Am J Physiol Regul Integr Comp Physiol 307: R347–R353, 2014. First published June 4, 2014; doi:10.1152/ajpregu.00125.2014.
Adrenocortical sensitivity to ACTH in neonatal rats: correlation of corticosterone responses and adrenal cAMP content Jonathan Bodager,1,2 Thomas Gessert,1 Eric D. Bruder,1 Ashley Gehrand,1 and Hershel Raff1,2 1 2
Endocrine Research Laboratory; Aurora St. Luke’s Medical Center, Aurora Research Institute, Milwaukee, Wisconsin; and Departments of Medicine, Surgery, and Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin
Submitted 20 March 2014; accepted in final form 28 May 2014
Bodager J, Gessert T, Bruder ED, Gehrand A, Raff H. Adrenocortical sensitivity to ACTH in neonatal rats: correlation of corticosterone responses and adrenal cAMP content. Am J Physiol Regul Integr Comp Physiol 307: R347–R353, 2014. First published June 4, 2014; doi:10.1152/ajpregu.00125.2014.—A coordinated hypothalamicpituitary-adrenal axis response is important for the survival of newborns during stress. We have previously shown that prior to postnatal day (PD) 5, neonatal rats exposed to hypoxia (one of the most common stressors effecting premature neonates) exhibit a large corticosterone response with a minimal increase in immunoassayable plasma ACTH and without a detectable increase in adrenal cAMP content (the critical second messenger). To explore the phenomenon of ACTH-stimulated steroidogenesis in the neonate, we investigated the adrenal response to exogenous ACTH in the normoxic neonatal rat. Rat pups at PD2 and PD8 were injected intraperitoneally with porcine ACTH at low, moderate, or high doses (1, 4, or 20 g/kg body wt). Trunk blood and whole adrenal glands were collected at baseline (before injection) and 15, 30, or 60 min after the injection. ACTH stimulated corticosterone release in PD2 and PD8 pups. In PD2 pups, plasma corticosterone at baseline and during the response to ACTH injection was greater than values measured in PD8 pups, despite lower adrenal cAMP content in PD2 pups. Specifically, the low and moderate physiological ACTH doses produced a large corticosterone response in PD2 pups without a change in adrenal cAMP content. At extremely high, pharmacological levels of plasma ACTH in PD2 pups (exceeding 3,000 pg/ml), an increase in adrenal cAMP was measured. We conclude that physiological increases in plasma ACTH may stimulate adrenal steroidogenesis in PD2 pups through a non-cAMPmediated pathway. corticosterone; adrenal gland; synthetic ACTH(1–39), second messenger; newborn THE ADRENOCORTICAL RESPONSE to stress in newborns is essential for survival (19). For example, acute episodes of hypoxia are common in neonates born prematurely (16, 27, 28, 30). Furthermore, these stressors can affect the developing neonatal brain via the disruption of metabolic and neurologic function and may have long-term endocrine and metabolic consequences (8, 10). A coordinated development of the hypothalamic-pituitary-adrenal (HPA) axis in neonates is important for promoting maturation of the immature lung, facilitating closing of the ductus arteriosis and improving vasoconstrictor response to catecholamines (12, 31, 51). Because the rate of premature births in the United States is increasing, it is important that we evaluate the mechanisms of steroidogenesis in the premature and full-term neonate (29).
Address for reprint requests and other correspondence: H. Raff, Endocrinology, Aurora St. Luke’s Medical Center, 2801 W KK River Pky., Suite 245, Milwaukee WI 53215 (e-mail: [email protected]). http://www.ajpregu.org
The neonatal rat pup is a useful model of prematurity in humans and has been used to investigate the development of the HPA axis (41, 45). Corticosterone (the primary glucocorticoid in rats) is produced in the zona fasciculata of the adrenal cortex. The production of corticosterone is stimulated by ACTH, which binds to, and activates the melanocortin 2 receptor (MC2R), leading to an increase in intracellular cyclic adenosine monophosphate (cAMP), together termed the ACTH-MC2R-cAMP cascade (17, 36, 40). Although it has been shown that there are other minor modulators of corticosterone production, such as calcium, cAMP remains the accepted obligatory second messenger of ACTH action (17). Increased cAMP leads to the activation of PKA and to a subsequent increase in the transport of free cholesterol from the cytosol across the mitochondrial membrane, in a process mediated by steroidogenic acute regulatory protein (StAR), the rate-limiting step in steroidogenesis (46). The ability of the adrenal gland to secrete corticosterone is present in most mammals at birth, but decreases during the first 1 to 2 wk of postnatal life. The adrenal gland in the developing rat displays an ACTH-hyporesponsive period between postnatal day (PD) 4 and PD14 (11, 25, 48, 49). We have previously performed ACTH stimulation testing in neonatal rats exposed to hypoxia from birth to 5–7 days. We found that there is an augmentation of the corticosterone, but not the aldosterone, response to ACTH, and that this increase is associated with an increase in StAR protein (37). Additionally, we have previously shown that up to PD5, pups exposed to acute hypoxia are capable of generating a large corticosterone response with a minimal increase in immunoassayable plasma ACTH and without a detectable increase in adrenal cAMP content (the critical second messenger) (23). The prior studies with hypoxia described above were the motivation for the current studies examining neonatal adrenal responses to exogenous ACTH in normoxic pups. Prior studies have not carefully investigated the adrenal response to ACTH in the very young (PD1– 4) neonatal rat; it has been assumed it is mediated in vivo by large increases in cytosolic cAMP, since adrenal cells from rats of this age respond to cAMP and ACTH in vitro (2). However, this does not establish that the adrenocortical response to ACTH or stress in vivo in the first few days of postnatal life is dependent on increases in adrenocortical cAMP as the second messenger. The primary goal of the present study was to determine whether exogenous ACTH stimulates corticosterone production from the adrenal gland in the PD2 rat and to investigate whether this process is mediated by the classic ACTH-MC2RcAMP cascade, as in the PD8 and adult rat adrenal gland (23). Specifically, we compared the corticosterone and adrenal cAMP responses of PD2 and PD8 rats using different doses of
0363-6119/14 Copyright © 2014 the American Physiological Society
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Statistical analyses. The hormone and cAMP data were analyzed by two-way ANOVA (factor 1: time, factor 2: age). Post hoc analysis was performed by Student-Newman-Keuls multiple-range test (P ⬍ 0.05) (SigmaPlot 11.0).
METHODS
RESULTS
Animal treatment and experimental protocol. The animal protocol was approved by the Institutional Animal Care and Use Committee of Aurora Health Care. Timed-pregnant Sprague-Dawley rats at gestational days 15–17 (n ⫽ 48) were obtained from Harlan Sprague Dawley (Indianapolis, IN), maintained on a standard diet and had water available ad libitum in a controlled environment (0600 –1800 lights on). Rats were allowed to deliver and care for their pups without interference until experimentation. Pups (both sexes; n ⫽ 574) were studied at postnatal days (PD) 2 and 8 (PD8). These ages were selected because we have previously shown that the dynamics of the HPA axis response to stress in PD8 rats are similar to a full-term human newborn, while the PD2 HPA axis response to stress models that of a premature human newborn (5, 6, 7, 18). On PD2 or PD8, at ⬃0800 h, rat pups were removed from the dams and placed in a small chamber with adequate bedding and were allowed free range of motion and room to huddle. A variable control heating pad (Moore Medical, Farmington, CT) was placed beneath the bedding and kept at the lowest setting required to maintain body temperature in normoxic rats at these ages (18). After 10 min in the chamber, at ⬃0830 h, rat pups were removed from the chamber and quickly weighed. Immediately after weighing, pups were killed (baseline) or were injected intraperitoneally, as described previously (37). Rats were injected with either vehicle (10 l/kg body wt of isotonic saline) or porcine ACTH (1–39) (Sigma Chemical, St. Louis, MO) in isotonic saline at low, moderate, or high doses (1, 4, or 20 g/kg body wt). After analyzing the plasma concentrations and corticosterone responses achieved with the low, moderate, and high doses of ACTH (see Figs. 1–3), we recognized that we needed a lower dose of ACTH to better evaluate the lower range of adrenocortical sensitivity. To obtain the most accurate dosing for this very low dose of exogenous ACTH, we conducted a pilot study and determined that an ACTH dose of 0.5 g/kg body wt in PD2 pups and 0.2 g/kg body wt in PD8 pups gave similar plasma ACTH concentrations. For vehicle and all doses of ACTH, pups were removed from the temporary holding chamber and decapitated at baseline (control), 15, 30, or 60 min postinjection. To evaluate the early (5 min) adrenal response to ACTH, an additional set of experiments were performed in which PD2 (n ⫽ 81) and PD8 (n ⫽ 55) pups were injected with low or moderate (1 or 4 g/kg body wt) doses of ACTH, and blood and adrenals were obtained and pooled (as described below) at 5 min after injection (n ⫽ 9 samples per age and dose). Blood and tissue collection. Trunk blood was collected at baseline (before ACTH injection) and at 15, 30, or 60 min after the injection, whereas adrenal glands were obtained at baseline and at 15 and 60 min after injection. Another set of experiments sampled at 5 min after ACTH injection. Trunk blood was pooled (2–3 pups/samples) in a tube with EDTA and treated as one sample for statistical purposes. Whole adrenals glands were pooled (4 – 6 adrenals/sample), flash frozen in liquid nitrogen, and stored along with the plasma samples at ⫺70°C until later analysis. Plasma hormone and adrenal cAMP assays. Plasma ACTH and corticosterone were measured by radioimmunoassay, as described previously (MP Biomedicals, Orangeburg, NJ) (34). Frozen adrenal samples were weighed and then pulverized using a liquid nitrogencooled minimortar (Bel-Art Products, Pequannock, NJ), reconstituted in cAMP assay buffer, and frozen at ⫺70°C until further analysis. Adrenal cAMP concentration was determined by ELISA, according to the manufacturer’s protocol (Arbor Assays, Ann Arbor, MI) as described previously (23).
Plasma corticosterone response to exogenous ACTH in PD2 and PD8 pups at baseline, 15, 30, and 60 min postinjection are shown in Fig. 1 (note the y-axis ranges are different for PD2 and PD8 results). Exogenous ACTH-stimulated corticosterone release in both PD2 and PD8 pups at 15, 30, and 60 min. The PD2 pups had higher baseline plasma corticosterone concentrations compared with the PD8 pups. The corticosterone response of the PD2 pups to exogenous ACTH was greater than the corticosterone response of the PD8 pups. Note the dose-response relationship between ACTH injection and corticosterone response at 60 min in PD2 rats. However, in PD8 rats, the low and moderate doses resulted in statistically similar increases in corticosterone. Baseline plasma ACTH was 46 ⫾ 3 pg/ml (n ⫽ 15) in PD2 pups and 37 ⫾ 2 pg/ml (n ⫽ 18) in PD8 pups (P ⫽ 0.015). The peak plasma ACTH concentrations achieved 30 min after ACTH injection were 277 ⫾ 42 pg/ml (n ⫽ 17), 720 ⫾ 150
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exogenous ACTH. We hypothesized that exogenous ACTH would stimulate corticosterone production in the PD2 rat, but that the increase may not be associated with an increase in adrenal cAMP content.
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Time (min) Fig. 1. Plasma corticosterone response to ACTH injection in postnatal day 2 (PD2) vs. PD8 rats. PD2 and PD8 pups were injected with porcine ACTH at low, moderate, and high doses (1, 4, or 20 g/kg body wt, respectively). aHigh dose significantly different from low dose (P ⬍ 0.05). bAll doses significantly different from each other (P ⬍ 0.05). cHigh dose significantly different from low and moderate dose (P ⬍ 0.05). Baseline (n ⫽ 15–18). Post-ACTH injection (n ⫽ 7–10). Data are presented as means ⫾ SE. Note the y-axis range is lower for PD8 pup results.
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Time (min) Fig. 2. Changes in adrenal cAMP content in PD2 and PD8 pups injected with three different doses of ACTH. PD2 and PD8 pups were injected with porcine ACTH at low, moderate, and high doses (1, 4, or 20 g/kg body wt, respectively). aSignificantly different from low dose (P ⬍ 0.05). bSignificantly different from low and moderate doses (P ⬍ 0.05). cSignificantly different from high dose by log transformation after the removal of one outlier (P ⬍ 0.05). n ⫽ 5. Data are presented as means ⫾ SE.
pg/ml (n ⫽ 16), and 6,198 ⫾ 901 pg/ml (n ⫽ 15) in the low, moderate, and high doses, respectively. The plasma ACTH concentrations achieved with low dose are comparable to those achieved in neonates injected with CRH (38). The concentrations achieved with the moderate dose are similar to those achieved in neonates exposed to intermittent hypoxia (7). The plasma ACTH concentrations achieved with the high dose of ACTH were clearly pharmacological. The PD2 and PD8 pups injected with vehicle showed no change in plasma ACTH, plasma corticosterone, or adrenal cAMP content (data not shown). The changes in adrenal cAMP content at 15 and 60 min after administration of ACTH are shown in Fig. 2. Adrenal cAMP increased significantly and proportionately with low, moderate, and high doses of ACTH in PD8 pups. In PD2 pups, low and moderate ACTH doses produced a large corticosterone response in the absence of a change in adrenal cAMP content. At high (pharmacological) doses of ACTH in PD2 pups (leading to plasma ACTH levels exceeding 3,000 pg/ml), increases in adrenal cAMP were detected. Baseline cAMP was 0.9 ⫾ 0.2 pmol/mg (n ⫽ 5) for PD2 adrenals and 1.1 ⫾ 0.1 pmol/mg (n ⫽ 5) for PD8 adrenals (not different). Figure 3 summarizes the cAMP response at 15 min and the corticosterone response at 60 min after low, moderate, and high
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doses of ACTH in PD2 and PD8 pups. The plasma corticosterone responses to ACTH were much greater in at PD2 compared with PD8. As the dose of ACTH increased, there was a clear and proportional increase in adrenal cAMP content in PD8 pups. This increase in cAMP was accompanied by increases in plasma corticosterone. In PD2 pups, there was a large corticosterone response at all three doses; however, adrenal cAMP content did not increase significantly in the PD2 pups at 15 min postinjection in response to low and moderate doses of ACTH. The highest dose of ACTH did produce an adrenal cAMP response in both PD2 and PD8 pups. At this highest dose, the adrenal cAMP content in PD8 pups was more than double than that of PD2 pups, despite a substantially smaller corticosterone response. After appreciating the data in Figs. 1–3, we decided to use a much lower dose of ACTH to achieve a more modest corticosterone response. We achieved equivalent plasma ACTH concentrations by giving PD2 pups a higher dose of exogenous ACTH (Fig. 4). The adrenal response to this very low dose ACTH followed a similar pattern to the higher doses, with the PD2 pups exhibiting a larger corticosterone response than PD8 pups (Fig. 4). There was no significant difference between the baseline corticosterone values in PD2 and PD8 pups, although there was a tendency for baseline corticosterone to be higher in
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Dose of ACTH (μg/kg) Fig. 3. Changes in adrenal cAMP content at 15 min vs. plasma corticosterone at 60 min (post-ACTH injection). PD2 and PD8 pups were injected with porcine ACTH at low, moderate, and high doses (1, 4, or 20 g/kg body wt, respectively). aPD2 significantly different from PD8 (P ⬍ 0.05). bSignificantly different from 1 g/kg body wt (low dose) (P ⬍ 0.05). cSignificantly different from 1 and 4 g/kg body wt (low and moderate doses, respectively) (P ⬍ 0.05). cAMP, n ⫽ 5; corticosterone, n ⫽ 7–10. Data are presented as means ⫾ SE.
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produce a larger corticosterone response in PD2 pups, compared with responses in PD8 pups. Furthermore, the response in the PD2 pup may not be accompanied by a sizeable increase in the obligate intracellular second messenger, cAMP. Our results confirmed this hypothesis by demonstrating 1) exogenous ACTH stimulated a larger increase in corticosterone in PD2 pups than in PD8 pups at each time point, 2) adrenal cAMP content in PD2 pups did not change when injected with low and moderate ACTH doses, 3) only high (pharmacological doses) of ACTH elicited a detectable increase in cAMP in PD2 pups, and 4) adrenal cAMP levels in PD8 pups increased significantly and proportionately with increasing doses of ACTH. The HPA axis of the neonatal rat undergoes a period of development that ultimately leads to a fully functional adrenal gland capable of producing corticosterone (33). In particular, it has been shown that the adrenal gland of the neonatal rat goes through a stress-hyporesponsive period (SHRP) (2). The neonatal rat SHRP is characterized by an attenuated corticosterone response to stress between PD4 and PD14 (11, 25, 48, 49). Previous studies have investigated the adrenal responsiveness in the neonatal rat during this hyporesponsive period (2). Additionally, we have focused on the adrenocortical response of neonatal rats during periods of hypoxia. The studies regarding the effects of acute and chronic hypoxia on the HPA axis of the developing neonatal rat are the motivation for the current
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PD2 pups. We were unable to detect a change in adrenal cAMP at 15 min after ACTH injection in either age group. Because of the cAMP data generated in Fig. 2, we were concerned that we had missed a small spike in cAMP occurring during the time between the injection of ACTH and the 15-min sampling time. Therefore, a new set of experiments was performed in which PD2 and PD8 pups were injected with the low or moderate dose of ACTH, and blood and adrenal glands were obtained at 5 min after injection. At the 5-min time point, plasma ACTH had increased to 142 ⫾ 4 pg/ml for the low dose and 515 ⫾ 69 pg/ml for the moderate dose of ACTH. Fig. 5 shows adrenal cAMP and plasma corticosterone levels at baseline and 5 min after injection of ACTH. The low dose of ACTH did not result in a statistically significant increase in cAMP or corticosterone at 5 min postinjection. The moderate dose of ACTH resulted in a significant increase in plasma corticosterone in the PD2 pups without a significant increase in adrenal cAMP, whereas adrenal cAMP increased in the PD8 pups without a change in plasma corticosterone. DISCUSSION
The present study evaluated the plasma corticosterone and adrenal cAMP response to exogenous ACTH injections in PD2 and PD8 rats. We hypothesized that exogenous ACTH would
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Fig. 5. Adrenal cAMP and plasma corticosterone 5 min after injection of low or moderate doses of ACTH in PD2 and PD8 pups. PD2 and PD8 pups were injected with porcine ACTH at low and moderate doses (1 or 4 g/kg body wt, respectively g/kg body wt, respectively). aSignificantly different from baseline (0 min).bSignificant difference between PD2 and PD8 pups (P ⬍ 0.05). n ⫽ 9. Data are presented as means ⫾ SE.
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study (5, 6, 37, 38, 39). Specifically, we continued the investigation of a phenomenon observed that, prior to PD5, when exposed to hypoxia, the neonatal rat exhibits a large corticosterone response without a drastic increase in immunoreactive plasma ACTH and without a detectable increase in adrenal cAMP content (the canonical second messenger) (23). These previous studies led us to question whether increases in ACTH in the normoxic neonatal rat stimulate corticosterone production via a rise in adrenal cAMP content or via another second messenger pathway. We have demonstrated that the PD2 adrenal has a much larger corticosterone response to ACTH compared with the PD8 pup, confirming what has been demonstrated previously in dispersed adrenal cells in vitro (2) and in the adrenal response to hypoxia in vivo (23). This was not due to changes in plasma corticosterone binding capacity since we have previously demonstrated that PD8 pups have higher corticosterone binding globulin levels compared with PD2 pups (23). The current study demonstrated a large corticosterone response to physiological doses of ACTH in PD2 pups without a measurable increase in adrenal cAMP. We understand that cAMP is the accepted intracellular second messenger for the adrenal response to ACTH in mature rats (17). Chronic ACTH stimulation can exert its steroidogenic effects through a noncAMP mechanism (26). In adult rats, it has been shown that ACTH-stimulated adrenal corticosterone content increases within 3 min without a change in adrenal cAMP content, particularly when the ACTH is injected in the morning (9). In addition, it has been suggested that adrenal cell responses to very low concentrations of ACTH are cAMP-independent (52) or that only very small amounts of cAMP are necessary to stimulate steroidogenesis (32). What, then, is mediating this large increase in ACTHstimulated corticosterone in the PD2 rat pup? There are other potential second messengers, such as calcium and cGMP; however, most studies do not support these as principal second messengers in this system (17, 23). There are other, less potent pathways that ACTH is capable of stimulating, including MAPK, inositol phosphate, and diacylglycerol (17). These other pathways will be considered for further investigation. Non-ACTH mechanisms for controlling steroidogenesis include sympathetic input via the splanchnic nerves (14, 47) and/or local factors, such as vasoactive intestinal peptide and catecholamines (3, 4). However, these non-ACTH-mediated pathways are unlikely to be the cause of increased corticosterone because we injected exogenous ACTH and because the vehicle injection group did not show an increase in corticosterone. It is thought that StAR protein is the principal mediator of cholesterol transport process essential for steroidogenesis (46). A previous study from the same group showed that exogenous ACTH and cAMP each resulted in a U-shaped curve of adrenal responsiveness in dispersed cells (2). In that study, the corticosterone responses to exogenous ACTH, cAMP, and a soluble (non-rate-limited) form of cholesterol were higher in PD1 adrenal cells compared with PD10, and responses increased again in cells from adult rats. It was concluded that it was unlikely that cholesterol transport served as a major contributing factor in hyporesponsiveness in neonatal adrenal cells (2) and, therefore, is unlikely to explain the results of our study.
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At first, we analyzed adrenal cAMP content at 15 min after injection of ACTH because previous studies have shown that the maximal effect of ACTH on cAMP production occurs after 15 min and returns to basal levels after ⬃40 min (17). Since, there is a possibility that very small increases in adrenal cAMP content in PD2 adrenals may have occurred earlier than 15 min (9), we repeated the low and moderate dose in PD2 and PD8 pups and sampled at 5 min. The 5-min results with the moderate dose of ACTH essentially validated the 15-min data shown in Figs. 1 and 2, as there was a corticosterone response in PD2 pups without a significant change in adrenal cAMP, whereas there was an adrenal cAMP response in PD8 pups without a change in plasma corticosterone. This confirms that the PD2 rat adrenal appears to have a non-cAMP-mediated increase in steroidogenesis, whereas steroidogenesis in the PD8 rat adrenal appears to be relatively insensitive to increases in cAMP stimulated by ACTH. It is also possible that very small increases in cAMP were generated, but only in the appropriate compartment to stimulate steroidogenesis (42). Our method measured total cAMP content, so a small change in a specific intracellular compartment may not have been detected. The possibility of cAMP independence is even more significant considering that the corticosterone response to a very low dose of ACTH did not correlate with an increase in adrenal cAMP content in PD8 pups (Fig. 4). Therefore, we conclude that either the PD2 adrenal does not require an increase in cAMP in the response to ACTH, or that it is ultrasensitive to very small, compartmental changes in adrenal cAMP content. Previous in vitro studies, however, do not suggest ultrasensitivity to cAMP in dispersed adrenal cells from PD1 compared with PD10 rat pups (2). On the basis of these findings, we still infer that another second messenger or intracellular mechanism may be involved in the large corticosterone responses to ACTH injection in the neonatal rat. Perspectives and Significance Our study has revealed a unique, but as yet, unidentified intracellular mechanism linking ACTH stimulation to an augmented increase in corticosterone production in the neonatal rat. This potentially novel phenomenon may have significant clinical implications. In particular, it is now established that transient, relative adrenal insufficiency is a significant clinical entity in the premature and low-birth weight human infant and may be associated with hypotension, respiratory failure, and increased morbidity (15, 20, 21, 34, 35, 43, 50). Furthermore, an altered adrenocortical response to physiological and pharmacological doses of exogenous ACTH is useful in the evaluation of adrenal function in the premature and low-birth weight infant (1, 13, 20, 22, 24, 43, 44). ACTH stimulation is an important diagnostic test of human adrenal function, the results of which can influence the implementation of glucocorticoid therapy (35, 44). It is important to evaluate the mechanism of ACTH action on the adrenal cortex using the rat model of human prematurity. An increased understanding of these mechanisms could lead to improved diagnostic and treatment strategies. The current study has provided evidence that ACTH may stimulate corticosterone production through an unidentified second messenger system in the very young neonatal rat. Future studies investigating the identity of this unknown intra-
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cellular mechanism will be helpful in gaining a full understanding of the development of adrenal function in neonates. ACKNOWLEDGMENTS The authors thank Kathan Chintamaneni and Barbara Jankowski for assistance in the laboratory. GRANTS J. Bodager is a participant in the Medical College of Wisconsin Medical Student Research Pathways program and received funding from the Dr. Michael J. Dunn Summer Research Fellowship Award. This study was supported, in part, by the Aurora Research Institute. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Author contributions: J.R.B., T.G.G., E.D.B., A.G., and H.R. conception and design of research; J.R.B., T.G.G., E.D.B., and A.G. performed experiments; J.R.B., T.G.G., E.D.B., and H.R. analyzed data; J.R.B., T.G.G., E.D.B., A.G., and H.R. interpreted results of experiments; J.R.B., T.G.G., E.D.B., A.G., and H.R. prepared figures; J.R.B. drafted manuscript; J.R.B., T.G.G., E.D.B., A.G., and H.R. edited and revised manuscript; J.R.B., T.G.G., E.D.B., A.G., and H.R. approved final version of manuscript. REFERENCES 1. Agwu JC, Bissenden J. The low-dose synacthen test: experience in well preterm infants. J Pediatr Endocrinol Metab 17: 1607–1612, 2004. 2. Arai M, Widmaier EP. Steroidogenesis in isolated adrenocortical cells during development in rats. Mol Cell Endocrinol 92: 91–97, 1993. 3. Bodnar M, Sarrieau A, Deschepper CF, Walker CD. Adrenal vasoactive intestinal peptide participates in neonatal corticosteroid production in the rat. Am J Physiol Regul Integr Comp Physiol 273: R1163–R1172, 1997. 4. Bornstein SR, EngelandWC, Ehrhart-Bornstein M, Herman JP. Dissociation of ACTH and glucocorticoids. Trends Endocrinol Metab 19: 175–180, 2008. 5. Bruder ED, Kamer KJ, Guenther MA, Raff H. Adrenocorticotropic hormone and corticosterone responses to acute hypoxia in the neonatal rat: effects of body temperature maintenance. Am J Physiol Regul Integr Comp Physiol 300: R708 –R715, 2011. 6. Bruder ED, Taylor JK, Kamer KJ, Raff H. Development of the ACTH and corticosterone response to acute hypoxia in the neonatal rat. Am J Physiol Regul Integr Comp Physiol 295: R1195–R1203, 2008. 7. Chintamaneni K, Bruder ED, Raff H. Effects of age on ACTH, corticosterone, glucose, insulin, and mRNA levels during intermittent hypoxia in the neonatal rat. Am J Physiol Regul Integr Comp Physiol 304: R782–R789, 2013. 8. Chintamaneni K, Bruder ED, Raff H. Programming of the hypothalamic-pituitary-adrenal axis by neonatal intermittent hypoxia: effects on adult male ACTH and corticosterone responses are stress-specific. Endocrinology 155: 1763–1770, 2014. 9. Dallman MF, Engeland WC, Rose JC, Wilkinson CW, Shinsako J, Siedenburg F. Nycthemeral rhythm in adrenal responsiveness to ACTH. Am J Physiol Regul Integr Comp Physiol 235: R210 –R219, 1978. 10. Davis DJ, Creery WD, Radziuk J. Inappropriately high plasma insulin levels in suspected perinatal asphyxia. Acta Paediatr 88: 76 –81, 1999. 11. Denenberg VH, Bell RW. Critical periods for the effects of infantile experience on adult learning. Science 131: 227–228, 1960. 12. Deruelle P, Houfflin-Debarge V, Magnenant E, Jaillard S, Riou Y, Puech F, Storme L. Effects of antenatal glucocorticoids on pulmonary vascular reactivity in the ovine fetus. Am J Obstet Gynecol 189: 208 –215, 2003. 13. El-Khuffash A, McNamara PJ, Lapointe A, Jain A. Adrenal function in preterm infants undergoing patent ductus arteriosus ligation. Neonatology 104: 28 –33, 2013. 14. Engeland WC. Functional innervation of the adrenal cortex by the splanchnic nerve. Horm Metab Res 30: 311–314, 1998. 15. Fernandez EF, Watterberg KL. Relative adrenal insufficiency in the preterm and term infant. J Perinatol 29: S44 –S40, 2009.
16. Frankel L, Stevenson DK. Metabolic emergencies of the newborn: hypoxemia and hypoglycemia. Compr Ther 13: 14 –19, 1987. 17. Gallo-Payet N, Payet MD. Mechanism of action of ACTH: beyond cAMP. Microsc Res Tech 61: 275–287, 2003. 18. Guenther MA, Bruder ED, Raff H. Effects of body temperature maintenance on glucose, insulin, and corticosterone responses to acute hypoxia in the neonatal rat. Am J Physiol Regul Integr Comp Physiol 302: R627–R633, 2012. 19. Hanukoglu A, Fried D, Nakash I, Hanukoglu I. Selective increases in adrenal steroidogenic capacity during acute respiratory disease in infants. Eur J Endocrinol 133: 552–556, 1995. 20. Hochwald O, Holsti L, Osiovich H. The use of an early ACTH test to identify hypoadrenalism-related hypotension in low birth weight infants. J Perinatol 32: 412–417, 2012. 21. Holsti L, Weinberg J, Whitfield MF, Grunau RE. Relationships between adrenocorticotropic hormone and cortisol are altered during clustered nursing care in preterm infants born at extremely low gestational age. Early Hum Dev 83: 341–348, 2007. 22. Huysman MWA, Hokken-Koelega ACS, De Ridder MAJ, Sauer PJJ. Adrenal function in sick very preterm infants. Pediatr Res 48: 629 –633, 2000. 23. Johnson K, Bruder ED, Raff H. Adrenocortical control in the neonatal rat: ACTH- and cAMP-independent corticosterone production during hypoxia. Physiol Rep 1: e00054, doi:10.1002/phy2.54, 2013. 24. Karlsson R, Kallio J, Irjala K, Ekblad S, Toppari J, Kero P. Adrenocorticotropin and corticotropin-releasing hormone tests in preterm infants. J Pediatr Endocrinol Metab 85: 4592–4595, 2000. 25. Levine S, Huchton DM, Wiener SG, Rosenfeld P. Time course of the effect of maternal deprivation on the hypothalamic-pituitary-adrenal axis in the infant rat. Dev Psychobiol 24: 547–558, 1991. 26. Liu H, Enyeart JA, Enyeart JJ. ACTH induces Cav3.2 current and mRNA by cAMP-dependent and cAMP-independent mechanisms. J Biol Chem 285: 20040 –20050, 2010. 27. Low JA, Froese AB, Galbraith RS, Smith JT, Sauerbrei EE, Derrick EJ. The association between preterm newborn hypotension and hypoxemia and outcome during the first year. Acta Paediatr 82: 433–437, 1993. 28. Martin RJ, Miller MJ, Carlo WA. Pathogenesis of apnea in preterm infants. J Pediatr 109: 733–741, 1986. 29. Martin JA, Hamilton BE, Sutton PD, Ventura SJ, Menacker F, Kirmeyer S, Munson ML. Births: final data for 2005. Natl Vital Stat Rep 56: 1–103, 2007. 30. Miller MJ, Martin RJ. Apnea of prematurity. Clin Perinatol 19: 789 – 808, 1992. 31. Morales P, Rastogi A, Bez ML, Akintorin SM, Pyati S, Andes SM, Pildes RS. Effect of dexamethasone therapy on the neonatal ductus arteriosus. Pediatr Cardiol 19: 225–229, 1998. 32. Moyle WR, Kong YC, Ramachandran J. Steroidogenesis and cyclic adenosine 3=, 5=-monophosphate accumulation in rat adrenal cells. J Biol Chem 248: 2409 –2417, 1973. 33. Nagaya M, Arai M, Widmaier EP. Ontogeny of immunoreactive and bioactive microsomal steroidogenic enzymes during adrenocortical development in the rat. Mol Cell Endocrinol 114: 27–34, 1995. 34. Nykänen P, Anttila E, Heinonen K, Hallman M, Voutilaninen R. Early hypoadrenalism in premature infants at risk for bronchopulmonary dysplasia or death. Acta Paediatr 96: 1600 –1605, 2007. 35. Quintos JB, Boney CM. The transient adrenal insufficiency in the premature newborn. Curr Opin Endocrinol Diabetes Obes 17: 8 –12, 2010. 36. Raff H, Findling JW. A physiologic approach to diagnosis of the Cushing syndrome. Ann Intern Med 138: 980 –991, 2003. 37. Raff H, Hong JJ, Oaks MK, Widmaier EP. Adrenocortical responses to ACTH in neonatal rats: effect of hypoxia from birth on corticosterone, StAR, and PBR. Am J Physiol Regul Integr Comp Physiol 284: R78 –R85, 2003. 38. Raff H, Jacobson L, Cullinan WE. Elevated corticosterone and inhibition of ACTH responses to CRH and ether in the neonatal rat: effect of hypoxia from birth. Am J Physiol Regul Integr Comp Physiol 285: R1224 –R1230, 2003. 39. Raff H, Lee JJ, Widmaier EP, Oaks MK, Engeland WC. Basal and adrenocorticotropin-stimulated corticosterone in the neonatal rat exposed to hypoxia from birth: modulation by chemical sympathectomy. Endocrinology 145: 79 –86, 2004. 40. Richards JS. New signaling pathways for hormones and cyclic adenosine 3=,5=-monophosphate action in endocrine cells. Mol Endocrinol 15: 209 – 218, 2001.
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ADRENAL SENSITIVITY TO ACTH 41. Romijn HJ, Hofmana MA, Gramsbergen A. At what age is the developing cerebral cortex of the rat comparable to that of the full-term newborn human baby? Early Hum Dev 26: 61–67, 1991. 42. Sala GB, Hayashi K, Catt KJ, Dufau ML. Adrenocorticotropin action in isolated adrenal cells. The intermediate role of cyclic AMP in stimulation of corticosterone synthesis. J Biol Chem 254: 3861–3865, 1979. 43. Sari FN, Dizdar EA, Oguz SS, Andiran N, Erdeve O, Uras R, Dilmen U. Baseline and stimulated cortisol levels in preterm infants: is there any clinical relevance? Horm Res Paediatr 77: 12–18, 2012. 44. Scott SM, Backstrom C, Bessman S. Effect of five days of dexamethasone therapy on ventilator dependence and adrenocorticotropic hormone-stimulated cortisol concentrations. J Perinatol 17: 24 –28, 1997. 45. Sheldon RA, Chuai J, Ferriero DM. A rat model for hypoxicischemic brain damage in very premature infants. Biol Neonate 60: 327–341, 1996. 46. Stocco DM, Clark BJ. The role of the steroidogenic acute regulatory protein in steroidogenesis. Steroids 62: 29 –36, 1997.
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47. Walker CD. Chemical sympathectomy and maternal separation affect neonatal stress responses and adrenal sensitivity to ACTH. Am J Physiol Regul Integr Comp Physiol 268: R1281–R1288, 1995. 48. Walker CD, Aubert ML. Effects of early undernutrition and handling on the adrenocortical activity of neonatal rats. Life Sci 43: 1983–1990, 1988. 49. Walker CD, Perrin M, Vale W, Rivier C. Ontogeny of the stress response in the rat: role of the pituitary and the hypothalamus. Endocrinology 118: 1445–1451, 1986. 50. Watterberg KL, Gerdes JS, Cook KL. Impaired glucocorticoid synthesis in premature infants developing chronic lung disease. Pediatr Res 50: 190 –195, 2001. 51. Watterberg KL, Gerdes JS, Gifford KL, Lin HM. Prophylaxis against early adrenal insufficiency to prevent chronic lung disease in premature infants. Pediatrics 104: 1258 –1263, 1999. 52. Yamazaki T, Kimoto T, Higuchi K, Ohta Y, Kawato S, Kominami S. Calcium ion as a second messenger for o-nitrophenylsulfenyl-adrenocorticotropin (NPS-ACTH) and ACTH in bovine adrenal steroidogenesis. Endocrinology 139: 4765–4771, 1998.
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Adrenocortical sensitivity to ACTH in neonatal rats: correlation of corticosterone responses and adrenal cAMP content Jonathan Bodager,1,2 Thomas Gessert,1 Eric D. Bruder,1 Ashley Gehrand,1 and Hershel Raff1,2 1 2
Endocrine Research Laboratory; Aurora St. Luke’s Medical Center, Aurora Research Institute, Milwaukee, Wisconsin; and Departments of Medicine, Surgery, and Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin
Submitted 20 March 2014; accepted in final form 28 May 2014
Bodager J, Gessert T, Bruder ED, Gehrand A, Raff H. Adrenocortical sensitivity to ACTH in neonatal rats: correlation of corticosterone responses and adrenal cAMP content. Am J Physiol Regul Integr Comp Physiol 307: R347–R353, 2014. First published June 4, 2014; doi:10.1152/ajpregu.00125.2014.—A coordinated hypothalamicpituitary-adrenal axis response is important for the survival of newborns during stress. We have previously shown that prior to postnatal day (PD) 5, neonatal rats exposed to hypoxia (one of the most common stressors effecting premature neonates) exhibit a large corticosterone response with a minimal increase in immunoassayable plasma ACTH and without a detectable increase in adrenal cAMP content (the critical second messenger). To explore the phenomenon of ACTH-stimulated steroidogenesis in the neonate, we investigated the adrenal response to exogenous ACTH in the normoxic neonatal rat. Rat pups at PD2 and PD8 were injected intraperitoneally with porcine ACTH at low, moderate, or high doses (1, 4, or 20 g/kg body wt). Trunk blood and whole adrenal glands were collected at baseline (before injection) and 15, 30, or 60 min after the injection. ACTH stimulated corticosterone release in PD2 and PD8 pups. In PD2 pups, plasma corticosterone at baseline and during the response to ACTH injection was greater than values measured in PD8 pups, despite lower adrenal cAMP content in PD2 pups. Specifically, the low and moderate physiological ACTH doses produced a large corticosterone response in PD2 pups without a change in adrenal cAMP content. At extremely high, pharmacological levels of plasma ACTH in PD2 pups (exceeding 3,000 pg/ml), an increase in adrenal cAMP was measured. We conclude that physiological increases in plasma ACTH may stimulate adrenal steroidogenesis in PD2 pups through a non-cAMPmediated pathway. corticosterone; adrenal gland; synthetic ACTH(1–39), second messenger; newborn THE ADRENOCORTICAL RESPONSE to stress in newborns is essential for survival (19). For example, acute episodes of hypoxia are common in neonates born prematurely (16, 27, 28, 30). Furthermore, these stressors can affect the developing neonatal brain via the disruption of metabolic and neurologic function and may have long-term endocrine and metabolic consequences (8, 10). A coordinated development of the hypothalamic-pituitary-adrenal (HPA) axis in neonates is important for promoting maturation of the immature lung, facilitating closing of the ductus arteriosis and improving vasoconstrictor response to catecholamines (12, 31, 51). Because the rate of premature births in the United States is increasing, it is important that we evaluate the mechanisms of steroidogenesis in the premature and full-term neonate (29).
Address for reprint requests and other correspondence: H. Raff, Endocrinology, Aurora St. Luke’s Medical Center, 2801 W KK River Pky., Suite 245, Milwaukee WI 53215 (e-mail: [email protected]). http://www.ajpregu.org
The neonatal rat pup is a useful model of prematurity in humans and has been used to investigate the development of the HPA axis (41, 45). Corticosterone (the primary glucocorticoid in rats) is produced in the zona fasciculata of the adrenal cortex. The production of corticosterone is stimulated by ACTH, which binds to, and activates the melanocortin 2 receptor (MC2R), leading to an increase in intracellular cyclic adenosine monophosphate (cAMP), together termed the ACTH-MC2R-cAMP cascade (17, 36, 40). Although it has been shown that there are other minor modulators of corticosterone production, such as calcium, cAMP remains the accepted obligatory second messenger of ACTH action (17). Increased cAMP leads to the activation of PKA and to a subsequent increase in the transport of free cholesterol from the cytosol across the mitochondrial membrane, in a process mediated by steroidogenic acute regulatory protein (StAR), the rate-limiting step in steroidogenesis (46). The ability of the adrenal gland to secrete corticosterone is present in most mammals at birth, but decreases during the first 1 to 2 wk of postnatal life. The adrenal gland in the developing rat displays an ACTH-hyporesponsive period between postnatal day (PD) 4 and PD14 (11, 25, 48, 49). We have previously performed ACTH stimulation testing in neonatal rats exposed to hypoxia from birth to 5–7 days. We found that there is an augmentation of the corticosterone, but not the aldosterone, response to ACTH, and that this increase is associated with an increase in StAR protein (37). Additionally, we have previously shown that up to PD5, pups exposed to acute hypoxia are capable of generating a large corticosterone response with a minimal increase in immunoassayable plasma ACTH and without a detectable increase in adrenal cAMP content (the critical second messenger) (23). The prior studies with hypoxia described above were the motivation for the current studies examining neonatal adrenal responses to exogenous ACTH in normoxic pups. Prior studies have not carefully investigated the adrenal response to ACTH in the very young (PD1– 4) neonatal rat; it has been assumed it is mediated in vivo by large increases in cytosolic cAMP, since adrenal cells from rats of this age respond to cAMP and ACTH in vitro (2). However, this does not establish that the adrenocortical response to ACTH or stress in vivo in the first few days of postnatal life is dependent on increases in adrenocortical cAMP as the second messenger. The primary goal of the present study was to determine whether exogenous ACTH stimulates corticosterone production from the adrenal gland in the PD2 rat and to investigate whether this process is mediated by the classic ACTH-MC2RcAMP cascade, as in the PD8 and adult rat adrenal gland (23). Specifically, we compared the corticosterone and adrenal cAMP responses of PD2 and PD8 rats using different doses of
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Statistical analyses. The hormone and cAMP data were analyzed by two-way ANOVA (factor 1: time, factor 2: age). Post hoc analysis was performed by Student-Newman-Keuls multiple-range test (P ⬍ 0.05) (SigmaPlot 11.0).
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Animal treatment and experimental protocol. The animal protocol was approved by the Institutional Animal Care and Use Committee of Aurora Health Care. Timed-pregnant Sprague-Dawley rats at gestational days 15–17 (n ⫽ 48) were obtained from Harlan Sprague Dawley (Indianapolis, IN), maintained on a standard diet and had water available ad libitum in a controlled environment (0600 –1800 lights on). Rats were allowed to deliver and care for their pups without interference until experimentation. Pups (both sexes; n ⫽ 574) were studied at postnatal days (PD) 2 and 8 (PD8). These ages were selected because we have previously shown that the dynamics of the HPA axis response to stress in PD8 rats are similar to a full-term human newborn, while the PD2 HPA axis response to stress models that of a premature human newborn (5, 6, 7, 18). On PD2 or PD8, at ⬃0800 h, rat pups were removed from the dams and placed in a small chamber with adequate bedding and were allowed free range of motion and room to huddle. A variable control heating pad (Moore Medical, Farmington, CT) was placed beneath the bedding and kept at the lowest setting required to maintain body temperature in normoxic rats at these ages (18). After 10 min in the chamber, at ⬃0830 h, rat pups were removed from the chamber and quickly weighed. Immediately after weighing, pups were killed (baseline) or were injected intraperitoneally, as described previously (37). Rats were injected with either vehicle (10 l/kg body wt of isotonic saline) or porcine ACTH (1–39) (Sigma Chemical, St. Louis, MO) in isotonic saline at low, moderate, or high doses (1, 4, or 20 g/kg body wt). After analyzing the plasma concentrations and corticosterone responses achieved with the low, moderate, and high doses of ACTH (see Figs. 1–3), we recognized that we needed a lower dose of ACTH to better evaluate the lower range of adrenocortical sensitivity. To obtain the most accurate dosing for this very low dose of exogenous ACTH, we conducted a pilot study and determined that an ACTH dose of 0.5 g/kg body wt in PD2 pups and 0.2 g/kg body wt in PD8 pups gave similar plasma ACTH concentrations. For vehicle and all doses of ACTH, pups were removed from the temporary holding chamber and decapitated at baseline (control), 15, 30, or 60 min postinjection. To evaluate the early (5 min) adrenal response to ACTH, an additional set of experiments were performed in which PD2 (n ⫽ 81) and PD8 (n ⫽ 55) pups were injected with low or moderate (1 or 4 g/kg body wt) doses of ACTH, and blood and adrenals were obtained and pooled (as described below) at 5 min after injection (n ⫽ 9 samples per age and dose). Blood and tissue collection. Trunk blood was collected at baseline (before ACTH injection) and at 15, 30, or 60 min after the injection, whereas adrenal glands were obtained at baseline and at 15 and 60 min after injection. Another set of experiments sampled at 5 min after ACTH injection. Trunk blood was pooled (2–3 pups/samples) in a tube with EDTA and treated as one sample for statistical purposes. Whole adrenals glands were pooled (4 – 6 adrenals/sample), flash frozen in liquid nitrogen, and stored along with the plasma samples at ⫺70°C until later analysis. Plasma hormone and adrenal cAMP assays. Plasma ACTH and corticosterone were measured by radioimmunoassay, as described previously (MP Biomedicals, Orangeburg, NJ) (34). Frozen adrenal samples were weighed and then pulverized using a liquid nitrogencooled minimortar (Bel-Art Products, Pequannock, NJ), reconstituted in cAMP assay buffer, and frozen at ⫺70°C until further analysis. Adrenal cAMP concentration was determined by ELISA, according to the manufacturer’s protocol (Arbor Assays, Ann Arbor, MI) as described previously (23).
Plasma corticosterone response to exogenous ACTH in PD2 and PD8 pups at baseline, 15, 30, and 60 min postinjection are shown in Fig. 1 (note the y-axis ranges are different for PD2 and PD8 results). Exogenous ACTH-stimulated corticosterone release in both PD2 and PD8 pups at 15, 30, and 60 min. The PD2 pups had higher baseline plasma corticosterone concentrations compared with the PD8 pups. The corticosterone response of the PD2 pups to exogenous ACTH was greater than the corticosterone response of the PD8 pups. Note the dose-response relationship between ACTH injection and corticosterone response at 60 min in PD2 rats. However, in PD8 rats, the low and moderate doses resulted in statistically similar increases in corticosterone. Baseline plasma ACTH was 46 ⫾ 3 pg/ml (n ⫽ 15) in PD2 pups and 37 ⫾ 2 pg/ml (n ⫽ 18) in PD8 pups (P ⫽ 0.015). The peak plasma ACTH concentrations achieved 30 min after ACTH injection were 277 ⫾ 42 pg/ml (n ⫽ 17), 720 ⫾ 150
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exogenous ACTH. We hypothesized that exogenous ACTH would stimulate corticosterone production in the PD2 rat, but that the increase may not be associated with an increase in adrenal cAMP content.
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Time (min) Fig. 1. Plasma corticosterone response to ACTH injection in postnatal day 2 (PD2) vs. PD8 rats. PD2 and PD8 pups were injected with porcine ACTH at low, moderate, and high doses (1, 4, or 20 g/kg body wt, respectively). aHigh dose significantly different from low dose (P ⬍ 0.05). bAll doses significantly different from each other (P ⬍ 0.05). cHigh dose significantly different from low and moderate dose (P ⬍ 0.05). Baseline (n ⫽ 15–18). Post-ACTH injection (n ⫽ 7–10). Data are presented as means ⫾ SE. Note the y-axis range is lower for PD8 pup results.
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pg/ml (n ⫽ 16), and 6,198 ⫾ 901 pg/ml (n ⫽ 15) in the low, moderate, and high doses, respectively. The plasma ACTH concentrations achieved with low dose are comparable to those achieved in neonates injected with CRH (38). The concentrations achieved with the moderate dose are similar to those achieved in neonates exposed to intermittent hypoxia (7). The plasma ACTH concentrations achieved with the high dose of ACTH were clearly pharmacological. The PD2 and PD8 pups injected with vehicle showed no change in plasma ACTH, plasma corticosterone, or adrenal cAMP content (data not shown). The changes in adrenal cAMP content at 15 and 60 min after administration of ACTH are shown in Fig. 2. Adrenal cAMP increased significantly and proportionately with low, moderate, and high doses of ACTH in PD8 pups. In PD2 pups, low and moderate ACTH doses produced a large corticosterone response in the absence of a change in adrenal cAMP content. At high (pharmacological) doses of ACTH in PD2 pups (leading to plasma ACTH levels exceeding 3,000 pg/ml), increases in adrenal cAMP were detected. Baseline cAMP was 0.9 ⫾ 0.2 pmol/mg (n ⫽ 5) for PD2 adrenals and 1.1 ⫾ 0.1 pmol/mg (n ⫽ 5) for PD8 adrenals (not different). Figure 3 summarizes the cAMP response at 15 min and the corticosterone response at 60 min after low, moderate, and high
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doses of ACTH in PD2 and PD8 pups. The plasma corticosterone responses to ACTH were much greater in at PD2 compared with PD8. As the dose of ACTH increased, there was a clear and proportional increase in adrenal cAMP content in PD8 pups. This increase in cAMP was accompanied by increases in plasma corticosterone. In PD2 pups, there was a large corticosterone response at all three doses; however, adrenal cAMP content did not increase significantly in the PD2 pups at 15 min postinjection in response to low and moderate doses of ACTH. The highest dose of ACTH did produce an adrenal cAMP response in both PD2 and PD8 pups. At this highest dose, the adrenal cAMP content in PD8 pups was more than double than that of PD2 pups, despite a substantially smaller corticosterone response. After appreciating the data in Figs. 1–3, we decided to use a much lower dose of ACTH to achieve a more modest corticosterone response. We achieved equivalent plasma ACTH concentrations by giving PD2 pups a higher dose of exogenous ACTH (Fig. 4). The adrenal response to this very low dose ACTH followed a similar pattern to the higher doses, with the PD2 pups exhibiting a larger corticosterone response than PD8 pups (Fig. 4). There was no significant difference between the baseline corticosterone values in PD2 and PD8 pups, although there was a tendency for baseline corticosterone to be higher in
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produce a larger corticosterone response in PD2 pups, compared with responses in PD8 pups. Furthermore, the response in the PD2 pup may not be accompanied by a sizeable increase in the obligate intracellular second messenger, cAMP. Our results confirmed this hypothesis by demonstrating 1) exogenous ACTH stimulated a larger increase in corticosterone in PD2 pups than in PD8 pups at each time point, 2) adrenal cAMP content in PD2 pups did not change when injected with low and moderate ACTH doses, 3) only high (pharmacological doses) of ACTH elicited a detectable increase in cAMP in PD2 pups, and 4) adrenal cAMP levels in PD8 pups increased significantly and proportionately with increasing doses of ACTH. The HPA axis of the neonatal rat undergoes a period of development that ultimately leads to a fully functional adrenal gland capable of producing corticosterone (33). In particular, it has been shown that the adrenal gland of the neonatal rat goes through a stress-hyporesponsive period (SHRP) (2). The neonatal rat SHRP is characterized by an attenuated corticosterone response to stress between PD4 and PD14 (11, 25, 48, 49). Previous studies have investigated the adrenal responsiveness in the neonatal rat during this hyporesponsive period (2). Additionally, we have focused on the adrenocortical response of neonatal rats during periods of hypoxia. The studies regarding the effects of acute and chronic hypoxia on the HPA axis of the developing neonatal rat are the motivation for the current
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PD2 pups. We were unable to detect a change in adrenal cAMP at 15 min after ACTH injection in either age group. Because of the cAMP data generated in Fig. 2, we were concerned that we had missed a small spike in cAMP occurring during the time between the injection of ACTH and the 15-min sampling time. Therefore, a new set of experiments was performed in which PD2 and PD8 pups were injected with the low or moderate dose of ACTH, and blood and adrenal glands were obtained at 5 min after injection. At the 5-min time point, plasma ACTH had increased to 142 ⫾ 4 pg/ml for the low dose and 515 ⫾ 69 pg/ml for the moderate dose of ACTH. Fig. 5 shows adrenal cAMP and plasma corticosterone levels at baseline and 5 min after injection of ACTH. The low dose of ACTH did not result in a statistically significant increase in cAMP or corticosterone at 5 min postinjection. The moderate dose of ACTH resulted in a significant increase in plasma corticosterone in the PD2 pups without a significant increase in adrenal cAMP, whereas adrenal cAMP increased in the PD8 pups without a change in plasma corticosterone. DISCUSSION
The present study evaluated the plasma corticosterone and adrenal cAMP response to exogenous ACTH injections in PD2 and PD8 rats. We hypothesized that exogenous ACTH would
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Fig. 4. Plasma ACTH and corticosterone after injection of a very low dose of ACTH in PD2 and PD8 pups. PD2 and PD8 pups were injected with porcine ACTH at very low doses (0.5 and 0.2 g/kg body wt, respectively). aSignificantly different from baseline (0 min).bSignificant difference in plasma corticosterone between PD2 and PD8 pups (P ⬍ 0.05). n ⫽ 5 or 6. Data are presented as means ⫾ SE.
Adrenal cAMP (pmol/mg)
Time (min)
a
100 80 60 40 b 20
b
b
0 Baseline
Low Dose
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Fig. 5. Adrenal cAMP and plasma corticosterone 5 min after injection of low or moderate doses of ACTH in PD2 and PD8 pups. PD2 and PD8 pups were injected with porcine ACTH at low and moderate doses (1 or 4 g/kg body wt, respectively g/kg body wt, respectively). aSignificantly different from baseline (0 min).bSignificant difference between PD2 and PD8 pups (P ⬍ 0.05). n ⫽ 9. Data are presented as means ⫾ SE.
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study (5, 6, 37, 38, 39). Specifically, we continued the investigation of a phenomenon observed that, prior to PD5, when exposed to hypoxia, the neonatal rat exhibits a large corticosterone response without a drastic increase in immunoreactive plasma ACTH and without a detectable increase in adrenal cAMP content (the canonical second messenger) (23). These previous studies led us to question whether increases in ACTH in the normoxic neonatal rat stimulate corticosterone production via a rise in adrenal cAMP content or via another second messenger pathway. We have demonstrated that the PD2 adrenal has a much larger corticosterone response to ACTH compared with the PD8 pup, confirming what has been demonstrated previously in dispersed adrenal cells in vitro (2) and in the adrenal response to hypoxia in vivo (23). This was not due to changes in plasma corticosterone binding capacity since we have previously demonstrated that PD8 pups have higher corticosterone binding globulin levels compared with PD2 pups (23). The current study demonstrated a large corticosterone response to physiological doses of ACTH in PD2 pups without a measurable increase in adrenal cAMP. We understand that cAMP is the accepted intracellular second messenger for the adrenal response to ACTH in mature rats (17). Chronic ACTH stimulation can exert its steroidogenic effects through a noncAMP mechanism (26). In adult rats, it has been shown that ACTH-stimulated adrenal corticosterone content increases within 3 min without a change in adrenal cAMP content, particularly when the ACTH is injected in the morning (9). In addition, it has been suggested that adrenal cell responses to very low concentrations of ACTH are cAMP-independent (52) or that only very small amounts of cAMP are necessary to stimulate steroidogenesis (32). What, then, is mediating this large increase in ACTHstimulated corticosterone in the PD2 rat pup? There are other potential second messengers, such as calcium and cGMP; however, most studies do not support these as principal second messengers in this system (17, 23). There are other, less potent pathways that ACTH is capable of stimulating, including MAPK, inositol phosphate, and diacylglycerol (17). These other pathways will be considered for further investigation. Non-ACTH mechanisms for controlling steroidogenesis include sympathetic input via the splanchnic nerves (14, 47) and/or local factors, such as vasoactive intestinal peptide and catecholamines (3, 4). However, these non-ACTH-mediated pathways are unlikely to be the cause of increased corticosterone because we injected exogenous ACTH and because the vehicle injection group did not show an increase in corticosterone. It is thought that StAR protein is the principal mediator of cholesterol transport process essential for steroidogenesis (46). A previous study from the same group showed that exogenous ACTH and cAMP each resulted in a U-shaped curve of adrenal responsiveness in dispersed cells (2). In that study, the corticosterone responses to exogenous ACTH, cAMP, and a soluble (non-rate-limited) form of cholesterol were higher in PD1 adrenal cells compared with PD10, and responses increased again in cells from adult rats. It was concluded that it was unlikely that cholesterol transport served as a major contributing factor in hyporesponsiveness in neonatal adrenal cells (2) and, therefore, is unlikely to explain the results of our study.
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At first, we analyzed adrenal cAMP content at 15 min after injection of ACTH because previous studies have shown that the maximal effect of ACTH on cAMP production occurs after 15 min and returns to basal levels after ⬃40 min (17). Since, there is a possibility that very small increases in adrenal cAMP content in PD2 adrenals may have occurred earlier than 15 min (9), we repeated the low and moderate dose in PD2 and PD8 pups and sampled at 5 min. The 5-min results with the moderate dose of ACTH essentially validated the 15-min data shown in Figs. 1 and 2, as there was a corticosterone response in PD2 pups without a significant change in adrenal cAMP, whereas there was an adrenal cAMP response in PD8 pups without a change in plasma corticosterone. This confirms that the PD2 rat adrenal appears to have a non-cAMP-mediated increase in steroidogenesis, whereas steroidogenesis in the PD8 rat adrenal appears to be relatively insensitive to increases in cAMP stimulated by ACTH. It is also possible that very small increases in cAMP were generated, but only in the appropriate compartment to stimulate steroidogenesis (42). Our method measured total cAMP content, so a small change in a specific intracellular compartment may not have been detected. The possibility of cAMP independence is even more significant considering that the corticosterone response to a very low dose of ACTH did not correlate with an increase in adrenal cAMP content in PD8 pups (Fig. 4). Therefore, we conclude that either the PD2 adrenal does not require an increase in cAMP in the response to ACTH, or that it is ultrasensitive to very small, compartmental changes in adrenal cAMP content. Previous in vitro studies, however, do not suggest ultrasensitivity to cAMP in dispersed adrenal cells from PD1 compared with PD10 rat pups (2). On the basis of these findings, we still infer that another second messenger or intracellular mechanism may be involved in the large corticosterone responses to ACTH injection in the neonatal rat. Perspectives and Significance Our study has revealed a unique, but as yet, unidentified intracellular mechanism linking ACTH stimulation to an augmented increase in corticosterone production in the neonatal rat. This potentially novel phenomenon may have significant clinical implications. In particular, it is now established that transient, relative adrenal insufficiency is a significant clinical entity in the premature and low-birth weight human infant and may be associated with hypotension, respiratory failure, and increased morbidity (15, 20, 21, 34, 35, 43, 50). Furthermore, an altered adrenocortical response to physiological and pharmacological doses of exogenous ACTH is useful in the evaluation of adrenal function in the premature and low-birth weight infant (1, 13, 20, 22, 24, 43, 44). ACTH stimulation is an important diagnostic test of human adrenal function, the results of which can influence the implementation of glucocorticoid therapy (35, 44). It is important to evaluate the mechanism of ACTH action on the adrenal cortex using the rat model of human prematurity. An increased understanding of these mechanisms could lead to improved diagnostic and treatment strategies. The current study has provided evidence that ACTH may stimulate corticosterone production through an unidentified second messenger system in the very young neonatal rat. Future studies investigating the identity of this unknown intra-
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cellular mechanism will be helpful in gaining a full understanding of the development of adrenal function in neonates. ACKNOWLEDGMENTS The authors thank Kathan Chintamaneni and Barbara Jankowski for assistance in the laboratory. GRANTS J. Bodager is a participant in the Medical College of Wisconsin Medical Student Research Pathways program and received funding from the Dr. Michael J. Dunn Summer Research Fellowship Award. This study was supported, in part, by the Aurora Research Institute. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Author contributions: J.R.B., T.G.G., E.D.B., A.G., and H.R. conception and design of research; J.R.B., T.G.G., E.D.B., and A.G. performed experiments; J.R.B., T.G.G., E.D.B., and H.R. analyzed data; J.R.B., T.G.G., E.D.B., A.G., and H.R. interpreted results of experiments; J.R.B., T.G.G., E.D.B., A.G., and H.R. prepared figures; J.R.B. drafted manuscript; J.R.B., T.G.G., E.D.B., A.G., and H.R. edited and revised manuscript; J.R.B., T.G.G., E.D.B., A.G., and H.R. approved final version of manuscript. REFERENCES 1. Agwu JC, Bissenden J. The low-dose synacthen test: experience in well preterm infants. J Pediatr Endocrinol Metab 17: 1607–1612, 2004. 2. Arai M, Widmaier EP. Steroidogenesis in isolated adrenocortical cells during development in rats. Mol Cell Endocrinol 92: 91–97, 1993. 3. Bodnar M, Sarrieau A, Deschepper CF, Walker CD. Adrenal vasoactive intestinal peptide participates in neonatal corticosteroid production in the rat. Am J Physiol Regul Integr Comp Physiol 273: R1163–R1172, 1997. 4. Bornstein SR, EngelandWC, Ehrhart-Bornstein M, Herman JP. Dissociation of ACTH and glucocorticoids. Trends Endocrinol Metab 19: 175–180, 2008. 5. Bruder ED, Kamer KJ, Guenther MA, Raff H. Adrenocorticotropic hormone and corticosterone responses to acute hypoxia in the neonatal rat: effects of body temperature maintenance. Am J Physiol Regul Integr Comp Physiol 300: R708 –R715, 2011. 6. Bruder ED, Taylor JK, Kamer KJ, Raff H. Development of the ACTH and corticosterone response to acute hypoxia in the neonatal rat. Am J Physiol Regul Integr Comp Physiol 295: R1195–R1203, 2008. 7. Chintamaneni K, Bruder ED, Raff H. Effects of age on ACTH, corticosterone, glucose, insulin, and mRNA levels during intermittent hypoxia in the neonatal rat. Am J Physiol Regul Integr Comp Physiol 304: R782–R789, 2013. 8. Chintamaneni K, Bruder ED, Raff H. Programming of the hypothalamic-pituitary-adrenal axis by neonatal intermittent hypoxia: effects on adult male ACTH and corticosterone responses are stress-specific. Endocrinology 155: 1763–1770, 2014. 9. Dallman MF, Engeland WC, Rose JC, Wilkinson CW, Shinsako J, Siedenburg F. Nycthemeral rhythm in adrenal responsiveness to ACTH. Am J Physiol Regul Integr Comp Physiol 235: R210 –R219, 1978. 10. Davis DJ, Creery WD, Radziuk J. Inappropriately high plasma insulin levels in suspected perinatal asphyxia. Acta Paediatr 88: 76 –81, 1999. 11. Denenberg VH, Bell RW. Critical periods for the effects of infantile experience on adult learning. Science 131: 227–228, 1960. 12. Deruelle P, Houfflin-Debarge V, Magnenant E, Jaillard S, Riou Y, Puech F, Storme L. Effects of antenatal glucocorticoids on pulmonary vascular reactivity in the ovine fetus. Am J Obstet Gynecol 189: 208 –215, 2003. 13. El-Khuffash A, McNamara PJ, Lapointe A, Jain A. Adrenal function in preterm infants undergoing patent ductus arteriosus ligation. Neonatology 104: 28 –33, 2013. 14. Engeland WC. Functional innervation of the adrenal cortex by the splanchnic nerve. Horm Metab Res 30: 311–314, 1998. 15. Fernandez EF, Watterberg KL. Relative adrenal insufficiency in the preterm and term infant. J Perinatol 29: S44 –S40, 2009.
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