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A further study of the effect of propranolol on the blockade of alpha adrenergic receptors
EUROPEAN JOURNAL OF PHARMACOLOGY 7 11969) 258-263. NORTH-HOLLAND PUBLISHING COMP., AMSTERDAM
A FURTHER STUDY OF THE EFFECT OF PROPRANOLOL THE BLOCKADE OF ALPHA ADRENERGIC RECEPTORS
ON
Henry I.YAMAMURA and Akira HORITA
Department of Pharmacology, School of Medicine, University of Washington, Seattle, Washington 98105 Accepted 21 May 1969
Received 11 April 1969
H.I.YAMAMURA and A.HORITA, A further study of the effect of propranolol on the blockade of alpha adrenergic receptors, European J. Pharmacol. 7 (1969) 258-263. The results reported herein have shown that the pressor responses of adrenergic agonists possessing only an alpha stimulatory property (oxymetazoline) were not restored to significant values after phenoxybenzamine and proprano!ol blockade. However, when the alpha agonistic property of oxymetazoline was combined with the beta stimulatory property of isoproterenol, significant recovery of the pressor response was noted after alpha and beta blockade. The results indicated that the pressor activity of the oxymetazoline plus isoproterenol mixture seemed to mimic the actions of a known alpha and beta agonist: epinephrine. The data also showed that time affected the antagonism of phenoxybenzamine by propranolol and of significance was the consistent finding that the phenoxybenzamine-induced alpha blockade reappeared after the effects of propranolol had worn off. These data provide further proof that only the agonists possessing both alpha and beta stimulatory activities can undergo the reversal after alpha receptor blockade. It is therefore postulated that the antagonism between alpha and beta blocking agents is associated with the activation of residual alpha receptors not blocked by phenoxybenzamine rather than the hypothesis of competitive displacement of phenoxybenzamine by propranoloi. Recovery of alpha-blockade
Oxymetazoline
1. INTRODUCTION Recently several reports have appeared in the literature (Hull et al., 1960; Gulati et al., 1965; Garrett et al., 1966; Sharma, 1966; Smith and Nash, 1968; Yamamura and Horita, 1968) describing an antagonistic action between the alpha and beta adrenergic blocking agents. Olivares et al. (1967) demonstrated the effect of propranolol on the blockade of alpha adrenergic receptors in the dog and on the isolated rabbit aortic strip. They provided evidence that once alpha blockade produced by phenoxybenzamine had been interrupted by propranolol0 the alpha blockade did not return after the wearing-off of the beta blocking agent. This suggested to them that propranolol had "unblocked" the alpha receptor site. Gulati et al. (1965) suggested that the antagonism of alpha adrenergic blockade is brought about by a competitive displacement mechanism. These results
Phenoxybenzamine
Propranolol
were acquired on the isolated rabbit aortic strip. Two possible hypotheses have been previously offered to explain the mechanism of this interaction. Hull et al. (1960) proposed that 1) the alpha blocking agent is displaced from the alpha receptor site by the beta blocking agent, or 2) the beta blocking agent, by combining with the beta receptors unmasks those spare alpha receptors sites assumed to have been spared by an inadequate dose of the alpha blocking agent. Neither of these hypotheses has been adequately proven. The purpose o f this study was to examine further the antagonistic action between the alpha and beta blocking agents. We have recently found that not all adrenergic amines undergo the same type of interaction after the administration of phenoxybenzamine and propranolol (Yamamura and Horita, 1968) The pure alpha agonistic actions of methoxamine and oxymetazoline on blood pressure were partially or completely blocked by phenoxybenzamine, and not
ALPHA ADRENERG1CRECEPTOR restored to any significant degree by propranolol. However, restoration of the pressor responses to epinephrine and norepinephrine did occur after the alpha and beta blocking agents. Since these results suggested that only the agonists possessing both alpha and beta stimulatory activities could undergo this reversal after alpha and beta adrenergic blockade, it was decided that in the present experiments the alpha agonistic property of oxymetazoline or methoxamine be combined with the beta agonistic property of isoproterenol to determine if an apparent recovery would occur after alpha and beta blockade.
2. METHODS Approximately 40 cats of either sex, weighing between 2.5 and 3.5 kg, were anesthetized with pentobarbital sodium (30 mg/kg) administered intraperitoneally. Blood pressure was recorded from the cannulated left femoral artery with a Physiograph Four recorder and an E and M pressure transducer (Model P-1000). The contractions of the nictitating membrane were recorded with an E and M myograph (Type B) force-displacement transducer with 5 g tension placed upon the membrane. A tracheal cannula was routinely inserted and artificial respiration was administered when necessary. In all experiments, a cat was given phenoxybenzamine (5 mg/kg), an insurmountable alpha receptor blocking agent, and propranolol (1.0 mg/kg), a surmountable beta receptor blocking agent. There was a 30 rain time lapse after the administration of the antagonists before the agonists were again given. Only one adrenergic agonist or mixture of agonists were administered to an animal. All statistical tests were carried out with Student's t test.
259
on the basis of their salts, except for epinephrine which is expressed as its free base. All drug administration to cats was done intravenously via a cannulated femoral vein.
3. RESULTS
3.1. Effects of oxymetazoline and oxymetazoline + isoproterenol Fig. 1 represents the responses of a typical experiment in cats given intravenous injections of epinephrine (10/~g/kg) under control, after phenoxybenzamine (5 mg/kg) and after propranolol (1.0 mg/kg) conditions. After administration of the alpha blocker, a depressor response to epinephrine was noticed. Upon subsequent administration of the beta blocker, the depressor response to epinephrine had reverted to a pressor response. On the nictitating membrane, epinephrine injections in control cats produced a contraction presumably through stimulation of the alpha adrenergic receptors. Once the insurmountable alpha antagonist was given, the response to epinephrine was almost totally inhibited and no amount of propranolol significantly restored the membrane contraction upon subsequent administration of epinephrine. Fig. 2. shows the results of the mean changes in pressor effects to oxymetazoline alone (30 #g/kg)
b
ca
c
0
E
E
E
2.1. Drugs The following drugs were used in this study: 1epinephrine bitartrate (Sigma Chemical Co.); Methoxamine HC1 (Burroughs Wellcome and Co.); oxymetazoline HC1 (Schering Corp.); dl-isoproterenol HC1 (Z.D. Gilman, Inc.); phenoxybenzamine HC1 solution (Smith, Kline and French Laboratories); and propranolol HC1 (Ayerst Laboratories). The concentrations of drugs used in all experiments are expressed
Phenoxybenzomlne (5 rng/Kg)
ptoprQnOlOI (I mc]l kq)
Fig. 1. The effect of propranolol (1 mg/kg) on the blockade by phenoxybenzamine (5 mg/kg) of the nictitating membrane and blood pressure of an anesthetized cat. The adrenergic agonist administered was epinephrine (E) (10 ug/kg). Time mark is 1 rain.
260
H.1.YAMAMURA and A.HORITA
under control, after phenoxybenzamine (5 mg/kg)and after propranolol (1.0 mg/kg) conditions. The results indicate that no significant increase in pressor response to o x y m e t a z o l i n e was seer ' l e n b e t a blockade was s u p e r i m p o s e d o n alpha b l o c k a d e . Similar re-
-& +8O
~, \
¢ -E <
I
0
g -2o
I~ Oxymetazohne Control [ ] O~ymetezoline + tsooroterenol
-4o
F:'-I Isoproterenol Control -60
Fig. 2. The effect of propranolol (1 mg/kg) on the blockade by phenoxybenzamine (5 mg/kg) of the mean change in blood pressure of an anesthetized cat. Adrenergic agonists administered were oxymetazoline (30 ug/kg), oxymetazoline + isoproterenol (30 gg/kg + 2 t~g/kg), and isoproterenol (2 #g/kg). Vertical bars are + S.E.
sults were seen o n t h e n i c t i t a t i n g m e m b r a n e to the a d m i n i s t r a t i o n o f o x y m e t a z o l i n e ( t a b l e 1). A n a t t e m p t was t h e n m a d e to d e t e r m i n e w h e t h e r a c o m p o u n d req u i r e d b o t h alpha a n d beta agonistic p r o p e r t i e s to exh i b i t this r e s t o r a t i o n o f p r e s s o r activity after alpha a n d beta b l o c k a d e . T h i s was d o n e b y c o m b i n i n g appropriate concentrations of oxymetazoline, a purely alpha agonist (Mujic a n d V a n R o s s u m , 1 9 6 5 ) w i t h isop r o t e r e n o l , a k n o w n beta s t i m u l a n t . T h i s m i x t u r e ( h e r e a f t e r d e s i g n a t e d as O X Y + ISO) was i n j e c t e d into cats b e f o r e a n d a f t e r p h e n o x y b e n z a m i n e , a n d after p r o p r a n o l o l t r e a t m e n t . T h e results s h o w t h a t a significant increase in t h e m e a n c h a n g e o f pressor response t o t h e O X Y + ISO m i x t u r e was seen w h e n beta b l o c k a d e was s u p e r i m p o s e d o n alpha b l o c k a d e (see fig. 2).
Table 1 Mean changes in blood pressure response (B.P.) and average peak tension of the nictitating membrane (N.M.) to i.v. injections of adrenergic agonists before and after phenoxybenzamine (5.0 mg/kg) and propranoloi (1.0 mg/kg). Control
After After phenoxybenzamine (A) propranolol (B)
Drugs Dose Qag/kg) Epinephrine Methoxamine Oxymetazoline
10
B.P. (mm Hg)
N,M. (g)
80 + 5.0 c (4) d 4.8 + 0.6 (4)
B.P. (mm Hg)
N.M. (g) B.P. (mm Hg)
- 2 5 + 3.1
0.25 + 0.2
N,M.(g)
61 +- 3.6 0.5 + 0.2
B-A
B.P. (mm Hg) 86 + 4.9 a
300
63+4.7
(6)
8.2-+0.8(3)
8+1.7
0
3-+0.9
0
-5-+5.5
30
58+4.2
(6)
8.2+1.4(3)
30-+3.1
0
25-+3.2
0
-5-+4.6
lsoproterenol
1
- 4 0 + 1.5 (3)
_e
- 3 8 -+ 2.2
-
- 3 -+ 3.3
-
34 -+ 5.2 b
lsoproterenol
2
- 4 4 + 3.8 (4)
_e
- 5 6 + 9.5
-
- 8 + 4.3
-
49 + 8.8 b
300+1
68+-4.7 (7)
8.4+-0.4(3)
-34+2.2
0
49 + 4.6 (10)
8.5 -+0.5 (5)
- 4 6 + 3.6
0
Methoxamine + isoproterenol Oxymetazoline + isoproterenol
30 + 2
6+-2.6
0
38-+2.4 a
33 -+ 3.4 0
79 -+4.8 a
a This represents the mean change in blood pressure response after propranolol minus the response after phenoxybenzamine (P < 0.01). b Same as footnote "a" except that (P < 0.05). c Values represent mean + S . E . d Value represents number of animals. e Nictitating membrane responses were not recorded with this agonist.
ALPHA ADRENERGIC RECEPTOR
Table 1 includes the mean changes in blood pressure and nictitating membrane responses to the various adrenergic amines under control, after phenoxybenzamine and after propranolol conditions. The mean value of the peak responses after propranolol minus the peak responses after phenoxybenzamine (hereafter designated as PROP-PBZ) for each amine was also calculated to aid in interpretation of the data. As can be seen from table 1, the PROP-PBZ value for the OXY + ISO combination was 79 mm Hg (S.E. = 4.8) P < 0.01. Cats were also given methoxamine (300/lg/kg), a known pure alpha agonist, as well as a methoxamine + isoproterenol (300 /ag/kg + 1 #g/kg) mixture. The results obtained from 6 methoxamine controls and 7 methoxamine + isoproterenol (METH + ISO) treated animals are summarized in table 1. Once alpha receptor blockade by phenoxybenzamine had occurred, propranolol at a dose of 1.0 mg/kg did cause a reappearance of a slight pressor effect to METH + ISO and
261
there was a significant PROP-PBZ value for the METH + ISO mixture (see table 1). On the nictitating membrane, both methoxamine and the METH + ISO mixture produced prolonged contractions. However, once the alpha blocker was administered no amount of propranolol could antagonize the blockade produced by phenoxybenzamine. 3.2. Effects of oxymetazoline and oxymetazoline +
isoproterenol after dissipation of propranolol action Experiments were also performed to determine whether time would affect the reversal phenomenon after phenoxybenzamine and propranolol. This was only possible because of phenoxybenzamine's prolonged duration of action and propranolol's effect being relatively short. The results obtained from three oxymetazoline controls and four OXY + ISO combinations are summarized graphically in fig. 3. After apparent recovery of the response to OXY + ISO after
IOOq
4°°1
4
}~°]_
6o E 40~ 2o-
"~ ~ 0
~
Methoxominecontrol
o-
~-20
-20. {
~40~$op~oterenOlcontrol
40[PBZ] [pRop] ~r I
.
1
-6o ~[p~z]
[PeOp] .
.
2
.
3
4
-~--
~
~
7
:
8
i
9
~
1
Hours
Fig. 3. The effect of adrenergic agonists after dissipation of propranolol action. White circles and white triangles represent the mean change in blood pressure to oxymetazoline (30 #g/kg) and isoproterenol (2 #g/kg), respectively, while the black circles represent the mean change in blood pressure to the oxymetazoline + isoproterenol (30 #g/kg + 2 t~g/kg) mixture before and after administration of phenoxybenzamine (5 mg/kg) and propranolol (1 mg/kg). After propranolol administration, the agonists were administered every 2 hr for 5 doses and a second dose of propranolol (l mg/kg) was given and the agonists were given once more. Each point represents a mean value of at least 3 cats.
~ [PRop]
I Hours
Fig. 4. The effect of adrenergic agonists after dissipation of propranolol action. White circles and white triangles represent the mean change in blood pressure to methoxamine (300 #g/kg) and isoproterenol (1 #g/kg), respectively, while the black circles represent the mean change in blood pressure to the methoxamine + isoproterenoi (300 pg/kg + 1 #g/kg) mixture before and after administration of phonoxybenzamine (5 mg/kg) and propranolol (1 mg/kg). After propranolol administration, the agonists were administered every 2 hr for 5 doses and a second dose of propranolol (1 mg/kg) was given and the agonists were given once more. Each point represents a mean values of 3 cats.
;62
ft.I.YAMAMURA and A.tIORITA
alpha and beta blockade, the experiment was continued until the effects of the beta blocking agent had terminated. Injections of isoproterenol (2 ~g/kg) were given in three control cats in order to verify the termination of the effects of the beta blocking agent. After partial termination of the effects of the beta blocking agent, the pressor response to OXY + ISO gradually reverted to a depressor response. A second dose of propranolot (1.0 mg/kg) was given 8-~4 hr later and upon subsequent administration of OXY t ISO, a re-establishment of the pressor response was seen. Results from fig. 3 also show that partial blockade to control doses of oxymetazoline (30/~g/kg) had occurred after phenoxybenzamine 15 mg/kg), but no significant recovery was seen after the administration of propranolol ( 1.0 mg/kg). Fig. 4 summarizes the effect of time on the mean changes in blood pressure to methoxamine (300/~g/ kg) and to the METH + ISO (300 ~g/kg + I /lg/kg) mixture. The graph indicates that after partial termination of the beta blocking agent, a depressor response gradually occurred with the METH + ISO mixture in 3 cats while in the control cats the mean response to methoxamine did not change. Injections of isoproterenol (1 /~g/kg) were given to 3 control cats to verify the termination of the effects of the betablocking agent.
4. DISCUSSION The results of these experiments indicate that the interaction between the alpha and beta blocking agents does not involve a displacement of one by the other as was hypothesized by Olivares et al. (1967), and Gulati et al. (1965). Of significance is the finding that the pressor actions of the pure alpha adrenergic agonists do not recover when beta blockade is superimposed on alpha blockade, but that if a pure alpha adrenergic agonist is combined with a beta adrenergic agonist a significant recovery does occur. The pressor responses to the OXY + ISO mixture which were antagonized by phenoxybenzamine exhibited significant recovery after propranolol; however, the results from the METH + ISO mixture were not as dramatic.
The results suggest that the apparent antagonism that is being observed with epinephrine and the OXY + ISO mixture after alpha and beta blockade is not due to a displacement mechanism but rather can be better explained as due to an inadequate blockade of the alpha receptor site by phenoxybenzamine. If it were possible to separate the alpha component of epinephrine from its beta component, we would possibly see that incomplete alpha blockade had occurred as seen in fig. 2 and fig. 3 in fire case of oxymetazotine. Mention was made previously that Olivares et at. ( 1967} noticed that once the alpha blockade was interrupted by a beta blocking agent, the alpha blockade did not reappear in dogs after the beta blocking agent had worn off, and that this suggested to them that there was unblocking of the alpha receptor site. ttowever, unlike them, we observed the reappearance of the alpha blockade which persists for a longer period than tile effects of beta blockade as produced by' propranolol. Upon administration of a second dose o f propranolol 8+~ hr later, there is again the apparent antagonism of alpha blockade, i.e., the restoratkm of a pressor response to the OXY + ISO mixture. We feel that since OXY + ISO ex ~ibits both alpha and beta agonistic actions, which mimic the action of epinephrine, its effect on blood pressure is a composite of the alpha and beta eSfects, part of which tend to raise and part attempt to lower blood pressure. With the dose of phenoxybenzamine used here a sufficient proportion of alpha receptors are blocked, which permits the beta effect, the vasodilator component, to predominate, thus resulting in the depressor effect. The administration of propranolol abolishes the beta component, and the spare alpha receptor effect of vasopressor activity again appears. However, as the beta blockade weakens with time the insurmountable alpha blockade of phenoxybenzamine becomes evident since the beta agonistic action of OXY + ISO reappears. An unexpected finding was that the recovery of METH + ISO mixture did not occur as dramatically as that seen with the OXY + ISO mixture after alpha and beta blockade. One probable reason for this discrepancy is that the METH + ISO mixture may have less efficacy for the alpha receptors than the OXY + ISO mixture and epinephrine, so that the METH +
ALPHA ADRENERGIC RECEPTOR ISO mixture requires attachment to a greater number o f spare alpha receptors to elicit a pressor response after alpha and beta blockade. Therefore, since there may be fewer spare alpha receptors available for the METH + ISO mixture to combine with, no significant recovery of a pressor response was seen. The concept o f activating spare alpha receptors, those not originally blocked by phenoxybenzamine, is further supported by the observation that only those agents possessing both alpha and beta agonistic properties, i.e., epinephrine or a combination of an alpha with a beta agonist, can exhibit this interaction. Pure alpha agonists, such as oxymetazoline, are partially blocked by phenoxybenzamine and the response after propranolol administration to oxymetazoline is not significantly greater than the response after phenoxybenzamine, indicating that "unblocking" o f alpha receptor sites is probably not taking place. If the view of Olivares et al. ( I 9 6 7 ) that the beta antagonist displaces the insurmountable alpha antagonist were correct, then the vasopressor response to oxymetazoline should have been restored to control values after alpha and beta~olockade.
ACKNOWLEDGEMENTS This research was supported by USPHS Research Grant MH-02435, National Institute of Mental Health; and by USPHS Training Grant GM-109 from the National Institutes of Health.
263
The authors are indebted to Ayerst Laboratories, Inc., New York, N.Y., for propranolol (Inderal ®), AY 64043; and to Smith, Kline and French Laboratories, Philadelphia, Pa., for phenoxybenzamine (Dibenzyline ®), SKF 688-A.
REFERENCES
Garrett, J., A.Malafaya-Baptistaand W.Osswald, 1966. Effects of pronethalol on the cardiovascular actions of catecholamines during blockade by phenoxybenzamine, Brit. J. Pharmacol. Chemotherap. 27,459-467. Gulati, O.D., S.D.Gokhale and B.P.Udwadia, 1965, Antagonism ofadrenergic blockade by pronethalol, Arch. intern. Pharmadyn. 156, 389-397. Hull, L.D., L.G.Eltherington, A.Horita, 1960, The antagonism ofadrenergLe blockade by dichloroisoproterenol(DCI) Experientia 16, 368-371. Mujic, M. and J.M.van Rossum, 1965, Comparative pharmacodynamics of sympathomimetic imidazolines; studies of intestinal smooth muscle of the rabbit and the cardiovascular system of the cat, Arch. intern. Pharmacodyn 155, 432-449. Olivares, G.J., N.T.Smith and L,Aronow, 1967, Effect of propranolol on alpha-adrenergic blockade in the dog and isolated rabbit aortic strip, Brit. J. Pharmacol. Chemotherap. 30, 240-250. Sharma, P.L., 1966, Interaction of adrenergic alpha and beta receptor blocking agents on the blood pressure response to adrenaline and noradrenaline, Quart. J. Exptl. Physiol. 51,256-261. Smith, R.D. and C.B.Nash, 1968, A comparative study of three beta adrenergic blocking agents in the anesthetized dog, Pharmacologist 10, 299. Yamamura, H.I. and A.Horita, 1968, Effect of propranolol on the blockade of alpha adrenergic receptors, J. Pharmacol; Exptl. Therap. 164, 82-89.
A FURTHER STUDY OF THE EFFECT OF PROPRANOLOL THE BLOCKADE OF ALPHA ADRENERGIC RECEPTORS
ON
Henry I.YAMAMURA and Akira HORITA
Department of Pharmacology, School of Medicine, University of Washington, Seattle, Washington 98105 Accepted 21 May 1969
Received 11 April 1969
H.I.YAMAMURA and A.HORITA, A further study of the effect of propranolol on the blockade of alpha adrenergic receptors, European J. Pharmacol. 7 (1969) 258-263. The results reported herein have shown that the pressor responses of adrenergic agonists possessing only an alpha stimulatory property (oxymetazoline) were not restored to significant values after phenoxybenzamine and proprano!ol blockade. However, when the alpha agonistic property of oxymetazoline was combined with the beta stimulatory property of isoproterenol, significant recovery of the pressor response was noted after alpha and beta blockade. The results indicated that the pressor activity of the oxymetazoline plus isoproterenol mixture seemed to mimic the actions of a known alpha and beta agonist: epinephrine. The data also showed that time affected the antagonism of phenoxybenzamine by propranolol and of significance was the consistent finding that the phenoxybenzamine-induced alpha blockade reappeared after the effects of propranolol had worn off. These data provide further proof that only the agonists possessing both alpha and beta stimulatory activities can undergo the reversal after alpha receptor blockade. It is therefore postulated that the antagonism between alpha and beta blocking agents is associated with the activation of residual alpha receptors not blocked by phenoxybenzamine rather than the hypothesis of competitive displacement of phenoxybenzamine by propranoloi. Recovery of alpha-blockade
Oxymetazoline
1. INTRODUCTION Recently several reports have appeared in the literature (Hull et al., 1960; Gulati et al., 1965; Garrett et al., 1966; Sharma, 1966; Smith and Nash, 1968; Yamamura and Horita, 1968) describing an antagonistic action between the alpha and beta adrenergic blocking agents. Olivares et al. (1967) demonstrated the effect of propranolol on the blockade of alpha adrenergic receptors in the dog and on the isolated rabbit aortic strip. They provided evidence that once alpha blockade produced by phenoxybenzamine had been interrupted by propranolol0 the alpha blockade did not return after the wearing-off of the beta blocking agent. This suggested to them that propranolol had "unblocked" the alpha receptor site. Gulati et al. (1965) suggested that the antagonism of alpha adrenergic blockade is brought about by a competitive displacement mechanism. These results
Phenoxybenzamine
Propranolol
were acquired on the isolated rabbit aortic strip. Two possible hypotheses have been previously offered to explain the mechanism of this interaction. Hull et al. (1960) proposed that 1) the alpha blocking agent is displaced from the alpha receptor site by the beta blocking agent, or 2) the beta blocking agent, by combining with the beta receptors unmasks those spare alpha receptors sites assumed to have been spared by an inadequate dose of the alpha blocking agent. Neither of these hypotheses has been adequately proven. The purpose o f this study was to examine further the antagonistic action between the alpha and beta blocking agents. We have recently found that not all adrenergic amines undergo the same type of interaction after the administration of phenoxybenzamine and propranolol (Yamamura and Horita, 1968) The pure alpha agonistic actions of methoxamine and oxymetazoline on blood pressure were partially or completely blocked by phenoxybenzamine, and not
ALPHA ADRENERG1CRECEPTOR restored to any significant degree by propranolol. However, restoration of the pressor responses to epinephrine and norepinephrine did occur after the alpha and beta blocking agents. Since these results suggested that only the agonists possessing both alpha and beta stimulatory activities could undergo this reversal after alpha and beta adrenergic blockade, it was decided that in the present experiments the alpha agonistic property of oxymetazoline or methoxamine be combined with the beta agonistic property of isoproterenol to determine if an apparent recovery would occur after alpha and beta blockade.
2. METHODS Approximately 40 cats of either sex, weighing between 2.5 and 3.5 kg, were anesthetized with pentobarbital sodium (30 mg/kg) administered intraperitoneally. Blood pressure was recorded from the cannulated left femoral artery with a Physiograph Four recorder and an E and M pressure transducer (Model P-1000). The contractions of the nictitating membrane were recorded with an E and M myograph (Type B) force-displacement transducer with 5 g tension placed upon the membrane. A tracheal cannula was routinely inserted and artificial respiration was administered when necessary. In all experiments, a cat was given phenoxybenzamine (5 mg/kg), an insurmountable alpha receptor blocking agent, and propranolol (1.0 mg/kg), a surmountable beta receptor blocking agent. There was a 30 rain time lapse after the administration of the antagonists before the agonists were again given. Only one adrenergic agonist or mixture of agonists were administered to an animal. All statistical tests were carried out with Student's t test.
259
on the basis of their salts, except for epinephrine which is expressed as its free base. All drug administration to cats was done intravenously via a cannulated femoral vein.
3. RESULTS
3.1. Effects of oxymetazoline and oxymetazoline + isoproterenol Fig. 1 represents the responses of a typical experiment in cats given intravenous injections of epinephrine (10/~g/kg) under control, after phenoxybenzamine (5 mg/kg) and after propranolol (1.0 mg/kg) conditions. After administration of the alpha blocker, a depressor response to epinephrine was noticed. Upon subsequent administration of the beta blocker, the depressor response to epinephrine had reverted to a pressor response. On the nictitating membrane, epinephrine injections in control cats produced a contraction presumably through stimulation of the alpha adrenergic receptors. Once the insurmountable alpha antagonist was given, the response to epinephrine was almost totally inhibited and no amount of propranolol significantly restored the membrane contraction upon subsequent administration of epinephrine. Fig. 2. shows the results of the mean changes in pressor effects to oxymetazoline alone (30 #g/kg)
b
ca
c
0
E
E
E
2.1. Drugs The following drugs were used in this study: 1epinephrine bitartrate (Sigma Chemical Co.); Methoxamine HC1 (Burroughs Wellcome and Co.); oxymetazoline HC1 (Schering Corp.); dl-isoproterenol HC1 (Z.D. Gilman, Inc.); phenoxybenzamine HC1 solution (Smith, Kline and French Laboratories); and propranolol HC1 (Ayerst Laboratories). The concentrations of drugs used in all experiments are expressed
Phenoxybenzomlne (5 rng/Kg)
ptoprQnOlOI (I mc]l kq)
Fig. 1. The effect of propranolol (1 mg/kg) on the blockade by phenoxybenzamine (5 mg/kg) of the nictitating membrane and blood pressure of an anesthetized cat. The adrenergic agonist administered was epinephrine (E) (10 ug/kg). Time mark is 1 rain.
260
H.1.YAMAMURA and A.HORITA
under control, after phenoxybenzamine (5 mg/kg)and after propranolol (1.0 mg/kg) conditions. The results indicate that no significant increase in pressor response to o x y m e t a z o l i n e was seer ' l e n b e t a blockade was s u p e r i m p o s e d o n alpha b l o c k a d e . Similar re-
-& +8O
~, \
¢ -E <
I
0
g -2o
I~ Oxymetazohne Control [ ] O~ymetezoline + tsooroterenol
-4o
F:'-I Isoproterenol Control -60
Fig. 2. The effect of propranolol (1 mg/kg) on the blockade by phenoxybenzamine (5 mg/kg) of the mean change in blood pressure of an anesthetized cat. Adrenergic agonists administered were oxymetazoline (30 ug/kg), oxymetazoline + isoproterenol (30 gg/kg + 2 t~g/kg), and isoproterenol (2 #g/kg). Vertical bars are + S.E.
sults were seen o n t h e n i c t i t a t i n g m e m b r a n e to the a d m i n i s t r a t i o n o f o x y m e t a z o l i n e ( t a b l e 1). A n a t t e m p t was t h e n m a d e to d e t e r m i n e w h e t h e r a c o m p o u n d req u i r e d b o t h alpha a n d beta agonistic p r o p e r t i e s to exh i b i t this r e s t o r a t i o n o f p r e s s o r activity after alpha a n d beta b l o c k a d e . T h i s was d o n e b y c o m b i n i n g appropriate concentrations of oxymetazoline, a purely alpha agonist (Mujic a n d V a n R o s s u m , 1 9 6 5 ) w i t h isop r o t e r e n o l , a k n o w n beta s t i m u l a n t . T h i s m i x t u r e ( h e r e a f t e r d e s i g n a t e d as O X Y + ISO) was i n j e c t e d into cats b e f o r e a n d a f t e r p h e n o x y b e n z a m i n e , a n d after p r o p r a n o l o l t r e a t m e n t . T h e results s h o w t h a t a significant increase in t h e m e a n c h a n g e o f pressor response t o t h e O X Y + ISO m i x t u r e was seen w h e n beta b l o c k a d e was s u p e r i m p o s e d o n alpha b l o c k a d e (see fig. 2).
Table 1 Mean changes in blood pressure response (B.P.) and average peak tension of the nictitating membrane (N.M.) to i.v. injections of adrenergic agonists before and after phenoxybenzamine (5.0 mg/kg) and propranoloi (1.0 mg/kg). Control
After After phenoxybenzamine (A) propranolol (B)
Drugs Dose Qag/kg) Epinephrine Methoxamine Oxymetazoline
10
B.P. (mm Hg)
N,M. (g)
80 + 5.0 c (4) d 4.8 + 0.6 (4)
B.P. (mm Hg)
N.M. (g) B.P. (mm Hg)
- 2 5 + 3.1
0.25 + 0.2
N,M.(g)
61 +- 3.6 0.5 + 0.2
B-A
B.P. (mm Hg) 86 + 4.9 a
300
63+4.7
(6)
8.2-+0.8(3)
8+1.7
0
3-+0.9
0
-5-+5.5
30
58+4.2
(6)
8.2+1.4(3)
30-+3.1
0
25-+3.2
0
-5-+4.6
lsoproterenol
1
- 4 0 + 1.5 (3)
_e
- 3 8 -+ 2.2
-
- 3 -+ 3.3
-
34 -+ 5.2 b
lsoproterenol
2
- 4 4 + 3.8 (4)
_e
- 5 6 + 9.5
-
- 8 + 4.3
-
49 + 8.8 b
300+1
68+-4.7 (7)
8.4+-0.4(3)
-34+2.2
0
49 + 4.6 (10)
8.5 -+0.5 (5)
- 4 6 + 3.6
0
Methoxamine + isoproterenol Oxymetazoline + isoproterenol
30 + 2
6+-2.6
0
38-+2.4 a
33 -+ 3.4 0
79 -+4.8 a
a This represents the mean change in blood pressure response after propranolol minus the response after phenoxybenzamine (P < 0.01). b Same as footnote "a" except that (P < 0.05). c Values represent mean + S . E . d Value represents number of animals. e Nictitating membrane responses were not recorded with this agonist.
ALPHA ADRENERGIC RECEPTOR
Table 1 includes the mean changes in blood pressure and nictitating membrane responses to the various adrenergic amines under control, after phenoxybenzamine and after propranolol conditions. The mean value of the peak responses after propranolol minus the peak responses after phenoxybenzamine (hereafter designated as PROP-PBZ) for each amine was also calculated to aid in interpretation of the data. As can be seen from table 1, the PROP-PBZ value for the OXY + ISO combination was 79 mm Hg (S.E. = 4.8) P < 0.01. Cats were also given methoxamine (300/lg/kg), a known pure alpha agonist, as well as a methoxamine + isoproterenol (300 /ag/kg + 1 #g/kg) mixture. The results obtained from 6 methoxamine controls and 7 methoxamine + isoproterenol (METH + ISO) treated animals are summarized in table 1. Once alpha receptor blockade by phenoxybenzamine had occurred, propranolol at a dose of 1.0 mg/kg did cause a reappearance of a slight pressor effect to METH + ISO and
261
there was a significant PROP-PBZ value for the METH + ISO mixture (see table 1). On the nictitating membrane, both methoxamine and the METH + ISO mixture produced prolonged contractions. However, once the alpha blocker was administered no amount of propranolol could antagonize the blockade produced by phenoxybenzamine. 3.2. Effects of oxymetazoline and oxymetazoline +
isoproterenol after dissipation of propranolol action Experiments were also performed to determine whether time would affect the reversal phenomenon after phenoxybenzamine and propranolol. This was only possible because of phenoxybenzamine's prolonged duration of action and propranolol's effect being relatively short. The results obtained from three oxymetazoline controls and four OXY + ISO combinations are summarized graphically in fig. 3. After apparent recovery of the response to OXY + ISO after
IOOq
4°°1
4
}~°]_
6o E 40~ 2o-
"~ ~ 0
~
Methoxominecontrol
o-
~-20
-20. {
~40~$op~oterenOlcontrol
40[PBZ] [pRop] ~r I
.
1
-6o ~[p~z]
[PeOp] .
.
2
.
3
4
-~--
~
~
7
:
8
i
9
~
1
Hours
Fig. 3. The effect of adrenergic agonists after dissipation of propranolol action. White circles and white triangles represent the mean change in blood pressure to oxymetazoline (30 #g/kg) and isoproterenol (2 #g/kg), respectively, while the black circles represent the mean change in blood pressure to the oxymetazoline + isoproterenol (30 #g/kg + 2 t~g/kg) mixture before and after administration of phenoxybenzamine (5 mg/kg) and propranolol (1 mg/kg). After propranolol administration, the agonists were administered every 2 hr for 5 doses and a second dose of propranolol (l mg/kg) was given and the agonists were given once more. Each point represents a mean value of at least 3 cats.
~ [PRop]
I Hours
Fig. 4. The effect of adrenergic agonists after dissipation of propranolol action. White circles and white triangles represent the mean change in blood pressure to methoxamine (300 #g/kg) and isoproterenol (1 #g/kg), respectively, while the black circles represent the mean change in blood pressure to the methoxamine + isoproterenoi (300 pg/kg + 1 #g/kg) mixture before and after administration of phonoxybenzamine (5 mg/kg) and propranolol (1 mg/kg). After propranolol administration, the agonists were administered every 2 hr for 5 doses and a second dose of propranolol (1 mg/kg) was given and the agonists were given once more. Each point represents a mean values of 3 cats.
;62
ft.I.YAMAMURA and A.tIORITA
alpha and beta blockade, the experiment was continued until the effects of the beta blocking agent had terminated. Injections of isoproterenol (2 ~g/kg) were given in three control cats in order to verify the termination of the effects of the beta blocking agent. After partial termination of the effects of the beta blocking agent, the pressor response to OXY + ISO gradually reverted to a depressor response. A second dose of propranolot (1.0 mg/kg) was given 8-~4 hr later and upon subsequent administration of OXY t ISO, a re-establishment of the pressor response was seen. Results from fig. 3 also show that partial blockade to control doses of oxymetazoline (30/~g/kg) had occurred after phenoxybenzamine 15 mg/kg), but no significant recovery was seen after the administration of propranolol ( 1.0 mg/kg). Fig. 4 summarizes the effect of time on the mean changes in blood pressure to methoxamine (300/~g/ kg) and to the METH + ISO (300 ~g/kg + I /lg/kg) mixture. The graph indicates that after partial termination of the beta blocking agent, a depressor response gradually occurred with the METH + ISO mixture in 3 cats while in the control cats the mean response to methoxamine did not change. Injections of isoproterenol (1 /~g/kg) were given to 3 control cats to verify the termination of the effects of the betablocking agent.
4. DISCUSSION The results of these experiments indicate that the interaction between the alpha and beta blocking agents does not involve a displacement of one by the other as was hypothesized by Olivares et al. (1967), and Gulati et al. (1965). Of significance is the finding that the pressor actions of the pure alpha adrenergic agonists do not recover when beta blockade is superimposed on alpha blockade, but that if a pure alpha adrenergic agonist is combined with a beta adrenergic agonist a significant recovery does occur. The pressor responses to the OXY + ISO mixture which were antagonized by phenoxybenzamine exhibited significant recovery after propranolol; however, the results from the METH + ISO mixture were not as dramatic.
The results suggest that the apparent antagonism that is being observed with epinephrine and the OXY + ISO mixture after alpha and beta blockade is not due to a displacement mechanism but rather can be better explained as due to an inadequate blockade of the alpha receptor site by phenoxybenzamine. If it were possible to separate the alpha component of epinephrine from its beta component, we would possibly see that incomplete alpha blockade had occurred as seen in fig. 2 and fig. 3 in fire case of oxymetazotine. Mention was made previously that Olivares et at. ( 1967} noticed that once the alpha blockade was interrupted by a beta blocking agent, the alpha blockade did not reappear in dogs after the beta blocking agent had worn off, and that this suggested to them that there was unblocking of the alpha receptor site. ttowever, unlike them, we observed the reappearance of the alpha blockade which persists for a longer period than tile effects of beta blockade as produced by' propranolol. Upon administration of a second dose o f propranolol 8+~ hr later, there is again the apparent antagonism of alpha blockade, i.e., the restoratkm of a pressor response to the OXY + ISO mixture. We feel that since OXY + ISO ex ~ibits both alpha and beta agonistic actions, which mimic the action of epinephrine, its effect on blood pressure is a composite of the alpha and beta eSfects, part of which tend to raise and part attempt to lower blood pressure. With the dose of phenoxybenzamine used here a sufficient proportion of alpha receptors are blocked, which permits the beta effect, the vasodilator component, to predominate, thus resulting in the depressor effect. The administration of propranolol abolishes the beta component, and the spare alpha receptor effect of vasopressor activity again appears. However, as the beta blockade weakens with time the insurmountable alpha blockade of phenoxybenzamine becomes evident since the beta agonistic action of OXY + ISO reappears. An unexpected finding was that the recovery of METH + ISO mixture did not occur as dramatically as that seen with the OXY + ISO mixture after alpha and beta blockade. One probable reason for this discrepancy is that the METH + ISO mixture may have less efficacy for the alpha receptors than the OXY + ISO mixture and epinephrine, so that the METH +
ALPHA ADRENERGIC RECEPTOR ISO mixture requires attachment to a greater number o f spare alpha receptors to elicit a pressor response after alpha and beta blockade. Therefore, since there may be fewer spare alpha receptors available for the METH + ISO mixture to combine with, no significant recovery of a pressor response was seen. The concept o f activating spare alpha receptors, those not originally blocked by phenoxybenzamine, is further supported by the observation that only those agents possessing both alpha and beta agonistic properties, i.e., epinephrine or a combination of an alpha with a beta agonist, can exhibit this interaction. Pure alpha agonists, such as oxymetazoline, are partially blocked by phenoxybenzamine and the response after propranolol administration to oxymetazoline is not significantly greater than the response after phenoxybenzamine, indicating that "unblocking" o f alpha receptor sites is probably not taking place. If the view of Olivares et al. ( I 9 6 7 ) that the beta antagonist displaces the insurmountable alpha antagonist were correct, then the vasopressor response to oxymetazoline should have been restored to control values after alpha and beta~olockade.
ACKNOWLEDGEMENTS This research was supported by USPHS Research Grant MH-02435, National Institute of Mental Health; and by USPHS Training Grant GM-109 from the National Institutes of Health.
263
The authors are indebted to Ayerst Laboratories, Inc., New York, N.Y., for propranolol (Inderal ®), AY 64043; and to Smith, Kline and French Laboratories, Philadelphia, Pa., for phenoxybenzamine (Dibenzyline ®), SKF 688-A.
REFERENCES
Garrett, J., A.Malafaya-Baptistaand W.Osswald, 1966. Effects of pronethalol on the cardiovascular actions of catecholamines during blockade by phenoxybenzamine, Brit. J. Pharmacol. Chemotherap. 27,459-467. Gulati, O.D., S.D.Gokhale and B.P.Udwadia, 1965, Antagonism ofadrenergic blockade by pronethalol, Arch. intern. Pharmadyn. 156, 389-397. Hull, L.D., L.G.Eltherington, A.Horita, 1960, The antagonism ofadrenergLe blockade by dichloroisoproterenol(DCI) Experientia 16, 368-371. Mujic, M. and J.M.van Rossum, 1965, Comparative pharmacodynamics of sympathomimetic imidazolines; studies of intestinal smooth muscle of the rabbit and the cardiovascular system of the cat, Arch. intern. Pharmacodyn 155, 432-449. Olivares, G.J., N.T.Smith and L,Aronow, 1967, Effect of propranolol on alpha-adrenergic blockade in the dog and isolated rabbit aortic strip, Brit. J. Pharmacol. Chemotherap. 30, 240-250. Sharma, P.L., 1966, Interaction of adrenergic alpha and beta receptor blocking agents on the blood pressure response to adrenaline and noradrenaline, Quart. J. Exptl. Physiol. 51,256-261. Smith, R.D. and C.B.Nash, 1968, A comparative study of three beta adrenergic blocking agents in the anesthetized dog, Pharmacologist 10, 299. Yamamura, H.I. and A.Horita, 1968, Effect of propranolol on the blockade of alpha adrenergic receptors, J. Pharmacol; Exptl. Therap. 164, 82-89.