Adrenal Epinephrine and Norepinephrine Content Following Denervation and Barbiturates Tissue Concentrations

ADRENAL EPINEPHRINE AND NOREPINEPHRINE CONTENT FOLLOWING DENERVATION AND BARBITURATES

DANIEL M. SILVER, A.B., JOHN F. SEATON, B.S., AND TIMOTHY S. HARRISON, M.D.

TISSUE CONCENTRATIONS of norepi- Another important consideration in the ex- nephrine in heart, striated muscle, kidney, and perimental approaches to adrenal medullary spleen [ 11,23,24] are known to depend on an physiology is the widely held conviction that intact postganglionic autonomic innervation. barbiturate anesthesia will acutely stimulate This is not true for the preganglionic sym- the massive release of catecholamines and pathetic fibers innervating these organs, and that immediate suppression of adrenal cate- division of the preganglionic neurons to these cholamine concentrations results. This was same tissues is not followed by a sustained first suggested by Lund in 1951 [20] on the change in their norepinephrine content [ll, basis of adrenal catecholamine levels of dogs 23, 241. killed by shooting. Because we cannot rec- Because the innervation of the adrenal oncile Lund’s findings with reports of barbi- medulla is by preganglionic fibers, and be- turate suppression of adrenal catecholamine cause of the occurrence in the adrenal medulla secretion rates [ 16, 261, we have examined of chromaffin cells rather than conventional this point in conscious dogs killed by rapid postganglionic neurons, it was of interest to exsanguination. determine if division of the preganglionic innervation of the adrenal gland resulted in any change in adrenal catecholamines. The METHODS unique efficiency of the adrenal medulla in completing the biosynthesis of epinephrine Two groups of 10 healthy adult mongrel from norepinephrine [I, 12, 171 made an dogs, weighing from 10 to 15 kg., and a third examination of the adrenal concentration of group of 5 dogs were used. In the first group epinephrine under these circumstances of the animals were given a large dose of barbi- particular interest, turate intravenously; the adrenals were immediately harvested, weighed, and homog- From the Department of Surgery, University of enized; and two extractions with 50 ml. of Michigan Medical School, .4nn Arbor, Mich. 10% trichloracetic acid were immediately This study was supported by Grant AM-07252 of the National Institutes of Health, and by a Michigan carried out. The extracts were neutralized; Heart Association Student Fellowship to Mr. Silver. the catecholamines were adsorbed onto Valuable technical assistance was provided by Mrs. alumina, and after elution with two 5-ml. Gretchen Crowley. Submitted for publication Apr. 24, 1967. portions of 0.2 N acetic acid were analyzed 177 JOURNAL OF SURGICAL RESEARCH VOL. 8 NO. 4, APRIL 1968 fluorometrically for epinephrine and norepi- medulla from surgery; moreover, significant nephrine following oxidation to their trihy- regeneration of the divided splanchnic nerve droxyindole forms by the method of Lund would not be expected at three weeks. [ 191, as modified by Crout [5] and von Euler To determine any influences of the barbi- and Lishajko [lo]. A Farrand photofluorom- turate anesthesia used in the previous experi- eter was used to measure fluorescence of ments a series of trained dogs were placed the samples, and two filter systems of fixed on a table and the right femoral artery ex- wavelengths were used for the differential posed after 1% local lidocaine (Xylocaine) estimation of epinephrine and norepinephrine. infiltration anesthesia. A large Teflon catheter Recovery of epinephrine and norepinephrine was passed into the artery and the animals added to the acid homogenate varied between bled rapidly and maximally into a flask at less 83% and 100%. No correction was made for than 1 atm. of pressure. The bleeding times the incomplete recovery. We have previously were 5 minutes or less. The adrenal glands used this technique for catecholamine deter- were immediately harvested and analyzed minations of monkey tissues and found it to for epinephrine and norepinephrine in a man- be suitably sensitive and reproducible [ 131. ner identical to that described above. In the second group the dogs were anes- thetized with 20 mg. per kilogram of intra- venous pentobarbital and the right adrenal RESULTS gland was surgically removed through a transverse upper abdominal incision. The innervation of the left adrenal gland was then The mean weights of the adrenal glands exposed and meticulously divided at a point studied were similar in all groups. As seen in midway between the diaphragm and the Table 1, the mean concentrations of epi- left adrenal gland in the manner described nephrine and norepinephrine from the right by Vogt [WI. Reflex excitation of the adrenal and left adrenal glands of the control group release of epinephrine and norepinephrine is of animals are comparable. The mean values completely destroyed by this technique and from the chronically denervated glands and on this basis we feel confident that virtually the corresponding controls are shown in complete adrenal denervation has been Table 1. The epinephrine and norepinephrine achieved in this study [ 141. The abdomen content of these two groups is comparable to was closed and the dog allowed to recover. the control values. The mean epinephrine Three weeks later the denervated left adrenal and norepinephrine contents seen in the was removed and the dog sacrificed by the surgically removed adrenals (Table 1) do same technique used in the control animals. not vary significantly from the controls. Three weeks was the period employed by In the ten adrenal glands removed after Vogt [25] in her studies on the denervated bleeding without barbiturate anesthesia both adrenal gland, and it is thought that this is epinephrine and norepinephrine levels are ample time for recovery of the adrenal comparable to those seen in the other groups.

Table 1. Mean Catecholamine Content of Right and Left Adrenal Glands in 10 Control Animals, 10 Surgically Excised Glands, 10 Chronically Denervated Glands, and 10 Glands After Bleeding (pg./gm. wet tissue, * 1 S.D.)

Right Left Surgically Chronically Exsanguination Control Control Removed Denervated Local Anesthesia

Norepinephrine 161? 65 166 +- 68 146 k 58 147 2 51 174 2 33 Epinephrine 752 t 173 819 ? 209 649 2 204 864 c 236 715 k 234 Group comparisons between each control group and the other three groupsreveal no significantdifferences.

178 SILVER ET AL.: ADRENAL EPINEPHRINE AND NOREPINEPHRINE

DISCUSSION stimulus of potassium salts. Any decrease in the adrenal medullary secretion of epineph- Up to this time there has been no separate rine and norepinephrine by the denervated measurement of the levels of both epinephrine adrenal gland such as that shown by Vogt and norepinephrine in the denervated adrenal [25] and Duner [6] cannot, therefore, be at- gland. This investigation has been conducted tributed to decreased tissue levels of these to furnish such data in order to interpret catecholamines. Restoration of epinephrine accurately the significance of decreased se- levels in the adrenal gland following reserpine cretory rates of catecholamines from the administration is not retarded in the de- denervated adrenal gland. We believe these nervated adrenal gland [3]. It is, therefore, data will be of eventual importance in help- probable that the biosynthesis of epinephrine ing to establish whether or not so-called by the chromaffin cells occurs after pregan- “direct” influences, acting independently from glionic nerve section. Our results are in agree- the nervous system, are important in the ment with this idea and with unpublished physiological secretion of the adrenal me- observations alluded to by von Euler [9]. dulla. Although one might deduce from ob- Of importance to the interpretation of adre- servations of Mirkin [21] and Callingham nal catecholamine levels in glands obtained and Mann [3], and indirectly from those of under anesthesia is the question of what Cammanni et al. [4], that the denervated influence barbiturates or other general anes- adrenal glands content of epinephrine and thetic agents might have on adrenal cate- norepinephrine is unchanged, separate mea- cholamine content. Old observations of surements of the adrenal levels of both of Elliott [7] indicate that ether anesthesia will these catecholamines after denervation have deplete the innervated adrenal medulla of not been recorded. It is well known from the epinephrine. More recently Elmes and Jeffer- observations of Bennett [2] and Lever [18] son [8] have shown that pentobarbital has that terminal preganglionic dendritic arbori- only a slight effect on adrenal catecholamines, zations are intimately applied to the individual and Price et al. [22] failed to demonstrate chromaffin cells of the adrenal medulla. With any secretory burst of catecholamines into the this in mind it has seemed of particular in- arterial plasma in human subjects given terest to determine if loss of neural activity barbiturate anesthesia. In spite of these ob- in these fibers will result in a decrease in the servations, well-known findings of Lund have concentration of epinephrine and norepineph- been cited frequently as evidence that barbi- rine in the chromaffin cells. It is probable turates acutely stimulate massive release of that the rate of biosynthesis of catecholamines epinephrine and norepinephrine and have by the adrenal medulla is increased by stimu- led to repeated criticism of tissue catechol- lation of the adrenal innervation, although amine levels obtained under anesthesia [20]. evidence used to support this contention is We are unable to confirm Lund’s results in somewhat crude [ 151. Whether or not pro- conscious dogs in the animals of this study longed absence of nervous stimuli to the which were sacrificed by sudden exsanguina- adrenal gland will result in a decreased adrenal tion. The adrenal secretory rates of catechol- catecholamine level does not appear to have amines in response to hemorrhage in conscious been examined rigorously. animals are not great enough [lS, 271 to ac- We feel that our observations demonstrate count for a depression of the magnitude sug- that the denervated adrenal gland will main- gested by Lund [20], and, furthermore, study tain physiological tissue levels of epinephrine of dogs under barbiturate anesthesia has and norepinephrine. This might also be in- demonstrated only suppression of catechol- ferred from old observations of Vogt’s [25], amine secretion in response to barbiturates in which chronically denervated cat adrenal [ 16, 261. The results reported in this study glands were able to release large quantities of indicate that there is no suppression of adre- catecholamines in response to the glandular nal epinephrine and norepinephrine con-

179 JOURNAL OF SURGICAL RESEARCH VOL. 8 NO. 4, APRIL 1968 tent in response to light barbiturate anesthesia suprarenal. Acta Physiol. Stand. 28 (Suppl. and that levels obtained under these condi- 102):3, 1953. tions accurately represent catecholamine 7. Elliott, T. R. The control of the suprarenal glands by the splanchnic nerves. J. Physiol. levels of the conscious animal. In all other (London) 44:374, 1912. respects the levels of adrenal catecholamines 8. Elmes, P. C., and Jefferson, A. A. The effect which we report in this study are comparable of anesthesia on the adrenaline content of the to those of the dogs studied by Lund [20]. suprarenal glands. J. Physiol. (London) 101:355, 1942. 9. Euler, U. S., von. Noradrenaline: Chemistry, physiology, pharmacology, and clinical aspects. SUMMARY Springfield, Ill.: Thomas, 1956. P. 125. 10. Euler, U. S., von, and Lishajko, F. Improved The concentration of epinephrine and nor- technique for the fluorometric estimation of catechol amines. Acta Physiol. Stand. 51:348, epinephrine in the adrenal gland of the dog is 1961. comparable in the left and right glands. De- 11. Goodall, M., and Kirshner, N. Effect of nervation of the left adrenal gland does not cervicothoracic ganglionectomy on adrenaline decrease the levels of these adrenal catechola- and noradrenaline content in mammalian heart. mines three weeks after denervation. These J. Clin. Invest. 35:649, 1956. 12. Goodall, M. C., and Kirshner, N. Biosynthesis findings are in keeping with the concept that of epinephrine and norepinephrine by sym- catecholamine biosynthesis by the adrenal pathetic nerves and ganglia. Cimztlation 17:366, gland is not dependent on adrenal innervation. 1958. Other evidence supporting this concept is 13. Harrison, T. S., and Seaton, J. F. Tissue con- reviewed. tent of epinephrine and norepinephrine following adrenal medullectomy. Amer. J. Physiol. In contrast to prevailing opinion we have 210:599, 1966. found that the catecholamine content of 14. Harrison, T. S., Seaton, J. F., and Bartlett, J. D., adrenal glands obtained from conscious dogs Jr. Adrenergic mechanisms in acute hypovo- is comparable to that in animals with general lemia. Surg. Forum 17366, 1966. barbiturate anesthesia. 15. Holland, W. C., and Schiimann, H. J. Forma- tion of catechol amines during splanchnic stimulation of the adrenal gland of the cat. Brit. J. Pharmacol. 11:449, 1956. REFERENCES 16. Hume, D. Some neurohumoral and endocrine aspects of shock. Fed. PTOC. 20 ( Suppl. 9) :87, 1. Axelrod, J. Purification and properties of 1961. phenylethanolamine-N-methyl transferase. J. 17. Kirshner, N., and Goodall, M. C. The forma- Biol. Chem. 237:1657, 1962. tion of adrenaline from noradrenaline. Biochim. 2. Bennett, N. S. Cytological manifestations in Biophys. Acta 24~658, 1957. the adrenal medulla of the cat. Amer. J. Anat. 18. Lever, J. D. Electron microscopic observations 69:333, 1941. on the normal and denervated adrenal medulla 3. Callingham, B. A., and Mann, M. Replace- of the rat. Endocrinology 57:621, 1955. ment of adrenaline and noradrenaline in the in- 19. Lund, A. Fluorometric determination of adren- nervated and denervated adrenal gland of the aline in blood. Acta Pharmacol. (Kobenhavn) rat, following depletion with reserpine. Natwe 5:231, 1949. (London) 182: 1020, 1958. 20. Lund, A. Release of adrenaline and noradrena- 4. Cammanni, F., Losana, O., Molinatti, G. M., line from the suprarenal gland. Acta PhaTmacol. and Olivetti, M. Effect of reserpine on the (Kobenhavn) 7:309, 1951. catecholamine content of the adrenal medulla 21. Mirkin, B. L. Catecholamine depletion in the of rats after denervation or administration of rat’s denervated adrenal gland following chronic iproniazid. Axh. Int. Pharmacodyn. 123:430, administration of reserpine. Nature (London) 1960. 182:113, 1958. 5. Crout, J. R. “Catechol Amines,” Standard 22. Price, H. L., Linde, H. W., Jones, R. E., Black, Methods of the American Association of Clinical G. W., and Price, M. L. Sympatho-adrenal Chemistry. Vol. III. New York: Academic, 1962. responses to general anesthesia in man and their P. 62. relation to hemodynamics. Anesthesiology 20: 6. Duner, H. Influence of blood glucose level on 563, 1959. secretion of adrenaline and noradrenaline from 23. Rehn, N. Effect of decentralization on the

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content of catechol amines in the spleen and 26. Walker, W. F., Zileli, M. S., Reutter, F. W., kidney of the cat. Acta Physiol. Scud 42:309, Shoemaker, W. C., and Moore, F. D. Factors 1958. influencing the “resting” secretion of the adrenal 24. Sedvall, G. Noradrenaline storage in skeletal medulla. Amer. J. Physiol. 197:765, 1959. muscle. Acta Physiol. Scud 60:39, 1964. 27. Walker, W. F., Zileli, M. S., Reutter, F. W., 25. Vogt, M. Secretion of the denervated adrenal Shoemaker, W. C., Friend, D., and Moore, F. D. medulla of the cat. Brit. J. Pharmacol. 7:325, Adrenal medullary secretion in hemorrhagic 1952. shock. Amer. J. Physiol. 197:773, 1959.

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