543
expanded dogs" goes back 50 years-fmdings that were further strengthened by parabiotic studies of volume-expanded rats.14 Reports that de Wardener’s
EDITORIALS
Welcome to ouabain—a steroid hormone
new
Life imitates art, so it is said. Recent discoveries might also allow one to proclaim that physiology imitates pharmacology. Identification of the brainendorphin system1 and recognition of nitrates within the vascular endothelium2 testify that endogenous factors have long pre-empted some of the most potent preparations in the pharmacopoeia. Now we leam3--7 that the long-sought digitalis-like factor in plasma is in fact ouabain-a naturally occurring adrenocortical product with a potential to affect sodium homoeostasis and blood pressure regulation. Digitalis and chemically related steroids are potent inhibitors of the plasma membrane enzyme Na-KA TPase,8 which is one of four systems that have been identified for transporting monovalent cations across the cell membrane.9 By pumping three sodium ions outward in exchange for two potassium ions moving into the cell, the Na-K-ATPase transport system is the main regulator of cation distribution between the intracellular and extracellular fluid compartments. The system responds to a rise in either intracellular sodium or extracellular potassium, and by this means helps to preserve the net negative charge within the cell and therefore the level of electrical excitability. Digitalis-like compounds, by binding to specific sites on the enzyme, inhibit the pump; the intracellular negative charge is reduced and the cell membrane becomes depolarised. In vascular smooth muscle cells (including those of the heart) depolarisation increases CaZ+ influx, which in turn increases inotropic function and potentiates vasoconstriction of arteriolar resistance vessels.1O Several studies in hypertensive patients have shown abnormalities of sodium transport, including reduced activity of Na-KATPase.ll The negative correlation between enzyme activity in red blood cells and mean arterial pressure across a wide range of blood pressuresl2 suggests that there is a circulating inhibitor of Na-K-ATPase. Evidence of a pressor substance in plasma of volume-
natriuretic factor15 was also an Na-K-ATPase inhibitor16 spawned an intensive search for a humoral agent with both pressor and natriuretic activity. Further stimulus to the quest was the finding that plasma from volume-expanded dogs contained an endogenous digitalis-like substance17 which was subsequently identified in human beings as well, especially in patients with hypertension associated with volume expansion. 18-20 In the hypotheses advanced2l to link these diverse findings the kidney was viewed as the culprit in not fully excreting a sodium load. The suggestion was that the resultant volume expansion was "sensed" (possibly by the hypothalamus), evoking an increase in plasma endogenous digitalis-like factor, which in turn promoted sodium excretion (by inhibiting renal tubular Na-K-ATPase) and raised vasomotor tone with an increase in blood pressure. Some researchers believed that those two mechanisms acted synergistically to enhance natriuresis but at the cost of a sustained increase in blood pressure.22 The news3 that human plasma contains a ouabainlike compound that seems to account for the endogenous digitalis-like factor measured in previous studies is an important step towards understanding the regulation of blood volume and vasomotor tone. From 85 litres of plasma (drawn from patients undergoing routine plasmapheresis), Hamlyn and colleagues isolated 12 llg of a compound identified as ouabain by mass spectrometry. Further proof of the identity of the steroid was provided by its ability to inhibit Na-K-ATPase and ouabain-sensitive 86Rb uptake by red cells and to displace 3H ouabain from its binding site. The same compound stimulated an inotropic response from guineapig atrium identical to that evoked by ouabain standards of plant origin. No other compound with these properties was identified in plasma extracts. Using a sensitive enzyme-linked immunosorbent assay6 to measure the ouabain-like compound in tissues and plasma, the researchers provided evidence that the steroid is an adrenocortical product. Adrenal tissue extracts contained the highest concentrations (500-fold those in plasma), and plasma concentrations in rats were more than halved by bilateral adrenalectomy. Preliminary results with cultured bovine adrenocortical cells show that the hormone is released in vitro and can be inhibited by potassium (40 mmol/1). In three species (rat, dog, and man), plasma concentrations ranged from 37 to 138 pmol/l, and concentrations increased 12-fold in rats given deoxycorticosterone implants. That patients receiving complete parenteral nutrition for a week had similar plasma concentrations of ouabain to those of normal subjects3,6excludes a dietary source of the steroid. Thus the endogenous digitalis-like factor in human plasma is ouabain, or an isomer of ouabain, the main
544
of which is likely to be the adrenal cortex. However, since the purification procedure relied on inhibition of ouabain-sensitive 86Rb uptake by red blood cells to detect active fractions, the findings do not exclude other (non-ouabain-like) circulating inhibitors of Na-K-ATPase. The antibody used to measure ouabain showed only 5% cross reactivity with digoxin; more important, some commercial digoxin antisera did not recognise the ouabain material purified from plasma.6 The precise nature of this digoxin immunoreactive material remains to be determined, but it seems to be an inconsistent marker of Na-K-ATPase inhibition. Apart from providing a firm basis for measurement of the circulating hormone, what is the clinical relevance of these findings and what do they tell us about the regulation of sodium and volumedependent blood pressure? Could ouabain be yet another adrenal "hypertensogenic" steroid? Perhaps ouabain deficiency compromises myocardial contractility and contributes to shock in primary adrenocortical deficiency. Another suggestion is that ouabain, by increasing intracellular Ca2 + , contributes to inhibition of renin secretion. A similar mechanism could explain ouabain’s stimulation of atrial natriuretic peptide release from rat atrial tissue.23 Before grappling with these and many related issues we need to know more about the place of ouabain in human physiology. Clear evidence of secretion of the hormone-ie, a significant gradient across the adrenal cortex-is essential, and it is important to determine whether ouabain is regulated and if so in what way. It will be more difficult to define the physiological action in man. Although the Kd (6-14 nmol/1) for ouabainNa-K-ATPase3seems to exceed the circulating plasma concentration in man (138 [SD43] pmoll), concentrations in plasma may be much higher in volume expanded states, as is already suggested by studies in rats.3Moreover, some isoforms of the enzyme, as reported in rat brain tissue, may have increased affinity for ouabain.24 Establishing normal, volume-manipulated, and pathological ranges for plasma ouabain should help to interpret the acute and chronic effects of ouabain infusions, as shown in studies of other newly identified natriuretic hormones such as atrial natriuretic peptide. It remains to be seen whether endogenous ouabain can fulfil all the expectations raised by speculation about endogenous digitalis. source
1. Lord J, Waterfield
AA, Hughes J, Kosterlitz HW. Endogenous opioid peptides: multiple agonists and receptors. Nature 1977; 276: 495-99. 2. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987; 327: 524-26. Bova S, et al. Identification and characterization of a ouabain-like compound from human plasma. Proc Natl Acad Sci USA 1991; 88: 6259-63. 4. Ludens JH, Clark MA, DuCharme DW, et al. Purification of an endogenous digitalis-like factor from human plasma for structural analysis. Hypertension 1991; 17: 923-29. 5. Mathews WR, DuCharme DW, Hamlyn JM, et al. Mass spectral charcterization of an endogenous digitalis-like factor from human plasma. Hypertension 1991; 17: 930-35.
3. Hamlyn JM, Blaustein MP,
6. Harris DW, Clark MA, Fisher JF, et al. Development of an immunoassay for endogenous digitalis-like factor. Hypertension 1991; 17: 936-43. 7. Bova S, Blaustein MP, Ludens JH, Harris DW, DuCharme DW, Hamlyn JM. Effects of an endogenous ouabain-like compound on heart and aorta. Hypertension 1991; 17: 944-50. 8. Langer GA. Mechanism of action of the cardiac glycosides on the heart. Biochem Pharmacol 1981; 30: 3261-64. 9. Aviv A, Lasker N. Proposed defects in membrane transport and intracellular ions as pathogenic factors in essential hypertension. In: Laragh JH, Brenner BM, eds. Hypertension: pathophysiology, diagnosis, and management. New York: Raven, 1990: 923-37. 10. Haddy FJ. Potassium effects on contraction in arterial smooth muscle mediated by Na+,K+-ATPase. Fed Proc 1983; 42: 239-45. 11. Hilton PJ. Cellular sodium transport in essential hypertension. N Engl J Med 1986; 314: 222-29. 12. Rygielski D, Reddi A, Kuriyama S, Lasker N, Aviv A. Erythrocyte ghost
Na+-K+-ATPase and blood pressure. Hypertension 1987; 10: 259-66. 13. Solandt DY, Nassim R, Cowan CR. Hypertensive effect of blood from hypertensive dogs. Lancet 1940; i: 873-74. 14. Dahl LK, Knudsen KD, Iwai J. Humoral transmission of hypertension: evidence from parabiosis. Circ Res 1969; 24 (suppl 1): I-21-33. 15. de Wardener HE, Mills IH, Clapham WG, Hayer CJ. Studies on the efferent mechanism of the sodium diuresis which follows the administration of intravenous saline in the dog. Clin Sci 1961; 21: 249-58. 16. Gonick HC, Kramer HJ, Paul W, Lu E. Circulating inhibitor of sodium-potassium activated adenosine triphosphatase after expansion of extracellular fluid volume in rats. Clin Sci Mol Med 1977; 53: 329-34. 17. Gruber KA, Whitaker JM, Buckalew VH. Endogenous digitalis-like substance in plasma volume-expanded dogs. Nature 1980; 287: 743-45. 18. Valdes R Jr. Endogenous digoxin-immunoactive factor in human subjects. Fed Proc 1985; 44: 2800-05. 19. Deray G, Rieu M, Devynck MA, et al. Evidence of an endogenous digitalis-like factor in the plasma of patients with acromegaly. N Engl J Med 1987; 316: 575-80. 20. Masugi F, Ogihara T, Hasegawa T, et al. Circulating factor with ouabain-like immunoreactivity in patients with primary aldosteronism. Biochem Biophys Res Commun 1986; 135: 41-45. 21. de Wardener HE, MacGregor GA. Dahl’s hypothesis that a saluretic substance may be responsible for a sustained rise in arterial pressure: its possible role in essential hypertension. Kidney Int 1980; 18: 1-9. 22. Haddy FJ. Endogenous digitalis-like factor or factors. N Engl J Med 1987; 316: 621-23. 23. Morise T, Takeuchi Y, Okamoto S, Takeda R. Stimulation of atrial natriuretic peptide secretion and synthesis by Na-K-ATPase inhibitors. Biochem Biophys Res Commun 1991; 176: 875-81. 24. Urayama O, Sweadner KJ. Ouabain sensitivity of alpha 3 isozyme of rat Na,K-ATPase. Biochem Biophys Res Commun 1988; 156: 796-800.
Medium chain
acyl CoA dehydrogenase deficiency Fatty acids
are an
important
source
of energy and
catabolised in mitochondria by progressive 0-oxidation. The first step of this pathway is the formation of a double bond in the 2-3 position by an acyl CoA dehydrogenase. Three distinct dehydrogenases have been identified with overlapping substrate specificity for long, medium, and short chain acyl CoA esters.1 Inborn errors of the 0-oxidation pathway are now well recognised, the most frequently reported being deficiency of medium chain acyl CoA dehydrogenase (MCAD).z2 Presentations of MCAD deficiency include acute encephalopathy and hepatomegaly sometimes resembling Reye’s syndrome, sudden and unexpected death, and near-miss cot death. During these episodes patients may be found to have hypoketotic hypoglycaemia.33 Mean age of presentation is 15 months, and as many as 25% of patients die during the initial illness.3Symptoms are commonly precipitated by metabolic stress such as fasting or infection; between episodes patients usually
are
expanded dogs" goes back 50 years-fmdings that were further strengthened by parabiotic studies of volume-expanded rats.14 Reports that de Wardener’s
EDITORIALS
Welcome to ouabain—a steroid hormone
new
Life imitates art, so it is said. Recent discoveries might also allow one to proclaim that physiology imitates pharmacology. Identification of the brainendorphin system1 and recognition of nitrates within the vascular endothelium2 testify that endogenous factors have long pre-empted some of the most potent preparations in the pharmacopoeia. Now we leam3--7 that the long-sought digitalis-like factor in plasma is in fact ouabain-a naturally occurring adrenocortical product with a potential to affect sodium homoeostasis and blood pressure regulation. Digitalis and chemically related steroids are potent inhibitors of the plasma membrane enzyme Na-KA TPase,8 which is one of four systems that have been identified for transporting monovalent cations across the cell membrane.9 By pumping three sodium ions outward in exchange for two potassium ions moving into the cell, the Na-K-ATPase transport system is the main regulator of cation distribution between the intracellular and extracellular fluid compartments. The system responds to a rise in either intracellular sodium or extracellular potassium, and by this means helps to preserve the net negative charge within the cell and therefore the level of electrical excitability. Digitalis-like compounds, by binding to specific sites on the enzyme, inhibit the pump; the intracellular negative charge is reduced and the cell membrane becomes depolarised. In vascular smooth muscle cells (including those of the heart) depolarisation increases CaZ+ influx, which in turn increases inotropic function and potentiates vasoconstriction of arteriolar resistance vessels.1O Several studies in hypertensive patients have shown abnormalities of sodium transport, including reduced activity of Na-KATPase.ll The negative correlation between enzyme activity in red blood cells and mean arterial pressure across a wide range of blood pressuresl2 suggests that there is a circulating inhibitor of Na-K-ATPase. Evidence of a pressor substance in plasma of volume-
natriuretic factor15 was also an Na-K-ATPase inhibitor16 spawned an intensive search for a humoral agent with both pressor and natriuretic activity. Further stimulus to the quest was the finding that plasma from volume-expanded dogs contained an endogenous digitalis-like substance17 which was subsequently identified in human beings as well, especially in patients with hypertension associated with volume expansion. 18-20 In the hypotheses advanced2l to link these diverse findings the kidney was viewed as the culprit in not fully excreting a sodium load. The suggestion was that the resultant volume expansion was "sensed" (possibly by the hypothalamus), evoking an increase in plasma endogenous digitalis-like factor, which in turn promoted sodium excretion (by inhibiting renal tubular Na-K-ATPase) and raised vasomotor tone with an increase in blood pressure. Some researchers believed that those two mechanisms acted synergistically to enhance natriuresis but at the cost of a sustained increase in blood pressure.22 The news3 that human plasma contains a ouabainlike compound that seems to account for the endogenous digitalis-like factor measured in previous studies is an important step towards understanding the regulation of blood volume and vasomotor tone. From 85 litres of plasma (drawn from patients undergoing routine plasmapheresis), Hamlyn and colleagues isolated 12 llg of a compound identified as ouabain by mass spectrometry. Further proof of the identity of the steroid was provided by its ability to inhibit Na-K-ATPase and ouabain-sensitive 86Rb uptake by red cells and to displace 3H ouabain from its binding site. The same compound stimulated an inotropic response from guineapig atrium identical to that evoked by ouabain standards of plant origin. No other compound with these properties was identified in plasma extracts. Using a sensitive enzyme-linked immunosorbent assay6 to measure the ouabain-like compound in tissues and plasma, the researchers provided evidence that the steroid is an adrenocortical product. Adrenal tissue extracts contained the highest concentrations (500-fold those in plasma), and plasma concentrations in rats were more than halved by bilateral adrenalectomy. Preliminary results with cultured bovine adrenocortical cells show that the hormone is released in vitro and can be inhibited by potassium (40 mmol/1). In three species (rat, dog, and man), plasma concentrations ranged from 37 to 138 pmol/l, and concentrations increased 12-fold in rats given deoxycorticosterone implants. That patients receiving complete parenteral nutrition for a week had similar plasma concentrations of ouabain to those of normal subjects3,6excludes a dietary source of the steroid. Thus the endogenous digitalis-like factor in human plasma is ouabain, or an isomer of ouabain, the main
544
of which is likely to be the adrenal cortex. However, since the purification procedure relied on inhibition of ouabain-sensitive 86Rb uptake by red blood cells to detect active fractions, the findings do not exclude other (non-ouabain-like) circulating inhibitors of Na-K-ATPase. The antibody used to measure ouabain showed only 5% cross reactivity with digoxin; more important, some commercial digoxin antisera did not recognise the ouabain material purified from plasma.6 The precise nature of this digoxin immunoreactive material remains to be determined, but it seems to be an inconsistent marker of Na-K-ATPase inhibition. Apart from providing a firm basis for measurement of the circulating hormone, what is the clinical relevance of these findings and what do they tell us about the regulation of sodium and volumedependent blood pressure? Could ouabain be yet another adrenal "hypertensogenic" steroid? Perhaps ouabain deficiency compromises myocardial contractility and contributes to shock in primary adrenocortical deficiency. Another suggestion is that ouabain, by increasing intracellular Ca2 + , contributes to inhibition of renin secretion. A similar mechanism could explain ouabain’s stimulation of atrial natriuretic peptide release from rat atrial tissue.23 Before grappling with these and many related issues we need to know more about the place of ouabain in human physiology. Clear evidence of secretion of the hormone-ie, a significant gradient across the adrenal cortex-is essential, and it is important to determine whether ouabain is regulated and if so in what way. It will be more difficult to define the physiological action in man. Although the Kd (6-14 nmol/1) for ouabainNa-K-ATPase3seems to exceed the circulating plasma concentration in man (138 [SD43] pmoll), concentrations in plasma may be much higher in volume expanded states, as is already suggested by studies in rats.3Moreover, some isoforms of the enzyme, as reported in rat brain tissue, may have increased affinity for ouabain.24 Establishing normal, volume-manipulated, and pathological ranges for plasma ouabain should help to interpret the acute and chronic effects of ouabain infusions, as shown in studies of other newly identified natriuretic hormones such as atrial natriuretic peptide. It remains to be seen whether endogenous ouabain can fulfil all the expectations raised by speculation about endogenous digitalis. source
1. Lord J, Waterfield
AA, Hughes J, Kosterlitz HW. Endogenous opioid peptides: multiple agonists and receptors. Nature 1977; 276: 495-99. 2. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987; 327: 524-26. Bova S, et al. Identification and characterization of a ouabain-like compound from human plasma. Proc Natl Acad Sci USA 1991; 88: 6259-63. 4. Ludens JH, Clark MA, DuCharme DW, et al. Purification of an endogenous digitalis-like factor from human plasma for structural analysis. Hypertension 1991; 17: 923-29. 5. Mathews WR, DuCharme DW, Hamlyn JM, et al. Mass spectral charcterization of an endogenous digitalis-like factor from human plasma. Hypertension 1991; 17: 930-35.
3. Hamlyn JM, Blaustein MP,
6. Harris DW, Clark MA, Fisher JF, et al. Development of an immunoassay for endogenous digitalis-like factor. Hypertension 1991; 17: 936-43. 7. Bova S, Blaustein MP, Ludens JH, Harris DW, DuCharme DW, Hamlyn JM. Effects of an endogenous ouabain-like compound on heart and aorta. Hypertension 1991; 17: 944-50. 8. Langer GA. Mechanism of action of the cardiac glycosides on the heart. Biochem Pharmacol 1981; 30: 3261-64. 9. Aviv A, Lasker N. Proposed defects in membrane transport and intracellular ions as pathogenic factors in essential hypertension. In: Laragh JH, Brenner BM, eds. Hypertension: pathophysiology, diagnosis, and management. New York: Raven, 1990: 923-37. 10. Haddy FJ. Potassium effects on contraction in arterial smooth muscle mediated by Na+,K+-ATPase. Fed Proc 1983; 42: 239-45. 11. Hilton PJ. Cellular sodium transport in essential hypertension. N Engl J Med 1986; 314: 222-29. 12. Rygielski D, Reddi A, Kuriyama S, Lasker N, Aviv A. Erythrocyte ghost
Na+-K+-ATPase and blood pressure. Hypertension 1987; 10: 259-66. 13. Solandt DY, Nassim R, Cowan CR. Hypertensive effect of blood from hypertensive dogs. Lancet 1940; i: 873-74. 14. Dahl LK, Knudsen KD, Iwai J. Humoral transmission of hypertension: evidence from parabiosis. Circ Res 1969; 24 (suppl 1): I-21-33. 15. de Wardener HE, Mills IH, Clapham WG, Hayer CJ. Studies on the efferent mechanism of the sodium diuresis which follows the administration of intravenous saline in the dog. Clin Sci 1961; 21: 249-58. 16. Gonick HC, Kramer HJ, Paul W, Lu E. Circulating inhibitor of sodium-potassium activated adenosine triphosphatase after expansion of extracellular fluid volume in rats. Clin Sci Mol Med 1977; 53: 329-34. 17. Gruber KA, Whitaker JM, Buckalew VH. Endogenous digitalis-like substance in plasma volume-expanded dogs. Nature 1980; 287: 743-45. 18. Valdes R Jr. Endogenous digoxin-immunoactive factor in human subjects. Fed Proc 1985; 44: 2800-05. 19. Deray G, Rieu M, Devynck MA, et al. Evidence of an endogenous digitalis-like factor in the plasma of patients with acromegaly. N Engl J Med 1987; 316: 575-80. 20. Masugi F, Ogihara T, Hasegawa T, et al. Circulating factor with ouabain-like immunoreactivity in patients with primary aldosteronism. Biochem Biophys Res Commun 1986; 135: 41-45. 21. de Wardener HE, MacGregor GA. Dahl’s hypothesis that a saluretic substance may be responsible for a sustained rise in arterial pressure: its possible role in essential hypertension. Kidney Int 1980; 18: 1-9. 22. Haddy FJ. Endogenous digitalis-like factor or factors. N Engl J Med 1987; 316: 621-23. 23. Morise T, Takeuchi Y, Okamoto S, Takeda R. Stimulation of atrial natriuretic peptide secretion and synthesis by Na-K-ATPase inhibitors. Biochem Biophys Res Commun 1991; 176: 875-81. 24. Urayama O, Sweadner KJ. Ouabain sensitivity of alpha 3 isozyme of rat Na,K-ATPase. Biochem Biophys Res Commun 1988; 156: 796-800.
Medium chain
acyl CoA dehydrogenase deficiency Fatty acids
are an
important
source
of energy and
catabolised in mitochondria by progressive 0-oxidation. The first step of this pathway is the formation of a double bond in the 2-3 position by an acyl CoA dehydrogenase. Three distinct dehydrogenases have been identified with overlapping substrate specificity for long, medium, and short chain acyl CoA esters.1 Inborn errors of the 0-oxidation pathway are now well recognised, the most frequently reported being deficiency of medium chain acyl CoA dehydrogenase (MCAD).z2 Presentations of MCAD deficiency include acute encephalopathy and hepatomegaly sometimes resembling Reye’s syndrome, sudden and unexpected death, and near-miss cot death. During these episodes patients may be found to have hypoketotic hypoglycaemia.33 Mean age of presentation is 15 months, and as many as 25% of patients die during the initial illness.3Symptoms are commonly precipitated by metabolic stress such as fasting or infection; between episodes patients usually
are
