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 receptors 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 isoprenaline 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 experiments 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 magnification 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 produced 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 blockers, 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.

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