(Hypertension. 1995;26:1173-1176.)
© 1995 American Heart Association, Inc.
Articles |
Stress-Induced Elevation of Ouabainlike Compound in Rat Plasma and Adrenal
From the Second Department of Internal Medicine, University of Tokyo (A.G., H.N., M.O.); Department of Human Dry Dock, Sanraku Hospital (K.Y.); and Department of Physiology, Osaka City University (Y.T.) (Japan).
Correspondence to Atsuo Goto, MD, Second Department of Internal Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract Recent observations demonstrate the presence of neurosteroids and their rapid increase in response to acute stress. In view of a steroidal nature of ouabainlike compound, we tested the hypothesis that ouabainlike compound may participate in a homeostatic response to acute stress. Male Wistar rats were subjected to acute stress by swimming in water (22°C) for 10 minutes. The levels of ouabainlike compound in plasma, hypothalamus, pituitary, and adrenal at 10, 40, and 70 minutes (n=8 for each) after the end of swim stress were compared with nonstressed control levels (n=10). Ouabainlike compound was measured by a radioimmunoassay for ouabain. Plasma levels of corticosterone and catecholamines were also measured. Plasma corticosterone concentrations increased rapidly at 10 minutes (P<.01) and then declined. A trend for a rise in plasma catecholamines was found at 10 minutes. Adrenal levels of ouabainlike compound concomitantly increased at 10 minutes (P<.01, control: 58.9±5.9 pmol ouabain equivalents per gram; 10 minutes: 92.5±4.8; 40 minutes: 47.3±9.6; 70 minutes: 45.1±6.3). In contrast, the response of plasma ouabainlike compound was slow and doubled at 40 minutes (P<.01, control: 115±12 pmol ouabain equivalents per liter; 10 minutes: 132±23; 40 minutes: 226±53; 70 minutes: 117±16). Ouabainlike compound levels in hypothalamus and pituitary remained unaltered. These findings suggest that ouabainlike compound may function as a stress hormone.
Key Words: stress • hypothalamo-hypophyseal system • hormones
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Endogenous digitalis-like factors or Na+,K+- ATPase inhibitors, which inhibit sodium pump activity and modulate sodium homeostasis and blood pressure, have been studied for almost three decades.1 2 3 4 5 A steroidal compound, ouabain or its isomer (ouabainlike compound [OLC]), has been purified from human plasma and bovine hypothalamus and is a strong candidate for endogenous digitalis-like factor.6 It is uncertain whether OLC could exert the presumed hypertensive and natriuretic actions. Although a contribution of OLC to several types of hypertension has been suggested,7 8 9 10 the natriuretic action of OLC is more controversial.11 The exact physiological and pathophysiological roles of OLC remain to be determined.
The body responds to increased physical or psychological stress with activation of the endocrine hypothalamo-pituitary-adrenal axis and sympathetic nervous system.12 Recent observations point to a link between OLC and these endocrine and nervous systems. Central adrenergic neurons may be involved in the OLC synthesis and release.13 Brain OLC in situ may play roles in the central regulation of the sympathetic nervous system and cardiovascular and renal functions.14 15 16 Furthermore, OLCs are exclusively distributed in the neural tissues such as hypothalamus and adrenal medulla.17 On the other hand, the presence of "neurosteroids" has been established, and rapid and robust increases of these steroids have been detected in the brain and plasma of rats after acute stress.18 These two lines of evidence prompted us to test the hypothesis that OLC may participate in a homeostatic response to acute stress. We subjected rats to acute swim stress and examined the time-related changes of OLC levels in plasma and the hypothalamo-pituitary-adrenal axis.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Male Wistar rats weighing 330 g were maintained on normal rat chow and tap water. All procedures were in accordance with the guidelines of the Animal Experiment Committee of the University of Tokyo. Experimental rats were subjected to acute stress by swimming for 10 minutes in ambient temperature water (22°C) in a cylindrical container filled to 75% capacity. At 10, 40, and 70 minutes after the end of swim stress, the rats (n=8 for each) were euthanatized by decapitation. So baseline values could be obtained, nonstressed control rats (n=10) were also killed by decapitation.
Trunk blood was collected into a tube containing Na2EDTA. The brain, pituitary, and right adrenal were removed, and the right hypothalamus was dissected out according to the method described by Glowinski and Iversen.19 The tissues were frozen within 2 minutes after decapitation and stored at -80°C until analysis. OLC levels in plasma, hypothalamus, pituitary, and adrenal were measured by radioimmunoassay for ouabain. Tissue samples were homogenized with 5 vol distilled water, and the homogenates were centrifuged at 15 000 rpm for 60 minutes. The resulting supernatant and the plasma sample (1.0 mL) mixed with an equal volume of 0.1% trifluoroacetic acid solution were applied to a Sep-Pak C18 cartridge (Waters Associates) that had been activated with methanol and equilibrated with distilled water. After complete washing with 30 mL distilled water, OLC was eluted with 3 mL of 25% acetonitrile in water. The eluant was evaporated and assayed for OLC based on a radioimmunoassay for ouabain as described previously.20 Because we eluted the OLC-containing fraction with 25% acetonitrile in water from a Sep-Pak C18 cartridge, it is unlikely that our OLC levels included common adrenocortical steroids such as corticosterone, cortisol, and aldosterone.20 These steroids are too hydrophobic to be eluted at this acetonitrile concentration. The antiserum showed minimum cross-reactivity with common steroids as described previously.20
Plasma corticosterone was measured by radioimmunoassay. Plasma epinephrine, norepinephrine, and dopamine were determined by high-performance liquid chromatography with electrochemical detection.
Statistical evaluations were made by one-way ANOVA followed by Fisher's protected least significant difference test. Values are presented as mean±SEM; a value of P<.05 was taken as significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
As expected, plasma corticosterone concentrations were increased at 10 minutes after swim stress (P<.01) and then declined (P<.05, Fig 1, Table). As shown in Fig 2 and the Table, a trend for a rise in plasma catecholamines was found at 10 minutes after swim stress.
|
|
|
In contrast, the response of plasma OLC was slower than those of corticosterone and catecholamines, with a significant twofold rise observed at 40 minutes (P<.05) after swim stress (Fig 3, Table).
|
Fig 4 shows the time-related changes of tissue OLC levels after swim stress. OLC levels in adrenal were elevated (P<.01) at 10 minutes after swim stress (Table). Although OLC levels in hypothalamus and pituitary tended to decrease at 10 minutes, the changes were not significant. Correlation analysis among measured parameters revealed that OLC levels in adrenal correlated positively with plasma epinephrine levels (Fig 5, r=.426, P<.05).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
A variety of neurosteroids are now thought to be formed de novo in mammalian brain via the classic mevalonate pathway to cholesterol.21 22 Enzymes present in the brain catalyze the side-chain cleavage of cholesterol to pregnenolone and the oxidation of pregnenolone to progesterone.21 22 Furthermore, 5
-reductase and
3-oxidoreductase present in neurons and/or glial cells may
form representative neurosteroids such as
allopregnanolone and
allotetrahydrodeoxycorticosterone.21 22 These neuroactive
steroids are rapidly increased in the brain and plasma of rats after
exposure to swim stress and modulate 
-aminobutyric acid type A
receptor function by novel nongenomic mechanisms.18 It is
likely that these steroids are responsible for the restoration of
central nervous system homeostasis during stress. A steroidal compound, OLC, has been identified as a major biologically active endogenous digitalis-like factor in the human circulation6 and is now under investigation for potential roles in cardiorenal function and blood pressure control. Recent observations demonstrate the presence of OLC in the central nervous system and point to possible roles of brain OLC in the regulation of the sympathetic nervous system. Although it is still a matter of debate whether OLC is an endogenous or exogenous substance,23 OLC shares the nongenomic mechanism with the above-mentioned neurosteroids. OLC acts on the cell membrane Na+,K+-ATPase but not on the intracellular steroid receptor. Taking into account this accumulated information on OLC, we hypothesized that OLC might also participate in a homeostatic response to acute stress. A previous study in which OLC was determined by radioreceptor assay indicated an increase of OLC in hypothalamus.24 It has been reported that digoxin-like immunoreactive factors in human serum increased during prolonged exercise to exhaustion.25
In the present study acute stress resulted in significant increases of OLC levels in plasma and adrenal. The rise in adrenal OLC was observed at 10 minutes and coincided with that in plasma corticosterone. On the other hand, the elevation of plasma OLC was slow and evident at 40 minutes. The stress-induced increases in plasma and adrenal OLC levels may have important consequences in cardiovascular and endocrine functions. Physiologically relevant OLC concentrations may influence the contractility of the human and rat vasculature and hence blood pressure. Several reports indicate a chronic in vivo hypertensive action of ouabain in rats.7 8 Ouabain at concentrations of 0.1 to 1 nmol/L, which were observed in rat plasma, based on our assay method, can augment caffeine-evoked contractions in rat resistance arteries.26 Lower concentrations of ouabain than previously thought can produce vasoconstriction in rabbit aorta.27
We found that adrenal OLC levels correlated positively with plasma epinephrine. Although considerable evidence suggests that OLC is synthesized and secreted by the adrenal cortex,28 29 Takahashi et al17 have shown with immunocytochemistry using a monoclonal ouabain antibody that OLC is exclusively distributed in the neural tissues such as hypothalamus and adrenal medulla in rats. It has been reported that ouabain modulates catecholamine secretion from adrenal chromaffin cells.30 31 OLC in adrenal may play autocrine and/or paracrine roles and modulate catecholamine release. Although a significant relation of OLC with corticosterone levels was not found in the present study, it has been suggested that OLC may exert autocrine and/or paracrine effects on aldosterone secretion.32 Consistent with this hypothesis, prolonged infusion of ouabain raises plasma aldosterone levels.5 These overall effects of OLC on cardiovascular and endocrine functions may represent adaptive responses of the organism to acute stress.
It is still unclear whether OLC is an endogenous or exogenous substance. However, consistent with our previous work13 and an accompanying article,33 the rapid responses of OLC in plasma and adrenal (within 80 minutes in the present study) suggest an endogenous nature of OLC. The response of plasma OLC was slower than those of corticosterone and catecholamines. Thus, the temporal profile of the increases in OLC and corticosterone levels was different, and there was no correlation between plasma OLC and corticosterone concentrations. A similar slower response of OLC has been found in humans to prolonged corticotropin infusion compared with that of cortisol.34 Since the increase in plasma OLC levels follows that in corticosterone concentration, OLC may reflect increased corticosterone metabolites. Polar metabolites of corticosterone and cortisol such as 6ß-OH-corticosterone and 6ß-OH-cortisol have polarities similar to that of ouabain, and our sample could contain these metabolites.35 36 However, our antibody did not cross-react with 6ß-OH-corticosterone or 6ß-OH-cortisol at the concentration of 10-4 to 10-10 mol/L (unpublished data, 1995). It is unlikely that OLC simply represents the cross-reactive glucocorticoids with antiouabain antibody.
After acute swim stress the brain content and plasma levels of neurosteroids markedly increase.18 By contrast, OLC levels in hypothalamus and pituitary remained unaltered despite the rise in OLC levels in adrenal and tended to be lower at 10 minutes after swim stress. Although we have previously provided evidence that brain OLC may be modulated by adrenergic neurons and that the central nervous system may be the source of circulating OLC,13 the data in the present study appear not to support our contention. The peak levels of OLC in adrenal after swim stress occur 30 minutes before the peak levels in plasma. It is more likely that the adrenal may be the origin of elevated circulating OLC after acute stress. A similar study in adrenalectomized rats remains to be carried out to identify conclusively the source of OLC. However, our present finding does not imply that the response of OLC would be the same and the adrenal would be the source for all types of stimuli.
In conclusion, our findings indicate that OLC levels in plasma and adrenal rise in response to acute stress in parallel to the expected stimulation of corticosterone and suggest that OLC may function as a stress hormone with a time course different from that of corticosterone.
Received June 18, 1995; first decision August 1, 1995; accepted August 18, 1995.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. De Wardener HE, Clarkson EM. Concept of natriuretic hormone. Physiol Rev. 1985;65:658-759.
2.
Blaustein MP. Physiological
effects of endogenous ouabain: control of intracellular
Ca2+ stores and cell responsiveness.
Am J Physiol. 1993;264:C1367-C1387.
3. Haddy F, Overbeck HW. The role of humoral agents in volume expanded hypertension. Life Sci. 1976;19:935-947. [Medline] [Order article via Infotrieve]
4. Goto A, Yamada K, Yagi N, Yoshioka M, Sugimoto T. Physiology and pharmacology of endogenous digitalis-like factors. Pharmacol Rev. 1992;44:377-399. [Medline] [Order article via Infotrieve]
5. Hamlyn JM, Manunta P. Ouabain, digitalis-like factors and hypertension. J Hypertens. 1992;10(suppl 7):S99-S111.
6.
Hamlyn JM, Blaustein MP, Bova S, DuCharme DW, Harris
DW, Mandel F, Mathews WR, Ludens JH. Identification and
characterization of a ouabain-like compound from human
plasma. Proc Natl Acad Sci U S A. 1991;88:6259-6263.
7.
Yuan C, Manunta P, Hamlyn JM, Chen S, Bohen E, Yeun J,
Haddy FJ, Pamnani M. Long-term ouabain administration
produces hypertension in rats.
Hypertension. 1993;22:178-187.
8. Manunta P, Rogowski AC, Hamilton BP, Hamlyn JM. Ouabain-induced hypertension in the rat: relationships among plasma and tissue ouabain and blood pressure. J Hypertens. 1994;12:569-576. [Medline] [Order article via Infotrieve]
9. Yamada K, Goto A, Nagoshi H, Chen Hui, Yagi N, Sasabe M, Omata M. Participation of ouabainlike compound in reduced renal mass–saline hypertension. Hypertension. 1994;23(suppl I):I-110-I-113.
10. Gomez-Sanchez EP, Gomez-Sanchez CE, Fort C. Immunization of Dahl SS/jr with an ouabain conjugate mitigates hypertension. Am J Hypertens. 1994;7:591-596. [Medline] [Order article via Infotrieve]
11. Ludens JH, Clark MA, Kolbasa KP, Hamlyn JM. Digitalis-like factor and ouabain-like compound in plasma of volume-expanded dogs. J Cardiovasc Pharmacol. 1993;22(suppl 2):S38-S41.
12.
Axelrod J, Reisine TD. Stress hormones: their
interaction and regulation. Science. 1984;224:452-459.
13. Yamada K, Goto A, Omata M. Modulation of the levels of ouabain-like compound by central catecholamine neurons in rats. FEBS Lett. 1995;360:67-69. [Medline] [Order article via Infotrieve]
14.
Huang BS, Harmsen E, Yu H, Leenen FHH. Brain
ouabain-like activity and the
sympathoexcitatory and pressor effects of
central sodium in rats. Circ Res. 1992;71:1059-1066.
15.
Yamada K, Goto A, Nagoshi H, Chen H, Omata M.
Role of brain ouabainlike compound in central nervous
system–mediated natriuresis in rats.
Hypertension. 1994;23:1027-1031.
16.
Huang B, Leenen FHH. Brain `ouabain,' sodium,
and arterial baroreflex in spontaneously hypertensive
rats. Hypertension. 1995;25:814-817.
17. Takahashi H, Ihara N, Terano Y, Yamada H, Nishimura M, Nakanishi T, Yamamoto K, Kinoshita Y, Yoshimura M. Ouabain-like immunoreactive substances exist in the hypothalamus and the adrenal medulla in rats. Pathophysiology. 1994;1:25-28.
18.
Purdy RH, Morrow AL, Moore PH, Paul SM.
Stress-induced elevations of -aminobutyric acid type A
receptor-active steroids in the rat brain. Proc Natl
Acad Sci U S A. 1991;88:4553-4557.
19. Glowinski J, Iversen LL. Catecholamine regional metabolism in rat brain. J Neurochem. 1966;13:655-669. [Medline] [Order article via Infotrieve]
20.
Yamada K, Goto A, Chen H, Yagi N, Nagoshi H, Sasabe M,
Sugimoto T. Role of ouabainlike compound in rats with
reduced renal mass-saline hypertension. Am J
Physiol. 1994;266:H1357-H1362.
21. Majewska MD. Steroids and brain activity. Biochem Pharmacol. 1987;36:3781-3788. [Medline] [Order article via Infotrieve]
22. Paul SM, Purdy RH. Neuroactive steroids. FASEB J. 1992;6:2311-2322. [Abstract]
23.
Doris PA, Jenkins LA, Stocco DM. Is ouabain an
authentic endogenous mammalian substance derived from the
adrenal? Hypertension. 1994;23:632-638.
24. Rauch AL, Callahan MF, Buckalew VM Jr. Influence of stress on plasma and tissue levels of an endogenous ligand to the digitalis receptor. Kidney Int. 1988;33:304. Abstract.
25. Valdes R Jr, Hagberg JM, Vaughan TE, Lau BWC, Seals DR, Ehsani AA. Endogenous digoxin-like immunoreactivity is increased during prolonged strenuous exercise. Life Sci. 1988;42:103-110. [Medline] [Order article via Infotrieve]
26.
Weiss DN, Blaustein MP. Nanomolar ouabain
augments caffeine-evoked contractions in rat arteries.
Am J Physiol. 1993;265:C1443-C1448.
27.
Stewart L, Hamilton C, Ingwall J, Naomi S, Graves S,
Canessa M, Williams G, Hollenberg N. Vascular smooth muscle
response to ouabain: relation of tissue Na+ to the
contractile response. Circ Res. 1993;73:1113-1120.
28.
Boulanger BR, Lilly MP, Hamlyn JM, Laredo J, Shurtleff
D, Gann DS. Ouabain is secreted by the adrenal gland in awake
dogs. Am J Physiol. 1993;264:E413-E419.
29. Laredo J, Hamilton BP, Hamlyn JM. Ouabain is secreted by bovine adrenocortical cells. Endocrinology. 1994;135:794-797. [Abstract]
30. Yanagihara N. Stimulatory effect of ouabain on catecholamine synthesis in bovine adrenal medullary slices. Tokushima J Exp Med. 1980;27:63-68. [Medline] [Order article via Infotrieve]
31. Yoshizumi M, Ishimura Y, Masuda Y, Houchi H, Kato I, Oka M. Inhibition by ouabain of palytoxin-induced catecholamine secretion and calcium influx into cultured bovine adrenal chromaffin cells. Biochem Pharmacol. 1994;48:1047-1049. [Medline] [Order article via Infotrieve]
32. Szalay KS. Ouabain: a local, paracrine, aldosterone synthesis regulating hormone? Life Sci. 1993;52:1777-1780. [Medline] [Order article via Infotrieve]
33. Yamada K, Goto A, Nagoshi H, Terano Y, Omata M. Ouabainlike compound as a defensive factor against excessive body sodium. Hypertension. 1995;25:1413. Abstract.
34. Cayosa LB, Hamilton BP, Manunta P, Hagen AS, Laredo J, Hamlyn JM. Effect of ACTH and angiotensin II on plasma ouabain. J Hypertens. 1994;12(suppl 3):S164. Abstract.
35. Watlington CO, Atkins JL, McNeil JS, Grogan WM, Johnson JP. Corticosterone is converted to 6ß-hydroxycorticosterone in rat: effects of the metabolite on urinary electrolyte excretion. J Steroid Biochem. 1988;31:947-954. [Medline] [Order article via Infotrieve]
36. Graves SW, Glatter KA, Lazarus JM, Williams GH, Hollenberg NK. Volume expansion in renal failure patients: a paradigm for a clinically relevant [Na,K]ATPase inhibitor. J Cardiovasc Pharmacol. 1993;22(suppl 2):S54-S57.
This article has been cited by other articles:
 |
|
 |
|
  A. Y. Bagrov, J. I. Shapiro, and O. V. Fedorova Endogenous Cardiotonic Steroids: Physiology, Pharmacology, and Novel Therapeutic Targets Pharmacol. Rev., March 1, 2009; 61(1): 9 - 38. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Schoner and G. Scheiner-Bobis Endogenous and exogenous cardiac glycosides: their roles in hypertension, salt metabolism, and cell growth Am J Physiol Cell Physiol, August 1, 2007; 293(2): C509 - C536. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Kotova, L. Al-Khalili, S. Talia, C. Hooke, O. V. Fedorova, A. Y. Bagrov, and A. V. Chibalin Cardiotonic Steroids Stimulate Glycogen Synthesis in Human Skeletal Muscle Cells via a Src- and ERK1/2-dependent Mechanism J. Biol. Chem., July 21, 2006; 281(29): 20085 - 20094. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Bauer, J. Muller-Ehmsen, U. Kramer, N. Hambarchian, C. Zobel, R. H.G. Schwinger, H. Neu, U. Kirch, E.-G. Grunbaum, and W. Schoner Ouabain-Like Compound Changes Rapidly on Physical Exercise in Humans and Dogs: Effects of {beta}-Blockade and Angiotensin-Converting Enzyme Inhibition Hypertension, May 1, 2005; 45(5): 1024 - 1028. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. V. Fedorova, D. E. Anderson, E. G. Lakatta, and A. Y. Bagrov Interaction of NaCl and behavioral stress on endogenous sodium pump ligands in rats Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2001; 281(1): R352 - R358. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Yamada, A. Goto, H. Nagoshi, Y. Terano, and M. Omata Elevation of Ouabainlike Compound Levels With Hypertonic Sodium Chloride Load in Rat Plasma and Tissues Hypertension, July 1, 1997; 30(1): 94 - 98. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||