Beta Adrenergic Receptor of Rabbit Thoracic Aorta in Relation to Age

Japan. J. Pharmacol. 23, 259-268 (1973) 259

BETA ADRENERGIC RECEPTOR OF RABBIT THORACIC AORTA IN RELATION TO AGE

O.D. GULATI, B.P. METHEW, ELM. PARIKH* and V.S.R. KRISHNAMURTY** Pharmacological Research Unit, Council of Scientific and Industrial Research and Department of Pharmacology, Medical College, Baroda, India Accepted October 16, 1972

Abstract-Isolated aortic strips from rabbits older than 6 months (1.7-2.5 kg) when

subjected to contraction by noradrenaline or adrenaline were relaxed by isoprenaline. The relaxation was not blocked by propranolol. When these strips were contracted

by potassium chloride, isoprenaline produced a small relaxation which was sensitive to propranolol. In aortic strips from rabbits 2-3 months old (0.75-0.95 kg), the

contraction produced by noradrenaline or potassium chloride was relaxed by isoprenaline; isoprenaline was equally active against both stimulants. Propranolol anta-

gonised the action of isoprenaline competitively, with a pA2 value, 7.05. In strips from rabbitsfrom a lower age group, the pA2 values of phentolamine against isoprenaline and methoxamine contractions were 7.61 and 7.50 respectively. It is concluded

that aortas of rabbits older than 6 months are almost devoid of beta adrenergic re-

ceptors whereas those of rabbits 2-3 months old contain both alpha and beta adrenergic receptors.

Vascular smooth muscle has both alpha and beta adrenergic receptors (1). Isoprena- line, the most potent vasodilator sympathomimetic amine (2) produces both vasocon- striction and vasodilation in rabbits (1). Furchgott (3, 4) and Furchgott and Bhadrakom

(5) first demonstrated that in high concentrations, isoprenaline (4 •~ 10-6 -2 •~ 10-3 M) caused contraction of isolated rabbit thoracic aorta similar to that produced by adrenaline and noradrenaline. These concentrations were higher than those of adrenaline and noradrenaline needed to produce the same effect. However, when a preparation was stimulated to contract with acetylcholine, histamine, adrenaline or noradrenaline, low concentrations of isoprenaline (4 •~ 10-6 M) caused it to relax. The stimulant effect of the higher concentrations of isoprenaline appeared to be mediated through alpha receptors since it was blocked by dibenamine like that of adrenaline or noradrenaline (4). The relaxant effect of lower concentrations was blocked by beta adrenergic receptor blocking agents

(6, 7).

During the course of our work on the alpha adrenergic receptor blocking action of beta adrenergic receptor blocking drugs (8), it was observed that the relaxant effect of isoprenaline was not blocked by beta adrenergic blockers in aortic strips from rabbits older

* Present address : Alembic Chemical Works Co . Ltd., Baroda, India. ** Present address: Department of Experimental Medicine, South Western Medical School, Dallas, Texas 75235, U.S.A. 260 O.D. GULATI ET AL.

than 6 months. Later, it was accidentally observed that the relaxant effect of isoprena-

line was blocked by propranolol in strips obtained from rabbits 2-3 months old. We

have, therefore, compared the age of the animal on the responses of rabbit aortic strip

preparations to a number of stimulant and relaxant drugs.

MATERIALS AND METHODS

Healthy albino rabbits (New Zealand breed) of both sexes weighing 0.75 to 2.5 kg

were used. The animals were divided into two groups according to age and weight. One

group was 2-3 months old (0.75-0.95 kg) and the other was older than 6 months (1.7-

2.5 kg). The animals were sacrificed by a blow on the head. The thoracic aorta between

the heart and the diaphragm of each rabbit was used for preparing two strips as described

by Furchgott and Bhadrakom (5). Each strip was approx. 3 cm in length (unstretched)

and was mounted in a 35 ml organ bath containing Krebs bicarbonate solution maintained

at 37•Ž •}0.5•Ž. The bicarbonate concentration (25.0 mM) employed maintained the

pH at approx. 7.4 when a mixture of oxygen (95 %) and carbon dioxide (5 %) was bubbled

through the solution in both the organ bath and the reservoir. In most of the experi-

ments responses were recorded on a smoked drum by an isotonic frontal writing lever

with a 10-fold magnification. The load on the lever was 3.5 g. In experiments made

to determine the pAx values of phentolamine an auxotonic lever with a 13.5-fold magni-

fication was employed. It has already been reported that results obtained with the two

levers in isolated rabbit aortic strip preparations are not significantly different (8). After

mounting, the strips were washed every 15 min for 2 hr before the start of investigation.

To study the effects of relaxant drugs, namely, isoprenaline or sodium nitrite, the

preparation was first stimulated to contract by adding potassium chloride, adrenaline,

noradrenaline, histamine, acetylcholine, angiotensin or barium chloride. The maximum

contraction of which the tissue was capable was determined by adding a very large dose

of one of these stimulants and following recovery, a concentration of stimulant which pro-

duced 75-85 % response of this maximum was added. After the contraction was fully

developed (7-8 min), the relaxant drug was added and allowed to act for 3 min which was

long enough for the preparation to have settled to a new level. Relaxation responses

were elicited either cumulatively, by adding further doses of the relaxant without washing

the preparation, or by allowing for recovery between doses i.e. washing the preparation,

replacing the stimulant and subsequently adding higher concentrations of the relaxant.

This was continued until no further relaxation could be elicited.

Dose-response lines were obtained by plotting % of maximal response on the ordi-

nate and log molar concentration of the drug on the abscissa.

In experiments designed to determine the pA2 values of alpha or beta adrenergic block-

ers, two preparations from the same aorta were set up simultaneously and it was possible

to study two doses of a blocker in one preparation so that all four doses could be studied in one aorta.

After eliciting control relaxant responses, the beta adrenergic blocker was added to ADRE'NERGIC RECEPTOR OF RABBIT AORTA 261

the bath 8 min before administering the stimulant drug. Responses to relaxant drugs

were elicited after the blocker had remained in the bath for 15 min. This was followed

by washouts and a rest period of 45 min. The procedure was repeated with one more

dose of the blocker.

For studying pA2 values of phentolamine, isoprenaline and methoxamine were used

as the agonists and responses to them were obtained cumulatively. The contact time

for agonists was 3 min and that for phentolamine was 45 min. The rest period before

the next addition of phentolamine was 15 min.

The degree of antagonism was calculated in terms of dose-ratio (ratio of equiactive

doses of agonist after and before the addition of antagonist) in the manner described by

Gaddum et al. (9). The pA2 values were determined by plotting log (dose ratio -1) against

the negative log of the molar concentration of antagonist (10).

The sensitivity of all the tissue preparations remained remarkably constant during the experimental period which lasted for 9-10 hr.

Drugs: The following drugs were used : (•})- isoprenaline hydrochloride, (•})- adrenaline hydrochloride, (-)- noradrenaline bitartrate, 5-hydroxytryptamine creatinine sulphate (5-HT), potassium chloride, methoxamine hydrochloride, sodium nitrite, histamine acid phosphate, angiotensin, barium chloride, propranolol, (•})-N-p-nitrophenyl- ethanolamine hydrochloride (INPEA), 1-(2-naphthyl)-2-isopropylaminoethanol hydrochloride (pronethalol), dichlorisoprenaline hydrochloride (DCI), phenoxybenzamine hydrochloride and phentolamine methane sulphonate. Fresh solutions were prepared for each experiment except for adrenaline and noradrenaline which were used as a stock solutions

(1 mg/ml) prepared in distilled water containing sodium metabisulphite (0.1 %).

RESULTS

Isolated aortic strips from rabbits older than 6 months

Contractions which were 75-85 % of maximal were obtained with noradrenaline (3.4 •~

10-7 M), adrenaline (1.6 •~ 10-6 M), potassium chloride (3.8 •~ 10-2 M), angiotensin (1.4 •~

10-7 M), barium chloride (2.4 •~ 10-2 M), histamine (2.2 •~ 10-6 M), 5-HT (1.9 •~ 10-6 M) and acetylcholine (9.4 •~ 10-6 M). They were fully developed after 7-8 min, were maintained for over 15 min and remained consistent for several hr.

Contractions induced by histamine and 5-HT were blocked by propranolol (1.5 •~

10-5 M; 6 experiments); those induced by acetylcholine were potentiated by propranolol

(3.4 •~ 10-5; 3 experiments) and those induced by other stimulants were not modified by propranolol, INPEA, pronethalol and DCI in the concentrations used.

Isoprenaline, added when the contraction produced by noradrenaline or adrenaline was fully developed, caused a graded relaxation which was potentiated by DCI (Fig. 1) and pronethalol but not by propranolol or INPEA (Table 1).

Isoprenaline had little effect on the contraction produced by potassium chloride. In two out of five experiments a very slight relaxation was observed with isoprenaline in lower concentrations (1.0 •~ 10-7-1.0 •~ 10-6 M) and a contraction additional to that produced 262 O.D. GULATI ET AL.

FIG. 1. Rabbit (1. 79 kg) aortic strip preparation : Effect of dichloroisoprenaline

(4.7 •~ 10-6M) on the relaxant responses to isoprenaline. Open columns show control responses while shaded columns show responses in the presence of

dichloroisoprenaline. Vertical bars represent S.E. of means.

by potassium chloride was consistently obtained in higher concentrations (4.0 •~ 10-6-

2.0 •~ 10-5 M). The relaxation was blocked by propranolol (6.8 •~ 10-6 M) and the additional contraction by phenoxybenzamine (3.0 •~ 10-6 M).

After exposure to phenoxybenzamine (8.2 •~ 10-8 M) for 45 min, noradrenaline (3.4 •~

10-6-3.4 •~ 10-5 M) no longer elicited contractions. However, angiotensin (5 experiments) and barium chloride (3 experiments) contracted the strips. Isoprenaline added at the height of the contraction induced by angiotensin produced a dose-dependant relaxation which was not modified by propranolol (Table 1). Isoprenaline failed to modify barium chloride induced contractions.

Aortic strips from rabbits 2-3 months old

Histamine, 5-HT, acetylcholine and barium chloride were not suitable as stimulants as the contractions with the former three were modified by propranolol and isoprenaline failed to produce relaxation with the latter. Angiotensin though found suitable was not used since the work could be satisfactorily concluded with noradrenaline and potassium chloride.

Isoprenaline was equally effective in relaxing the contractions induced by noradrenaline or potassium chloride. The relaxation was dose-related (Table 1) and was blocked by propranolol. Propranolol did not affect the contractions. The slopes of the dose- response (relaxation) lines of isoprenaline with the two stimulants were not significantly

(P>0.5) different (Table 1).

The magnitude of relaxation in response to the same concentration of isoprenaline ADRENERGIC RECEPTOR OF RABBIT AORTA 263 264 G.D. GULATI ET AL.

in strips stimulated with potassium chloride was 15-25 times greater than that obtained in strips from rabbits older than 6 months.

Effect of propranolol against isoprenaline and sodium nitrite-induced relaxation

Only noradrenaline was used as the stimulant for quantitative work. Propranolol

(1.5 •~ 10-5-1.2 •~ 10-4 M) inhibited the relaxation produced by isoprenaline given at the

FIG. 2. Rabbit (0.82 kg) aortic strip preparations : Cumulative log dose-response curves for the relaxant effect of isoprenaline in the absence of (control, average of 2 observations ; •œ•\•\•œ and in presence of 1.5 x 10-5M (0-0) ; 3.0 x 10-51VI (+ •\•\ +) ; 6.0 •~ 10-5M (•¢•\•\•¢ A) and 1.2 •~ 10-4M (A-A) of propranolol. Propranolol caused a parallel shift of the dose-response curves to the right giving dose ratios of 6.7, 14.1, 24.2 and 31.6 respectively.

TABLE 2. Antagonistic effects of propranolol and phentolamine against isoprenaline- induced and of phentolamine against methoxamine-induced responses of rabbit aortic strip preparations.

Figures in parentheses indicate the number of experiments. (1) Negative log M of the antagonist was plotted on the abscissa and log (dose ratio -1)was plotted on the ordinate. ADRENERGIC RECEPTOR OF RABBIT AORTA 265 height of noradrenaline-induced contraction. The dose-response curves for isoprenaline were shifted to the right without any alteration in their slopes. Maximal responses to isoprenaline remained unaffected (Fig. 2). The regression lines of plots of log (dose ratio -1) versus -log M propranolol were significantly different from zero (P<0.001). The regression coefficient of -1.11 •}0.10 was not significantly (P>0.5) different from the theoretical value of -1.0 for competitive antagonism. The pA2 value of propranolol was 7.05. Data are summarised in Table 2.

Sodium nitrite (8.3 •~ 10-4 M) elicited reproducible relaxation of the aortic strip contracted by potassium chloride (3.8 •~ 10-2 M). Propranolol (3.4 •~ 10-5 M and 6.8 •~ 10-5

M) did not block relaxation due to sodium nitrite but that due to isoprenaline (1.1 •~ 10-7

M) elicited in the paired preparations was totally blocked (4 experiments; Fig. 3).

FIG. 3. Paired aortic strips (A and B) of rabbit (0.8 kg). Potassium chloride (3.8 ×10-2M) kept the strips submaximally contracted at the plateau level (P). Sodium nitrite (8.3 •~ 10-4M) and isoprenaline (1.1 •~ 10-7 M) were added at NIT and ISO respectively. The left hand panels show control responses. The

responses shown in right hand panels were obtained in the presence of pro-

pranolol (3.4 •~ 10-4M) added at arrows. Time mark, 1 min.

Effect of phentolamine against isoprenaline and tnethoxamine induced contraction Cumulative administration of increasing concentrations of isoprenaline (2.2 x 10-6 M) or methoxamine elicited dose-related contractile responses. The slopes of the dose- response regression lines for isoprenaline and methoxamine were not significantly dif- 266 O.D. GULATI ET AL. ferent (P>0.1) (Table 1). Phentolamine (1.2 •~ 10-7-1.0 •~ 10 M) induced parallel shift of the agonist dose-response line. The regression lines of plots of log (dose ratio -1) versus -log M phentolamine were significantly different from zero (P<0.01). The pA2 values of phentolamine against isoprenaline and methoxamine were 7.61 and 7.56 respectively. The slope values obtained in the present experiments were not significantly different (P>0.5) from the theoretical value (-1) of slope for competitive antagonism. Data are given in Table 2.

DISCUSSION

Strips obtained from rabbits older than 6 months

The present study has shown that in aortic strips contracted by noradrenaline or adrenaline, isoprenaline produced relaxation which was not blocked by propranolol. This ruled out the relaxation having been mediated through activation of beta adrenergic receptors. This finding could be related to the use of (+)-isoprenaline. (+) Isoprena- line possesses alpha adrenergic receptor blocking action (11).

No satisfactory explanation can be provided for potentiation of the isoprenaline- induced relaxation by DCI and pronethalol.

Since isoprenaline acts on alpha receptors also, these were completely blocked by phenoxybenzamine, an irreversible alpha receptor blocker. Contractions in these preparations were induced by angiotensin and barium chloride since other non-adrenergic stimulants like histamine, acetylcholine, 5-HT and potassium chloride are also blocked by beta-haloalkylamines (12-16). In strips treated with phenoxybenzamine and contracted by angiotensin, the relaxant effect of isoprenaline was not blocked by propranolol.

The slope of the dose-response regression line was 73.6•}3.0. This slope was significantly higher (P<0.001) than the slope (35.5•}5.1) of regression line for isoprenaline in strips contracted by noradrenaline, implying distinct but admittedly not beta adrenergic receptor mediated mechanisms for the inhibitory effects of isoprenaline in the two cases.

Isoprenaline inhibited to a very minor extent contraction induced by potassium chloride. This small relaxant effect was blocked by propranolol and, therefore, must be due to activation of beta adrenergic receptors.

Aortic strips from rabbits 2-3 months old

The slope values of log dose-response curves of isoprenaline for relaxing aortic strips against noradrenaline and potassium chloride induced contractions were identical, sug- gesting that interaction between isoprenaline and noradrenaline at the alpha adrenergic receptor did not mediate relaxation. A perusal of Table 1 indicates that the highest concentration of isoprenaline for inducing relaxation was less than the lowest concentration necessary for inducing contraction pointing to the activation of beta adrenergic receptors with lower concentrations and alpha adrenergic receptors with higher concentrations.

The blocking action of propranolol was specific, since isoprenaline was blocked and sodium nitrite was not blocked, the block being competitive in nature.

A competitive reversible antagonist would have the same dissociation constant (KB), ADRENERGIC RECEPTOR OF RABBIT AORTA 267 in different tissue preparations, if it reacts with closely similar receptors (11, 17-20). It has thus been possible to identify the alpha adrenergic receptors in different tissues since the K. values for alpha receptor antagonists do not differ significantly in these tissues.

On the other hand, there are considerable differences in KB or pA2 values of beta receptor antagonists obtained with different tissue preparations (6, 21). The rabbit thoracic aorta and guinea pig trachea have been grouped under one head as the K. values of pronethalol are identical (6). The pA2 value of propranolol of 7.05 obtained in our experiments is closely similar to the value of 7.2 with guinea pig tracheal muscle reported by Takagi &

Takayanagi (21). Thus it can be concluded that the receptors activated by isoprenaline and blocked by propranolol in the aortic strips obtained from the younger rabbits are beta adrenergic receptors.

In strips obtained from younger rabbits, isoprenaline in the same concentration range produced relaxation which was 15-25 times greater than that obtained in strips from older rabbits. It appears likely that beta adrenergic receptor effect of isoprenaline in rabbit aortic strip is age dependent and that there is a decrease in the beta receptor population in thoracic aorta of older animals. Similar conclusions have very recently been drawn by Fleish et al. (22).

Further work must determine why beta adrenergic receptors disappear from rabbit aorta with age. We have not been able to obtain any evidence for the presence of beta adrenergic receptors in carotid arterial strips from adult cats (2-3 kg) and femoral arterial strips from adult dogs (10-14 kg), although aortic strips from adult rats (150-250 g) do show the presence of beta receptors (unpublished observations).

In experiments with strips obtained from younger rabbits, the slopes of the regression lines for the contractile responses to isoprenaline and methoxamine were not significantly different (P>0.1). The pA2 values of phentolamine for blocking contractile responses to isoprenaline and methoxamine were 7.61 and 7.56 respectively and closely agree with the value of 7.52 reported by Furchgott (19). Thus, alpha adrenergic receptors of the aortic smooth muscle of younger rabbits are similar to those of older rabbits.

Acknowledgements: It is a pleasure to acknowledge the gifts of pronethalol and propranolol by Dr. S.A. Stephen of Imperial Chemical Industries Ltd. (Cheshire); of dichloroisoprenaline by Eli lilly and Company (Indianapolis); of (•})-N-isopropyl-p-nitro- phenylethanol-amine by Dr. W. Murmann of Selvi and Co. (Milan); of noradrenaline and isoprenaline by Dr. R.C. Clinton of Sterling-Winthrop Research Institute (Rensselaer); of 5-hydroxytryptamine by Sandoz Ltd., (Basle); of methoxamine by Burroughs Wellcome

& Co. (Bombay); of phentolamine and angiotensin by Ciba-Geigy Pharmaceutical Com- pany (Basle); and of phenoxybenzamine by Mr. Gordon Wilfon of Smith Kline & French

Laboratories (Philadelphia).

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