Endogenous digitalis-like factors

TiPS - ]wne 1991lVol. 121 It is clear however, that downregulation of a detectable fraction of the G-pmtcin population is not observed in all tissues in response to all agonists. This may be a reflection of the relative amounts of receptors and G proteins. Furthermore, the findings have, to date, provided more questions than answers in relation to the mechanistic details of the observed phenomena. Alterations in the rate of expression, the stability of expressed mRNA and the rate of pmtcin turnover may all contribute to the final effects (see Fig. 1). Combinations of these factors may be mutually reinforcing or ic in terms of the final pmduced2). It is clear, however, that al1 of these factors winill have to be assessed within a single system to allow elucidation of how agonists at G-proteinlinked mceptom can regulate celiuhw G-protein levels and hence contml cellular responsiveness.

209 11 LonBabaugh,J.P., Didsbwy, J., Spiegel, A. and Stiles, C. L. (1969) Mot. Pharmacot. 36, 6B1-69B 12 Green, A., Mill&an, C. and Belt, S. E. (1991) Biochrm.Sot. Trans. 19,212~ 13 Mochly-Rosen, D. et al. (1988) Natarc 333,646-Bxl 14 Nlgy. L E Diamond, I., Casso, D. J., Franklin, C. and Cordon, A. S. (1990) J. Biol. Chrm. 26!5,1%6-19S1 15 Gordon, A. S., Nagy, L, Mochly-Rosen. D. and Diamond, 1. (1990) Biochem.SW. Symp. S6,llF136 16 McKenzie, F. R. and Mill&an, C. (1990) J. Btol. Chcm.265,171X&17093 17 McKenzie, F. R., Adie, E. J. and Milligan, C. (1991) Biochtm.Sot. Trans. 19,81S

18 Milligan. C.. Unson. C. G. and Wakelam, M. J. 0. (1969) Eiorhfm./. 262, 643-649 19 Carr. C.. Loney. C.. Unson,C., Knowler, J. T. and Milligan, G. (1990) Eur. /. Mol. Pharmacol. Socfion.188, Phannacol. 203-m 20 Chaq, F. H. and Boume, H. R. (1969) J. Biol. Chra. 264,5352-5357 21 Hadmck. J. R.. Ros, M., Watkins, D. C. and Malbon, C. C. (1990) J. Biol. Chcm. 265,147&l-14790 22 Reitbmann, C., Gierschik, P., Sidiropoubs, D., Werdan. K. and yk&,lKiz;. (1989) Ear. 1. Pkarmacol. , 23 Malbon, C. C. et at. (1990) BiochemSot. Symp. 56.155-164

Endogenousdigitaiis-likefactors

The understanding that cardiac glycosides (e.g. the natural products dieitalis and ouabain) act as specifit inhibitors of Ni+jK+ATPase (the sodium pump) to induce their therapeutic and toxic Work in the authors’ laboraeffects has long begged the question as to why a receptor site tories is supported by the Medical for a toxin should be so conserved Reseat& Council, the Agricultural through evolution. The existence and Food Research Council, the British Heart Foundation (G. M.) of this receptor again suggests and the National Institutes of that an endogenous ligand might exist - and much research has He&h (A. G.). been directed towards identifying an ‘endogenous digitalis-like subCRAEME NILWGAN AND stance’. To some extent this effort ALLAN GREEN* has been rewarded, and several Mokcatar Plmrm~olqy Gmup, Departendogenous ligands manta of Btochemistryand Pharmacotogy. candidate w8mtty of chqow, Glaqora c12 BQQ, have been reported in the literaUK aad Vkparhaents of Intcmal Medicine ture. and Phamicdogy,Lhiiventty of Texas Inhibitors of the Na+ pump M&l Bmnch, Galwston, TX 77550, USA. which contribute to reduction in renal Na+ reabsorption have been identified in human biological fluids’. These inhibitors are found 1 L&owttz, R. J., Hauadorff, W. P. and Caron, M. C. (1990) Trends Pharmacol. in increased concentrations in the set. 11,1%194 blood of patients with essential 2 Raptejko, P. J., Watktns, D. C., Ros, M. hypertension and variol:s types of and Mdbon, C. C. (1969) J. Biol. &a~. Na+ imbalance. Inhibition-of Na’ 264,16183-16169 transport leads indirectly to an 3 L&M, M. A. cl al. (1990) J. Biot. Chrm. 265,355%%60 increase in intracellular Ca2+ via 4 S&o, N. d at. (19%‘) Proc. Nat1 Acad. the Na+-Ca’+ exchanger, and 5c1. USA 86.~3910 consequently to vascular contrac5 Mtlligan, &and Sager&m, E. D. (1990) tion and a rise in blood pressure. Bt0~km. I. 2m. 765-769 Raised levels of a substance that 6 Pawns, W. J. and S&, G. L. (19B7) J. Bid. Chum.262, B41-B47 crossreacts with anti-digitalis 7 Gmen, A. (1987) 1. Biol. Chcm. 262, antibodies are observed in women l5792-Lvo7 with pregnancy-induced hyper8 Simonds, W. F., Goldsmith, P. K., tension. in amniotic fluid and in Cod#iu, J., Unson, C. C. and Spiegel, A. M. (19B9) proc. Natt Acad. Sri. USA the plasma of newborn babies’. B6,78&Bi3 Substances that crossreact with 9 b(d
urine of rats and humans loaded with Na+ J-5. Immunohistochemicd studies with anti-digoxin antibodies show high activity in the paraventricuhr and supraoptic nuclei of the brain, areas involved in water and electrolyte control. The therapeutic concentration of digoxin in plasma is in the range 13nM. Concentrations of endogenous digitalis-like factor in plasma have been estimated by various methods: measurement of inhibition of “Rb+ uptake into human red blood cells*; competition with 13H)ouabain for its binding site on Nat/K+-ATPase’; and immunological croasreactivity with ouabain antibodie~~~~. Concentrations determined by the two former methods are 20-1OOpM ouabain equivalents9,“, while the concentration range estimated immunologically was determined to be 50 plus and 600-1100 pm’> in different studies. Volume expansion in humans increases the concentrations of endogenous digitalis-like factor aknost tenfold, from 80~~ to 900 pk(“. Patients suffering from essential hypertension have an almost threefold elevation in concentrations in endogenous digitalis-like activity”. Purified digitalis-like factors Endogenous digitalis-like substances have recently been purified from human plasma7~y*~1*, hemofiltrate”, human urine”. bovine plasmaIs, hypothalamu8’, and adrenals”. hypophysis” Chmmatographic, immunological and enzymatic data support the

TPS - june 1991 [Vol. 221

Hd 11,13-dihydroxy-14-octadecaenoicacid Fig. l.sfmcmsd-~~-lsle~.

showed identical properties to digoxin in NhIK and mass of spectroscopyM* Estimates serum concentration were in the a Endoponsly synthesized oua10-50p~ range, too low to be b&z. Similar procedures can be physiologicaIIy reIevantm. The used to purify substances of hysite of biosynthesis is not yet drophilic character from human known. fluids and bovine adrenals’“~u-14*17. ?? A digoxin-like substance, which Recently, the group of Hamlyn is not identical to authentic together with Ludens and his cocardenolides, has also been puworkers purified a substance from rified from hypothaIamus16akz. human plasma which was found This inhibitor, isolated by the to be identical to ouabains. High groups of Haupert and Sancho, is concentrations of this substance an acid-resistant zwitterion, and were found in adrenals’s. The differs from cardiac glycosides in serum concentrations of the enthat it is not selective for Na+iK+dogenously synthesized ouabain ATPase but also inhibits sarcoin normotensives have been estiplasmic reticular, but not lasma mated by an immunoassay to be Almembrane Ca’+-AT&s J. 53 + 8 PM”. To date most of the though th; hypothalamic inhibidata are consistent with the view tor binds to the cardiac glycoside that endogenously synthesized receptor site located MraceIMarly ouabain is the substance that on Na+/K+-ATPase”, its hydroexerts the physiological functions phobic character also allows it to ascribed to the circulating entraverse liposomal membraneP. dogenous digitalis-like factor. It increases the force of contrac?? Etnfogenonsly synthesized tion in beating rat heart cells and digotia Cloix had previously raises intracellular Ca2+ concenshown that human urine contains trations to levels exceeding those a hydrophobic substance that associated with ouabain-induced crossreacts with anti-digoxin antitoxicity; this rise has been specubocW9. More recently, Cot0 et lated to be due to inhibition of the al. have isolated a substance that Ca’+ pump by the factor at an crossreacts with anti-digoxin antiintracellular locatior?. This hypobodies from human urine. This thalamic factor also leads reverscould not be distinguished from ibly to contractions of vascular digoxin in three different HPLC rings of the rat aorta. However, and three TLC systems, and there is as yet no evidence that it

existence of at least three different types of endogenous digitalis-like substance (see Fig. 1):

circulates in bIood, and it is not

Otherfactomhaveakobcen claimed to have disihlir_like

aLu isolated the ara&icl~& acid derivative U(n)-hycIruxy5,&10,14eicosatehrenoic acid from beef cornea. AII these subetanees have been shown to act as inI-dbitctrs of Na+/K+-ATPase. The studies pubbshed so far raise more questions than can be answered. The physioIogieaI role, sites of bioqntbe& and m&anisms of controI of the biosynthesis of the various digitrll-wre substances are unknown. It is not clear whether the serum concentrations are sufficient to induce physioIogica1 effectsz for example, the dissociation constant of the ouabain receptor site of the human heart muscle is 2.51~. Assuming the concentration of formed ouabain to ~dcz&Sy rn * normotensives, only 2% of the cardiac giycoside binding sites would be occupied (and consequently 2% of Na+/K+ATPase inhibited). The threefold increase in this factor that has

Tip.5- ]une 1992 fI-‘ol.121 been measured in patients with essential hypertension” would allow 5.6% of the Pumps to be inhibited. Would this limited inhibition be sufficient to induce a rise in blood pressure? Isoenzymes of Na+/K+-ATPase exist, and these may differ in their affinity for cardiac glycosides in different tissues. It is conceivable that these isoenzymes might show a varying specificity for different cardiac glycosides - this could at least allow a concept of tissue specificity to provide some answers to some of the questions. WILHELM SCHONER lnstihr: fir Biochcmir und Endokrinologic, FachbcrcichVcterimirmcdizin.&&LicbigUnivcnitHt G&en, Frankfurter Sfr. 100, D63BBGicsscn,FRC.

References 1 De Wardener, H. E. and Clarkson, E. M. (19BS) Physiof.Rev. 65,6!%8-759

2 l’oston, L. (1990) Cordiovarc.DrugsTher. 4.351-356 3 Takahashi. H. cl al. (19%) Therapeutic Rcs.9,60-77 4 Masugi. F., Ogihara. T., Hasezawa, T. and ffumaha;, Y. (19B7) C&. .t.rp. Hypertension Theory Practice A9, X83-1242 5 Masugi,F. et al. (1996) B&hem. Biophys. Rcs. Commrn. 135,41-45 6 Hamlyn, j. M. (1990) in Diuretics 111 (llh& B&zreenbeq, A., eds),

, 7 Goto A. et al. (1988) B&hem. Biophys. Rcs. Common. 152.322-327 8 Harris, D. W., Clark, M. A., Ludens, 1. H., DuChrmw, D. W. and Ham+, J. M. (1990) ffypertension 16, 337, I?? (abstr.) 9 Hamlyn, J. M., Harris. D. W. and Ludens, J. H. (1988) FASEBJ. 2, A239, 194 (abstr.) 10 Goto, A. et al. (1988) Biochcm.Biophys. Rcs. Commun.152,322-327 11 Schoner, W., Moreth, K., Kuske, R. and Renner, D. (1966) in Cardiac Clycosidcs 1785-1985. Biochcmisfry.Pharmrcology. Clinical R&t once (Mmann, @., Greet, K. and Skau, 1. C., eds), pp. 13E142, Springer-Veclag 12 Ham&n, J. M., Harris, D. W. and Ludens, 1. H. (19B9) 1. Bio!. Chcm. 264,

Polymophhisrnin human Nacetyltransferase - the case of the miming allele It has been recognized for more than 30 years that N-acetylation of xenobiotics is subject to genetic variation in humans. While early observations had indicated a simple mend&n inheritance pattern for the acetyktor phenotype, with fast acetylation being an autosomel dominant characteristic’**, several subsequent studies had not been obviously compatible with such a system. Very recent molecular studks, however, have now clarified this issue by SUPporting the original observations and by offering explanations for the apparently aberrant results. The antitubercular drug isoniazid is a mono-substituted hydrazine which is N-acetylated through transfer of an acetyl group from acetyl coenzyme A. The original studies into N-acetylation were polymorphism prompted by the important clinical consideration of the rehtionship between the antitubercular effectiveness of isoniazid and its rate of inactivation, which paral-

lels the rate of acetylation of the drug. The antihypertensivc drug hydralazine is, like isoniazid, a mono-substituted hydrazine, and is also metabolized by N-acetylation. Again, polymorphism in its N-acetylation has clinical implications, since slow acetyktors show a greater reduction in blood pressure than fast acetyktors in response to hydralazine therapg. The slow acetylator phenotype also shows a strong association with the toxic side effects of both drugs’. Other substrates of Nacetyltransferase which are subject to inter-individual variation in acetylation indude the arylamines sulphamethazine (an antibiotic) and procainamide (an antiarrhythmic drug). The carcinogens 2-aminofluorene and henzidine also show polymorphic and benzidineN-acetyiation, induced bladder cancer has been associated with the slow acetylatoc phenotvpes. Since these early studies on the genetics of N-acetyltransferase,

7395-7404 13 Wenzel, M. et al. (1990) Physiol. Bohcmoslovaca 39,79-i% 14 Goto, A. et al. (1988) Biochm. Biophys. Rcs. Commun.154. B47-8S3 i5 Liihts:,-iz, D. c! a! (1991) 1. Endocnnol. 128.71-78 16 lllescas, M., Ricote, M., Mendez, E., Robles, R. G. and Sancho,J. (1990) FEBS Lctt. 261, 435-+&l 17 Tamun, M., Lam, T-T. and InaBami, T. (19BB)BiochemistryW, 4244-4253 18 Hamlyn, J. et 01. (1990) Hypcrtcnsion16, 316, Abstr. 10 19 Crabos, M., Grichois, M-L., Wainer, I. W. and Cloix, J-F. (1987) Eur. 1. Biochcm.162,129-135 20 Goto, A. et al. (1990) Biocbcm.Btophys. Rcs.Commun.173,1093-1101 21 Car%, C. T., Beme, M., Cantky, L. C. and Hauprt, C. T. (19BS)j. Biol. Chcm. 260,1027-1031 22 Anner, B. M., Rey, H. C., Mocsmayer, M., Mesmely, I. and Haupert, G. J. (1990) Am. 1. Physiol.258, F144-Fl53 23 HaUaq, H. A. and Haupert, G. T. (1989) Pmt. Nad Acad. Sri. USA 86. ltxxUxx4 24 Morgan, K. et al. (19B5) 1. Biof. Chcm. 260,13595-13600 25 schwa&man, M. L et al. (1987) Proc. Nat1 Acrd. Sci. USA 84,8125-B129

many investigations have considered this polymorphism at the protein level both in humans and in animal models. The genetic basis of N-acetyltransferase polymorphism has been confirmed in studies of fast and slow acetylating strains of rabbits, mice and hamsters’. Studies on animal models of N-acetyltransferase polymorphism have been important in providing protein sequence inforn@ion’j which has led to the identification of Nacetyltransferase genes in humans. These animal studies have, however, added some confusion, since the mechanism of generation of the slow acetylator phenotype in rabbits (through gene dektion? is different from the mechanism of generation of N-acetyltransferase polymorphism in humans. Early studies suggested a inheritance simple mendelian pattern, with the fast acetylator phenotype being a dominant characteristic. However, certain aspects of N-acetyltransferase a~tivity in humans have been difficult to reconcik with this. For exampk, the distribution of fast and slow acetyktor phm varies among diierent ethnic groups. Also, certain substrates of N-eEetylmetabolized transferase are