Biol. Neonate 36 : 119 127 (1979)
Plasma Renin Activity and Adrenal Angiotensin II Receptors in Fetal, Newborn, Adult and Pregnant Rabbits M.G. Pernollet, M.A. Devynck, G.J. Macdonald and P. Meyer Renal and Vascular Physiology and Pharmacology Unit, Inscrm U 7, Hôpital Necker, Paris
Key Words. Plasma renin activity • Adrenal angiotensin II receptors • Ontogenesis ■Pregnant rabbit • Hormonal ‘self-regulation’
Introduction
Increased activity of the renin-angiotensin system and raised plasma aldosterone concen trations have been reported in the new-born of various species (7, 12, 18, 20, 22, 24). Since angiotensin II is one of the most potent stimuli of aldosterone synthesis, a causal relationship has been proposed between elevations of plas ma renin activity and aldosterone concentra tion. However, little is known about the sensi tivity of the adrenal cortex to angiotensin in the newborn. We have thus investigated the ontogeny of the adrenal receptors which recog nize and interact specifically with angiotensin II and trigger off the cellular response. Such
angiotensin II receptors have been previously identified with the use of a radioactive hor mone in rat (10, 21) and bovine (9, 10) adrenal cortex and in rabbit isolated glomerulosa cells (12). As observed with many other polypeptidic and steroidogenic hormones and neurotrans mitters, angiotensin II adrenal receptors have been shown to be modified by sustained changes in circulating angiotensin II levels: an giotensin receptors appear to be down- and up-regulated by a chronic increase and decrease of circulating angiotensin, respectively (21). They have also been observed to be modulated by dietary electrolytes (6, 8), and their in creased number after dietary sodium deficiency may contribute to the enhancement of the
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/1/2018 7:12:08 AM
Abstract. Plasma renin activity (PRA) and adrenal angiotensin II receptors have been studied simultaneously in the rabbit at various stages of development. In the fetus both parameters show very low values but increase rapidly during the last 4 days of gestation. PRA reaches a maximal level in the early post-natal period but the concentration of adrenal angiotensin II receptors continues to increase further up to the adult state. In adult females, pregnancy results in an initial rise in PRA and adrenocortical angiotensin II receptors. However, PRA remains at a high level throughout the gestation period whereas the number of adrenocortical angiotensin II receptors decreases progressively as pregnancy progresses. The role of circulating angiotensin in the regulation of the concentration and affinity of its receptor sites is discussed.
Pernollet/Devynck/Macdonald/Meyer
120
( 1).
This investigation of adrenal angiotensin II receptor development was associated with a study of these receptors in the mother during pregnancy which is characterized by both a moderate increase in sodium status and high plasma renin and aldosterone levels (11, 28, 30). It was found that in the rabbit fetus the adrenal receptors for angiotensin II become de tectable only in the last days of gestation and that their appearance is synchronous to that of plasma renin activity. This suggests either an independent but simultaneous maturation, or an induction of the receptors by angiotensin. However, in contrast to the plasma renin activ ity which reached a maximal level in the early post-natal period, adrenal angiotensin II recep tors continue to increase in number up to the adult state. In the pregnant mother during the first part of gestation, the number of adreno cortical receptors for angiotensin II was ob served to be higher than in the non-pregnant female rabbit. However, the receptor number decreased at the end of pregnancy. Methods Female New Zealand rabbits received tap water to drink and a standard diet (0.14% Na*, 1.25% K‘) (diet 111-UAR, Villemoisson, France). Male rabbits from the same strain were used as mates. The duration of gestation is 30-31 days in the rabbit. Adult and newborn rabbits were killed by air embolism and fetuses were extracted from uterine horns in less than 5 min. Blood was collected by cardiac puncture and the adrenal glands removed. Whole adrenal glands from 5-11 fetuses were pooled to allow the study of angiotensin II binding. It has not been possible to sample adrenal glands from fetuses younger than 20 days (0.65 of term). Newborn rabbits less than 48 h old were not studied to avoid the influence of parturition stress (24). In adults, whole
adrenal glands or isolated adrenal cortex was studied as indicated in the text. The preparation of adrenal membranes was per formed after homogenization and differential centri fugation as previously reported (21). Tritiated angiotensin II (70Ci/mmol) prepared by S. Fernandjian and P. Fromageot (Service de Bio chimie, CEN, Saclay, France) was used for binding studies. Its purity and intact biological activity were assessed as previously described (19). Membranes were incubated with tritiated hormone at 29 °C in 100 mAf KC1, 5 mM MgCl2, 10 mM histidine buffer pH 6.8. Bound and free hormones were separated with the use of Milliporc filters HAWP 0.45 jun as previously de scribed (21). Specific binding was taken as the differ ence between total and non-specific binding measured as the radioactivity bound to membranes in the ab sence and presence of a 1,000-fold excess of un labelled angiotensin II, respectively. The non-specific binding was extremely low and accounted for less than 2-3% of the total binding. The concentration of membrane protein the sample was determined by the method of Lowry et al. (16). The various angiotensin analogues used in the speci ficity studies were synthesized and kindly supplied by Dr. F.M Bumpus and Dr. M.C. Khosla (Research Division, Cleveland Clinic, Cleveland, Ohio). Plasma renin activity was assayed by a modifica tion of the method of Macdonald et al. (17). Phenylmethylsulphonyl fluoride 0.02% was used as an ad ditional inhibitor of angiotensin I conversion and the reaction was stopped by rapid cooling rather than boiling. Radioimmunoassay of angiotensin I was per formed immediately at 4 °C. Results
Specific Angiotensin II Binding to Adreno-Cortical Particulate Fraction o f Adult Rabbit The characterization of angiotensin IIbinding sites as hormonal receptors has been performed as a first step of this study. The kinetic constants of 3H-angiotensin II specific binding on the membraneous fraction from adrenal cortex were as follows: the association and dissociation rate constants determined at
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/1/2018 7:12:08 AM
aldosterone-releasing effect of angiotensin II
29 °C were 1.0 X 10s A T1-sec-1 and 1.7 X 10-3 sec-1, reflecting the rapid and reversible 3H-angiotensin II binding. Figure 1 illustrates the binding at two initial concentrations of 3H-angiotensin II (5 X 10- 11 and 2 X 10-10M). Equilibrium was obtained 25 min after addition of tritiated angiotensin to the membrane sus pension. The bound radioactive fraction re mained stable for at least 45 min (the longest period studied), suggesting that there is no degradation of cell membrane suspension or of the hormone within that time. 30 min was chosen as the usual incubation time for studies of concentration-dependent binding. From 2 X 10-10 to 6 X 10_9 Af, a single type of binding site was observed (see fig. 3) with an apparent Kd of 3.8 ± 0.3 X 10~9 M and a mean number of sites of 2.4 ± 0.3 pmol-mg-1 for adult rabbit (mean ± SEM of 11 determinations). Therefore, the apparent dissociation constant Kd determined from Scatchard plots of con centration-dependent binding studies was very close to that obtained from the ratio of dissocia tion and association rate constants (k-j /k ,). The specificity was studied by the inhibitory effect of increasing concentrations of angio tensin derivatives and fragments of the 3Hangiotensin-specific binding. (The initial 3Hangiotensin II concentration was (1.0 ± 0.2] X 10- 9 Af). The decapeptide angiotensin I, the 1 -7 heptapeptide, and the 3 -8 angiotensin II fragments have a weak inhibitory effect, indi cating a low affinity for angiotensin II binding sites (fig. 2). The inhibitory effect of the com petitive antagonists Sar’-Ala8 angiotensin II and Sar1-He8 angiotensin II (3, 5, 15, 25) was at least as potent as the agonist angiotensin II. The agonist des-Asp1 angiotensin II (2 -8 angioten sin II peptide or angiotensin III) has an affinity for 3H-angiotensin II binding sites 2—3 times lower than Asn'-Val5 angiotensin II. These re sults are extremely close to those previously
121
Fig. 1. Time course of 5H-angiotensin II binding to a membrane fraction from rabbit adrenal cortex. Initial angiotensin II concentrations were 5 X 10"" (circles) and 2 X 10"10Ai (triangles). Total and non specific binding were measured in the absence (*, •) and presence (a, o) of 10*‘ M unlabelled angiotensin, respectively.
observed in bovine adrenal cortex (10) and rat adrenal cortex (21): only the peptides exhibiting a high agonist or antagonist effect are potent competitors of 3H-angiotensin II binding. The overall parallel of binding inhibitory potency and physiological activity of angiotensin ana logues indicates that these binding sites are probably the hormonal receptors triggering the biological response. Angiotensin II Binding to Adrenal Particulate Fraction from Immature or Pregnant Rabbits Affinity o f Receptors. Similar measurements have been performed on particulate fractions of whole adrenals from fetuses and newborn rab-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/1/2018 7:12:08 AM
Renin and Angiotensin II Receptors
PernoUet/Devynck/Macdonald/Meycr
122
T a b l e 1. Plasma renin activity (ng angiotensin I - m l ' - l f receptors during development and pregnancy (mean ± SEM)
Gestation days
Whole adrenal Fetus
Newborn Adult Adrenal cortex Adult
i
10
20
30
>
Fetus (days of gestation)
Fig. 5. Variation of the binding capacity of adreno cortical angiotensin II receptor sites during pregnancy. Individual values are given as percentage of the density measured concomitantly in the non-pregnant rabbit.
angiotensin system, since both renin concentra tion and kinetics, and substrate concentration are modified during gestation, in the mother and the fetus (23, 30). Figure 4 compares the variation of PRA and the number of adrenal angiotensin 11 binding sites during fetal develop ment and growth. Significant renin activity appeared in the last 4 days of gestation in fetal plasma, simultaneously with angiotensin II adrenal receptors. It reached the highest values in young rabbits less than 4 weeks old and then decreased in the adult rabbits whereas the number of adrenal angiotensin II receptors in creased further with aging. As expected, pregnancy increased plasma renin activity (table I). The mean value during pregnancy was 25.7 ± 4.0 ng angiotensin I • ml“ ‘ *h-1 (19 determinations, mean ± SEM). This value differs significantly from that mea sured in the non-pregnant rabbit (17.1 ±2.6 ng angiotensin I ,ml_ I'h ~ l , mean ± SEM, 11 deter minations p
Plasma Renin Activity and Adrenal Angiotensin II Receptors in Fetal, Newborn, Adult and Pregnant Rabbits M.G. Pernollet, M.A. Devynck, G.J. Macdonald and P. Meyer Renal and Vascular Physiology and Pharmacology Unit, Inscrm U 7, Hôpital Necker, Paris
Key Words. Plasma renin activity • Adrenal angiotensin II receptors • Ontogenesis ■Pregnant rabbit • Hormonal ‘self-regulation’
Introduction
Increased activity of the renin-angiotensin system and raised plasma aldosterone concen trations have been reported in the new-born of various species (7, 12, 18, 20, 22, 24). Since angiotensin II is one of the most potent stimuli of aldosterone synthesis, a causal relationship has been proposed between elevations of plas ma renin activity and aldosterone concentra tion. However, little is known about the sensi tivity of the adrenal cortex to angiotensin in the newborn. We have thus investigated the ontogeny of the adrenal receptors which recog nize and interact specifically with angiotensin II and trigger off the cellular response. Such
angiotensin II receptors have been previously identified with the use of a radioactive hor mone in rat (10, 21) and bovine (9, 10) adrenal cortex and in rabbit isolated glomerulosa cells (12). As observed with many other polypeptidic and steroidogenic hormones and neurotrans mitters, angiotensin II adrenal receptors have been shown to be modified by sustained changes in circulating angiotensin II levels: an giotensin receptors appear to be down- and up-regulated by a chronic increase and decrease of circulating angiotensin, respectively (21). They have also been observed to be modulated by dietary electrolytes (6, 8), and their in creased number after dietary sodium deficiency may contribute to the enhancement of the
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/1/2018 7:12:08 AM
Abstract. Plasma renin activity (PRA) and adrenal angiotensin II receptors have been studied simultaneously in the rabbit at various stages of development. In the fetus both parameters show very low values but increase rapidly during the last 4 days of gestation. PRA reaches a maximal level in the early post-natal period but the concentration of adrenal angiotensin II receptors continues to increase further up to the adult state. In adult females, pregnancy results in an initial rise in PRA and adrenocortical angiotensin II receptors. However, PRA remains at a high level throughout the gestation period whereas the number of adrenocortical angiotensin II receptors decreases progressively as pregnancy progresses. The role of circulating angiotensin in the regulation of the concentration and affinity of its receptor sites is discussed.
Pernollet/Devynck/Macdonald/Meyer
120
( 1).
This investigation of adrenal angiotensin II receptor development was associated with a study of these receptors in the mother during pregnancy which is characterized by both a moderate increase in sodium status and high plasma renin and aldosterone levels (11, 28, 30). It was found that in the rabbit fetus the adrenal receptors for angiotensin II become de tectable only in the last days of gestation and that their appearance is synchronous to that of plasma renin activity. This suggests either an independent but simultaneous maturation, or an induction of the receptors by angiotensin. However, in contrast to the plasma renin activ ity which reached a maximal level in the early post-natal period, adrenal angiotensin II recep tors continue to increase in number up to the adult state. In the pregnant mother during the first part of gestation, the number of adreno cortical receptors for angiotensin II was ob served to be higher than in the non-pregnant female rabbit. However, the receptor number decreased at the end of pregnancy. Methods Female New Zealand rabbits received tap water to drink and a standard diet (0.14% Na*, 1.25% K‘) (diet 111-UAR, Villemoisson, France). Male rabbits from the same strain were used as mates. The duration of gestation is 30-31 days in the rabbit. Adult and newborn rabbits were killed by air embolism and fetuses were extracted from uterine horns in less than 5 min. Blood was collected by cardiac puncture and the adrenal glands removed. Whole adrenal glands from 5-11 fetuses were pooled to allow the study of angiotensin II binding. It has not been possible to sample adrenal glands from fetuses younger than 20 days (0.65 of term). Newborn rabbits less than 48 h old were not studied to avoid the influence of parturition stress (24). In adults, whole
adrenal glands or isolated adrenal cortex was studied as indicated in the text. The preparation of adrenal membranes was per formed after homogenization and differential centri fugation as previously reported (21). Tritiated angiotensin II (70Ci/mmol) prepared by S. Fernandjian and P. Fromageot (Service de Bio chimie, CEN, Saclay, France) was used for binding studies. Its purity and intact biological activity were assessed as previously described (19). Membranes were incubated with tritiated hormone at 29 °C in 100 mAf KC1, 5 mM MgCl2, 10 mM histidine buffer pH 6.8. Bound and free hormones were separated with the use of Milliporc filters HAWP 0.45 jun as previously de scribed (21). Specific binding was taken as the differ ence between total and non-specific binding measured as the radioactivity bound to membranes in the ab sence and presence of a 1,000-fold excess of un labelled angiotensin II, respectively. The non-specific binding was extremely low and accounted for less than 2-3% of the total binding. The concentration of membrane protein the sample was determined by the method of Lowry et al. (16). The various angiotensin analogues used in the speci ficity studies were synthesized and kindly supplied by Dr. F.M Bumpus and Dr. M.C. Khosla (Research Division, Cleveland Clinic, Cleveland, Ohio). Plasma renin activity was assayed by a modifica tion of the method of Macdonald et al. (17). Phenylmethylsulphonyl fluoride 0.02% was used as an ad ditional inhibitor of angiotensin I conversion and the reaction was stopped by rapid cooling rather than boiling. Radioimmunoassay of angiotensin I was per formed immediately at 4 °C. Results
Specific Angiotensin II Binding to Adreno-Cortical Particulate Fraction o f Adult Rabbit The characterization of angiotensin IIbinding sites as hormonal receptors has been performed as a first step of this study. The kinetic constants of 3H-angiotensin II specific binding on the membraneous fraction from adrenal cortex were as follows: the association and dissociation rate constants determined at
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/1/2018 7:12:08 AM
aldosterone-releasing effect of angiotensin II
29 °C were 1.0 X 10s A T1-sec-1 and 1.7 X 10-3 sec-1, reflecting the rapid and reversible 3H-angiotensin II binding. Figure 1 illustrates the binding at two initial concentrations of 3H-angiotensin II (5 X 10- 11 and 2 X 10-10M). Equilibrium was obtained 25 min after addition of tritiated angiotensin to the membrane sus pension. The bound radioactive fraction re mained stable for at least 45 min (the longest period studied), suggesting that there is no degradation of cell membrane suspension or of the hormone within that time. 30 min was chosen as the usual incubation time for studies of concentration-dependent binding. From 2 X 10-10 to 6 X 10_9 Af, a single type of binding site was observed (see fig. 3) with an apparent Kd of 3.8 ± 0.3 X 10~9 M and a mean number of sites of 2.4 ± 0.3 pmol-mg-1 for adult rabbit (mean ± SEM of 11 determinations). Therefore, the apparent dissociation constant Kd determined from Scatchard plots of con centration-dependent binding studies was very close to that obtained from the ratio of dissocia tion and association rate constants (k-j /k ,). The specificity was studied by the inhibitory effect of increasing concentrations of angio tensin derivatives and fragments of the 3Hangiotensin-specific binding. (The initial 3Hangiotensin II concentration was (1.0 ± 0.2] X 10- 9 Af). The decapeptide angiotensin I, the 1 -7 heptapeptide, and the 3 -8 angiotensin II fragments have a weak inhibitory effect, indi cating a low affinity for angiotensin II binding sites (fig. 2). The inhibitory effect of the com petitive antagonists Sar’-Ala8 angiotensin II and Sar1-He8 angiotensin II (3, 5, 15, 25) was at least as potent as the agonist angiotensin II. The agonist des-Asp1 angiotensin II (2 -8 angioten sin II peptide or angiotensin III) has an affinity for 3H-angiotensin II binding sites 2—3 times lower than Asn'-Val5 angiotensin II. These re sults are extremely close to those previously
121
Fig. 1. Time course of 5H-angiotensin II binding to a membrane fraction from rabbit adrenal cortex. Initial angiotensin II concentrations were 5 X 10"" (circles) and 2 X 10"10Ai (triangles). Total and non specific binding were measured in the absence (*, •) and presence (a, o) of 10*‘ M unlabelled angiotensin, respectively.
observed in bovine adrenal cortex (10) and rat adrenal cortex (21): only the peptides exhibiting a high agonist or antagonist effect are potent competitors of 3H-angiotensin II binding. The overall parallel of binding inhibitory potency and physiological activity of angiotensin ana logues indicates that these binding sites are probably the hormonal receptors triggering the biological response. Angiotensin II Binding to Adrenal Particulate Fraction from Immature or Pregnant Rabbits Affinity o f Receptors. Similar measurements have been performed on particulate fractions of whole adrenals from fetuses and newborn rab-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/1/2018 7:12:08 AM
Renin and Angiotensin II Receptors
PernoUet/Devynck/Macdonald/Meycr
122
T a b l e 1. Plasma renin activity (ng angiotensin I - m l ' - l f receptors during development and pregnancy (mean ± SEM)
Gestation days
Whole adrenal Fetus
Newborn Adult Adrenal cortex Adult
i
10
20
30
>
Fetus (days of gestation)
Fig. 5. Variation of the binding capacity of adreno cortical angiotensin II receptor sites during pregnancy. Individual values are given as percentage of the density measured concomitantly in the non-pregnant rabbit.
angiotensin system, since both renin concentra tion and kinetics, and substrate concentration are modified during gestation, in the mother and the fetus (23, 30). Figure 4 compares the variation of PRA and the number of adrenal angiotensin 11 binding sites during fetal develop ment and growth. Significant renin activity appeared in the last 4 days of gestation in fetal plasma, simultaneously with angiotensin II adrenal receptors. It reached the highest values in young rabbits less than 4 weeks old and then decreased in the adult rabbits whereas the number of adrenal angiotensin II receptors in creased further with aging. As expected, pregnancy increased plasma renin activity (table I). The mean value during pregnancy was 25.7 ± 4.0 ng angiotensin I • ml“ ‘ *h-1 (19 determinations, mean ± SEM). This value differs significantly from that mea sured in the non-pregnant rabbit (17.1 ±2.6 ng angiotensin I ,ml_ I'h ~ l , mean ± SEM, 11 deter minations p
