Endogenous digitalis: a review of the evidence

286 v i d e s evidence that EDRF is a spontaneously-released chemical moiety 3,4. The finding of F6rsterm a n n et al. s that EDRFs b i n d extensively to serum a l b u m i n is also of interest since it supports the idea that these agents are fatty acid derivatives. Thus, certain epoxides, aldehydes and h y d r o peroxides that might be formed from arachidonate, via a noncyclo-oxygenase p a t h w a y , represent good EDRF candidates 1,s-7. PAF-acether (1-0-alkyl-2-acetylsn - glyceryl- 3 - p h o s p h o c h o l i n e ) , which also b i n d s to a l b u m i n s and which produces e n d o t h e l i u m - i n d e p e n d e n t vasorelaxation, at least in some canine arteries 9, represents another EDRF-candidate. I read the interesting abstract of Long and Stone 2 while m y letter 1° was in press, and agree that they have p r o v i d e d good evidence for Ca2+-dependency of EDRF release in rat aorta. However, species-dif-

TIPS - July 1985 ferences a p p e a r to exist with regard to the nature of EDRF s. The half-life of EDRF has b e e n s h o w n to be a b o u t 24 s for r a b b i t aorta and about 49 s for canine femoral artery, and the half-lives of EDRFs a p p e a r to d e p e n d u p o n the pO2 of the b a t h i n g (perfusion) m e d i u m that is employed s. Thus, the shorter half-life of EDRF of r a b b i t aorta (about 6 s) reported b y Griffith et al. 1 could have been related to their use of a m e d i u m which had a high pO2 (Ref. 5). Q u e s t i o n s that r e m a i n are" is EDRF release Ca2+-dependent in all species, in all blood vessels? What is the optimal b a t h i n g m e d i u m (perfusion solution) for extending EDRF half-life, so that its chemical nature m i g h t be determined?

F. V. DEFEUDIS

Universitd Louis Pasteur, Facultd de Mddecine, 67084 Strasbourg Cedex, France

References 1 Griffith, T. M., Edwards, D. H., Lewis, M.J., Newby, A.C. and Henderson, A.H. (1984) Nature (London) 308, 645647 2 Long, C. J. and Stone, T. W. (1985) Br. J. Pharmacol. 84, 152P 3 Furchgott, R. F., Cherry, P. D., Zawadski, J. V. and lothianandan, D. (1984) J. Cardiovasc. Pharmacol. 6, $336-$343 4 Martin, W., ViUani, G. M., Jothianandan, D. and Furchgott, R. F. J. Pharmacol. Exp. Ther. (in press) 5 F6rstermann, U., Trogisch, G. and Busse, R. (1985). Eur. J. Pharmacol. 106, 639-643 6 DeFeudis, F.V. Med. Hypotheses (in press) 7 Davies, J.M., Hensby, C.N., Loginbuhl, B. and Williams, K.I. (1985) in Proc. Br. Pharmacol. Soc., Cardiff, 10-12 April 1985, p. 63 8 Rainsford, K. D. (1985) Trends Pharmacol. Sci. 6, 95-97 9 Houston, D.S. and Vanhoutte, P.M. (1984) Physiologist 27, 282 10 DeFeudis, F.V. (1985). Trends Pharmacol. Sci. 6, 63

W i t h e r i n g ' s observations on exogenous foxglove, a n d in the light of the discovery of ANP, to review the evidence for e n d o g e n o u s digitalis.

Endogenous digitalis: a review of the evidence M. R. Wilkins There is considerable support for the concept that the membrane-bound enzyme Na+ /K+-ATPase acts as a receptor for the drug digoxin. This begs the question 'Why should mammalian cells bear a receptor for a vegetable extract?'. In this article Martin Wilkins reviews the evidence for a circulating 'endogenous digitalis' with a role in blood volume regulation.

For over twenty years researchers have p u r s u e d the i d e n t i t y of a humoral factor, distinct from aldosterone, with a role in Na + excretion and extracellular fluid regulation. Secreted in response to central hypervolaemia, this factor is thought to circulate in plasma and stimulate a natriuresis to restore Na + and water balance (Fig. 1). There are currently two candidates for this role. During the last five years, a p e p t i d e called atrial natriuretic p e p t i d e (ANP) w i t h p o t e n t M. R. Wilkins is a Lecturer in Therapeutics and Clinical Pharmacology, Medical School, Edgbaston, Birmingham 15, UK.

natriuretic properties has been found in m a m m a l i a n atria and its a m i n o acid sequence has been d e t e r m i n e d 1. R a d i o i m m u n o a s s a y s for circulating h u m a n and animal A N P have been d e v e l o p e d and a rise in ANP-like i m m u n o r e a c t i v i t y has recently been d e m o n s t r a t e d in the plasma of v o l u m e - e x p a n d e d rats 2. More controversial is the second candidate - a digitalis-like substance thought to be secreted b y the h y p o t h a l a m u s and i n h i b i t Na + transport in renal tubules b y i n h i b i t i o n of N a + / K + - A T P a s e 3,4. The very existence of this factor is in some doubt. It seems appropriate in this anniversary year of

1985,ElsevierSciencePublishersB.V.,Amsterdam 0165- 6147/85/$02.00

Evidence for a humoral factor Pressure-induced natriuresis and the r e n i n - a n g i o t e n s i n - a l d o sterone system are well recognized m e c h a n i s m s for regulating Na + excretion. The principal evidence that a third factor - a humoral agent other than aldosterone - also modifies urinary N a + o u t p u t comes from experiments in which the blood volume of an animal p r e d o s e d w i t h large amounts of salt-retaining steroids is rapidly increased 3. It seems i m p o r t a n t that the e x p a n d i n g fluid is blood w h i c h has been allowed time to equilibrate w i t h that of the recipient animal; experiments using other fluids, such as saline, are accomp a n i e d b y h a e m o d i l u t i o n which complicates analysis of the results. The natriuretic response is assayed b y a d e n e r v a t e d k i d n e y left in situ or a k i d n e y perfused in a saline bath. In b o t h assay systems the perfusion pressure is controlled. A u t h o r s report rises in urinary N a + excretion of 50-100% (Ref. 3). Intrathoracic blood volume is the d o m i n a n t stimulus, rather than total b l o o d volume. Immersion of h u m a n s to the neck in water leads to d i s p l a c e m e n t of fluid from the

287

TIPS - J u l y 1985

lower extremities and to a 10% rise in intrathoracic blood volume 4 effectively, an autotransfusion of 500 ml of blood. A sustained increase in u r i n a r y N a + excretion occurs d e s p i t e a gradual decrease in total blood volume. Extracts from urine passed during this natriuresis are significantly more natriuretic in a rat b i o a s s a y than similar extracts of control urine s .

Intra-thoracic blood volume

J

-ve

/

s

Na + excretion

/

Experimental evidence for an endogenous inhibitor of Na+/K+-ATPase O n a philosophical level, comp a r i s o n s have been m a d e b e t w e e n e n d o g e n o u s digitalis and the end o g e n o u s opioids. The enzyme N a + / K + - A T P a s e has a high affinity b i n d i n g site for the cardiac glycosides and there is good evidence that the e n z y m e acts as a receptor for these drugs. This begs the q u e s t i o n - w h y should m a m malian cells bear receptors for vegetable extracts such as digoxin and digitoxin? In the example of the o p i o i d story the identification of receptors for exogenous opiates p r e c e d e d the discovery of the endorphins. Isolated reports over the past 16 years have p r o v i d e d more tangible evidence of an e n d o g e n o u s inhibitor of Na+/K+-ATPase. Kramer a n d colleagues 6 d e m o n s t r a t e d a slight b u t significant reduction in cortical N a + / K + - A T P a s e activity in the k i d n e y s of rats subjected to 90 m i n of m a i n t a i n e d extracellular fluid v o l u m e expansion. Gonick et al. 7 extracted a low tool. wt factor from the plasma of hypervolaemic rats w h i c h i n h i b i t e d Na + transport across frog skin and toad bladder, largely b y i n h i b i t i o n of N a + / K + - A T P a s e activity. G r u b e r et al. 8 used v o l u m e - e x p a n d e d dogs and extracted a small tool. wt factor from their plasma which i n h i b i t e d hog-brain Na+/K+-ATPase in v i t r o and cross-reacted with two different digoxin antibodies. De W a r d e n e r a n d colleagues 9 changed

Kidney

etch

receptors

-"

Release of natriuretic factors (? ANP; ? endogenous

digitalis)

Fig. 1. The regulation of blood volume - the proposed role of atria/nattiureticpeptide and

endogenous digitalis, Increased intrathoracic blood volume leads to stretching of receptors and release of natriuretic factors which promote a natnuresis.

a group of healthy volunteers from a low to a high salt diet and, using a sensitive cytochemical assay, d e m o n s t r a t e d a 25-fold increase in p l a s m a levels of a circulating inhibitor of Na+/K+-ATPase.

Pathological volume expansion If volume expansion is the stimulus to secretion of e n d o g e n o u s digitalis, one might expect to find increased circulating levels in pathologically volume expanded states. In 1964, Welt et al. 1° reported raised intraceUular Na + concentrations and reduced membrane ouabain-sensitive 42 K uptake in erythrocytes from patients with chronic renal failure; furthermore, this abnormality could be transferred to erythrocytes from healthy subjects b y incubating their cells in plasma from those uraemic patients with the highest intracellular Na + concentrations. These findings have since been

TABLE I. A comparison of the properties of endogenous digitalis and atrial natriuretic peptide Endogenous digitalis

Atrial natriuretic peptide

Structure Site of production

? (<500 mol. wt) ? Hypothalamus

28 amino acid peptide Secretory granules in cardiac atria

Biochemical action

Inhibits Na+/K+-ATPase

? (Does not inhibit Na+/K+.ATPase)

Action on vascular bed Elimination

Vasoconstriction Urinary excretion

Vasodilation Degradation in plasma

confirmed by others 11. Edmondson et al. 12 observed an impairment of glycoside-sensitive 22Na effiux from leucocytes from patients in renal failure and noted that this abnormality improved during regular haemodialysis. The authors suggest that their results are compatible with a small dialysable molecule in uraemic plasma affecting Na + transport. More recently Graves et al. 13 have reported false positive digoxin tests in 63% of 54 patients with chronic renal failure. This finding of cross-reactivity of uraemic plasma with digoxin antibodies complements the observations of Gruber et al. s using v o l u m e - e x p a n d e d dogs. An i m p a i r m e n t of leucocyte ouabain-sensitive Na + transport has been demonstrated in hepatic failure 13, another volume-expanded state. It is of interest that Nanji and Greenway 13 have recently reported falsely raised digoxin concentrations in six patients with chronic liver disease. De Wardener and MacGregor 16 and others have argued that essential hypertension is a condition in which, at least transiently, there is a phase of central hypervolaemia. Although this concept has been contested 17, there are numerous reports of reduced Na + transport in erythrocytes and leucocytes from patients with essential hy-

288 pertension. Poston et al. TM demonstrated that this i m p a i r m e n t could be induced in leucocytes from normotensive patients w h e n these ceils were incubated with the plasma of hypertensive patients. Two groups, MacGregor et al. 19 and H a m l y n et al. 2°, u s i n g different assays, have reported increased concentrations of a circulating inhibitor of Na+/K+-ATPase in plasma from hypertensive patients compared to normotensive controis. Rather surprisingly, perhaps, considering previous reports, Hamlyn et al. 2° could find no crossreactivity with digoxin antibodies. More recently Devynck et al. 21 have demonstrated a factor in the plasma of patients with essential hypertension which competes with radiolabelled ouabain for its b i n d ing site on erythrocytes.

Properties of endogenous digitalis Taken together these reports provide impressive circumstantial evidence for an e n d o g e n o u s digitalis-like substance. However, despite these specific sightings over the years, the i n h i b i t o r has remained elusive and its precise nature u n k n o w n . It is excreted into urine and fractionation of plasma and urine on Sephadex columns have demonstrated both high and low mol. wt factors with i n h i b i t o r y properties 4. The high mol. wt factor (around 30 000) is less potent and thought to be a precursor of the smaller factor (mol. wt ~500). The inhibitory activity of plasma is reputed to increase initially if it is left on the bench after v e n e p u n c ture (due to cleavage of the smaller factor from its precursor?) b u t subsequently it diminishes, perhaps due to degradation (Wilkins, M. R., unpublished observations). It is preserved be deproteinating the sample by boiling21 or acidification 2°, and so is both heat and acid stable, and it is best stored at -70°C. Unfortunately, attempts at further characterization have produced contradictory results 4,7.

Endogenous digitalis and/or ANP Since we have ANP, do we need to look for endogenous digitalis? ANP does not i n h i b i t Na+/K +-

T I P S - J u l y 1985

ATPase and so does not explain experiments in which volume exp a n s i o n has produced a rise in plasma i n h i b i t o r y activity. Furthermore, early experiments have suggested that the effects of ANP are not transferred to the recipient animal in cross-circulation protocols; this may be a consequence of its short half-life or a dependence on changes in perfusion pressure for its natriuretic effect. Conversely, cross-circulation experiments 22 suggest that the contribution of e n d o g e n o u s digitalis to the overall natriuretic response to acute volu m e expansion is relatively small; and the difference cannot wholly be explained by changes in renal arterial or venous pressure or aldosterone levels. Thus there is room for two natriuretic factors. Endogenous digitalis is thought to have a vasoconstrictor effect on peripheral vasculature whereas ANP is a vasodilator. Sagneila a n d MacGregor 23 have suggested that the existence of two natriuretic substances, one a vasopressor and the other a vasodilator, makes good sense and would allow the integrated control of both electrolyte and blood pressure homeostasis. [] [] [] There is good evidence for a humoral factor other than aldosterone with a role in Na + homeostasis. Evidence in favour of ANP is gathering and it would seem only a matter of time before the factors governing its secretion and mode of action are more clearly defined. Collectively, other reports point to a digitalis-like substance. The two natriuretic factors are not identical (Table I) and may be complementary. However, some doubts remain about e n d o g e n o u s digitalis. The principal reservation is that despite several years of study its precise nature remains u n k n o w n . If all the reports are true, this factor shares m a n y properties with exogenous foxglove: i n h i b i tion of Na+/K+-ATPase i n h i b i tion of Na + transport across ceil membranes, i n h i b i t i o n of radiolabelled glycoside b i n d i n g and cross-reactivity with digoxin anti-

bodies. This should aid its identification in plasma and plasma fractions. Considering then the rapidity with which ANP was identified, isolated and sequenced, one cannot help v i e w i n g with some suspicion the delay in characterizing e n d o g e n o u s digitalis.

References 1 Mills,I. H. (1984)Br. Med. J. 289,210-211 2 Lang, R.E., Tholken, H., Ganten, D., Luft, F. C., Ruskoaho,H. and Unger,Th. (1985) Nature (London) 314, 264-266 3 De Wardener,H. E. (1977)Clin. Sci. Mol. Med. 1-8

4 De Wardener,H. E. and Clarkson,E. M. (1982) Clin. Sci. 63, 415-420 5 Epstein, M., Bricker, N.S. and Bourgoignie, J. J. (1978) Kidney Int. 13, 152158 6 Kramer, H.J., Gonick, H. C., Paul, W. and Lu, E. (1969) Abstr. 4th Int. Congr. Nephrol. p. 373 7 Gonick, H. C., Kramer, H.J., Paul, W. and Lu, E. (1977) Clin. Sci. 53, 329-334 8 Gruber, K.A., Whitaker, J.M. and Buckalew, V. M. (1980) Nature (London) 287, 743--745 9 De Wardener, H. E., MacGregor, G. A., Clarkson, E. M., Alaghband-Zadeh, J., Bitensky,L. and Chayen,J. (1981)Lancet i, 411--412 10 Welt, L. G., Sachs, J. R. and McManus, T. J. (1964)Trans. Assoc. Amer. Physicians 77, 169-181 11 Kramer, H.J., Gospodinov, D. and Kruck, F. (1976)Nephron 16, 344-358 12 Edmondson,R. P. S., Hilton,P. J., Jones, N.F., Patrick, J. and Thomas, R.D. (1975) Clin. Sci. Mol. Med. 49, 213-216 13 Graves,S. W., Broiwn,B. and Valdes,R. (1983) Ann. Intern. Med. 99, 604-608 14 Jewell,R. B., Poston,L., Wilkinson,S. P. and Williams,R. (1984)Clin. Sci. 66, 741744 15 Nanjiu, A.A. and Greenway, D.C. (1985) Br. Med. J. 290, 432-433 16 De Wardener, H.E. and MacGregor, G. A. (1982) Lancet i, 1450-1454 17 Heagerty, A.M., Thurston, H., Bing, R. F. and Swales,J. D. (1983)Lancet i, 591 18 Poston, L., Sewell, R.B., Wilkinson, S.P., Richardson, P.J., Williams, R., Clarkson, E. M., MacGregor, G. A. and De Wardener,H. E. (1981)Br. Med. J. 282, 847M349 19 MacGregor, G.A., Fenton, S., Alaghband-Zadeh, J., Markandu, N.D., Roulston, J. E. and De Wardener, H. E. (1981) Br. Med. ]. 283, 1355-1357 20 Hamlyn, J. M., Ringel, R., Schaeffer, J., Levinson, P.D., Hamilton, B.P., Kowarski, A.A. and Blaustein, M.P. (1982) Nature (London) 300, 650~52 21 Devynck, M.A., Pernollet, M.G., Rosenfeld, J. B. and Meyer, P. (1983)Br. Med. J. 287, 631~34 22 Sonnenberg, H., Veress, A.T. and Pearce, J.W. (1972) ]. Clin. Invest. 51, 2631-2644 23 Sagnella, G.A. and MacGregor, G.A. (1984) Nature (London) 309, 666~67