + exchange by amiloride and EIPA in rabbit erythrocytes and the inhibitory effect of EIPA on intracellular acidification activated Na+/H + exchange, in cardiac myocytes. Methods Sodium [email protected] into erythrocytes New Zealand white rabbits (Ivanovas) were fed a rabbit standard chow with 2% cholesterol for six weeks to activate the Na + /H + exchange [ 131. This facilitates measurement of sodium influx via Na + /H + exchange by flame photometry in the erythrocytes. Blood was drawn from the ear artery of the rabbits and prevented from coagulating by the addition of 25 W/ml potassium heparin. Hematocrit was determined in duplicate by centrifugation. The initial sodium content of the erythrocytes was measured in 1001.11 aliquots. To determine the amiloride sensitive sodium influx into erythrocytes, 100~1 of blood were diluted into 5ml of buffer (in mmol/l: NaCl 140, KC1 3, sucrose 150, ouabain 0.1, Trishydroxy-methylaminomethane 20, pH 7.4) made hyperosmolar by the addition of sucrose and incubated for 60 min at 37’C. Subsequently, the erythrocytes were washed three centrifugation in icecold times by MgC$ouabain-solution (in mmol/l: MgC12 112, ouabain 0.1). For determination of intracellular Na+ content, the cells were hemolysed in distilled water
in a final volume of 2 .O ml, the cell membranes were centrifuged and the sodium concentration of the supernatant was measured by flame photometry with an Eppendorf flame photometer (Netheler + Hinz GmbH Hamburg) at a wavelength of 589nm. Net influx of sodium into the erythrocytes was calculated from the difference between the initial sodium content and the sodium content after the incubation. Amiloride sensitive Na+-influx was calculated from the difference between Na+ content of erythrocytes incubated with and without amiloride 3 x 10 - 4 mol/l.
Sodium-in+
into cardiac myocytes
Rat myocardial cells were isolated by trypsin digestion of hearts from neonatal rats. The cells were cultured on 35mm dishes and grown to confluency in Dulbeco’s Minimum Essential Medium (DMEM, GIBCOR) in an atmosphere with 10% CO1. After confluency the cells were used for measurement of 22Na+ influx. The cells were washed twice with Krebs-Ringer-solution buffered with Hepes/Tris (KRB) in which sodium chloride had been replaced by choline chloride (in mmoyl: choline chloride, 130, CaC12 1.5, KC1 5, MgCl, 1, Hepes 20, pH7.0 with Tris) and then incubated for twenty minutes at 37’C in the same buffer with added 0.1% bovine serum albumin (BSA) and lOmmol/l glucose. The culture dishes were then incubated for another 10min with Na+-propionate for cytosolic acidification and stimulation of Na+/H + exchange. The compounds were dissolved in 500pl/dish KRB in which 50% of the sodium chloride had been replaced by choline chloride containing additionally P@i/ml **Na+-bicarbonate, and 5-(N-ethyl-N-isopropyl)amiloride (EIPA). After the stimulation period sodium influx was terminated by washing the cells twice with an icecold stop solution (in mmol/l: MgCl, 0.1, Tris 10, pH 7.0). Subsequently, the cells were lyzed with 250~1 trichloric acid and scraped from the dishes. Radioactivity was determined in a Packard gamma counter. Measurement of Na + /H + exchange was performed with n = 6. Results are given in mean f S.D.
Na+/H+
Exchange
Isolated working rut heart preparation Isolated working rat heart preparation from Wistar rats of both sexes weighing 280-300 g were used in all experiments as described before [ 141. Animals were injected intraperitoneally with sodium heparin (500 U/kg) 1 h before killing. The hearts were excised and placed in icecold perfusion medium until contraction had ceased, approximately 5 s. The isolated hearts were then perfused via the aorta at a constant perfusion pressure equivalent to 65 mmHg (15) with a modified Krebs-Henseleit buffer of the following composition (mmol/l: NaCl 113.8, NaHC03 22, KC1 4.7, KH2P04 1.2, MgS04 1.1, CaC12 2.5, glucose 11.0, Napyruvate 2.0). The medium was gassed with 95% 02 plus 5% CO* at 37°C and pH7.4. The perfusate was not recirculated. A silicone balloon was then introduced into the left ventricle through the mitral valve. Preload was held at 7.4 mmHg and afterload as well as coronary perfusion were kept constant at 65 mmHg. Amiloride and EIPA were present in the respective experiments throughout the perfusion period. The compounds were dissolved in the medium without the use of any solvent. After the perfusion had commenced the hearts immediately began to pump and to perform pressure volume work (for detailed description see 14). The hearts were perfused for an initial period of 20min (control perfusion period). Acute regional myocardial ischemia was then produced by clamping the left anterior descending coronary artery close to its origin for 15 min (ischemic period). Thereafter, the clip was reopened and changes during reperfusion were monitored for 30min (reperfusion period). The following parameters were measured: left ventricular presure (LVP) via a Statham pressure transducer P23 Db, which on differentiation yielded LV dPldt,,, and heart rate (HR). Coronary flow (CF) was determined by an electromagnetic flow probe in the aortic cannula. An epicardial electrocardiogram recording was obtained during the reperfusion period via two silver electrodes attached to the heart. All parameters were recorded on a Brush 2600 recorder. Lactate release, lactate dehydrogenase (LDH) and creatine kinase (CK) activities
in Cardiac
Ischemia
733
were measured by spectrophotometrical measurements in the perfusate [II] collected from the coronary effluent. All heart weights are given in g of wet tissue. Wet weight of the hearts (approximately 1.2 g) was determined immediately after excision from the rats and before they were mounted in the perfusion chamber. Dry tissue weight (approximately 240 mg) was measured after drying the hearts for 12h at 110%. After the experiments, hearts were rapidly frozen in liquid nitrogen and stored at - 80%. For tissue analysis of lactate, glycogen and high energy phosphates (HEP) 500 mg of the left ventricle were put into 5 ml ice-cold HC104 (0.6 mol/l) and homogenized with an Ultra-Turrax (Junke & Kunkel, IkaWerk, Typ TP). Glycogen was hydrolized to glucose with amyloglycosidase (pH 4.8) [ 141. Lactate was assayed spectrophotometrically according to F. No11 [16]. ATP and creatine phosphate (CP) were measured according to Trauschold et al. and Heinz et al. published in Methods of Enzymatic Analysis [ 17, 181, Statistical analysis of the data obtained was performed with Student’s t-test for independent groups. Differences were considered significant if PcO.05. Results are given as mean values? S.E.M.
Anaesthetized rat Male Sprague-Dawley rats weighing 280444g were anaesthetized with sodium pentobarbital (60 mg/kg i.p.). The rats were intubated and respirated with room air lo-13 ml/kg at 54 breaths/min by a Rhema@ respirator (Frankfurt, Germany). A catheter was inserted into the left carotid artery and connected to a pressure transducer (Type P23Dd, Statham, Hato Ray, Puerto Rico) to record blood pressure with a Brush Mark 220 polygraph. The ECG was recorded from lead 2 and arrhythmias were evaluated according to the guidelines of the Lambeth conventions [19\ where: VPB’s (ventricular premature beats) are defined as discrete and identifiable premature QRS complexes (premature in relation to the P wave). All the VPB’s occurring in the ischemic period (30min) were counted. VT (ventricular tachycardia) was defined as a
734
w. Scholz et al.
run of four or more consecutive ventricular premature beats. The duration of VT, in seconds, was measured. VF (ventricular fibrillation) was defined as a signal for which individual QRS deflections could no longer be distinguished from one another. The duration of VF, in seconds, was measured. The heart rate was counted from the ECG. Thoracotomy for coronary artery ligation was performed at the fifth intercostal space. During an equilibration period of 15 min rats with spontaneous arrhythmias or a systolic blood pressure below 70 mmHg were rejected. The left coronary artery was then ligated for 30mins. For intravenous applications a catheter was placed into the right jugular vein. Amiloride (30 mg/kg) was given intraperitoneally 15 min before coronary artery occlusion. EIPA (1 mg/kg) was given intravenously as a bolus 5 min before coronary artery occlusion, followed by a constant infusion of 0.01 mg/kg/min. The results are presented as mean + S.E.M. Differences between mean values were analysed with the Student’s t-test and differences between numbers of animals with arrhythmia were analysed with the x2-test.
Results Sodium inJEux into erythrocytes As shown in Figure 1 both Na+/H+ exchange inhibitors decreased sodium influx into rabbit erythrocytes in a dose dependent manner. The IC,, of amiloride was 1.5 x 10e5 mol/l.
3-
(mmolll) FIGURE 1. Concentration dependent inhibition at Na’ influx via Na’ /H + exchange by amiloride and EIPA in rabbit erythrocytes (mean + s E.M., n = 6) (- - -: Amiloride; EIPA).
The derivative of amiloride, EIPA, was about 500 times more potent in this assay with an ICsO of 5 x lo-* mol/l.
Sodium influx into rat myocardial cells Rat cardiac myocytes acidified with sodium propionate showed a marked increase of Na* influx during the incubation period as compared with controls (Fig. 2). While there was hardly any inhibition of Na+ influx by 10 ’ mol/l EIPA under control conditions, the increase of Na + influx caused by propionate was totally inhibited by EIPA 10 m6 mol/l. Almost no inhibition of Na+ influx in propionate treated myocytes could be observed with 10-j mol/l EIPA.
Isolated working
rat heart preparation
To investigate a possible protective role of Na + /H + exchange inhibition under ischemic conditions isolated rat hearts were subjected to ischemia and reperfusion. In these experiments all six untreated hearts suffered ventricular fibrillation with an incidence of 100% and a mean duration of about 17min during the reperfusion period of 30 min (Fig. 3). Perfusion of the isolated hearts with lo-’ mol/l amiloride or lo-’ mol/l EIPA did not cause any change. However, when the concentration of amiloride or EIPA was increased in the perfusion medium to 10 - ’ or 3 x 10 ’ mol/l, respectively, the incidence of ventricular fibrillation as well as the duration of the fibrillation was markedly decreased. EIPA at 10 m6 mol/l totally suppressed ventricular fibrillation (Fig. 3). During ischemia LV dPldt was significantly increased by about 35 %, while coronary flow (CF) was slightly decreased with 10. ‘, mol/l amiloride in comparison with control hearts (Fig. 4). The release of LDH and CK from the hearts into the venous effluent was slightly diminished during ischemia (Fig. 4). Analysis of the energy stores of the treated hearts at the end of the experiments showed significant differences in the levels of glycogen, ATP and CP as compared to control hearts. Tissue contents of glycogen, ATP and CP were increased by 48%) 200% and 200% respectively from 88.4 + 3.4, 5.3 + 0.5 and
Na+/H+
Exchange
in
Cardiac
735
Ischemia
1000 900 600 aJ 700 'j h 600 2xX3 bM 300 200 100 o_.
_.
control
Proplonote
+EIPA 10-S
FIGURE 2. Inhibition ation by Na+ propionate.
2oi T
of “Na+ Relative
mflux 22 Na+
Propkmote +EIPA lo-’
(mol/l)
into rat cardiac myocytes by EIPA with and without intracellular acidificinflux given as counts per min per culture plate (mean + s D , n = 6).
T
( Cardiodynamics
1Venous
effhxnt
( -dial
tii
FIGURE 5. Effect of EIPA 10 -” mol/l on isolated working rat hearts during &hernia and rcperfusion. Cardiodynamics during ischemia, enzymes in the venous effluent at the beginning of reperfusion and HEP stores after &hernia and reperfusion (mean + s E M , R = 6 in each group, ‘PCO.05).
MolA FIGURE 3. Effect of am&ride duration of ventricular fibrillation hearts during reperfusion (mean group) (I: Control: a: Amiloride;
Proplonote +EIPA 10-s
and EIPA on the in isolated working rat f S.E M , R = 6 in each 0: EIPA).
/enwseffluent
Myoa7rdiol tissue I
*
FI(GURE 4. Effect of amiloride 10 - ’ mol/l on isolated working rat hearts during ischemia and reperfusion Cardiodynamirs during &hernia, enzymes in the venous effluent at the beginning of reperfusion and HEP stores after ix-hernia and reperfusion (meant s E M . n = 6 in each group, *P
