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The slopes of cumulative and non-cumulative dose-response curves for nonadrenaline and isoprenaline
EUROPEAN JOURNAL OF PHARMACOLOGY 17 (1972) 44-49. NORTH-HOLLAND PUBLISHING COMPANY
THE SLOPES OF CUMULATIVE AND NON-CUMULATIVE DOSE-RESPONSE NORADRENALINE AND ISOPRENALINE *
CURVES FOR
S.GUIMARAES Department o f Pharmacology, Medical Faculty, Porto, Portugal Received 11 February 1971
Accepted 7 September 1971
S. GUIMAR,~ES, The slopes of cumulative and non-cumulative dose-response curves for noradrenaline and isoprenaline, European J. Pharmaco117 (1972) 44-49. On guinea pig aortic and dog saphenous vein strips, cumulative and non-cumulative dose-response curves were determined for noradrenaline and isoprenaline. Cumulative dose-response curves for noradrenaline were significantly flatter than non-cumulative curves. However, for isoprenaline cumulative and non-cumulative dose-response curves were identical, and their slopes corresponded to that of the non-cumulative dose-response curve for noradrenaline. After pretreatment with cocaine plus iproniazid plus tropolone, cumulative dose-response curves for noradrenaline were as steep as non-cumulative curves. Experiments with the oil immersion technique showed that isoprenaline was much more slowly inactivated than noradrenaline. There are two possible explanations for the observed changes in slopes of dose-response curves: (a) during .cumulative dose-response curves more of the noradrenaline can be inactivated by the tissue (if the agonist is inactivated at a high rate) than during non-cumulative dose-response curves; (b) during cumulative dose-response curves desensitization develops to noradrenaline in the absence but not in the presence of cocaine p~s'iproniazid plus tropolone; however, desensitization does not develop to isoprenaline. Noradrenaline Isoprenaline
Dose-response curves Slope
1. INTRODUCTION When noradrenaline and isoprenaline were used on isolated guinea pig aortic strips, comparison of conventional dose-response curves (individual doses) with cumulative dose-response curves led to two observations: (1) if cumulative dose-response curves were determined for both amines, the slopes of the curves were not identical, the curve for noradrenaline being flatter than that for isoprenaline; (2) curves for noradrenaline calculated by the two different methods were not parallel, the cumulative dose-response curve being flatter than the conventional curve (Guimar~es, 1968). A study was undertaken to explain the mechanism * Supported by a grant from Instituto de Alta Cultura, Research contract nr. 2-2917.
Cumulative way Non-cumulative way
underlying these facts. Experiments were carried out with two different isolated vascular smooth muscle preparations, the isolated guinea pig aortic strip and the isolated dog saphenous vein strip.
2. MATERIALS AND METHODS Mongrel dogs ( 6 - 1 7 kg) were anesthetized with pentobartital, 30 mg/kg, i.v. After careful dissection the venous segment selected was removed and placed in a .porcelain dish containing oxygenated K r e b s Henseleit solution. Four helically cut strips (about 25 m m X3 m m each) were prepared as described by Furchgott and Bhadrakom (1953) and Guimar~es and Osswald (1969). Guinea pigs, body weight 3 2 0 - 6 0 0 g, were killed by a blow on the head. The thoracic aorta was re-
S. Guimar#es, Slopes of dose-response curves moved and placed in a porcelain dish containing oxygenated Krebs-Henseleit solution. Two helically cut strips (about 30 mm × 2.5 mm each) were prepared as described by Furchgott and Bhadrakom (1953). The preparations were set up in a 25 ml organ bath filled with Krebs-Henseleit solution of the following composition ( g / 1 distilled water): NAC1,6.94; KC1, 0.35; CaC12, 0.28; KH2PO4, 0.16; MgSO4. 7H20, 0.29; NaHCO3, 2.10; glucose, 1.81. This solution contained 10-SM EDTA. The solution in the bath was maintained at 37°C and bubbled with 95% O 2 - 5 % CO2. The preparations were allowed to equilibrate for 2 hr with repeated changes of the medium at optimum resting tensions, which were about 1 g for aortic strips and about 1.2 g for saphenous vein strips. The contractions were recorded on a smoked drum with a lever giving a 5× magnification or on a E & M Physiograph Four ink-writing recorder after suitable amplification by means of an isotonic myograph (E & M 91-100-73). Dose-response curves for only one amine were determined per preparation. When cumulative d o s e response curves were determined the amines were added cumulatively in such a way that the final concentration in the bath was increased by a factor of 2 in the case of the guinea pig aortic strips, and by a factor of 2, 3 or 4 in the case of the dog saphenous vein strips, whenever the response attained a steady state. When non-cumulative dose-response curves were determined, repeated washings (at least three) were made between drug additions. The doses of the drug added to the bath increased by a factor of 2 in the case of the guinea pig aortic strips and by a factor of 2, 3 or 4 in the case of the dog saphenous vein strips. Inactivation of noradrenaline or isoprenaline was determined from the rate of relaxation of isolated strips in mineral oil after contractions produced in Krebs-Henseleit medium as described by Kalsner and Nickerson (1968) and Osswald et al. (1971). Results are expressed in terms of the mean values + S.E.M. Significance of differences between the slopes of dose-response curves was calculated by Student's t-test after determining the regression lines obtained from the probit transformation of the d o s e response curves; the slope of the curves was calculated from the points corresponding to probits 4 and 6 (16% and 84%). p values of 0.05 or less were considered significant.
45
The following drugs were used: cocaine hydrochloride (Bios), iproniazid phosphate (F. H o f f m a n n La Roche), (-)-isoprenaline (Cilag Chemie AG), ( - ) noradrenaline (Ht~chst) and tropolone (Aldrich Chemical Co.,Inc.). The mineral oil used was a U.S.P. quality liquid paraffin. Stock solutions of isoprenaline and noradrenaline were prepared in 0.9% NaC1 solution (acidified by concentrated HC1 in order to obtain an approximately 0.05 N solution) and renewed once a week. All solutions were refrigerated when not in use. When needed, final solutions of drugs were made with Krebs-Henseleit solutions immediately before use. Concentrations of drugs are expressed as molar concentrations.
3. RESULTS
3.1. Guinea pig aortic strips: slopes of dose-response curves for noradrenaline and isoprenaline 30 dose-response curves were determined for noradrenaline (15 cumulative and 15 non-cumulative, see Materials and Methods) and 24 for isoprenaline (12 cumul~itive and 12 non-cumulative). The geometric sequence 1 - 2 - 4 - 8 - e t c . (factor of 2) of increasing the dose was used for both cumulative and non-cumulative dose-response curves. Fig: 1 shows the four mean curves of the total of 54 dose-response curves calculated. The difference between the slopes of the two regression lines obtained from the dose-response curves for noradrenaline, one calculated by noncumulative additions and the other by cumulative additions, is highly significant (p=0.01). On the other hand, the regression line of the cumulative d o s e response curve for noradrenaline is significantly flatter than that for isoprenaline determined in the same way (p<0.01). Isoprenaline regression lines (one resuiting from cumulative dose-response curves and the other from the curves determined in the conventional manner have identical slopes. 3.2. Dog saphenous vein strips 3.2.1. Slopes of dose-response curves for noradrenaline and isoprenaline. As described in Materials and Methods, d o s e response curves obtained either in the cumulative manner or in the non-cumulative manner were calculated by increasing the concentration by a factor of 2,
S. Guimar~es, Slopes o f dose-response curves
46
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,~ flo
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= eL,
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1~ e
I
lo-7
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~66
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16"
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Fig. 1. M e a n d o s e - r e s p o n s e c u r v e s o b t a i n e d w i t h i s o l a t e d g u i n e a pig a o r t i c strips, using the f a c t o r o f 2 for i n c r e a s i n g a g o n i s t c o n c e n t r a t i o n . N - c u r v e s for n o r a d r e n a l i n e : o o c u m u l a t i v e curve ( n = 1 5 ) ; o - - - o , n o n - c u m u l a t i v e curve ( n = 1 5 ) ; I - curves for i s o p r e n a l i n e : z~ ~, n o n - c u m u l a t i v e curve ( n = 1 2 ) ; z ~ _ _ _ q n o n - c u m u l a t i v e curve (n=12). T h e d i f f e r e n c e . b e t w e e n the slopes of o o and o---o and between o o a n d z~ z~are h i g h l y significant, p v a l u e s b e i n g r e s p e c t i v e l y =0.01 a n d < 0 . 0 1
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Fig. 2. Mean d o s e - r e s p o n s e c u r v e s o b t a i n e d w i t h i s o l a t e d d o g s a p h e n o u s vein strips. N - curves for n o r a d r e n a l i n e : o o, c u m u l a t i v e c u r v e w i t h f a c t o r o f 2 for i n c r e a s i n g c o n c e n t r a t i o n s ( n = 1 2 ) ; o - - - o c u m u l a t i v e curve w i t h f a c t o r o f 3 (n=5); o-.-.-.% c u m u l a t i v e curve w i t h f a c t o r o f 4 (n=5); o ...... o, n o n - c u m u l a t i v e curve w i t h f a c t o r o f 2 (n=10). I - curves for i s o p r e n a l i n e ; z~ % c u m u l a t i v e curve, w i t h f a c t o r o f 2 (n=6); ~ . . . . ~, n o n - c u m u l a t i v e curve, w i t h f a c t o r o f 2 (n=7). T h e difference between the slopes of o o a n d o ..... o a n d b e t w e e n o o and ~ zxare h i g h l y significant. T h e values o f p w e r e <0.01 and <0.02 respectively.
47
S. GuimaNles, Slopes o f dose-response curves
3 or 4. The mean dose-response curves for noradrenaline and for isoprenaline (only 2) are shown in fig. 2. This figure shows that the regression lines of d o s e response curves for noradrenaline determined by the cumulative method have different slopes depending on the factor used: the curve obtained using the factor 2 was flatter that the curve obtained using the factor 3 and this in turn was flatter than the curve obtained using the factor 4. When dose-response curves for noradrenaline are considered, the difference between the slope of the regression lines resulting from the cumulative dose-response curve using the factor 2 and from the non-cumulative d o s e response curve using the same factor, is highly significant. On the other hand, the difference between the slope of the regression line corresponding to the cumulative dose-response curve for noradrenaline using the factor 2 and the slope of the regression line of the cumulative dose-response curve for isoprenaline using the same factor is also highly significant (p<0.02). Regression lines of the dose-response curves for isoprenaline have identical slopes, independent of the method used for determining the curves and of the factor used. The slopes of non-cumulative dose-
response curves for noradrenaline were not affected by the factor by which the concentration was increased. 3.2.2. Slopes of noradrenaline curves after iproniazid plus cocaine plus tropolone Four strips were obtained from each vein as described in Materials and Methods. Two strips without pretreatment served as controls; the other two strips were exposed to iproniazid (7X 10-4 M) for 30 min, the drug was then washed out and an additional 30 min period (during which 8 washings were made) was allowed to elapse. The strips were exposed to tropolone (2 X 10-4 M) and cocaine (10 s M) for 30 min. Then cumulative dose-response curves for noradrenaline were determined with tropolone and cocaine still present in the bath. The addition of noradrenaline was made, in this case, to both pretreated and control strips in such a way that the final concentration in the bath was increased by a factor of 2. As fig. 3 shows, iproniazid plus tropolone plus cocaine treatment caused a highly significant (P<0.01) shift of the dose-response curve to the left and a highly significant change in the slope (p<0.02), the curve becoming steeper than in the control. Comparison of
100
ao u m o 60 =,¢
=o
40
¢=
_= 20
I
I
i
I
i
,64M Fig. 3. Mean cumulative dose-response curves for noradrenaline (using the factor 2) without (o e) o) (n=24) and after (e (n=8) treatment with iproniazid plus cocaine plus tropolone. The curve is steeper for pretreated strips than for the control strips, the change in the slope being significant (p<0.02); pretreatment also shifted the curve to the left. The numerals on the broken lines refers to the enhancement of noradrenaline effect at different levels of the dose-response curves.
48
S. Guimar~es, Slopes of dose-response curves
the EDs o values of noradrenaline in the controls and in the strips pretreated with this combination of drugs shows a 23 + 1.8-fold enhancement of the action of noradrenaline. The curves determined after treatment with iproniazid plus cocaine plus tropolone became parallel to the non-cumulative dose-response curves determined in the absence of this treatment. 3.2.3. Inactivation of noradrenaline and isoprenaline With the technique of Kalsner and Nickerson (1968), maximal doses of noradrenaline (10 -s M) or isoprenaline (1.8×10 -4 M) were added to the bath; after the response had reached a stable plateau, the Krebs solution was rapidly replaced with warm and oxygenated mineral oil. The rate of relaxation was then measured and considered as indicating the rate of inactivation of the drug under study. In the case of noradrenaline, the time necessary for a 50% relaxation was 9.5 -+ 0.8 rain. In the case of isoprenaline this value could not be calculated because the strip did not relax after 2 hr of observation. 4. DISCUSSION Experiments in guinea pig aortic and dog saphenous vein strips showed that cumulative doseresponse curves for noradrenaline and isoprenaline were not parallel; this was not expected since it is known that the two sympathomimetic amines activate the same receptor system in these tissues (Guimar~es, 1968; Guimar~es and Osswald, 1969). However, cumulative dose-response curves for noradrenaline are flatter than non-cumulative dose-response curves for the same amine. Two possibilities can be considered to explain these facts. According to the first hypothesis, the inactivation of a significant amount of noradrenaline due to a long time of contact between the amine and the inactivation mechanisms, during the determination of cumulative dose-response curves, could account for the flat slope of noradrenaline d o s e response curves obtained in this way. In the case of isoprenaline, the presence of a bulky group on the nitrogen of isoprenaline caused a marked reduction in its affinity for MAO (Blaschko, 1952); moreover, isoprenaline uptake by the nerve endings is very low (And~n et al., 1964; Hertting, 1964; Iversen, 1965); its inactivation depends almost entirely on metabo-
lism by COMT. Since this enzyme represents a minor pathway for the disposition of catecholarnines in venous tissue (Osswald et al, 1971)a pronounced difference between the rate of inactivation of noradrenaline and that of isoprenaline was expected. Results obtained with the oil immersion technique of Kalsner and Nickerson (1968) confirm that the inactivation of isoprenaline in this tissue is much slower than that of noradrenaline. When uptake by nerve endings, oxidation by MAO and O-meth3flation was blocked by cocaine plus iproniazid plus tropolone, the cumulative dose-response curves for noradrenaline were steeper than the control curves and were parallel to the non-cumulative dose-response curves for the same amine. Saturation of uptake is one of the factors influencing the slope of dose-response curves as shown by Blinks (1967) for guinea pig atria and by Langer and Trendelenburg (1969) for the nictitating membrane. This working hypothesis is in partial but not in full agreement with results recently obtained from the measurement of the removal of noradrenaline from the medium by isolated dog saphenous strips (Guimar~'es et al., 1971). In these experiments, the venous strips were incubated for 60 min in a medium (0.4 ml for 10 mg of tissue) containing 250 ng/ml of (-)-noradrenaline; since the average weight of the tissues was about 75 mg, the bath volume was approx. 3 ml. Under these conditions, a venous strip removed about 150 ng of noradrenaline per hr from the medium. If it is assumed that an identical removal is obtained with a bath volume of 25 ml (as used in the present experiments), the concentration in the medium should fall from 250 to 244 ng/ml of noradrenaline. Such a fall is too small to account for the degree of subsensitivity of the strips subjected to cumulative doseresponse curves (see fig. 2, and note that 250 ng/ml corresponds to the ED4 s ). However, such calculations are subject to considerable error, since removal must be assumed to be directly related to the concentration in the medium; this fell much more when the bath volume was small (Guimar~es et al., 1971)than in the present experiments with a 25 ml bath. Moreover, the present experiments involved stepwise increases in the bath concentrations rather than a prolonged exposure to a single concentration. Hence, an exact comparison of the results is not possible. There is a second possible explanation for our re-
S. Guimarifes, Slopes o f dose-response curves suits: desensitization to, or autoinhibition of an agonist may develop during prolonged exposure to the agonist (Gaddum, 1953). Desensitization to noradrenaline has been postulated to occur in the rat vas deferens, and cocaine has been claimed to prevent desensitization to noradrenaline (Barnett et al., 1968). However, it is difficult to reconcile this proposal with the present observation that the p h e n o m e n o n was not observed for isoprenaline. One would postulate that there is no desensitization to isoprenaline although this amine has a low potency and has to be administered in concentrations which are higher than those requ!red for dose-response curves for noradrenaline. Van. Rossum and Ari~ns (1959) observed that the abo~e m e n t i o n e d sympathomimetic amine had virtually identical dose-response curves which were independent of the method of calculation used and Ari~ns and Simonis (1960) considered that the vas deferens of the rat or guinea pig is suitable for studying a-sympathomimetic amines by using cumulative dose-response curves. Our results led to different conclusions, perhaps because the contractile response has a very slow development; in the dog saphenous vein preparation the time elapsing between the first addition tO the bath and the point at which the maximal effect is attained, when the cumulative method and the factor of 2 were used, ranged between 50 and 65 min. Thus there was prolonged contact between the preparation and the amine permitting considerable metabolism of the amine to occur (if the rate of metabolism is high), and desensitization or autoinhibition to develop (if this develops to the amine under study). In conclusion we assume that the method of individual doses (non-cumulative) is preferred to the cumulative method whenever the comparison of slopes of dose-response curves is an important aim of the investigation.
ACKNOWLEDGEMENTS We are indebted to Prof. J. Maia for valuable help in the statistical analysis of the results and to Prof. U. Trendelenburg (Wiirzburg) for his most helpful criticism and revision of the manuscript.
49
REFERENCES And~n, N.-E., H. Corrodi, M. Ettles, E. Gustafsson and H. Persson, 1964, Selective uptake of some catecholamines by isolated heart and its inhibition by cocaine and phenoxybenzamine, Acta Pharmacol. Toxicol. 21, 247-259. Ari~ns, E.J. and A.M. Simonis, 1960, Autonomic drugs and their receptors, Arch. Intern. Pharmacodyn. 127, 479-507. Barnett, A., D.D. Greenhouse and R.I. Taber, 1968, A new type of drug enhancement: increased maximum response to cumulative noradrenaline in the isolated rat vas deferens, Brit. J. Pharmacol. 33, 171-176. Blaschko, H., 1952, Amine oxidase and amine metabolism, Pharmacol. Rev. 4, 415-478. Blinks, J.R., 1967, Evaluation of the cardiac effects of several beta adrenergic blocking drugs, Ann. N.Y. Acad. Sci. 139, 673-685. Furchgott, R.F. and S. Bhadrakom, 1953, Reactions of strips of rabbit aorta to epinephrine, isopropylarterenol, sodium nitrite and other drugs, J. Pharmacol. Exptl. Therap. 108, 129-143. Gaddum, J.H., 1953, Tryptamine receptors, J. Physiol. 119, 363-368. Guimar~es, S., 1968, Receptores adren~rgicos, Tese, Porto. Guimaraes, S. and W. Osswald, 1969, Adrenergic receptors in the veins of the dog, European J. Pharmacol. 5, 133-140. Guimar~es, S., W. Osswald, W. Cardoso and D. Branco, 1971, The effects of cocaine and denervation on the sensitivity to noradrenaline, its uptake and termination of action in isolated venous tissue, Arch. Pharmakol. 271,262-273. Hertting, G., 1964, The fate of 3H-isoproterenol in the rat, Biochem. Pharmacol. 13, 1119-1128. Iversen, L.L., 1965, The uptake of catechol amines at high perfusion concentrations in the rat isolated heart: a novel catechol amine uptake process, Brit. J. Pharmacol. 24, 18-33. Kalsner, S. and M. Nickerson, 1968, A method for study of mechanisms of drug disposition in smooth muscle, Can. J. Physiol. Pharmacol. 46,719-730. Langer, S. and U. Trendelenburg, 1969, The effect of a saturable uptake mechanism on the slopes of dose-response curves for sympathomimetic amines and on the shifts of dose-response curves produced by competitive antagonist, J. Pharmacol. Exptl. Therap. 167, 117-142. Osswald, W., S. Guimar~es and A. Coimbra, 1971, The termination of action of catecholamines in the isolated venous tissue of the dog, Arch. Pharmakol. 269, 15-31. Van Rossum, J.M. and E.J. Ari~ns, 1959, Pharmacodynamics of parasympathetic drugs, Arch. Intern. Pharmacodyn. 118, 418-447.
THE SLOPES OF CUMULATIVE AND NON-CUMULATIVE DOSE-RESPONSE NORADRENALINE AND ISOPRENALINE *
CURVES FOR
S.GUIMARAES Department o f Pharmacology, Medical Faculty, Porto, Portugal Received 11 February 1971
Accepted 7 September 1971
S. GUIMAR,~ES, The slopes of cumulative and non-cumulative dose-response curves for noradrenaline and isoprenaline, European J. Pharmaco117 (1972) 44-49. On guinea pig aortic and dog saphenous vein strips, cumulative and non-cumulative dose-response curves were determined for noradrenaline and isoprenaline. Cumulative dose-response curves for noradrenaline were significantly flatter than non-cumulative curves. However, for isoprenaline cumulative and non-cumulative dose-response curves were identical, and their slopes corresponded to that of the non-cumulative dose-response curve for noradrenaline. After pretreatment with cocaine plus iproniazid plus tropolone, cumulative dose-response curves for noradrenaline were as steep as non-cumulative curves. Experiments with the oil immersion technique showed that isoprenaline was much more slowly inactivated than noradrenaline. There are two possible explanations for the observed changes in slopes of dose-response curves: (a) during .cumulative dose-response curves more of the noradrenaline can be inactivated by the tissue (if the agonist is inactivated at a high rate) than during non-cumulative dose-response curves; (b) during cumulative dose-response curves desensitization develops to noradrenaline in the absence but not in the presence of cocaine p~s'iproniazid plus tropolone; however, desensitization does not develop to isoprenaline. Noradrenaline Isoprenaline
Dose-response curves Slope
1. INTRODUCTION When noradrenaline and isoprenaline were used on isolated guinea pig aortic strips, comparison of conventional dose-response curves (individual doses) with cumulative dose-response curves led to two observations: (1) if cumulative dose-response curves were determined for both amines, the slopes of the curves were not identical, the curve for noradrenaline being flatter than that for isoprenaline; (2) curves for noradrenaline calculated by the two different methods were not parallel, the cumulative dose-response curve being flatter than the conventional curve (Guimar~es, 1968). A study was undertaken to explain the mechanism * Supported by a grant from Instituto de Alta Cultura, Research contract nr. 2-2917.
Cumulative way Non-cumulative way
underlying these facts. Experiments were carried out with two different isolated vascular smooth muscle preparations, the isolated guinea pig aortic strip and the isolated dog saphenous vein strip.
2. MATERIALS AND METHODS Mongrel dogs ( 6 - 1 7 kg) were anesthetized with pentobartital, 30 mg/kg, i.v. After careful dissection the venous segment selected was removed and placed in a .porcelain dish containing oxygenated K r e b s Henseleit solution. Four helically cut strips (about 25 m m X3 m m each) were prepared as described by Furchgott and Bhadrakom (1953) and Guimar~es and Osswald (1969). Guinea pigs, body weight 3 2 0 - 6 0 0 g, were killed by a blow on the head. The thoracic aorta was re-
S. Guimar#es, Slopes of dose-response curves moved and placed in a porcelain dish containing oxygenated Krebs-Henseleit solution. Two helically cut strips (about 30 mm × 2.5 mm each) were prepared as described by Furchgott and Bhadrakom (1953). The preparations were set up in a 25 ml organ bath filled with Krebs-Henseleit solution of the following composition ( g / 1 distilled water): NAC1,6.94; KC1, 0.35; CaC12, 0.28; KH2PO4, 0.16; MgSO4. 7H20, 0.29; NaHCO3, 2.10; glucose, 1.81. This solution contained 10-SM EDTA. The solution in the bath was maintained at 37°C and bubbled with 95% O 2 - 5 % CO2. The preparations were allowed to equilibrate for 2 hr with repeated changes of the medium at optimum resting tensions, which were about 1 g for aortic strips and about 1.2 g for saphenous vein strips. The contractions were recorded on a smoked drum with a lever giving a 5× magnification or on a E & M Physiograph Four ink-writing recorder after suitable amplification by means of an isotonic myograph (E & M 91-100-73). Dose-response curves for only one amine were determined per preparation. When cumulative d o s e response curves were determined the amines were added cumulatively in such a way that the final concentration in the bath was increased by a factor of 2 in the case of the guinea pig aortic strips, and by a factor of 2, 3 or 4 in the case of the dog saphenous vein strips, whenever the response attained a steady state. When non-cumulative dose-response curves were determined, repeated washings (at least three) were made between drug additions. The doses of the drug added to the bath increased by a factor of 2 in the case of the guinea pig aortic strips and by a factor of 2, 3 or 4 in the case of the dog saphenous vein strips. Inactivation of noradrenaline or isoprenaline was determined from the rate of relaxation of isolated strips in mineral oil after contractions produced in Krebs-Henseleit medium as described by Kalsner and Nickerson (1968) and Osswald et al. (1971). Results are expressed in terms of the mean values + S.E.M. Significance of differences between the slopes of dose-response curves was calculated by Student's t-test after determining the regression lines obtained from the probit transformation of the d o s e response curves; the slope of the curves was calculated from the points corresponding to probits 4 and 6 (16% and 84%). p values of 0.05 or less were considered significant.
45
The following drugs were used: cocaine hydrochloride (Bios), iproniazid phosphate (F. H o f f m a n n La Roche), (-)-isoprenaline (Cilag Chemie AG), ( - ) noradrenaline (Ht~chst) and tropolone (Aldrich Chemical Co.,Inc.). The mineral oil used was a U.S.P. quality liquid paraffin. Stock solutions of isoprenaline and noradrenaline were prepared in 0.9% NaC1 solution (acidified by concentrated HC1 in order to obtain an approximately 0.05 N solution) and renewed once a week. All solutions were refrigerated when not in use. When needed, final solutions of drugs were made with Krebs-Henseleit solutions immediately before use. Concentrations of drugs are expressed as molar concentrations.
3. RESULTS
3.1. Guinea pig aortic strips: slopes of dose-response curves for noradrenaline and isoprenaline 30 dose-response curves were determined for noradrenaline (15 cumulative and 15 non-cumulative, see Materials and Methods) and 24 for isoprenaline (12 cumul~itive and 12 non-cumulative). The geometric sequence 1 - 2 - 4 - 8 - e t c . (factor of 2) of increasing the dose was used for both cumulative and non-cumulative dose-response curves. Fig: 1 shows the four mean curves of the total of 54 dose-response curves calculated. The difference between the slopes of the two regression lines obtained from the dose-response curves for noradrenaline, one calculated by noncumulative additions and the other by cumulative additions, is highly significant (p=0.01). On the other hand, the regression line of the cumulative d o s e response curve for noradrenaline is significantly flatter than that for isoprenaline determined in the same way (p<0.01). Isoprenaline regression lines (one resuiting from cumulative dose-response curves and the other from the curves determined in the conventional manner have identical slopes. 3.2. Dog saphenous vein strips 3.2.1. Slopes of dose-response curves for noradrenaline and isoprenaline. As described in Materials and Methods, d o s e response curves obtained either in the cumulative manner or in the non-cumulative manner were calculated by increasing the concentration by a factor of 2,
S. Guimar~es, Slopes o f dose-response curves
46
lOO ,,~)
N
I
/
8O
,~ flo
/
i 4C
l
= eL,
2C
I
I
1~ e
I
lo-7
I
~66
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16"
~o-4 M
Fig. 1. M e a n d o s e - r e s p o n s e c u r v e s o b t a i n e d w i t h i s o l a t e d g u i n e a pig a o r t i c strips, using the f a c t o r o f 2 for i n c r e a s i n g a g o n i s t c o n c e n t r a t i o n . N - c u r v e s for n o r a d r e n a l i n e : o o c u m u l a t i v e curve ( n = 1 5 ) ; o - - - o , n o n - c u m u l a t i v e curve ( n = 1 5 ) ; I - curves for i s o p r e n a l i n e : z~ ~, n o n - c u m u l a t i v e curve ( n = 1 2 ) ; z ~ _ _ _ q n o n - c u m u l a t i v e curve (n=12). T h e d i f f e r e n c e . b e t w e e n the slopes of o o and o---o and between o o a n d z~ z~are h i g h l y significant, p v a l u e s b e i n g r e s p e c t i v e l y =0.01 a n d < 0 . 0 1
N
m
I
IIC
•w
.°/
i
20
•
I
167
I
I~ 6
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16 5
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164
I
1~ M
Fig. 2. Mean d o s e - r e s p o n s e c u r v e s o b t a i n e d w i t h i s o l a t e d d o g s a p h e n o u s vein strips. N - curves for n o r a d r e n a l i n e : o o, c u m u l a t i v e c u r v e w i t h f a c t o r o f 2 for i n c r e a s i n g c o n c e n t r a t i o n s ( n = 1 2 ) ; o - - - o c u m u l a t i v e curve w i t h f a c t o r o f 3 (n=5); o-.-.-.% c u m u l a t i v e curve w i t h f a c t o r o f 4 (n=5); o ...... o, n o n - c u m u l a t i v e curve w i t h f a c t o r o f 2 (n=10). I - curves for i s o p r e n a l i n e ; z~ % c u m u l a t i v e curve, w i t h f a c t o r o f 2 (n=6); ~ . . . . ~, n o n - c u m u l a t i v e curve, w i t h f a c t o r o f 2 (n=7). T h e difference between the slopes of o o a n d o ..... o a n d b e t w e e n o o and ~ zxare h i g h l y significant. T h e values o f p w e r e <0.01 and <0.02 respectively.
47
S. GuimaNles, Slopes o f dose-response curves
3 or 4. The mean dose-response curves for noradrenaline and for isoprenaline (only 2) are shown in fig. 2. This figure shows that the regression lines of d o s e response curves for noradrenaline determined by the cumulative method have different slopes depending on the factor used: the curve obtained using the factor 2 was flatter that the curve obtained using the factor 3 and this in turn was flatter than the curve obtained using the factor 4. When dose-response curves for noradrenaline are considered, the difference between the slope of the regression lines resulting from the cumulative dose-response curve using the factor 2 and from the non-cumulative d o s e response curve using the same factor, is highly significant. On the other hand, the difference between the slope of the regression line corresponding to the cumulative dose-response curve for noradrenaline using the factor 2 and the slope of the regression line of the cumulative dose-response curve for isoprenaline using the same factor is also highly significant (p<0.02). Regression lines of the dose-response curves for isoprenaline have identical slopes, independent of the method used for determining the curves and of the factor used. The slopes of non-cumulative dose-
response curves for noradrenaline were not affected by the factor by which the concentration was increased. 3.2.2. Slopes of noradrenaline curves after iproniazid plus cocaine plus tropolone Four strips were obtained from each vein as described in Materials and Methods. Two strips without pretreatment served as controls; the other two strips were exposed to iproniazid (7X 10-4 M) for 30 min, the drug was then washed out and an additional 30 min period (during which 8 washings were made) was allowed to elapse. The strips were exposed to tropolone (2 X 10-4 M) and cocaine (10 s M) for 30 min. Then cumulative dose-response curves for noradrenaline were determined with tropolone and cocaine still present in the bath. The addition of noradrenaline was made, in this case, to both pretreated and control strips in such a way that the final concentration in the bath was increased by a factor of 2. As fig. 3 shows, iproniazid plus tropolone plus cocaine treatment caused a highly significant (P<0.01) shift of the dose-response curve to the left and a highly significant change in the slope (p<0.02), the curve becoming steeper than in the control. Comparison of
100
ao u m o 60 =,¢
=o
40
¢=
_= 20
I
I
i
I
i
,64M Fig. 3. Mean cumulative dose-response curves for noradrenaline (using the factor 2) without (o e) o) (n=24) and after (e (n=8) treatment with iproniazid plus cocaine plus tropolone. The curve is steeper for pretreated strips than for the control strips, the change in the slope being significant (p<0.02); pretreatment also shifted the curve to the left. The numerals on the broken lines refers to the enhancement of noradrenaline effect at different levels of the dose-response curves.
48
S. Guimar~es, Slopes of dose-response curves
the EDs o values of noradrenaline in the controls and in the strips pretreated with this combination of drugs shows a 23 + 1.8-fold enhancement of the action of noradrenaline. The curves determined after treatment with iproniazid plus cocaine plus tropolone became parallel to the non-cumulative dose-response curves determined in the absence of this treatment. 3.2.3. Inactivation of noradrenaline and isoprenaline With the technique of Kalsner and Nickerson (1968), maximal doses of noradrenaline (10 -s M) or isoprenaline (1.8×10 -4 M) were added to the bath; after the response had reached a stable plateau, the Krebs solution was rapidly replaced with warm and oxygenated mineral oil. The rate of relaxation was then measured and considered as indicating the rate of inactivation of the drug under study. In the case of noradrenaline, the time necessary for a 50% relaxation was 9.5 -+ 0.8 rain. In the case of isoprenaline this value could not be calculated because the strip did not relax after 2 hr of observation. 4. DISCUSSION Experiments in guinea pig aortic and dog saphenous vein strips showed that cumulative doseresponse curves for noradrenaline and isoprenaline were not parallel; this was not expected since it is known that the two sympathomimetic amines activate the same receptor system in these tissues (Guimar~es, 1968; Guimar~es and Osswald, 1969). However, cumulative dose-response curves for noradrenaline are flatter than non-cumulative dose-response curves for the same amine. Two possibilities can be considered to explain these facts. According to the first hypothesis, the inactivation of a significant amount of noradrenaline due to a long time of contact between the amine and the inactivation mechanisms, during the determination of cumulative dose-response curves, could account for the flat slope of noradrenaline d o s e response curves obtained in this way. In the case of isoprenaline, the presence of a bulky group on the nitrogen of isoprenaline caused a marked reduction in its affinity for MAO (Blaschko, 1952); moreover, isoprenaline uptake by the nerve endings is very low (And~n et al., 1964; Hertting, 1964; Iversen, 1965); its inactivation depends almost entirely on metabo-
lism by COMT. Since this enzyme represents a minor pathway for the disposition of catecholarnines in venous tissue (Osswald et al, 1971)a pronounced difference between the rate of inactivation of noradrenaline and that of isoprenaline was expected. Results obtained with the oil immersion technique of Kalsner and Nickerson (1968) confirm that the inactivation of isoprenaline in this tissue is much slower than that of noradrenaline. When uptake by nerve endings, oxidation by MAO and O-meth3flation was blocked by cocaine plus iproniazid plus tropolone, the cumulative dose-response curves for noradrenaline were steeper than the control curves and were parallel to the non-cumulative dose-response curves for the same amine. Saturation of uptake is one of the factors influencing the slope of dose-response curves as shown by Blinks (1967) for guinea pig atria and by Langer and Trendelenburg (1969) for the nictitating membrane. This working hypothesis is in partial but not in full agreement with results recently obtained from the measurement of the removal of noradrenaline from the medium by isolated dog saphenous strips (Guimar~'es et al., 1971). In these experiments, the venous strips were incubated for 60 min in a medium (0.4 ml for 10 mg of tissue) containing 250 ng/ml of (-)-noradrenaline; since the average weight of the tissues was about 75 mg, the bath volume was approx. 3 ml. Under these conditions, a venous strip removed about 150 ng of noradrenaline per hr from the medium. If it is assumed that an identical removal is obtained with a bath volume of 25 ml (as used in the present experiments), the concentration in the medium should fall from 250 to 244 ng/ml of noradrenaline. Such a fall is too small to account for the degree of subsensitivity of the strips subjected to cumulative doseresponse curves (see fig. 2, and note that 250 ng/ml corresponds to the ED4 s ). However, such calculations are subject to considerable error, since removal must be assumed to be directly related to the concentration in the medium; this fell much more when the bath volume was small (Guimar~es et al., 1971)than in the present experiments with a 25 ml bath. Moreover, the present experiments involved stepwise increases in the bath concentrations rather than a prolonged exposure to a single concentration. Hence, an exact comparison of the results is not possible. There is a second possible explanation for our re-
S. Guimarifes, Slopes o f dose-response curves suits: desensitization to, or autoinhibition of an agonist may develop during prolonged exposure to the agonist (Gaddum, 1953). Desensitization to noradrenaline has been postulated to occur in the rat vas deferens, and cocaine has been claimed to prevent desensitization to noradrenaline (Barnett et al., 1968). However, it is difficult to reconcile this proposal with the present observation that the p h e n o m e n o n was not observed for isoprenaline. One would postulate that there is no desensitization to isoprenaline although this amine has a low potency and has to be administered in concentrations which are higher than those requ!red for dose-response curves for noradrenaline. Van. Rossum and Ari~ns (1959) observed that the abo~e m e n t i o n e d sympathomimetic amine had virtually identical dose-response curves which were independent of the method of calculation used and Ari~ns and Simonis (1960) considered that the vas deferens of the rat or guinea pig is suitable for studying a-sympathomimetic amines by using cumulative dose-response curves. Our results led to different conclusions, perhaps because the contractile response has a very slow development; in the dog saphenous vein preparation the time elapsing between the first addition tO the bath and the point at which the maximal effect is attained, when the cumulative method and the factor of 2 were used, ranged between 50 and 65 min. Thus there was prolonged contact between the preparation and the amine permitting considerable metabolism of the amine to occur (if the rate of metabolism is high), and desensitization or autoinhibition to develop (if this develops to the amine under study). In conclusion we assume that the method of individual doses (non-cumulative) is preferred to the cumulative method whenever the comparison of slopes of dose-response curves is an important aim of the investigation.
ACKNOWLEDGEMENTS We are indebted to Prof. J. Maia for valuable help in the statistical analysis of the results and to Prof. U. Trendelenburg (Wiirzburg) for his most helpful criticism and revision of the manuscript.
49
REFERENCES And~n, N.-E., H. Corrodi, M. Ettles, E. Gustafsson and H. Persson, 1964, Selective uptake of some catecholamines by isolated heart and its inhibition by cocaine and phenoxybenzamine, Acta Pharmacol. Toxicol. 21, 247-259. Ari~ns, E.J. and A.M. Simonis, 1960, Autonomic drugs and their receptors, Arch. Intern. Pharmacodyn. 127, 479-507. Barnett, A., D.D. Greenhouse and R.I. Taber, 1968, A new type of drug enhancement: increased maximum response to cumulative noradrenaline in the isolated rat vas deferens, Brit. J. Pharmacol. 33, 171-176. Blaschko, H., 1952, Amine oxidase and amine metabolism, Pharmacol. Rev. 4, 415-478. Blinks, J.R., 1967, Evaluation of the cardiac effects of several beta adrenergic blocking drugs, Ann. N.Y. Acad. Sci. 139, 673-685. Furchgott, R.F. and S. Bhadrakom, 1953, Reactions of strips of rabbit aorta to epinephrine, isopropylarterenol, sodium nitrite and other drugs, J. Pharmacol. Exptl. Therap. 108, 129-143. Gaddum, J.H., 1953, Tryptamine receptors, J. Physiol. 119, 363-368. Guimar~es, S., 1968, Receptores adren~rgicos, Tese, Porto. Guimaraes, S. and W. Osswald, 1969, Adrenergic receptors in the veins of the dog, European J. Pharmacol. 5, 133-140. Guimar~es, S., W. Osswald, W. Cardoso and D. Branco, 1971, The effects of cocaine and denervation on the sensitivity to noradrenaline, its uptake and termination of action in isolated venous tissue, Arch. Pharmakol. 271,262-273. Hertting, G., 1964, The fate of 3H-isoproterenol in the rat, Biochem. Pharmacol. 13, 1119-1128. Iversen, L.L., 1965, The uptake of catechol amines at high perfusion concentrations in the rat isolated heart: a novel catechol amine uptake process, Brit. J. Pharmacol. 24, 18-33. Kalsner, S. and M. Nickerson, 1968, A method for study of mechanisms of drug disposition in smooth muscle, Can. J. Physiol. Pharmacol. 46,719-730. Langer, S. and U. Trendelenburg, 1969, The effect of a saturable uptake mechanism on the slopes of dose-response curves for sympathomimetic amines and on the shifts of dose-response curves produced by competitive antagonist, J. Pharmacol. Exptl. Therap. 167, 117-142. Osswald, W., S. Guimar~es and A. Coimbra, 1971, The termination of action of catecholamines in the isolated venous tissue of the dog, Arch. Pharmakol. 269, 15-31. Van Rossum, J.M. and E.J. Ari~ns, 1959, Pharmacodynamics of parasympathetic drugs, Arch. Intern. Pharmacodyn. 118, 418-447.