TIPS - May 1985

L a w o f m a s s action a n d b i n d i n g ecp,ilihrium constant are often m i s q u o t e d

Imprecision in presentation of binding studies The clarity of presenta t ion of re- sults, in part icular those from b i n d i n g studies, might be im- proved if words were used more precisely.

There seems to have deve loped a w i desp read tendency to use the term 'Law of Mass Act ion ' as though it were synonymous wi th ' the Langmui r b ind ing equat ion at equ i l ib r ium' . This is, of course, qui te wrong. The Law of Mass Action is an empirical rule that states that the rate at which a react ion proceeds is directly pro- por t ional to the product of the reactant concentrat ions, the pro- por t iona l i ty constant be ing refer- red to as the rate constant for the reaction. It is thus a law about the

rates of reactions. Contrary to wha t one often reads, it does not assume that this reaction is at equ i l ib r ium (on the contrary it is the usual tool for exploring the rate of approach to equilibrium1), or that the reactant concentrations are constant , or that b ind ing sites are all identical and independent , or that the reaction is reversible, or that the receptor is not changed by agonist b inding . In practice it may, of course, be convenient to make some assumpt ions about one or more of these points, but these assumpt ions have got no th ing to do wi th the Law of Mass Action.

Another common error that still persists concerns the confusion

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be tween a microscopic b ind ing constant and what is actually measured in a b ind ing experi- ment. The simplest example is perhaps the wel l -known mechan- ism of Castillo and Katz 2 for b ind ing of an agonist (A) to a receptor (R), followed by isomeri- zation of the receptor to an active state, R*, wi th the rate constants shown on the arrows.

k+l f~ A + R ~ A R ~ A R *

k-1 0~

The b ind ing equi l ibr ium constant (K = k_l/k+l) is not measured by a b ind ing experiment; the ex- per iment will actually measure the effective b ind ing constant K/(1 + ~/(x), which corresponds to a h igher affinity than K itself.

D. C O L Q U H O U N

Department of Pharmacology, University College, London, Gower Street, London WC16 6BT, UK.

References 1 Hill , A. V. (1909) ]. Physiol. (London) 39,

361-373 2 Cast iUo, J. del a n d Katz , B. (1957) Proc.

Roy. Soc. B146, 369-381

Hydralazine inhibits covalent binding of complement component C4; its acetylated metabolites do not

More drugs interact with the complement system I was very pleased to read Pro- fessor Vogt 's recent article 'Drugs and the Complement System' (TIPS March 1985, 114-119). I would like to draw at tention to another area in which toxic side effects of drugs are likely to involve interact ion with the com- p lement system.

Hydra laz ine and isoniazid, both of which induce systemic lupus erythematosus (SLE)-like condi t ions 1, inhib i t the covalent- b ind ing reaction of the comple- ment component C4, whils t the acetylated metabol i tes of these drugs do not 2. The toxic SLE-like side effects of hydralazine 3 and isoniaz id 4 are known to occur almost exclusively in indiv iduals of the slow acetylator phenotype. That the drugs rather than their metabol i tes inhib i t C4 would be consis tent wi th these clinical ob- servations.

Professor Vogt ment ions the

correlation between genetic defi- ciencies of C4 and immune com- plex diseases including SLE. As he states, the link between the two condit ions is likely to be due to inhibi t ion of C3-dependent solu- bi l izat ion of immune complexes, wi th a resulting increase in size and deposi t ion of immune com- plexes in vascular epi thel ia for example 5. Any effect which inhi- bits C4 activity, such as hydral- azine inhibi t ion, would be ex- pected to predispose susceptible indiv iduals to SLE.

It has recently been observed that when hydralazine inhibi ts the covalent b ind ing of C4, the drug itself becomes covalently bound to the thiol-ester site in activated C4b 6. This occurs only when the drug is present dur ing activation of C4 and may partly explain the f indings that com- plement levels are not usually decreased 7 in drug- induced SLE.

The role of C4 inhib i t ion in hydra laz ine- induced SLE has been ques t ioned 8 on the grounds that measured plasma concentra- tions of hydralazine are too low and Professor Vogt referred to this problem with other compounds which interfere with the comple- ment system. However, hydral- az ine- induced SLE only develops after long-term treatment wi th the drug and it has been suggested that a cumulative dose of 100 g is required 9 for development of toxic side effect. To my knowledge, there have been no reports of plasma hydralazine concentra- tions in these circumstances (i.e. after more than a year of hydral- azine therapy). Values around 1 ~M have been measured but after only one 1° to five 11 oral doses of I mg kg-1; these experimental doses would not induce SLE.

It may be relevant that hydra- z ine- induced SLE has been rec- orded and hydraz ine is a known potent inhibi tor of the covalent b ind ing reaction of C4 and C312.

EDITH SIM

Wellcome Immunotoxicology Unit, University Department of Pharmacology, South Parks Road, Oxford, OX1 3QT, UK.

1985, Elsevier Science Publishers B.V., Amsterdam 0165 - 6147/85/$02.00

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References 1 Harpey, J. P. (1973) Adverse Drug React.

Bull. 43, 140-143 2 Sim, E., Gill, E. W. and Sim, R. B. (1984)

Lancet ii, 422--424 3 Perry, H. M., Tan, E. M., Carmody, S.

and Sakamoto, A. (1970) J. Lab. Clin, Med. 76, 114--125

4 Godeau, P., Aukert, M., Imbert, |-C.

and Herreman, G. (1974) Ann. Med. Interne 124, 181-186

5 Lachmann, P. J. (1984) Philos. Trans. R. Soc, London Ser. B. 306, 419--430

6 Sire, E. and Law, S. K. A. (1985) FEBS Lett. (in press)

7 Perry, H. M. (1973) Am. J. Med. 54, 58--72 8 Christophidis, N. (1984) Lancet ii, 868 9 Hughes, G. R. V. in Connective Tissue

TIPS - May 1985

Disease. (1977) p. 44, Blackwell 10 Ludden, T. M., McNay, J. L., Shepherd,

A. M. M. and Lin, M. S. (1981) Arthritis Rheum. 24, 987-994

11 Reece, P. A., Cozamanis, l. and Zacest, R. (1980) Clin. Pharmacol. Ther. 28, 769-- 778

12 Durant, P. J. and Harris, R. A. (1980) N. Engl. J, Med. 303, 584-585

Possible role for a calci u m-activated, phospholipid-dependent protein kinase in mode of action of DSCG Ronit Sagi-Eisenberg

Ronit Sagi-Eisenberg suggests tha t the anti-allergic drug disodium cromoglycate (DSCG) exerts its inhibitory activity on mast cell degranulation by interacting with a Ca t+- and phospholipid-dependent protein kinase C involved in the stimulus-secretion coupling of these cells. Her hypothesis is based on similarities between the conditions required to activate this kinase and those needed to evoke secretion. In addition, binding of DSCG to mast cells leads to protein phosphorylation; protein kinase C has been shown to play a dual role in the activation and termination of the secretory process in RBL- 2H3 cells. Hence, the Ca 2+ phospholipid-dependent protein kinase C appears to be an attractive candidate for the protective action of DSCG.

The anti-allergic drug disodium cromoglycate (DSCG), widely used as a prophylactic drug in the treatment of bronchial asthma, has been subjected to extensive studies in attempts to resolve its mode of action. Clearly, elucida- tion of the molecular mechanism by which this drug exerts its protective effect is of both phar- macological and biochemical im- portance as it might shed new light on the mechanism of allergy and asthma.

Early studies established the inhibitory effect of DSCG on the allergic response in human lung in vivo, provided it is administered before the allergen. Subsequent in-vitro experiments demonstrat-

Ronit Sagi-Eisenberg is a post-doctoral fellow in the Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot, Israel

ed that DSCG inhibits the release of the mediators of immediate- type hypersensitivity from sensi- tized lung fragments as well as from isolated rat peritoneal mast cells 1. On the basis of these obser- vations, it has been suggested that DSCG exhibits its pharmacologi- cal activity by stabilizing the mast cell membrane, thereby prevent- ing degranulation. However, al- though DSCG markedly reduces degranulation of mast cells in certain tissues, in others no activity is demonstrated 2. Differences in membrane structure or composi- tion of these ceils may account for this reduced drug-cell interaction. In fact mast cells from different species or even from individual tissues within a single animal were shown to vary significantly in their response to given hist- amine releases 2.

Inhibition of mast cells degran-

1985, Elsevier Science Publishers B.V., Amsterdam 0165 - 6147/85/$02.00

ulation by DSCG is undoubtedly of major importance in its protec- tive mode of action although its very particular effectiveness against asthma raises the possibil- ity that cells other than mast cells are also involved in the mode of action of this drug.

Characteristics of the DSCG-mast cell interaction

Inhibition of mast cell degranu- lation by DSCG has been shown to be accompanied by blocking of antigen-induced Ca 2+ influx 3. Since Ca 2+ influx is essential for antigen-induced cell degranula- tion 4, the inhibitory action of DSCG may result from interfer- ence with the cellular Ca 2+ gating mechanism. Indeed, low concen- trations of DSCG which complete- ly inhibit the antigen-induced release (10-50 ~tM), fail to signifi- cantly affect release induced by the Ca 2+ ionophore A23187 (cali- mycin) which bypasses the Ca 2+ gating mechanisms 5. Further- more, a specific, Ca2+-dependent, binding site for DSCG on the outer membrane of mast cells has been demonstrated 6. This has been subsequently shown to be a membrane protein, and has been isolated from rat basophilic leu- kemia cells (RBL-2H3). Possible involvement of this protein in the Ca 2+ gating mechanism of these cells is implied by its ability to restore Ca 2+ uptake activity of RBL variants impaired in both b inding of DSCG and Ca 2+ up- take 7. However, recent reports have revealed that high concentra- tions of DSCG (> 100 ~u~4) do inhi- bit release induced by sub-opti- mal concentrations of A23187 (Ref. 8). It therefore appears that this drug has additional modes of ac- tion. The divergent dose-response suggests that either there are at least two different receptors for DSCG or that the cellular target for this drug 's action is multifunc- tional.

A clue to the molecular mechan- ism by which DSCG might exert its activity has been recently ob-