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Divergent effects of propranolol and furosemide pretreatment on acute cardiomyopathy induced by minoxidil in beagle dogs
Toxicology, 20 (1981) 155--164 © Elsevier/North-Holland Scientific Publishers Ltd.
DIVERGENT EFFECTS OF PROPRANOLOL AND FUROSEMIDE PRETREATMENT ON ACUTE CARDIOMYOPATHY INDUCED BY MINOXIDIL IN BEAGLE DOGS
EUGENE YOUNG
H. H E R M A N ,
T I B O R BALAZS, V I C T O R J. F E R R A N S
and R O B E R T
S.K.
Division of Drug Biology, Food and Drug Administration, Washington, D C 20204; and Pathology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20205 (U.S.A.) (Received November 21st, 1980) (Accepted February 9th, 1981)
SUMMARY
Previous studies have shown that oral administration of minoxidil on 2 consecutive days produces an increase in heart rate and myocardial necrosis in Beagle dogs. Propranolol treatment (1.0 or 3.0 mg/kg every 8 h) did not abolish tachycardia and did not alter the incidence or severity of minoxidilinduced necrosis. In the present studies, pretreatment with either furosemide (10 mg/kg) or hydrochlorothiazide (250 mg/kg) reduced serum potassium levels. However, only furosemide (for 11 days) reduced significantly the incidence of minoxidil-induced necrosis; only 2 of 10 animals (20%) developed myocardial lesions compared to 11 of 14 (79%) in the non-treated group. The incidence and severity of lesions in hearts from animals treated with furosemide for 3 days or hydrochlorothiazide for 11 days were essentially the same as in animals given minoxidil alone. Thus, furosemide, under certain conditions, can reduce the incidence of acute ventricular lesions induced by minoxidil.
INTRODUCTION
Minoxidil is a potent long-acting vasodilating drug which lowers arterial pressure in normotensive animals [1] and hypertensive patients [2--4]. Clinical reports indicate that minoxidil-induced hypotension is associated with signs of sympathetic activation such as tachycardia, increased venous Address correspondence to: Miss Helen L. Reynolds, Technical Editor, HFF-40, Food and Drug Administration, 200 C Street, S.W., Washington, D.C. 20204, U.S.A. Address reprint requests to: Dr. Eugene H. Herman, Division of Drug Biology, Food and Drug Administration, 200 C Street, S.W., Washington, D.C. 20204, U.S.A.
155
return, and increased cardiac o u t p u t [ 4 , 5 ] . Electrocardiogram (ECG) changes showing left ventricular strain have also been noted [ 6 ] . Experimentally, in addition to marked sympathetic stimulation, minoxidil has been found to cause at least 2 types of pathologic alterations in the heart: acute left ventricular myocardial necrosis [7] and acute and chronic right atrial hemorrhage, arteritis, degeneration o f muscle cells and proliferation of blood vessels and connective tissue [1,7]. Minoxidil continues to be used in patients w h o are simultaneously receiving diuretics and ~-adrenergic blocking agents [2--4,6,8]. These drug combinations are claimed to provide more effective blood pressure control along with attenuation of the adverse cardiovascular effects of minoxidil. The present studies were initiated to determine whether simultaneous treatment with diuretics or ~-adrenergic blocking drugs would alter the cardiostimulation and acute left ventricular necrosis observed in Beagle dogs following minoxidil administration. MATERIALS AND METHODS Unanesthetized adult male Beagle dogs weighing between 8 and 12 kg were used in all experiments. Lead II of the ECG was monitored from subcutaneous electrodes before and during each drug pretreatment period. In addition the ECG was recorded 2--4 h after the first minoxidil dose and 24 h after the second minoxidil dose. The following drugs, doses, and pretreatment schedules were used in the studies.
dl-Propranolol Three groups of 5 dogs each were given 3 mg/kg minoxidil orally on each of 2 consecutive days. One group received 3 mg/kg (+)-propranolol and a second group 1 mg/kg (+)-propranolol orally 1 h before minoxidil and every 8 h thereafter during the 2
Furosemide and hydrochlorothiazide The effect of diuretic pretreatment on minoxidil cardiotoxicity was examined in 2 experimental series. In 1 series, 10 mg/kg furosemide or 250 mg/kg hydrochlorothiazide was given orally twice daily over an l l < l a y period to 12 and 6 animals, respectively. On days 10 and 11 of diuretic therapy, all b u t 2 dogs from each group received a 3 mg/kg oral dose of minoxidil. Ten additional non-pretreated dogs received only the 2 doses o f minoxidil. In the second series, furosemide (10 mg/kg) was given to 4 dogs twice daffy over a 3~lay period. Minoxidil (3 mg/kg) was given to these dogs on the second and third day of diuretic therapy and to 4 additional nonpretreated animals. Blood samples were taken before, during and on the last day of diuretic 156
therapy in each o f the experimental groups for serum potassium determinations. Experiments were terminated 24 h after the last dose of minoxidil, and necropsies on all animals were done immediately. The hearts were examined grossly and fixed in buffered 10% formalin. Histologic sections were prepared from anterior and posterior left ventricular papillary muscles; anterior, middle and posterior right ventricular papillary muscles; left anterior and posterior ventricular walls; and right and left atria. The sections were stained with hematoxylin-eosin (H&E) and Masson trichrome. Myocardial necrosis was graded to reflect t h e extent of lesion visible on microsocopic examination as follows: (1) minimal; (2) mild; (3) moderate; (4) moderately severe; and (5) severe. The necrosis score was totalled for each animal and the mean + S.E. calculated for each experimental series. Statistics Where results are given as mean + standard error {mean + S.E.), the student's t-test was utilized for determination o f statistical significance. Paired t-test was used to compare pre- and post-treatment values. The incidence of myocardial lesions was evaluated b y chi-square contingency tables. For all data, a value of P < 0.05 was considered significant. RESULTS
( +)-Propranolol Pretreatment with either 1.0 or 3.0 mg/kg (+)-propranolol reduced the resting heart rate by 10--20 beats/min. Heart rates increased approximately 100% in dogs dosed with minoxidil alone and in dogs given the combination of (+)-propranolol and minoxidfl (Fig. 2A). The absolute increases in heart rates 24 h after the second dose of minoxidil were significantly lower (P < 0.05) in the drug combination groups. Myocardial necrosis (essentially limited to the left anterior and posterior papillary muscles, particularly the posterior one) was found in propranololtreated and non-treated groups of animals. Grossly, the necrotic lesions were weU
157
Fig. 1. Photomicrograph of left ventricular papillary muscle lesion observed in a Beagle dog 24 h after second daily dose of 3 mg/kg minoxidil. (A) Low magnification view of hemorrhage and necrosis. H&E, X 80. (B) Higher magnification of subendocardial area showing hemorrhage and infiltration with inflammatory cells. H&E, x 150.
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Fig. 2. (A) Mean heart rate in Beagle dogs before dosing, 1 h after I or 3 mg/kg propranolol, 2 h after the first dose, and 24 h after the second dose of 3.0 mg/kg minoxidil. (B) Incidence and histopathological severity of myocardial necrosis in control dogs and dogs treated with 1.0 or 3.0 mg/kg propranolol (every 8 h) 24 h after the second dose of 3.0 mg/kg minoxidil. The necrosis score was totaled for each animal and the mean ± S.E. calculated for each series.
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Fig. 3. (A) Mean heart rates in control Beagle dogs and dogs treated w i t h f u r o s e m i d e ( 1 0 mg/kg, h y d r o c h l o r o t h i a z i d e ( 2 5 0 mg/kg, t w i c e daily) for 11 days before m i n o x i d i l , 2 h after the first dose, 3.0 m g / k g m i n o x i d i l . Each point represents the m e a n ± S.E. (B) I n c i d e n c e and severity o f necrosis in furosemide or h y d r o c h l o r o t h i a z i d e 24 h after the s e c o n d dose o f 3.0 m g / k g m i n o x i d i l . The necrosis and the mean ± S.E. calculated for each series.
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Fig. 4. Photomicrograph of left ventricular papillary muscle taken from a Beagle dog dosed with furosemide (10 mg/kg, twice daily) for 11 days and sacrificed 24 h after the second daily dose of 3 mg/kg minoxidil. (A) Histologically normal myocardium. H&E, × 80. (B) Small focus of myocardial necrosis in the same animal showing minute size of lesions. H&E x 150.
161
Furosemide and hydrochlorothiazide Furosemide (10 mg/kg, twice daily) given for 3 days decreased serum potassium from 5.0 + 0.1 to 4.0 -+ 0.1 mEq/liter (P < 0.05), while in the l l ~ l a y treatment group the level fell from 4.7 + 0.1 to 3.7 + 0.2 mEq/ liter (P < 0.05). Similarly, a decrease in serum potassium from 4.9 +- 0.1 to 3.7 + 0.2 mEq/liter (P < 0.05) was f o u n d in animals given hydrochlorothiazide (250 mg/kg, twice daily) for 11 days. In non-treated animals there was little variation in serum potassium concentration (mean initial value: 5.0 + 0.1 mEq/liter) over the duration of the experimental period. ECGs recorded during the diuretic t r e a t m e n t did not differ from those obtained from control animals over the same time intervals. Minoxidil increased heart rate in all groups by 70--100%. The absolute heart rates 2 h after minoxidil were slightly but n o t significantly less in the 2 furosemide-treated groups than in the non-treated groups (Fig. 3A). Tachycardia was still present 24 h after the second dose of minoxidil in all 4 drug t r e a t m e n t groups. However, at this time the magnitude of the heart rate in the l l < l a y furosemide-treated animals was significantly less (P < 0.05) t h a n in the 3 other minoxidil-treated groups. The incidence of necrosis after minoxidil was reduced in those animals receiving furosemide for 11 days. Only 2 o f 10 animals (20%) treated with furosemide for 11 days developed myocardial necrosis compared to 11 of 14 (79%) in the non-treated group (Fig. 3B). The reduced incidence of necrosis in the furosemide-treated animals was f o u n d to be highly significant (P < 0.005 by chi-square analysis). The severity of the lesions in the 2 furosemide-treated animals was less in one (Fig. 4) and comparable in the second to that observed in the non-pretreated group. Hemorrhage was observed in 2 of 10 furosemide-treated dogs and 5 of 14 from the non-treated group. The incidence and severity of lesions in the hearts from animals treated with furosemide for 3 days and minoxidil were essentially the same as in the animals given minoxidil alone (Fig. 3B). Hydrochlorothiazide therapy for 11 days did not significantly alter the incidence or severity of minoxidil lesions (Fig. 3B). DISCUSSION
Oral administration of minoxidil on 2 consecutive days produces myocardial necrosis in dogs [7; this paper]. The most frequent sites of necrosis are in the subendocardium and particularly in the left ventricular papillary muscles. The subendocardial areas of the myocardium have been found to be the most susceptible to ischemia [9--11]. Several anatomical and functional factors further increase the susceptibility of the papillary muscles to ischemia [ 1 2 ] . Minoxidil m a y cause myocardial ischemia by at least 2 mechanisms [7]. The hypotensive action of minoxidil is due to a relaxation of the arterioles [ 1 ]. A decrease in arterial blood pressure would be reflected in the coronary arteries and ultimately could result in a decrease in coronary perfusion pressure. A second consequence of the minoxidil-induced hypoten-
162
sion is the simultaneous occurrence o f p r o n o u n c e d reflex tachycardia. Tachycardia increases myocardial oxygen demand and decreases the diastolic period, when most perfusion occurs. Thus, the pharmacological mechanisms, as well as the particular site of the lesion induced b y minoxidil, imply an ischemic origin of the cardiac necrosis. Propranolol has been used in combination with either hydralazine or minoxidil in hypertensive patients to enhance the antihypertensive effects of these agents and decrease adverse cardiac effects [3,4]. Propranolol, when given over a 12-h period, decreased infarct size following acute coronary occlusion in dogs [ 1 3 ] . The results of our studies, where we used a combination of propranolol and minoxidil, are n o t entirely consistent with either the clinical or experimental findings mentioned above. Although reflex tachycardia was n o t abolished b y either dose of propranolol, the absolute heart rates were less in both low and high dose treatment groups 24 h after the second minoxidil dose. There are observations that propranolol may n o t be equally effective against all types of tachycardia [ 1 4 ] . Of 4 types examined, the tachycardia due to isoproterenol was most susceptible and that due to exercise most resistant to block b y propranolol [ 1 4 ] . The tachycardia elicited by minoxidil administration appears to be mediated to a significant degree by parasympathetic withdrawal [ 1 5 ] . Thus, even though adequate ~-adrenergic blocking doses were utilized, propranolol might be expected to exert only limited suppression of minoxidil-induced tachycardia. This lack of suppression of tachycardia was probably one reason w h y propranolol did n o t alter the incidence or severity of minoxidil-induced necrosis. The incidence of minoxidil-induced necrosis was significantly reduced b y the l l < l a y furosemide administration b u t n o t b y hydrochlorothiazide given over the same period of time. The protective effect of the l l - d a y furosemide treatment appears to be independent of potassium depletion since potassium levels were also equally reduced after l l < l a y hydrochlorothiazide and 3
numbers of rats pretreated with furosemide survived periods of renal ischemia t h a n d i d n o n - t r e a t e d c o n t r o l a n i m a l s [ 1 9 ] . T h e r e is a l s o s o m e e v i d e n c e t h a t the protective effect of furosemide might be due to a specific metabolic inhibition of mitochondria in renal cells causing a decrease in oxygen cons u m p t i o n a n d m e t a b o l i c d e m a n d [ 2 0 , 2 1 ] . A t p r e s e n t , i t is n o t k n o w n w h e t h e r s i m i l a r e f f e c t s o c c u r in o t h e r t i s s u e s s u c h a s t h e m y o c a r d i u m o r whether such an action could reduce the ischemic effects of minoxidil. Further experiments are necessary to delineate the mechanisms involved and the possible usefulness of the beneficial effects of furosemide.
ACKNOWLEDGEMENTS The authors express their gratitude to Mr. Donald Gates for his technical assistance during the course of this study. REFERENCES 1 D.W. DuCharme, W.A. Freyburger, B.E. Graham and R.G. Carlson, J. Pharmacol. Exp. Ther., 184 (1973) 622. 2 E. Gilmore, J. Weil and C. Chidsey, N. Engl. J. Med., 282 (1970) 521. 3 T.B. Gottlieb, F.H. Katz and C.A. Chidsey, Circulation, 45 (1972) 571. 4 W.A. Pettinger and H.C. Mitchell, N. Engl. J. Med., 289 (1973) 167. 5 J. Koch-Weser, Arch. Intern. Med., 133 (1974) 1017. 6 D. Hall, F. Charocopos, K.L. Froer and W. Rudolph, Arch. Intern. Med., 139 (1979) 790. 7 E.H. Herman, T. Balazs, R. Young, F.J. Earl, S. Krop and V.J. Ferrans, Toxicol. Appl. Pharmacol., 47 (1979) 493. 8 R.L. Wilburn, A. Blaufuss and C.M. Bennett, Circulation, 52 (1975) 706. 9 R.F. Debusk and D.C. Harrison, N. Engl. J. Med., 281 (1969) 1458. 10 J.R. Bell and A.C. F o x , Am. J. Med. Sci., 268 (1974) 3. 11 J.I.E. Hoffman and G.D. Buckberg, Br. Med. J., 1 (1975) 76. 12 W.C. Roberts and L.S. Cohen, Circulation, 46 (1972) 138. 13 P.R. Maroko, J.K. Kjekhus, B.E. Sobel, T. Watanabe, J.W. Corell, J. Ross Jr. and E. Braunwald, Circulation, 43 (1971) 67. 14 H.D. Lehman, Naunyn Schmiedebergs Arch. Pharmacol., 293 (Suppl) (1976) R29. 15 M. Gerold, Experientia, 33 (1977) 804. 16 R.R. Blair-West, M.J. McKinley and J.S. McKenzie, J. Pharm. Pharmacol., 24 (1972) 442. 17 J.P. Mtabaji, M.S. Manku and D.F. Horrobin, Can. J. Physiol. Pharmacol., 54 (1976) 357. 18 P. Panijayanond, S.I. Cho, F. Ulrich and D.C. Nabseth, Surgery, 73 (1973) 368. 19 Y. Rikukawa and E.S. Lindsey, Furosemide as a protective agent in renal ischemia. I. Preliminary investigations in rats, in J.S. Norman (Ed.), Conference on Organ Perfusion and Preservation, Appleton-Century-Crofts, New York, 1968, pp. 131-135. 20 H.J. Mohr, Submicroscopic alterations of tubular epithelia induced by furosemide, in G. Peters and F. Roch-Ramel (Eds.), Progress in Nephrology, Springer-Verlag, Berlin, 1969, pp. 281--289. 21 W. Herms and F. Kersting, Studies on oxygen consumption and changes of tissue cation concentration in K ÷ depleted rabbit kidney slices, under the influence of diuretic agents, p-chloromercuribenzoate and N-ethylmalemide, in G. Peters and F. Roch-Ramel (Eds.), Progress in Nephrology, Springer-Verlag, Berlin, 1969, pp. 290--295.
164
DIVERGENT EFFECTS OF PROPRANOLOL AND FUROSEMIDE PRETREATMENT ON ACUTE CARDIOMYOPATHY INDUCED BY MINOXIDIL IN BEAGLE DOGS
EUGENE YOUNG
H. H E R M A N ,
T I B O R BALAZS, V I C T O R J. F E R R A N S
and R O B E R T
S.K.
Division of Drug Biology, Food and Drug Administration, Washington, D C 20204; and Pathology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20205 (U.S.A.) (Received November 21st, 1980) (Accepted February 9th, 1981)
SUMMARY
Previous studies have shown that oral administration of minoxidil on 2 consecutive days produces an increase in heart rate and myocardial necrosis in Beagle dogs. Propranolol treatment (1.0 or 3.0 mg/kg every 8 h) did not abolish tachycardia and did not alter the incidence or severity of minoxidilinduced necrosis. In the present studies, pretreatment with either furosemide (10 mg/kg) or hydrochlorothiazide (250 mg/kg) reduced serum potassium levels. However, only furosemide (for 11 days) reduced significantly the incidence of minoxidil-induced necrosis; only 2 of 10 animals (20%) developed myocardial lesions compared to 11 of 14 (79%) in the non-treated group. The incidence and severity of lesions in hearts from animals treated with furosemide for 3 days or hydrochlorothiazide for 11 days were essentially the same as in animals given minoxidil alone. Thus, furosemide, under certain conditions, can reduce the incidence of acute ventricular lesions induced by minoxidil.
INTRODUCTION
Minoxidil is a potent long-acting vasodilating drug which lowers arterial pressure in normotensive animals [1] and hypertensive patients [2--4]. Clinical reports indicate that minoxidil-induced hypotension is associated with signs of sympathetic activation such as tachycardia, increased venous Address correspondence to: Miss Helen L. Reynolds, Technical Editor, HFF-40, Food and Drug Administration, 200 C Street, S.W., Washington, D.C. 20204, U.S.A. Address reprint requests to: Dr. Eugene H. Herman, Division of Drug Biology, Food and Drug Administration, 200 C Street, S.W., Washington, D.C. 20204, U.S.A.
155
return, and increased cardiac o u t p u t [ 4 , 5 ] . Electrocardiogram (ECG) changes showing left ventricular strain have also been noted [ 6 ] . Experimentally, in addition to marked sympathetic stimulation, minoxidil has been found to cause at least 2 types of pathologic alterations in the heart: acute left ventricular myocardial necrosis [7] and acute and chronic right atrial hemorrhage, arteritis, degeneration o f muscle cells and proliferation of blood vessels and connective tissue [1,7]. Minoxidil continues to be used in patients w h o are simultaneously receiving diuretics and ~-adrenergic blocking agents [2--4,6,8]. These drug combinations are claimed to provide more effective blood pressure control along with attenuation of the adverse cardiovascular effects of minoxidil. The present studies were initiated to determine whether simultaneous treatment with diuretics or ~-adrenergic blocking drugs would alter the cardiostimulation and acute left ventricular necrosis observed in Beagle dogs following minoxidil administration. MATERIALS AND METHODS Unanesthetized adult male Beagle dogs weighing between 8 and 12 kg were used in all experiments. Lead II of the ECG was monitored from subcutaneous electrodes before and during each drug pretreatment period. In addition the ECG was recorded 2--4 h after the first minoxidil dose and 24 h after the second minoxidil dose. The following drugs, doses, and pretreatment schedules were used in the studies.
dl-Propranolol Three groups of 5 dogs each were given 3 mg/kg minoxidil orally on each of 2 consecutive days. One group received 3 mg/kg (+)-propranolol and a second group 1 mg/kg (+)-propranolol orally 1 h before minoxidil and every 8 h thereafter during the 2
Furosemide and hydrochlorothiazide The effect of diuretic pretreatment on minoxidil cardiotoxicity was examined in 2 experimental series. In 1 series, 10 mg/kg furosemide or 250 mg/kg hydrochlorothiazide was given orally twice daily over an l l < l a y period to 12 and 6 animals, respectively. On days 10 and 11 of diuretic therapy, all b u t 2 dogs from each group received a 3 mg/kg oral dose of minoxidil. Ten additional non-pretreated dogs received only the 2 doses o f minoxidil. In the second series, furosemide (10 mg/kg) was given to 4 dogs twice daffy over a 3~lay period. Minoxidil (3 mg/kg) was given to these dogs on the second and third day of diuretic therapy and to 4 additional nonpretreated animals. Blood samples were taken before, during and on the last day of diuretic 156
therapy in each o f the experimental groups for serum potassium determinations. Experiments were terminated 24 h after the last dose of minoxidil, and necropsies on all animals were done immediately. The hearts were examined grossly and fixed in buffered 10% formalin. Histologic sections were prepared from anterior and posterior left ventricular papillary muscles; anterior, middle and posterior right ventricular papillary muscles; left anterior and posterior ventricular walls; and right and left atria. The sections were stained with hematoxylin-eosin (H&E) and Masson trichrome. Myocardial necrosis was graded to reflect t h e extent of lesion visible on microsocopic examination as follows: (1) minimal; (2) mild; (3) moderate; (4) moderately severe; and (5) severe. The necrosis score was totalled for each animal and the mean + S.E. calculated for each experimental series. Statistics Where results are given as mean + standard error {mean + S.E.), the student's t-test was utilized for determination o f statistical significance. Paired t-test was used to compare pre- and post-treatment values. The incidence of myocardial lesions was evaluated b y chi-square contingency tables. For all data, a value of P < 0.05 was considered significant. RESULTS
( +)-Propranolol Pretreatment with either 1.0 or 3.0 mg/kg (+)-propranolol reduced the resting heart rate by 10--20 beats/min. Heart rates increased approximately 100% in dogs dosed with minoxidil alone and in dogs given the combination of (+)-propranolol and minoxidfl (Fig. 2A). The absolute increases in heart rates 24 h after the second dose of minoxidil were significantly lower (P < 0.05) in the drug combination groups. Myocardial necrosis (essentially limited to the left anterior and posterior papillary muscles, particularly the posterior one) was found in propranololtreated and non-treated groups of animals. Grossly, the necrotic lesions were weU
157
Fig. 1. Photomicrograph of left ventricular papillary muscle lesion observed in a Beagle dog 24 h after second daily dose of 3 mg/kg minoxidil. (A) Low magnification view of hemorrhage and necrosis. H&E, X 80. (B) Higher magnification of subendocardial area showing hemorrhage and infiltration with inflammatory cells. H&E, x 150.
158
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Fig. 2. (A) Mean heart rate in Beagle dogs before dosing, 1 h after I or 3 mg/kg propranolol, 2 h after the first dose, and 24 h after the second dose of 3.0 mg/kg minoxidil. (B) Incidence and histopathological severity of myocardial necrosis in control dogs and dogs treated with 1.0 or 3.0 mg/kg propranolol (every 8 h) 24 h after the second dose of 3.0 mg/kg minoxidil. The necrosis score was totaled for each animal and the mean ± S.E. calculated for each series.
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Fig. 3. (A) Mean heart rates in control Beagle dogs and dogs treated w i t h f u r o s e m i d e ( 1 0 mg/kg, h y d r o c h l o r o t h i a z i d e ( 2 5 0 mg/kg, t w i c e daily) for 11 days before m i n o x i d i l , 2 h after the first dose, 3.0 m g / k g m i n o x i d i l . Each point represents the m e a n ± S.E. (B) I n c i d e n c e and severity o f necrosis in furosemide or h y d r o c h l o r o t h i a z i d e 24 h after the s e c o n d dose o f 3.0 m g / k g m i n o x i d i l . The necrosis and the mean ± S.E. calculated for each series.
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225
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Fig. 4. Photomicrograph of left ventricular papillary muscle taken from a Beagle dog dosed with furosemide (10 mg/kg, twice daily) for 11 days and sacrificed 24 h after the second daily dose of 3 mg/kg minoxidil. (A) Histologically normal myocardium. H&E, × 80. (B) Small focus of myocardial necrosis in the same animal showing minute size of lesions. H&E x 150.
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Furosemide and hydrochlorothiazide Furosemide (10 mg/kg, twice daily) given for 3 days decreased serum potassium from 5.0 + 0.1 to 4.0 -+ 0.1 mEq/liter (P < 0.05), while in the l l ~ l a y treatment group the level fell from 4.7 + 0.1 to 3.7 + 0.2 mEq/ liter (P < 0.05). Similarly, a decrease in serum potassium from 4.9 +- 0.1 to 3.7 + 0.2 mEq/liter (P < 0.05) was f o u n d in animals given hydrochlorothiazide (250 mg/kg, twice daily) for 11 days. In non-treated animals there was little variation in serum potassium concentration (mean initial value: 5.0 + 0.1 mEq/liter) over the duration of the experimental period. ECGs recorded during the diuretic t r e a t m e n t did not differ from those obtained from control animals over the same time intervals. Minoxidil increased heart rate in all groups by 70--100%. The absolute heart rates 2 h after minoxidil were slightly but n o t significantly less in the 2 furosemide-treated groups than in the non-treated groups (Fig. 3A). Tachycardia was still present 24 h after the second dose of minoxidil in all 4 drug t r e a t m e n t groups. However, at this time the magnitude of the heart rate in the l l < l a y furosemide-treated animals was significantly less (P < 0.05) t h a n in the 3 other minoxidil-treated groups. The incidence of necrosis after minoxidil was reduced in those animals receiving furosemide for 11 days. Only 2 o f 10 animals (20%) treated with furosemide for 11 days developed myocardial necrosis compared to 11 of 14 (79%) in the non-treated group (Fig. 3B). The reduced incidence of necrosis in the furosemide-treated animals was f o u n d to be highly significant (P < 0.005 by chi-square analysis). The severity of the lesions in the 2 furosemide-treated animals was less in one (Fig. 4) and comparable in the second to that observed in the non-pretreated group. Hemorrhage was observed in 2 of 10 furosemide-treated dogs and 5 of 14 from the non-treated group. The incidence and severity of lesions in the hearts from animals treated with furosemide for 3 days and minoxidil were essentially the same as in the animals given minoxidil alone (Fig. 3B). Hydrochlorothiazide therapy for 11 days did not significantly alter the incidence or severity of minoxidil lesions (Fig. 3B). DISCUSSION
Oral administration of minoxidil on 2 consecutive days produces myocardial necrosis in dogs [7; this paper]. The most frequent sites of necrosis are in the subendocardium and particularly in the left ventricular papillary muscles. The subendocardial areas of the myocardium have been found to be the most susceptible to ischemia [9--11]. Several anatomical and functional factors further increase the susceptibility of the papillary muscles to ischemia [ 1 2 ] . Minoxidil m a y cause myocardial ischemia by at least 2 mechanisms [7]. The hypotensive action of minoxidil is due to a relaxation of the arterioles [ 1 ]. A decrease in arterial blood pressure would be reflected in the coronary arteries and ultimately could result in a decrease in coronary perfusion pressure. A second consequence of the minoxidil-induced hypoten-
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sion is the simultaneous occurrence o f p r o n o u n c e d reflex tachycardia. Tachycardia increases myocardial oxygen demand and decreases the diastolic period, when most perfusion occurs. Thus, the pharmacological mechanisms, as well as the particular site of the lesion induced b y minoxidil, imply an ischemic origin of the cardiac necrosis. Propranolol has been used in combination with either hydralazine or minoxidil in hypertensive patients to enhance the antihypertensive effects of these agents and decrease adverse cardiac effects [3,4]. Propranolol, when given over a 12-h period, decreased infarct size following acute coronary occlusion in dogs [ 1 3 ] . The results of our studies, where we used a combination of propranolol and minoxidil, are n o t entirely consistent with either the clinical or experimental findings mentioned above. Although reflex tachycardia was n o t abolished b y either dose of propranolol, the absolute heart rates were less in both low and high dose treatment groups 24 h after the second minoxidil dose. There are observations that propranolol may n o t be equally effective against all types of tachycardia [ 1 4 ] . Of 4 types examined, the tachycardia due to isoproterenol was most susceptible and that due to exercise most resistant to block b y propranolol [ 1 4 ] . The tachycardia elicited by minoxidil administration appears to be mediated to a significant degree by parasympathetic withdrawal [ 1 5 ] . Thus, even though adequate ~-adrenergic blocking doses were utilized, propranolol might be expected to exert only limited suppression of minoxidil-induced tachycardia. This lack of suppression of tachycardia was probably one reason w h y propranolol did n o t alter the incidence or severity of minoxidil-induced necrosis. The incidence of minoxidil-induced necrosis was significantly reduced b y the l l < l a y furosemide administration b u t n o t b y hydrochlorothiazide given over the same period of time. The protective effect of the l l - d a y furosemide treatment appears to be independent of potassium depletion since potassium levels were also equally reduced after l l < l a y hydrochlorothiazide and 3
numbers of rats pretreated with furosemide survived periods of renal ischemia t h a n d i d n o n - t r e a t e d c o n t r o l a n i m a l s [ 1 9 ] . T h e r e is a l s o s o m e e v i d e n c e t h a t the protective effect of furosemide might be due to a specific metabolic inhibition of mitochondria in renal cells causing a decrease in oxygen cons u m p t i o n a n d m e t a b o l i c d e m a n d [ 2 0 , 2 1 ] . A t p r e s e n t , i t is n o t k n o w n w h e t h e r s i m i l a r e f f e c t s o c c u r in o t h e r t i s s u e s s u c h a s t h e m y o c a r d i u m o r whether such an action could reduce the ischemic effects of minoxidil. Further experiments are necessary to delineate the mechanisms involved and the possible usefulness of the beneficial effects of furosemide.
ACKNOWLEDGEMENTS The authors express their gratitude to Mr. Donald Gates for his technical assistance during the course of this study. REFERENCES 1 D.W. DuCharme, W.A. Freyburger, B.E. Graham and R.G. Carlson, J. Pharmacol. Exp. Ther., 184 (1973) 622. 2 E. Gilmore, J. Weil and C. Chidsey, N. Engl. J. Med., 282 (1970) 521. 3 T.B. Gottlieb, F.H. Katz and C.A. Chidsey, Circulation, 45 (1972) 571. 4 W.A. Pettinger and H.C. Mitchell, N. Engl. J. Med., 289 (1973) 167. 5 J. Koch-Weser, Arch. Intern. Med., 133 (1974) 1017. 6 D. Hall, F. Charocopos, K.L. Froer and W. Rudolph, Arch. Intern. Med., 139 (1979) 790. 7 E.H. Herman, T. Balazs, R. Young, F.J. Earl, S. Krop and V.J. Ferrans, Toxicol. Appl. Pharmacol., 47 (1979) 493. 8 R.L. Wilburn, A. Blaufuss and C.M. Bennett, Circulation, 52 (1975) 706. 9 R.F. Debusk and D.C. Harrison, N. Engl. J. Med., 281 (1969) 1458. 10 J.R. Bell and A.C. F o x , Am. J. Med. Sci., 268 (1974) 3. 11 J.I.E. Hoffman and G.D. Buckberg, Br. Med. J., 1 (1975) 76. 12 W.C. Roberts and L.S. Cohen, Circulation, 46 (1972) 138. 13 P.R. Maroko, J.K. Kjekhus, B.E. Sobel, T. Watanabe, J.W. Corell, J. Ross Jr. and E. Braunwald, Circulation, 43 (1971) 67. 14 H.D. Lehman, Naunyn Schmiedebergs Arch. Pharmacol., 293 (Suppl) (1976) R29. 15 M. Gerold, Experientia, 33 (1977) 804. 16 R.R. Blair-West, M.J. McKinley and J.S. McKenzie, J. Pharm. Pharmacol., 24 (1972) 442. 17 J.P. Mtabaji, M.S. Manku and D.F. Horrobin, Can. J. Physiol. Pharmacol., 54 (1976) 357. 18 P. Panijayanond, S.I. Cho, F. Ulrich and D.C. Nabseth, Surgery, 73 (1973) 368. 19 Y. Rikukawa and E.S. Lindsey, Furosemide as a protective agent in renal ischemia. I. Preliminary investigations in rats, in J.S. Norman (Ed.), Conference on Organ Perfusion and Preservation, Appleton-Century-Crofts, New York, 1968, pp. 131-135. 20 H.J. Mohr, Submicroscopic alterations of tubular epithelia induced by furosemide, in G. Peters and F. Roch-Ramel (Eds.), Progress in Nephrology, Springer-Verlag, Berlin, 1969, pp. 281--289. 21 W. Herms and F. Kersting, Studies on oxygen consumption and changes of tissue cation concentration in K ÷ depleted rabbit kidney slices, under the influence of diuretic agents, p-chloromercuribenzoate and N-ethylmalemide, in G. Peters and F. Roch-Ramel (Eds.), Progress in Nephrology, Springer-Verlag, Berlin, 1969, pp. 290--295.
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