Relationships Between the Release and Tissue Depletion of Norepinephrine from the Heart by Guanethidine and Reserpine

Transcription

1 Relationships Between the Release and Tissue Depletion of Norepinephrine from the Heart by Guanethidine and Reserpine By Donald C. Harrison, M.D., Charles A. Chidsey, M.D., Robert Goldman, M.D., and Eugene Braunwald, M.D. Although depletion of myoeardial norepinephrine stores has been noted following the administration of guanethidine 1 ' 2 and reserpine, 3-8 the mechanism by which this depletion occurs has not been defined. The intravenous administration of guanethidine has been shown to result in a transient sympathomimetie response, and it has been suggested that this may result from the release of norepinephrine from the sympathetic nerve endings. c> 7 A similar, but less marked response has been noted following the intravenous administration of reserpine. 5 ' s ' 9 In a recent study it was demonstrated that the administration of a number of aromatic amines results in the release of norepinephrine from the heart of intact anesthetized dogs X1 An incidental observation in this survey was the finding, in one dog, that guanethidine also produced a measurable release of norepinephrine from the heart. 11 Accordingly, the present investigation was undertaken to examine the time course of the release of norepinephrine from the heart following guanethidine and to correlate this release into the blood with the norepinephrine content of cardiac tissue. Similar studies were carried out with reserpine in order to define the role of norepinephrine release into the blood in the depletion of norepinephrine from the heart by these two drugs. Methods Sixteen mongrel dogs, 20 to 38 kg were anesthetized with pentobarbital (30 mg/kg) given intravenously and ventilated with a Harvard respirator. After the chest was opened, a no. 8 birdseye catheter was placed in the coronary sinus via the jugular vein 12 and a Walton-Brodie strain From the Cardiology Branch, National Heart Institute, U. S. Public Health Service, Bethesda, Maryland. Eeceived for publication September 27, gauge arch was sutured to the right ventricular wall to record contractile force. 13 Bilateral vagotomy was performed in all animals to prevent the compensatory vagal inhibition to pressor responses. Femoral arterial pressure was recorded by means of a Statham transducer and the heart rate was determined from the electrocardiogram. Prior to the administration of each drug, blood samples were taken simultaneously from the coronary sinus and femoral artery and a portion of the right atrial appendage was biopsied. In four dogs guanethidine sulfate (15 mg/kg of the free base) was given intravenously and in four other dogs reserpine (3 mg/kg of the free base) was similarly administered. Blood samples of 30 ml were taken simultaneously from the coronary sinus and femoral artery at 2, 15, 60,, 180, and 240 minutes after administration o the drug. After each sample the blood removed was replaced with fresh donor blood which had been mixed with the experimental dog's blood prior to the study in order to prevent any reduction in blood volume. Biopsies from either atrial appendage were obtained at 60,, and 240 minutes after the drug was administered. Arterial pressure (femoral), cardiac rate and contractile force were monitored throughout the period of study. Five control dogs were anesthetized in a manner similar to the experimental animals and after the thoracotomy and vagotomy the right atrial appendage was biopsied. No further manipulations were performed in these animals and the left atrial appendage was biopsied at four hours. In three other dogs guanethidine sulfate (15 mg/kg of the free base) was given intravenously and 24 hours later the atrial appendages were obtained and analyzed for norepinephrine. The blood samples for eateeholamine analysis were heparinized and immediately refrigerated; the measurements were begun -within one hour of the time they were obtained. Plasma was separated by centrifugation for 30 minutes at 25,000 X G at 4 C in a refrigerated centrifuge. The eatecholamines were isolated from 15 ml of plasma by adsorption of alumina which results in isolation only of compounds with a catechol structure. The amines were eluted with acetic acid 14 and after oxidation with potassium ferricyanide were 256 Circulation Research, Volume XII, March 19GS

2 NOREPINEPHRINE DEPLETION FROM HEART 257 measured fluoroinetrically in the cluate as the trihydroxyiudole. 15 Norepinephi'ine and epinephrine were determined simultaneously by measuring fluorescence at two different wave lengths of activation and fluorescence in an Aminco-Bowman Spectrophotofluoronieter. 16 The production of the trihydroxyindole by this method of oxidation and its selective fluorometric determination both lend a high degree of specificity to the analytic procedure. The values presented for norepinephrine concentration in plasma were not coi-reeted for recovery which averaged 80.7% (SD ± 10.4%) when 0.05 fxg of norepinephrine was added to the plasma sample. The tissue samples were homogenized with approximately 20 volumes of 5% trichloracetic acid and the catecholamine determined in the tissue extract as norepinephrine equivalents. The results were not corrected for recovery which averaged 88.5% (SD ± 7.6%) when 1 /j,g of norepinephrine was added to the homogenate. These recoveries which were performed with every analysis are within the range expected for this type of procedure. The difference between the average plasma and tissue recoveries is due to the larger volume of solution used in the alumina adsorption step in the tissue analyses. Results The intravenous administration of guanethidine resulted in an initial short hypotensive period followed by an elevation above control levels of the systemic arterial pressure ; heart rate and contractile force rose immediately in the four dogs studied after injection of the drug. The increases in pressure, force, and rate persisted for 60 to minutes and a gradual progressive reduction in these variables occurred during the four hours in which observations were continued (fig. 1). The average difference between coronary sinus and arterial (CS-FA) norepinephrine concentration was /ng/liter during the control period. Following the administration of guanethidine, norepinephrine was released from the heart as evidenced by an increase of the CS-FA norepinephrine difference to an average of ^.g/liter at two minutes, /xg/liter at 15 minutes, /j.g/liter at one hour, and /ig/liter at two hours. Thereafter the average CS-FA became negative, 0.08 /tg/liter at three hours and 0.63 /xg/liter at four hours. This release of norepinephrine into the coronary circulation cor- Circulation Research, Volume XII, March 106S PHYSIOLOGICAL * GUANETHIOINE GUANETHIOINE Il5mg/Kg) GUANETHIDINE DATA AFTER SrSTOLIC PRESSUHE H PULSE 3 4 HOURS RATE AVERAGE % CHANGE IN CONTRACTILE FORCE FIGURE 1 The upper two figures show the mean and standard errors of the systolic arterial pressure and heart rate in 4 dogs following the administration of guarnethidine (15 mg/kg). The loicer figure shows the average per cent changes in myocardial contractile force in these 4 dogs. responded temporally with the adrenergic response observed in the cardiovascular system. The absolute concentrations of plasma norepinephrine in each of the four dogs are shown in table 1. It is apparent that the duration of the norepinephrine release was variable among the four dogs and ranged from 30 to 180 minutes, a period comparable in each animal to the elevation in systolic arterial pressure (table 1). The arterial norepinephrine concentration rose immediately after the

3 258 HARRISON, CHIDSEY, GOLDMAN, BRAUNWALD TABLE 1 Arterial and Coronary Sinus Norepinephrine Concentration and Systolic Arterial Pressures in Guanethidine Treated Animals 1G 2G 3G 4G Time of determination Control (minutes) Arterial norepinephrine concentration in Ag/liter. Coronary sinus norepinephrine in fig/liter. Systolic arterial pressure in mm Hg. * Sample lost ] administration of the drug and remained elevated for the duration of the experiment. The norepinephrine concentrations in the atrial appendages of these dogs obtained before and at various time intervals after guanethidine are shown on the bottom half of figure 2. The average control value, 2.14 /j-g/g, was unchanged at one hour (average = 2.27 jug/g), but four hours after the drug, the concentration was reduced to an average of 1.62 //.g/g. This value was significantly lower (P <0.05) when compared to the control levels. In the control dogs which received no drugs, the norepinephrine concentration of atrial appendage averaged 2.23 jug/g initially and four hours later averaged 2.52 ixg/g (table 2). The mean changes and standard errors in systemic arterial pressure, pulse rate, and contractile force in the four dogs following intravenous reserpine (3 mg/kg) are shown in figure 3. There was no initial hypotension and the increases in pressure, heart rate, and contractile force occurred later and were smaller than after guanethidine. These changes persisted for 60 minutes and were followed by a gradual decrease in these variables. Although these responses were noted in all four dogs following reserpine administration, an initial measurable release of norepinephrine from the heart into the coronary sinus blood was observed in only a single sample in one experiment (table 3, 1R). The mean of the CS-FA norepinephrine differences and the mean content of norepinephrine in the atrial appendages in these dogs are shown in figure 4. The average control value of norepinephrine in the atrial appendage in dogs receiving reserpine was 2.73 /xg/g, and at one hour the average value was 2.55 /xg/g. The average concentrations were significantly lower, 1.74 ^g/g and 1.16 /j-g/g at two and four hours after the drug (P <0.05). When the concentrations of norepinephrine in the atrial appendage four hours -after guanethidine were compared with those after reserpine by an analysis of eovariance, the values after reserpiue were significantly lower (P <0.05). The arterial levels of epinephrine and norepinephrine and the arterial systolic pressure Circulation Research, Volume XII, March 1963

4 NOREPINEPHRINE DEPLETION FROM HEART 259 PLASMA NOREPINEPHRINE (CORONARY SINUS - ) DIFFERENCE PHYSIOLOGICAL DATA AFTER RESERPINE SYSTOLIC PRESSURE HESERPINE (3mg/Kg) [ GUANETHIDINE (I5mg/Kg) 40 HOURS PULSE RATE ATRIAL APPENDAGE NOREPINEPHRINE RESERPiNE 3mg/Kgl TISSUE (pg/gm) i AVERAGE % CHANGE IN CONTRACTILE FORCE HOURS GUANETHIDINE (ISmg/Kg) FIGURE 2 The mean values for the venoarterial (CS-FA) norepinephrine differences before and after the administration of guanethidine are shown in the upper figure. The lower figure demonstrates the mean values of the concentration of norepinephrine in atrial appendages at intervals after the administration of guanethidine to 4 dogs. following guanethidine are shown in the left half of figure 5, and after reserpine in the right half of this figure. During the period of elevation of arterial pressure, the arterial epinephrine concentration showed little change, but when the pressure fell below control levels, a consistent and progressive elevation of the arterial epinephrine concentration occurred, which was more marked after guanethidine than after reserpine. Discussion The results of these studies with guanethidine demonstrate that this drug given intravenously in this dose consistently results in the release of measurable quantities of nor- CiTctdation Research, Volume XII. March 1903 FIGURE 3 The upper two figures demonstrate the mean and standard errors of the mean of the systolic arterial pressure and heart rale follotving the administration of reserpine (3.0 mg/kg). The lotver figure shows the average per cent changes in contractile force in these 4 dogs. epinephrine into the coronary sinus blood. This period of release was terminated three hours after injection in all dogs and its duration corresponded closely with the augmentation of arterial systolic pressure, heart rate, and contractile force. The increases of these variables were similar to those noted by other workers 5 " 7 who have suggested that they are mediated by the release of norepinephrine. Guanethidine, in doses comparable to those employed here, has been shown previously to produce a reduction of the norepinephrine concentration of the heart to less than 10% of the control values 18 hours after adminis-

5 260 HAEEISON, CHIDSEY, GOLDMAN, BRATJNWALD TABLE 2 Norepinephrine Concentration in Atrial Appendage in Control and Drug Treated Dogs Control dogs Guanethidine treated dogs Reserpine treated dogs Dog No i 5 Mean 1G 2G 3G 4G Mean 1R 2R 3R 4R Mean Control /73 1 Hour Hours ]T29 (P <.1O) (-P <-05) 4 Hours (P <-05) (P <.0o) All values expressed as /ig/gram of tissue. The control dogs had their chest open for four hours but received no drug. The guanethidine treated dogs received guanethidine sulfate 15 mg/kg and the reserpine treated dogs received reserpine 3.0 mg/kg, intravenously. TABLE 3 Arterial amd Coronary Sinus Norepinephrine Concentration and Systolic Arterial Pressure in Reserpine Treated Animals 1R 2R 33 4R AET. NE AET. NE Time Control of determination (minutes) Arterial norepinephrine concentration in ^g/liter. Coronary sinus norepinephrine in Systolic arterial pressure in mm Hg S tration in the rat, cat, dog, and rabbit. 1 ' 2 ' 17 In the present study intravenous guanethidine (15 jug/kg) was given to three dogs and reduced the average concentrations of norepinephrine in the atrial appendages to 0.13 fig/g (0.05 to 0.18) in 24 hours. How- Circulation Research, Volume XII, March 196S

6 NOREPINEPHRINE DEPLETION FROM HEART 261 ever, at the time when release of norepinephrine as the free amine from the heart had apparently ceased, the norepinephrine content of atrial appendages was decreased only to between 0.97 and 2.28 /xg/g, representing an average decrease of 24% of control levels (table 2). It is apparent, therefore, that a major portion of the reduction of norepinephrine in the heart produced by guanethidine occurred after the termination of the period during which a measurable release of norepinephrine (as the amine) into the coronary venous blood had taken place. The observation that the norepinephrine releasing effect of guanethidine was terminated at a time when a major fraction of the myocardial amine stores persisted suggests that this initial release may be divorced from the later, more profound reduction of tissue concentrations. These results are similar to those observed following prolonged infusion of tyramine, when the norepinephrine release had apparently ceased in spite of the slight reductions of myocardial norepinephrine stores which had taken place. The experimental techniques, such as the duration of anesthesia and of thoracotomy necessarily differed in the animals studied immediately after and those studied 24 hours following guanethidine. However, it appears that guanethidine has an initial norepinephrine releasing action similar to tyramine, and a tissue-depleting action which occurs later and which is not accompanied by release of measurable quantities of norepinephrine as the catecholamine into the blood. Thus, it may be concluded that metabolism and/or decreased synthesis must account for the later reduction in tissue norepinephrine. In these experiments the reduction in atrial norepinephrine content following reserpine was somewhat greater than after guanethidine, but the adrenergic response following reserpine was less pronounced. These findings are similar to those noted by Krayer et al. is These investigators suggested that the difference is due to a greater sensitivity to released norepinephrine in the guanethidine treated animals/ a conclusion based on the finding Circulation Research, Volume Xll, March 196S jig/'jter 2- TISSUE (jjg/gm) PLASMA NOREPINEPHRINE (CORONARY SINUS - ) DIFFERENCE HOURS ATRIAL APPENDAGE NOREPINEPHRINE RESERPINE I(3mg/Kg) FIGURE 4 The upper figure shows the mean of the venoarterial (CS-FA) norepinephrine dijfe.rp.nc.es before and after the administration of reserpine: the lower figure shoios the mean values of the concentration of norepinephrine in atrial appendages at intervals after the administration of reserpine to 4 dogs. that those animals were supersensitive to infused norepinephrine. However, the present observations clearly demonstrate that, with the doses of the drugs employed, a greater release of norepinephrine from the heart into the blood occurs after guanethidine than after reserpine. In view of the finding that the concentration of norepinephrine in atrial appendage is reduced more rapidly after reserpine than after guanethidine and since norepinephrine did not appear in the coronary sinus blood after reserpine, it may be reasoned that metabolism of norepinephrine, perhaps associated with reduced synthesis within the heart, is responsible for the reduction in tissue concentration observed after this drug. An incidental finding of interest in this investigation was the progressive elevation of

7 262 HARBISON, CHIDSEY, GOLDMAN, BRATJNWALD RESPONSE TO GUANETHIDINE RESPONSE TO RESERPINE NOR-EPINEPHRINE NOR-EPINEPHRINE 0-S/L) AUINE (jig/l) 0 I 2 1 GUANETHIDINE I (ISmg/Kg) \ RESERPINE I CJmg/Kg) EPINEPHRINE EPINEPHRINE K** i 6 I i I GUANETHIDINE (ISmj/Kgl I (Sing/Kg) SYSTOLIC PRESSURE COO- SYSTOLIC PRESSURE f OUANETHIDINE 1 (ISntg/Kg) mm. L Mg ZOO RESERPINE Omg/Kg) ISO- IOOt *-i I 2 i «HOURS * a «HOURS FIGURE 5 The mean and standard errors of the arterial norepinephrine and epinephrine concentrations and the systolic arterial pressures following the intravenous administration of guamethidine are shown in the left half of this figure and those following reserpine administration in the right half of the figure. Dorepinephrine and epinephrine in arterial blood after the administration of both drugs. This was an unexpected finding because it occurred at a time when arterial blood pressure and the myocardial contractile force were declining. These observations raise the possibility that the ability of the effector organs to respond to circulatory catecholamines had become impaired. Summary The release of norepinephrine from the heart of anesthetized open-chest dogs has been determined both after the intravenous administration of guancthidine (15 mg/kg) and after reserpine (3 mg/kg) by measuring the plasma norepinephrine concentration with the fiuorometrie method (THI) in bloods obtained simultaneously from the coronary sinus and femoral artery. In four dogs following the administration of guanethidine, norepinephrine release into the coronary sinus blood occurred and persisted for two to three hours. This release of norepinephrine was accompanied by an adrenergic response of similar duration. The norepinephrine content of atrial appendage was reduced by 24% of Circulation Research, Volume XII, March 19BS

8 NOREPINEPHRI1TE DEPLETION FEOM HEART 263 control at four hours in these dogs and to extremely low levels in three other dogs 24 hours after guanethidine administration. Thus, the reduction of the tissue content of norepinephrine continued after measurable release of norepinephrine into the coronary sinus blood had ceased. In four dogs following reserpine administration the adrenergic response was consistently smaller, and detectable release of norepinephrine into the coronary sinus blood was found transiently only in one dog. However, the norepinephrine content of atrial appendage was reduced by an average of 65% of control values at four hours. These findings suggest a difference in the mechanism of norepinephrine depletion in the period immediately after administration of guanethidine and reserpine. References 1. CASK, K.., KTJNTZMAK, R., AND BRODIE, B. B.: Norepinephrine depletion as a possible mechanism of action of guanethidine (SU 5864) a new hypotensive agent. Proe. Soe. Exp. Biol. Med. 103: 871, CASS, R., AND SPMGGS, T. L. B.: Tissue amine levels and sympathetic blockade after guanethidine and bretylium. Brit. J. Pharmacol. 17: 442, BERTLER, A., CARLSSON, A., AND ROSENGREN, E.: Release by reserpine of eatecholamines from rabbit hearts. Naturwissenschaftcn 43: 521, i. WAUD, D. R., KOTTEGODA, S. B., AND KRAYER, O.: Threshold dose and time course of norepinephrine depletion of the mammalian heart by reserpiae. J. Pharmacol. Exp. Therap. 124: 340, KRAYER, O., ALPER, M. H., AND PAASONEN, M. K.: Action of guanethidine and reserpine upon the isolated mammalian heart. J. Pharmacol. Exp. Therap. 133: 164, MCCUBBIN, J. W., KANCKO, Y., AND PAGE, I. H.: Peripheral cardiovascular actions of guanethidine in dogs. J. Pharnmcol. Exp. Therap. 131: 346, GAFFNEY, T. E.: Effect of guanethidine and bretylium on the dog heart-lung preparation. Circulation Research 9: 83, DOMINO, E. F., AND R.ECH, R. H.: Observations on the initial hypertensive response to reserpine. J. Pharmacol. Exp. Therap. 121: 173, MAXWELL, B. A., Ross, S. D., PT.TJMMER, A. J., AND SiGG, E. B.: A peripheral action of reserpine. J. Pharmacol. Exp. Therap. 119: 69, HARRISON, D. H., CniDSEr, C. A., AND BRATJN- WALD, E.: The release of norepinephrine from the heart by vasoaetive amines. Clin. Res. 9: 328, CHIDSEY, C. A., HARRISON, D. H., AND BRAUN- WALD, E.: Release of norepinephrine from the heart by vasoaetive amines. Proc. Soc. Exp. Biol. Med. 109: 488, GOODALE, W. T., TJTJBIN, M., EOKENHOFr, J. E., HAFKENSCHIEL, J. H., AND BANFIELD, W. G., JR. : Coronary sinus cathetcrization for studying coronary blood flow and myocardial metabolism. Am. J. Physiol. 152: 340, COTTEN, M. EE~V., AKD BAT, E.: Direct measurement of changes in cardiac contractile force. Am. J. Physiol. 187: 122, CROTJT, J. R., CREVELING, C. R., AND TJDENFRIEND, S.: Norepinephrine metabolism in rat brain and heart. J. Pharmacol. Exp. Therap. 132: 269, EULEB, U. S. v., AND LISHAJKO, F.: Improved technique for the fluorometric estimation of catecholamines. Acta Physiol. Seand. 51: 348, BERTLER, A., CARLSSQN, A., AND ROSENOREN, E.: A method for the fluorometric determination of adrenaline and noradrsnaline in tissues. Acta Physiol. Scand. 44: 273, BTJTTERFIELD, J. TJ., AND RICHARDSON, J. A.: Acute effects of guanethidine on myocardial contractility and catecholamine levels. Proc. Soc. Exp. Biol. Med. 106: 259, Circulation Research, Volume XII. March 1963