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A quantitative study of beta-adrenergic receptors in rabbit atria
EUROPEAN JOURNAL OF PHARMACOLOGY 12 (1970) 120-123. NORTH-HOLLAND PUBLISHING COMPANY
Short communication
A QUANTITATIVE
STUDY OF BETA-ADRENERGIC
RECEPTORS
IN RABBIT ATRIA * M. BRISTOW and R.D. GREEN Department of Pharmacology, University of Illinois at the Medical Center, 901 South Wolcott Avenue, P.O.Box 6998, Chicago, lllinois 60680, USA
Received 18 May 1970
Accepted 24 June 1970
M. BRISTOW and R.D. GREEN, A quantitative study of beta-adrenergic receptors in rabbit atria, European J. Pharmacol. 12 (1970) 120-123. The effects of propranolol, AY 21,011 and quinterenol on the responses of spontaneously beating right atrium and electrically driven left atrium to isoproterenol have been determined. The dissociation constants (KB values) of these antagonists were the same for antagonizing the inotropic and chronotropic effects of isoproterenol. These results give further support to the hypothesis that the inotropic and chronotropic responses to isoproterenol are mediated via the same receptors. Cardiac beta receptors,
Beta receptor antagonists,
1. INTRODUCTION Recent evidence has indicated that beta-receptors in different tissues are not the same (Levy, 1964; VanDeripe and Moran, 1965 ; Furchgott, 1967; Lands et al., 1967; Bristow et al., 1970). The apparent dissociation constants (K B values) of certain competitive/3-adrenergic blocking agents have been shown to be markedly different in several tissues of the rabbit (Bristow et al., 1970). For example, the K B of AY 21,011 in rabbit aortic strips is about 150-fold that in rabbit atrium (chronotropic response), i.e., AY 21,011 is about 150 times more potent in blocking the atrial chronotropic response to isoproterenol than the relaxant response to isoproterenol in rabbit aorta. Propranolol, however, is about equally potent in antagonizing the agonistic effect of isoproterenol in these two preparations.
* This investigation was supported by U.S. Public Health Service Research Grant NB 07964-01 from the National Institute of Neurological Diseases and Blindness.
KB values
Blinks (1967) showed that the potency of propranolol, as measured by its pA 2 value, is similar for antagonizing the chronotropic and inotropic effects of propranolol on guinea-pig atria. This is the strongest evidence that the inotropic and chronotropic effects of catechotamines are mediated via the same receptors. This evidence has been considerably weakened by the discovery that although the K B of propranolol may be the same in two tissues, the K B of another antagonist such as AY21,011 may be markedly different in these same two tissues. The present study was undertaken to reinvestigate the postulate that the chronotropic and inotropic effects o f adrenergic amines are mediated via the same receptors. Dose-response curves for both the chronotropic and inotropic effects of isoproterenol have been determined in the absence and presence of competitive antagonists and K B values calculated. Three antagonists have been studied: propranolol, AY 21,011, and quinterenol. The latter two agents have been reported to have selective activity in vivo (Dunlop and Shanks, 1968; Scriabine et al., 1968) and in vitro (Bristow et al., 1970).
M.Bristow, R.D. Green, Cardiac beta-receptors
2. METHODS Rabbits of either sex weighing between 1.5 and 2 kg were sacrificed by a blow on the head and the heart rapidly removed. Two preparations were studied: spontaneously beating right atrium and electrically driven left atrium. The tissues were mounted in isolated organ baths containing modified KrebsHenseleit solution saturated with 95% O2 and 5% CO2 and maintained at 37 to 38°C. The composition of the modified Krebs-Henseleit solution in (mM) was: NaC1 110; KC1 4.8; CaC12 2.5; KH2PO4 1.2; MgSO4 1.2; NaHCOa 25; glucose 11; and EDTA 0.002. Responses were recorded on a Grass polygraph via Grass force displacement transducers (FT 03). Grass model SD 5 stimulators were employed to provide the driving stimulus for the left atrial preparations. All responses were assumed to be maximum when a threefold increase in agonist concentration failed to produce an increased response.
3. RIGHT AND LEFT ATRIUM PREPARATIONS Right and left atria were isolated by incisions through the atrioventricular and interatrial septa. The atria were mounted side by side in the same 60 ml muscle bath. The right atria beat simultaneously the left atrium was driven electrically by means of a bi-punctate electrode delivering a threshold stimulus of 5-msec duration at a rate of 2/see. The muscle baths and electrodes employed were similar to those described by Blinks (1965). The resting tension of the right atrium was adjusted to 500 mg; the left atrial resting tension was adjusted to a level that achieved maximum force of contraction (usually a resting tension of 1-1.5 g).
4. K B DETERMINATIONS
The K B values or apparent dissociation constants of competitive antagonists were calculated from formula(l) (Paton, 1961; Furchgott, 1967)
[a] KB = dose ratio -
i
(1)
121
where B = concentration of antagonist. K B values are the means calculated for two or three concentrations of antagonist in each experiment. The applicability of this equation was shown by demonstrating that n in equation (2) was approximately - 1 (Arunlakshana and Schild, 1959): 1 - npAx logl0(x-1) = log~-~B
(2)
where x = dose ratio, pA x = - logto of the antagonist concentration and n is a constant and equals the slope of the regression line when loglo(X-1) is plotted against pA x (-logB). This, of course, shows that the calculated K B is not related to the concentration of antagonist studied. When K B values are determined in atria all concentrations of the antagonist have to be studied on the same atria by performing successive dose-response curves in the presence of increasing concentrations of antagonist. For this reason preliminary experiments were conducted to see if significant desensitization of the preparations occurs over the five-hour period and four dose-response curves needed to complete the experiment. A significant degree of desensitization was found to occur when the right and left atrial preparations were mounted together in the same muscle bath. Moreover, the degree of desensitization was not the same for the two preparations. The mean desensitization dose ratios for ten experiments were 1.4, 1.8 and 2.5 for chronotropic responses at 1, 2 and 4½ hr after the initial dose-response curve and 1.1, 1.4 and 1.8 for the inotropic responses at the same time periods. The dose ratios used in both equation (1) and (2) were, therefore, corrected for changes in sensitivity by use of the appropriate mean desensitization dose ratio (true dose ratio equals experimental dose ratio/desensitization dose ratio). The following drugs were used: dl-isoproterenol HC1, propranolol HCI, AY 21,011 (4-(2-hydroxy3-isopropylamine-propoxy)acetanilid) and quinterenol (1-5-(8-hydroxyquinolyl)-2-isopropylaminoethanol). All concentrations are expressed on a molar basis. AY21,011 was dissolved in distilled water. dl-Isoproterenol HCI, propranolol HC1, and quinterenol were dissolved in saline to which 1 mg/ml ascorbic acid was added. All solutions were made just prior to use. The significance of differences between groups was assessed with Student's t test.
122
M.Bristow, R.D. Green, Cardiac beta-receptors
5. RESULTS 4-
Pr,01xm'elol
The effects o f three competitive beta-adrenergic blocking agents, propranolol, AY 21,011 and quinterenol on the inotropic and chronotropic dose-response curves to isoproterenol were determined. Fig. 1 shows the mean dose-response curves to isoproterenol before and after three concentrations o f each o f these antagonists. These dose-response curves are not corrected for the small changes in sensitivity which occur during the experiment. It is evident, however, that these antagonists affect the inotropic and chronotropic effects o f isoproterenol similarly. Ioo -
"~"
~
25 -
,4," /
A/1< "/ W ,
.~ I00-
,
~z 75
--
~k o
2-
\2",,
PA2 868 B61 685 6 85 6.19 627
",, %\
%
-LOgo {~togonist} i
Fig. 2. Plot of eqn: ]oglo(x-l) = log KB-- - n p A x. Ordinate: ]ogio(x-l); Abscissa: -Ioglo antagonist concentration (PAx); C = chronotropic; ] =inotropic. Points shown are mean values for log lo(x-l) for each concentration of antagonist studied.
,,.
B
~,
Aff,,i r
cL 50
,oo
,,
5-
Slope 097 103 095 092 096 I OI
~
/[
ii
,o_
,'~T .,z~
Ay 21,011
C I o--o C I b.- -,e,
4,//" /
,o
25
-LOG~o .........
Individual dose ratios from each experiment with each concentration o f antagonist were corrected for desensitization and plots o f l o g l o ( X - 1 ) vs. - l O g l 0 B were constructed. The regression lines were calculated b y the m e t h o d o f least squares. These plots along with the slopes of the regression lines and pA2 values are given in fig. 2. (The points shown are the mean values for l o g l o ( X - 1 ) for each concentration of antagonist studied.) As the slopes of these plots were close to the theoretical value o f - 1 , K B values were calculated and are given in table 1. The K B of each antagonist was the same for its antagonism of the inotropic and chronotropic responses.
Table 1
:]::SOPROTERENOL CHRONOTROPIC INOTROPIC
Fig. 1. The effects of propranolol, AY 21,011 and quinterenol on the chronotropic and inotropic dose response curves to isoproterenol. Ordinate: response as percent of maximum response. Abscissa: -log 10 concentration of isoproterenol. Control, o; A) Propranolol, % 1 X 10-8 M;A, 1 X 10-7;A, 1 X 10-6 M; B) AY 21.011, o, 1 X 10-6 M; A, 1 X 10-5 M; % 1 X 10..4 M; C) Quinterenol, o, 1 X 10-6 M; A, 1 X 10-s M;% 1 X 10"4M.
KB a X 109 + S.E.M.
Antagonist dt-Propranolol AY 21,011 Quinterenol
Inotropic
Chronotropic
2.67 + 0.35 259 + 91 617 + 195
2.40 + 0.42 265 + 111 905 + 242
a Each group is the mean of 5 determinations.
M.Bristow, R.D. Green, Cardiac beta-receptors
6. DISCUSSION In 1967 Blinks found that the pA2 o f propranolol for antagonizing the chronotropic and inotropic effects o f isoproterenol in guinea-pig atria were very similar b u t were statistically significantly different. Blinks questioned the biological significance o f this small difference and concluded that the same receptors mediate these responses. In the present experiments the K B values o f propranolol, quinterenol and AY 21,011 for antagonizing these two responses were not significantly different and thus support Blink's conclusion. The latter two agents have previously been shown to indicate differences in/3-receptors o f different tissues of the rabbit which were undetected b y propranolol (Dunlop and Shanks, 1968; Scriabine et al., 1968; Bristow et al., 1970).
ACKNOWLEDGEMENTS The authors gratefully acknowledge the careful and excellent technical assistance of Mrs. Elaine Shays and wish to express their gratitude to Ayerst Laboratories (New York, N.Y.) for the propranolol and AY 21,011, to Dr. Rex Pinson of Chas. Pfizer & Co., Inc. (Brooklyn, N.Y.) for the quinterenol used in these experiments.
REFERENCES Arunlakshana, O. and H.O. Schild, 1959, Some quantitative uses of drug antagonist, Brit. J. Pharmacol. 14, 48-58.
123
BUnks, J.R., 1965, Convenient apparatus for recording contractions of isolated heart muscle, J. Appl. Physiol. 20, 755-757. Blinks, J.R., 1967, Evaluation of the cardiac effects of several /3-adrenergic blocking agents, Ann. N.Y. Acad. Sci. 139, 673-685. Bristow, M., T.R. Sherrod and R.D. Green, 1970, Analysis of beta receptor drug interactions in isolated rabbit atrium, aorta, stomach and trachea, J. Pharmacol. Exptl. Therap. 171, 52-61. Dunlop, D. and R.G. Shanks, 1968, Selective blockade of adrenoceptive beta-receptors in the heart, Brit. J. Pharmacol. 32,201-218. Furchgott, R.F., 1967, The pharmacological differentiation of adrenergic receptors, Ann. N.Y. Acad. Sci. 139,553570. Lands, A.M., A. Arnold, J.P. McAuliff, F.P. Luduena and T.G. Brown, 1967, Differentiation of receptor systems activated by sympathomimetic amines, Nature 214, 597598. Levy, B., 1964, Alterations of adrenergic responses by N-isopropyimethoxamine, J. Pharmacol. Exptl. Therap. 146, 129-138. Paton, W.D.M., 1961, A theory of drug action based on the rate of drug receptor combination, Proc. Roy. Soc. London Set. B 154, 21-69. Scriabine, A., P.F. Moore, I.C. Iorio, I.M. Goldman, W.K. McShane and K.D. Booker, 1968, Quinterenol, a new beta-adrenergic stimulant, J. Pharmacol. Exptl. Therap. 162, 60-69. VanDeripe, D.R. and N.C. Moran, 1965, Comparison of cardiac and vasodilator adrenergic blocking activity of DC1 and four analogs, Federation Proc. 24, 712.
Short communication
A QUANTITATIVE
STUDY OF BETA-ADRENERGIC
RECEPTORS
IN RABBIT ATRIA * M. BRISTOW and R.D. GREEN Department of Pharmacology, University of Illinois at the Medical Center, 901 South Wolcott Avenue, P.O.Box 6998, Chicago, lllinois 60680, USA
Received 18 May 1970
Accepted 24 June 1970
M. BRISTOW and R.D. GREEN, A quantitative study of beta-adrenergic receptors in rabbit atria, European J. Pharmacol. 12 (1970) 120-123. The effects of propranolol, AY 21,011 and quinterenol on the responses of spontaneously beating right atrium and electrically driven left atrium to isoproterenol have been determined. The dissociation constants (KB values) of these antagonists were the same for antagonizing the inotropic and chronotropic effects of isoproterenol. These results give further support to the hypothesis that the inotropic and chronotropic responses to isoproterenol are mediated via the same receptors. Cardiac beta receptors,
Beta receptor antagonists,
1. INTRODUCTION Recent evidence has indicated that beta-receptors in different tissues are not the same (Levy, 1964; VanDeripe and Moran, 1965 ; Furchgott, 1967; Lands et al., 1967; Bristow et al., 1970). The apparent dissociation constants (K B values) of certain competitive/3-adrenergic blocking agents have been shown to be markedly different in several tissues of the rabbit (Bristow et al., 1970). For example, the K B of AY 21,011 in rabbit aortic strips is about 150-fold that in rabbit atrium (chronotropic response), i.e., AY 21,011 is about 150 times more potent in blocking the atrial chronotropic response to isoproterenol than the relaxant response to isoproterenol in rabbit aorta. Propranolol, however, is about equally potent in antagonizing the agonistic effect of isoproterenol in these two preparations.
* This investigation was supported by U.S. Public Health Service Research Grant NB 07964-01 from the National Institute of Neurological Diseases and Blindness.
KB values
Blinks (1967) showed that the potency of propranolol, as measured by its pA 2 value, is similar for antagonizing the chronotropic and inotropic effects of propranolol on guinea-pig atria. This is the strongest evidence that the inotropic and chronotropic effects of catechotamines are mediated via the same receptors. This evidence has been considerably weakened by the discovery that although the K B of propranolol may be the same in two tissues, the K B of another antagonist such as AY21,011 may be markedly different in these same two tissues. The present study was undertaken to reinvestigate the postulate that the chronotropic and inotropic effects o f adrenergic amines are mediated via the same receptors. Dose-response curves for both the chronotropic and inotropic effects of isoproterenol have been determined in the absence and presence of competitive antagonists and K B values calculated. Three antagonists have been studied: propranolol, AY 21,011, and quinterenol. The latter two agents have been reported to have selective activity in vivo (Dunlop and Shanks, 1968; Scriabine et al., 1968) and in vitro (Bristow et al., 1970).
M.Bristow, R.D. Green, Cardiac beta-receptors
2. METHODS Rabbits of either sex weighing between 1.5 and 2 kg were sacrificed by a blow on the head and the heart rapidly removed. Two preparations were studied: spontaneously beating right atrium and electrically driven left atrium. The tissues were mounted in isolated organ baths containing modified KrebsHenseleit solution saturated with 95% O2 and 5% CO2 and maintained at 37 to 38°C. The composition of the modified Krebs-Henseleit solution in (mM) was: NaC1 110; KC1 4.8; CaC12 2.5; KH2PO4 1.2; MgSO4 1.2; NaHCOa 25; glucose 11; and EDTA 0.002. Responses were recorded on a Grass polygraph via Grass force displacement transducers (FT 03). Grass model SD 5 stimulators were employed to provide the driving stimulus for the left atrial preparations. All responses were assumed to be maximum when a threefold increase in agonist concentration failed to produce an increased response.
3. RIGHT AND LEFT ATRIUM PREPARATIONS Right and left atria were isolated by incisions through the atrioventricular and interatrial septa. The atria were mounted side by side in the same 60 ml muscle bath. The right atria beat simultaneously the left atrium was driven electrically by means of a bi-punctate electrode delivering a threshold stimulus of 5-msec duration at a rate of 2/see. The muscle baths and electrodes employed were similar to those described by Blinks (1965). The resting tension of the right atrium was adjusted to 500 mg; the left atrial resting tension was adjusted to a level that achieved maximum force of contraction (usually a resting tension of 1-1.5 g).
4. K B DETERMINATIONS
The K B values or apparent dissociation constants of competitive antagonists were calculated from formula(l) (Paton, 1961; Furchgott, 1967)
[a] KB = dose ratio -
i
(1)
121
where B = concentration of antagonist. K B values are the means calculated for two or three concentrations of antagonist in each experiment. The applicability of this equation was shown by demonstrating that n in equation (2) was approximately - 1 (Arunlakshana and Schild, 1959): 1 - npAx logl0(x-1) = log~-~B
(2)
where x = dose ratio, pA x = - logto of the antagonist concentration and n is a constant and equals the slope of the regression line when loglo(X-1) is plotted against pA x (-logB). This, of course, shows that the calculated K B is not related to the concentration of antagonist studied. When K B values are determined in atria all concentrations of the antagonist have to be studied on the same atria by performing successive dose-response curves in the presence of increasing concentrations of antagonist. For this reason preliminary experiments were conducted to see if significant desensitization of the preparations occurs over the five-hour period and four dose-response curves needed to complete the experiment. A significant degree of desensitization was found to occur when the right and left atrial preparations were mounted together in the same muscle bath. Moreover, the degree of desensitization was not the same for the two preparations. The mean desensitization dose ratios for ten experiments were 1.4, 1.8 and 2.5 for chronotropic responses at 1, 2 and 4½ hr after the initial dose-response curve and 1.1, 1.4 and 1.8 for the inotropic responses at the same time periods. The dose ratios used in both equation (1) and (2) were, therefore, corrected for changes in sensitivity by use of the appropriate mean desensitization dose ratio (true dose ratio equals experimental dose ratio/desensitization dose ratio). The following drugs were used: dl-isoproterenol HC1, propranolol HCI, AY 21,011 (4-(2-hydroxy3-isopropylamine-propoxy)acetanilid) and quinterenol (1-5-(8-hydroxyquinolyl)-2-isopropylaminoethanol). All concentrations are expressed on a molar basis. AY21,011 was dissolved in distilled water. dl-Isoproterenol HCI, propranolol HC1, and quinterenol were dissolved in saline to which 1 mg/ml ascorbic acid was added. All solutions were made just prior to use. The significance of differences between groups was assessed with Student's t test.
122
M.Bristow, R.D. Green, Cardiac beta-receptors
5. RESULTS 4-
Pr,01xm'elol
The effects o f three competitive beta-adrenergic blocking agents, propranolol, AY 21,011 and quinterenol on the inotropic and chronotropic dose-response curves to isoproterenol were determined. Fig. 1 shows the mean dose-response curves to isoproterenol before and after three concentrations o f each o f these antagonists. These dose-response curves are not corrected for the small changes in sensitivity which occur during the experiment. It is evident, however, that these antagonists affect the inotropic and chronotropic effects o f isoproterenol similarly. Ioo -
"~"
~
25 -
,4," /
A/1< "/ W ,
.~ I00-
,
~z 75
--
~k o
2-
\2",,
PA2 868 B61 685 6 85 6.19 627
",, %\
%
-LOgo {~togonist} i
Fig. 2. Plot of eqn: ]oglo(x-l) = log KB-- - n p A x. Ordinate: ]ogio(x-l); Abscissa: -Ioglo antagonist concentration (PAx); C = chronotropic; ] =inotropic. Points shown are mean values for log lo(x-l) for each concentration of antagonist studied.
,,.
B
~,
Aff,,i r
cL 50
,oo
,,
5-
Slope 097 103 095 092 096 I OI
~
/[
ii
,o_
,'~T .,z~
Ay 21,011
C I o--o C I b.- -,e,
4,//" /
,o
25
-LOG~o .........
Individual dose ratios from each experiment with each concentration o f antagonist were corrected for desensitization and plots o f l o g l o ( X - 1 ) vs. - l O g l 0 B were constructed. The regression lines were calculated b y the m e t h o d o f least squares. These plots along with the slopes of the regression lines and pA2 values are given in fig. 2. (The points shown are the mean values for l o g l o ( X - 1 ) for each concentration of antagonist studied.) As the slopes of these plots were close to the theoretical value o f - 1 , K B values were calculated and are given in table 1. The K B of each antagonist was the same for its antagonism of the inotropic and chronotropic responses.
Table 1
:]::SOPROTERENOL CHRONOTROPIC INOTROPIC
Fig. 1. The effects of propranolol, AY 21,011 and quinterenol on the chronotropic and inotropic dose response curves to isoproterenol. Ordinate: response as percent of maximum response. Abscissa: -log 10 concentration of isoproterenol. Control, o; A) Propranolol, % 1 X 10-8 M;A, 1 X 10-7;A, 1 X 10-6 M; B) AY 21.011, o, 1 X 10-6 M; A, 1 X 10-5 M; % 1 X 10..4 M; C) Quinterenol, o, 1 X 10-6 M; A, 1 X 10-s M;% 1 X 10"4M.
KB a X 109 + S.E.M.
Antagonist dt-Propranolol AY 21,011 Quinterenol
Inotropic
Chronotropic
2.67 + 0.35 259 + 91 617 + 195
2.40 + 0.42 265 + 111 905 + 242
a Each group is the mean of 5 determinations.
M.Bristow, R.D. Green, Cardiac beta-receptors
6. DISCUSSION In 1967 Blinks found that the pA2 o f propranolol for antagonizing the chronotropic and inotropic effects o f isoproterenol in guinea-pig atria were very similar b u t were statistically significantly different. Blinks questioned the biological significance o f this small difference and concluded that the same receptors mediate these responses. In the present experiments the K B values o f propranolol, quinterenol and AY 21,011 for antagonizing these two responses were not significantly different and thus support Blink's conclusion. The latter two agents have previously been shown to indicate differences in/3-receptors o f different tissues of the rabbit which were undetected b y propranolol (Dunlop and Shanks, 1968; Scriabine et al., 1968; Bristow et al., 1970).
ACKNOWLEDGEMENTS The authors gratefully acknowledge the careful and excellent technical assistance of Mrs. Elaine Shays and wish to express their gratitude to Ayerst Laboratories (New York, N.Y.) for the propranolol and AY 21,011, to Dr. Rex Pinson of Chas. Pfizer & Co., Inc. (Brooklyn, N.Y.) for the quinterenol used in these experiments.
REFERENCES Arunlakshana, O. and H.O. Schild, 1959, Some quantitative uses of drug antagonist, Brit. J. Pharmacol. 14, 48-58.
123
BUnks, J.R., 1965, Convenient apparatus for recording contractions of isolated heart muscle, J. Appl. Physiol. 20, 755-757. Blinks, J.R., 1967, Evaluation of the cardiac effects of several /3-adrenergic blocking agents, Ann. N.Y. Acad. Sci. 139, 673-685. Bristow, M., T.R. Sherrod and R.D. Green, 1970, Analysis of beta receptor drug interactions in isolated rabbit atrium, aorta, stomach and trachea, J. Pharmacol. Exptl. Therap. 171, 52-61. Dunlop, D. and R.G. Shanks, 1968, Selective blockade of adrenoceptive beta-receptors in the heart, Brit. J. Pharmacol. 32,201-218. Furchgott, R.F., 1967, The pharmacological differentiation of adrenergic receptors, Ann. N.Y. Acad. Sci. 139,553570. Lands, A.M., A. Arnold, J.P. McAuliff, F.P. Luduena and T.G. Brown, 1967, Differentiation of receptor systems activated by sympathomimetic amines, Nature 214, 597598. Levy, B., 1964, Alterations of adrenergic responses by N-isopropyimethoxamine, J. Pharmacol. Exptl. Therap. 146, 129-138. Paton, W.D.M., 1961, A theory of drug action based on the rate of drug receptor combination, Proc. Roy. Soc. London Set. B 154, 21-69. Scriabine, A., P.F. Moore, I.C. Iorio, I.M. Goldman, W.K. McShane and K.D. Booker, 1968, Quinterenol, a new beta-adrenergic stimulant, J. Pharmacol. Exptl. Therap. 162, 60-69. VanDeripe, D.R. and N.C. Moran, 1965, Comparison of cardiac and vasodilator adrenergic blocking activity of DC1 and four analogs, Federation Proc. 24, 712.