0013-7227/91/128l-0226$02.00/0 Endocrinology Copyright© 1991 by The Endocrine Society
Vol. 128, No. 1 Printed in U.S.A.
ZHENG-PEI ZENGf, MITSUHIDE NARUSE, KIYOKO NARUSE, YUKO KATO, YI-FAN SHI, HIROSHI DEMURA, AND KAZUO SHIZUME Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women's Medical College, Tokyo 162, Japan; and the Department of Endocrinology, Peking Union Medical College Hospital ((Y.-F.S.), Beijing 100730, China
with a significant increase in plasma levels of ANP. Administration of ANP-specific antiserum significantly impaired the return to normal sodium balance as well as the augmented kaliuresis that were observed on the second day after injection of DOCA. No significant effect was observed on either sodium or potassium balance after the injection of normal rabbit serum. These results suggest that plasma ANP plays an important role in mineralocorticoid escape. {Endocrinology 128: 226-230, 1991)
ABSTRACT. To elucidate the physiological role of atrial natriuretic peptide (ANP) in plasma during mineralocorticoid escape, we investigated the effects of passive immunization with ANP-specific antiserum on deoxycorticosterone (DOCA)treated rats. Sodium was retained in excess of intake during the first day after treatment with DOCA, and sodium balance returned to control values by the second day, whereas the excretion of potassium exceeded the intake during all days after DOCA treatment. These changes in electrolyte balance were associated
E
XOGENOUS administration of mineralocorticoid results in a transient accumulation of sodium chloride, but the balance between sodium intake and output returns to the basal level after a certain period of time. This phenomenon has been termed mineralocorticoid escape (1). Various factors have been suggested as being involved in the phenomenon, namely increases in systemic arterial pressure, glomerular filtration rate, renal arterial pressure, renal interstitial hydrostatic pressure, and renal prostaglandins as well as decreases in levels of angiotensin-II and renal adrenergic activity (2-6). However, details of the mechanism responsible for this phenomenon have not been elucidated. Atrial natriuretic peptide (ANP), discovered in mammalian atrial tissue, possesses potent natriuretic and vasorelaxant activities (for reviews, see Refs. 7 and 8). The importance of the peptide has been implied in various physiological and pathological conditions associated with abnormalities in blood pressure and/or body fluid
volume: natriuresis during saline loading (9), supraventricular tachycardia (10), congestive heart failure (11), chronic renal failure (12), and various types of hypertension (13). In addition, increased plasma levels of ANP have been demonstrated during mineralocorticoid escape in animals (14, 15) and humans (16-19). This latter observation suggests a possible role for the peptide in the escape phenomenon, although no direct evidence for its involvement has been presented. The present study was undertaken to examine the role of plasma ANP during mineralocorticoid escape by application of a passive immunization technique with ANPspecific antiserum. We found that the ANP-specific antiserum abolished mineralocorticoid escape in rats treated with deoxycorticosterone acetate (DOCA). Materials and Methods Animals and experimental protocols Adult male Wistar rats, weighing 250-300 g, were kept under standard light in individual metabolic cages for several days before experiments. Rats were fed a special low sodium diet that included the following components: water, 10.3%; sodium, 7.41 mg/100 g; potassium, 401 mg/100 g; calcium, 630 mg/100 g; phosphorus, 530 mg/100 g; and magnesium, 28.1 mg/100 g. Sodium chloride was provided in drinking water (Na, 25.5 mmol/liter), such that animals consumed 0.7-0.9 mmol sodium/day.
Received June 11,1990. Address all correspondence and requests for reprints to: Dr. Mitsuhide Naruse, Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women's Medical College, Tokyo 162, Japan. * This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture and a research grant from the Foundation for Growth Science in Japan. t Current address: Department of Endocrinology, Peking Union Medical College Hospital, Beijing, China. 226
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Antiserum against Homologous Atrial Natriuretic Peptide Diminishes the Natriuretic Response during Mineralocorticoid Escape in Rats*
ANP IN MINERALOCORTICOID ESCAPE Experimental protocols
it returned to the control level on the second and subsequent days after DOCA injection (Fig. 1). The fractional excretion of sodium decreased significantly from 5.85 ± 3.33% to 1.00 ± 0.57% during the first 24-h period and increased significantly to 13.11 ± 3.90% during the second 24-h period after the injection of DOCA. Urine volumes increased significantly from 18.3 ± 0.7 ml/24 h during the control period to 53.8 ± 4.6 ml/24 h during the second 24-h period after the administration of DOCA, whereas no significant change was observed during the first 24-h period. The potassium balance was significantly decreased each day after the injection of DOCA (Fig. 2). By contrast, there were no significant changes in urine volume (data not shown), sodium balance, or potassium balance throughout the experimental period in control rats (Figs. 1 and 2). In addition, no significant differences DO : A
•o 31-0
e balance o
*T-
T
en
Blood samples
+
Assay methods Concentrations of sodium and potassium in serum and urine were determined by flame photometry. The sodium and potassium balances for each 24-h period were calculated by subtracting the urinary output of each from the respective intake. Concentrations of creatinine in serum and urine were measured with commercially available kits (Eiken Immunochemical Laboratories, Tokyo, Japan). Plasma concentrations of ANP were determined by RIA after extraction with a Sep-Pak C18 cartridge (Waters, Milford, CA) (21). PRA was determined by RIA after generation of angiotensin-I (20). Plasma aldosterone concentrations (PAC) were measured with RIA kits (Dainabot RI Laboratories, Tokyo, Japan).
Sodiu
F
**
1 **t i
2
3
3
Trunk blood was collected immediately after decapitation into chilled test tubes, either with or without Na2EDTA (5 mM) plus aprotinin (500 IU/ml). The plasma and serum samples obtained by centrifugation at 4 C were kept at -20 C until assayed.
Control period
1
Time ( days) FIG. 1. Effects of a single injection of DOCA (DOCA group; • ) or vehicle (control group; • ) on sodium balance in rats. Values are the mean ± SE (n = 16 during the control period, n = 12 on the first day, n = 8 on the second day, and n = 4 on the third day). *, P < 0.01 vs. control period. **, P < 0.01 vs. the first day after the injection of DOCA. f, P < 0.05 vs. control group, ft, P < 0.01 vs. control group. |2.0
Statistical analysis All data are presented as the mean ± SE of results of multiple experiments. Data were analyzed by analysis of variance followed by Student's t test or by Duncan's new multiple range test. Significance was presumed if P < 0.05.
Results Although sodium balance was significantly increased during the first 24-h period after the injection of DOCA,
Control period
1 '
9 *•
Time (days) FIG. 2. Effects of a single injection of DOCA (DOCA group; B) or vehicle (control group; • ) on potassium balance in rats. Values are the mean ± SE (n = 16 during the control period, n = 12 on the first day, n = 8 on the second day, and n = 4 on the third day). *, P < 0.01 vs. control period, f, P < 0.01 vs. control group.
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Body weight and daily intake of food and water were measured every morning throughout the experiment. Urine was also collected daily and was used to measure rates of excretion of sodium, potassium, and creatinine. After a 3-day control period, the first group of rats (DOCA group; n = 16) was sc injected with 10 mg DOCA in 0.1 ml sesame oil at 0900 h on the fourth day of the experiment, and these rats were killed by decapitation at 0 h (n = 4), 24 h (n = 4), 48 h (n = 4), and 72 h (n = 4) after the injection of DOCA. The second group of rats (control group; n = 16) was sc injected with 0.1 ml sesame oil alone, and these rats were also killed by decapitation 0 h (n = 4), 24 h (n = 4), 48 h (n = 4), and 72 h (n = 4) after injection. The third group of rats (DOCA plus ANP-specific antiserum group; n = 5) and the fourth group of rats (DOCA plus normal rabbit serum group; n = 5) were injected with 100 ^1 antiserum raised against rat ANP (Ab 420; 1:100) (20) and normal rabbit serum (NRS; 1:100), respectively, through the jugular vein under pentobarbital anesthesia 24 h after the administration of DOCA. Forty-eight hours after the injection of DOCA, rats were killed by decapitation. Based upon the extent of the binding of 125I-labeled ANP, the capacity of the antiserum to bind ANP was estimated to be about 1 ng/ 1 (A antiserum.
227
ANP IN MINERALOCORTICOID ESCAPE
228
Discussion Sodium balance was significantly increased during the first 24-h period after a single injection of DOCA and was restored during the next 24-h period. The initial retention of sodium was the result of the mineralocorticoid effects of DOCA. The rate of onset of the escape has been reported to be related to the extent of sodium intake (22, 23). Mohring and Mohring (21) showed that escape occurs within 48 h after the administration of DOCA for 5-7 days in rats given less than 2.5 mmol sodium/day. However, Ballermann et al. (14) demonstrated that retention of sodium during the first 24 h after the administration of mineralocorticoid can be most
reliably demonstrated with a sodium intake of 0.5-1.0 mmol/day, and in this case the escape occurs within 48 h. The chronological changes in sodium balance in the present study, in which 0.7-0.9 mmol/day sodium was taken up, are in agreement with the timing of the mineralocorticoid escape observed by Ballerman et al. (14). Administration of DOCA was accompanied by changes in plasma levels of ANP. In agreement with the results of previous studies (14-19), plasma ANP levels were significantly elevated 24 h after the injection of DOCA and remained high throughout the experimental period. The increase seems to be sufficiently large to suggest the involvement of ANP in mineralocorticoid escape. Since the passive immunization technique has been effectively used to elucidate the biological significance of plasma ANP in rats (20), we investigated the effects of ANPspecific antiserum on mineralocorticoid escape in DOCAtreated rats. In contrast to that in control rats injected with NRS, a significant inhibition of the restoration of the sodium balance during the second 24-h period after the injection of DOCA was observed in rats given ANPspecific antiserum. These results provide direct evidence that increased levels of endogenous ANP in plasma play a key role in the natriuretic response during mineralocorticoid escape. Potassium balance was decreased during the first 24h period after the administration of DOCA, and this decrease can be attributed to the mineralocorticoid action of DOCA. Furthermore, in contrast to the changes in sodium balance, potassium balance decreased further during the second 24-h period after the administration of DOCA. This result is in agreement with the previously reported finding that the kidney escapes from the sodium-retaining effect of mineralocorticoids, but not from the loss of potassium (1, 24-26). The discrepancies in the changes in rates of excretion of sodium and potassium as well as the absence of any escape from the sodium-retaining effects in the gastrointestinal tract and
TABLE 1. Changes in plasma levels of ANP, PRA, and PAC before and after injection of DOCA Group ANP (pg/ml) Control DOCA PRA(ngAngI/mlh) Control DOCA PAC (ng/dl) Control DOCA
Days after DOCA
Control period 1
2
3
142.5 ± 9.0 168.4 ± 19.0
104.4 ± 17.8 361.4 ± 49.5°
177.6 ± 23.5 431.8 ± 87.8c'rf
221.9 ± 26.1 303.9 ± 55.2"
4.0 ± 0.5 5.5 ± 0.8
4.3 ± 0.3 1.6 ± 0.6°
4.6 ± 0.2 2.0 ± 0.3"
4.7 ± 0.5 1.9 ± 0.5°
13.7 ± 2.0 17.3 ± 0.9
9.7 ± 3.4 1.1 ± 0.9*
12.4 ± 3.6 0.6 ± 0.5*
8.4 ± 3.3 0.5 ± 0.2*
Data are the mean ± SE of results from four rats. " P < 0.05 us. control period; P < 0.05 us. control group. b P< 0.01 us. control period; P < 0.01 vs. control group.
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in creatinine clearance were found between groups or during experiments. Changes in plasma ANP levels, PRA, and PAC are shown in Table 1. In the DOCA group, plasma ANP levels increased approximately 2-fold, whereas PRA and PAC were suppressed. By contrast, in the control group, plasma ANP concentrations, PRA, and PAC did not show significant changes over the experimental period. It appeared from the changes in sodium balance and plasma levels of ANP in the first series of experiments that mineralocorticoid escape occurs from the second day after the administration of DOCA. In the second series of experiment with NRS or antiserum, therefore, sodium and potassium balance were determined for 2 days after the injection of DOCA. The chronological changes in these parameters were essentially the same as those in the DOCA-injected group described above. The restoration of the sodium balance during the second 24-h period after the administration of DOCA was abolished by the iv administration of ANP-specific antiserum (Fig. 3). In addition, the decrease in potassium balance during the second 24-h period was significantly diminished by the injection of antiserum against rat ANP (Pig. 4).
Endo • 1991 Voll28-Nol
ANP IN MINERALOCORTICOID ESCAPE
DOCA
Ab or NRS
I
)
1.5
T
Control period
*< !
1 Time (days)
2
FIG. 3. Effects of iv administration of ANP-specific antiserum (Ab; B) or NRS (•) on sodium balance in DOCA-treated rats. Each point represents the mean ± SE of results from five rats. *, P < 0.01 us. control period. **, P < 0.05 us. the first day after DOCA. f, P < 0.01 us. DOCA plus NRS group.
-0.5t 1 2 Time (days)
FlG. 4. Effects of iv administration of ANP-specific antiserum (Ab; H) or NRS (•) on potassium balance in DOCA-treated rats. Each point represents the mean ± SE of results from five rats. *, P < 0.01 us. control period, t, P < 0.01 us. DOCA plus NRS group.
salivary or sweat glands (22, 27, 28) support the hypothesis that the escape is not due to a circulating antagonists) of mineralocorticoids (22). However, ANP is well known to have a kaliuretic activity in vivo in rats (7, 8). Administration of ANP-specific antiserum resulted in a significant increase in potassium balance in the present study. These results suggest that the kaliuretic response .Abserved during the second 24-h period after the injection of DOCA can also be attributed at least in part to the increased plasma ANP levels. In conclusion, passive immunization with ANP-specific antiserum resulted in a significant suppression of the decrease in both the sodium and the potassium balance that was otherwise observed during the second 24-h period after the injection of DOCA. It is, therefore, suggested that plasma ANP plays an important role in the mineralocorticoid escape phenomenon. The results provide further evidence to support the physiological significance of this cardiac peptide hormone in the regulation of body fluid homeostasis.
References 1. August JT, Nelson DH, Thorn GW 1958 Response of normal subjects to large amounts of aldosterone. J Clin Invest 37:1549 2. Knox FG, Burnett Jr JC, Kohan DE, Spielman WS, Strand JC 1980 Escape from the sodium-retaining effects of mineralocorticoids. Kidney Int 17:263 3. Hall JE, Montani JP, Woods LL, Mizelle HL 1986 Renal escape from vasopressin: role of pressure diuresis. Am J Physiol 250:F907 4. Hall JE, Granger JP, Smith MJ, Premen AJ 1984 Role of renal hemodynamics and atrial pressure in aldosterone "escape." Hypertension [Suppl 1] 6:1183 5. Hall JE, Granger JP, Hester RL, Coleman TG, Smith Jr MJ, Cross RB 1984 Mechanisms of escape from sodium retention during angiotensin II hypertension. Am J Physiol 246:F627 6. Burnett Jr JC, Haas JA, Larson MS 1985 Renal interstitial pressure in mineralocorticoid escape. Am J Physiol 249:F396 7. Cantin M, Genest J 1985 The heart and the atrial natriuretic factor. Endocr Rev 6:107 8. De Bold AJ 1985 Atrial natriuretic factor: a hormone produced by the heart. Science 230:767 9. Lang RE, Tholken H, Ganten D, Luft FC, Ruskoacho H, Unger TH 1985 Atrial natriuretic factor-a circulating hormone stimulated by volume loading. Nature 314:264 10. Naruse M, Ohnishi S, Naruse K, Kasanuki H, Shibasaki T, Demura H, Hirosawa K, Inagami T, Shizume K 1989 Increased atrial natriuretic peptide and decreased vasopressin during supraventricular tachycardia. In: Brenner BM, Laragh JH (eds) Progress in Atrial Peptide Research. American Society of Hypertention Symposium Series. Raven Press, New York, vol 3:483 11. Burnett JC, Kao PC, Hu DC, Heser DW, Heublin D, Granger JP, Opgenorth TJ, Reeder GS 1986 Atrial natriuretic peptide elevation in congestive heart failure in the human. Science 231:1145 12. Anderson JV, Raine AEG, Proudler A, Bloom SR 1986 Effect of hemodyalysis on plasma concentrations of atrial natriuretic peptide in adult patients with chronic renal failure. J Endocrinol 110:193 13. Genest J, Larochelle P, Cusson JR, Gutkowska J, Cantin M 1988 The atrial natriuretic factor in hypertension. Hypertension [Suppl 1] 11:13 14. Ballermann BJ, Bloch KD, Seidmen JG, Brenner BM 1986 Atrial natriuretic peptide transcription, secretion and glomerular receptor activity during mineralocorticoid escape in the rat. J Clin Invest 78:840 15. Metzler LH, Gardner DG, Keil LC, Baxter JD, Ramsay DJ 1987 Increased synthesis and release of atrial peptide during DOCA escape in conscious dogs. Am J Physiol 252:R188 16. Miyamori I, Ikeda M, Matsubara T, Okamoto S, Koshida H, Morise T, Takeda R 1987 Human atrial natriuretic polypeptide during escape from mineralocorticoid excess in man. Clin Sci 73:431 17. Zimmerman RS, Edwards BS, Schwab TR, Heublein DM, Burnett Jr JC 1987 Atrial natriuretic peptide during mineralocorticoid escape in the human. J Clin Endocrinol Metab 64:624 18. Gaillard CA, Koomans HA, Rabelink TJ, Braam B, Boer P, Mees D 1988 Enhanced natriuretic effect of atrial natriuretic factor during mineralocorticoid escape in humans. Hypertension 12:450 19. Weidmann P, Matter DR, Matter EE, Gnadinger MP, Uehlinger DE, Shaw S, Hess C 1988 Glucocorticoid and mineralocorticoid stimulation of atrial natriuretic peptide release in man. J Clin Endocrinol Metab 66:1233 20. Naruse M, Obana K, Naruse K, Sugino N, Demura H, Shizume K, Inagami T 1985 Antisera to atrial natriuretic factor reduces urinary sodium excretion and increases plasma renin activity in rats. Biochem Biophys Res Commun 132:954 21. Mohring J, Mohring B 1972 Reevaluation of DOCA escape phenomenon. Am J Physiol 223:1237 22. Naruse M, Naruse K, Obana K, Kurimoto F, Sakurai H, Honda T, Higashida T, Demura H, Inagami T, Shizume K 1986 Immunoreactive-human atrial natriuretic polypeptide in human plasma. Peptides 7:141 23. Mulrow PJ, Forman BH 1972 The tissue effect of mineralocorticoids. Am J Med 53:561
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s las
1
229
230
ANP IN MINERALOCORTICOID ESCAPE
24. Ellinghaus K 1971 Sodium and potassium balance during the administration of desoxycorticosterone in dogs with differing sodium intakes. Pfluegers Arch Ges Physiol 322:347 25. Bartter FC, Biglieri EG 1958 Primary aldosteronism: clinical staff conference at the National Institutes of Health. Ann Intern Med 48:647 26. Conn JW, Knopf RF, Nesbit RM 1964 Primary aldosteronism: present evaluation of its clinical characteristics and of the results
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of surgery. In: Baulieu EE, Robel P (eds) Aldosterone. Blackwell, Oxford, p 327 27. Lauler DP, Hickler RB, Thorn GW 1962 The salivary sodiumpotassium ratio: a useful "screening test" for aldosteronism in hypertensive subjects. N Engl J Med 267:1136 28. Conn JW 1963 Aldosteronism in man. I. Some clinical and climatological aspects. JAMA 183:775
On May 23-24, 1991, the 34th Symposium of the JOURNEES INTERNATIONALES HENRI-PIERRE KLOTZ D'ENDOCRINOLOGIE CLINIQUE on: Endocrinology of Puberty will be held in Paris. Main lectures and free communications in English and French are programmed. The deadline for submitting abstracts is January 31, 1991. For further information, please contact the Scientific Secretariat: Dr. G. Copinschi, Dr. A. Caufriez, Laboratoire de Medecine Experimentale, Faculte de Medecine, U.L.B., 2 rue Evers, B-1000 Brussels, Belgium.
Endocrinologie de la Puberte Les 23 et 24 mai 1991 auront lieu a Paris les 34emes Journees Internationales Henri-Pierre Klotz d'Endocrinologie Clinique consacre a: Endocrinologie de la Puberte. Des exposes magistraux et des communications libres en franc, ais et en anglais sont prevus. La date limite pour l'envoi des resumes est le 31 Janvier 1991. Si vous desirez recevoir des informations supplementaires, veuillez ecrire au Secretariat Scientifique: Dr. G. Copinschi, Dr. A. Caufriez, Laboratoire de Medecine Experimentale, Faculte de Medecine, U.L.B., 2 rue Evers, B-1000 Bruxelles, Belgique.
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Endocrinology of Puberty
Vol. 128, No. 1 Printed in U.S.A.
ZHENG-PEI ZENGf, MITSUHIDE NARUSE, KIYOKO NARUSE, YUKO KATO, YI-FAN SHI, HIROSHI DEMURA, AND KAZUO SHIZUME Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women's Medical College, Tokyo 162, Japan; and the Department of Endocrinology, Peking Union Medical College Hospital ((Y.-F.S.), Beijing 100730, China
with a significant increase in plasma levels of ANP. Administration of ANP-specific antiserum significantly impaired the return to normal sodium balance as well as the augmented kaliuresis that were observed on the second day after injection of DOCA. No significant effect was observed on either sodium or potassium balance after the injection of normal rabbit serum. These results suggest that plasma ANP plays an important role in mineralocorticoid escape. {Endocrinology 128: 226-230, 1991)
ABSTRACT. To elucidate the physiological role of atrial natriuretic peptide (ANP) in plasma during mineralocorticoid escape, we investigated the effects of passive immunization with ANP-specific antiserum on deoxycorticosterone (DOCA)treated rats. Sodium was retained in excess of intake during the first day after treatment with DOCA, and sodium balance returned to control values by the second day, whereas the excretion of potassium exceeded the intake during all days after DOCA treatment. These changes in electrolyte balance were associated
E
XOGENOUS administration of mineralocorticoid results in a transient accumulation of sodium chloride, but the balance between sodium intake and output returns to the basal level after a certain period of time. This phenomenon has been termed mineralocorticoid escape (1). Various factors have been suggested as being involved in the phenomenon, namely increases in systemic arterial pressure, glomerular filtration rate, renal arterial pressure, renal interstitial hydrostatic pressure, and renal prostaglandins as well as decreases in levels of angiotensin-II and renal adrenergic activity (2-6). However, details of the mechanism responsible for this phenomenon have not been elucidated. Atrial natriuretic peptide (ANP), discovered in mammalian atrial tissue, possesses potent natriuretic and vasorelaxant activities (for reviews, see Refs. 7 and 8). The importance of the peptide has been implied in various physiological and pathological conditions associated with abnormalities in blood pressure and/or body fluid
volume: natriuresis during saline loading (9), supraventricular tachycardia (10), congestive heart failure (11), chronic renal failure (12), and various types of hypertension (13). In addition, increased plasma levels of ANP have been demonstrated during mineralocorticoid escape in animals (14, 15) and humans (16-19). This latter observation suggests a possible role for the peptide in the escape phenomenon, although no direct evidence for its involvement has been presented. The present study was undertaken to examine the role of plasma ANP during mineralocorticoid escape by application of a passive immunization technique with ANPspecific antiserum. We found that the ANP-specific antiserum abolished mineralocorticoid escape in rats treated with deoxycorticosterone acetate (DOCA). Materials and Methods Animals and experimental protocols Adult male Wistar rats, weighing 250-300 g, were kept under standard light in individual metabolic cages for several days before experiments. Rats were fed a special low sodium diet that included the following components: water, 10.3%; sodium, 7.41 mg/100 g; potassium, 401 mg/100 g; calcium, 630 mg/100 g; phosphorus, 530 mg/100 g; and magnesium, 28.1 mg/100 g. Sodium chloride was provided in drinking water (Na, 25.5 mmol/liter), such that animals consumed 0.7-0.9 mmol sodium/day.
Received June 11,1990. Address all correspondence and requests for reprints to: Dr. Mitsuhide Naruse, Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women's Medical College, Tokyo 162, Japan. * This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture and a research grant from the Foundation for Growth Science in Japan. t Current address: Department of Endocrinology, Peking Union Medical College Hospital, Beijing, China. 226
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Antiserum against Homologous Atrial Natriuretic Peptide Diminishes the Natriuretic Response during Mineralocorticoid Escape in Rats*
ANP IN MINERALOCORTICOID ESCAPE Experimental protocols
it returned to the control level on the second and subsequent days after DOCA injection (Fig. 1). The fractional excretion of sodium decreased significantly from 5.85 ± 3.33% to 1.00 ± 0.57% during the first 24-h period and increased significantly to 13.11 ± 3.90% during the second 24-h period after the injection of DOCA. Urine volumes increased significantly from 18.3 ± 0.7 ml/24 h during the control period to 53.8 ± 4.6 ml/24 h during the second 24-h period after the administration of DOCA, whereas no significant change was observed during the first 24-h period. The potassium balance was significantly decreased each day after the injection of DOCA (Fig. 2). By contrast, there were no significant changes in urine volume (data not shown), sodium balance, or potassium balance throughout the experimental period in control rats (Figs. 1 and 2). In addition, no significant differences DO : A
•o 31-0
e balance o
*T-
T
en
Blood samples
+
Assay methods Concentrations of sodium and potassium in serum and urine were determined by flame photometry. The sodium and potassium balances for each 24-h period were calculated by subtracting the urinary output of each from the respective intake. Concentrations of creatinine in serum and urine were measured with commercially available kits (Eiken Immunochemical Laboratories, Tokyo, Japan). Plasma concentrations of ANP were determined by RIA after extraction with a Sep-Pak C18 cartridge (Waters, Milford, CA) (21). PRA was determined by RIA after generation of angiotensin-I (20). Plasma aldosterone concentrations (PAC) were measured with RIA kits (Dainabot RI Laboratories, Tokyo, Japan).
Sodiu
F
**
1 **t i
2
3
3
Trunk blood was collected immediately after decapitation into chilled test tubes, either with or without Na2EDTA (5 mM) plus aprotinin (500 IU/ml). The plasma and serum samples obtained by centrifugation at 4 C were kept at -20 C until assayed.
Control period
1
Time ( days) FIG. 1. Effects of a single injection of DOCA (DOCA group; • ) or vehicle (control group; • ) on sodium balance in rats. Values are the mean ± SE (n = 16 during the control period, n = 12 on the first day, n = 8 on the second day, and n = 4 on the third day). *, P < 0.01 vs. control period. **, P < 0.01 vs. the first day after the injection of DOCA. f, P < 0.05 vs. control group, ft, P < 0.01 vs. control group. |2.0
Statistical analysis All data are presented as the mean ± SE of results of multiple experiments. Data were analyzed by analysis of variance followed by Student's t test or by Duncan's new multiple range test. Significance was presumed if P < 0.05.
Results Although sodium balance was significantly increased during the first 24-h period after the injection of DOCA,
Control period
1 '
9 *•
Time (days) FIG. 2. Effects of a single injection of DOCA (DOCA group; B) or vehicle (control group; • ) on potassium balance in rats. Values are the mean ± SE (n = 16 during the control period, n = 12 on the first day, n = 8 on the second day, and n = 4 on the third day). *, P < 0.01 vs. control period, f, P < 0.01 vs. control group.
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Body weight and daily intake of food and water were measured every morning throughout the experiment. Urine was also collected daily and was used to measure rates of excretion of sodium, potassium, and creatinine. After a 3-day control period, the first group of rats (DOCA group; n = 16) was sc injected with 10 mg DOCA in 0.1 ml sesame oil at 0900 h on the fourth day of the experiment, and these rats were killed by decapitation at 0 h (n = 4), 24 h (n = 4), 48 h (n = 4), and 72 h (n = 4) after the injection of DOCA. The second group of rats (control group; n = 16) was sc injected with 0.1 ml sesame oil alone, and these rats were also killed by decapitation 0 h (n = 4), 24 h (n = 4), 48 h (n = 4), and 72 h (n = 4) after injection. The third group of rats (DOCA plus ANP-specific antiserum group; n = 5) and the fourth group of rats (DOCA plus normal rabbit serum group; n = 5) were injected with 100 ^1 antiserum raised against rat ANP (Ab 420; 1:100) (20) and normal rabbit serum (NRS; 1:100), respectively, through the jugular vein under pentobarbital anesthesia 24 h after the administration of DOCA. Forty-eight hours after the injection of DOCA, rats were killed by decapitation. Based upon the extent of the binding of 125I-labeled ANP, the capacity of the antiserum to bind ANP was estimated to be about 1 ng/ 1 (A antiserum.
227
ANP IN MINERALOCORTICOID ESCAPE
228
Discussion Sodium balance was significantly increased during the first 24-h period after a single injection of DOCA and was restored during the next 24-h period. The initial retention of sodium was the result of the mineralocorticoid effects of DOCA. The rate of onset of the escape has been reported to be related to the extent of sodium intake (22, 23). Mohring and Mohring (21) showed that escape occurs within 48 h after the administration of DOCA for 5-7 days in rats given less than 2.5 mmol sodium/day. However, Ballermann et al. (14) demonstrated that retention of sodium during the first 24 h after the administration of mineralocorticoid can be most
reliably demonstrated with a sodium intake of 0.5-1.0 mmol/day, and in this case the escape occurs within 48 h. The chronological changes in sodium balance in the present study, in which 0.7-0.9 mmol/day sodium was taken up, are in agreement with the timing of the mineralocorticoid escape observed by Ballerman et al. (14). Administration of DOCA was accompanied by changes in plasma levels of ANP. In agreement with the results of previous studies (14-19), plasma ANP levels were significantly elevated 24 h after the injection of DOCA and remained high throughout the experimental period. The increase seems to be sufficiently large to suggest the involvement of ANP in mineralocorticoid escape. Since the passive immunization technique has been effectively used to elucidate the biological significance of plasma ANP in rats (20), we investigated the effects of ANPspecific antiserum on mineralocorticoid escape in DOCAtreated rats. In contrast to that in control rats injected with NRS, a significant inhibition of the restoration of the sodium balance during the second 24-h period after the injection of DOCA was observed in rats given ANPspecific antiserum. These results provide direct evidence that increased levels of endogenous ANP in plasma play a key role in the natriuretic response during mineralocorticoid escape. Potassium balance was decreased during the first 24h period after the administration of DOCA, and this decrease can be attributed to the mineralocorticoid action of DOCA. Furthermore, in contrast to the changes in sodium balance, potassium balance decreased further during the second 24-h period after the administration of DOCA. This result is in agreement with the previously reported finding that the kidney escapes from the sodium-retaining effect of mineralocorticoids, but not from the loss of potassium (1, 24-26). The discrepancies in the changes in rates of excretion of sodium and potassium as well as the absence of any escape from the sodium-retaining effects in the gastrointestinal tract and
TABLE 1. Changes in plasma levels of ANP, PRA, and PAC before and after injection of DOCA Group ANP (pg/ml) Control DOCA PRA(ngAngI/mlh) Control DOCA PAC (ng/dl) Control DOCA
Days after DOCA
Control period 1
2
3
142.5 ± 9.0 168.4 ± 19.0
104.4 ± 17.8 361.4 ± 49.5°
177.6 ± 23.5 431.8 ± 87.8c'rf
221.9 ± 26.1 303.9 ± 55.2"
4.0 ± 0.5 5.5 ± 0.8
4.3 ± 0.3 1.6 ± 0.6°
4.6 ± 0.2 2.0 ± 0.3"
4.7 ± 0.5 1.9 ± 0.5°
13.7 ± 2.0 17.3 ± 0.9
9.7 ± 3.4 1.1 ± 0.9*
12.4 ± 3.6 0.6 ± 0.5*
8.4 ± 3.3 0.5 ± 0.2*
Data are the mean ± SE of results from four rats. " P < 0.05 us. control period; P < 0.05 us. control group. b P< 0.01 us. control period; P < 0.01 vs. control group.
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in creatinine clearance were found between groups or during experiments. Changes in plasma ANP levels, PRA, and PAC are shown in Table 1. In the DOCA group, plasma ANP levels increased approximately 2-fold, whereas PRA and PAC were suppressed. By contrast, in the control group, plasma ANP concentrations, PRA, and PAC did not show significant changes over the experimental period. It appeared from the changes in sodium balance and plasma levels of ANP in the first series of experiments that mineralocorticoid escape occurs from the second day after the administration of DOCA. In the second series of experiment with NRS or antiserum, therefore, sodium and potassium balance were determined for 2 days after the injection of DOCA. The chronological changes in these parameters were essentially the same as those in the DOCA-injected group described above. The restoration of the sodium balance during the second 24-h period after the administration of DOCA was abolished by the iv administration of ANP-specific antiserum (Fig. 3). In addition, the decrease in potassium balance during the second 24-h period was significantly diminished by the injection of antiserum against rat ANP (Pig. 4).
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ANP IN MINERALOCORTICOID ESCAPE
DOCA
Ab or NRS
I
)
1.5
T
Control period
*< !
1 Time (days)
2
FIG. 3. Effects of iv administration of ANP-specific antiserum (Ab; B) or NRS (•) on sodium balance in DOCA-treated rats. Each point represents the mean ± SE of results from five rats. *, P < 0.01 us. control period. **, P < 0.05 us. the first day after DOCA. f, P < 0.01 us. DOCA plus NRS group.
-0.5t 1 2 Time (days)
FlG. 4. Effects of iv administration of ANP-specific antiserum (Ab; H) or NRS (•) on potassium balance in DOCA-treated rats. Each point represents the mean ± SE of results from five rats. *, P < 0.01 us. control period, t, P < 0.01 us. DOCA plus NRS group.
salivary or sweat glands (22, 27, 28) support the hypothesis that the escape is not due to a circulating antagonists) of mineralocorticoids (22). However, ANP is well known to have a kaliuretic activity in vivo in rats (7, 8). Administration of ANP-specific antiserum resulted in a significant increase in potassium balance in the present study. These results suggest that the kaliuretic response .Abserved during the second 24-h period after the injection of DOCA can also be attributed at least in part to the increased plasma ANP levels. In conclusion, passive immunization with ANP-specific antiserum resulted in a significant suppression of the decrease in both the sodium and the potassium balance that was otherwise observed during the second 24-h period after the injection of DOCA. It is, therefore, suggested that plasma ANP plays an important role in the mineralocorticoid escape phenomenon. The results provide further evidence to support the physiological significance of this cardiac peptide hormone in the regulation of body fluid homeostasis.
References 1. August JT, Nelson DH, Thorn GW 1958 Response of normal subjects to large amounts of aldosterone. J Clin Invest 37:1549 2. Knox FG, Burnett Jr JC, Kohan DE, Spielman WS, Strand JC 1980 Escape from the sodium-retaining effects of mineralocorticoids. Kidney Int 17:263 3. Hall JE, Montani JP, Woods LL, Mizelle HL 1986 Renal escape from vasopressin: role of pressure diuresis. Am J Physiol 250:F907 4. Hall JE, Granger JP, Smith MJ, Premen AJ 1984 Role of renal hemodynamics and atrial pressure in aldosterone "escape." Hypertension [Suppl 1] 6:1183 5. Hall JE, Granger JP, Hester RL, Coleman TG, Smith Jr MJ, Cross RB 1984 Mechanisms of escape from sodium retention during angiotensin II hypertension. Am J Physiol 246:F627 6. Burnett Jr JC, Haas JA, Larson MS 1985 Renal interstitial pressure in mineralocorticoid escape. Am J Physiol 249:F396 7. Cantin M, Genest J 1985 The heart and the atrial natriuretic factor. Endocr Rev 6:107 8. De Bold AJ 1985 Atrial natriuretic factor: a hormone produced by the heart. Science 230:767 9. Lang RE, Tholken H, Ganten D, Luft FC, Ruskoacho H, Unger TH 1985 Atrial natriuretic factor-a circulating hormone stimulated by volume loading. Nature 314:264 10. Naruse M, Ohnishi S, Naruse K, Kasanuki H, Shibasaki T, Demura H, Hirosawa K, Inagami T, Shizume K 1989 Increased atrial natriuretic peptide and decreased vasopressin during supraventricular tachycardia. In: Brenner BM, Laragh JH (eds) Progress in Atrial Peptide Research. American Society of Hypertention Symposium Series. Raven Press, New York, vol 3:483 11. Burnett JC, Kao PC, Hu DC, Heser DW, Heublin D, Granger JP, Opgenorth TJ, Reeder GS 1986 Atrial natriuretic peptide elevation in congestive heart failure in the human. Science 231:1145 12. Anderson JV, Raine AEG, Proudler A, Bloom SR 1986 Effect of hemodyalysis on plasma concentrations of atrial natriuretic peptide in adult patients with chronic renal failure. J Endocrinol 110:193 13. Genest J, Larochelle P, Cusson JR, Gutkowska J, Cantin M 1988 The atrial natriuretic factor in hypertension. Hypertension [Suppl 1] 11:13 14. Ballermann BJ, Bloch KD, Seidmen JG, Brenner BM 1986 Atrial natriuretic peptide transcription, secretion and glomerular receptor activity during mineralocorticoid escape in the rat. J Clin Invest 78:840 15. Metzler LH, Gardner DG, Keil LC, Baxter JD, Ramsay DJ 1987 Increased synthesis and release of atrial peptide during DOCA escape in conscious dogs. Am J Physiol 252:R188 16. Miyamori I, Ikeda M, Matsubara T, Okamoto S, Koshida H, Morise T, Takeda R 1987 Human atrial natriuretic polypeptide during escape from mineralocorticoid excess in man. Clin Sci 73:431 17. Zimmerman RS, Edwards BS, Schwab TR, Heublein DM, Burnett Jr JC 1987 Atrial natriuretic peptide during mineralocorticoid escape in the human. J Clin Endocrinol Metab 64:624 18. Gaillard CA, Koomans HA, Rabelink TJ, Braam B, Boer P, Mees D 1988 Enhanced natriuretic effect of atrial natriuretic factor during mineralocorticoid escape in humans. Hypertension 12:450 19. Weidmann P, Matter DR, Matter EE, Gnadinger MP, Uehlinger DE, Shaw S, Hess C 1988 Glucocorticoid and mineralocorticoid stimulation of atrial natriuretic peptide release in man. J Clin Endocrinol Metab 66:1233 20. Naruse M, Obana K, Naruse K, Sugino N, Demura H, Shizume K, Inagami T 1985 Antisera to atrial natriuretic factor reduces urinary sodium excretion and increases plasma renin activity in rats. Biochem Biophys Res Commun 132:954 21. Mohring J, Mohring B 1972 Reevaluation of DOCA escape phenomenon. Am J Physiol 223:1237 22. Naruse M, Naruse K, Obana K, Kurimoto F, Sakurai H, Honda T, Higashida T, Demura H, Inagami T, Shizume K 1986 Immunoreactive-human atrial natriuretic polypeptide in human plasma. Peptides 7:141 23. Mulrow PJ, Forman BH 1972 The tissue effect of mineralocorticoids. Am J Med 53:561
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s las
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of surgery. In: Baulieu EE, Robel P (eds) Aldosterone. Blackwell, Oxford, p 327 27. Lauler DP, Hickler RB, Thorn GW 1962 The salivary sodiumpotassium ratio: a useful "screening test" for aldosteronism in hypertensive subjects. N Engl J Med 267:1136 28. Conn JW 1963 Aldosteronism in man. I. Some clinical and climatological aspects. JAMA 183:775
On May 23-24, 1991, the 34th Symposium of the JOURNEES INTERNATIONALES HENRI-PIERRE KLOTZ D'ENDOCRINOLOGIE CLINIQUE on: Endocrinology of Puberty will be held in Paris. Main lectures and free communications in English and French are programmed. The deadline for submitting abstracts is January 31, 1991. For further information, please contact the Scientific Secretariat: Dr. G. Copinschi, Dr. A. Caufriez, Laboratoire de Medecine Experimentale, Faculte de Medecine, U.L.B., 2 rue Evers, B-1000 Brussels, Belgium.
Endocrinologie de la Puberte Les 23 et 24 mai 1991 auront lieu a Paris les 34emes Journees Internationales Henri-Pierre Klotz d'Endocrinologie Clinique consacre a: Endocrinologie de la Puberte. Des exposes magistraux et des communications libres en franc, ais et en anglais sont prevus. La date limite pour l'envoi des resumes est le 31 Janvier 1991. Si vous desirez recevoir des informations supplementaires, veuillez ecrire au Secretariat Scientifique: Dr. G. Copinschi, Dr. A. Caufriez, Laboratoire de Medecine Experimentale, Faculte de Medecine, U.L.B., 2 rue Evers, B-1000 Bruxelles, Belgique.
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Endocrinology of Puberty
