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REPERFUSED ISOLATED WORKING RAT HEARTS: INTERACTION WITH GLIBENCLAMIDE
Pharmacological Research, Vol. 39, No. 3, 1999 Article No. phrs.1998.0430, available online at http:rrwww.idealibrary.com on
CARDIOPROTECTIVE EFFECT OF ENANTIOMERS OF CICLETANINE ( BN50417, BN50418 ) IN ISCHAEMIC r REPERFUSED ISOLATED WORKING RAT HEARTS: INTERACTION WITH GLIBENCLAMIDE b ´ P. FERDINANDY a,U , Z. SZILVASSY , T. CSONT a , M. KOLTAI c,& and M.T. DROY-LEFAIX c a
Department of Biochemistry, Albert Szent-Gyorgyi Uni¨ ersity, Medical School, PO Box 415, ¨ H-6701 Szeged, Hungary, bDepartment of Medicine, Albert Szent-Gyorgyi Uni¨ ersity, Medical ¨ School, PO Box 415, H-6701 Szeged, Hungary, and c IPSEN-Beaufour, Paris, France Accepted 13 No¨ ember 1998
In the present study, interaction of the ATP-sensitive Kq-channel blocker glibenclamide with enantiomers of the antihypertensive drug, cicletanine, was studied on ischaemic myocardial function, lactate-dehydrogenase ŽLDH. release, and early reperfusion-induced ventricular fibrillation ŽVF.. Isolated working rat hearts subjected to 10-min coronary artery occlusion followed by 2-min reperfusion were perfused with 1.5= 10y5 ]6.0= 10y5 M D-cicletanine wqx ŽBN50417. and L-cicletanine wyx ŽBN50418., respectively. Their interaction with 10y7 M glibenclamide was also studied. The most effective concentration of BN50418 Ž3 = 10y5 M. increased ischaemic aortic flow ŽAF. from its non-treated control value of 20.3" 1.16 to 30.3" 2.6 ml miny1 Ž P- 0.01., decreased left ventricular end-diastolic pressure ŽLVEDP. from 1.81" 0.05 to 0.97" 0.08 kPa Ž P- 0.001., attenuated ischaemiainduced increase in LDH leakage from 164 " 41 to 14.8" 20 mU miny1 gy1 wet wt. Ž P- 0.01. at the 10th-min of coronary occlusion, and reduced VF upon reperfusion. Glibenclamide did not considerably affect cardiac performance, however, it inhibited the anti-ischaemic but not the antiarrhythmic effect of BN50418. BN50417 Ž3 = 10y5 M. tended to improve ischaemic AF to 24.2" 1.1 ml miny1 , and significantly attenuated ischaemiainduced increase in LVEDP to 1.3" 0.08 kPa Ž P- 0.01., relative increase in LDH release to 29.4" 44 mU miny1 gy1 Ž P- 0.05., and alleviated reperfusion-induced VF. Glibenclamide abolished the anti-ischaemic and antiarrhythmic effect of BN50417. The cardioprotective effect of both enantiomers of cicletanine involves a glibenclamide-sensitive mechanism, however, the antiarrhythmic effect of BN50418 is not glibenclamide sensitive. BN50418 is the more potent enantiomer of cicletanine in terms of its cardioprotective effect. Q 1999 The Italian Pharmacological Society KEY WORDS: cicletanine, enantiomers, glibenclamide, myocardial function, ventricular fibrillation, coronary occlusion.
INTRODUCTION Consequences of sustained arterial hypertension Že.g. ischaemic heart disease, etc.. are among the major causes of mortality in the civilised society. Therefore, development of antihypertensive agents with potential cardioprotective effect is of particular interest. Cicletanine racemate w1,3-dihydro-3-Ž4-chloU
Corresponding author. Cardiovascular Disease Research Group, 4-62 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta, T6G 2S2, Canada. & Deceased. 1043]6618r99r030225]07r$30.00r0
rophenyl.-7-hydroxy-6-methylfuro Ž3,4-C. pyridinex an antihypertensive agent was shown to possess anti-ischaemic, antiarrhythmic and tissue protective properties in the ischaemicrreperfused myocardium in animal models w1]4x. The drug is known to have several pharmacological actions. It has been shown to inhibit low K m Ca2q-calmodulin-dependent cGMP phosphodiesterase ŽPDE. and cGMP-selective PDE w5, 6x, to increase myocardial cGMP-content w7x, and to stimulate prostacyclin release w8x. Cyclic GMP was shown to modulate several potassium channels w9, 10x. We have recently shown that the anti-ischaemic but not the antiarrhythmic effect of cicletanine raceQ1999 The Italian Pharmacological Society
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mate involves activation of ATP-sensitive potassium channels ŽK ATP ., and that the combination of cicletanine with cromakalim, a potassium channel opener, produces a marked anti-ischaemic and antiarrhythmic effect in the isolated working rat heart w11x. Opening of K ATP -channels leads to a significant hypotensive effect via relaxation of the vascular smooth muscle w12, 13x, and improves functional recovery of ischaemicrreperfused myocardium w14]16x. These effects are antagonised by glibenclamide, a blocker of K ATP . Nevertheless, the therapeutic potential and the mechanism of action of enantiomers of cicletanine have not been studied previously. Therefore, the goals of the present study were Ži. to assess the potential cardioprotective effect of enantiomers of cicletanine; and Žii. to examine the role of a glibenclamide sensitive mechanism in the anti-ischaemic and antiarrhythmic effects of cicletanine enantiomers.
MATERIALS AND METHODS The investigation conforms with the Guide for the care and use of laboratory animals published by the US National Institutes of Health ŽNIH publication No 85-23, revised 1985..
Chemicals
Enantiomers of cicletanine ŽD-Cicletaninewqx ŽBN50417. and L-Cicletanine wyx ŽBN50418., Institute Henri Beaufour, Paris, France., and glibenclamide ŽSigma, St Louis, MO. were dissolved in dimethyl-sulfoxide ŽDMSO. and added to the perfusion fluid throughout the experiments. The final concentration of DMSO was 2 = 10y2 % Žvrv. in each group. Other chemicals used were of analytical grade.
Isolated heart preparation Male Wistar rats Ž300]360-g. fed standard laboratory chow and tap water ad libitum were anaesthetised with diethylether and injected intravenously with 500 U kgy1 heparin. After 30 s, hearts were excised and cannulated through the aorta, and perfused in the Langendorff mode at a constant perfusion pressure of 100 cm water Ž9.8 kPa. for 10 min. During this period, the left atrium was cannulated, and a suture was placed around the left main coronary artery close to its origin, allowing regional ischaemia and reperfusion to be induced as described earlier w1x. The heart was then converted to a working preparation w17, 18x for 10 min. The preparation was perfused at 378C with Krebs]Henseleit bicarbonate buffer containing Žin mM. NaCl 118, KCl 4.3, CaCl 2 2.4, NaHCO3 25, KH 2 PO4 1.2, MgSO4 1.2 and glucose 11.1, gassed with 95% O 2
and 5% CO 2 Ž P O 2 : 63.3" 6.0 kPa; P CO 2 : 4.0" 0.19 kPa., where the buffer first enters the cannulated left atrium at a pressure equivalent to 17 cm water Ž1.7 kPa., then passes to the left ventricle, from which it is spontaneously ejected through the aortic cannula against a pressure equivalent to 100 cm water Ž9.8 kPa..
Measurements Heart rate ŽHR. derived from the left ventricular pressure curve, coronary flow ŽCF. measured by collecting effluent from the right atrium in a measuring cylinder for a timed period, aortic flow ŽAF. measured by a calibrated rotameter ŽKDG Flowmeters, Sussex, England., left ventricular developed pressure ŽLVDP. counted as peak systolic pressure minus left ventricular end-diastolic pressure ŽLVEDP., qd Prdt max , and LVEDP were recorded. Ventricular pressure was measured by a pressure transducer ŽB. Braun, Melsungen, Germany. connected to a small polyethylene catheter inserted into the left ventricle through the left atrial cannula as described w11x. Epicardial electrogram was obtained by using two silver electrodes attached directly to the heart. Ventricular fibrillation ŽVF. was determined from the epicardial electrogram and the left ventricular pressure curve according to the recommendations of Lambeth Conventions w19x. Data were on line digitalised, recorded, and stored within an IBM PC. Coronary effluents were assayed for lactate dehydrogenase ŽLDH. activity by means of an automatic analyser Ž Hitachi-911 . using Boehringer]Mannheim kits. LDH release was calculated as ŽLDH ischaemic y LDH preischaemic . = CFrheart wet wt. and expressed as mU miny1 gy1 as described earlier w20x.
Experimental design Hearts were divided into ten groups and perfused Ž1. with the appropriate dilution of the solvent DMSO Žcontrol, n s 12.; Ž2. 10y7 M Ž n s 9. glibenclamide; Ž3. 1.5= 10y5 M Ž n s 9.; Ž4. 3 = 10y5 M Ž n s 8.; Ž5. 6 = 10y5 M Ž n s 10. BN50417; Ž6. 1.5= 10y5 M Ž n s 9.; Ž7. 3 = 10y5 M Ž n s 9.; Ž8. 6 = 10y5 y7 M Ž n s 10. BN50418; the combination of Ž9. 10 M y5 glibenclamide with 6 = 10 M BN50417 Ž n s 10.; and Ž10. with 6 = 10y5 M BN50418 Ž n s 10.. The concentration range of enantiomers of cicletanine was determined according to our previous studies w1, 2, 4, 7, 11x. To study the involvement of K ATP channels in the action of enantiomers of cicletanine, 10y7 M glibenclamide was used, since this concentration of the drug alone did not significantly affect myocardial function, however, it abolished the cardioprotective effect of the most effective concentration of the K ATP opener cromakalim in this model as shown in our previous studies w11x. After 10 min of aerobic working perfusion hearts of all groups were subjected to 10-min coronary
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Table I Effects of enantiomers of cicletanine (BN50417, BN50418), glibenclamide, and their combination on heart rate (HR), coronary flow (CF), and Id P r d t max before ischaemia and at the 10th-min of coronary occlusion in isolated working rat hearts Treatment
Solvent Glibenclamide BN50417 BN50418 BN50417q glibenclamide BN50418q glibenclamide
Concentration
10y7 M 1.5y 10y5 M 3.0y 10y5 M 6.0= 10y5 M 1.5= 10y5 M 3.0= 10y5 M 6.0= 10y5 M 3.0= 10y5 M 10y7 M 3.0= 10y5 M 10y7 M
Before ischaemia
n
Coronary occlusion
HR Ž(b.p.m.)
CF (ml miny 1)
ydP r dtma x (kPa sy 1)
12 9 9 8 10 9 9 10 10
273 " 5 283 " 3 281 " 6 290 " 7 282 " 4 276 " 10 274 " 11 290 " 6 278 " 6
22.5" 1.2 22.3" 1.0 22.0" 1.0 24.6" 1.4 24.4" 1.5 22.2" 1.6 22.7" 1.9 24.3" 0.9 22.7" 0.8
422 " 25 458 " 20 424 " 26 408 " 17 376 " 22 403 " 30 424 " 33 450 " 27 478 " 24
10
283 " 5
24.2" 0.8
455 " 22
HRCF (ml miny 1)
ydP r dtma x (kPa sy 1)
265 " 4 273 " 7 270 " 4 262 " 6 266 " 3 264 " 7 264 " 8 275 " 6 271 " 4
15.4" 1.7 13.5" 0.5 14.6" 1.5 18.8" 2.6 14.7" 0.8 17.7" 2.2 18.4" 2.9 16.8" 0.5 16.2" 0.5
274 " 10 325 " 18 320 " 17 328 " 20 288 " 13 411 " 27U 424 " 26U 320 " 13 302 " 9
268 " 5
17.3" 1.0
312 " 6
(b.p.m.)
U Data are means " SEM; Ž P- 0.05. show significant difference compared to solvent-treated hearts.
occlusion followed by 2-min reperfusion. Ten-minute coronary occlusion was chosen for assessing myocardial function, because this short-term regional ischaemia induced considerable deterioration of myocardial function, but it did not result in ischaemiainduced arrhythmias that would have disturbed measures of myocardial function w1x. However, reperfusion after a 10-min regional ischaemia triggered high incidence of reperfusion-induced VF, thus allowing us to examine any antiarrhythmic effect of the drugs against early reperfusion-induced VF.
Statistical analysis
Data were expressed as means " SE of the mean ŽSEM.. A one-way analysis of variance ŽANOVA. was initially carried out to test any difference between the mean values of all groups. If a difference was established, all groups were compared to the control solvent-treated group using a modified t-test according to the Bonferroni method for simultaneous multiple comparisons w21x. An analogue procedure was followed for distribution of binomially distributed variables, such as VF. An overall x 2-test for a 2 = n table was constructed followed by a sequence of 2 = 2 x 2-tests to compare individual groups.
RESULTS
Cardiac function As compared with normoxic values, 10-min regional ischaemia slightly reduced HR, and markedly decreased CF ŽTable I. in the solvent treated group. Neither concentration of enantiomers of cicletanine, glibenclamide, or their combination influenced HR and CF. Enantiomers of cicletanine or glibenclamide did not affect AF significantly before ischaemia, except
the highest dose of BN50417, which moderately decreased non-ischaemic AF. AF was markedly deteriorated by coronary occlusion in the solvent-treated control hearts. BN50417 and glibenclamide did not change ischaemic AF. BN50418 Ž1.5= 10y5 and 3.0 = 10y5 M. significantly improved the parameter, however, the combination of the most effective concentration of BN50418 with glibenclamide did not show any beneficial effect ŽFig. 1.. Similarly to that found with AF, enantiomers of cicletanine or glibenclamide did not affect LVDP significantly before ischaemia, except the highest dose of BN50417, which moderately decreased nonischaemic LVDP. Ischaemic LVDP deteriorated by coronary occlusion was markedly improved by 3.0= 10y5 M BN50417 and by 1.5= 10y5 M and 3.0= 10y5 M BN50418. Glibenclamide, however, abolished the protective effect of the most effective concentration of both enantiomers of cicletanine ŽFig. 1.. Before ischaemia, index of cardiac contractility, qd Prdt max , was moderately decreased by 6 = 10y5 y5 M BN50417. During coronary occlusion, 3 = 10 M BN50417, and all concentrations of BN50418 improved qd Prdt max . Glibenclamide inhibited the effect of 3 = 10y5 M BN50417 and BN50418, respectively ŽFig. 2.. A similar pattern was seen with yd Prdt max ŽTable I.. LVEDP was not influenced by the drugs before ischaemia, however, 3 = 10y5 M BN50417 and all concentrations of BN50418 significantly attenuated elevation of LVEDP due to coronary occlusion. Glibenclamide abolished the beneficial effect of the drugs ŽFig. 2..
LDH release LDH release, a marker of tissue damage during ischaemic stress, was studied with the most effective concentrations of enantiomers of cicletanine. Both
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Fig. 1. Effects of different concentrations of enantiomers of cicletanine, i.e. BN50417 ŽDC, m M., BN50418 ŽLC, m M., and 10y7 M glibenclamide ŽG., as well as their combination on aortic flow ŽAF. and left ventricular developed pressure ŽLVDP. before ischaemia and at the 10th-min of coronary occlusion in isolated working rat hearts. U Ž P- 0.05. designates significant changes of the parameters when compared to solvent-treated control ŽCont. hearts.
3 = 10y5 M BN50417 and 3 = 10y5 M BN50418 significantly decreased LDH leakage during coronary occlusion, however, in the presence of 10y7 M glibenclamide, this effect was not seen ŽFig. 3..
Reperfusion-induced VF Reperfusion after a 10-min coronary artery occlusion resulted in 100% incidence of VF, which occurred immediately upon reflow in the solventtreated hearts ŽFig. 4.. BN50417, in concentrations of 3 = 10y5 and 6 = 10y5 M, significantly decreased the incidence of VF, and glibenclamide abolished its antiarrhythmic effect. All concentrations of BN50418 markedly attenuated occurrence of VF, however, glibenclamide did not significantly influence the antiarrhythmic effect of 3 = 10y5 M BN50418.
DISCUSSION Our results show that both enantiomers of the antihypertensive drug, cicletanine, significantly improve myocardial function deteriorated by 10-min coronary
occlusion, markedly decrease ischaemic LDH release, and reduce the incidence of VF upon early reperfusion. The concentration of 3 = 10y5 M is the most effective in the case of both enantiomers, however, BN50418 shows more potent anti-ischaemic and antiarrhythmic effect. The anti-ischaemic effect of both enantiomers is blocked by glibenclamide, however, in contrast to the slight antiarrhythmic effect of BN50417, the potent antiarrhythmic effect of BN50418 is not sensitive to glibenclamide treatment. The present study is in accordance with our previous observations in isolated working rat heart w1x and in conscious rabbits w7x demonstrating that cicletanine racemate attenuates functional consequences of coronary occlusion and pacing-induced global myocardial ischaemia, and confirms our earlier study showing the involvement of a glibenclamide-sensitive mechanism in the action of cicletanine racemate w11x. This study revealed, however, that BN50418 is the more potent enantiomer of cicletanine. We have selected the concentration of 10y7 M of glibenclamide for studying the interaction of K ATP block-
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Fig. 2. Effects of different concentrations of enantiomers of cicletanine, i.e. BN50417 ŽDC, m M., BN50418 ŽLC, m M., and 10y7 M glibenclamide ŽG., as well as their combination on qd Prdt max and left ventricular end-diastolic pressure ŽLVEDP. before ischaemia and at the 10th-min of coronary occlusion in isolated working rat hearts. U Ž P- 0.05. designates significant changes of the parameters when compared to solvent-treated control ŽCont. hearts.
Fig. 3. Effects of enantiomers of cicletanine, i.e. 3 = 10y5 y5 M BN50417 ŽDC30., 3 = 10 M BN50418 ŽLC30., and y7 Ž . 10 M glibenclamide G , as well as their combination on lactate dehydrogenase ŽLDH. release at the 10th-min of coronary occlusion in isolated working rat hearts. U Ž P 0.05. designates significant change of the parameter when compared to solvent-treated control ŽCont. group.
Fig. 4. Effects of different concentrations of enantiomers of cicletanine, i.e. BN50417 ŽDC, m M., BN50418 ŽLC, m M., and 10y7 M glibenclamide ŽG., as well as their combination on the incidence of reperfusion-induced ventricular fibrillation ŽVF. in isolated working rat hearts. U Ž P- 0.05. designates significant change of the parameter when compared to solvent-treated control ŽCont. group.
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ade with enantiomers of cicletanine, since we have previously showed, that this concentration did not considerably influence cardiac performance, but abolished the antiischaemic effect of the optimal concentration Ž10y6 M. of the K ATP opener cromakalim, thereby suggesting a blockade of K ATP in the myocardium produced by 10y7 M glibenclamide in the isolated working rat heart w11x. Although glibenclamide is widely used and accepted as the most specific blocker of K ATP , it should be noted that the effects of the drug are likely not solely related to its actions on K ATP w22, 23x, therefore, the results with glibenclamide should be interpreted very cautiously. Our present study shows that 10y7 M glibenclamide abolishes the beneficial effects of both enantiomers of cicletanine during ischaemia, but does not affect the marked antiarrhythmic effect of BN50418, however, inhibits the slight antiarrhythmic effect of BN50417. This finding indicates that opening of K ATP may be involved in the anti-ischaemic, but not in the antiarrhythmic effect of the BN50418. Cidetanine racemate was shown to possess intracellular Ca2q antagonistic property and prostacyclin releasing effect w8, 24, 25x, therefore, the antiarrhythmic effect of BN50418 may be attributed to these actions. Both the anti-ischaemic and antiarrhythmic effect of the less active cicletanine enantiomer, BN50417, seems to be based at least in part on glibenclamide-sensitive K ATP channel activation. Potassium channel opener agents have been shown to possess dual effect on cardiac arrhythmias. Shortening of action potential due to K ATP -opening may lead to an increased susceptibility to re-entry arrhythmias, and may decrease the probability of occurrence of early after-depolarisation. Thus, K ATP openers may exert either arrhythmogenic or antiarrhythmic action depending on the experimental model used w26]28x. Our present results indicate that in contrast to the marked anti-ischaemic effect of enantiomers of cicletanine, the K ATP activator effect of the compounds may have minor contribution to the potent antiarrhythmic effect of cicletanine racemate. The mechanism by which enantiomers of cicletanine may lead to opening of the glibenclamide-sensitive K ATP channel is not known. The effects of enantiomers of cicletanine in the isolated heart, especially that of BN50418, on AF, LVDP, qd Prdt max , and LVEDP is similar to that of cromakalim w11, 13, 14x in the same experimental model, and glibenclamide blocks the effect of both drugs, however, in contrast to cromakalim, enantiomers of cicletanine does not increase CF. This might indicate, that the possible K ATP -opener effect of enantiomers of cicletanine may be more specific on myocardial cells than on smooth muscle cells. Accordingly, opening of K ATP produced by cicletanine racemate on smooth muscle cells is not supported by Noak and Deitmer w24x, since no cicletanine-induced
whole-cell Kq currents were observed in guinea pig portal vein smooth muscle cell, and glibenclamide did not significantly block the cicletanine-induced relaxation in rat, guinea-pig, and rabbit portal vein preparations. Enantiomers of cicletanine may open glibenclamide-sensitive K ATP channels indirectly through modulation of myocardial cyclic nucleotide metabolism. Cicletanine racemate has been shown to inhibit low K m Ca-calmodulin-dependent PDE ŽPDE I. and cGMP specific PDE ŽPDE V. w5, 6x, thereby leading to an increase in cardiac cGMP level w7x. Increased cGMP concentration may directly stimulate potassium channels w9, 10x, or in a certain cell compartment it may give rise to cAMPdependent phosphorylation of K ATP w29x through inhibition of cGMP-inhibited cAMP-specific PDE ŽPDE III., an enzyme of high activity in myocardial cells w30x. Nevertheless, the cardioprotective effect of myocardial cGMP is well established w31]33x. In summary, this is the first study on enantiomers of the antihypertensive drug cicletanine. Our present results suggest that both enantiomers of the drug show anti-ischaemic and antiarrhythmic effect in the rat heart, and that L-cicletanine ŽBN50418. is the more active enantiomer. The anti-ischaemic but not the antiarrhythmic effect of BN50418 may involve a glibenclamide-sensitive mechanism, probably opening of K ATP . BN50418, the active enantiomer of cicletanine, may confer beneficial effects on patients suffering from hypertension and consequent ischaemic heart disease.
ACKNOWLEDGEMENTS This study was supported by grants form IPSEN, Paris, France; National Scientific Research Found ŽOTKA F19946, D23736., National Technical Development Committee ŽOMFB 05201950616., Ministry of Education ŽFKFP 1284r1997., and the Hungarian Space Research Office, Budapest, Hungary.
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CARDIOPROTECTIVE EFFECT OF ENANTIOMERS OF CICLETANINE ( BN50417, BN50418 ) IN ISCHAEMIC r REPERFUSED ISOLATED WORKING RAT HEARTS: INTERACTION WITH GLIBENCLAMIDE b ´ P. FERDINANDY a,U , Z. SZILVASSY , T. CSONT a , M. KOLTAI c,& and M.T. DROY-LEFAIX c a
Department of Biochemistry, Albert Szent-Gyorgyi Uni¨ ersity, Medical School, PO Box 415, ¨ H-6701 Szeged, Hungary, bDepartment of Medicine, Albert Szent-Gyorgyi Uni¨ ersity, Medical ¨ School, PO Box 415, H-6701 Szeged, Hungary, and c IPSEN-Beaufour, Paris, France Accepted 13 No¨ ember 1998
In the present study, interaction of the ATP-sensitive Kq-channel blocker glibenclamide with enantiomers of the antihypertensive drug, cicletanine, was studied on ischaemic myocardial function, lactate-dehydrogenase ŽLDH. release, and early reperfusion-induced ventricular fibrillation ŽVF.. Isolated working rat hearts subjected to 10-min coronary artery occlusion followed by 2-min reperfusion were perfused with 1.5= 10y5 ]6.0= 10y5 M D-cicletanine wqx ŽBN50417. and L-cicletanine wyx ŽBN50418., respectively. Their interaction with 10y7 M glibenclamide was also studied. The most effective concentration of BN50418 Ž3 = 10y5 M. increased ischaemic aortic flow ŽAF. from its non-treated control value of 20.3" 1.16 to 30.3" 2.6 ml miny1 Ž P- 0.01., decreased left ventricular end-diastolic pressure ŽLVEDP. from 1.81" 0.05 to 0.97" 0.08 kPa Ž P- 0.001., attenuated ischaemiainduced increase in LDH leakage from 164 " 41 to 14.8" 20 mU miny1 gy1 wet wt. Ž P- 0.01. at the 10th-min of coronary occlusion, and reduced VF upon reperfusion. Glibenclamide did not considerably affect cardiac performance, however, it inhibited the anti-ischaemic but not the antiarrhythmic effect of BN50418. BN50417 Ž3 = 10y5 M. tended to improve ischaemic AF to 24.2" 1.1 ml miny1 , and significantly attenuated ischaemiainduced increase in LVEDP to 1.3" 0.08 kPa Ž P- 0.01., relative increase in LDH release to 29.4" 44 mU miny1 gy1 Ž P- 0.05., and alleviated reperfusion-induced VF. Glibenclamide abolished the anti-ischaemic and antiarrhythmic effect of BN50417. The cardioprotective effect of both enantiomers of cicletanine involves a glibenclamide-sensitive mechanism, however, the antiarrhythmic effect of BN50418 is not glibenclamide sensitive. BN50418 is the more potent enantiomer of cicletanine in terms of its cardioprotective effect. Q 1999 The Italian Pharmacological Society KEY WORDS: cicletanine, enantiomers, glibenclamide, myocardial function, ventricular fibrillation, coronary occlusion.
INTRODUCTION Consequences of sustained arterial hypertension Že.g. ischaemic heart disease, etc.. are among the major causes of mortality in the civilised society. Therefore, development of antihypertensive agents with potential cardioprotective effect is of particular interest. Cicletanine racemate w1,3-dihydro-3-Ž4-chloU
Corresponding author. Cardiovascular Disease Research Group, 4-62 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta, T6G 2S2, Canada. & Deceased. 1043]6618r99r030225]07r$30.00r0
rophenyl.-7-hydroxy-6-methylfuro Ž3,4-C. pyridinex an antihypertensive agent was shown to possess anti-ischaemic, antiarrhythmic and tissue protective properties in the ischaemicrreperfused myocardium in animal models w1]4x. The drug is known to have several pharmacological actions. It has been shown to inhibit low K m Ca2q-calmodulin-dependent cGMP phosphodiesterase ŽPDE. and cGMP-selective PDE w5, 6x, to increase myocardial cGMP-content w7x, and to stimulate prostacyclin release w8x. Cyclic GMP was shown to modulate several potassium channels w9, 10x. We have recently shown that the anti-ischaemic but not the antiarrhythmic effect of cicletanine raceQ1999 The Italian Pharmacological Society
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mate involves activation of ATP-sensitive potassium channels ŽK ATP ., and that the combination of cicletanine with cromakalim, a potassium channel opener, produces a marked anti-ischaemic and antiarrhythmic effect in the isolated working rat heart w11x. Opening of K ATP -channels leads to a significant hypotensive effect via relaxation of the vascular smooth muscle w12, 13x, and improves functional recovery of ischaemicrreperfused myocardium w14]16x. These effects are antagonised by glibenclamide, a blocker of K ATP . Nevertheless, the therapeutic potential and the mechanism of action of enantiomers of cicletanine have not been studied previously. Therefore, the goals of the present study were Ži. to assess the potential cardioprotective effect of enantiomers of cicletanine; and Žii. to examine the role of a glibenclamide sensitive mechanism in the anti-ischaemic and antiarrhythmic effects of cicletanine enantiomers.
MATERIALS AND METHODS The investigation conforms with the Guide for the care and use of laboratory animals published by the US National Institutes of Health ŽNIH publication No 85-23, revised 1985..
Chemicals
Enantiomers of cicletanine ŽD-Cicletaninewqx ŽBN50417. and L-Cicletanine wyx ŽBN50418., Institute Henri Beaufour, Paris, France., and glibenclamide ŽSigma, St Louis, MO. were dissolved in dimethyl-sulfoxide ŽDMSO. and added to the perfusion fluid throughout the experiments. The final concentration of DMSO was 2 = 10y2 % Žvrv. in each group. Other chemicals used were of analytical grade.
Isolated heart preparation Male Wistar rats Ž300]360-g. fed standard laboratory chow and tap water ad libitum were anaesthetised with diethylether and injected intravenously with 500 U kgy1 heparin. After 30 s, hearts were excised and cannulated through the aorta, and perfused in the Langendorff mode at a constant perfusion pressure of 100 cm water Ž9.8 kPa. for 10 min. During this period, the left atrium was cannulated, and a suture was placed around the left main coronary artery close to its origin, allowing regional ischaemia and reperfusion to be induced as described earlier w1x. The heart was then converted to a working preparation w17, 18x for 10 min. The preparation was perfused at 378C with Krebs]Henseleit bicarbonate buffer containing Žin mM. NaCl 118, KCl 4.3, CaCl 2 2.4, NaHCO3 25, KH 2 PO4 1.2, MgSO4 1.2 and glucose 11.1, gassed with 95% O 2
and 5% CO 2 Ž P O 2 : 63.3" 6.0 kPa; P CO 2 : 4.0" 0.19 kPa., where the buffer first enters the cannulated left atrium at a pressure equivalent to 17 cm water Ž1.7 kPa., then passes to the left ventricle, from which it is spontaneously ejected through the aortic cannula against a pressure equivalent to 100 cm water Ž9.8 kPa..
Measurements Heart rate ŽHR. derived from the left ventricular pressure curve, coronary flow ŽCF. measured by collecting effluent from the right atrium in a measuring cylinder for a timed period, aortic flow ŽAF. measured by a calibrated rotameter ŽKDG Flowmeters, Sussex, England., left ventricular developed pressure ŽLVDP. counted as peak systolic pressure minus left ventricular end-diastolic pressure ŽLVEDP., qd Prdt max , and LVEDP were recorded. Ventricular pressure was measured by a pressure transducer ŽB. Braun, Melsungen, Germany. connected to a small polyethylene catheter inserted into the left ventricle through the left atrial cannula as described w11x. Epicardial electrogram was obtained by using two silver electrodes attached directly to the heart. Ventricular fibrillation ŽVF. was determined from the epicardial electrogram and the left ventricular pressure curve according to the recommendations of Lambeth Conventions w19x. Data were on line digitalised, recorded, and stored within an IBM PC. Coronary effluents were assayed for lactate dehydrogenase ŽLDH. activity by means of an automatic analyser Ž Hitachi-911 . using Boehringer]Mannheim kits. LDH release was calculated as ŽLDH ischaemic y LDH preischaemic . = CFrheart wet wt. and expressed as mU miny1 gy1 as described earlier w20x.
Experimental design Hearts were divided into ten groups and perfused Ž1. with the appropriate dilution of the solvent DMSO Žcontrol, n s 12.; Ž2. 10y7 M Ž n s 9. glibenclamide; Ž3. 1.5= 10y5 M Ž n s 9.; Ž4. 3 = 10y5 M Ž n s 8.; Ž5. 6 = 10y5 M Ž n s 10. BN50417; Ž6. 1.5= 10y5 M Ž n s 9.; Ž7. 3 = 10y5 M Ž n s 9.; Ž8. 6 = 10y5 y7 M Ž n s 10. BN50418; the combination of Ž9. 10 M y5 glibenclamide with 6 = 10 M BN50417 Ž n s 10.; and Ž10. with 6 = 10y5 M BN50418 Ž n s 10.. The concentration range of enantiomers of cicletanine was determined according to our previous studies w1, 2, 4, 7, 11x. To study the involvement of K ATP channels in the action of enantiomers of cicletanine, 10y7 M glibenclamide was used, since this concentration of the drug alone did not significantly affect myocardial function, however, it abolished the cardioprotective effect of the most effective concentration of the K ATP opener cromakalim in this model as shown in our previous studies w11x. After 10 min of aerobic working perfusion hearts of all groups were subjected to 10-min coronary
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Table I Effects of enantiomers of cicletanine (BN50417, BN50418), glibenclamide, and their combination on heart rate (HR), coronary flow (CF), and Id P r d t max before ischaemia and at the 10th-min of coronary occlusion in isolated working rat hearts Treatment
Solvent Glibenclamide BN50417 BN50418 BN50417q glibenclamide BN50418q glibenclamide
Concentration
10y7 M 1.5y 10y5 M 3.0y 10y5 M 6.0= 10y5 M 1.5= 10y5 M 3.0= 10y5 M 6.0= 10y5 M 3.0= 10y5 M 10y7 M 3.0= 10y5 M 10y7 M
Before ischaemia
n
Coronary occlusion
HR Ž(b.p.m.)
CF (ml miny 1)
ydP r dtma x (kPa sy 1)
12 9 9 8 10 9 9 10 10
273 " 5 283 " 3 281 " 6 290 " 7 282 " 4 276 " 10 274 " 11 290 " 6 278 " 6
22.5" 1.2 22.3" 1.0 22.0" 1.0 24.6" 1.4 24.4" 1.5 22.2" 1.6 22.7" 1.9 24.3" 0.9 22.7" 0.8
422 " 25 458 " 20 424 " 26 408 " 17 376 " 22 403 " 30 424 " 33 450 " 27 478 " 24
10
283 " 5
24.2" 0.8
455 " 22
HRCF (ml miny 1)
ydP r dtma x (kPa sy 1)
265 " 4 273 " 7 270 " 4 262 " 6 266 " 3 264 " 7 264 " 8 275 " 6 271 " 4
15.4" 1.7 13.5" 0.5 14.6" 1.5 18.8" 2.6 14.7" 0.8 17.7" 2.2 18.4" 2.9 16.8" 0.5 16.2" 0.5
274 " 10 325 " 18 320 " 17 328 " 20 288 " 13 411 " 27U 424 " 26U 320 " 13 302 " 9
268 " 5
17.3" 1.0
312 " 6
(b.p.m.)
U Data are means " SEM; Ž P- 0.05. show significant difference compared to solvent-treated hearts.
occlusion followed by 2-min reperfusion. Ten-minute coronary occlusion was chosen for assessing myocardial function, because this short-term regional ischaemia induced considerable deterioration of myocardial function, but it did not result in ischaemiainduced arrhythmias that would have disturbed measures of myocardial function w1x. However, reperfusion after a 10-min regional ischaemia triggered high incidence of reperfusion-induced VF, thus allowing us to examine any antiarrhythmic effect of the drugs against early reperfusion-induced VF.
Statistical analysis
Data were expressed as means " SE of the mean ŽSEM.. A one-way analysis of variance ŽANOVA. was initially carried out to test any difference between the mean values of all groups. If a difference was established, all groups were compared to the control solvent-treated group using a modified t-test according to the Bonferroni method for simultaneous multiple comparisons w21x. An analogue procedure was followed for distribution of binomially distributed variables, such as VF. An overall x 2-test for a 2 = n table was constructed followed by a sequence of 2 = 2 x 2-tests to compare individual groups.
RESULTS
Cardiac function As compared with normoxic values, 10-min regional ischaemia slightly reduced HR, and markedly decreased CF ŽTable I. in the solvent treated group. Neither concentration of enantiomers of cicletanine, glibenclamide, or their combination influenced HR and CF. Enantiomers of cicletanine or glibenclamide did not affect AF significantly before ischaemia, except
the highest dose of BN50417, which moderately decreased non-ischaemic AF. AF was markedly deteriorated by coronary occlusion in the solvent-treated control hearts. BN50417 and glibenclamide did not change ischaemic AF. BN50418 Ž1.5= 10y5 and 3.0 = 10y5 M. significantly improved the parameter, however, the combination of the most effective concentration of BN50418 with glibenclamide did not show any beneficial effect ŽFig. 1.. Similarly to that found with AF, enantiomers of cicletanine or glibenclamide did not affect LVDP significantly before ischaemia, except the highest dose of BN50417, which moderately decreased nonischaemic LVDP. Ischaemic LVDP deteriorated by coronary occlusion was markedly improved by 3.0= 10y5 M BN50417 and by 1.5= 10y5 M and 3.0= 10y5 M BN50418. Glibenclamide, however, abolished the protective effect of the most effective concentration of both enantiomers of cicletanine ŽFig. 1.. Before ischaemia, index of cardiac contractility, qd Prdt max , was moderately decreased by 6 = 10y5 y5 M BN50417. During coronary occlusion, 3 = 10 M BN50417, and all concentrations of BN50418 improved qd Prdt max . Glibenclamide inhibited the effect of 3 = 10y5 M BN50417 and BN50418, respectively ŽFig. 2.. A similar pattern was seen with yd Prdt max ŽTable I.. LVEDP was not influenced by the drugs before ischaemia, however, 3 = 10y5 M BN50417 and all concentrations of BN50418 significantly attenuated elevation of LVEDP due to coronary occlusion. Glibenclamide abolished the beneficial effect of the drugs ŽFig. 2..
LDH release LDH release, a marker of tissue damage during ischaemic stress, was studied with the most effective concentrations of enantiomers of cicletanine. Both
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Fig. 1. Effects of different concentrations of enantiomers of cicletanine, i.e. BN50417 ŽDC, m M., BN50418 ŽLC, m M., and 10y7 M glibenclamide ŽG., as well as their combination on aortic flow ŽAF. and left ventricular developed pressure ŽLVDP. before ischaemia and at the 10th-min of coronary occlusion in isolated working rat hearts. U Ž P- 0.05. designates significant changes of the parameters when compared to solvent-treated control ŽCont. hearts.
3 = 10y5 M BN50417 and 3 = 10y5 M BN50418 significantly decreased LDH leakage during coronary occlusion, however, in the presence of 10y7 M glibenclamide, this effect was not seen ŽFig. 3..
Reperfusion-induced VF Reperfusion after a 10-min coronary artery occlusion resulted in 100% incidence of VF, which occurred immediately upon reflow in the solventtreated hearts ŽFig. 4.. BN50417, in concentrations of 3 = 10y5 and 6 = 10y5 M, significantly decreased the incidence of VF, and glibenclamide abolished its antiarrhythmic effect. All concentrations of BN50418 markedly attenuated occurrence of VF, however, glibenclamide did not significantly influence the antiarrhythmic effect of 3 = 10y5 M BN50418.
DISCUSSION Our results show that both enantiomers of the antihypertensive drug, cicletanine, significantly improve myocardial function deteriorated by 10-min coronary
occlusion, markedly decrease ischaemic LDH release, and reduce the incidence of VF upon early reperfusion. The concentration of 3 = 10y5 M is the most effective in the case of both enantiomers, however, BN50418 shows more potent anti-ischaemic and antiarrhythmic effect. The anti-ischaemic effect of both enantiomers is blocked by glibenclamide, however, in contrast to the slight antiarrhythmic effect of BN50417, the potent antiarrhythmic effect of BN50418 is not sensitive to glibenclamide treatment. The present study is in accordance with our previous observations in isolated working rat heart w1x and in conscious rabbits w7x demonstrating that cicletanine racemate attenuates functional consequences of coronary occlusion and pacing-induced global myocardial ischaemia, and confirms our earlier study showing the involvement of a glibenclamide-sensitive mechanism in the action of cicletanine racemate w11x. This study revealed, however, that BN50418 is the more potent enantiomer of cicletanine. We have selected the concentration of 10y7 M of glibenclamide for studying the interaction of K ATP block-
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Fig. 2. Effects of different concentrations of enantiomers of cicletanine, i.e. BN50417 ŽDC, m M., BN50418 ŽLC, m M., and 10y7 M glibenclamide ŽG., as well as their combination on qd Prdt max and left ventricular end-diastolic pressure ŽLVEDP. before ischaemia and at the 10th-min of coronary occlusion in isolated working rat hearts. U Ž P- 0.05. designates significant changes of the parameters when compared to solvent-treated control ŽCont. hearts.
Fig. 3. Effects of enantiomers of cicletanine, i.e. 3 = 10y5 y5 M BN50417 ŽDC30., 3 = 10 M BN50418 ŽLC30., and y7 Ž . 10 M glibenclamide G , as well as their combination on lactate dehydrogenase ŽLDH. release at the 10th-min of coronary occlusion in isolated working rat hearts. U Ž P 0.05. designates significant change of the parameter when compared to solvent-treated control ŽCont. group.
Fig. 4. Effects of different concentrations of enantiomers of cicletanine, i.e. BN50417 ŽDC, m M., BN50418 ŽLC, m M., and 10y7 M glibenclamide ŽG., as well as their combination on the incidence of reperfusion-induced ventricular fibrillation ŽVF. in isolated working rat hearts. U Ž P- 0.05. designates significant change of the parameter when compared to solvent-treated control ŽCont. group.
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ade with enantiomers of cicletanine, since we have previously showed, that this concentration did not considerably influence cardiac performance, but abolished the antiischaemic effect of the optimal concentration Ž10y6 M. of the K ATP opener cromakalim, thereby suggesting a blockade of K ATP in the myocardium produced by 10y7 M glibenclamide in the isolated working rat heart w11x. Although glibenclamide is widely used and accepted as the most specific blocker of K ATP , it should be noted that the effects of the drug are likely not solely related to its actions on K ATP w22, 23x, therefore, the results with glibenclamide should be interpreted very cautiously. Our present study shows that 10y7 M glibenclamide abolishes the beneficial effects of both enantiomers of cicletanine during ischaemia, but does not affect the marked antiarrhythmic effect of BN50418, however, inhibits the slight antiarrhythmic effect of BN50417. This finding indicates that opening of K ATP may be involved in the anti-ischaemic, but not in the antiarrhythmic effect of the BN50418. Cidetanine racemate was shown to possess intracellular Ca2q antagonistic property and prostacyclin releasing effect w8, 24, 25x, therefore, the antiarrhythmic effect of BN50418 may be attributed to these actions. Both the anti-ischaemic and antiarrhythmic effect of the less active cicletanine enantiomer, BN50417, seems to be based at least in part on glibenclamide-sensitive K ATP channel activation. Potassium channel opener agents have been shown to possess dual effect on cardiac arrhythmias. Shortening of action potential due to K ATP -opening may lead to an increased susceptibility to re-entry arrhythmias, and may decrease the probability of occurrence of early after-depolarisation. Thus, K ATP openers may exert either arrhythmogenic or antiarrhythmic action depending on the experimental model used w26]28x. Our present results indicate that in contrast to the marked anti-ischaemic effect of enantiomers of cicletanine, the K ATP activator effect of the compounds may have minor contribution to the potent antiarrhythmic effect of cicletanine racemate. The mechanism by which enantiomers of cicletanine may lead to opening of the glibenclamide-sensitive K ATP channel is not known. The effects of enantiomers of cicletanine in the isolated heart, especially that of BN50418, on AF, LVDP, qd Prdt max , and LVEDP is similar to that of cromakalim w11, 13, 14x in the same experimental model, and glibenclamide blocks the effect of both drugs, however, in contrast to cromakalim, enantiomers of cicletanine does not increase CF. This might indicate, that the possible K ATP -opener effect of enantiomers of cicletanine may be more specific on myocardial cells than on smooth muscle cells. Accordingly, opening of K ATP produced by cicletanine racemate on smooth muscle cells is not supported by Noak and Deitmer w24x, since no cicletanine-induced
whole-cell Kq currents were observed in guinea pig portal vein smooth muscle cell, and glibenclamide did not significantly block the cicletanine-induced relaxation in rat, guinea-pig, and rabbit portal vein preparations. Enantiomers of cicletanine may open glibenclamide-sensitive K ATP channels indirectly through modulation of myocardial cyclic nucleotide metabolism. Cicletanine racemate has been shown to inhibit low K m Ca-calmodulin-dependent PDE ŽPDE I. and cGMP specific PDE ŽPDE V. w5, 6x, thereby leading to an increase in cardiac cGMP level w7x. Increased cGMP concentration may directly stimulate potassium channels w9, 10x, or in a certain cell compartment it may give rise to cAMPdependent phosphorylation of K ATP w29x through inhibition of cGMP-inhibited cAMP-specific PDE ŽPDE III., an enzyme of high activity in myocardial cells w30x. Nevertheless, the cardioprotective effect of myocardial cGMP is well established w31]33x. In summary, this is the first study on enantiomers of the antihypertensive drug cicletanine. Our present results suggest that both enantiomers of the drug show anti-ischaemic and antiarrhythmic effect in the rat heart, and that L-cicletanine ŽBN50418. is the more active enantiomer. The anti-ischaemic but not the antiarrhythmic effect of BN50418 may involve a glibenclamide-sensitive mechanism, probably opening of K ATP . BN50418, the active enantiomer of cicletanine, may confer beneficial effects on patients suffering from hypertension and consequent ischaemic heart disease.
ACKNOWLEDGEMENTS This study was supported by grants form IPSEN, Paris, France; National Scientific Research Found ŽOTKA F19946, D23736., National Technical Development Committee ŽOMFB 05201950616., Ministry of Education ŽFKFP 1284r1997., and the Hungarian Space Research Office, Budapest, Hungary.
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