- Email: [email protected]
Adrenocorticotropin-Induced Hypertension in Rats
AJH 1997; 10:403 – 408
Adrenocorticotropin-Induced Hypertension in Rats Role of Ouabain-like Compound Kaoru Yamada, Atsuo Goto, and Masao Omata
We examined the role of ouabain-like compound ( OLC ) in hypertension associated with corticotropin ( ACTH ) excess in rats. Physiological saline solution ( 1 mL / kg ) or synthetic ACTH-Z ( 0.5 mg / kg ) was injected intramuscularly for 15 days to 14 control and 13 male Wistar rats. Significant increases in blood pressure and plasma sodium/ potassium ratio, and decreases in plasma potassium concentration and urinary sodium/ potassium ratio were observed in ACTHtreated rats. The plasma OLC level was higher in ACTH-treated group ( control; 76 { 13, ACTH; 202 { 48 pmol / L, P õ .05 ) . Plasma OLC level correlated with systolic blood pressure ( SPB; r Å 0.53, P õ .01 ) . Urinary OLC excretion was also higher in ACTH-treated group ( control; 0.95 { 0.01, ACTH; 3.32 { 0.67 pmol / day, P õ .01 ) . A
I
njection of corticotropin ( ACTH ) produces increases in blood pressure, together with a variety of electrolyte and metabolic effects in humans, 1,2 sheep, 3,4 and rats.5 – 9 ACTH-induced hypertension is dependent on the adrenal cortex and is prevented by adrenalectomy.1,6 Although ACTH-induced
Received April 22, 1996. Accepted September 30, 1996. From the Department of Human Dry Dock, Sanraku Hospital ( KY ) and Second Department of Internal Medicine, University of Tokyo ( AG, MO ) , Tokyo, Japan. This study was supported in part by a Grant-in-Aid ( 07457164 ) from the Ministry of Education, Science, and Culture, Japan and a Grant from Japan Cardiovascular Research Foundation. Address correspondence and reprint requests to Kaoru Yamada, MD, Department of Human Dry Dock, Sanraku Hospital, 2-5 Kanda-surugadai, Chiyoda-ku, Tokyo 101, Japan.
q 1997 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
/ 2e15$$2180
03-10-97 11:33:06
significant relation was also found between urinary OLC excretion and SBP ( r Å 0.66, P õ .01 ) . Plasma potassium concentration negatively correlated with SBP ( r Å 00.48, P õ .01 ) and urinary sodium/ potassium ratio also correlated inversely with urinary OLC excretion ( r Å 00.55, P õ .01 ) . Measurement of OLC levels after the fractionation of urine by reverse-phase high performance liquid chromatography showed that the major OLC peak in urine from both groups coincided with that of authentic ouabain. These results suggest the contribution of OLC to ACTHinduced hypertension in rats. q 1997 American Journal of Hypertension, Ltd. Am J Hypertens 1997; 10:403 – 408 KEY WORDS: Ouabain-like compound, corticotropin ( ACTH ) , potassium.
hypertension is not simply a form of ‘‘mineralocorticoid’’ hypertension, several studies have demonstrated an essential component of mineralocorticoid hypertension in the onset of ACTH hypertension. Several mineralocorticoid antagonists, including spironolactone, blocked the development of ACTH hypertension in sheep.4 In human ectopic ACTH syndrome, hypertension has mainly been attributed to cortisol inactivation overload giving rise to mineralocorticoidtype hypertension through cortisol occupancy of mineralocorticoid receptors.10 It has been proposed that sodium retention stimulates release of a sodium pump inhibitor with a digitalis-like action, causing vasoconstriction and hypertension in the state of mineralocorticoid excess.11 – 13 Consistent with this view, ACTH increased blood
eajha
EL: AJH
0895-7061/97/$17.00 PII S0895-7061(96)00449-9
404
AJH–APRIL 1997–VOL. 10, NO. 4, PART 1
YAMADA ET AL
pressure in parallel with plasma levels of digoxinlike substances ( DLS ) in rats and there was a positive correlation between DLS and blood pressure. 14 Because a major biologically active sodium pump inhibitor in circulation may be a ouabain-like compound ( OLC ) , 15,16 we recently measured plasma OLC levels in a hypertensive patient with ectopic ACTH syndrome and found a marked increase in plasma OLC levels and its correlation with blood pressure.17 In the present study, we tested the hypothesis that OLC may contribute to ACTH-induced hypertension in rats.
METHODS Experimental Animals and Protocol This study was approved by the Animal Experiment Committee of the University of Tokyo. Male Wistar rats weighing 130 to 140 g were raised in a room with alternating 12-h cycles of light and given free access to a normal rat chow containing 0.8% NaCl ( Oriental Kobo Co., Tokyo, Japan ) and tap water. Rats were divided into two groups. Thirteen rats were given 0.5 mg / kg / day ( 1 mL / kg ) synthetic ACTH-Z ( Daiichi Pharmaceutical Co., Tokyo, Japan ) intramuscularly at 4 PM for 15 days. Control rats ( n Å 14 ) received an intramuscular injection of physiological saline solution ( 1 mL / kg ) for 15 days. Body weight ( BW ) and systolic blood pressure ( SBP ) by the tail-cuff method were measured on days 6 and 16. Eight rats from each group were placed in individual metabolic cages for 24 h after the last injections on day 15. Water intake and urine volume were measured. Urine samples were used for the measurements of OLC and electrolytes concentrations. Twenty-four hours after the last injection, the animals were anesthetized by pentobarbital sodium ( 50 mg / kg ) , and the trunk blood was collected for the measurement of plasma OLC, electrolyte concentrations, and serum corticosterone levels. The brain, pituitary, heart, right kidney, and right adrenal were removed, and the right hypothalamus was dissected out according to the method described by Glowinski and Iversen.18 The tissues were weighed and OLC levels were measured in hypothalamus, pituitary, and adrenal. Measurement of OLC The tissue samples were homogenized with 5 volumes of distilled water and the homogenates were centrifuged at 15,000 rpm for 60 min. The resulting supernatant, urine sample ( 1 mL ) , or plasma sample ( 1 mL ) , mixed with the equal volume of 0.1% TFA solution, was applied to the Sep-Pak C18 cartridge that had been activated with methanol and equilibrated with distilled water. After complete washing with 30 mL of distilled water, OLC was eluted with 3 mL of 25% acetonitrile in water. The eluent was evaporated and assayed for OLC as described in our previous report.19 The antiserum used for the radioimmunoassay showed minimum cross-reactivity with common
/ 2e15$$2180
03-10-97 11:33:06
steroids ( cortisol, cortisone, deoxycorticosterone, testosterone, progesterone, dehydroepiandrosterone sulfate, and b-estradiol ) . Polar metabolites of corticosterone and cortisol such as 6b-OH-corticosterone and 6bOH-cortisol have a similar polarity to that of ouabain, and our sample may have contained these metabolites. However, our antibody did not cross-react with 6bOH-corticosterone and 6b-OH-cortisol at the concentration of 10 04 to 10 010 mol / L ( unpublished data, 1995 ) .
OLC Measurement in Urine After HPLC Analysis We measured OLC levels in urine after the fractionation by reverse-phase high performance liquid chromatography ( HPLC ) . Collected urine ( 100 mL ) from control and ACTH-treated rats were applied to the Sep-Pak C18 cartridge that had been activated with methanol and equilibrated with distilled water. The fraction that was eluted by 25% acetonitrile after complete washing with distilled water was analyzed on a R-ODS-5 column ( Yamamura Chemical Co., Kyoto, Japan ) , with a linear gradient of acetonitrile in water ( 0% to 30% ) for 30 min after washing with water for 9 min at 1 mL /min. Oneminute fractions were collected and assessed for OLC. Measurements of Hormone and Electrolytes Plasma corticosterone levels were measured by radioimmunoassay. Sodium and potassium concentrations were measured by flame photometry. Statistical Analysis The data, expressed as mean { SEM, were analyzed by the unpaired Student’s t test. A P value õ .05 was considered significant. RESULTS Sham injection was associated with an increase in SBP from 124 { 2 to 136 { 2 on day 6 and to 133 { 2 mm Hg at the end of the injection period ( P õ .01 ) . ACTH injection significantly increased SBP from 122 { 3 to 146 { 3 on day 6 and to 153 { 4 on day 16 ( P õ .01 ) . The effect of ACTH treatment was significantly greater ( P õ .01 ) than that of sham injection, both on days 6 and 16. Although both groups gained significant weight over the period of study, the BW of the ACTH-treated
TABLE 1. BODY WEIGHT AND TISSUE WEIGHTS OF CONTROL AND ACTH-TREATED RATS Control (n Å 14) Body weight (g) Cardiac weight (mg/100 g BW) Renal weight (mg/100 g BW) Adrenal weight (mg/100 g BW) Pituitary weight (mg/100 g BW)
218 305 422 9.5 3.7
{ { { { {
6 5 10 0.4 0.1
ACTH (n Å 13) 171 373 508 38.0 4.4
{ { { { {
7* 9* 10* 3.3* 0.2*
Values are mean { SEM. ACTH, adrenocorticotrophin; BW, body weight. *P õ .01 compared with control.
eajha
EL: AJH
AJH–APRIL 1997–VOL. 10, NO. 4, PART 1
ACTH-INDUCED HYPERTENSION AND OUABAIN-LIKE COMPOUND
TABLE 2. EFFECTS OF ACTH TREATMENT ON MEASURED PARAMETERS
Water intake (mL/day) Urine volume (mL/day) Urinary Na excretion (mmol/day) Urinary K excretion (mmol/day) Na/K ratio in urine Plasma Na concentration (mmol/L) Plasma K concentration (mmol/L) Na/K ratio in plasma
Control (n Å 14)
ACTH (n Å 13)
47.5 { 21.9 31.9 { 12.5
57.9 { 19.9 30.8 { 5.9
0.44 { 0.05
0.30 { 0.11
1.03 { 0.09 0.43 { 0.04
1.20 { 0.22 0.22 { 0.03*
138.1 { 0.4 5.5 { 0.1 25.3 { 0.6
139.4 { 0.7 4.2 { 0.2* 33.8 { 1.6*
Values are mean { SEM. ACTH, adrenocorticotrophin; BW, body weight; OLC, ouabain-like compound. *P õ .01 compared with control. Water intake and urine data were obtained from eight rats in each group.
group was significantly lower than that of the control group ( P õ .01, Table 1 ) . Effects of ACTH treatment on electrolyte homeostasis were summarized in Table 2. Water intake, urine volume, and urinary sodium and potassium excretions were not different between the two groups. Sodium/ potassium ratio in urine was significantly lower in ACTH-treated groups than in the control group ( P õ .01 ) . Plasma sodium concentrations were not different between the two groups, but ACTH treatment significantly decreased plasma potassium concentration and increased plasma sodium/ potassium ratio ( P õ .01 ) . ACTH treatment increased cardiac, renal, adrenal, and pituitary weights as compared with sham injection ( P õ 0.01, Table 1 ) .
405
Table 3 shows the effects of ACTH treatment on hormonal parameters. Urinary OLC excretion was significantly higher in the ACTH-treated group ( control, 0.95 { 0.10; ACTH, 3.32 { 0.67 pmol / day, P õ .01 ) . The plasma OLC level was also significantly higher in the ACTH-treated group ( control 76 { 13; ACTH, 202 { 48 pmol / L, P õ .05 ) . OLC levels in the hypothalamus, pituitary, and adrenal, as well as plasma corticosterone levels, showed no significant differences between the two groups. Plasma OLC levels correlated with SBP ( Figure 1A; r Å 0.53, P õ .01 ) . A significant relationship also was found between urinary OLC excretion and SBP ( Figure 1B; r Å 0.66, P õ .01 ) . Plasma potassium concentrations negatively correlated with SBP ( Figure 2A; r Å 00.48, P õ .01 ) , and sodium/ potassium ratios in urine showed inverse correlations with urinary OLC excretion ( Figure 2B, r Å 00.55, P õ .01 ) . The major OLC peak in urine from both groups appeared at 29 to 30 min and coincided with that of authentic ouabain on HPLC ( Figure 3 ) . Another broad immunoreactive peak was found in fractions corresponding to more hydrophobic material.
TABLE 3. HORMONAL PARAMETERS OF CONTROL AND ACTH-TREATED RATS
Urinary OLC excretion (pmol/day) Plasma OLC level (pmol/L) OLC level in hypothalamus (pmol/g) OLC level in pituitary (pmol/g) OLC level in adrenal (pmol/g) Serum corticosterone concentration (ng/mL)
Control (n Å 14)
ACTH (n Å 13)
0.95 { 0.10
3.32 { 0.67†
76 { 13
202 { 48*
0.87 { 0.16
0.79 { 0.22
4.71 { 0.76
7.67 { 0.67
2.39 { 0.65
1.75 { 0.36
330 { 57
373 { 89
Values are mean { SEM. ACTH, adrenocorticotrophin; OLC, ouabain-like compound. *P õ .05, †P õ .01 compared with control. Data of urinary OLC excretion were obtained from eight rats in each group.
/ 2e15$$2180
03-10-97 11:33:06
FIGURE 1. Relations between systolic blood pressure ( SBP ) and OLC levels in plasma ( A ) or urinary OLC excretion ( B ) . There was a significant correlation between SBP and OLC levels in plasma ( r Å 0.53, P õ .01 ) . Urinary OLC excretion also was correlated with SBP ( r Å 0.66, P õ .01 ) .
eajha
EL: AJH
406
AJH–APRIL 1997–VOL. 10, NO. 4, PART 1
YAMADA ET AL
FIGURE 2. Relations between plasma potassium concentration and systolic blood pressure ( A ) , and between sodium/ potassium ratio in urine and urinary OLC excretion ( B ) . There was a significant inverse correlation between plasma potassium concentration and systolic blood pressure ( r Å 00.48, P õ .05 ) . Sodium/ potassium ratio in urine was negatively correlated with urinary OLC excretion ( r Å 00.55, P õ .05 ) .
excretion. Whitworth et al have recently shown that ACTH administration in the rat produces an adrenally dependent rise in blood pressure, fall in body weight and plasma potassium concentration, and increases in water intake, urine output, urinary sodium, and potassium excretion as well as in adrenal, kidney, and cardiac weight.6 Because we measured the parameters of water and electrolyte homeostasis only on the final day, we cannot rule out the possibility that the rats were not accustomed to metabolic cages. Differences in sampling day also may explain the discrepancy between the results of our study and those of previous studies. ACTH-induced hypertension appears to be adrenally dependent and has been attributed to adrenocortical steroids. In this study, ACTH treatment significantly decreased plasma potassium concentration, increased plasma sodium/ potassium ratio, and decreased urine sodium/ potassium ratio as compared with ratios for control rats, suggesting an excess mineralocorticoid state in ACTH-treated rats. In our study, serum concentrations of corticosterone, the major glucocorticoid in rats, were not significantly increased in ACTH-treated rats. Since plasma corticosterone concentration was higher in ACTH-treated rats in previous studies, 8 differences in administration method ( injection v continuous infusion ) , anesthesia, dose, duration, or sampling time may account for this discrepancy. Although we did not measure serum levels of other mineralocorticoids such as deoxycorticosterone, these mineralocorticoid effects are well known consequences of ACTH administration. In this study, we have shown that both plasma OLC levels and urinary OLC excretions were threefold higher in ACTH-treated rats, and both correlated with SBP. Li et al have shown that ACTH increased plasma
DISCUSSION In this study, we measured OLC in plasma, urine, and several tissues to examine the contribution of OLC to ACTH-induced hypertension in rats. We found that ACTH administration increased threefold the OLC levels in plasma and urine in association with a rise in blood pressure. Furthermore, blood pressure levels correlated positively with both plasma and urine OLC levels. In agreement with previous reports, 5 – 9 daily injection of ACTH over 15 days induced the development of hypertension in our rats. In addition to a rise in blood pressure, we found reduced BW gain, a fall in plasma potassium concentration, and increases in adrenal, kidney, pituitary, and cardiac weights with ACTH administration. These observations are similar to previous results.6 On the other hand, there were no significant differences in water intake, urine volume, plasma sodium concentration, and urinary electrolyte
/ 2e15$$2180
03-10-97 11:33:06
FIGURE 3. Reverse phase HPLC of C18 extracts from 100 mL of pooled rat urine. Bound materials were eluted with a linear acetonitrile gradient ( 0% to 30% in 30 min ) after washing with water. One-minute fractions were collected and assayed for ouabain immunoreactivity. The arrow indicates the elution position of authentic ouabain on an R-ODS-5 column under the conditions used.
eajha
EL: AJH
AJH–APRIL 1997–VOL. 10, NO. 4, PART 1
ACTH-INDUCED HYPERTENSION AND OUABAIN-LIKE COMPOUND
DLS and have found a positive correlation between DLS and blood pressure in rats.14 Several groups of investigators have demonstrated that chronic inhibition of sodium pump with ouabain leads to the development of hypertension in rats.20,21 Recent studies suggest that the immunization against ouabain partly blocked the development of reduced renal mass-saline hypertension and sodium-induced hypertension in Dahl salt-sensitive rats.22,23 These findings suggest that OLC may exert a hypertensinogenic action. Furthermore, Rossi et al have shown that plasma OLC levels are elevated in primary aldosteronism and essential hypertension, and that removal of adenoma lowered blood pressure in patients in whom plasma OLC levels normalized after surgery.24 We recently found a marked increase in plasma OLC level and its correlation with blood pressure in a hypertensive patient with ectopic ACTH syndrome.17 Taken together, our findings support the possibility that OLC may contribute in part to ACTH-induced hypertension in rats. Several concerns in particular have recently been raised about circulating OLC levels, OLC measurement, and the putative roles of OLC.25,26 In this study, we confirmed the presence of ouabain-immunoreactive peak coincident with authentic ouabain on reverse-phase HPLC system, using a sufficient volume of rat urine. This observation is in agreement with the results of other investigators.27 We cannot exclude the presence of minor peaks, but the peak corresponding to authentic ouabain is the major and distinct ouabainimmunoreactivity in rat urine. This is also the case with human urine and plasma, both in our experience and in recent communications.13,17,28,29 It is now thought that OLC is an isomer of ouabain and that the recognition of OLC may depend on the particular anti-ouabain antibodies used. Furthermore, OLC may possess different and more potent actions in vivo than those of plant ouabain. Recent findings suggest that even plant ouabain evokes contractions at lower concentrations than previously thought, and at the concentrations of OLC ( 0.1 to 1 nmol / L ) normally present in the circulation.30,31 The physiological or pathophysiological actions of OLC remain to be determined, although it appears likely that most actions of this steroid are mediated by binding to the a-2 or a3 isoform of the mammalian Na / , K / -ATPase. However, our data do not provide direct evidence that the concentration obtained in this study is relevant enough to influence the contractility of rat vasculature and, hence, blood pressure. We clearly must await the availability of compounds able to block the OLC actions in vivo. It remains to be determined what are the mechanism( s ) that led to the hypersecretion of OLC, and which tissue might be its production site in the ACTH-
/ 2e15$$2180
03-10-97 11:33:06
407
treated rats. It is generally thought that sodium retention and volume expansion stimulate OLC release. Although BW gain was smaller in the ACTH-treated rats in our study, we cannot exclude the contribution of volume expansion, taking into account the catabolic effects of ACTH. Moreover, a rise in sodium concentration and / or a fall in potassium concentration may increase plasma OLC level in rats, according to the result of Wellard and Adam. 32 The inverse correlation of urine OLC levels with urine sodium/ potassium ratio in this study may indicate the relationship of sodium retention or potassium depletion with OLC production. Although several studies point to a close connection between OLC and serum cation concentration, 33,34 the direct connection between OLC regulation and cation concentration remains to be clarified. A recent report indicated that ACTH stimulates the OLC secretion from adrenal glands.35 Furthermore, in some individuals, primary adrenal overproduction of OLC may account for elevated plasma levels and high blood pressure.24 Although OLC levels in the hypothalamus, pituitary, and adrenals showed no significant difference between control and ACTH-treated rats, adrenal glands were enlarged fourfold, and total tissue content in the adrenals was significantly increased in ACTH-treated rats. It is possible that the site of OLC production is the adrenals, and that the increase in plasma OLC levels can be ascribed to direct stimulation by ACTH. However, further studies clearly are needed to resolve the precise origin of OLC. In conclusion, ACTH administration in rats increased OLC levels in plasma and urine in parallel with blood pressure levels. In addition, blood pressure levels were positively correlated with both plasma and urine OLC levels. These results suggest that OLC may play a role in ACTH-induced hypertension in rats.
ACKNOWLEDGMENT We are indebted to Ms Akiko Kusuhara for technical assistance.
REFERENCES 1. Whitworth JA, Saines D, Scoggins BA: Blood pressure and metabolic effects of ACTH in normotensive man. Clin Exp Hypertens 1983; 5:501 – 522. 2. Whitworth JA: Adrenocorticotrophin and steroid-induced hypertension in humans. Kidney Int 1992; 41:S41 – S37. 3. Scoggins BA, Denton DA, Whitworth JA, et al: ACTH dependent hypertension. Clin Exp Hypertens 1984; 6:A599 – A646. 4. Reid AF, Coghlan LP, Spence CD, et al: Studies on spironolactone steroid antagonists in ACTH-induced hypertension in sheep. J Steroid Biochem 1989; 33:1213 – 1221. 5. Vazir H, Whitehouse BJ, Vinson GP, et al: Effects of
eajha
EL: AJH
408
AJH–APRIL 1997–VOL. 10, NO. 4, PART 1
YAMADA ET AL
prolonged ACTH treatment on adrenal steroidogenesis and blood pressure in rats. Acta Endocrinol 1981; 97:533 – l542. 6. Whitworth JA, Hewitson TD, Li M, et al: Adrenocorticotrophin-induced hypertension in the rat: haemodynamic, metabolic and morphological characteristics. J Hypertens 1990; 8:27 – 36. 7. Whitworth JA, Hewitson TD, Cornwallis C: Corticotropin effects on blood pressure and fluid electrolyte homeostasis in five strains of rats. Am J Hypertens 1992; 5:302 – 313.
bain administration produces hypertension in rats. Hypertension 1993; 22:178 – 187. 22. Yamada K, Goto A, Nagoshi H, et al: Participation of ouabainlike compound in reduced renal mass-saline hypertension. Hypertension 1994; 23 ( suppl 1 ) :I110 – I113. 23. 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.
8. Gamallo A, Alario P, Villanua MA, et al: Effect of chronic stress in the blood pressure in the rat: ACTH administration. Horm Metabol Res 1988; 20:336 – 338.
24. Rossi GP, Manunta P, Hamlyn JM, et al: Immunoreactive endogenous ouabain in primary aldosteronism and essential hypertension: relationship with plasma renin, aldosterone and blood pressure levels. J Hypertens 1995; 13:1181 – 1191.
9. Freeman RH, Davis JO, Fullerton D: Chronic ACTH administration and the development of hypertension in rats. Proc Soc Exp Biol Med 1980; 163:473 – 477.
25. Doris PA, Jenkins LA, Stocco DM: Is ouabain an authentic endogenous mammalian substance derived from the adrenal? Hypertension 1994; 23:632 – 638.
10. Ulick S, Wang JZ, Blumenfeld JD, et al: Cortisol inactivation overload: a mechanism of mineralocorticoid hypertension in the ectopic adrenocorticotropin syndrome. J Clin Endocrinol Metab 1992; 74:963 – 967.
26. Lewis LK, Yandle TG, Lewis JG et al: Ouabain is not detectable in human plasma. Hypertension 1994; 23:632 – 638.
11. Haddy F, Overbeck HW: The role of humoral agents in volume expanded hypertension. Life Sci 1976; 19:935 – 947. 12. Blaustein MP: Physiological effects of endogenous ouabain: control of intracellular Ca 2/ stores and cell responsiveness. Am J Physiol 1993; 264:C1367 – C1387. 13. Goto A, Yamada K, Yagi N, et al: Physiology and pharmacology of endogenous digitalis-like factors. Pharmacol Rev 1992; 44:377 – 399. 14. Li M, Wang KS, Martin A, et al: Adrenocorticotrophininduced hypertension: role of progesterone and digoxin-like substances. Am J Hypertens 1994; 7:59 – 68. 15. Hamlyn JM, Blaustein MP, Bova S, et al: Identification and characterization of a ouabain-like compound from human plasma. Proc Natl Acad Sci USA 1991; 88:6259 – 6263. 16. Zhao N, Lo LC, Berova N, et al: Na,K-ATPase inhibitors from bovine hypothalamus and human plasma are different from ouabain: nanogram scale CD structural analysis. Biochemistry 1995; 34:9893 – 9896. 17. Goto A, Yamada K, Hazama H, et al: Ouabainlike compound in hypertension associated with ectopic corticotropin syndrome. Hypertension 1996; 28:421 – 425. 18. Glowinski J, Iversen LL: Catecholamine regional metabolism in rat brain. J Neurochem 1966; 13:655 – 669. 19. Yamada K, Goto A, Hui C, et al: Role of ouabainlike compound in rats with reduced renal mass-saline hypertension. Am J Physiol 1994; 266:H1357 – H1362. 20. Manunta P, Rogowski AC, Hamilton BP, et al: Ouabain-induced hypertension in the rat: relationships among plasma and tissue ouabain and blood pressure. J Hypertens 1994; 12:569 – 576. 21. Yuan C, Manunta P, Hamlyn JM, et al: Long-term oua-
/ 2e15$$2180
03-10-97 11:33:06
27. Komiyama K, Mori T, Okuda K, et al: Effects of intracerebroventricular administration of 6-hydroxydopamine on ouabain-like immunoreactivity in plasma and the hypothalamo-pituitary axis in rats. J Hypertens 1996; 14:447 – 452. 28. Di Bartolo V, Balzan S, Pieraccini L, et al: Evidence for an endogenous ouabain-like immunoreactive factor in human newborn plasma coeluted with ouabain on HPLC. Life Sci 1995; 57:1417 – 1425. 29. Worgall S, Hnze J, Wagner R, et al: Characterization of ouabain-like immunoreactivity in human urine. J Hypertens 1996; 14:623 – 628. 30. Weiss DN, Blaustein MP: Nanomolar ouabain augments caffein-evoked contractions in rat arteries. Am J Physiol 1993; 265:C1443 – C1448. 31. Stewart L, Hamilton C, Ingwall J, et al: Vascular smooth muscle response to ouabain: relation of tissue Na / to the contractile response. Circ Res 1993; 73:1113 – 1120. 32. Wellard RM, Adam WR: Water depletion, not oral sodium loading, increases levels of sodium, potassiumdependent adenosine triphosphatase inhibitors in rat plasma. Clin Sci 1987; 73:87 – 92. 33. Szylman P, Wolach B, Winaver J, et al: Nonazotemic hyperkalemia with renal and extrarenal defects in potassium transport: association with high levels of digoxin-like immunoreactive factor. J Lab Clin Med 1990; 116:315 – 326. 34. Bagrov AY, Kuznetsova EA, Fedorova OV: Endogenous digoxin-like factor in acute myocardial infarction. J Int Med 1994; 235:63 – 67. 35. Laredo J, Hamilton BP, Hamlyn JM: Secretion of endogenous ouabain from bovine adrenocortical cells: role of the zonaglomerulosa and zona fasciculata. Biochem Biophys Res Commun 1995; 212:487 – 493.
eajha
EL: AJH
Adrenocorticotropin-Induced Hypertension in Rats Role of Ouabain-like Compound Kaoru Yamada, Atsuo Goto, and Masao Omata
We examined the role of ouabain-like compound ( OLC ) in hypertension associated with corticotropin ( ACTH ) excess in rats. Physiological saline solution ( 1 mL / kg ) or synthetic ACTH-Z ( 0.5 mg / kg ) was injected intramuscularly for 15 days to 14 control and 13 male Wistar rats. Significant increases in blood pressure and plasma sodium/ potassium ratio, and decreases in plasma potassium concentration and urinary sodium/ potassium ratio were observed in ACTHtreated rats. The plasma OLC level was higher in ACTH-treated group ( control; 76 { 13, ACTH; 202 { 48 pmol / L, P õ .05 ) . Plasma OLC level correlated with systolic blood pressure ( SPB; r Å 0.53, P õ .01 ) . Urinary OLC excretion was also higher in ACTH-treated group ( control; 0.95 { 0.01, ACTH; 3.32 { 0.67 pmol / day, P õ .01 ) . A
I
njection of corticotropin ( ACTH ) produces increases in blood pressure, together with a variety of electrolyte and metabolic effects in humans, 1,2 sheep, 3,4 and rats.5 – 9 ACTH-induced hypertension is dependent on the adrenal cortex and is prevented by adrenalectomy.1,6 Although ACTH-induced
Received April 22, 1996. Accepted September 30, 1996. From the Department of Human Dry Dock, Sanraku Hospital ( KY ) and Second Department of Internal Medicine, University of Tokyo ( AG, MO ) , Tokyo, Japan. This study was supported in part by a Grant-in-Aid ( 07457164 ) from the Ministry of Education, Science, and Culture, Japan and a Grant from Japan Cardiovascular Research Foundation. Address correspondence and reprint requests to Kaoru Yamada, MD, Department of Human Dry Dock, Sanraku Hospital, 2-5 Kanda-surugadai, Chiyoda-ku, Tokyo 101, Japan.
q 1997 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
/ 2e15$$2180
03-10-97 11:33:06
significant relation was also found between urinary OLC excretion and SBP ( r Å 0.66, P õ .01 ) . Plasma potassium concentration negatively correlated with SBP ( r Å 00.48, P õ .01 ) and urinary sodium/ potassium ratio also correlated inversely with urinary OLC excretion ( r Å 00.55, P õ .01 ) . Measurement of OLC levels after the fractionation of urine by reverse-phase high performance liquid chromatography showed that the major OLC peak in urine from both groups coincided with that of authentic ouabain. These results suggest the contribution of OLC to ACTHinduced hypertension in rats. q 1997 American Journal of Hypertension, Ltd. Am J Hypertens 1997; 10:403 – 408 KEY WORDS: Ouabain-like compound, corticotropin ( ACTH ) , potassium.
hypertension is not simply a form of ‘‘mineralocorticoid’’ hypertension, several studies have demonstrated an essential component of mineralocorticoid hypertension in the onset of ACTH hypertension. Several mineralocorticoid antagonists, including spironolactone, blocked the development of ACTH hypertension in sheep.4 In human ectopic ACTH syndrome, hypertension has mainly been attributed to cortisol inactivation overload giving rise to mineralocorticoidtype hypertension through cortisol occupancy of mineralocorticoid receptors.10 It has been proposed that sodium retention stimulates release of a sodium pump inhibitor with a digitalis-like action, causing vasoconstriction and hypertension in the state of mineralocorticoid excess.11 – 13 Consistent with this view, ACTH increased blood
eajha
EL: AJH
0895-7061/97/$17.00 PII S0895-7061(96)00449-9
404
AJH–APRIL 1997–VOL. 10, NO. 4, PART 1
YAMADA ET AL
pressure in parallel with plasma levels of digoxinlike substances ( DLS ) in rats and there was a positive correlation between DLS and blood pressure. 14 Because a major biologically active sodium pump inhibitor in circulation may be a ouabain-like compound ( OLC ) , 15,16 we recently measured plasma OLC levels in a hypertensive patient with ectopic ACTH syndrome and found a marked increase in plasma OLC levels and its correlation with blood pressure.17 In the present study, we tested the hypothesis that OLC may contribute to ACTH-induced hypertension in rats.
METHODS Experimental Animals and Protocol This study was approved by the Animal Experiment Committee of the University of Tokyo. Male Wistar rats weighing 130 to 140 g were raised in a room with alternating 12-h cycles of light and given free access to a normal rat chow containing 0.8% NaCl ( Oriental Kobo Co., Tokyo, Japan ) and tap water. Rats were divided into two groups. Thirteen rats were given 0.5 mg / kg / day ( 1 mL / kg ) synthetic ACTH-Z ( Daiichi Pharmaceutical Co., Tokyo, Japan ) intramuscularly at 4 PM for 15 days. Control rats ( n Å 14 ) received an intramuscular injection of physiological saline solution ( 1 mL / kg ) for 15 days. Body weight ( BW ) and systolic blood pressure ( SBP ) by the tail-cuff method were measured on days 6 and 16. Eight rats from each group were placed in individual metabolic cages for 24 h after the last injections on day 15. Water intake and urine volume were measured. Urine samples were used for the measurements of OLC and electrolytes concentrations. Twenty-four hours after the last injection, the animals were anesthetized by pentobarbital sodium ( 50 mg / kg ) , and the trunk blood was collected for the measurement of plasma OLC, electrolyte concentrations, and serum corticosterone levels. The brain, pituitary, heart, right kidney, and right adrenal were removed, and the right hypothalamus was dissected out according to the method described by Glowinski and Iversen.18 The tissues were weighed and OLC levels were measured in hypothalamus, pituitary, and adrenal. Measurement of OLC The tissue samples were homogenized with 5 volumes of distilled water and the homogenates were centrifuged at 15,000 rpm for 60 min. The resulting supernatant, urine sample ( 1 mL ) , or plasma sample ( 1 mL ) , mixed with the equal volume of 0.1% TFA solution, was applied to the Sep-Pak C18 cartridge that had been activated with methanol and equilibrated with distilled water. After complete washing with 30 mL of distilled water, OLC was eluted with 3 mL of 25% acetonitrile in water. The eluent was evaporated and assayed for OLC as described in our previous report.19 The antiserum used for the radioimmunoassay showed minimum cross-reactivity with common
/ 2e15$$2180
03-10-97 11:33:06
steroids ( cortisol, cortisone, deoxycorticosterone, testosterone, progesterone, dehydroepiandrosterone sulfate, and b-estradiol ) . Polar metabolites of corticosterone and cortisol such as 6b-OH-corticosterone and 6bOH-cortisol have a similar polarity to that of ouabain, and our sample may have contained these metabolites. However, our antibody did not cross-react with 6bOH-corticosterone and 6b-OH-cortisol at the concentration of 10 04 to 10 010 mol / L ( unpublished data, 1995 ) .
OLC Measurement in Urine After HPLC Analysis We measured OLC levels in urine after the fractionation by reverse-phase high performance liquid chromatography ( HPLC ) . Collected urine ( 100 mL ) from control and ACTH-treated rats were applied to the Sep-Pak C18 cartridge that had been activated with methanol and equilibrated with distilled water. The fraction that was eluted by 25% acetonitrile after complete washing with distilled water was analyzed on a R-ODS-5 column ( Yamamura Chemical Co., Kyoto, Japan ) , with a linear gradient of acetonitrile in water ( 0% to 30% ) for 30 min after washing with water for 9 min at 1 mL /min. Oneminute fractions were collected and assessed for OLC. Measurements of Hormone and Electrolytes Plasma corticosterone levels were measured by radioimmunoassay. Sodium and potassium concentrations were measured by flame photometry. Statistical Analysis The data, expressed as mean { SEM, were analyzed by the unpaired Student’s t test. A P value õ .05 was considered significant. RESULTS Sham injection was associated with an increase in SBP from 124 { 2 to 136 { 2 on day 6 and to 133 { 2 mm Hg at the end of the injection period ( P õ .01 ) . ACTH injection significantly increased SBP from 122 { 3 to 146 { 3 on day 6 and to 153 { 4 on day 16 ( P õ .01 ) . The effect of ACTH treatment was significantly greater ( P õ .01 ) than that of sham injection, both on days 6 and 16. Although both groups gained significant weight over the period of study, the BW of the ACTH-treated
TABLE 1. BODY WEIGHT AND TISSUE WEIGHTS OF CONTROL AND ACTH-TREATED RATS Control (n Å 14) Body weight (g) Cardiac weight (mg/100 g BW) Renal weight (mg/100 g BW) Adrenal weight (mg/100 g BW) Pituitary weight (mg/100 g BW)
218 305 422 9.5 3.7
{ { { { {
6 5 10 0.4 0.1
ACTH (n Å 13) 171 373 508 38.0 4.4
{ { { { {
7* 9* 10* 3.3* 0.2*
Values are mean { SEM. ACTH, adrenocorticotrophin; BW, body weight. *P õ .01 compared with control.
eajha
EL: AJH
AJH–APRIL 1997–VOL. 10, NO. 4, PART 1
ACTH-INDUCED HYPERTENSION AND OUABAIN-LIKE COMPOUND
TABLE 2. EFFECTS OF ACTH TREATMENT ON MEASURED PARAMETERS
Water intake (mL/day) Urine volume (mL/day) Urinary Na excretion (mmol/day) Urinary K excretion (mmol/day) Na/K ratio in urine Plasma Na concentration (mmol/L) Plasma K concentration (mmol/L) Na/K ratio in plasma
Control (n Å 14)
ACTH (n Å 13)
47.5 { 21.9 31.9 { 12.5
57.9 { 19.9 30.8 { 5.9
0.44 { 0.05
0.30 { 0.11
1.03 { 0.09 0.43 { 0.04
1.20 { 0.22 0.22 { 0.03*
138.1 { 0.4 5.5 { 0.1 25.3 { 0.6
139.4 { 0.7 4.2 { 0.2* 33.8 { 1.6*
Values are mean { SEM. ACTH, adrenocorticotrophin; BW, body weight; OLC, ouabain-like compound. *P õ .01 compared with control. Water intake and urine data were obtained from eight rats in each group.
group was significantly lower than that of the control group ( P õ .01, Table 1 ) . Effects of ACTH treatment on electrolyte homeostasis were summarized in Table 2. Water intake, urine volume, and urinary sodium and potassium excretions were not different between the two groups. Sodium/ potassium ratio in urine was significantly lower in ACTH-treated groups than in the control group ( P õ .01 ) . Plasma sodium concentrations were not different between the two groups, but ACTH treatment significantly decreased plasma potassium concentration and increased plasma sodium/ potassium ratio ( P õ .01 ) . ACTH treatment increased cardiac, renal, adrenal, and pituitary weights as compared with sham injection ( P õ 0.01, Table 1 ) .
405
Table 3 shows the effects of ACTH treatment on hormonal parameters. Urinary OLC excretion was significantly higher in the ACTH-treated group ( control, 0.95 { 0.10; ACTH, 3.32 { 0.67 pmol / day, P õ .01 ) . The plasma OLC level was also significantly higher in the ACTH-treated group ( control 76 { 13; ACTH, 202 { 48 pmol / L, P õ .05 ) . OLC levels in the hypothalamus, pituitary, and adrenal, as well as plasma corticosterone levels, showed no significant differences between the two groups. Plasma OLC levels correlated with SBP ( Figure 1A; r Å 0.53, P õ .01 ) . A significant relationship also was found between urinary OLC excretion and SBP ( Figure 1B; r Å 0.66, P õ .01 ) . Plasma potassium concentrations negatively correlated with SBP ( Figure 2A; r Å 00.48, P õ .01 ) , and sodium/ potassium ratios in urine showed inverse correlations with urinary OLC excretion ( Figure 2B, r Å 00.55, P õ .01 ) . The major OLC peak in urine from both groups appeared at 29 to 30 min and coincided with that of authentic ouabain on HPLC ( Figure 3 ) . Another broad immunoreactive peak was found in fractions corresponding to more hydrophobic material.
TABLE 3. HORMONAL PARAMETERS OF CONTROL AND ACTH-TREATED RATS
Urinary OLC excretion (pmol/day) Plasma OLC level (pmol/L) OLC level in hypothalamus (pmol/g) OLC level in pituitary (pmol/g) OLC level in adrenal (pmol/g) Serum corticosterone concentration (ng/mL)
Control (n Å 14)
ACTH (n Å 13)
0.95 { 0.10
3.32 { 0.67†
76 { 13
202 { 48*
0.87 { 0.16
0.79 { 0.22
4.71 { 0.76
7.67 { 0.67
2.39 { 0.65
1.75 { 0.36
330 { 57
373 { 89
Values are mean { SEM. ACTH, adrenocorticotrophin; OLC, ouabain-like compound. *P õ .05, †P õ .01 compared with control. Data of urinary OLC excretion were obtained from eight rats in each group.
/ 2e15$$2180
03-10-97 11:33:06
FIGURE 1. Relations between systolic blood pressure ( SBP ) and OLC levels in plasma ( A ) or urinary OLC excretion ( B ) . There was a significant correlation between SBP and OLC levels in plasma ( r Å 0.53, P õ .01 ) . Urinary OLC excretion also was correlated with SBP ( r Å 0.66, P õ .01 ) .
eajha
EL: AJH
406
AJH–APRIL 1997–VOL. 10, NO. 4, PART 1
YAMADA ET AL
FIGURE 2. Relations between plasma potassium concentration and systolic blood pressure ( A ) , and between sodium/ potassium ratio in urine and urinary OLC excretion ( B ) . There was a significant inverse correlation between plasma potassium concentration and systolic blood pressure ( r Å 00.48, P õ .05 ) . Sodium/ potassium ratio in urine was negatively correlated with urinary OLC excretion ( r Å 00.55, P õ .05 ) .
excretion. Whitworth et al have recently shown that ACTH administration in the rat produces an adrenally dependent rise in blood pressure, fall in body weight and plasma potassium concentration, and increases in water intake, urine output, urinary sodium, and potassium excretion as well as in adrenal, kidney, and cardiac weight.6 Because we measured the parameters of water and electrolyte homeostasis only on the final day, we cannot rule out the possibility that the rats were not accustomed to metabolic cages. Differences in sampling day also may explain the discrepancy between the results of our study and those of previous studies. ACTH-induced hypertension appears to be adrenally dependent and has been attributed to adrenocortical steroids. In this study, ACTH treatment significantly decreased plasma potassium concentration, increased plasma sodium/ potassium ratio, and decreased urine sodium/ potassium ratio as compared with ratios for control rats, suggesting an excess mineralocorticoid state in ACTH-treated rats. In our study, serum concentrations of corticosterone, the major glucocorticoid in rats, were not significantly increased in ACTH-treated rats. Since plasma corticosterone concentration was higher in ACTH-treated rats in previous studies, 8 differences in administration method ( injection v continuous infusion ) , anesthesia, dose, duration, or sampling time may account for this discrepancy. Although we did not measure serum levels of other mineralocorticoids such as deoxycorticosterone, these mineralocorticoid effects are well known consequences of ACTH administration. In this study, we have shown that both plasma OLC levels and urinary OLC excretions were threefold higher in ACTH-treated rats, and both correlated with SBP. Li et al have shown that ACTH increased plasma
DISCUSSION In this study, we measured OLC in plasma, urine, and several tissues to examine the contribution of OLC to ACTH-induced hypertension in rats. We found that ACTH administration increased threefold the OLC levels in plasma and urine in association with a rise in blood pressure. Furthermore, blood pressure levels correlated positively with both plasma and urine OLC levels. In agreement with previous reports, 5 – 9 daily injection of ACTH over 15 days induced the development of hypertension in our rats. In addition to a rise in blood pressure, we found reduced BW gain, a fall in plasma potassium concentration, and increases in adrenal, kidney, pituitary, and cardiac weights with ACTH administration. These observations are similar to previous results.6 On the other hand, there were no significant differences in water intake, urine volume, plasma sodium concentration, and urinary electrolyte
/ 2e15$$2180
03-10-97 11:33:06
FIGURE 3. Reverse phase HPLC of C18 extracts from 100 mL of pooled rat urine. Bound materials were eluted with a linear acetonitrile gradient ( 0% to 30% in 30 min ) after washing with water. One-minute fractions were collected and assayed for ouabain immunoreactivity. The arrow indicates the elution position of authentic ouabain on an R-ODS-5 column under the conditions used.
eajha
EL: AJH
AJH–APRIL 1997–VOL. 10, NO. 4, PART 1
ACTH-INDUCED HYPERTENSION AND OUABAIN-LIKE COMPOUND
DLS and have found a positive correlation between DLS and blood pressure in rats.14 Several groups of investigators have demonstrated that chronic inhibition of sodium pump with ouabain leads to the development of hypertension in rats.20,21 Recent studies suggest that the immunization against ouabain partly blocked the development of reduced renal mass-saline hypertension and sodium-induced hypertension in Dahl salt-sensitive rats.22,23 These findings suggest that OLC may exert a hypertensinogenic action. Furthermore, Rossi et al have shown that plasma OLC levels are elevated in primary aldosteronism and essential hypertension, and that removal of adenoma lowered blood pressure in patients in whom plasma OLC levels normalized after surgery.24 We recently found a marked increase in plasma OLC level and its correlation with blood pressure in a hypertensive patient with ectopic ACTH syndrome.17 Taken together, our findings support the possibility that OLC may contribute in part to ACTH-induced hypertension in rats. Several concerns in particular have recently been raised about circulating OLC levels, OLC measurement, and the putative roles of OLC.25,26 In this study, we confirmed the presence of ouabain-immunoreactive peak coincident with authentic ouabain on reverse-phase HPLC system, using a sufficient volume of rat urine. This observation is in agreement with the results of other investigators.27 We cannot exclude the presence of minor peaks, but the peak corresponding to authentic ouabain is the major and distinct ouabainimmunoreactivity in rat urine. This is also the case with human urine and plasma, both in our experience and in recent communications.13,17,28,29 It is now thought that OLC is an isomer of ouabain and that the recognition of OLC may depend on the particular anti-ouabain antibodies used. Furthermore, OLC may possess different and more potent actions in vivo than those of plant ouabain. Recent findings suggest that even plant ouabain evokes contractions at lower concentrations than previously thought, and at the concentrations of OLC ( 0.1 to 1 nmol / L ) normally present in the circulation.30,31 The physiological or pathophysiological actions of OLC remain to be determined, although it appears likely that most actions of this steroid are mediated by binding to the a-2 or a3 isoform of the mammalian Na / , K / -ATPase. However, our data do not provide direct evidence that the concentration obtained in this study is relevant enough to influence the contractility of rat vasculature and, hence, blood pressure. We clearly must await the availability of compounds able to block the OLC actions in vivo. It remains to be determined what are the mechanism( s ) that led to the hypersecretion of OLC, and which tissue might be its production site in the ACTH-
/ 2e15$$2180
03-10-97 11:33:06
407
treated rats. It is generally thought that sodium retention and volume expansion stimulate OLC release. Although BW gain was smaller in the ACTH-treated rats in our study, we cannot exclude the contribution of volume expansion, taking into account the catabolic effects of ACTH. Moreover, a rise in sodium concentration and / or a fall in potassium concentration may increase plasma OLC level in rats, according to the result of Wellard and Adam. 32 The inverse correlation of urine OLC levels with urine sodium/ potassium ratio in this study may indicate the relationship of sodium retention or potassium depletion with OLC production. Although several studies point to a close connection between OLC and serum cation concentration, 33,34 the direct connection between OLC regulation and cation concentration remains to be clarified. A recent report indicated that ACTH stimulates the OLC secretion from adrenal glands.35 Furthermore, in some individuals, primary adrenal overproduction of OLC may account for elevated plasma levels and high blood pressure.24 Although OLC levels in the hypothalamus, pituitary, and adrenals showed no significant difference between control and ACTH-treated rats, adrenal glands were enlarged fourfold, and total tissue content in the adrenals was significantly increased in ACTH-treated rats. It is possible that the site of OLC production is the adrenals, and that the increase in plasma OLC levels can be ascribed to direct stimulation by ACTH. However, further studies clearly are needed to resolve the precise origin of OLC. In conclusion, ACTH administration in rats increased OLC levels in plasma and urine in parallel with blood pressure levels. In addition, blood pressure levels were positively correlated with both plasma and urine OLC levels. These results suggest that OLC may play a role in ACTH-induced hypertension in rats.
ACKNOWLEDGMENT We are indebted to Ms Akiko Kusuhara for technical assistance.
REFERENCES 1. Whitworth JA, Saines D, Scoggins BA: Blood pressure and metabolic effects of ACTH in normotensive man. Clin Exp Hypertens 1983; 5:501 – 522. 2. Whitworth JA: Adrenocorticotrophin and steroid-induced hypertension in humans. Kidney Int 1992; 41:S41 – S37. 3. Scoggins BA, Denton DA, Whitworth JA, et al: ACTH dependent hypertension. Clin Exp Hypertens 1984; 6:A599 – A646. 4. Reid AF, Coghlan LP, Spence CD, et al: Studies on spironolactone steroid antagonists in ACTH-induced hypertension in sheep. J Steroid Biochem 1989; 33:1213 – 1221. 5. Vazir H, Whitehouse BJ, Vinson GP, et al: Effects of
eajha
EL: AJH
408
AJH–APRIL 1997–VOL. 10, NO. 4, PART 1
YAMADA ET AL
prolonged ACTH treatment on adrenal steroidogenesis and blood pressure in rats. Acta Endocrinol 1981; 97:533 – l542. 6. Whitworth JA, Hewitson TD, Li M, et al: Adrenocorticotrophin-induced hypertension in the rat: haemodynamic, metabolic and morphological characteristics. J Hypertens 1990; 8:27 – 36. 7. Whitworth JA, Hewitson TD, Cornwallis C: Corticotropin effects on blood pressure and fluid electrolyte homeostasis in five strains of rats. Am J Hypertens 1992; 5:302 – 313.
bain administration produces hypertension in rats. Hypertension 1993; 22:178 – 187. 22. Yamada K, Goto A, Nagoshi H, et al: Participation of ouabainlike compound in reduced renal mass-saline hypertension. Hypertension 1994; 23 ( suppl 1 ) :I110 – I113. 23. 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.
8. Gamallo A, Alario P, Villanua MA, et al: Effect of chronic stress in the blood pressure in the rat: ACTH administration. Horm Metabol Res 1988; 20:336 – 338.
24. Rossi GP, Manunta P, Hamlyn JM, et al: Immunoreactive endogenous ouabain in primary aldosteronism and essential hypertension: relationship with plasma renin, aldosterone and blood pressure levels. J Hypertens 1995; 13:1181 – 1191.
9. Freeman RH, Davis JO, Fullerton D: Chronic ACTH administration and the development of hypertension in rats. Proc Soc Exp Biol Med 1980; 163:473 – 477.
25. Doris PA, Jenkins LA, Stocco DM: Is ouabain an authentic endogenous mammalian substance derived from the adrenal? Hypertension 1994; 23:632 – 638.
10. Ulick S, Wang JZ, Blumenfeld JD, et al: Cortisol inactivation overload: a mechanism of mineralocorticoid hypertension in the ectopic adrenocorticotropin syndrome. J Clin Endocrinol Metab 1992; 74:963 – 967.
26. Lewis LK, Yandle TG, Lewis JG et al: Ouabain is not detectable in human plasma. Hypertension 1994; 23:632 – 638.
11. Haddy F, Overbeck HW: The role of humoral agents in volume expanded hypertension. Life Sci 1976; 19:935 – 947. 12. Blaustein MP: Physiological effects of endogenous ouabain: control of intracellular Ca 2/ stores and cell responsiveness. Am J Physiol 1993; 264:C1367 – C1387. 13. Goto A, Yamada K, Yagi N, et al: Physiology and pharmacology of endogenous digitalis-like factors. Pharmacol Rev 1992; 44:377 – 399. 14. Li M, Wang KS, Martin A, et al: Adrenocorticotrophininduced hypertension: role of progesterone and digoxin-like substances. Am J Hypertens 1994; 7:59 – 68. 15. Hamlyn JM, Blaustein MP, Bova S, et al: Identification and characterization of a ouabain-like compound from human plasma. Proc Natl Acad Sci USA 1991; 88:6259 – 6263. 16. Zhao N, Lo LC, Berova N, et al: Na,K-ATPase inhibitors from bovine hypothalamus and human plasma are different from ouabain: nanogram scale CD structural analysis. Biochemistry 1995; 34:9893 – 9896. 17. Goto A, Yamada K, Hazama H, et al: Ouabainlike compound in hypertension associated with ectopic corticotropin syndrome. Hypertension 1996; 28:421 – 425. 18. Glowinski J, Iversen LL: Catecholamine regional metabolism in rat brain. J Neurochem 1966; 13:655 – 669. 19. Yamada K, Goto A, Hui C, et al: Role of ouabainlike compound in rats with reduced renal mass-saline hypertension. Am J Physiol 1994; 266:H1357 – H1362. 20. Manunta P, Rogowski AC, Hamilton BP, et al: Ouabain-induced hypertension in the rat: relationships among plasma and tissue ouabain and blood pressure. J Hypertens 1994; 12:569 – 576. 21. Yuan C, Manunta P, Hamlyn JM, et al: Long-term oua-
/ 2e15$$2180
03-10-97 11:33:06
27. Komiyama K, Mori T, Okuda K, et al: Effects of intracerebroventricular administration of 6-hydroxydopamine on ouabain-like immunoreactivity in plasma and the hypothalamo-pituitary axis in rats. J Hypertens 1996; 14:447 – 452. 28. Di Bartolo V, Balzan S, Pieraccini L, et al: Evidence for an endogenous ouabain-like immunoreactive factor in human newborn plasma coeluted with ouabain on HPLC. Life Sci 1995; 57:1417 – 1425. 29. Worgall S, Hnze J, Wagner R, et al: Characterization of ouabain-like immunoreactivity in human urine. J Hypertens 1996; 14:623 – 628. 30. Weiss DN, Blaustein MP: Nanomolar ouabain augments caffein-evoked contractions in rat arteries. Am J Physiol 1993; 265:C1443 – C1448. 31. Stewart L, Hamilton C, Ingwall J, et al: Vascular smooth muscle response to ouabain: relation of tissue Na / to the contractile response. Circ Res 1993; 73:1113 – 1120. 32. Wellard RM, Adam WR: Water depletion, not oral sodium loading, increases levels of sodium, potassiumdependent adenosine triphosphatase inhibitors in rat plasma. Clin Sci 1987; 73:87 – 92. 33. Szylman P, Wolach B, Winaver J, et al: Nonazotemic hyperkalemia with renal and extrarenal defects in potassium transport: association with high levels of digoxin-like immunoreactive factor. J Lab Clin Med 1990; 116:315 – 326. 34. Bagrov AY, Kuznetsova EA, Fedorova OV: Endogenous digoxin-like factor in acute myocardial infarction. J Int Med 1994; 235:63 – 67. 35. Laredo J, Hamilton BP, Hamlyn JM: Secretion of endogenous ouabain from bovine adrenocortical cells: role of the zonaglomerulosa and zona fasciculata. Biochem Biophys Res Commun 1995; 212:487 – 493.
eajha
EL: AJH