ACTH and cortisol responses to hypotension in fetal sheep after a prior CRF injection DEWANA R. KERR, MARIA I. CASTRO, NAYEL M. RAWASHDEH, AND JAMES
NANCY K. VALEGO, C. ROSE
Departments of Physiology and Pharmacology and Obstetrics and Gynecology and the Perinutal Laboratories, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27103; and Department of Physiology and Pharmacology, Jordan University of Science and Technology, Irbid, Jordan Kerr, DeWana It., Maria I. Castro, Nancy K. Valego, Nayel M. Rawashdeh, and James C. Rose. ACTH and cortisol responses to hypotension in fetal sheep after a prior CRF injection. Am. J. Physiol. 262 (Endocrirud. Metab. 25): E325-E329,1992.-To determine whether an ovine corticotropin-releasing factor (oCRF) injection modifies adrenocorticotropic hormone (ACTH) and cortisol responses to hypotension and whether the effect of any interactions between these stimuli changes across gestation, we studied chronically cannulated fetal lambs of 103-113 (“immature”) and 133-139 days gestation (“mature”). Experimental groups received 500 rig/kg oCRF injections and 6 h later had arterial pressure reduced 20% for 10 min with nitroprusside. Blood samples were obtained before and after each manipulation. Controls received vehicle instead of oCRF. The oCRF increased plasma cortisol levels from 2.1 * 0.4 to 14.2 * 4.7 (SE) rig/ml in immature and 44.9 =f=2.2 to 102.8 =f: 15 rig/ml in mature animals. In mature fetuses the oCRF did not alter plasma ACTH and cortisol increases due to hypotension. In immature animals ACTH increases were normal but cortisol increases were eliminated. This suggests that the CRF caused maximal stimulation of the adrenal gland. In older fetuses, it appears that the action of ACTH-releasing factors, secreted in response to arterial hypotension, can overcome the negative feedback effects of elevations in endogenous cortisol. hypothalamus; pituitary; adrenal; adrenocorticotropic hormone; corticotropin-releasing factor THE LOW CIRCULATING LEVELS of glucocorticoids
observed before -120 days gestation [0.83 gestation (G)] in fetal sheep are primarily of maternal origin; subsequently the fetal adrenal increases basal glucocorticoid output (25). The length of gestation in sheep is -145 days. Various stresses such as hypotension increase circulating levels of adrenocorticotropic hormone (ACTH) and cortisol in fetal sheep, and the magnitude of the responses changes across gestation (6,21,22). Elevating circulating cortisol levels by the administration of exogenous cortisol inhibits ACTH responses to nitroprusside-induced hypotension or ovine corticotropin-releasing factor (oCRF) injections (20, 27). Also, inhibiting maternal cortisol production before 120 days gestation decreases circulating fetal cortisol levels and increases plasma immunoreactive ACTH levels. Likewise, decreasing fetal cortisol production after 120 days gestation results in elevated basal ACTH levels (23). These studies suggest that cortisol inhibitory feedback mechanisms exist in the fetus. Therefore it is possible that elevations in endogenous cortisol would alter ACTH and cortisol responses to a stress in the late-gestation ovine fetus and that the extent of such an effect would increase after -0.83 G. The 0193-1849/92
Research
experiments reported here were designed to determine whether, at different periods of development, an injection of oCRF changes the ACTH and cortisol responses to a subsequent period of hypotension. An oCRF challenge was employed because CRF stimulates ACTH release from the fetal pituitary (7, 11, 19, 20). Arterial hypotension was used as the second stimulus because it is a stimulus, effective in causing the release of ACTH, that may occur in utero. It mobilizes various ACTH-releasing factors, including CRF and arginine vasopressin (AVP) (22). Nitroprusside was used to induce hypotension because it has no known direct effects on hormone secretion or on the central nervous system (24). A 6-h interval between challenges was chosen because studies in adult animals frequently reveal inhibition of ACTH and cortisol responses to another stimulus applied hours later, depending on the intensity and duration of the stimulus and resultant plasma ACTH and cortisol increases (14). MATERIALS AND METHODS All surgical and experimental protocols were approved by the Animal Care and Use Committee of the Bowman Gray School of Medicine. We studied two groups of animals. One group (“immature,” n = 5) was studied at 103-113 days gestation (0.75 G) when the adrenal is relatively unresponsive to ACTH. The other group (“mature,” n = 5) was studied in late gestation at l33139 days gestation (0.95 G) when the adrenal is extremely responsive to ACTH. These were paired studies with animals serving as their own controls. Experimental and control studies were conducted randomly in each animal with at least 2 days separating experiments. Surgery. Ewes with known single insemination dates in which pregnancy was confirmed at 50 days gestation by ultrasound were brought to the vivarium ~5 days before surgery to acclimate them to the environment. They were fasted 48 h, and water was restricted 24 h before surgery. The surgery procedure for maternal and fetal vascular catheterization is described in detail in the companion paper (15). Daily, the fetal and maternal vascular catheters were drained of heparin, flushed with sterile saline, filled with heparin, and plugged to maintain patency. Postoperatively, the ewes were housed in metabolic carts in air-conditioned rooms, with a 12:12-h light-dark cycle. Experiment. After a minimum 4day recovery period from surgery, the animals were studied, with the ewe in the portable metabolic cart into which she was placed after surgery. Food and water were withdrawn l-2 h before the experiment. Each experiment was preceded by 80 mg gentamicin administered intravenously to the ewe. All experiments were initiated between 1100 and 1300 h. Care was taken to conduct the experiments in a quiet environment. An aliquot of lyophilized oCRF (Bachem) was diluted with a
$2.00 Copyright 0 1992 the American Physiological
Society
Downloaded from www.physiology.org/journal/ajpendo at Lunds Univ Medicinska Fak Biblio (130.235.066.010) on February 12, 2019.
E325
E326
ACTH
AND
CORTISOL
RESPONSES
solution of 0.1% bovine serum albumin (BSA) in normal saline (BSA-saline) immediately before each experiment. Fetal weight was estimated based on gestational age (12) and was later determined by back calculation from birth or postmortem weight, assuming a 2% weight gain per day. Two control arterial blood samples were obtained 15 min apart. Then a 1-min injection of synthetic oCRF (500 rig/kg iv) in 2 ml BSA-saline or vehicle was administered to the experimental or control groups, respectively. Arterial blood sampling followed. Fetal blood pressure, heart rate, and amniotic fluid pressure were monitored from 330 through 420 min after the initial injection of the study, with the use of Statham P23 DB transducers connected to a Hewlett-Packard or Gould recorder. All fetal blood pressures were corrected for amniotic fluid pressure. Immediately after the 360-min sampling, sodium nitroprusside (Elkins-Sinn, Cherry Hill, NJ) in 5% dextrose water at a concentration of 125 pg/ml was infused intravenously to control and experimental fetuses to achieve a 20% drop in mean arterial pressure (MAP). Once a 20% drop was achieved it was maintained for 10 min by adjusting the nitroprusside infusion rate. Blood samples were obtained at 5 and 10 min after induction of hypotension. A final blood sample was obtained 20 min after hypotension was terminated by discontinuing the nitroprusside infusion. Arterial blood samples were taken at all sample times from the ewe and fetus for cortisol, blood gases, and hematocrit. In the fetus, blood samples were also taken for ACTH at these times. In all ewes and in mature fetuses, 1.5 ml blood were removed at each sample time for hormone analysis, whereas only 1.0 ml was taken in immature fetuses as an adjustment for the smaller total fetoplacental blood volume. In all animals, a total of 0.5 ml blood was obtained for each blood gas and hematocrit determination. Hormone assays.ACTH and cortisol were measured by radioimmunoassays, the characteristics of which have been described in detail elsewhere (21, 22). Statistical procedures. Analysis of variance (ANOVA) for repeated measures and Dunnett’s test were used for preliminary evaluation of the changes in hormone levels with time. Due to heteroscedasticity in ACTH and cortisol responses, log transformation was executed before these analyses. Paired t tests or, when appropriate, the nonparametric equivalent (Wilcoxon) were also used in evaluating certain differences in ACTH and cortisol values between control and experimental groups. Independent groups t tests were used for comparisons between the basal and maximum ACTH and cortisol levels of immature and mature groups. The P values reported are from t tests unless otherwise indicated. Data are reported as means -I- SE. A significance level of P < 0.05 was used. ANOVA for repeated measures was employed to examine fetal arterial blood gases, hematocrit, and MAP levels, in addition to maternal plasma cortisol values. Basal values in control and experimental groups were compared by paired t test. RESULTS
Arterial blood gases and hematocrits are shown in Table 1. Basal hematocrit, Pco~, and Po2 were similar between experimental and control groups at both ages. In the mature group, a small difference in basal arterial pH was detectable between control and experimental groups. In the immature group, control and experimental groups exhibited similar slight decreases in hematocrit (F = 42.250, P c 0.01) and pH (F = 20.129, P < 0.02). In the mature fetuses, similar small decreases in pH (F = 9.274, P < 0.04) were detectable in experimental and
TO
REPEATED
STIMULI
Table 1. Arterial
blood gases and hematocrit mmHg Hematocrit
PH
Pco*
PO*
Basal values Immature Experimental Control Mature Experimental Control
3Ok2 29&l
7.354t0.010 7.351t0.007
47.7k1.2 47.9kl.O
21.8k0.9 20.9t1.0
30t2 32tl
7.343t0.008’ 7.369kO.0011
46.4rt1.8 45.9k1.5
19.2zk1.4 20.5zkO.8
End hypotension Immature Experimental Control Mature Experimental Control Values
are means
t
2721 27t2
7.342t0.011 7.339kO.002
47.8k1.4 48.6k1.4
20.7t0.9 19.8k1.4
31*1 29t2
7.329kO.004 7.351t0.013
48.8k1.9 44.8t 1.4
19.6k2.1 20.8t0.8
SE. * P c 0.05,
experimental
vs. control.
Table 2. Mean arterial pressure Hypotension Before
Immature Experimental Control Mature Experimental Control Values are means corticotropin-releasing P c 0.05.
Mid
End
38&l 38tl
27&l* 27kl*
27kl* 27t2*
35t3 36k2
4523 5Ok2
43t’ 36t2’
36t2’ 38tl”
52k4 51t2
k SE in mmHg. Experimental factor; control group received
20 Min after
group received saline. * Less than
control groups. In immature fetuses, MAP levels (Table 2) at 360 min were 38 k 1 mmHg in control and experimental groups. In the mature animals, control and experimental group MAP at 360 min were not different, and the average was 47 t 2 mmHg. At both ages, MAP levels in experimental and control studies decreased similarly (20% hypotension) in response to nitroprusside infusion. The average maternal plasma cortisol value was 17.8 k 4.8 rig/ml. Maternal plasma cortisol levels were not different between groups and did not change significantly across time. Plasma ACTH levels in immature fetuses are shown in Fig. lA. The CRF injection increased ACTH concentration in the experimental group during the first 6 h (F = 107.6, P < 0.01) of the experiment, and levels were not back to baseline at 360 min (P < 0.05). The increases (A) in response to hypotension were similar between control and experimental groups. Plasma cortisol levels are shown in Fig. 1B. Cortisol increased in the experimental group after CRF (F = 24.2, P < 0.01). Levels in the experimental group remained elevated at 360 min (P < 0.01). The changes (A) in cortisol after hypotension were different (F = 20.3, P < 0.01) in the two groups. Twenty m .inutes after the end of hypotension, the plasma cortisol concentrations of the control group remained elevated above basal levels (Dunnett’s, P < 0.01). Plasma ACTH responses mature fetuses are shown
Downloaded from www.physiology.org/journal/ajpendo at Lunds Univ Medicinska Fak Biblio (130.235.066.010) on February 12, 2019.
ACTH 800
AND CORTISOL
RESPONSES
TO REPEATED
TASAL
1
200-
L
NANCY K. VALEGO, C. ROSE
Departments of Physiology and Pharmacology and Obstetrics and Gynecology and the Perinutal Laboratories, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27103; and Department of Physiology and Pharmacology, Jordan University of Science and Technology, Irbid, Jordan Kerr, DeWana It., Maria I. Castro, Nancy K. Valego, Nayel M. Rawashdeh, and James C. Rose. ACTH and cortisol responses to hypotension in fetal sheep after a prior CRF injection. Am. J. Physiol. 262 (Endocrirud. Metab. 25): E325-E329,1992.-To determine whether an ovine corticotropin-releasing factor (oCRF) injection modifies adrenocorticotropic hormone (ACTH) and cortisol responses to hypotension and whether the effect of any interactions between these stimuli changes across gestation, we studied chronically cannulated fetal lambs of 103-113 (“immature”) and 133-139 days gestation (“mature”). Experimental groups received 500 rig/kg oCRF injections and 6 h later had arterial pressure reduced 20% for 10 min with nitroprusside. Blood samples were obtained before and after each manipulation. Controls received vehicle instead of oCRF. The oCRF increased plasma cortisol levels from 2.1 * 0.4 to 14.2 * 4.7 (SE) rig/ml in immature and 44.9 =f=2.2 to 102.8 =f: 15 rig/ml in mature animals. In mature fetuses the oCRF did not alter plasma ACTH and cortisol increases due to hypotension. In immature animals ACTH increases were normal but cortisol increases were eliminated. This suggests that the CRF caused maximal stimulation of the adrenal gland. In older fetuses, it appears that the action of ACTH-releasing factors, secreted in response to arterial hypotension, can overcome the negative feedback effects of elevations in endogenous cortisol. hypothalamus; pituitary; adrenal; adrenocorticotropic hormone; corticotropin-releasing factor THE LOW CIRCULATING LEVELS of glucocorticoids
observed before -120 days gestation [0.83 gestation (G)] in fetal sheep are primarily of maternal origin; subsequently the fetal adrenal increases basal glucocorticoid output (25). The length of gestation in sheep is -145 days. Various stresses such as hypotension increase circulating levels of adrenocorticotropic hormone (ACTH) and cortisol in fetal sheep, and the magnitude of the responses changes across gestation (6,21,22). Elevating circulating cortisol levels by the administration of exogenous cortisol inhibits ACTH responses to nitroprusside-induced hypotension or ovine corticotropin-releasing factor (oCRF) injections (20, 27). Also, inhibiting maternal cortisol production before 120 days gestation decreases circulating fetal cortisol levels and increases plasma immunoreactive ACTH levels. Likewise, decreasing fetal cortisol production after 120 days gestation results in elevated basal ACTH levels (23). These studies suggest that cortisol inhibitory feedback mechanisms exist in the fetus. Therefore it is possible that elevations in endogenous cortisol would alter ACTH and cortisol responses to a stress in the late-gestation ovine fetus and that the extent of such an effect would increase after -0.83 G. The 0193-1849/92
Research
experiments reported here were designed to determine whether, at different periods of development, an injection of oCRF changes the ACTH and cortisol responses to a subsequent period of hypotension. An oCRF challenge was employed because CRF stimulates ACTH release from the fetal pituitary (7, 11, 19, 20). Arterial hypotension was used as the second stimulus because it is a stimulus, effective in causing the release of ACTH, that may occur in utero. It mobilizes various ACTH-releasing factors, including CRF and arginine vasopressin (AVP) (22). Nitroprusside was used to induce hypotension because it has no known direct effects on hormone secretion or on the central nervous system (24). A 6-h interval between challenges was chosen because studies in adult animals frequently reveal inhibition of ACTH and cortisol responses to another stimulus applied hours later, depending on the intensity and duration of the stimulus and resultant plasma ACTH and cortisol increases (14). MATERIALS AND METHODS All surgical and experimental protocols were approved by the Animal Care and Use Committee of the Bowman Gray School of Medicine. We studied two groups of animals. One group (“immature,” n = 5) was studied at 103-113 days gestation (0.75 G) when the adrenal is relatively unresponsive to ACTH. The other group (“mature,” n = 5) was studied in late gestation at l33139 days gestation (0.95 G) when the adrenal is extremely responsive to ACTH. These were paired studies with animals serving as their own controls. Experimental and control studies were conducted randomly in each animal with at least 2 days separating experiments. Surgery. Ewes with known single insemination dates in which pregnancy was confirmed at 50 days gestation by ultrasound were brought to the vivarium ~5 days before surgery to acclimate them to the environment. They were fasted 48 h, and water was restricted 24 h before surgery. The surgery procedure for maternal and fetal vascular catheterization is described in detail in the companion paper (15). Daily, the fetal and maternal vascular catheters were drained of heparin, flushed with sterile saline, filled with heparin, and plugged to maintain patency. Postoperatively, the ewes were housed in metabolic carts in air-conditioned rooms, with a 12:12-h light-dark cycle. Experiment. After a minimum 4day recovery period from surgery, the animals were studied, with the ewe in the portable metabolic cart into which she was placed after surgery. Food and water were withdrawn l-2 h before the experiment. Each experiment was preceded by 80 mg gentamicin administered intravenously to the ewe. All experiments were initiated between 1100 and 1300 h. Care was taken to conduct the experiments in a quiet environment. An aliquot of lyophilized oCRF (Bachem) was diluted with a
$2.00 Copyright 0 1992 the American Physiological
Society
Downloaded from www.physiology.org/journal/ajpendo at Lunds Univ Medicinska Fak Biblio (130.235.066.010) on February 12, 2019.
E325
E326
ACTH
AND
CORTISOL
RESPONSES
solution of 0.1% bovine serum albumin (BSA) in normal saline (BSA-saline) immediately before each experiment. Fetal weight was estimated based on gestational age (12) and was later determined by back calculation from birth or postmortem weight, assuming a 2% weight gain per day. Two control arterial blood samples were obtained 15 min apart. Then a 1-min injection of synthetic oCRF (500 rig/kg iv) in 2 ml BSA-saline or vehicle was administered to the experimental or control groups, respectively. Arterial blood sampling followed. Fetal blood pressure, heart rate, and amniotic fluid pressure were monitored from 330 through 420 min after the initial injection of the study, with the use of Statham P23 DB transducers connected to a Hewlett-Packard or Gould recorder. All fetal blood pressures were corrected for amniotic fluid pressure. Immediately after the 360-min sampling, sodium nitroprusside (Elkins-Sinn, Cherry Hill, NJ) in 5% dextrose water at a concentration of 125 pg/ml was infused intravenously to control and experimental fetuses to achieve a 20% drop in mean arterial pressure (MAP). Once a 20% drop was achieved it was maintained for 10 min by adjusting the nitroprusside infusion rate. Blood samples were obtained at 5 and 10 min after induction of hypotension. A final blood sample was obtained 20 min after hypotension was terminated by discontinuing the nitroprusside infusion. Arterial blood samples were taken at all sample times from the ewe and fetus for cortisol, blood gases, and hematocrit. In the fetus, blood samples were also taken for ACTH at these times. In all ewes and in mature fetuses, 1.5 ml blood were removed at each sample time for hormone analysis, whereas only 1.0 ml was taken in immature fetuses as an adjustment for the smaller total fetoplacental blood volume. In all animals, a total of 0.5 ml blood was obtained for each blood gas and hematocrit determination. Hormone assays.ACTH and cortisol were measured by radioimmunoassays, the characteristics of which have been described in detail elsewhere (21, 22). Statistical procedures. Analysis of variance (ANOVA) for repeated measures and Dunnett’s test were used for preliminary evaluation of the changes in hormone levels with time. Due to heteroscedasticity in ACTH and cortisol responses, log transformation was executed before these analyses. Paired t tests or, when appropriate, the nonparametric equivalent (Wilcoxon) were also used in evaluating certain differences in ACTH and cortisol values between control and experimental groups. Independent groups t tests were used for comparisons between the basal and maximum ACTH and cortisol levels of immature and mature groups. The P values reported are from t tests unless otherwise indicated. Data are reported as means -I- SE. A significance level of P < 0.05 was used. ANOVA for repeated measures was employed to examine fetal arterial blood gases, hematocrit, and MAP levels, in addition to maternal plasma cortisol values. Basal values in control and experimental groups were compared by paired t test. RESULTS
Arterial blood gases and hematocrits are shown in Table 1. Basal hematocrit, Pco~, and Po2 were similar between experimental and control groups at both ages. In the mature group, a small difference in basal arterial pH was detectable between control and experimental groups. In the immature group, control and experimental groups exhibited similar slight decreases in hematocrit (F = 42.250, P c 0.01) and pH (F = 20.129, P < 0.02). In the mature fetuses, similar small decreases in pH (F = 9.274, P < 0.04) were detectable in experimental and
TO
REPEATED
STIMULI
Table 1. Arterial
blood gases and hematocrit mmHg Hematocrit
PH
Pco*
PO*
Basal values Immature Experimental Control Mature Experimental Control
3Ok2 29&l
7.354t0.010 7.351t0.007
47.7k1.2 47.9kl.O
21.8k0.9 20.9t1.0
30t2 32tl
7.343t0.008’ 7.369kO.0011
46.4rt1.8 45.9k1.5
19.2zk1.4 20.5zkO.8
End hypotension Immature Experimental Control Mature Experimental Control Values
are means
t
2721 27t2
7.342t0.011 7.339kO.002
47.8k1.4 48.6k1.4
20.7t0.9 19.8k1.4
31*1 29t2
7.329kO.004 7.351t0.013
48.8k1.9 44.8t 1.4
19.6k2.1 20.8t0.8
SE. * P c 0.05,
experimental
vs. control.
Table 2. Mean arterial pressure Hypotension Before
Immature Experimental Control Mature Experimental Control Values are means corticotropin-releasing P c 0.05.
Mid
End
38&l 38tl
27&l* 27kl*
27kl* 27t2*
35t3 36k2
4523 5Ok2
43t’ 36t2’
36t2’ 38tl”
52k4 51t2
k SE in mmHg. Experimental factor; control group received
20 Min after
group received saline. * Less than
control groups. In immature fetuses, MAP levels (Table 2) at 360 min were 38 k 1 mmHg in control and experimental groups. In the mature animals, control and experimental group MAP at 360 min were not different, and the average was 47 t 2 mmHg. At both ages, MAP levels in experimental and control studies decreased similarly (20% hypotension) in response to nitroprusside infusion. The average maternal plasma cortisol value was 17.8 k 4.8 rig/ml. Maternal plasma cortisol levels were not different between groups and did not change significantly across time. Plasma ACTH levels in immature fetuses are shown in Fig. lA. The CRF injection increased ACTH concentration in the experimental group during the first 6 h (F = 107.6, P < 0.01) of the experiment, and levels were not back to baseline at 360 min (P < 0.05). The increases (A) in response to hypotension were similar between control and experimental groups. Plasma cortisol levels are shown in Fig. 1B. Cortisol increased in the experimental group after CRF (F = 24.2, P < 0.01). Levels in the experimental group remained elevated at 360 min (P < 0.01). The changes (A) in cortisol after hypotension were different (F = 20.3, P < 0.01) in the two groups. Twenty m .inutes after the end of hypotension, the plasma cortisol concentrations of the control group remained elevated above basal levels (Dunnett’s, P < 0.01). Plasma ACTH responses mature fetuses are shown
Downloaded from www.physiology.org/journal/ajpendo at Lunds Univ Medicinska Fak Biblio (130.235.066.010) on February 12, 2019.
ACTH 800
AND CORTISOL
RESPONSES
TO REPEATED
TASAL
1
200-
L
