Effect of a melanocortin type 2 receptor (MC2R) antagonist on the corticosterone response to hypoxia and ACTH stimulation in the neonatal rat

Abstract

The adrenal stress response in the neonatal rat shifts from ACTH-independent to ACTH-dependent between postnatal days 2 (PD2) and 8 (PD8). This may be due to an increase in an endogenous, bioactive, nonimmunoreactive ligand to the melanocortin type 2 receptor (MC2R). GPS1574 is a newly described MC2R antagonist that we have shown to be effective in vitro. Further experimentation with GPS1574 would allow better insight into this seemingly ACTH-independent steroidogenic response in neonates. We evaluated the acute corticosterone response to hypoxia or ACTH injection following pretreatment with GPS1574 (32 mg/kg) or vehicle for GPS1574 in PD2, PD8, and PD15 rat pups. Pretreatment with GPS1574 decreased baseline corticosterone in PD2 pups but increased baseline corticosterone in PD8 and PD15 pups. GPS1574 did not attenuate the corticosterone response to hypoxia in PD2 pups and augmented the corticosterone response in PD8 and PD15 pups. GPS1574 augmented the corticosterone response to ACTH in PD2 and PD15 pups but had no significant impact on the response in PD8 pups. Baseline adrenal Mrap and Star mRNA increased from PD2 to PD15, whereas Mrap2 mRNA expression was low and did not change with age. The data suggest that GPS1574 is not a pure MC2R antagonist, but rather acts as a biasing agonist/antagonist. Its ability to attenuate or augment the adrenal response may depend on the ambient plasma ACTH concentration and/or developmental changes in early transduction steroidogenic pathway genes.

INTRODUCTION

The perinatal development of the hypothalamic-pituitary-adrenal (HPA) axis plays a key role in lung maturation, closure of the ductus arteriosus, and potentiating the response to vasoconstriction (11, 19, 30). Because of lung immaturity, premature neonates often suffer from acute episodes of hypoxia, which can have long-term endocrine and metabolic consequences (1, 9, 10, 16, 29) As the rate and global burden of premature births continue to rise (12), it becomes progressively more important to understand the mechanisms of steroidogenesis in the adrenal gland of premature and full-term neonates. The neonatal rat pup is a useful model for premature human neonates and has been used to study the development of the HPA axis (21, 22, 24, 26).

The primary glucocorticoid in the rat is corticosterone, which is produced in the zona fasciculata of the adrenal cortex. Corticosterone synthesis is stimulated by ACTH, which binds the melanocortin type 2 receptor (MC2R), causing an increase in intracellular cAMP (13, 14, 22, 23). The increase in intracellular cAMP activates protein kinase A (PKA) and subsequently the steroidogenic acute regulatory (StAR) protein. The StAR protein regulates the rate-limiting step of steroidogenesis and functions to increases the movement of free cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane (28).

In previous studies, we have shown that the postnatal day 2 (PD2) rat pup exposed to acute hypoxia demonstrates a dramatic increase in corticosterone production that appears to be independent of significant increases both plasma ACTH and intracellular adrenal cAMP (6, 17). PD8 pups also generate an increase in corticosterone in response to hypoxia, but it is associated with a classic increase in immunoassayable ACTH and adrenal cAMP (4, 6, 8). The mechanism of corticosterone production in the PD2 rat pup is of much interest, as it appears to be independent of significant increases in ACTH and cAMP. It is possible that there is a bioactive form of ACTH in the PD2 pup that is undetected via radioimmunoassay. Alternatively, a posttranslational product of the proopiomelanocortin (POMC) gene other than ACTH-(1–39) could be responsible for this phenomenon (18). It is important to further explore the mechanisms of corticosterone production in the neonatal rat pup because of its usefulness as a model for human prematurity (24, 26).

If the corticosterone response to hypoxia in the PD2 rat pup can be blocked by antagonism of the MC2R receptor, this would suggest that there is an ACTH-like ligand activating the MC2R receptor. If antagonizing the receptor does not attenuate corticosterone production, then the mechanism is most likely independent of the MC2R, unless the antagonist is ineffective. GPS1574 is a newly described MC2R antagonist (3). We have previously shown that GPS1574 attenuates the corticosterone response to ACTH in PD2, PD8, and adult adrenal cells in vitro (20). A similar compound, GPS1573, was shown to attenuate the response in vitro even more (20). However, when these two compounds were used in vivo (given as ip injections at 4 and 8 mg/kg body wt), the corticosterone response to ACTH injection was attenuated by GPS1574, but surprisingly enhanced by GPS1573 (20). This difference is thought to be due to the ring-structure of GPS1574 [Nle- (E-f-R-w-F-K)-A-V-G-K-K-R-R NH₂)], which GPS1573 lacks (3). Because of these findings, we decided to further investigate the effect of GPS1574 in vivo.

The purpose of the current study is to explore the effect of pretreatment with GPS1574 on the corticosterone response to ACTH injection or hypoxia in neonatal rat pups at PD2, PD8, and PD15. We also evaluated developmental changes in baseline expression of early transduction steroidogenic pathway gene mRNAs to correlate with the adrenal responses. We hypothesize that GPS1574 will attenuate the corticosterone response to hypoxia in the PD8 and PD15 rat pups but will not attenuate this response in PD2 pups, due to the immaturity of their adrenal function. Furthermore, we hypothesize that GPS1574 will attenuate the response to ACTH injection in all pups.

MATERIALS AND METHODS

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 14–17 (n = 22) were acquired from Harlan Sprague Dawley (Indianapolis, IN), housed in a controlled environment (0600–1800 lights on), and maintained on a standard diet with water available ad libitum. Dams delivered and cared for pups without interference until experimentation. The MC2R antagonist GPS1574 was synthesized by Genepep (St. Jean de Védas, France) and reconstituted as previously described (3).

GPS1574 studies in the neonatal rat in vivo.

Pups were studied on the mornings of postnatal days 2, 8, and 15 (PD2, PD8, and PD15). Immediately before experimentation, the pups were removed from their dams and placed into a chamber with adequate bedding and a variable control heating pad (Moore Medical, Farmington, CT) set at the lowest setting required to maintain body temperature as described previously (15). The chamber permitted free range of motion and room to huddle. For dosing purposes, an average pup weight was calculated by calculating the mean weight of three sentinel pups from each experimental group.

PD2, PD8, and PD15 pups were randomly divided into eight groups: Vehicle for GPS1574 pretreatment followed by baseline, vehicle for ACTH injection, ACTH injection, or hypoxia; or GPS1574 pretreatment followed by baseline, vehicle for ACTH injection, ACTH injection, or hypoxia. Pups were given intraperitoneal injections of either GPS1574 (32 mg/kg body wt, diluted in isotonic saline) or vehicle for GPS1574 (10 μl/kg body wt of isotonic saline), then placed back into their respective chambers (21% O_2; room air). Sixty minutes after GPS1574 or vehicle for GPS1574 injection, one group of GPS1574 pups and one group of vehicle for GPS1574 pups were euthanized and trunk blood was collected (baseline, time 0). At this time (0 min), the remaining six subsets of pups for each age were subjected to one of the following treatments: ACTH injection [1 μg/kg body wt ip; Sigma porcine ACTH as described in (2)], vehicle for ACTH injection (10 μl/kg body wt isotonic saline ip), or hypoxic exposure [chamber vented with 8% O₂ as described in Ref. 4]. Thirty minutes following these treatments, pups were euthanized and trunk blood was collected.

Blood and tissue collection.

All experimental groups were euthanized via decapitation, and trunk blood was collected in EDTA tubes (2 pups/sample for PD2 and 1 pup/sample for PD8 and PD15 pups), processed to plasma, and frozen at −20°C. Adrenal glands were removed and frozen in liquid nitrogen for real-time PCR mRNA expression analysis.

Plasma hormone assays, adrenal RNA isolation and real-time PCR (qPCR), and statistical analyses.

Radioimmunoassay was utilized to measure plasma ACTH and corticosterone as previously described (MP Biomedicals, Orangeburg, NJ) (22). Because GPS1574 cross-reacts in the plasma ACTH assay, we only report plasma ACTH results from animals receiving the vehicle for GPS1574.

Total RNA was isolated from previously flash-frozen adrenal glands as previously described (6, 8). RNA concentrations were determined by spectrophotometry using a Nanodrop 2000 (ThermoFisher Scientific, Grand Island, NY), with an acceptable 260/280 range of 1.9–2.1. One-hundred fifty nanograms of isolated RNA was synthesized into cDNA using the High-Capacity RNA-to-cDNA Reverse Transcription kit with Multiscribe enzyme (ThermoFisher) following the manufacturer’s instructions. All cDNA was diluted 1:10 in nuclease-free water before real-time PCR analysis. The following genes were analyzed using the TaqMan gene expression assay method (ThermoFisher): Mc2r (Rn02082290_s1), Mrap (Rn01477212_m1), Mrap2 (Rn01445736_m1), and Star (Rn00580695_m1). Real-time PCR was performed using Roche LightCycler Probes Master (Roche, Indianapolis, IN) and TaqMan gene expression assays (FAM fluorophore). The final reaction volume of 20 µl consisted of 1X Probes Master Mix, 1X TaqMan primer/probe, and 5 µl of previously diluted cDNA. Real-time PCR was performed using the Roche LightCycler 480 II real-time PCR machine using the following thermal cycler conditions: 95°C for 10 min followed by 45 cycles at 95°C for 10 s, 60°C for 30 s, and 72°C for 1 s. Samples were assayed in triplicate. Nuclease-free water and randomly selected RNA isolations (instead of diluted cDNA) were used as negative controls. Rpl19 (Rn00821265_g1) was run as reference gene for each cDNA sample.

Data were analyzed by two-way ANOVA. Post hoc analysis was performed by Holm-Sidak multiple-range test (P < 0.05) (SigmaPlot 11.0). Data are presented as means ± SE.

RESULTS

ACTH injections.

Figure 1 shows the plasma corticosterone concentrations at baseline and after vehicle for ACTH or ACTH injection in PD2, PD8, and PD15 pups. In PD2 pups, pretreatment with GPS1574 resulted in a significant decrease in baseline plasma corticosterone (Fig. 1, top). Administration of vehicle for ACTH in PD2 pups did not alter plasma corticosterone. Administration of ACTH to pups pretreated with vehicle for GPS1574 resulted in a significant increase in plasma corticosterone; this response to ACTH was significantly augmented by pretreatment with GPS1574. In the pups pretreated with vehicle for GPS1574, plasma ACTH was unchanged after administration of vehicle for ACTH but increased significantly with ACTH administration (Table 1, row 1).

Fig. 1.

Plasma corticosterone levels at baseline and in response to ACTH or vehicle for ACTH injection in postnatal day 2 (PD2) (top), PD8 (middle), and PD15 rats (bottom) pretreated with either GPS1574 (GPS injection) or vehicle for GPS1574 (GPS vehicle) injection. ^aSignificantly different from vehicle for GPS1574. ^bSignificantly different from baseline (0 min) within GPS1574 treatment group. ^cSignificantly different from vehicle for ACTH within GPS1574 treatment group. ^dSignificantly different from vehicle for GPS1574 by t-test. ^eSignificantly different from baseline (0 min) by t-test. N = 5–14 pups per group for PD2; N = 8–15 pups per group for PD8; N = 6–15 pups per group for PD15.

Table 1.

Plasma ACTH (pg/ml) concentrations for pups receiving vehicle for GPS1574 by experimental group

		Experiment
Age	Baseline	ACTH Vehicle	ACTH Stim	Hypoxia
PD2	51.2 ± 2.7	69.3 ± 1.1	338.0 ± 25.5a	76.2 ± 2.5
PD8	97.1 ± 14.7	195.0 ± 33.3	462.8 ± 90.3a	311.4 ± 15.2a,b
PD15	147.9 ± 26.8	223.6 ± 32.5	465.2 ± 98.2a	555.0 ± 28.7a,b,c

Values are means ± SE. For N values, see the legends for Figs. 1 and 2. PD2, PD8, and PD15 are postnatal days 2, 8, and 15, respectively.

^aDifferent from baseline in same row;

^bdifferent from PD2 in same column;

^cdifferent from PD8 in same column.

In PD8 pups (Fig. 1, middle) the range of the y-axis (plasma corticosterone) is much less than in PD2 pups. In PD8 pups, pretreatment with GPS1574 resulted in a significant increase in baseline plasma corticosterone (by t-test). Administration of vehicle for ACTH in PD8 pups pretreated with vehicle for GPS1574 resulted in a significant increase in plasma corticosterone. This effect was augmented by pretreatment with GPS1574. Administration of ACTH in pups pretreated with vehicle for GPS1574 or GPS1574 displayed a significant increase in plasma corticosterone compared with baseline; this was not significantly different from administration of vehicle for ACTH. Although plasma ACTH after administration of vehicle for ACTH in pups pretreated with vehicle for GPS1574 was not significantly increased, there was a tendency for an increase (Table 1, row 2). Administration of ACTH to these pups resulted in a large, significant increase in plasma ACTH.

In PD15 pups, pretreatment with GPS1574 resulted in a significant increase in plasma corticosterone that was statistically similar in baseline, vehicle for ACTH, and ACTH injection groups (Fig. 1, bottom). In pups pretreated with vehicle for GPS1574, plasma corticosterone was not significantly increased after vehicle for ACTH but was increased after ACTH injection (but only by t-test). Although plasma ACTH after administration of vehicle for ACTH in pups pretreated with vehicle for GPS1574 was not significantly increased, there was a tendency for an increase (Table 1, row 3). Administration of ACTH to these pups resulted in a large, significant increase in plasma ACTH.

Hypoxia.

The baseline corticosterone data in Fig. 2 are repeated from Fig. 1 for comparison to the responses to hypoxia. In PD2 pups (Fig. 2, top), there was a statistically significant plasma corticosterone response to hypoxia the magnitude of which was not different between vehicle for GPS1574 and GPS1574 pretreatment. This increase in plasma corticosterone during hypoxia occurred despite no statistically significantly change in plasma ACTH (Table 1, row 1). In PD8 pups (Fig. 2, middle), hypoxia resulted in a significant increase in plasma corticosterone in the vehicle-for-GPS1574 pretreated pups; this response was augmented by pretreatment with GPS1574. Hypoxia results in a significant increase in plasma ACTH compared with baseline (Table 1, row 2). In PD15 pups pretreated with vehicle for GPS1574, plasma ACTH (Table 1, row 3) and plasma corticosterone (Fig. 2, bottom) increased significantly during exposure to hypoxia. Because of the higher baseline plasma corticosterone in pups pretreated with GPS1574, hypoxia did not result in a statistically significant increase in plasma corticosterone by the multiple-range test, but it was significant by t-test.

Fig. 2.

Plasma corticosterone levels at baseline and in response to 30 min of hypoxia (8% O₂) in postnatal day 2 (PD2) (top), PD8 (middle), and PD15 rats (bottom) pretreated with either GPS1574 (GPS injection) or vehicle for GPS1574 (GPS vehicle) injection. Baseline data are repeated from Fig. 1 for comparison. ^aSignificantly different from vehicle for GPS1574. ^bSignificantly different from baseline (0 min) within GPS1574 treatment group. ^eSignificantly different from baseline (0 min) by t-test. N = 8 pups per group for PD2; N = 8–9 pups per group for PD8; N = 6–7 pups per group for PD15.

Expression of early transduction steroidogenic pathway genes.

Table 2 shows the baseline mRNA data at different ages for critical early transduction pathway genes evaluated in adrenal glands removed from pups 1 h after injection of GPS1574 or its vehicle. Mc2r (ACTH receptor) mRNA did not change between PD2 and PD8 but increased (decreased C_t) between PD8 and P15. Mrap (melanocortin 2 receptor accessory protein) and Star (steroidogenic acute regulatory protein) showed increases in mRNA between PD2 and PD8 as well as between PD8 and PD15. The expression of Mrap2 (melanocortin 2 receptor accessory protein 2) mRNA was very low (high C_t) and did not change over time. It is important to note that Rpl19 mRNA expression (reference gene) did increase slightly in the adrenal glands from vehicle-treated PD15 pups so the associated data should be interpreted with caution. Treatment with GPS1574 (compared with vehicle for GPS1574) had no effect within gene at each age.

Table 2.

mRNA by qPCR (cycles to threshold; C_t) at baseline (60 min after GPS1574 or vehicle for GPS1574) for critical early transduction steroidogenic pathway genes

		GPS1574 (Given 60 min Before)
Age	Gene	32 mg/kg	Vehicle
PD2	Mc2r	29.11 ± 0.34	29.15 ± 0.22
	Mrap	26.85 ± 0.25	26.68 ± 0.07
	Mrap2	35.22 ± 0.23	35.16 ± 0.34
	Star	25.49 ± 0.40	24.91 ± 0.11
	Rpl19	24.48 ± 0.06	24.54 ± 0.10
PD8	Mc2r	28.96 ± 0.06	29.13 ± 0.12
	Mrap	25.86 ± 0.14*	26.14 ± 0.26
	Mrap2	34.75 ± 0.18	35.41 ± 0.18
	Star	23.27 ± 0.18*	23.39 ± 0.26*
	Rpl19	24.31 ± 0.07	24.59 ± 0.09
PD15	Mc2r	28.15 ± 0.12*	27.97 ± 0.35*
	Mrap	24.08 ± 0.21*	24.41 ± 0.20*
	Mrap2	34.90 ± 0.14	35.08 ± 0.23
	Star	21.82 ± 0.22*	22.00 ± 0.22*
	Rpl19	23.98 ± 0.07	23.76 ± 0.24*

Values are means ± SE; N = 4–5. A decrease in C_t indicates an increase in mRNA. Mc2r, Melanocortin 2 receptor (ACTH receptor); Mrap,- melanocortin 2 receptor accessory protein; Mrap2,- melanocortin 2 receptor accessory protein 2; Star, steroidogenic acute regulatory protein; Rpl19, ribosomal protein L19 (reference gene). PD2, PD8, and PD15 are postnatal days 2, 8, and 15, respectively.

^*Specific gene mRNA expression different from next younger age within column (P < 0.05).

There were no differences between columns within genes.

DISCUSSION

We have previously shown that PD2 rat pups exhibit a large corticosterone response to hypoxia without a significant increase in plasma ACTH measured by immunoassay. This effect requires an intact, perfused adrenal gland in vivo since the ACTH sensitivity of adrenal cells in vitro is not increased by physiological decreases in oxygen concentration (5). Our aim in the current study was to determine if this response could be mediated through the MC2R by using a new MC2R receptor antagonist, GPS1574, which has been shown to be potent in vitro (3, 20). In the current study, we confirmed the apparently ACTH-independent increase in plasma corticosterone in response to hypoxia in PD2 pups (17). Our main findings were 1) GPS1574 decreased baseline corticosterone in PD2 pups but paradoxically increased baseline plasma corticosterone in PD8 and PD15 pups, 2) pretreatment with GPS1574 in PD2 pups surprisingly augmented the plasma corticosterone response to ACTH injection, 3) pretreatment with GPS1574 did not attenuate the plasma corticosterone response to hypoxia, and 4) expression of critical early transduction steroidogenic pathway genes (Mrap and Star) increased markedly between PD2 and PD15.

We demonstrated that GPS1574 decreased baseline corticosterone in PD2 pups but increased baseline corticosterone in PD8 and PD15 pups. What could explain this difference? It could be that GPS1574 acts as a biasing agonist in vivo, in which its MC2R agonistic versus antagonistic activity depends on the ambient plasma ACTH concentration or adrenal development (25). Our baseline data show that GPS1574 acted like an agonist in the presence of higher plasma ACTH, as in PD8 and PD15 pups. However, when plasma ACTH was lower, as in PD2 pups, it acted like an antagonist.

The mechanism for GPS1574 acting as a biasing agonist is currently unknown; however, studies have shown that the affinity of ACTH for MC2R is regulated by two accessory proteins, MRAP and MRAP2 (25). MRAP promotes ACTH binding and signal transduction. MRAP2 may act in a dominant-negative fashion, altering ACTH binding and signal transduction depending on the conditions present (25). It is possible that these regulatory mechanisms are influenced by changes in plasma ACTH concentration or by the presence of GPS1574. This could potentially lead to an inhibition of adrenal production of corticosterone in some conditions (e.g., baseline in PD2 pups) and an augmentation in others (e.g., after ACTH injection in PD2 pups). We did find substantial age-related increases in Mrap mRNA that may account for the paradoxical effect of GPS1574 we observed. Mrap2 mRNA expression was very low and did not change with age, suggesting it is probably not involved in the phenomena we describe. However, StAR protein gene expression did increase with age. Since StAR protein is involved in early steroidogenesis (13, 14, 28), it is possible it was somehow involved in the paradoxical effect we observed, although the mechanism of such an effect is difficult to hypothesize at this point. It is clear that the changes in mRNA expression in these critical pathways did not explain the lack of effectiveness of GPS1574 as an MC2R antagonist at high concentrations of ACTH nor does it reveal the mechanism of the apparently ACTH-independent corticosterone response to hypoxia in PD2 rats.

Could the divergent effects of GPS1574 be explained by a difference in the MC2R expression profile among neonates of various ages? We did not detect an increase in Mc2r mRNA between PD2 and PD8, which is consistent with our previous studies (6, 8) and suggests that this is probably not the mechanism. There was a small increase in Mc2r mRNA expression between PD8 and PD15, although these data should be interpreted with caution since Rpl19 mRNA (reference gene) also slightly increased. It is known that the MC2R is essential for adrenal gland development and steroidogenesis. A study of MC2R-knockout mice showed that most pups with MC2R gene knockout died shortly after birth (7). Furthermore, the few pups that survived had severe adrenal hypoplasia, particularly in the zona fasciculata, and undetectable levels of corticosterone with a compensatory elevation in ACTH. Our data are a more subtle evaluation of MC2R in the transduction of ACTH stimulation of early pathway steroidogenesis, so correlation with a knockout model is challenging.

It is also possible that GPS1574 could affect the physiology of the HPA axis via a CNS-mediated process. The synthetic ACTH-like peptide, ebiratide, can cross the blood-brain barrier (BBB) in its intact form in rats and therefore was developed as a potential treatment for Alzheimer’s disease (27). Ebiratide is a six-amino acid peptide with the following structure: [H-Met-(O₂)-Glu-His-Phe-D-Lys-Phe-NH(CH₂)₈-NH₂]. Like ebiratide, GPS1574 is a small peptide that shares homology with ACTH; therefore, it is possible that GPS1574 crosses the BBB which may have an (undetermined) impact on our findings.

GPS1574 significantly augmented the corticosterone response to ACTH injection in PD2 and PD15 pups. As described earlier, this augmentation may be due to the tendency for GPS1574 to act like an MC2R agonist in the presence of higher plasma ACTH concentrations. The most likely explanation for the lack of augmentation in PD8 pups is that these pups tend to be hyporesponsive to ACTH injection, as we have previously shown (2). It is likely that the PD8 pups had already maximized their corticosterone production in the absence of GPS1574. When comparing vehicle for ACTH with ACTH injection in PD8 pups, one can reasonably infer that the stress of the second injection was enough of a stimulus to maximize the corticosterone response, regardless of which injection the pups received.

We have previously shown that PD2 pups subjected to hypoxia have a significant increase in plasma corticosterone compared with baseline without a significant increase in ACTH (17). Because this response appears to be ACTH independent, we hypothesized that GPS1574 would not attenuate the corticosterone response to hypoxia in PD2 pups. Our data confirmed a lack of attenuation. Furthermore, we hypothesized that GPS1574 would attenuate the corticosterone response to hypoxia in PD8 and PD15 pups. Our results indicate that GPS1574 did not attenuate the response in PD15, and actually augmented the corticosterone response to hypoxia in PD8 pups. Once again, a possible explanation for this finding is that the high levels of ACTH in PD8 (311 ± 15.2 pg/ml) and PD15 (555.0 ± 28.7 pg/ml) pups caused GPS1574 to act more like an agonist rather than an antagonist.

One potential drawback of this study is the inability to accurately measure ACTH in pups pretreated with GPS1574. As stated earlier, GPS1574 cross-reacts in the radioimmunoassay for ACTH, leading to unreliable ACTH measurements in these pups. Obtaining these ACTH values would allow for a more thorough analysis of the corticosterone response. Another potential drawback is the incomplete understanding of the pharmacological properties of GPS1574. Other than its amino acid sequence, little is known about the behavior of this peptide in vivo such as whether it interacts with serum proteases or alters processing of other [pro]hormones. It is possible that this drug could alter the metabolism (i.e., half-life) or absorption of ACTH. We have previously utilized intraperitoneal injection for GPS1574 due to its structural homology with ACTH (20); however, it remains to be completely understood if GPS1574 is absorbed well through intraperitoneal injections. The fact that plasma ACTH measurements were confounded after GPS1574 injection suggests the compound was absorbed into the blood. One final drawback is that PD8 pups, as explained above, demonstrate a stress response to a second injection regardless of whether the injection is vehicle for ACTH or ACTH itself. It is possible that the maximal stress response to the second injection in PD8 pups confounds the interpretation of the corticosterone data.

In conclusion, it appears that GPS1574 is not a useful MC2R antagonist in vivo, despite its effectiveness in vitro (20). In vivo, it tends to act as an agonist in the presence of high ACTH concentrations and an antagonist at low ACTH concentrations. The mechanism of this finding remains to be understood but is likely related to the activity of StAR protein and the aforementioned MC2R accessory protein, MRAP, but probably not MRAP2 (25).

Perspectives and Significance

Our data support an ACTH-independent corticosterone response to hypoxia in PD2 pups that may be relevant to adaptation in the newborn human (19, 30). However, it remains possible that PD2 pups utilize a form of ACTH not detected by our immunoassay. Despite this notion, our findings indicate that PD2 pups have a strong corticosterone response to ACTH injection required for normal adaptation to extrauterine life (11). The increase in expression of the Mc2r, Mrap, and Star genes during neonatal life is a significant observation considering the importance of steroidogenesis in survival during the perinatal period. These novel observations further highlight the usefulness of our rat model of human prematurity (21, 22, 24, 26). An important point is the critical nature of performing preclinical studies in the appropriate animal model since small peptide receptor antagonists that are potent in vitro clearly have unpredictable effects in vivo.

GRANTS

This study was funded, in part, by the Aurora Research Institute, the Aurora Metro Staff Summer Research Fellowship, and by National Heart, Lung, and Blood Institute Training Grant T35-HL-072483(25).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.J.G., A.L.G., and H.R. conceived and designed research; A.J.G., A.L.G., E.W., M.J., B.H., and H.R. performed experiments; A.J.G., A.L.G., E.W., M.J., B.H., and H.R. analyzed data; A.J.G., A.L.G., E.W., M.J., B.H., and H.R. interpreted results of experiments; A.J.G. and H.R. prepared figures; A.J.G. and H.R. drafted manuscript; A.J.G. and H.R. edited and revised manuscript; A.J.G., A.L.G., E.W., M.J., B.H., and H.R. approved final version of manuscript.