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The Uptake and Release of Catecholamines and the Effect of Drugs JULIUS AXELROD Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Maryland (U.S.A.)
During the past few years our laboratory has been engaged in studies on the metabolism, uptake and release of noradrenaline and adrenaline and the effect of drugs on these processes. In these investigations, we used radioactive catecholamines of high specific activity. The availability of these radioactive compounds not only made it possible to work with physiological amounts of the hormones but also enabled us to isolate and characterize several major and minor metabolic products and investigate in a more precise manner the fate of circulating and bound catecholamines in the body. The following is an account of some of this work.
UPTAKE O F CIRCULATJNG CATECHOLAMINES
Cats were given 3H-noradrenaline or 3H-adrenaline intravenously and the uptake of these amines was measured immediately after injection and for various time intervals thereafter (Axelrod et al., 1959; Whitby et a/., 1961). Within 2 min after their administration, both amines were taken up in unequal amounts by various tissues. The greatest quantities of the radioactive catecholamines were found in the heart, spleen and glandular tissues; skeletal muscle took up the least. High levels of catecholamines in the heart, spleen and adrenal glands were maintained for many hours which indicated that these hormones can be held in tissue in a physiologically inactive form for long periods of time until they are released. The large amounts of circulating catecholamines bound in the heart suggest that the adrenal gland could supply the heart with adrenaline and noradrenaline. Binding serves to protect the catecholamines from attack by enzymes until they are released. The uptake and retention of circulating noradrenaline and adrenaline by tissues may be an important mechanism for the physiological inactivation of these hormones. Within minutes after the administration of radioactive noradrenaline or adrenaline, large amounts of 3H-normetanephrine or 3H-metanephrine, the 0-methylated metabolites, were found in most tissues. About 90 % of the injected radioactivity was accounted for as unchanged catecholamines or their corresponding 0-methylated metabolites. These findings indicate that O-methReferences p . 87-89
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ylation plays an important role in terminating the action of the circulating hormones. After the intravenous administration of radioactive adrenaline and noradrenaline, only small quantities of these compounds were present in the brain (Weil-Malherbe et al., 1959; Weil-Malherbe et al., 1961). The hypothalamus took up a small amount of the catecholamines whle the pituitary gland and the pineal body contained larger quantities presumably because there is no blood-brain barrier in these structures. The brain contains relatively large amounts of endogenous noradrenaline (Vogt, 1954). Catecholamines in the brain are probably formed from precursors that are capable of crossing the blood-brain barrier such as dopa. Using a broken cell preparation, monoamine oxidase activity was shown to be higher in the rat brain than catechol-0-methyltransferase (Crout et al., 1961). As a result of these observations, it was postulated that monoamine oxidase was mainly involved in the inactivation of noradrenaline in the brain. After the administration of pyrogallol, a catechol-0methyltransferase inhibitor (Axelrod and Laroche, 1959), into the lateral ventricle of the rabbit, there was a threefold elevation in the endogenous noradrenaline in the brain (Masami et al., 1962). The administration of 14C-noradrenaline into the lateral ventricle of cats resulted in the formation of normetanephrine and other 0-methylated products as major metabolites (Mannarino et a/., 1962). From these observations it would appear that 0-methylation is a n important enzymatic process in the metabolism of noradrenaline in nervous tissue. It still remains to be established whether binding, monoamine oxidase or catechol-0-methyltransferase or a combination of these mechanisms are involved in the initial inactivation of noradrenaline in the brain. The isolated-perfused rat heart also can take up and retain SH-noradrenaline (Axelrod et al., 1962a). The bound catecholamine is then released in a multiphasic fashion which indicates that there are several types of binding (Kopin et al., 1962). In rat hearts, noradrenaline was found to be inactivated principally by binding. The released noradrenaline in the isolated-perfused heart is metabolized by 0-methylation and deamination. The changes in the released metabolites with time suggest that 0methylation is the main pathway for the metabolism of the easily releasable noradrenaline and deamination is the main pathway for the tightly bound catecholamines. Although noradrenaline and adrenaline are taken up, stored, and metabolized in a similar manner, there are quantitative differences in their disposition (Whitby et a]., 1961). More 3H-noradrenaline was taken up in tissues and held for longer periods of time. There were more 0-methylated metabolites found after adrenaline than after noradrenaline. These findings indicate that binding is quantitatively a more important mechanism for the inactivation of noradrenaline while enzymatic 0methylation is more important for adrenaline. The higher levels of circulating endogenous noradrenaline might be explained in terms of the differences in the degree of binding of these hormones rather than in terms of a greater release of noradrenaline into the circulation. The subcellular localization of catecholamines was studied in heart, salivary and adrenal glands (Potter and Axelrod, 1962). About 80% of the circulating 3H-noradrenaline and 3H-adrenaline was taken up by a particulate fraction associated with
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the microsomes of the heart and salivary gland. Endogenous catecholamines were also found in this fraction of the cell. 3H-Dopamine, 3H-dopa, and 3H-normetanephrine, on the other hand, were present only in the cell sap. In the adrenal medulla, 3H-noradrenaline was found in the microsomal fraction while 3H-adrenaline, 3Hdopamine, and endogenous catecholamines were localized in the chromafin granules. 3H-Dopa was present in the supernatant fraction of the adrenal medulla and in the ‘pinched-off nerve endings’ of the brain described by Whittaker (1959). Using brain slices, 3H-noradrenaline was also found to be taken up in this layer. It was demonstrated that circulating noradrenaline and adrenaline were taken up by sympathetic nerve endings (Hertting et a/., 1961a). When sympathetic nerves were destroyed, they were unable to retain the catecholamines. Supersensitivity to noradrenaline results partly because the sympathetic nerves cannot inactivate the catecholamines by binding. The uptake of radioactive noradrenaline by sympathetic nerves made it possible to visualize sympathetic nerves by radioautographic techniques (Wolfe et a/., 1962). Electron microscopy showed a striking localization of radioactive grains only within sympathetic nerves associated with dense core vesicles 40 to 50 mp thick. These experiments provide conclusive evidence that noradrenaline can be taken up from the circulation into sympathetic nerves and stored there until it is released. RELEASE OF N O R A D R E N A L I N E A N D A D R E N A L I N E
After the administration of noradrenaline and adrenaline, these catecholamines disappeared from the whole animal in two phases (Axelrod et al., 1959; Whitby et al., 1961). In the first few minutes, there was a rapid decline followed by a slow disappearance of the catecholamines. The initial phase is due to enzymatic destruction of the catecholamines and their uptake and binding by tissues; the second to slow release and metabolism. In the first few minutes, about two-thirds of the adrenaline and one-half of the noradrenaline were metabolized. Almost all of the catecholamines that disappeared in the first few minutes could be accounted for as 0-methylated metabolites, normetanephrine and metanephrine. There was a slower decline of noradrenaline as compared with adrenaline during the second phase which indicated that noradrenaline is more tightly bound and more slowly released. Bound noradrenaline was released from the heart over a period of days and the half-life of noradrenaline became progressively longer (Axelrod et al., 1961a). These observations indicated that noradrenaline became more tightly bound with time. Tyramine has been shown to act by releasing noradrenaline (Burn and Rand, 1958). Using tyramine as a tool, it was demonstrated that noradrenaline is bound with different degrees of tenacity (Potter et al., 1962). After the first injection, tyramine released about 30% of both endogenous and 3H-noradrenaline. With repeated injections of tyramine, the amount of endogenous and exogenous noradrenaline liberated from the heart became less and less. There was no further release of noradrenaline and no blood pressure elevation after the third or fourth injection of tyramine. Yet considerable quantities of the catecholamines were still present in the heart. It was apparent from these Rejerentes p . 87-89
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experiments that noradrenaline is bound in two pools, one which is easily released and another which is more firmly held. The 3H-noradrenaline recently taken up by the heart can be released by tyramine more easily. With the passage of time, tyramhe releases less and less 3H-noradrenaline. The longer the noradrenaline remained in the heart the more firmly bound it became. This is due to the different rates in which the pools were turning over. The more easily releasable pool was found to have a half-life of several hours and the more tightly bound pool has a half-life of about one day. In contrast to the finding with tyramine, reserpine releases noradrenaline from both stores equally well (Potter and Axelrod, 1963). It has been shown that the noradrenaline released by reserpine is metabolized by inonoamine oxidase while the noradrenaline released by tyramine is metabolized by catechol-0-methyltransferase (Kopin et al., 1962). Although reserpine releases large amounts of the catecholamine, it produces only a slight physiological effect while tyramine releases much less noradrenaline and produces a marked physiological response. The noradrenaline released by reserpine appears to be metabolized by monoamine oxidase within the nerve and it leaves the nerve as an inactive deaminated metabolite. Tyramine discharges noradrenaline in a physiologically active form and once it is liberated the neurohumor is inactivated by 0-methylation. It has been demonstrated that inhibition of monoamine oxidase does not prolong the physiological actions of noradrenaline (Griesemer et a]., 1953). Consequently, this enzyme appears to act within the sympathetic nerves, and metabolizes the neurohumor before it becomes physiologically active. Once noradrenaline leaves the nerve, monoamine oxidase plays a negligible role in the subsequent fate of the catecholamine. Because sympathetic nerves are capable of taking up and retaining radioactive noradrenaline it was possible to introduce 3H-noradrenaline into these nerves and study its fate when the neurohumor is discharged (Hertting and Axelrod, 1961). Cats were given 3H-noradrenaline and the spleen which contained large amounts of the radioactive catecholamine was isolated and perfused with blood free of radioactive material. After stimulation of the splenic nerve, 3H-noradrenaline and its metabolites were measured in the venous outflow. There was a decided increase in the 3H-noradrenalinc in the venous outflow after each stimulation. There was also a smaller increase in thc 3H-normetanephrine which was directly related to the 3H-noradrenaline liberated. These results again demonstrated that noradrenaline can be taken up from the circulation by sympathetic nerves and on stimulation, discharged. When liberated, the noradrenaline interacts with the receptor and part is released into the blood stream, part 0-methylated by catechol-0-methyltransferase, and part returns to the sympathetic nerves to be bound and used again. EFFECT OF D R U G S O N THE U P T A K E , RELEASE A N D METABOLISM OF N O R A D R E N A L I N E
Drugs affecting behavior also alter the uptake, release and metabolism of catecholamines. Antidepressant drugs inhibit monoamine oxidase and raise the concentration of catecholamines in the heart and brain of certain species (Shore et al., 1957);
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reserpine depletes catecholamines from tissues (Holzbauer and Vogt, 1956). In our laboratory, we have used radioactive catecholamines to study the effect of drugs on uptake, release and metabolism of these hormones. Reserpine and chlorpromazine have been shown to increase the rate of destruction of circulating catecholamines (Axelrod and Tomchick, 1959). These drugs prevented the uptake of the catecholamines, thus interfering with the protective binding. The hormones were then exposed to enzymatic attack and more rapid metabolism. As pointed out above, reserpine also releases the bound noradrenaline within the nerve where it is deaminated by monoamine oxidase. Chlorpromazine blocked the uptake of noradrenaline by the nerve (Axelrod et al., 1961b). Once the neurohumor was bound within the nerve, this drug could not release it (Axelrod et al., 1962b). Chlorpromazine is a drug of many actions: It is an antihistamine, tranquilizer, and an adrenergic blocking agent. The blocking of the uptake of noradrenaline was found to reside only in the antiadrenergic activity of chlorpromazine (Rose11 and Axelrod, 1963). Other antiadrenergic drugs such as Dibenzyline and dichlorisoproterenol affect the uptake and release of noradrenaline (Axelrod et al., 1962b). Like chlorpromazine, the antidepressant drug imipramine increases the metabolism of catecholamines and blocks the uptake of these hormones. Many antidepressant drugs also inhibit monoamine oxidase and elevate the concentration of catecholamines in the brain, heart, and other tissues (Shore et al., 1957). These enzyme inhibitors, however, do not prolong the physiological response to catecholamines and appear to play a minor role in the inactivation of circulating catecholamines. As pointed out above, monoamine oxidase operates mainly within the nerves, inactivating the noradrenaline before it leaves the nerve (Kopin et al., 1962). In the presence of monoamine oxidase inhibitors the released noradrenaline returns to the storage vesicle or escapes into the circulation. Monoamine oxidase inhibitors also blocked the spontaneous release of noradrenaline from stores (Axelrod et al., 1961a), and this also serves to elevate the endogenous catecholamine levels in tissues. Bretylium and ganglionic blocking agents prevent the spontaneous release of 3H-noradrenaline (Hertting et al., 1962a, b). Although these compounds do not inhibit monoamine oxidase, they increase the endogenous catecholamine levels (Hertting et al., 1962b; Ryd, 1962). The releasing action of guanethidine and reserpine was found to be blocked by monoamine oxidase inhibitors, bretylium and ganglionic blocking agents. When noradrenaline is liberated from the easily releasable pool by sympathomimetic amines neither monoamine oxidase inhibitors nor bretylium blocked its release (Potter and Axelrod, 1963). Monoamine oxidase inhibitors, bretylium, guanethidine and reserpine are hypotensive drugs which affect the uptake, release and metabolism of catecholamines. The actions of reserpine and monoamine oxidase inhibitors on noradrenaline have been described above. Bretylium and guanethidine have many actions in common and also differ in their effects on circulating and bound noradrenaline (Hertting et al., 1962a). Both compounds when injected produced a brief rise in blood pressure as well as an immediate release of noradrenaline. These drugs also inhibited the uptake of noradrenaline and potentiated the effects of the hormone. Bretylium and guanethiReferences p . 87-89
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dine blocked the liberation of noradrenaline as well as the physiological effects when sympathetic nerves were stimulated. When the noradrenaline was bound in tissues, bretylium prevented its spontaneous release while guanethidine caused a slow but continuous release of the neurohumor. Many drugs such as cocaine, guanethidine, bretylium, imipramine, chlorpromazine, and Dibenzyline cause supersensitivity to catecholamines. All of these drugs prevented the uptake of circulating catecholamines by tissues (Whitby et al., 1960; Axelrod et al., 1961b; Hertting et al., 1961b). The concentration of catecholamines in the blood was elevated when the animals were pretreated with these drugs. Supersensitivity resulted because these drugs prevented inactivation of the hormone by binding and a higher concentration of the free and active catecholamines was present at the receptor site. Chronic denervation of sympathetically innervated organs also caused supersensitivity to catecholamines. When the nerves degenerate, the vesicles which bind and inactivate noradrenaline are destroyed. Consequently, the amount of unbound and active catecholamines in the vicinity of the receptors in denervated tissues would be expected to persist for longer periods, thus resulting in supersensitivity. Sympathomimetic amines not only liberated catecholamines (Burn and Rand, 1958) but also prevented their uptake and increased the rate of metabolism (Axelrod and Tomchick, 1960; Hertting et al., 1961b). After repeated administration of tyramine
Fig. I . The fate of noradrenaline at the sympathetic nerve endings and the effect of drugs. Bret bretylium; GBO ganglionic blockers; MAOJ = monoamine oxidase inhibitors; COMT -- catechol-0-meth yltransferase.
and other sympathomimetic amines, there was successive decrease in the amount of noradrenaline released from the heart and a concomitant decrease in physiological response (Potter et al., 1962). When there was no further liberation of noradrenaline
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by tyramine, tachyphylaxis (loss of responsiveness) resulted. At this time, there is a considerable amount of noradrenaline still present in the heart. The action of sympathomimetic amines could be restored after an infusion of noradrenaline (Cowan et af., 1961). Tachyphylaxis appears to be a result of the depletion of the bound catecholamines easily available for release. Fig. 1 shows a working model of the sympathetic nerve which is consistent with its morphological appearance and would explain differences in storage, metabolism and the action of drugs on noradrenaline. The majority of the storage vesicles are deep within the nerves. Reserpine and guanethidine release noradrenaline from the deep vesicles. The catecholamines are deaminated by monoamine oxidase in the mitochondria and leave the nerve as inactive metabolites. Bretylium appears to block the releasing action of these drugs from the storage vesicles and monoamine oxidase inhibitors prevent the metabolism of noradrenaline liberated by reserpine and guanethidine. A smaller number of vesicles may be close to the synaptic terminals where sympathomimetic amines or nerve impulses release noradrenaline from the nerve cell. Once released, the noradrenaline reacts with the receptor and is metabolized by catechol-0-methyltransferase, discharged into the circulation, or returns to the storage vesicle. Bretylium and guanethidine block the release of noradrenaline by nerve impulses. Noradrenaline is also released from the stores spontaneously and this release is blocked by monoamine oxidase inhibitors, bretylium, and ganglionic blocking agents. The storage vesicles can also take up noradrenaline from the circulation. Cocaine, chlorpromazine, imipramine, bretylium, guanethidine, and reserpine block this uptake thus causing supersensitivity. SUMMARY
Circulating noradrenaline and adrenaline are mainly inactivated by catechol-0methyltransferase or they are taken up and bound in dense core vesicles in sympathetic nerves. When the catecholamines are released they are metabolized by monoamine oxidase within the nerve or by catechol-0-methyltransferase outside the nerve. A part of the catecholamines are also inactivated by being bound again or by diffusing into the circulation. Noradrenaline is stored in an easily releasable or firmly bound form. Many drugs such as cocaine, chlorpromazine, imipramine, reserpine, guanethidine, bretylium and sympathomimetic amines interfere with the uptake, storage and release of catecholamines. REFERENCES AXELROD, J., GORDON, E., HERTTING, G., KOPIN,I. J., AND POTTER, L. T., (1962a); On the mechanism of tachyphylaxis to tyramine in the isolated rat heart. Brit. J. Pharmacol., 19, 56-63. AXELROD, J., HERTTING, G., AND PATRICK,R. W., (1961a); Inhibition of H3-norepinephrine release by monoamine oxidase inhibitors. J . Pharmacol. exp. Ther., 134, 325-328. AXELROD, J., HERTTING, G., AND POTTER,L. T., (1962b); Effect of drugs on the uptake and release of 3H-norepinephrine in the rat heart. Nature (Loncl.), 194, 297.
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AXELROD, J., AND LAROCHE, M . J., (1959); Inhibitor of 0-methylation of epinephrine and norepinephrine in vitro and in vivo. Science, 130, 800. AXELROD, J., AND TOMCHICK, R., (1959); Activation and inhibition of adrenaline metabolism. Nature (Lond.), 184, 2027. AXELROD, J., AND TOMCHICK, R., (1960); lncreased rate of metabolism of epinephrine and norepinephrine by sympathomimetic amines. J . Pharmacol. exp. Ther., 130,367-369. H., AND TOMCHICK, R., (1959); The physiological disposition of AXELROD, J., WEIL-MALHERBE, H3-epinephrine and its metabolite metanephrine. J . Pharmacol. exp. Ther., 127, 251-256. AXELROD, J., WHITEY, L. G., AND HERTTING, G., (1961b); Effect of psychotropic drugs on the uptake of H3-norepinephrine by tissues. Science, 133, 383-384. BURN,J. H., AND RAND,M. J., (1958); The action of sympathomimetic amines in animals treated with reserpine, J . Physiol. (Lond.), 144, 314-336. COWAN,F. F., CANNON, C., KOPPANYI, T., AND MAENGWYN-DAVIES, G. D., (1961); Reversal of phenylalkylamine tachyphylaxis by norepinephrine. Science, 134, 1069-1070. CROUT,J. R., CREVELING, C. R., A N D UDENFRIEND, S., (1961); Norepinephrine metabolism in rat brain and heart. J . Pharmacol. exp. Ther., 132, 269-271. GRIESEMER, E. C., BARSKY,J., DRAGSTEDT, C. A., WELLS, J. A., A N D ~ E L L E R E.A.,(1953); , Potentiating effect of iproniazid on the pharmacological action of sympathomimetic amines. Proc. Soc. exp. Biol. ( N . Y . ) , 84, 699-701. HERTTINC, G., AND AXELROD, J., (1961); Fate of tritiated noradrenaline at the sympathetic nerve endings. Nature (Lond.), 192, 172-173. HERTTING, G., AXELROD, J., KOPIN,1. J., AND WHITBY, L. G., (1961a); Lack of uptake of catecholamines after chronic denervation of sympathetic nerves. Nature (Lond.), 189, 66. HERTTING, G., AXELROD, J., AND PATRICK, R. W., (1962a); Actions of bretylium and guanethidine on the uptake and release of (3H)-noradrenaline. Brit. J . Pharmacol., 18, 161-166. HERTTING, G., AXELROD, J., A N D WHITBY, L. G., (1961 b); Effect of drugs on the uptake and metabolism of H%orepinephrine. J . Pharmacol. exp. Ther., 134, 146-1 53. HERTTING, G., POTTER, L. T., AND AXELROD, J., (1962b); Effect of decentralization and ganglionic blocking agents on the spontaneous release of H3-norepinephrine. J . Pharmacol. exp. Ther., 136, 289-292. HOLZBAUER, M., AND VOGT,M . , (1956); Depression by reserpine of the noradrenaline concentration in the hypothalamus of the cat. J . Neurochetn., 1, 8-1 1. KOPIN,I. J . , HERTTING, G., AND GORDON, E. K., (1962); Fate of norepinephrine-H3 in the isolated perfused rat heart. J . Pharmacol. exp. Ther., 138, 34-40. MANNARINO, E., KIRSHNER, N . , AND NASHOLD, JR., B. S.,(1962); Themetabolism of noradrenaline-C'd by cat brain in vivo. Fed. Proc., 21, 182. MASAMI, M., HIROSHI, Y.,ANDREIJI, I . , (1962); Effect of pyrogallol on the catecholamine content of the rabbit brain. Biochem. Pharmacol., 11, 1109-11 10. POTTER, L. T., AND AXELROD, J . , (1962); Intracehlar localization of catecholamines in tissues of the rat. Nature (Lond.), 194, 581-582. POTTER, L. T., AND AXELROD, J., (1963); Studies on the storage of norepinephrine and the effect of drugs. J . Pharmacol. exp. Ther., 140, 199-206. POTTER, L. T., AXELROD, J., AND KOPIN,I. J., (1962); Differential binding and release of norepinephrine and tachyphylaxis. Biochem. Pharmacol., 11, 254-256. ROSELL, S., A N D AXELROD, S., (1963); Relation between blockade of 3H-noradrenaline uptake and pharmacological actions produced by phenothiazine derivatives. Experientia (Easel), 19, 3 18. RYD, G., (1962); Protective effect of bretylium on noradrenaline stores in organs. Acra physiol. scand., 56, 90-93. SHORE, P. A., MEAD,J. A. R., KUNTZMAN, R. G., SPECTOR, S., AND BRODIE, B. B., (1957); On the physiologic significance of monoamine oxidase in brain. Science, 126, 1063-1064. VOGT,M., (1954); The concentration of sympathin in different parts of the central nervous system under normal conditions and after the administration of drugs. J. Physiol. (Lond.), 123, 451-481. WEIL-MALHERBE, H., AXELROD, J., AND TOMCHICK, R., (1959); Blood-brain barrier for adrenalinc. Science, 129, 1226-1227. WEIL-MALHERBE, H., WHITBY, L. G . , AND AXELROD, J., (1961); The blood-brain barrier for catecholamines in different regions of the brain. Regional Neurochemistry. S. S . Kety and J. Elkes, Editors. Oxford, Pergamon Press (p. 284-292). WHITBY, L. G., AXELROD, J., AND WEIL-MALHERBE, H., (1961); The fate of H3-norepinephrine in animals. J . Pharmacol. exp. Ther., 132, 193-201.
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WHITBY,L. G., HERTTING, G., AND AXELROD, J., (1960); Effect of cocaine on the disposition of noradrenaline labelled with tritium. Nature (Lonil.), 187, 604-605. WHITTAKER, V. P., (1959); The isolation and characterization of acetylcholine-containing particles from brain. Biochern. J., 72, 694-706. WOLFE,D. E., POTTER,L. T., RICHARDSON, K. C., A N D AXELROD, J. (1962); Localizing tritiated norepinephrine in sympathetic axons by electron microscopic autoradiography. Science, 138, 440-442.
The Uptake and Release of Catecholamines and the Effect of Drugs JULIUS AXELROD Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Maryland (U.S.A.)
During the past few years our laboratory has been engaged in studies on the metabolism, uptake and release of noradrenaline and adrenaline and the effect of drugs on these processes. In these investigations, we used radioactive catecholamines of high specific activity. The availability of these radioactive compounds not only made it possible to work with physiological amounts of the hormones but also enabled us to isolate and characterize several major and minor metabolic products and investigate in a more precise manner the fate of circulating and bound catecholamines in the body. The following is an account of some of this work.
UPTAKE O F CIRCULATJNG CATECHOLAMINES
Cats were given 3H-noradrenaline or 3H-adrenaline intravenously and the uptake of these amines was measured immediately after injection and for various time intervals thereafter (Axelrod et al., 1959; Whitby et a/., 1961). Within 2 min after their administration, both amines were taken up in unequal amounts by various tissues. The greatest quantities of the radioactive catecholamines were found in the heart, spleen and glandular tissues; skeletal muscle took up the least. High levels of catecholamines in the heart, spleen and adrenal glands were maintained for many hours which indicated that these hormones can be held in tissue in a physiologically inactive form for long periods of time until they are released. The large amounts of circulating catecholamines bound in the heart suggest that the adrenal gland could supply the heart with adrenaline and noradrenaline. Binding serves to protect the catecholamines from attack by enzymes until they are released. The uptake and retention of circulating noradrenaline and adrenaline by tissues may be an important mechanism for the physiological inactivation of these hormones. Within minutes after the administration of radioactive noradrenaline or adrenaline, large amounts of 3H-normetanephrine or 3H-metanephrine, the 0-methylated metabolites, were found in most tissues. About 90 % of the injected radioactivity was accounted for as unchanged catecholamines or their corresponding 0-methylated metabolites. These findings indicate that O-methReferences p . 87-89
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ylation plays an important role in terminating the action of the circulating hormones. After the intravenous administration of radioactive adrenaline and noradrenaline, only small quantities of these compounds were present in the brain (Weil-Malherbe et al., 1959; Weil-Malherbe et al., 1961). The hypothalamus took up a small amount of the catecholamines whle the pituitary gland and the pineal body contained larger quantities presumably because there is no blood-brain barrier in these structures. The brain contains relatively large amounts of endogenous noradrenaline (Vogt, 1954). Catecholamines in the brain are probably formed from precursors that are capable of crossing the blood-brain barrier such as dopa. Using a broken cell preparation, monoamine oxidase activity was shown to be higher in the rat brain than catechol-0-methyltransferase (Crout et al., 1961). As a result of these observations, it was postulated that monoamine oxidase was mainly involved in the inactivation of noradrenaline in the brain. After the administration of pyrogallol, a catechol-0methyltransferase inhibitor (Axelrod and Laroche, 1959), into the lateral ventricle of the rabbit, there was a threefold elevation in the endogenous noradrenaline in the brain (Masami et al., 1962). The administration of 14C-noradrenaline into the lateral ventricle of cats resulted in the formation of normetanephrine and other 0-methylated products as major metabolites (Mannarino et a/., 1962). From these observations it would appear that 0-methylation is a n important enzymatic process in the metabolism of noradrenaline in nervous tissue. It still remains to be established whether binding, monoamine oxidase or catechol-0-methyltransferase or a combination of these mechanisms are involved in the initial inactivation of noradrenaline in the brain. The isolated-perfused rat heart also can take up and retain SH-noradrenaline (Axelrod et al., 1962a). The bound catecholamine is then released in a multiphasic fashion which indicates that there are several types of binding (Kopin et al., 1962). In rat hearts, noradrenaline was found to be inactivated principally by binding. The released noradrenaline in the isolated-perfused heart is metabolized by 0-methylation and deamination. The changes in the released metabolites with time suggest that 0methylation is the main pathway for the metabolism of the easily releasable noradrenaline and deamination is the main pathway for the tightly bound catecholamines. Although noradrenaline and adrenaline are taken up, stored, and metabolized in a similar manner, there are quantitative differences in their disposition (Whitby et a]., 1961). More 3H-noradrenaline was taken up in tissues and held for longer periods of time. There were more 0-methylated metabolites found after adrenaline than after noradrenaline. These findings indicate that binding is quantitatively a more important mechanism for the inactivation of noradrenaline while enzymatic 0methylation is more important for adrenaline. The higher levels of circulating endogenous noradrenaline might be explained in terms of the differences in the degree of binding of these hormones rather than in terms of a greater release of noradrenaline into the circulation. The subcellular localization of catecholamines was studied in heart, salivary and adrenal glands (Potter and Axelrod, 1962). About 80% of the circulating 3H-noradrenaline and 3H-adrenaline was taken up by a particulate fraction associated with
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the microsomes of the heart and salivary gland. Endogenous catecholamines were also found in this fraction of the cell. 3H-Dopamine, 3H-dopa, and 3H-normetanephrine, on the other hand, were present only in the cell sap. In the adrenal medulla, 3H-noradrenaline was found in the microsomal fraction while 3H-adrenaline, 3Hdopamine, and endogenous catecholamines were localized in the chromafin granules. 3H-Dopa was present in the supernatant fraction of the adrenal medulla and in the ‘pinched-off nerve endings’ of the brain described by Whittaker (1959). Using brain slices, 3H-noradrenaline was also found to be taken up in this layer. It was demonstrated that circulating noradrenaline and adrenaline were taken up by sympathetic nerve endings (Hertting et a/., 1961a). When sympathetic nerves were destroyed, they were unable to retain the catecholamines. Supersensitivity to noradrenaline results partly because the sympathetic nerves cannot inactivate the catecholamines by binding. The uptake of radioactive noradrenaline by sympathetic nerves made it possible to visualize sympathetic nerves by radioautographic techniques (Wolfe et a/., 1962). Electron microscopy showed a striking localization of radioactive grains only within sympathetic nerves associated with dense core vesicles 40 to 50 mp thick. These experiments provide conclusive evidence that noradrenaline can be taken up from the circulation into sympathetic nerves and stored there until it is released. RELEASE OF N O R A D R E N A L I N E A N D A D R E N A L I N E
After the administration of noradrenaline and adrenaline, these catecholamines disappeared from the whole animal in two phases (Axelrod et al., 1959; Whitby et al., 1961). In the first few minutes, there was a rapid decline followed by a slow disappearance of the catecholamines. The initial phase is due to enzymatic destruction of the catecholamines and their uptake and binding by tissues; the second to slow release and metabolism. In the first few minutes, about two-thirds of the adrenaline and one-half of the noradrenaline were metabolized. Almost all of the catecholamines that disappeared in the first few minutes could be accounted for as 0-methylated metabolites, normetanephrine and metanephrine. There was a slower decline of noradrenaline as compared with adrenaline during the second phase which indicated that noradrenaline is more tightly bound and more slowly released. Bound noradrenaline was released from the heart over a period of days and the half-life of noradrenaline became progressively longer (Axelrod et al., 1961a). These observations indicated that noradrenaline became more tightly bound with time. Tyramine has been shown to act by releasing noradrenaline (Burn and Rand, 1958). Using tyramine as a tool, it was demonstrated that noradrenaline is bound with different degrees of tenacity (Potter et al., 1962). After the first injection, tyramine released about 30% of both endogenous and 3H-noradrenaline. With repeated injections of tyramine, the amount of endogenous and exogenous noradrenaline liberated from the heart became less and less. There was no further release of noradrenaline and no blood pressure elevation after the third or fourth injection of tyramine. Yet considerable quantities of the catecholamines were still present in the heart. It was apparent from these Rejerentes p . 87-89
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experiments that noradrenaline is bound in two pools, one which is easily released and another which is more firmly held. The 3H-noradrenaline recently taken up by the heart can be released by tyramine more easily. With the passage of time, tyramhe releases less and less 3H-noradrenaline. The longer the noradrenaline remained in the heart the more firmly bound it became. This is due to the different rates in which the pools were turning over. The more easily releasable pool was found to have a half-life of several hours and the more tightly bound pool has a half-life of about one day. In contrast to the finding with tyramine, reserpine releases noradrenaline from both stores equally well (Potter and Axelrod, 1963). It has been shown that the noradrenaline released by reserpine is metabolized by inonoamine oxidase while the noradrenaline released by tyramine is metabolized by catechol-0-methyltransferase (Kopin et al., 1962). Although reserpine releases large amounts of the catecholamine, it produces only a slight physiological effect while tyramine releases much less noradrenaline and produces a marked physiological response. The noradrenaline released by reserpine appears to be metabolized by monoamine oxidase within the nerve and it leaves the nerve as an inactive deaminated metabolite. Tyramine discharges noradrenaline in a physiologically active form and once it is liberated the neurohumor is inactivated by 0-methylation. It has been demonstrated that inhibition of monoamine oxidase does not prolong the physiological actions of noradrenaline (Griesemer et a]., 1953). Consequently, this enzyme appears to act within the sympathetic nerves, and metabolizes the neurohumor before it becomes physiologically active. Once noradrenaline leaves the nerve, monoamine oxidase plays a negligible role in the subsequent fate of the catecholamine. Because sympathetic nerves are capable of taking up and retaining radioactive noradrenaline it was possible to introduce 3H-noradrenaline into these nerves and study its fate when the neurohumor is discharged (Hertting and Axelrod, 1961). Cats were given 3H-noradrenaline and the spleen which contained large amounts of the radioactive catecholamine was isolated and perfused with blood free of radioactive material. After stimulation of the splenic nerve, 3H-noradrenaline and its metabolites were measured in the venous outflow. There was a decided increase in the 3H-noradrenalinc in the venous outflow after each stimulation. There was also a smaller increase in thc 3H-normetanephrine which was directly related to the 3H-noradrenaline liberated. These results again demonstrated that noradrenaline can be taken up from the circulation by sympathetic nerves and on stimulation, discharged. When liberated, the noradrenaline interacts with the receptor and part is released into the blood stream, part 0-methylated by catechol-0-methyltransferase, and part returns to the sympathetic nerves to be bound and used again. EFFECT OF D R U G S O N THE U P T A K E , RELEASE A N D METABOLISM OF N O R A D R E N A L I N E
Drugs affecting behavior also alter the uptake, release and metabolism of catecholamines. Antidepressant drugs inhibit monoamine oxidase and raise the concentration of catecholamines in the heart and brain of certain species (Shore et al., 1957);
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reserpine depletes catecholamines from tissues (Holzbauer and Vogt, 1956). In our laboratory, we have used radioactive catecholamines to study the effect of drugs on uptake, release and metabolism of these hormones. Reserpine and chlorpromazine have been shown to increase the rate of destruction of circulating catecholamines (Axelrod and Tomchick, 1959). These drugs prevented the uptake of the catecholamines, thus interfering with the protective binding. The hormones were then exposed to enzymatic attack and more rapid metabolism. As pointed out above, reserpine also releases the bound noradrenaline within the nerve where it is deaminated by monoamine oxidase. Chlorpromazine blocked the uptake of noradrenaline by the nerve (Axelrod et al., 1961b). Once the neurohumor was bound within the nerve, this drug could not release it (Axelrod et al., 1962b). Chlorpromazine is a drug of many actions: It is an antihistamine, tranquilizer, and an adrenergic blocking agent. The blocking of the uptake of noradrenaline was found to reside only in the antiadrenergic activity of chlorpromazine (Rose11 and Axelrod, 1963). Other antiadrenergic drugs such as Dibenzyline and dichlorisoproterenol affect the uptake and release of noradrenaline (Axelrod et al., 1962b). Like chlorpromazine, the antidepressant drug imipramine increases the metabolism of catecholamines and blocks the uptake of these hormones. Many antidepressant drugs also inhibit monoamine oxidase and elevate the concentration of catecholamines in the brain, heart, and other tissues (Shore et al., 1957). These enzyme inhibitors, however, do not prolong the physiological response to catecholamines and appear to play a minor role in the inactivation of circulating catecholamines. As pointed out above, monoamine oxidase operates mainly within the nerves, inactivating the noradrenaline before it leaves the nerve (Kopin et al., 1962). In the presence of monoamine oxidase inhibitors the released noradrenaline returns to the storage vesicle or escapes into the circulation. Monoamine oxidase inhibitors also blocked the spontaneous release of noradrenaline from stores (Axelrod et al., 1961a), and this also serves to elevate the endogenous catecholamine levels in tissues. Bretylium and ganglionic blocking agents prevent the spontaneous release of 3H-noradrenaline (Hertting et al., 1962a, b). Although these compounds do not inhibit monoamine oxidase, they increase the endogenous catecholamine levels (Hertting et al., 1962b; Ryd, 1962). The releasing action of guanethidine and reserpine was found to be blocked by monoamine oxidase inhibitors, bretylium and ganglionic blocking agents. When noradrenaline is liberated from the easily releasable pool by sympathomimetic amines neither monoamine oxidase inhibitors nor bretylium blocked its release (Potter and Axelrod, 1963). Monoamine oxidase inhibitors, bretylium, guanethidine and reserpine are hypotensive drugs which affect the uptake, release and metabolism of catecholamines. The actions of reserpine and monoamine oxidase inhibitors on noradrenaline have been described above. Bretylium and guanethidine have many actions in common and also differ in their effects on circulating and bound noradrenaline (Hertting et al., 1962a). Both compounds when injected produced a brief rise in blood pressure as well as an immediate release of noradrenaline. These drugs also inhibited the uptake of noradrenaline and potentiated the effects of the hormone. Bretylium and guanethiReferences p . 87-89
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dine blocked the liberation of noradrenaline as well as the physiological effects when sympathetic nerves were stimulated. When the noradrenaline was bound in tissues, bretylium prevented its spontaneous release while guanethidine caused a slow but continuous release of the neurohumor. Many drugs such as cocaine, guanethidine, bretylium, imipramine, chlorpromazine, and Dibenzyline cause supersensitivity to catecholamines. All of these drugs prevented the uptake of circulating catecholamines by tissues (Whitby et al., 1960; Axelrod et al., 1961b; Hertting et al., 1961b). The concentration of catecholamines in the blood was elevated when the animals were pretreated with these drugs. Supersensitivity resulted because these drugs prevented inactivation of the hormone by binding and a higher concentration of the free and active catecholamines was present at the receptor site. Chronic denervation of sympathetically innervated organs also caused supersensitivity to catecholamines. When the nerves degenerate, the vesicles which bind and inactivate noradrenaline are destroyed. Consequently, the amount of unbound and active catecholamines in the vicinity of the receptors in denervated tissues would be expected to persist for longer periods, thus resulting in supersensitivity. Sympathomimetic amines not only liberated catecholamines (Burn and Rand, 1958) but also prevented their uptake and increased the rate of metabolism (Axelrod and Tomchick, 1960; Hertting et al., 1961b). After repeated administration of tyramine
Fig. I . The fate of noradrenaline at the sympathetic nerve endings and the effect of drugs. Bret bretylium; GBO ganglionic blockers; MAOJ = monoamine oxidase inhibitors; COMT -- catechol-0-meth yltransferase.
and other sympathomimetic amines, there was successive decrease in the amount of noradrenaline released from the heart and a concomitant decrease in physiological response (Potter et al., 1962). When there was no further liberation of noradrenaline
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by tyramine, tachyphylaxis (loss of responsiveness) resulted. At this time, there is a considerable amount of noradrenaline still present in the heart. The action of sympathomimetic amines could be restored after an infusion of noradrenaline (Cowan et af., 1961). Tachyphylaxis appears to be a result of the depletion of the bound catecholamines easily available for release. Fig. 1 shows a working model of the sympathetic nerve which is consistent with its morphological appearance and would explain differences in storage, metabolism and the action of drugs on noradrenaline. The majority of the storage vesicles are deep within the nerves. Reserpine and guanethidine release noradrenaline from the deep vesicles. The catecholamines are deaminated by monoamine oxidase in the mitochondria and leave the nerve as inactive metabolites. Bretylium appears to block the releasing action of these drugs from the storage vesicles and monoamine oxidase inhibitors prevent the metabolism of noradrenaline liberated by reserpine and guanethidine. A smaller number of vesicles may be close to the synaptic terminals where sympathomimetic amines or nerve impulses release noradrenaline from the nerve cell. Once released, the noradrenaline reacts with the receptor and is metabolized by catechol-0-methyltransferase, discharged into the circulation, or returns to the storage vesicle. Bretylium and guanethidine block the release of noradrenaline by nerve impulses. Noradrenaline is also released from the stores spontaneously and this release is blocked by monoamine oxidase inhibitors, bretylium, and ganglionic blocking agents. The storage vesicles can also take up noradrenaline from the circulation. Cocaine, chlorpromazine, imipramine, bretylium, guanethidine, and reserpine block this uptake thus causing supersensitivity. SUMMARY
Circulating noradrenaline and adrenaline are mainly inactivated by catechol-0methyltransferase or they are taken up and bound in dense core vesicles in sympathetic nerves. When the catecholamines are released they are metabolized by monoamine oxidase within the nerve or by catechol-0-methyltransferase outside the nerve. A part of the catecholamines are also inactivated by being bound again or by diffusing into the circulation. Noradrenaline is stored in an easily releasable or firmly bound form. Many drugs such as cocaine, chlorpromazine, imipramine, reserpine, guanethidine, bretylium and sympathomimetic amines interfere with the uptake, storage and release of catecholamines. REFERENCES AXELROD, J., GORDON, E., HERTTING, G., KOPIN,I. J., AND POTTER, L. T., (1962a); On the mechanism of tachyphylaxis to tyramine in the isolated rat heart. Brit. J. Pharmacol., 19, 56-63. AXELROD, J., HERTTING, G., AND PATRICK,R. W., (1961a); Inhibition of H3-norepinephrine release by monoamine oxidase inhibitors. J . Pharmacol. exp. Ther., 134, 325-328. AXELROD, J., HERTTING, G., AND POTTER,L. T., (1962b); Effect of drugs on the uptake and release of 3H-norepinephrine in the rat heart. Nature (Loncl.), 194, 297.
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AXELROD, J., AND LAROCHE, M . J., (1959); Inhibitor of 0-methylation of epinephrine and norepinephrine in vitro and in vivo. Science, 130, 800. AXELROD, J., AND TOMCHICK, R., (1959); Activation and inhibition of adrenaline metabolism. Nature (Lond.), 184, 2027. AXELROD, J., AND TOMCHICK, R., (1960); lncreased rate of metabolism of epinephrine and norepinephrine by sympathomimetic amines. J . Pharmacol. exp. Ther., 130,367-369. H., AND TOMCHICK, R., (1959); The physiological disposition of AXELROD, J., WEIL-MALHERBE, H3-epinephrine and its metabolite metanephrine. J . Pharmacol. exp. Ther., 127, 251-256. AXELROD, J., WHITEY, L. G., AND HERTTING, G., (1961b); Effect of psychotropic drugs on the uptake of H3-norepinephrine by tissues. Science, 133, 383-384. BURN,J. H., AND RAND,M. J., (1958); The action of sympathomimetic amines in animals treated with reserpine, J . Physiol. (Lond.), 144, 314-336. COWAN,F. F., CANNON, C., KOPPANYI, T., AND MAENGWYN-DAVIES, G. D., (1961); Reversal of phenylalkylamine tachyphylaxis by norepinephrine. Science, 134, 1069-1070. CROUT,J. R., CREVELING, C. R., A N D UDENFRIEND, S., (1961); Norepinephrine metabolism in rat brain and heart. J . Pharmacol. exp. Ther., 132, 269-271. GRIESEMER, E. C., BARSKY,J., DRAGSTEDT, C. A., WELLS, J. A., A N D ~ E L L E R E.A.,(1953); , Potentiating effect of iproniazid on the pharmacological action of sympathomimetic amines. Proc. Soc. exp. Biol. ( N . Y . ) , 84, 699-701. HERTTINC, G., AND AXELROD, J., (1961); Fate of tritiated noradrenaline at the sympathetic nerve endings. Nature (Lond.), 192, 172-173. HERTTING, G., AXELROD, J., KOPIN,1. J., AND WHITBY, L. G., (1961a); Lack of uptake of catecholamines after chronic denervation of sympathetic nerves. Nature (Lond.), 189, 66. HERTTING, G., AXELROD, J., AND PATRICK, R. W., (1962a); Actions of bretylium and guanethidine on the uptake and release of (3H)-noradrenaline. Brit. J . Pharmacol., 18, 161-166. HERTTING, G., AXELROD, J., A N D WHITBY, L. G., (1961 b); Effect of drugs on the uptake and metabolism of H%orepinephrine. J . Pharmacol. exp. Ther., 134, 146-1 53. HERTTING, G., POTTER, L. T., AND AXELROD, J., (1962b); Effect of decentralization and ganglionic blocking agents on the spontaneous release of H3-norepinephrine. J . Pharmacol. exp. Ther., 136, 289-292. HOLZBAUER, M., AND VOGT,M . , (1956); Depression by reserpine of the noradrenaline concentration in the hypothalamus of the cat. J . Neurochetn., 1, 8-1 1. KOPIN,I. J . , HERTTING, G., AND GORDON, E. K., (1962); Fate of norepinephrine-H3 in the isolated perfused rat heart. J . Pharmacol. exp. Ther., 138, 34-40. MANNARINO, E., KIRSHNER, N . , AND NASHOLD, JR., B. S.,(1962); Themetabolism of noradrenaline-C'd by cat brain in vivo. Fed. Proc., 21, 182. MASAMI, M., HIROSHI, Y.,ANDREIJI, I . , (1962); Effect of pyrogallol on the catecholamine content of the rabbit brain. Biochem. Pharmacol., 11, 1109-11 10. POTTER, L. T., AND AXELROD, J . , (1962); Intracehlar localization of catecholamines in tissues of the rat. Nature (Lond.), 194, 581-582. POTTER, L. T., AND AXELROD, J., (1963); Studies on the storage of norepinephrine and the effect of drugs. J . Pharmacol. exp. Ther., 140, 199-206. POTTER, L. T., AXELROD, J., AND KOPIN,I. J., (1962); Differential binding and release of norepinephrine and tachyphylaxis. Biochem. Pharmacol., 11, 254-256. ROSELL, S., A N D AXELROD, S., (1963); Relation between blockade of 3H-noradrenaline uptake and pharmacological actions produced by phenothiazine derivatives. Experientia (Easel), 19, 3 18. RYD, G., (1962); Protective effect of bretylium on noradrenaline stores in organs. Acra physiol. scand., 56, 90-93. SHORE, P. A., MEAD,J. A. R., KUNTZMAN, R. G., SPECTOR, S., AND BRODIE, B. B., (1957); On the physiologic significance of monoamine oxidase in brain. Science, 126, 1063-1064. VOGT,M., (1954); The concentration of sympathin in different parts of the central nervous system under normal conditions and after the administration of drugs. J. Physiol. (Lond.), 123, 451-481. WEIL-MALHERBE, H., AXELROD, J., AND TOMCHICK, R., (1959); Blood-brain barrier for adrenalinc. Science, 129, 1226-1227. WEIL-MALHERBE, H., WHITBY, L. G . , AND AXELROD, J., (1961); The blood-brain barrier for catecholamines in different regions of the brain. Regional Neurochemistry. S. S . Kety and J. Elkes, Editors. Oxford, Pergamon Press (p. 284-292). WHITBY, L. G., AXELROD, J., AND WEIL-MALHERBE, H., (1961); The fate of H3-norepinephrine in animals. J . Pharmacol. exp. Ther., 132, 193-201.
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WHITBY,L. G., HERTTING, G., AND AXELROD, J., (1960); Effect of cocaine on the disposition of noradrenaline labelled with tritium. Nature (Lonil.), 187, 604-605. WHITTAKER, V. P., (1959); The isolation and characterization of acetylcholine-containing particles from brain. Biochern. J., 72, 694-706. WOLFE,D. E., POTTER,L. T., RICHARDSON, K. C., A N D AXELROD, J. (1962); Localizing tritiated norepinephrine in sympathetic axons by electron microscopic autoradiography. Science, 138, 440-442.