A method for pharmacological studies on adrenaline release from the rat adrenal.

Study on adrenaline release from the rat adrenal was performed by means of fluorometric determination of adrenaline in adrenal-venous blood specimens. The basal rate of adrenaline secretion under ether anesthesia was about 10 times that under pentobarbital anesthesia. Splanchnicotomy and spinal-transection prevented the secretion. Increase of adrenaline output was observed at doses of 0.125 to 0.5 mg/kg i.v. of nicotine under pentobarbital anesthesia, but under ether anesthesia increase of adrenaline secretion was not detected. Ether prevented the nicotine induced ad renaline release from splanchnicotomized rat adrenal, and this action was stronger than that of pentobarbital anesthesia. Morphine caused a marked adrenaline secre tion under ether anesthesia and the action was dose responsive from 30 to 120 mg/kg i.p. as the hydrochloride salt. Splanchnicotomy largely prevented the increase of adrenaline release in response to morphine. These results provide a method for the study of drug effects on adrenaline release from rat adrenals. Udenfriend et al. (1) calculated the basal rate of secretion of adrenaline from rat ad renals from the turnover rate of adrenaline in the glands. Cession-Fossion (2) has used perfusion in situ to study drug effects on adrenaline release from rat adrenals. Rohr (3) observed the adrenaline release caused by muscarone, acetylcholine and histamine by means of histochemical methods. Roffi et al. (4) reported adrenaline releasing effects of ACTH and corticosteroids by bioassay of adrenaline in adrenal-venous blood of hypophysecto mized rats under pentobarbital anesthesia. Few observations have been made, however, on the rat as compared to dogs and cats. The present paper describes a method for the study of effects of drugs on adrenaline release from rat adrenal gland, which revealed the basal rate of adrenaline secretion in certain conditions, the effects of nicotine and morphine and the influence of anesthetics on the adrenal response to nicotine. MATERIALS AND METHODS Experiments were carried out with male rats of Sprague-Dawley strain weighing 400 600 g. The animals were usually anesthetized by ether or by an i.p. administration of 50 mg/kg of pentobarbital sodium; a cannula being inserted in the saphenous vein for drug injection and infusion of fluids. The abdomen was opened by a horizontal incision and the animal was heparinized. The left renal vein was ligated at both terminals and cannu lated for collection of adrenal-venous blood. The abdomen was closed after coupling the two cannulas. During collection of adrenal-venous blood, Ringer's solution contain ing 0.1 % of glucose was usually infused, 3.0 to 4.0 ml per 15 min. Splanchnicotomy was performed just under the diaphragm 25 min prior to the onset of the blood collection. Spinal-transection was performed between cervical VII and tho racic I, 3 to 6 hr prior to the experiment. Adrenal-venous blood was collected for 15 min and immediately centrifuged. One ml of the blood plasma was put into a glass-stoppered centrifuge tube containing 9 ml of acidified butanol (0.85 nil conc. HCI added to one liter of n-butanol). The tube was shaken by hand, allowed to stand for 30 min in an ice bath and then centrifuged. The supernatant was transferred to another glass-stoppered tube; about 2 mg of sodium metabi sulfite was added, the tube shaken and then stored at 20'C until used. Adrenaline contents of the stored samples were determined by the method of Chang (5) with a slight modification. Eight ml of acidified butanol extract was pipetted into a glass-stoppered tube containing 15 ml of n-heptane and 5 nil of water. The mixture was mechanically shaken for 5 min and centrifuged. The solvent layer was removed and 4 ml of the aqueous phase was transferred to a glass-stoppered centrifuge tube containing 0.2 g of alumina, adjusted to pH 7.0-7.5 by addition of I ml of 2 M sodium acetate and a suit able volume of 0.1 N NaOH, and shaken for 10 min to absorb the adrenaline. The tube was centrifuged and the supernatant fluid removed by aspiration. The precipitated alu mina was washed with 2 nil of distilled water; the tube was shaken for 5 nun, centrifuged and the supernatant was removed. Adrenaline was eluted from the alumina by shaking with 2.5 ml of 0.06 N HCI for 10 min. One nil of the eluate was transferred into a test tube and 0.2 nil of 0.1 N NaOH and 0.3 nil of distilled water were added, bringing the sample to about pH 4. To this sample was added 0.2 nil of 0.1 M EDTA reagent (37.2 g of disodium ethylenediamine tetraacetate dehydrate, dissolved in I M sodium acetate, the pH being adjusted to 6.7-7.0 by the addition of 5 N NaOH to a final volume of I liter) and thoroughly mixed. Then, 0.1 nil of 0.1 N iodine (1.27 g of iodine in 100 nil of 99.5;, ethanol) was added to oxidize adrenaline, and after exactly 2 min the oxidation was stop ped by the addition of 0.2 nil of alkaline sulfite (250 nig of Na,,SO,, in 1 ml of distilled water, diluted with 9 ml of 5 N NaOH just before use) and exactly 2 min later the solution was adjusted to pH 5.4 by addition of 0.2 nil of 5 N acetic acid. Ten min after the addition of acetic acid, the fluorescence (activation 410 m1t, fluorescence 500 nice) of the sample was read on Aniinco-Bowman Spectrophotofluoronieter. Standards were prepared by carrying out the procedure with known amounts of adrenaline, about 0.2 /tg,/tube. Nicotine (Tokyo Kasei) was dissolved in 0.9% saline and 0.5 ml/kg of the solution was injected in the saphenous vein. Morphine hydrochloride (Shionogi) was administered i.p. as an aqueous solution. RESULTS The basal rate of adrenaline release from the left adrenal of the rat is shown in Table 1. The adrenaline secretion from the gland when transfused with Ringer's solution con taining 0.1 % of glucose was not so different from that from a gland transfused with fresh heparinized rat blood under ether anesthesia. The adrenaline secretion under ether anes thesia was about 10 times that under pentobarbital anesthesia. Splanclinicotoniy and spinal-transection decreased the ether-caused adrenaline output to a trace. The effect of nicotine on adrenaline secretion is shown in Table 2. Nicotine did not cause hypersecretion of adrenaline when utilizing ether anesthesia, but with pentobarbital anesthesia it increased adrenaline secretion from the gland at doses of 0.125 to 0.5 mg,/kg. Inhibitory effects of anesthetics on the adrenaline release in response to nicotine were tested in the splanchnicotomized rat (Table 3). Nicotine caused a marked increase of adrenaline release under the non-anesthetic condition at a dose of 0.5 mg/kg in spinal transected rats, however, under ether anesthesia no increase in adrenaline release was ob served. Adrenaline releasing action of nicotine was observed under pentobarbital anes thesia, but the action was weaker than that in the absence of anesthesia. At a dose of 0.125 mg/kg, increase of adrenaline output was not observed under non-anesthetic condi tions. TABLE 1. Basic release of adrenaline from intact and denervated left adrenal in rats. Data represent mean ± S.E. Number of experiments indicated in brackets. TABLE 2. Effect of nicotine on adrenaline release from left adrenal of rats under anesthesia. Data represent mean + S.E. Number of experiments indicated in brackets. TABLE 3. Effects of anesthetics on adrenaline release in response to nicotine from splanchnicotomized rat left adrenal. Data represent mean ± S.E. Number of experiments indicated in brackets. (1) Spinal-transection was performed 3 to 6 hr prior to experiment. TABLE 4. Effect of morphine on adrenaline release from rat left adrenal under ether anesthesia. Morphine hydrochloride, 120 mg, kg, was administered i.p. Data represent mean±S.E. FIG. 1. Effect of morphine on adrenaline release from left adrenal in ether anesthetized rats. All points represent a. mean of 5 experiments. Bars are S.E. of the mean. FIG. 2. Effect of splanchnicotomy on adrenaline release caused by morphine from rat adrenal under ether anesthesia. Adrenal-venous blood was collected from 40 min to 55 min after 120 mg/kg of morphine hydrochloride i.p. Bars are S.E. of the mean. The effect of morphine on adrenaline secretion from the rat adrenal was also pursued. Morphine hydrochloride, 120 mg/kg, was administered i.p., after which the rat was anes thetized with ether and the adrenal-venous blood was collected. Hypersecretion of adre naline was observed from 30 min after morphine administration (Table 4). The effect of morphine on adrenaline secretion was dose-responsive from 30 to 120 mg/kg at 40 min (Fig. 1). Increase of adrenaline output after administration of morphine was largely pre vented by splanchnicotomy (Fig. 2). DISCUSSION Adrenaline and noradrenaline are the main catecholamines in the adrenal medulla. Each s ,ecies has its own ratio of these compounds, and adrenaline is the major compound in the rat (6). Westfall and Anderson (7) reported, in their study on urinary excretion of catecholamines and metabolites, that adrenaline was the major catecholamine secreted by nicotine from rat adrenals. As fluorometric determination is not sensitive enough to distinguish adrenaline and noradrenaline in samples containing very small amounts of the compounds, we did not perform simultaneous determination of the two. For the assay of adrenaline, Chang's method (5) was used with some modifications. Anton and Sayre (8) recommended addition of sodium metabisulfite to samples as a pre servative. Acidified butanol, instead of 0.4 N perchloric acid, was used as extraction solvent but it made the recovery of adrenaline poor when sodium metabisulfite was present in sample plasma. The addition of sodium metabisulfite to the acidified butanol extract, however, gave a better result. There was still a slight loss of adrenaline during long stor age, making it desirable to assay the adrenaline as soon as possible. The step of washing the alumina with distilled water was added in order to remove any residual sodium metabi sulfite. One of the factors affecting the adrenaline release from adrenal glands is blood pres sure (9). As quite an adequate volume of blood is required for adrenaline determination, big rats were used in our experiments. It is advisable that blood withdrawn be compen sated by transfusion of fluids. Heparinized fresh rat blood was compared with Ringer's solution containing 0.1 % of glucose in regard to adrenaline secretion and little difference in the action was found between the two transfusions. Ringer's solution was used through out, since there is the possibility that blood transfusion will cause a synthesis of histamine or bradykinin which will stimulate adrenaline secretion from the glands. Udenfriend et al. (1) observed that the basal rate of adrenaline secretion from rat adrenal glands was 0.002 to 0.004 1zg/kg/min. In our experiments, adrenaline output under pentobarbital anesthesia and that in spinal-sectioned rats in non-anesthetic condi tions approximated these values. Satake et al. (10) measured the basal secretion of cate cholamines in non-anesthetized posterior rhizotomized dogs. Houssay and Rapela (11) also reported these values in dogs under pentobarbital anesthesia. The secretion rate in the dog is about 10 times that in the rat, while the basal secretion rate of catecholamines from cat adrenal glands (12, 13) is greater than that in the dog. Adrenaline secretion rate of rats under ether anesthesia was about 10 times that under pentobarbital anesthesia. The rate was decreased to almost zero by splanchnicotomy or spinal-transection and it is concluded that the action site of ether in the rat is the central nervous system. Nicotine causes hypersecretion of adrenaline (14), which is mainly due to direct action on medullary cells (15, 16). De Schaepdryver (17) reported that 0.1 mg/kg of nicotine induced a substantial increase of catecholamine secretion from the dog adrenal up to nearly 100 times the basal value, while higher doses provoked a less marked increase under chlo ralosane anesthesia. In the experiments herein, nicotine caused little increase of adre naline secretion at 0.125 mg/kg and only about 13 to 28 times the basal rate at 0.5 mg/kg under non-anesthesia or pentobarbital anesthesia. These findings suggest that the re sponsiveness of the rat adrenal medulla to nicotine is weaker than that of the dog. Barbiturates have a curariform peripheral action and reduce the response of skeletal muscle to nerve stimulation. Neuromuscular blockade by ether anesthesia is stronger than that by barbiturates (18). In this experiment ether anesthesia prevented adrenaline secretion from splanchnicotomized adrenal in response to nicotine. The inhibitory effect of pentobarbital anesthesia was weaker than that of ether. These results suggest that inhibition of the nicotine induced adrenaline secretion by anesthetics is in proportion to the degree of neuromuscular blockade. It is reported that the hypersecretion of adrenaline caused by morphine is markedly diminished by splanchnieotomy in dogs (19). Recently, Kayaalp and Kaymakcalan (20) reported that the initial site of action in dogs is the spinal cord. In our experiment, marked adrenaline secretion was observed after i.p. injection of morphine and the action was pre vented by splanchnicotomy. This indicates that the site of action of morphine in the rat also is mainly the central nervous system. It is concluded that the response of the rat adrenal medulla to the stimulation of splan chnic nerve is strong despite of the weak direct action of drugs on the glands. Acknowledgement: The author thanks Dr. Y. Ogawa of the Shionogi Research La boratory for many helpful discussions and suggestions.