- Email: [email protected]
Plasma atrial natriuretic peptide levels during the rat estrous cycle, pregnancy, and puerperium
Plasma atrial natriuretic peptide levels during the rat estrous cycle, pregnancy, and puerperium Lony C. Castro, MD, Chander Arora, PhD, Simone Parvez, PhD, Hassan Parvez, PhD, Guillermo Valenzuela, MD, and Calvin J. Hobel, MD Los Angeles, California, and Paris, France The rat has been used as a model for studying the changes that occur in maternal blood volume and renal function during pregnancy. The role, if any, that atrial natriuretic peptide plays in regulating these changes is unknown, and little information is available on atrial natriuretic peptide levels at different stages of gestation in the rat. In this study we measured plasma atrial natriuretic peptide levels by radioimmunoassay in the rat at each stage of the estrous cycle, during the last 2 weeks of pregnancy, and in the early postpartum period. Atrial natriuretic peptide levels did not change during the estrous cycle. Atrial natriuretic peptide levels were low on days 10 to 15 of gestation but rose to become significantly higher than nonpregnant levels on days 16 to 18. On day 21 shortly before delivery, levels were similar to nonpregnant values. Postpartum, atrial natriuretic peptide levels rose immediately and remained elevated for the next 48 hours. These findings suggest that factors other than blood volume may mediate plasma atrial natriuretic peptide levels during pregnancy and the postpartum period. (AM J OBSTET GVNECOL 1989;160:15-9.)
Key words: Atrial natriuretic peptide, pregnancy, puerperium, rat
Atrial natriuretic peptide appears to be involved in the regulation of blood volume, blood pressure, and renal function. Changes in atrial stretch, such as those induced by acute blood volume expansion, stimulate the secretion of atrial natriuretic peptide from mammalian cardiac atria.' In turn atrial natriuretic peptide acts on the kidney to increase the glomerular filtration rate and suppresses basal and stimulated aldosterone secretion. 3 Atrial natriuretic peptide has also been shown to relax precontracted vascular beds.' The end result of these diverse actions is to decrease the blood volume sensed by the atria. During normal human pregnancy there is a significant increase in maternal blood volume,' as well as an increase in the glomerular filtration rate 6 and altered vascular sensitivity to pressor hormones. 7 These changes also occur in pregnant rats and they have served as a model to study the pregnancy-induced alterations in blood volume and renal function. Whether atrial natriuretic peptide is involved in regulating these changes is unclear, since little has been published conFrom the DIVISIOns of Maternal-Fetal-Medicine, Cedars-Sinal Medical Center and Lama Linda University, and the Neuropharmacology Umt, UnIVersity of Paris. Supported by BwmedicalResearch Support Grant No. S07RR05486. Presented at the Thirty-fifth Annual Meeting of the Society for Gynecologic Investigation, Baltimore, Maryland, March 17-20, 1988. Reprint requests: Lony C. Castro, MD, Department of Obstetrics/Gynecology, Rm. 3210, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048.
cerning atrial natriuretic peptide in the pregnant rat. The purpose of this study was to describe the changes that occur in plasma atrial natriuretic peptide levels during the rat estrous cycle, pregnancy, and the early postpartum period to determine if the changes parallel the known pregnancy-induced alterations in blood volume and renal function.
Material and methods Animals studied. These experiments were done on 49 time-dated pregnant or postpartum SpragueDawley rats and 34 age-matched virgin control rats that were 10 to 12 weeks old (Simonsen, Gilroy, Calif.). Weights ranged from 160 to 219 gm (virgin), 190 to 360 gm (pregnant), and 230 to 285 gm (postpartum). The experimental protocol was approved in advance by the institutions' animal research committee. The animals were housed at least 6 days before study in a facility controlled for temperature and light. They had free access to standard laboratory chow and water. In the nonpregnant animals, the stage of the estrous cycle was determined by vaginal smear on the morning of study. The pregnant animals were studied on days 10 to 15, 16 to 18, and 21 of gestation. Day 1 of pregnancy was determined by the presence of a cervical mucus plug. Each group of pregnant animals had an average of 12 ± 1 fetuses. Postpartum animals were allowed to suckle. They were studied 0 to 3 and 8 to 48 hours after delivery. In all animals, between 2 and 2.5 ml of blood was obtained from the abdominal aorta im-
15
16 Castro et al.
January 1989 Am J Obstet Gynecol
B/B o 1 .0
PLASMA EXTRACTS
1:32 116 1:8 1:4 1:2 ~----41~----~1------+1----~1 I
0.8 0.6 0.4
0.2
10
100
rat ANP (pg) Fig. 1. Rat atrial natriuretic peptide (ANP) standard curve (e--e) and serial dilutions of rat plasma extracts: pregnant (0--0), postpartum (. - - . ) , and nonpregnant (0 --0).
mediately after cervical dislocation. The blood was transferred into chilled tubes containing ethylenediaminetetraacetic acid (1 mg/ml) and aprotinin (500 kallikrein inhibitor units per milliliter) and centrifuged at 2000 x g for 10 minutes at 4° C. The plasma was aspirated and stored at - 50° C until extracted. Plasma extraction. Sep-Pak C-18 cartridges (Waters Associates, Milford, Mass.) were preactivated with 10 ml of methanol and washed with 10 ml of triethylamine acetate buffer (20 mmol/L, pH 4.0); 100 fLl of 1 mollL hydrochloric acid was added to each milliliter of plasma. The plasma was then centrifuged and 0.5 to 1 ml was loaded onto each Sep-Pak cartridge. The cartridge was then washed with 4 ml of triethylamine acetate buffer, and the adsorbed atrial natriuretic peptide was eluted with 4 ml of 80% methanol, 20% triethylamine acetate buffer. The eluate was dried in a Speed Vac concentrator (Savant Instruments, Hicksville, N.Y.). The dried extracts were stored at - 50° C and resuspended in buffer on the day of assay. Atrial natriuretic peptide radioimmunoassay. A double antibody radioimmunoassay was used to measure plasma atrial natriuretic peptide concentrations. Rabbit antiatrial natriuretic polypeptide antiserum (Peninsula Laboratories, Belmont, Calif.) was used (Lot No. 009325-10). This antiserum recognizes the carboxyl terminus of the 28-amino-acid atrial natriuretic peptide molecule. Cross-reactivities have been previously reported S and were: 1 to 28 human atrial natri-
uretic peptide (100%), 1 to 28 rat atrial natriuretic peptide (100%), 5 to 28 rat atriopeptin III (100%), 5 to 27 rat atriopeptin II (5%), and 5 to 25 Tat atriopeptin I (1.7%). Five fLCi of iodine 125-rat atrial natriuretic peptide (Peninsula Laboratories) were resuspended in distilled water to yield a solution containing 14,000 counts per minute per 100 fLl. Standard curves were constructed with 1 to 28 rat atrial natriuretic peptide (rat atrial natriuretic peptide, Peninsula Laboratories) dissolved in radioimmunoassay buffer over a concentration range of 1 to 250 pgltube. The assay buffer consisted of 19 mmollL monobasic and 81 mmollL dibasic sodium phosphate, 0.05 mollL sodium chloride, 0.1 % bovine serum albumin, 0.1 % Triton X-100, and 0.01 % sodium azide (pH 7.4). The assay was performed in 12 by 75 mm polystyrene tubes; 100 fLl of standard or unknown was added to 100 fLl of rehydrated antisera and incubated overnight at 4° C. The 100 fLl of 1251_rat atrial natriuretic peptide was then added, mixed, and incubated overnight at 4° C. Bound and free fractions were separated by means of diluted normal rabbit serum and goat antirabbit ,,(-globulin (Peninsula Laboratories). After 2 hours of incubation at room temperature, 0.5 ml of assay buffer was added to each tube, and the tubes were centrifuged at 2000 x gat 4° C for 20 minutes. The supernatant was then aspirated, and the precipitate was counted in a ,,(-counter. Statistical analysis. All values are reported as mean ± standard error of the mean. Differences between groups were analyzed by one-way analysis of var-
Atrial natriuretic peptide in the pregnant rat
Volume 160 Number I
-
150 -
0'
-
17
E
"
c.. 100 ....., 0..
Z
E
T
T
T
T
50 -
en
0
c..
0
metestrus diestrus proestrus n=9 n=10 n=7
estrus n=8
Fig. 2. Plasma atrial natriuretic peptide (ANP) levels in nonpregnant rats during each stage of the estrous cycle.
iance with the Student-Newman-Keuls test. A p value <0.05 was considered significant. To eliminate any interas say variability, all atrial natriuretic peptide values reported are from samples run in a single assay.
Results A typical standard curve plotted as B/Bo versus picograms of rat atrial natriuretic peptide is shown in Fig. 1. The sensitivity of the assay, the least amount distinguishable from 0 with 95% confidence, ranged from 1 to 2 pg. Multiple dilutions of pregnant, nonpregnant (metestrus), and postpartum rat plasma extracts,were approximately parallel to the rat atrial natriuretic peptide standard curve. The extraction recovery of 100 pg of added synthetic rat atrial natriuretic peptide in pregnant and nonpregnant rat plasma was similar (88% ± 0.07%, n = 4 and 93% ± 0.03%, n = 4, respectively). Duplicate measurements of five replicate extractions of a single plasma sample were used to calculate the intraassay coefficients of variation at 76% and 35% binding, which were 6.5% and 3%, respectively. The plasma atrial natriuretic peptide levels obtained in nonpregnant rats during each stage of the estrous cycle are shown in Fig. 2. Atrial natriuretic peptide levels did not change significantly during the estrous cycle, and the average atrial natriuretic peptide value for this group was 76 ± 6 pg/ml. The changes in plasma atrial natriuretic peptide levels obtained during pregnancy are depicted in Fig. 3, A. Plasma atrial natriuretic peptide levels were low at 10 to 15 days of pregnancy (37 ± 4 pg/ml) when compared with values at 16 to 18 days of pregnancy
but were not significantly lower than nonpregnant levels or levels on day 21 of pregnancy. On days 16 to 18 of gestation, plasma atrial natriuretic peptide levels had increased to 163 ± 32 pg/ml and were significantly greater than levels obtained in either the nonpregnant group or at 10 to 15 days' gestation (p <: 0.01). By day 21 of pregnancy (shortly before delivery), atrial natriuretic peptide levels had decreased to 85 ± 21 pg I ml and were similar to nonpregnant levels. Post partum, plasma atrial natriuretic peptide levels rose to 167 ± 31 pg/ml during the first 3 hours after delivery (Fig. 3, B) and were significantly higher than maternal levels obtained just before delivery (p < 0.05) and on days 10 to 15 of pregnancy (p < 0.01), as well as higher than nonpregnant levels (p < 0.01). Plasma atrial natriuretic peptide levels remained elevated as long as 48 hours after delivery.
Comment Stimulation of atrial stretch receptors, such as that caused by short- and long-term changes in blood volume,9 appears to be the primary determinant of atrial natriuretic peptide release in vivo. There is additional evidence that other factors such as changes in myocardial work 'o and hormonal stimuli (e.g., glucocorticoids," catecholamines,12 and vasopressin '3 ) may also be involved in regulating atrial natriuretic peptide release either directly or through secondary hemodynamic changes. In rats pregnancy is accompanied by an increase in blood volume of greater than 50% at term. II Plasma volume has been found to be elevated as early as day
18
Castro et al.
January 1989 Am J Obstet Gynecol
10-15 days n=34
n=IO
16-18 days n = II
0-3
21 days
hours
n=9
n=9
8-48 haurs n =10
Fig. 3. A, Plasma atrial natriuretic peptide (ANP) levels in nonpregnant (0) and pregnant rats (I§:j) at different stages of gestation. B, Plasma atrial natriuretic peptide levels in postpartum rats (~). **P < 0.01 vs nonpregnant and 10 to 15 days' pregnant, ++ p < 0.01 vs 21 days' pregnant, *P < 0.05 vs nonpregnant and 10 to 15 days' pregnant, +p < 0.05 vs 21 days' pregnant.
615 and to progressively rise until term, with about 50% of the increase occurring during the third trimester. 11 We postulated a rise in plasma atrial natriuretic peptide levels during pregnancy that would parallel the known changes in blood volume. Our results did not confirm this. The fact that plasma atrial natriuretic peptide levels were not increased until days 16 to 18 suggests that the expanded blood volume is not great enough to stimulate atrial stretch receptors until the third trimester. The finding that plasma atrial natriuretic peptide levels decreased at term and were similar to virgin control rats was unexpected, since blood volume expansion is maximal at this time. This result is consistent with a report by Nadel et al.,lb which showed that plasma atrial natriuretic peptide levels in chronically catheterized pregnant rats on days 19 and 20 of pregnancy were not elevated compared with virgin control rats despite significant increases in plasma volume. Kristensen et al. 17 also reported that atrial natriuretic peptide levels in term rat atria (measured by a bioassay) were similar to virgin controls. Collectively, these studies suggest that in the near-term and term pregnant rat, the enlarged maternal vascular compartment is sensed by the atria as normal. One possible explanation for this is that blood pressure does not appear to decline until day 19 of pregnancylS and remains low until term. This could result in a decrease in myocardial work (a proposed determinant of atrial natriuretic peptide se-
cretion)lO and a consequent lowering of atrial natriuretic peptide levels from day 19 of pregnancy on. In the present study most of the term animals were in labor, and one could postulate that the endocrine changes associated with parturition may have suppressed atrial natriuretic peptide levels. We are unaware of any detailed studies that examined the effects of estrogen, progesterone, or prolactin on atrial natriuretic peptide secretion. In this study the changes in estrogen and progesterone that occur during the estrous cycle were not associated with any significant changes in plasma atrial natriuretic peptide levels. On the other hand, glucocorticoids" and oxytocin l3 have been reported to stimulate atrial natriuretic peptide levels in vivo and in vitro. Our finding that plasma atrial natriuretic peptide levels increased within I hour of delivery and remained elevated for the next 48 hours is also consistent with the study by Nadel et al. 16 In human pregnancies it has recently been shown that plasma atrial natriuretic peptide levels also increase during the first few days postpartum. 19 As suggested by these studies and ours, the decrease in maternal vascular capacitance that occurs postpartum may be associated with an increase in the "effective blood volume" (despite the decrease in absolute blood volume), which in turn stimulates atrial natriuretic peptide release. The increase in blood pressure observed postpartum may also increase myocardial
Volume 160 \\;umber 1
work, causing an elevation in plasma atrial natriuretic peptide levels. Alternatively, one can speculate that the profound changes in maternal endocrine status that occur in the early postpartum period may either directly or indirectly stimulate atrial natriuretic peptide release. We cannot say definitively whether the increase in plasma atrial natriuretic peptide levels observed were associated with any alterations in renal function since we made no measurements of this. Most investigators conclude that in the third trimester, the glomerular filtration rate is elevated, with debate centered on how early in pregnancy the glomerular filtration rate rises and whether it declines at term?O 21 The increase in atrial natriuretic peptide levels on days 16 to 18 suggests that atrial natriuretic peptide may be at least partially responsible for the elevated glomerular filtration rate present at that time. The postpartum rise in plasma atrial natriuretic peptide levels is consistent with reports of an increase in glomerular filtration rate accompanying lactation in the rat"2 and suggests that atrial natriuretic peptide may be involved in the diuresis that occurs after birth. We are grateful to Diana French for her expert secretarial assistance. REFERENCES 1. Lang R, Tholken H, Ganten D, et al. Atrial natriuretic factor-a circulating hormone stimulated by volume loading. Nature 1985;314:264. 2. Maack T, Camargo MJ, Kleinert HD, Laragh JH, Atlas SA. Atrial natriuretic factor: structure and functional properties. Kidney Int 1985;27:607. 3. Laragh JH. Atrial natriuretic hormone, the renin aldosterone axis and blood pressure-electrolyte homeostasis. N EnglJ Med 1985;313:1330. 4. Garcia R, Thibault G, Cantin M, Genest J. Effect of a purified atrial natriuretic factor on rat and rabbit vascular strips and vascular beds. AmJ Physiol 1984;234:R34. 5. Chesley LC. Plasma and red cell volumes during pregnancy. AM J OBSTET GYNECOL 1972; 112:440.
Atrial natriuretic peptide in the pregnant rat
19
6. DavisonJM. Dunlap W. Renal hemodynamics and tubular function in normal human pregnancy. Kidney Int 1980; 18: 152. 7. Schwarz R, Retzke U. Cardiovascular response to infusion of angiotensin II in pregnant women. Obstet Gynecol 1971;38:714. 8. Castro LC, Lam RW, Ross MG, et al. Atrial natriuretic peptide in the sheep. J Dev Phvsiol 1988; I 0:235. 9. Kohno M, Clegg K, Sambhi M. Effects of volume change on circulating immunoreactive atnal natriuretic factor in rats. Hypertension 1987; 10: 171. 10. Rankin AJ. Mechanisms for the release of atrial natriuretic peptIde. Can J Physiol Pharmacol 1987;65: 1673. 11. Matsubara H, Hirata Y, Yoshimi H, et al. Effects of steroid and thyroid hormones on synthesis of atrial natriuretic peptide by cultured atrial myocytes of rat. Biochem Biophys Res Commun 1987;145:336. 12. Toth M, Ruskoaho H, Lang RE. Regulation of atrial natriuretic peptide secretion. J Hypertens 1986;4(suppl 6):S538. 13. Manning P, Schwarz D. Katsube N, Holmberg S, Needleman P. Vasopressin-stimulated release of atriopeptin: endocrine antagonists in fluid homeostasis. Science 1985; 229:395. 14. Barron WM. Volume homeostasis during pregnancy in the rat. Am J Kidney Dis 1987;9:296. 15. Atherton JC, Dark JM, Garland HO, et al. Changes in water and electrolyte balance, plasma volume and composition during pregnancy in the rat. J Physiol 1982; 330:81. 16. Nadel AS, Ballerman BJ, Anderson S, Brenner BM. Interrelationships among atrial peptIdes, renin and blood volume in pregnant rats. Am J Physiol 1988;254:R793. 17. Kristensen CG, Nakagawa Y, Coe FL, Lindheimer MD. Effect of atrial natriuretIc factor in rat pregnancy. Am J Physiol 1986;250:R589. 18. Pike R. Sodium requirement of the rat during pregnancy. In: Lindheimer MD, Katz AI, Zuspan FP, eds. Hypertension III pregnancy. New York: John Wiley & Sons, 1976: 207-15. 19. Steegers EAP, Hein PR, Groeneveld EAM, et al. Atrial natriuretic peptide concentrations during pregnancy. Lancet 1987; I: 1267. 20. Baylis C. Renal hemodynamics and volume control during pregnancy in the rat. Semin Nephrol 1984;4:208. 21. Lindheimer MD, Katz AI. Kidney function in the pregnant rat. J Lab Clin Med 1971 ;78:633. 22. Arthur SK, Green R. Renal function during lactation in the rat. J Physiol 1983;334:379.
Key words: Atrial natriuretic peptide, pregnancy, puerperium, rat
Atrial natriuretic peptide appears to be involved in the regulation of blood volume, blood pressure, and renal function. Changes in atrial stretch, such as those induced by acute blood volume expansion, stimulate the secretion of atrial natriuretic peptide from mammalian cardiac atria.' In turn atrial natriuretic peptide acts on the kidney to increase the glomerular filtration rate and suppresses basal and stimulated aldosterone secretion. 3 Atrial natriuretic peptide has also been shown to relax precontracted vascular beds.' The end result of these diverse actions is to decrease the blood volume sensed by the atria. During normal human pregnancy there is a significant increase in maternal blood volume,' as well as an increase in the glomerular filtration rate 6 and altered vascular sensitivity to pressor hormones. 7 These changes also occur in pregnant rats and they have served as a model to study the pregnancy-induced alterations in blood volume and renal function. Whether atrial natriuretic peptide is involved in regulating these changes is unclear, since little has been published conFrom the DIVISIOns of Maternal-Fetal-Medicine, Cedars-Sinal Medical Center and Lama Linda University, and the Neuropharmacology Umt, UnIVersity of Paris. Supported by BwmedicalResearch Support Grant No. S07RR05486. Presented at the Thirty-fifth Annual Meeting of the Society for Gynecologic Investigation, Baltimore, Maryland, March 17-20, 1988. Reprint requests: Lony C. Castro, MD, Department of Obstetrics/Gynecology, Rm. 3210, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048.
cerning atrial natriuretic peptide in the pregnant rat. The purpose of this study was to describe the changes that occur in plasma atrial natriuretic peptide levels during the rat estrous cycle, pregnancy, and the early postpartum period to determine if the changes parallel the known pregnancy-induced alterations in blood volume and renal function.
Material and methods Animals studied. These experiments were done on 49 time-dated pregnant or postpartum SpragueDawley rats and 34 age-matched virgin control rats that were 10 to 12 weeks old (Simonsen, Gilroy, Calif.). Weights ranged from 160 to 219 gm (virgin), 190 to 360 gm (pregnant), and 230 to 285 gm (postpartum). The experimental protocol was approved in advance by the institutions' animal research committee. The animals were housed at least 6 days before study in a facility controlled for temperature and light. They had free access to standard laboratory chow and water. In the nonpregnant animals, the stage of the estrous cycle was determined by vaginal smear on the morning of study. The pregnant animals were studied on days 10 to 15, 16 to 18, and 21 of gestation. Day 1 of pregnancy was determined by the presence of a cervical mucus plug. Each group of pregnant animals had an average of 12 ± 1 fetuses. Postpartum animals were allowed to suckle. They were studied 0 to 3 and 8 to 48 hours after delivery. In all animals, between 2 and 2.5 ml of blood was obtained from the abdominal aorta im-
15
16 Castro et al.
January 1989 Am J Obstet Gynecol
B/B o 1 .0
PLASMA EXTRACTS
1:32 116 1:8 1:4 1:2 ~----41~----~1------+1----~1 I
0.8 0.6 0.4
0.2
10
100
rat ANP (pg) Fig. 1. Rat atrial natriuretic peptide (ANP) standard curve (e--e) and serial dilutions of rat plasma extracts: pregnant (0--0), postpartum (. - - . ) , and nonpregnant (0 --0).
mediately after cervical dislocation. The blood was transferred into chilled tubes containing ethylenediaminetetraacetic acid (1 mg/ml) and aprotinin (500 kallikrein inhibitor units per milliliter) and centrifuged at 2000 x g for 10 minutes at 4° C. The plasma was aspirated and stored at - 50° C until extracted. Plasma extraction. Sep-Pak C-18 cartridges (Waters Associates, Milford, Mass.) were preactivated with 10 ml of methanol and washed with 10 ml of triethylamine acetate buffer (20 mmol/L, pH 4.0); 100 fLl of 1 mollL hydrochloric acid was added to each milliliter of plasma. The plasma was then centrifuged and 0.5 to 1 ml was loaded onto each Sep-Pak cartridge. The cartridge was then washed with 4 ml of triethylamine acetate buffer, and the adsorbed atrial natriuretic peptide was eluted with 4 ml of 80% methanol, 20% triethylamine acetate buffer. The eluate was dried in a Speed Vac concentrator (Savant Instruments, Hicksville, N.Y.). The dried extracts were stored at - 50° C and resuspended in buffer on the day of assay. Atrial natriuretic peptide radioimmunoassay. A double antibody radioimmunoassay was used to measure plasma atrial natriuretic peptide concentrations. Rabbit antiatrial natriuretic polypeptide antiserum (Peninsula Laboratories, Belmont, Calif.) was used (Lot No. 009325-10). This antiserum recognizes the carboxyl terminus of the 28-amino-acid atrial natriuretic peptide molecule. Cross-reactivities have been previously reported S and were: 1 to 28 human atrial natri-
uretic peptide (100%), 1 to 28 rat atrial natriuretic peptide (100%), 5 to 28 rat atriopeptin III (100%), 5 to 27 rat atriopeptin II (5%), and 5 to 25 Tat atriopeptin I (1.7%). Five fLCi of iodine 125-rat atrial natriuretic peptide (Peninsula Laboratories) were resuspended in distilled water to yield a solution containing 14,000 counts per minute per 100 fLl. Standard curves were constructed with 1 to 28 rat atrial natriuretic peptide (rat atrial natriuretic peptide, Peninsula Laboratories) dissolved in radioimmunoassay buffer over a concentration range of 1 to 250 pgltube. The assay buffer consisted of 19 mmollL monobasic and 81 mmollL dibasic sodium phosphate, 0.05 mollL sodium chloride, 0.1 % bovine serum albumin, 0.1 % Triton X-100, and 0.01 % sodium azide (pH 7.4). The assay was performed in 12 by 75 mm polystyrene tubes; 100 fLl of standard or unknown was added to 100 fLl of rehydrated antisera and incubated overnight at 4° C. The 100 fLl of 1251_rat atrial natriuretic peptide was then added, mixed, and incubated overnight at 4° C. Bound and free fractions were separated by means of diluted normal rabbit serum and goat antirabbit ,,(-globulin (Peninsula Laboratories). After 2 hours of incubation at room temperature, 0.5 ml of assay buffer was added to each tube, and the tubes were centrifuged at 2000 x gat 4° C for 20 minutes. The supernatant was then aspirated, and the precipitate was counted in a ,,(-counter. Statistical analysis. All values are reported as mean ± standard error of the mean. Differences between groups were analyzed by one-way analysis of var-
Atrial natriuretic peptide in the pregnant rat
Volume 160 Number I
-
150 -
0'
-
17
E
"
c.. 100 ....., 0..
Z
E
T
T
T
T
50 -
en
0
c..
0
metestrus diestrus proestrus n=9 n=10 n=7
estrus n=8
Fig. 2. Plasma atrial natriuretic peptide (ANP) levels in nonpregnant rats during each stage of the estrous cycle.
iance with the Student-Newman-Keuls test. A p value <0.05 was considered significant. To eliminate any interas say variability, all atrial natriuretic peptide values reported are from samples run in a single assay.
Results A typical standard curve plotted as B/Bo versus picograms of rat atrial natriuretic peptide is shown in Fig. 1. The sensitivity of the assay, the least amount distinguishable from 0 with 95% confidence, ranged from 1 to 2 pg. Multiple dilutions of pregnant, nonpregnant (metestrus), and postpartum rat plasma extracts,were approximately parallel to the rat atrial natriuretic peptide standard curve. The extraction recovery of 100 pg of added synthetic rat atrial natriuretic peptide in pregnant and nonpregnant rat plasma was similar (88% ± 0.07%, n = 4 and 93% ± 0.03%, n = 4, respectively). Duplicate measurements of five replicate extractions of a single plasma sample were used to calculate the intraassay coefficients of variation at 76% and 35% binding, which were 6.5% and 3%, respectively. The plasma atrial natriuretic peptide levels obtained in nonpregnant rats during each stage of the estrous cycle are shown in Fig. 2. Atrial natriuretic peptide levels did not change significantly during the estrous cycle, and the average atrial natriuretic peptide value for this group was 76 ± 6 pg/ml. The changes in plasma atrial natriuretic peptide levels obtained during pregnancy are depicted in Fig. 3, A. Plasma atrial natriuretic peptide levels were low at 10 to 15 days of pregnancy (37 ± 4 pg/ml) when compared with values at 16 to 18 days of pregnancy
but were not significantly lower than nonpregnant levels or levels on day 21 of pregnancy. On days 16 to 18 of gestation, plasma atrial natriuretic peptide levels had increased to 163 ± 32 pg/ml and were significantly greater than levels obtained in either the nonpregnant group or at 10 to 15 days' gestation (p <: 0.01). By day 21 of pregnancy (shortly before delivery), atrial natriuretic peptide levels had decreased to 85 ± 21 pg I ml and were similar to nonpregnant levels. Post partum, plasma atrial natriuretic peptide levels rose to 167 ± 31 pg/ml during the first 3 hours after delivery (Fig. 3, B) and were significantly higher than maternal levels obtained just before delivery (p < 0.05) and on days 10 to 15 of pregnancy (p < 0.01), as well as higher than nonpregnant levels (p < 0.01). Plasma atrial natriuretic peptide levels remained elevated as long as 48 hours after delivery.
Comment Stimulation of atrial stretch receptors, such as that caused by short- and long-term changes in blood volume,9 appears to be the primary determinant of atrial natriuretic peptide release in vivo. There is additional evidence that other factors such as changes in myocardial work 'o and hormonal stimuli (e.g., glucocorticoids," catecholamines,12 and vasopressin '3 ) may also be involved in regulating atrial natriuretic peptide release either directly or through secondary hemodynamic changes. In rats pregnancy is accompanied by an increase in blood volume of greater than 50% at term. II Plasma volume has been found to be elevated as early as day
18
Castro et al.
January 1989 Am J Obstet Gynecol
10-15 days n=34
n=IO
16-18 days n = II
0-3
21 days
hours
n=9
n=9
8-48 haurs n =10
Fig. 3. A, Plasma atrial natriuretic peptide (ANP) levels in nonpregnant (0) and pregnant rats (I§:j) at different stages of gestation. B, Plasma atrial natriuretic peptide levels in postpartum rats (~). **P < 0.01 vs nonpregnant and 10 to 15 days' pregnant, ++ p < 0.01 vs 21 days' pregnant, *P < 0.05 vs nonpregnant and 10 to 15 days' pregnant, +p < 0.05 vs 21 days' pregnant.
615 and to progressively rise until term, with about 50% of the increase occurring during the third trimester. 11 We postulated a rise in plasma atrial natriuretic peptide levels during pregnancy that would parallel the known changes in blood volume. Our results did not confirm this. The fact that plasma atrial natriuretic peptide levels were not increased until days 16 to 18 suggests that the expanded blood volume is not great enough to stimulate atrial stretch receptors until the third trimester. The finding that plasma atrial natriuretic peptide levels decreased at term and were similar to virgin control rats was unexpected, since blood volume expansion is maximal at this time. This result is consistent with a report by Nadel et al.,lb which showed that plasma atrial natriuretic peptide levels in chronically catheterized pregnant rats on days 19 and 20 of pregnancy were not elevated compared with virgin control rats despite significant increases in plasma volume. Kristensen et al. 17 also reported that atrial natriuretic peptide levels in term rat atria (measured by a bioassay) were similar to virgin controls. Collectively, these studies suggest that in the near-term and term pregnant rat, the enlarged maternal vascular compartment is sensed by the atria as normal. One possible explanation for this is that blood pressure does not appear to decline until day 19 of pregnancylS and remains low until term. This could result in a decrease in myocardial work (a proposed determinant of atrial natriuretic peptide se-
cretion)lO and a consequent lowering of atrial natriuretic peptide levels from day 19 of pregnancy on. In the present study most of the term animals were in labor, and one could postulate that the endocrine changes associated with parturition may have suppressed atrial natriuretic peptide levels. We are unaware of any detailed studies that examined the effects of estrogen, progesterone, or prolactin on atrial natriuretic peptide secretion. In this study the changes in estrogen and progesterone that occur during the estrous cycle were not associated with any significant changes in plasma atrial natriuretic peptide levels. On the other hand, glucocorticoids" and oxytocin l3 have been reported to stimulate atrial natriuretic peptide levels in vivo and in vitro. Our finding that plasma atrial natriuretic peptide levels increased within I hour of delivery and remained elevated for the next 48 hours is also consistent with the study by Nadel et al. 16 In human pregnancies it has recently been shown that plasma atrial natriuretic peptide levels also increase during the first few days postpartum. 19 As suggested by these studies and ours, the decrease in maternal vascular capacitance that occurs postpartum may be associated with an increase in the "effective blood volume" (despite the decrease in absolute blood volume), which in turn stimulates atrial natriuretic peptide release. The increase in blood pressure observed postpartum may also increase myocardial
Volume 160 \\;umber 1
work, causing an elevation in plasma atrial natriuretic peptide levels. Alternatively, one can speculate that the profound changes in maternal endocrine status that occur in the early postpartum period may either directly or indirectly stimulate atrial natriuretic peptide release. We cannot say definitively whether the increase in plasma atrial natriuretic peptide levels observed were associated with any alterations in renal function since we made no measurements of this. Most investigators conclude that in the third trimester, the glomerular filtration rate is elevated, with debate centered on how early in pregnancy the glomerular filtration rate rises and whether it declines at term?O 21 The increase in atrial natriuretic peptide levels on days 16 to 18 suggests that atrial natriuretic peptide may be at least partially responsible for the elevated glomerular filtration rate present at that time. The postpartum rise in plasma atrial natriuretic peptide levels is consistent with reports of an increase in glomerular filtration rate accompanying lactation in the rat"2 and suggests that atrial natriuretic peptide may be involved in the diuresis that occurs after birth. We are grateful to Diana French for her expert secretarial assistance. REFERENCES 1. Lang R, Tholken H, Ganten D, et al. Atrial natriuretic factor-a circulating hormone stimulated by volume loading. Nature 1985;314:264. 2. Maack T, Camargo MJ, Kleinert HD, Laragh JH, Atlas SA. Atrial natriuretic factor: structure and functional properties. Kidney Int 1985;27:607. 3. Laragh JH. Atrial natriuretic hormone, the renin aldosterone axis and blood pressure-electrolyte homeostasis. N EnglJ Med 1985;313:1330. 4. Garcia R, Thibault G, Cantin M, Genest J. Effect of a purified atrial natriuretic factor on rat and rabbit vascular strips and vascular beds. AmJ Physiol 1984;234:R34. 5. Chesley LC. Plasma and red cell volumes during pregnancy. AM J OBSTET GYNECOL 1972; 112:440.
Atrial natriuretic peptide in the pregnant rat
19
6. DavisonJM. Dunlap W. Renal hemodynamics and tubular function in normal human pregnancy. Kidney Int 1980; 18: 152. 7. Schwarz R, Retzke U. Cardiovascular response to infusion of angiotensin II in pregnant women. Obstet Gynecol 1971;38:714. 8. Castro LC, Lam RW, Ross MG, et al. Atrial natriuretic peptide in the sheep. J Dev Phvsiol 1988; I 0:235. 9. Kohno M, Clegg K, Sambhi M. Effects of volume change on circulating immunoreactive atnal natriuretic factor in rats. Hypertension 1987; 10: 171. 10. Rankin AJ. Mechanisms for the release of atrial natriuretic peptIde. Can J Physiol Pharmacol 1987;65: 1673. 11. Matsubara H, Hirata Y, Yoshimi H, et al. Effects of steroid and thyroid hormones on synthesis of atrial natriuretic peptide by cultured atrial myocytes of rat. Biochem Biophys Res Commun 1987;145:336. 12. Toth M, Ruskoaho H, Lang RE. Regulation of atrial natriuretic peptide secretion. J Hypertens 1986;4(suppl 6):S538. 13. Manning P, Schwarz D. Katsube N, Holmberg S, Needleman P. Vasopressin-stimulated release of atriopeptin: endocrine antagonists in fluid homeostasis. Science 1985; 229:395. 14. Barron WM. Volume homeostasis during pregnancy in the rat. Am J Kidney Dis 1987;9:296. 15. Atherton JC, Dark JM, Garland HO, et al. Changes in water and electrolyte balance, plasma volume and composition during pregnancy in the rat. J Physiol 1982; 330:81. 16. Nadel AS, Ballerman BJ, Anderson S, Brenner BM. Interrelationships among atrial peptIdes, renin and blood volume in pregnant rats. Am J Physiol 1988;254:R793. 17. Kristensen CG, Nakagawa Y, Coe FL, Lindheimer MD. Effect of atrial natriuretIc factor in rat pregnancy. Am J Physiol 1986;250:R589. 18. Pike R. Sodium requirement of the rat during pregnancy. In: Lindheimer MD, Katz AI, Zuspan FP, eds. Hypertension III pregnancy. New York: John Wiley & Sons, 1976: 207-15. 19. Steegers EAP, Hein PR, Groeneveld EAM, et al. Atrial natriuretic peptide concentrations during pregnancy. Lancet 1987; I: 1267. 20. Baylis C. Renal hemodynamics and volume control during pregnancy in the rat. Semin Nephrol 1984;4:208. 21. Lindheimer MD, Katz AI. Kidney function in the pregnant rat. J Lab Clin Med 1971 ;78:633. 22. Arthur SK, Green R. Renal function during lactation in the rat. J Physiol 1983;334:379.