Moiec~iar and Cellular Endocrinology, 78 (1991) C95-C98 0 1991 Elsevier Scientific Publishers Ireland, Ltd. 0303-~207/91/$03.50
At the Cutting
kidneys, sweet roots and dirty drugs Carl &fonder
Population Council, New York, NY 10021-6399, (Accepted
Key tvor&r Glycyrrhetinic Gastric ulcc~
The origins of the healing powers of licorice are lost somewhere in the dim recesses of Oriental and mid-Eastern folk medicine (Davis and Morris, 1991). Licorice, the extract of the roots of Glycyrrhiza glabru, is referred to in old herbals as a ‘protection against the acrimony of food’, as a rejuvenator, expectorant, and flavoring agent. The formal Linnaean designation for the plant derives from g/y.+ rhiza, literally ‘sweet root’ in Greek. The term Licorice is a corruption of the original word. Long before it was introduced into the pharmacopeia for the treatment of dyspepsia, it is likely that extract of Licorice root was used as a condiment. In some countries, the amount of licorice ingested in candies, flavored liquors or tobacco is great enough to have become a matter of serious medical concern. The pharmacologically important ingredient, glycyrrhizin, has been found to be frequenty toxic, causing high blood pressure, heart failure, edema, hypokalemia, polyuria, polydypsia and muscle weakness. The isolation and identification of glycyrrhetinic acid (GA), or enoxolone, the biologically active form of glycyrrhizin, led to the development of synthetic derivatives, the most important of which was glycyrrhetinic acid hemisuccinate, designated carbenoxolone. This substance proved to
Address for correspondence: Carl Monder, Population Council, 1230 York Avenue, New York, NY 10021-6399, U.S.A.
be the first practical therapy for gastric and duodenal ulcers. The cluster of toxic side effects of carbenoxolone (CA) were the same as those caused by GA. Both caused symptoms characteristic of aldosteronism; unexpectedly a rise in circulating aldosterone did not occur. It was found on the contrary that both agents decreased aldosterone secretion and excretion, and it was deduced that the aldosterone-like behavior elicited by these compounds were properties of the agents themselves, or that they augmented the mineralcorticoid actions of even diminished levels of aldosterone. The conclusion that GA amplifies aldosterone action was based on the observation that, without adrenal glands, neither rats nor patients could be maintained on GA or CA alone. This was reinforced by the observation that GA had a synergistic effect on aldosterone enhanced active sodium transport in toad skin (Porter, 1970). It was postulated that aldosterone was displaced by GA from non-specific binding sites, and was thus made available to ~neralocorticoid receptors (Humphey et al., 1972). An alternative mechanism which would also increase the biological availability of aldosterone was proposed by Tamura et al. (1979), and later amplified by Latif et al. (1990). GA and CA inhibit steroid SD-reduetase and 3~-hydroxysteroid dehydrogenase, retarding the inactivation of aldosterone and glucocorticoids, decreasing their metabolic clearance. and prolonging their biological half-life.
The idea of increasing the potency of a steroid by slowing its rate of degradation is not new. Synthetic variants of the natural steroids were originally created in order to defeat the actions of catabolic enzymes and thus extend their effective lifetimes. GA, by inhibiting specific enzymes, provides an alternative approach to achieve the same end with the natural steroids. The results are not always beneficial, as the following example illustrates. The most actively discussed mechanism of licorice induced hypertension depends on the inhibition of [email protected]
dehydrogenase by GA (Funder. 1990; Shackleton and Stewart, 1990; Stewart and Edwards 1990). Kidney mineralocorticoid receptors (MR) are activated by aldosterone as part of a process that is central to blood pressure control (Monder, 1990). Cortisol and corticosterone are capable of competing with aldosterone for receptor binding and thus functioning as mineralocorticoids. There is so much more circulating glucocorticoid than mineralocorticoid that unless some mechanism existed to inactivate the excess glucocorticoid, aldosterone would not gain access to MR. The consequence would be persistent MR activation by glucocorticoid, resulting in unrelieved hypertension. Normally, glucocorticoids are oxidized by lip-hydroxysteroid dehydrogenase to inactive 11-0x0 metabolites that do not compete with aldosterone for MR. However, when the organism is exposed to GA (or CA) at sufficiently high concentration over a long period of exposure, inhibition of 11/3-hydroxysteroid dehydrogenase blocks glucocorticoid inactivation, leading to an undesirable local renal accumulation of active glucocorticoid, which results in the clinical picture of mineralocorticoid excess, despite a sharp drop in circulating aldosterone. GA is a potent inhibitor of cortisol oxidation, but inhibits cortisone reduction comparatively poorly. This selective inhibition is seen with rat kidney preparations in vitro and in vivo. In humans, the effects of GA ingestion on cortisol metabolism reflects a significant net decrease in its oxidation to cortisone (MacKenzie et al., 1990). CA, though similar to GA in its kinetic behavior, appears to show differences in its in vivo behavior in patients. In contrast with GA, CA does not
initiate kaluresis in spite of sodium retention and hypokalemia. In addition, urinary (THF + allo THF)/THE is unchanged despite evidence that cortisol oxidation is inhibited. The basis for the selectivity is not yet fully understood. It is possible, though yet to be rigorously established, that CA in vivo inhibits both renal ll/%dehydrogenation and ll-oxoreduction (Stewart and Edwards, 1990). Since GA delays the metabolic clearance of glucocorticoids and prolongs their metabolic halflives, it should potentiate glucocorticoid activity in corticosterone dependent tissues. The expressed effects of GA would therefore be anti-inflammatory and ulcerogenic. Descriptions of the anti-inflammatory activity of GA or CA and of their ability to enhance the activities of glucocorticoids have appeared intermittently in the literature since 1958. However, the effects are minor. The impact of GA and CA on mineralocorticoid action is far more dramatic than on glucocorticoid action. despite the obvious fact that it is the metabolism of the latter that is mainly altered by these agents. A full exploration of the range of dehydrogenases inhibited by GA has not yet been made. In addition to the enzymes already discussed, it has recently been found that GA and CA are inhibitors of bacterial 3a,20/?-hydroxysteroid dehydrogenase (W.L. Duax, personal communication) and testicular [email protected]
dehydrogenase (Sakamoto and Wakabayashi. 1988). The effects on the various dehydrogenases may in part reflect the steroid-like structures of enoxolones and their derivatives, and in part their non-specific absorption to and partial disruption of protein molecules. The importance of these compounds as structural analogues is illustrated by their participation as competitive inhibitor of 1 I/?-hydroxysteroid dehydrogenase (Monder et al., 1989) and non-competitive inhibitor of SP-reductase (Latif et al., 1990). The possible non-specific effects are shown by their disruption of the three-dimensional structure of of 3a,20P_hydroxysteroid dehydrogenase (W.L. Duax, personal communication). The dramatically greater potency of GA and CA as inhibitors of purified llj%hydroxysteroid dehydrogenase when compared with enzyme in intact cells or homogenates may be due to non-
specifc sequestration of GA and CA to proteins in the latter more complex en~ronments, and their consequent decreased availability for enzyme binding. Because enoxolones stick to membranes and proteins, extrapolation of in vitro observations on the effects of GA or CA on enzyme activity to the in vivo environment must be made cautiously. In vitro, CA is an uncoupler of oxidative phosphorylation (Whitehouse et al., 1967). but in vivo it is ineffective, since it binds to proteins and is not accessible to the mitochondria. In humans, the magnitude of sequestration of the drugs in the circulation and tissues varies between individuals. The greater the degree of binding, the less likely it is that side effects will be caused by specific enzyme inhibition. The variability of non-specific binding from individual to individual may explain why the hypertensive side effects of GA or CA are seen in fewer than 50% of random treated populations. The enzyme that are affected by GA or CA include a number that do not use steroids as substrates. This lack of specificity complicates the problems of explaining how these enoxolones function. To illustrate, GA and CA have been reported to inhibit 5- and 12-lipoxygenase and cyclooxygenase (Ohuchi et al., 1981). They inhibit histamine synthesis and release from mast cells, a process that may be related to their anti-ulcer activity (Jones, 1974; Imanishi et al., 1989). It has been suggested that GA and its derivatives, by extending the biological half-lives of the glucocorticoids, and increasing gluco~orticoid receptor occupancy, depress prostaglandin synthesis, and thus raise Na-K-ATPase activity. It has been proposed that this process could affect the electrochemical gradient for sodium and potassium in transporting epithelia (Itoh et al., 1989). The likelihood that GA could contribute significantly to salt metabolism in vivo by this mechanism is tempered by the observation that the effective half-concentration for inhibition of Na-K-ATPase in dog kidney homogenates is about 50 WM. Whether GA and CA are themselves mineralocorticoids still remains a matter of dispute. Evidence has been presented that shows no mineralocorticoid activity in adrenalectomized experimental animals or humans. Based on in vitro studies with mononuclear leukocytes (MNL), mea-
suring transcellular salt exchange, Armanini has concluded that GA is a weak mineralocorticoid, and that CA must be hydrolyzed to GA to express its mineralocorticoid-mimetic properties (Armanini et al., 1989). Consistent with its observed behavior in this model system, GA binds to MR, though the ligand-receptor binding constant is only IO-‘-fold that of aldosterone. It has been stated that the levels of circulating GA in licorice toxicity are sufficiently high to permit GA to express its mineralocorticoid properties. Calculations to support this argument are based on the assumption that all of the drug is freely circulating and accessible to MR. However, several studies have concluded that GA and CA bind so completely to circulating proteins that less than 1% (Pinder et al., 1976) (in one study, less than 0.05%, Ishida et al., 1988) is available for MR binding. A further confounding factor is introduced by the fact that, by inhibiting IljShydroxysteroid dehydrogenase, GA increases the accessibility to MR of the very glucocorticoids with which it competes, and thus diminishes its accessibility to the receptor even further. Glycyrrhetinic acid and its derivatives have proved to be important tools for exploring significant metabolic questions with serious clinical implications. The recognition that these pharmacological agents are potent inhibitors of ll/?-hydroxysteroid dehydrogenase has contributed to our understanding of the role of this enzyme in blood pressure control, and has led to the emergence of insights that could have been made in no other way. The purpose of this essay is in part to reexamine the role of llj3-hydroxysteroid dehydrogenase as the key element in GA mediated hypertension. It is, in my view, necessary to emphasize that GA and CA have multiple effects, and to question may commitment to a unitary interpretation of these effects. It is likely that the mineralocorticoid behavior produced by GA and CA is the result of the convergence of many inputs, including their effects on ll/&hydroxysteroid dehydrogenase, A-ring reduction, interaction with MR, synergistic effects with other steroids. These are not all equally important. The artifices of in vitro experiments have not provided us with insights sufficient to weigh the potential alternative mechanisms in a physiological context. At the start of
the essay, I emphasized that Ga and CA have been the subjects of intense study because of their medicinal properties. Many studies have been published directed to attempts to explain how CA heals gastric and duodenal ulcers. The hypertensive side effects were of secondary interest. Studies devised to explain their therapeutic properties have revealed that GA and CA bind readily to membranes and serum proteins. In some instances, such as with pepsin, non-specific binding with CA results in protease inhibition (Walker and Taylor, 1974). Targeted effects, including increased gastric mucus synthesis, decreased rate of DNA synthesis and cell turnover in gastic epithelium have been reported. Clearly, the effects of these compounds are broad ranging, and possibly tissue specific, It would be worthwhile to explore the effects of GA and CA on the epithelium elsewhere, as in the colon, salivary glands, or kidney. The range of specific and non-specific processes affected by GA and CA are so great that caution should be exercised in concluding that any particular process explains what they do, and how they do it. References Armanini, D., Wehling, M. and Weber, P.C. (1989) J. Endocrinol. Invest. 12, 303-306. Davis. EA. and Morris, D.J. (1991) Mol. Cell. Endocrinol. 78, l-6. Funder, J.W. (1990) TEM 1, 145-148. Humphrey. M.J., Lindup, W.E.. Parke, D.V. and Chakraborty, J. (19’72) Biochem. J. 130, 87P.
Imanishi, N.. Kawai, H., Hayashi, Y., Yatsunami. K. and Ichikawa, A. (1989) Biochem. Pharmacol. 38, 2421-2526. Ishids, S., Ichikawa, T. and Sakiya, Y. (1988) Chem. Pharm. Bull. 36, 440443. Itoh, K., Hara, T., Shiraishi, T.. Tanigucbi, K.. Morimoto, S. and Onishi, T. (1989) Biochem. Int. 18, 81-89. Jones, F.A. (1974) in Fourth Symposium on Carbenoxolone (Jones. F.A. and Parke. D.V., eds.). pp. 173-187. Butterworths, London. Latif, S.A.. Conca, T.J. and Morris, D.J. (1990) Steroids 55, 52-58. MacKenzie, M.A.. Hoefnagels, W.H.L.. Jansen, R.W.M.M., Benraad, T.J. and Klopenborg, P.W.C. (1990) .I. Clin. Endocrinol. Metab. 70, 1637-1643. Monder, C. (1991) FASEB J.. (in press), Mender, C., Stewart, P.M., Lakshmi, V., Valentine, R., Burt. D. and Edwards. C.R.W. (1989) Endocrinology 125, 1046 1053. Ohuchi, K.. Kamada, Y., Levine, L. and Tsurufuji, S. (1981) Prostaglandins and medicine. FASEB J. Pinder, R.M., Brogden, R.N., Sawyer, P.R., Speight. T.M., Spencer, R. and Avery, G.S. (1976) Drugs 11, 245-307. Porter, G.A. (1970) in Carbenoxolone Sodium (Baron, J.H. and Sullivan, F.M., eds.), pp. 33-47. Butterworths, London. Sakamoto, K. and Wakabayashi, K. (1988) Endocrinol. Jpn. 35, 333-342. Shackleton, C.H.L. and Stewart, P.M. (1990) in Endocrine Hypertension (Biglieri, E.G. and Melby, J.C., eds.), pp. 155-173, Raven Press, New York. Stewart, P.M. and Edwards, C.R.W. (1990) TEM 1. 225-230. Tamura, Y., Nishikawa, T.. Yamada, K., Yamamoto, M. and Kumagai, A. (1979) Arzneim.-Forschung./Drug Res. 29, 64-l-649. Walker, V. and Taylor, W.H. (1974) in Fourth Symposium on Carbenoxolone (Jones, F.A. and Parke, D.V.. eds.). pp. 55-69, Butte~orths, London. Whitehouse, M.W.. Dean, P.D.G. and HalsaIl. T.G. (1967) J. Pharm. Pharmacol. 19. 533-543.