Alteration of norepinephrine responses in the dog by dual adrenergic blockade

EUROPEAN JOURNAL OF PHARMACOLOGY 8 (1969) 310-314. NORTH-HOLLAND PUBLISHING COMP., AMSTERDAM

ALTERATION

OF NOREPINEPHRINE

RESPONSES

BY DUAL ADRENERGIC

BLOCKADE

IN THE DOG

Clinton B. NASH and Ronald D. SMITH Department o f Pharmacology, University o f Tennessee Medical Units, 62 South Dunlap Street, Memphis, Tennessee 38103, USA

Received 15 July 1969

Accepted 19 September 1969

C.B.NASH and R.D.SMITH, Alteration o f norepinephrine responses in the dog by dual adrenergic blockade, European J. Pharmacol. 8 (1969) 310-314. Norepinephrine was infused into anesthetized dogs at a rate of 10 ug/kg]min for 2 hr and the animals sacrificed after an additional 2 hr. Measurements were made in the presence and absence of alpha blockade (phenoxybenzamine, 7.5 mg/kg), beta blockade (propranolol, 2 mg/kg), and dual adrenergic blockade (phenoxybenzamine, 7.5 + propranolol, 2 mg]kg). At these dose levels beta blockade alone did not prevent the decrease in blood pH; the increase in hematocrit, serum potassium, serum phosphorus, and blood lactic acid; or the production of pericardial effusion, cardiac hemorrhage, cardiac arrhythmias and ECG voltage depression. Alpha blockade alone blocked ttematocrit, serum phosphorus, pericardial effusion effects and deaths, and reversed the rise in serum potassium. Dual blockade was unique in eliminating cardiac arrhythmias, decrease in blood pH, changes in serum potassium, rise in blood lactate, and largely reducing blood pressure changes. These data support the concept that certain effects of catecholamines are mediated by a combination of alpha plus beta stimulation. Phenoxybenzamine Propranolol Blood lactic acid

Norepinephrine post-infusional hypotension

Myocardial hemorrhage Cardiac arrhythmias Serum potassium

1 : INTRODUCTION

2. METHODS

Many of the responses to norepinephrine are known to be mediated via either alpha or beta receptors (vasoconstriction, cardiac stimulation, etc.); however, the dependence o f other responses such as acidosis, hyperlactic acidemia, hyperpotassemia, etc., upon specific norepinephrine receptors is far from clear (Ellis, 1956). A concept that has received some attention holds that certain effects o f epinephrine are m6diated via b o t h alpha and beta receptors (Ahlquist and Levy, 1959; Nash, 1968). The present study is an attempt to identify responses to norepinephrine that may involve a simultaneous agonistic action on b o t h alpha and beta receptors.

Both male and female dogs were anesthetized with pentobarbital sodium, 30 mg/kg intravenously. The animals were surgically prepared for recording blood pressure from the c o m m o n carotid artery with a Statham P23-AC strain gauge, respiration from the trachea with a Grass PT5 volumetric transducer, and ECG Limb Lead II b y subcutaneous needle electrodes. These measurements were recorded on a Grass Polygraph. Blood samples were taken at intervals from the femoral vein for determinations o f pH (Beckman Zeromatic meter with blood electrodes), hematocrit, lactic acid (Horn et al., 1956), potassium (flame photometer), and phosphorus (Fiske and Subbarow, 1925).

NOREPINEPHRINEAND DUAL ADRENERGIC BLOCKADE All animals received the same infusion rate of norepinephrine and were divided into 4 groups: (1) controis (2) alpha blockade with phenoxybenzamine (3) beta blockade with propranolol, and (4) dual adrenergic blockade with phenoxybenzamine plus proprano1ol. Phenoxybenzamine HC1 was injected intravenously in a dose of 7.5 mg/kg 45 rain prior to the beginning of the norepinephrine infusion. Propranolol HC1, a beta adrenergic blocking agent, was injected intravenously in a dose of 2 mg/kg 10 min prior to the norepinephrine infusion. Norepinephrine bitartrate was dissolved in 0.9% sodium chloride solution and the concentration adjusted so that an infusion rate of 0.123 ml/min by a Harvard syringe pump would deliver 10 ~g/kg/min (as the base) into the femoral vein. Following control recordings, the appropriate blocking agent or agents were given as described above and the norepinephrine infusion was started. The infusion was discontinued at the end of 2 hr and a 2-hr recovery period was allowed before sacrificing the surviving animals.

3. RESULTS 3.1. Blood pressure responses

In the control dogs receiving only norepinephrine infusion, mean blood pressure initially rose sharply from a control of 158+6.7 to a peak of 262-+11.2 mm Hg, and this was followed almost immediately by a progressive fall which brought blood pressure below the control value at the end of the infusion (fig. 1). When the infusion of norepinephrine was discontinued, blood pressure fell rapidly to 80-+7.0 mm Hg and did not recover during the remaining time. The propranolol pretreated group responded in a similar manner (149 -+9.8 to 249 -+9.7 mm Hg) with the notable exception that when the infusion was ended, the blood pressure fell drastically and 6 of 7 animals died within a few minutes. In the presence of alpha blockade by phenoxybenzamine, norepinephrine infusion caused a fall in blood pressure of approximately 40 mm Hg (99 -+ 13.2 to 60 -+ 2.8 mm Hg) and the pressure was stable throughout the remainder of the infusion period. In contrast to the other groups, those animals receiving dual adrenergic blockade exhibited only a

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moderate and quite transient increase in blood pressure and throughout most of the infusion period blood pressure was stable and virtually the same as the control value. At the conclusion of the infusion, blood pressure tended to rise rather than fall. 3.2. Blood lactate levels

Blood lactic acid changes are shown in fig. 2. Con...... Norepi only - - Pba 7.5 ..... Prop 2 .........Pba 7.5 + Prop 2

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312

C.B.NASH and R.D.SMITH Table 1 Effect of norepinephrine infusion on blood pH, hematocrit, potassium, and phosphorus levels with and without adrenergic blockade. Treatment a

Parameters

Control norepi, inf.

Blood pH 0hr 2hr 4 hr

Propranolol + norepi, inf.

7.32 +0.01 6.97 +0.04 c 6.98 _+0.05 c

Hematocrit 0hr 2 hr 4 hr

40 57 58

Serum potassium b 0hr 2hr 4hr Serum phosphorus b Ohr 2hr 4hr

Phenoxybenzamine + norepi, inf.

7.32 +0.01 7.01 +0.04 c _

±2.6 + 3.7 c + 2.7 c

7.33 +0.003 7.16 +0.01 c 7.22 +0.04 c

39 57 _

±3.0 ± 4.8 c

38 36 41

15.7 +0.6 20.6 ±1.4 c 27.4 _+2.3 c

14.1 34.2 -

±0.7 +2.0 c

15.6 _+0.4 11.6 _+0.4 c 11.9 _+1.0 c

5.3 9.1 10.8

5.0 ±0.3 11.0 +1.5 c -

±0.4 +1.2 c _+2.1 c

Dual blockade + norepi, inf.

7.33 ±0.01 7.32 ±0,01 7.37 +0.01

+1.4 _+0.7 _+1.3

5.4 +0.5 5.2 _+0.5 5.9 _+0.6

39 40 42

+2.1 + 2.5 ±2.6

15.9 16.4 16.5

±0.5 ±1.2 ±1.4

5.7 _+0.4 6.0 +0.5 5.7 +0.6

a All dogs received an infusion of norepinephrine, 10 #g/kg/min for 2 hr and were observed for 2 hr more. Doses of phenoxybenzamine and propranolol were 7.5 and 2 mg/kg i.v. respectively. Tabular values are expressed as means + S.E. b Expressed in rag/100 ml. c Compared to zero hour reading, p ~ 0.05.

trol infusions o f n o r e p i n e p h r i n e increased lactic acid levels nearly ten fold. N e i t h e r p h e n o x y b e n z a m i n e n o r p r o p r a n o l o l p r e t r e a t m e n t h a d an a p p r e c i a b l e e f f e c t to

3.3. Blood pH, hematocrit, serum potassium, and phosphorus

p r e v e n t the m a r k e d

acidemia, w h i c h was u n c h a n g e d b y p r o p r a n o l o l , 15artly b l o c k e d b y p h e n o x y b e n z a m i n e , and totally

initial increase in lactic acid.

One o f t h e early e f f e c t s o f n o r e p i n e p h r i n e was

H o w e v e r , dual b l o c k a d e c o m p l e t e l y b l o c k e d the rise in lactic' acid, resulting in stable lactate values

b l o c k e d b y dual b l o c k a d e (table 1). T h e sharp in-

t h r o u g h o u t the entire e x p e r i m e n t a l p e r i o d .

crease in h e m a t o c r i t w h i c h was seen in b o t h t h e c o n -

Table 2 Effects of dual adrenergic blockade on some toxic effects of norepinephrine infusion * Incidence of Pretreatment

Dose (mg/kg)

None Propranolol Phenoxybenzamine Propranolol + phenoxybenzamine

2 7.5 2 7.5

Pericardial effusion

Cardiac hemorrhage

Cardiac arrhythmias

7/7 7/7 0/5 0/7

7/7 7/7

7/7 7/7

5]5

3/5

7/7

0/7

* Norepinephrine infused for 2 hr at 10 ~g/kg/min and dogs observed for 2 hr more.

ECG R-wave voltage % decrease (mean _+S.E.) 77 81 32 36

± ± ± ±

9 9 7 8

Mortality rate

1/7 6/7

0[5 0/7

NOREPINEPHRINEAND DUAL ADRENERGIC BLOCKADE trol and propranolol treated groups was effectively prevented by either phenoxybenzamine or dual blockade. The rise in serum phosphorus was unaffected by propranolol and was blocked by either phenoxybenzamine or dual blockade. The control hyperpotassemia was increased still more by beta blockade. It was reversed by phenoxybenzamine to a hypopotassemia and completely blocked by dual blockade. 3.4. Cardiac effects and mortality Pericardial effusion was a consistent finding in the control animals (table 2). The volume of blood-tinged serous fluid in the pericardial sac ranged from 25 to 45 ml. Hemorrhagic areas were grossly visible and generally covered more than half of the surface area of the left and right endocardium, sometimes extending through the ventricular wall to appear on the epicardium. Cardiac arrhythmias were present in every control dog, consisting principally of ventricular tachycardia and ventricular ectopic beats. A prominent effect of norepinephrine was a marked reduction in voltage of the ECG. This decrease began within a few minutes and was maximal near the end of the norepinephrine infusion. Beta blockade had little or no action to change the control responses with the notable exception that mortality was increased from 1/7 in controls to 6/7 in the propranolol pretreated group. Alpha blockade improved this picture somewhat in that all animals survived and pericardial effusion was prevented. However, cardiac hemorrhage, arrhythmias, and ECG voltage depression were still present. Dual blockade eliminated pericardial effusion, cardiac arrhythmias, and death, but failed to prevent myocardial hemorrhage and only partly blocked the ECG voltage depression.

4. DISCUSSION The effects of blocking simultaneously both alpha and beta adrenergic receptors have received scant attention in the literature. In the patient with pheochromocytoma, Pritchard and Ross (1966) and Crago et al. (1967)have provided impressive evidence by the use of combined alpha and beta blockade for the control of sudden pressor and depressor responses resulting from the manipulation of the tumor. Our data in

313

the dog show that with an appropriate ratio of alpha/ beta blockade, the pressor effects of large amounts of norepinephrine can be limited to moderate and transient changes. In addition, the post-infusional hypotension which was always seen in control dogs did not develop when the infusion was discontinued. It has been shown that phenoxybenzamine does not prevent the increase in lactic acid produced by epinephrine in the dog (Mayer, 1961), whereas, pmpranolol does block hyperlactic acidemia induced by catecholamines in the rat (Salvador et al., 1967). The present study confirms the ineffe.ctiveness of phenoxybenzamine in a dose of 7.5 mg/kg, and also shows that in the dog propranolol in a dose of 2 mg/kg, likewise had no action on the lactate rise. The complete blockade that occurred when phenoxybenzamine and propranolol were combined, emphasizes that dual blockade has potentialities beyond those of either agent alone at the same dose level. Although the literature is fairly consistent in categorizing lactate responses to catecholamines as a beta function and in showing that alpha blockade is without effect, our data emphasize that when an ineffective dose of propranolol is combined with an ineffective dose of phenoxybenzamine, a solid blockade of the lactic acid response is produced, indicating that both alpha and beta receptors are very likely involved in this particular catecholamine response. In control dogs serum potassium and phosphorus (table 1) were significantly increased by norepinephrine, and the elevated levels persisted beyond the period of infusion, in agreement with reports by Muirhead (1954) and Morris et al. (1966). The increase in serum phosphorus by norepinephrine is in contrast to the decrease produced by epinephrine and has been noted previously (Ellis, 1956). Brewer et al. (1938) found that epinephrine produced first an increase in serum potassium followed by a decrease in both dog and man. In our hands phenoxybenzamine not only prevented the norepinephrine-induced increase in serum potassitma but actually decreased it below control. In contradistinction, beta blockade resulted in a still greater rise. Thus, the presence of both alpha and beta blockade was required to prevent serum potassium from varying from control. Beta blockade had no effect on the rise in phosphorus while either alpha blockade or dual blockade eliminated changes in serum phosphorus, suggesting that

314

C.B.NASH and R.D.SMITH

alpha receptors may be more important in this respect than beta receptors. The relationship of the cardiac effects of catecholamines to specific receptors has so far eluded a complete description. Tachycardia and the increase in contractile force now seem to be related to the activation of a type of beta receptor designated by Lands (1967) as beta-1. In the present study pericardial effusion was prevented by phenoxybenzamine or dual blockade, but not by propranolol; however, the importance of specific receptor blockade in this response is unknown, since the effusion may have been an indirect effect of the catecholamine. Cardiac arrhythmias, which were still present after either alpha blockade or beta blockade, were entirely eliminated by dual blockade. The myocardial hemorrhage produced b y norepinephrine was not prevented b y alpha, beta, or dual blockade; in contrast to the myocardial hemorrhage resulting from epinephrine infusion which was eliminated by dual blockade (Nash, 1968), thus illustrating another distinction between epinephrine and norepinephrine. Deaths in this study were due to a gradual deterioration of the cardiovascular system which was increased in the presence of beta blockade and appeared to be decreased by alpha blockade. The importance of the ECG effects is unknown, but the marked decrease in R-wave voltage was a consistent finding in the absence of phenoxybenzamine. Acidemia is known to inhibit the pressor response to catecholamines (Wood et al., 1963), and the blockade of blood pH and lactic acid changes by dual blockade may partly explain the stable blood pressure seen in dual blocked animals.

ACKNOWLEDGEMENT The authors wish to thank Mrs. Shirley Hillman for her excellent technical asistance. This work was supported in part by a University of Tennessee Institutional Grant.

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