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Endocrinology Vol. 140, No. 1 71-78
Copyright © 1999 by The Endocrine Society

ARTICLES

Urocortin Is Not a Significant Regulator of Intermittent Electrofootshock-Induced Adrenocorticotropin Secretion in the Intact Male Rat1

Andrew V. Turnbull2, Joan Vaughan, Jean E. Rivier, Wylie W. Vale and Catherine Rivier

The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037

Address all correspondence and requests for reprints to: C. Rivier, The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: catherine_rivier{at}qm.salk.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Urocortin (Ucn) is a newly identified mammalian member of the CRF family of peptides. Ucn activates CRF receptors (both CRF-R1 and CRF-R2) with greater potency than CRF itself, suggesting that Ucn may play an endogenous role in eliciting (at least some) CRF receptor-mediated events. Because the most characterized physiological function of CRF receptors is the activation of pituitary ACTH secretion, we have compared the effects and potential endogenous roles of CRF and Ucn in regulating plasma ACTH concentrations in intact male rats. Synthetic rat Ucn injected iv (0.09–9.0 nmol/kg) elicited ACTH secretion in a dose-dependent manner, causing greater ACTH secretion than CRF at each dose tested. The increases in plasma ACTH concentrations produced by CRF or Ucn were virtually abolished by pretreatment with the CRF receptor antagonist, astressin (3 mg/kg), and were partially attenuated (by 27–37%) by an anti-arginine vasopressin serum. These data indicate that both Ucn and CRF elicit ACTH secretion via CRF receptor-dependent mechanisms, and that the ACTH-releasing activities of both CRF and Ucn are potentiated by endogenous arginine vasopressin. Intravenous administration of rabbit anti-Ucn serum, which inhibited ACTH secretion produced by Ucn, but not CRF, had no statistically significant effect on either resting (midday) plasma ACTH concentrations or the rise in ACTH levels elicited by 30 min of intermittent electrofootshocks. By contrast, treatment with a rabbit anti-CRF serum that specifically inhibited the ACTH response to CRF lowered plasma concentrations in control unstressed rats and largely prevented the plasma ACTH response to electrofootshocks. These data indicate that although Ucn is a more potent ACTH secretagogue than CRF in the intact male rat, it is not a major endogenous regulator of pituitary ACTH secretion under basal (midday) conditions or during acute footshock stress.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CRF IS A 41-amino acid mammalian neurohormone that is best known as the major physiological regulator of pituitary ACTH secretion and, in addition, stimulates complimentary stress-related endocrine, autonomic, and behavioral responses (1, 2, 3, 4, 5). CRF belongs to a family of structurally related peptides, including amphibian sauvagine and piscine urotensin I (4, 5). The effects of CRF-related peptides in mammalian systems are mediated via interaction with a number of distinct CRF receptors that have distinct pharmacologies and fairly exclusive anatomical distributions (6, 7, 8, 9, 10). CRF receptor type I (CRF-R1) messenger RNA (mRNA) is expressed in the brain and pituitary (6, 11, 12, 13, 14, 15). Within the pituitary CRF-R1 mRNA is colocalized with ACTH immunoreactivity, indicating that CRF-R1 is expressed by corticotropes (6). CRF-R2 has at least two splice variants; in the rat, 2{alpha} mRNA is found only in the brain, whereas 2ß is present in both the brain and peripheral tissues (heart, skeletal muscle), but not the pituitary (7, 8, 9, 16, 17, 18, 19). In addition to interacting with CRF receptors, CRF is bound with high affinity by a binding protein (CRF-BP) that can inhibit the biological activity of CRF (20). In the rat CRF-BP is present in both the brain and pituitary (20, 21, 22), where it may limit the availability of CRF for interaction with its receptors.

Urocortin (Ucn) is a 40-amino acid mammalian peptide originally identified in rat brain via screening a complementary DNA library constructed from mRNA derived from a urotensin I-immunoreactive region, the Edinger-Westphal nucleus (23). Subsequently, human Ucn has also been cloned and characterized (24). Rat Ucn displays 63%, 45%, and 35% amino acid sequence homology with carp urotensin I, rat/human CRF, and frog sauvagine, respectively (23). Ucn mRNA is expressed in discrete regions of the rat brain: predominantly in the Edinger-Westphal nucleus and lateral superior olive, with weaker expression also apparent in certain motor nuclei (facial, hypoglossal, and ambiguual nuclei) and in the lateral hypothalamus and supraoptic nucleus (23). Ucn is also present in a number of peripheral tissues, with Ucn mRNA and immunoreactivity having been detected in the rat and human pituitary (25, 26, 27), human placenta and fetal membranes (28), human lymphocytes (29), and rat cardiac myocytes (30). Urotensin-like immunoreactivity is also present in the rat duodenum (23).

Ucn is a more potent activator of CRF receptors (in particular, CRF-R2) than CRF itself (23, 24). Like CRF receptors, the CRF-BP has greater affinity for Ucn than CRF (23, 24). Ucn has been shown to produce physiological/pharmacological effects that, in general, are similar to those produced by CRF (4, 23, 24, 31, 32, 33, 34, 35, 36, 37), with Ucn commonly being found to be more potent than CRF. Ucn stimulates ACTH secretion from rat anterior pituitary cell cultures with an approximately 7-fold lower EC50 than CRF (23, 24). Similarly, Ucn is a potent ACTH secretagogue in vivo in both the rat (4, 23, 31) and sheep (34). Reports indicating expression of Ucn mRNA or protein in tissues (hypothalamic paraventricular nucleus, anterior pituitary) relevant to the control of pituitary ACTH secretion have suggested that Ucn may be an endogenous ACTH secretagogue (25, 26, 27, 38, 39, 40). The purpose of the present study was, firstly, to compare the effects and mechanisms of Ucn- and CRF-induced ACTH secretion in the rat. Using specific, passive immunoneutralization, we then determined the respective contributions of Ucn and CRF to the regulation of ACTH secretion under resting conditions and in response to acute physical stress (electrofootshock).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male Sprague-Dawley rats (220–240 g) were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN), and housed in animal facilities adjacent to the experimental rooms (ambient temperature, 22 C). They were maintained on a 12-h light, 12-h dark cycle (lights on at 0600 h) and provided rat chow (Harlan-Teklad, Madison, WI) and water ad libitum. All procedures described were approved by The Salk Institute animal use and care committee. Forty-eight hours before experimentation the rats were equipped with indwelling jugular venous catheters (41) for the purposes of blood sampling and drug injection.

Peptides and antisera
Rat Ucn, rat/human CRF, arginine vasopressin (AVP), and the potent CRF receptor antagonist, astressin, were synthesized by solid phase methodologies (42, 43). Each peptide was first dissolved in sterile water, and then diluted to the desired concentration using sterile 0.9% saline containing 0.1% BSA.

CRF, Ucn, and [Arg8]vasopressin antisera were raised in rabbits using methods previously described for CRF (44). Antigens used were as follows: rat/human CRF-(1–41) conjugated to human {alpha}-globulins via bisdiazotized benzidine (anti-CRF, code PBL rC69), rat Ucn-(23–40) conjugated to human {alpha}-globulins via bisdiazotized benzidine (anti-Ucn, code PBL 5833), rat Ucn-(1–40) conjugated to human {alpha}-globulins via gluteraldehyde (anti-Ucn, code PBL 5779), and [Arg8]vasopressin conjugated to bovine thyroglobulins via carbodiimide (anti-AVP, code PBL 5408). All antisera used were high titer, high affinity, and extremely selective for their respective peptide immunogens. Antisera against CRF and urocortin predominantly recognize the divergent mid- to C-terminal portion of these peptides. CRF (PBL rC69) and Ucn (PBL 5779 and PBL 5833) antisera were highly selective for their own category of peptide; cross-reactivity with known related CRF superfamily members, if it exists, is less than 0.01%. Normal rabbit serum (NRS; Colorado Serum Co., Denver, CO) was used as a control injection. The appropriate doses of antisera required to inhibit the effects of their respective peptide immunogens were determined empirically within this study. The total amount of serum administered to rats varied between treatment groups, but serum was always administered in a total volume of 0.4 ml/rat, and dilutions were made with sterile 0.9% saline containing 0.1% BSA.

Sample collection
The day before the experiments the rats were placed in experimental buckets and provided with food and water (41). On the morning of each experiment, indwelling iv catheters were attached to sampling syringes at 0800 h and left undisturbed for 3–4 h before taking a control, basal blood sample (at 1100–1200 h). A maximum of 0.3 ml blood/sample was drawn for up to seven serial samples, each blood sample being replaced with 0.2 ml sterile heparinized saline. This sampling regimen does not produce significant elevations in plasma ACTH concentrations due to the sampling procedure itself (41, 45). Blood samples were collected into chilled tubes containing EDTA as anticoagulant and centrifuged, and the plasma was decanted and stored at -20 C until ACTH assay.

Electrofootshock
The rats were placed in experimental buckets as described above. After collection of a control basal blood sample, animals were injected (iv) with either NRS or antiserum and transferred immediately to the electrofootshock apparatus (dimensions: 30 cm wide x 26 cm deep x 26 cm high). The rats were naive to these chambers and remained conscious and unrestrained during the footshock procedure. One shock of 1-mA amplitude and 1-sec duration was randomly applied in each 30-sec period for a total of 30 min.

Plasma ACTH measurements
Plasma ACTH concentrations were measured using a commercial immunoradiometric assay (Allegro, Nichols Institute Diagnostics, San Juan Capistrano, CA), as described previously (46). In these experiments, within- and between-assay coefficients of variation at 44 pg/ml were 7% and 12%, respectively, and at 360 pg/ml were 3% and 5%, respectively. The detection limit of this assay was 5 pg/ml on all occasions, and plasma ACTH concentrations that were undetectable were assigned this value for the purposes of statistical analysis.

Data presentation and statistical analyses
The majority of data are presented as the mean ± SEM, and the numbers of subjects in each experimental group are indicated on either the figures themselves or in the figure legends. Statistical analyses of these data were performed using unpaired Student’s t test, one-way ANOVA (followed by Dunnett’s multiple comparison test), or two-way ANOVA, as appropriate, and were performed on either 1) absolute plasma ACTH concentrations for single time point studies or 2) integrated plasma ACTH levels over time (area under the curve) for multiple time point studies. In one series of experiments, basal plasma ACTH concentrations were statistically compared, and in some of these groups a substantial number of animals (>20%) had ACTH levels below the detection limit of the ACTH assay. Consequently, these were additionally statistically analysed using a nonparametric test (Mann-Whitney U test). In all analyses a two-tailed probability of less than 5% (i.e. P < 0.05) was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Comparison of the temporal profiles of plasma ACTH concentrations after treatment with either CRF or Ucn
Plasma ACTH concentrations immediately before treatment were low (group means, 7–15 pg/ml) and did not differ significantly between subsequent treatment groups. Injection of the vehicle (0.9% saline-0.1% BSA) at 1200 h produced a small elevation at 10 min after treatment (39 ± 8 pg/ml), but levels returned toward preinjection values by 60 min (16 ± 6 pg/ml; see Fig. 1Go). Plasma ACTH concentrations in vehicle-treated rats began to rise again at 3 h and reached 42 ± 9 pg/ml at 4 h, which is consistent with the normal elevations that we routinely observe at this time of day (2 h before lights out) (45).



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  		Figure 1. The effect of either CRF or Ucn (0.09–9 nmol/kg, iv) on plasma ACTH concentrations in intact male rats. Values are the mean ± SEM of five or six animals per group. The data presented were obtained from a single experiment, but in the interests of clarity the effects of three different doses are represented in three separate graphs. The vehicle-treated group from this experiment is presented on each graph to permit comparison to control animals. Statistical analysis (two-way ANOVA) of the integrated plasma ACTH levels (picograms per h/ml) over the 240-min test period indicated significant dose (P < 0.001) and peptide (P < 0.001) effects (0.09 nmol/kg: CRF, 132 ± 10; Ucn, 243 ± 34; 0.9 nmol/kg: CRF, 483 ± 123; Ucn, 914 ± 156; 9 nmol/kg: CRF, 899 ± 83; Ucn, 2127 ± 336).

 
CRF injected iv (0.09–9.0 nmol/kg) produced a dose-dependent increase in plasma ACTH concentrations (Fig. 1Go). Elevations were apparent at 10 min and lasted for 60–120 min, depending on the dose. The peak in plasma ACTH levels occurred at 10 min after 0.09 nmol/kg CRF administration, and ACTH levels at this time point were maximally elevated by 0.9 nmol/kg CRF. The higher dose of CRF (9 nmol/kg) produced a later peak (30–60 min) than the two lower doses.

Administration of Ucn produced elevations in plasma ACTH concentrations that were more marked than CRF at each dose tested (see Fig. 1Go). Statistical analysis (two-factor ANOVA) indicated significant single effects of both dose (P < 0.001) and treatment group (CRF vs. Ucn, P < 0.001). As with CRF, Ucn-induced elevations in the plasma ACTH concentration at 10 min were already maximal at a dose of 0.9 nmol/kg, with the higher dose of Ucn producing a later peak (at 60 min) and a more sustained elevation (still increased compared with controls at 240 min). At each dose of peptide tested, the plasma ACTH response to Ucn was more prolonged than that produced by CRF. This apparent difference seems likely to reflect the greater potency of Ucn (rather than an inherent difference in the actions of these ACTH secretagogues), because the temporal profiles of plasma ACTH levels in rats treated with a dose of CRF (9 nmol/kg) or Ucn (0.9 nmol/kg) that elicited similar peak plasma ACTH levels were virtually superimposable (see Fig. 2Go).



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  		Figure 2. Comparison of the temporal profiles of plasma ACTH concentrations in intact male rats treated with doses of CRF (9 nmol/kg) and Ucn (0.9 nmol/kg) that elicit similar peak (CRF, 637 ± 122 pg/ml; Ucn, 612 ± 154 pg/ml) and integrated (CRF, 899 ± 83 pg/h·ml; Ucn, 914 ± 156 pg/h·ml) plasma ACTH levels. Values are the mean ± SEM of five animals per group.

 
Ucn produces ACTH secretion by CRF receptor- dependent mechanisms
To determine whether Ucn elicits ACTH secretion via CRF receptor-dependent mechanisms, the effects of a potent CRF receptor antagonist, astressin (43), on CRF- and Ucn-stimulated ACTH secretion were determined. After iv administration of only vehicle, plasma ACTH concentrations remained low (10 ± 5 to 21 ± 6 pg/ml). The mean plasma ACTH concentrations in rats treated with astressin alone (3 mg/kg = 0.8 µmol/kg) were generally lower than those in animals treated with vehicle (e.g. 30 and 60 min vehicle-treated, 14 ± 4 and 15 ± 5 pg/ml, respectively; astressin-treated, <5 and 9 ± 4 pg/ml, respectively).

CRF or Ucn (0.9 nmol/kg, iv, in each case) produced increases in plasma ACTH concentrations (see Fig. 3Go) similar to those reported in the previous experiment (see Fig. 1Go). Administration of astressin 1 min before CRF or Ucn produced a marked inhibition of the plasma ACTH response to either peptide (Fig. 3Go). In either CRF- or Ucn-injected rats, astressin completely prevented elevations in plasma ACTH concentrations at 10–30 min, with only small increases in plasma ACTH being apparent at 60 and 120 min (Fig. 3Go). Statistical analysis (two-way ANOVA) of the integrated plasma ACTH concentrations over time indicated significant single effects of both pretreatment (astressin vs. vehicle, P < 0.001) and peptide treatment (CRF vs. vehicle, P < 0.001; Ucn vs. vehicle, P < 0.001) and a significant interaction between pretreatment and peptide treatment (astressin x CRF, P < 0.001; astressin x Ucn, P < 0.001). Astressin proved equally as effective at inhibiting ACTH secretion induced by either CRF (94 ± 2% inhibition) or Ucn (95 ± 1% inhibition).



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  		Figure 3. The effect of the CRF receptor antagonist astressin (0.8 µmol/kg; 3 mg/kg) on the rise in plasma ACTH concentrations induced by either CRF (0.9 nmol/kg) or Ucn (0.9 nmol/kg) in intact male rats. Values are the mean ± SEM of four to six rats. The data presented were obtained from a single experiment, but in the interests of clarity are separated into two graphs. The vehicle only and astressin- plus vehicle-treated groups from this experiment are presented on each graph to permit comparison to control animals. Statistical analysis (two-way ANOVA) of the integrated plasma ACTH levels (picograms per h/ml) over the 120-min test period indicated a highly significant (P < 0.001) interaction between CRF and astressin (vehicle alone, 14 ± 13; astressin alone, 3 ± 3; CRF alone, 429 ± 41; astressin plus CRF, 26 ± 11) and between Ucn and astressin (Ucn alone, 1191 ± 131; Ucn plus astressin, 62 ± 12).

 
Endogenous AVP potentiates ACTH secretion produced by CRF or Ucn
Because AVP and CRF synergistically stimulate ACTH secretion (47, 48, 49, 50, 51, 52), we determined whether Ucn-induced ACTH secretion is influenced by the presence or absence of endogenous AVP. This was achieved by passive immunoneutralization of rats with an antiserum to AVP. Administration of 0.4 ml anti-AVP serum (PBL 5408) produced no effect on plasma ACTH levels compared with those in rats treated with the same dose of NRS (10 min NRS-treated, 22 ± 5 pg/ml; anti-AVP-treated, 30 ± 11 pg/ml), but pretreatment with this antiserum 2 min before AVP (1.1 nmol/kg) completely prevented AVP-induced elevations in plasma ACTH concentrations (10 min after AVP: NRS-pretreated, 373 ± 93 pg/ml; anti-AVP-pretreated, 23 ± 8 pg/ml; Fig. 4Go).



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  		Figure 4. The effect of rabbit anti-AVP serum (0.4 ml/rat) on the rise in plasma ACTH concentrations induced by AVP (1.1 nmol/kg), CRF (0.9 nmol/kg), or Ucn (0.9 nmol/kg) in intact male rats. NRS (0.4 ml/rat) was used as a control serum injection. Values are the mean ± SEM of four to six rats. The data presented were obtained from a single experiment, but in the interests of clarity are separated into three graphs. The NRS-plus vehicle-treated and anti-AVP-plus vehicle-treated groups from this experiment are presented on each graph to permit comparison to control animals. Statistical analysis (two-way ANOVA) of the integrated plasma ACTH levels (picograms per h/ml) over the 120-min test period indicated significant interactions between anti-AVP and AVP treatments (NRS plus vehicle, 38 ± 14; anti-AVP plus vehicle, 35 ± 17; NRS plus AVP, 134 ± 28; anti-AVP plus AVP, 32 ± 20; P < 0.01), anti-AVP and CRF treatments (NRS plus CRF, 537 ± 50; anti-AVP plus CRF, 360 ± 44; P < 0.05), and anti-AVP and Ucn treatments (NRS plus Ucn, 987 ± 63; anti-AVP plus Ucn, 734 ± 76; P < 0.05).

 
Anti-AVP serum significantly (by two-way ANOVA, P < 0.05 in each case) reduced integrated plasma ACTH levels after the administration of either CRF or Ucn. At 10 min after either CRF or Ucn, plasma ACTH levels were similar whether animals had been pretreated with only NRS or with the anti-AVP serum (Fig. 4Go). However, plasma ACTH levels fell more sharply in the anti-AVP-treated groups. The reduction of integrated plasma ACTH levels over time produced by anti-AVP pretreatment was similar whether animals had been treated with CRF (37 ± 9%) or Ucn (27 ± 8%).

Effects of anti-CRF and anti-Ucn sera on basal and electrofootshock-induced ACTH secretion
Antisera raised against CRF and Ucn were tested for their abilities to inhibit CRF- and Ucn-induced elevations in plasma ACTH concentrations. Two minutes before administration of 0.9 nmol/kg CRF or Ucn, rats were pretreated with either 0.4 ml NRS or 0.015–0.4 ml anti-CRF (PBL rC69) or anti-Ucn (PBL 5833 or PBL 5779). Ten minutes after administration of either CRF or Ucn, plasma ACTH concentrations in rats pretreated with only NRS were markedly elevated (250–600 pg/ml; see Fig. 5Go; compare to untreated controls, 15–30 pg/ml). Anti-CRF (PBL rC69) specifically inhibited the elevation in ACTH secretion produced by CRF, and doses as low as 15 µl significantly reduced ACTH levels. Doses of PBL rC69 as high as 400 µl did not affect Ucn-stimulated ACTH secretion (Fig. 5AGo). Conversely, the two anti-Ucn sera specifically inhibited the plasma ACTH response to Ucn, but not that to CRF (Fig. 5Go, B and C). In each case, inhibition of the plasma ACTH response to either CRF or Ucn appeared to be maximal with 0.133 ml of the respective antiserum.



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  		Figure 5. The effect of anti-CRF (PBL rC69) and anti-Ucn sera (PBL 5833, PBL 5779) on plasma ACTH concentrations in intact male rats 10 min after injection of either CRF or Ucn. Animals were pretreated with antisera 2 min before administration (iv) of 0.9 nmol/kg of either CRF or Ucn. Administration of 0.4 ml NRS served as a control. Values are the mean ± SEM of four to seven rats. Mean plasma ACTH concentrations in control animals ranged from 15–30 pg/ml (not shown). **, P < 0.01 compared with NRS plus respective peptide treatments, by Dunnett’s multiple comparison test (by one-way ANOVA, P < 0.001 in each case).

 
To determine the contributions of endogenous CRF and Ucn to basal plasma ACTH secretion, we examined the effects of the different CRF and Ucn antisera on plasma ACTH levels (at 1130–1300 h) in rats that were otherwise untreated. After a basal blood sample, 0.133 ml NRS, anti-CRF, or anti-Ucn was administered iv, and another blood sample was taken 30 min later. Plasma ACTH concentrations immediately before and 30 min after administration of NRS were similar (means ranging from 12–22 pg/ml). Administration of only anti-CRF serum produced a statistically significant (P = 0.002, by Mann-Whitney U test) effect on plasma ACTH levels (Fig. 6AGo). Indeed, the plasma ACTH concentrations in 10 of 11 rats treated with anti-CRF were undetectable (<5 pg/ml; compared with only 3 of 13 undetectable in the NRS-treated group). By contrast, neither of the two anti-Ucn sera produced a statistically significant (PBL 5833, P = 0.76; PBL 5779, P = 0.80, by Mann-Whitney U test) effect on plasma ACTH concentrations (Fig. 6Go, B and C).



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  		Figure 6. The effect of anti-CRF (PBL rC69) and anti-Ucn (PBL 5833, PBL 5779) sera on plasma ACTH levels in otherwise untreated intact male rats. After a basal blood sample was taken (means plasma ACTH concentrations ranging from 15–20 pg/ml), animals were treated (iv) with 0.133 ml of either NRS or antisera. Values are plasma ACTH concentrations from individual rats 30 min after the administration of either NRS or antiserum. The numbers (n) per group are indicated on the graph. The broken line indicates the detection limit of the assay (5 pg/ml). Statistical analyses indicated that PBL rC69 significantly lowered the plasma ACTH concentration compared with that in NRS-treated rats (P = 0.002, by Mann-Whitney U test; P < 0.001, by unpaired Student’s t test), whereas no significant effect was detected with either PBL 5833 (P = 0.76, by Mann-Whitney U test; P = 0.93, by unpaired Student’s t test) or PBL 5779 (P = 0.80, by Mann-Whitney U test; P = 0.46, by unpaired Student’s t test).

 
Rats pretreated with only NRS (0.133 ml, iv) and subsequently stressed by electrofootshock exhibited marked elevations in plasma ACTH levels, with mean plasma ACTH concentrations for the different experiments ranging from 23–32 pg/ml before treatment to 709–875 pg/ml after 30 min of footshock. Pretreatment with anti-CRF (PBL rC69, 0.133 ml, iv) virtually abolished electrofootshock-induced elevations in plasma ACTH concentrations (PBL rC69 plus 30 min electrofootshock, 46 ± 6 pg/ml; Fig. 7AGo). In contrast, rats pretreated with either NRS or PBL 5833 (anti-Ucn) exhibited plasma ACTH profiles that were virtually superimposable (Fig. 7BGo).



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  		Figure 7. Effect of either anti-CRF (PBL rC69) or anti-Ucn (PBL 5833, PBL 5779) on plasma ACTH concentrations in intact male rats after exposure to electrofootshock (see Materials and Methods for parameters). A basal blood sample was taken (0 min), and rats were immediately injected iv with 0.133 ml NRS, PBL rC69 (anti-CRF), PBL 5833 (anti-Ucn), or PBL 5779 (anti-Ucn) and transferred to electrofootshock chambers. Values are the mean ± SEM of 5–6 rats (A), 8–11 rats (B), and 29–32 rats (C). The values presented in C are pooled data from three separate experiments.

 
The effect of anti-Ucn (PBL 5779) on the plasma ACTH response to electrofootshock was tested on three separate occasions, and Fig. 7CGo illustrates the pooled data from all three experiments. The majority of anti-Ucn (PBL 5779)-pretreated rats exhibited plasma ACTH profiles in response to footshock that were not discernibly different from those of animals pretreated with only NRS. However, of a total of 32 rats pretreated with anti-Ucn (PBL 5779), three animals exhibited markedly lower plasma ACTH levels after electrofootshock (i.e. <100 pg/ml; compared with all 29 NRS-pretreated rats, which exhibited plasma ACTH concentrations >200 pg/ml at each time point after footshock). Consequently, the mean plasma ACTH concentrations in PBL 5779-pretreated rats were slightly lower both 15 and 30 min after commencement of electrofootshock compared with those in rats pretreated with only NRS. However, there was no significant difference between integrated plasma ACTH levels (picograms per h/ml) in NRS-pretreated and PBL 5779-pretreated rats after electrofootshock whether analyzed as three separate experiments (by unpaired Student’s t test, P = 0.687, 0.336, and 0.199) or with the data from all experiments pooled (by unpaired Student’s t test P = 0.144). Similarly, repeated measures ANOVA indicated no statistical difference between NRS- and PBL 5779-treated animals whether treated as three separate experiments (P = 0.659, 0.290, and 0.255) or with data from all experiments pooled (P = 0.148).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CRF receptors play key roles in regulating endocrine, behavioral, and autonomic nervous system responses to stress. The recent identification of Ucn, a new putative ligand for CRF receptors in mammals (23, 24), suggests that in addition to CRF itself, Ucn may play important endogenous roles in activation of CRF receptors. The discovery of at least one additional mammalian CRF-like peptide (Ucn) means that data from previous studies using either CRF receptor antagonists or antiserum raised against CRF to probe the role of endogenous CRF in physiological/stress responses have to be interpreted with at least a degree of caution. Clearly, CRF receptor antagonists may inhibit the effects of not only CRF itself, but possibly other CRF-like peptides that signal via the same receptors. Indeed, in the present studies the potent peptide CRF receptor antagonist, astressin (43), inhibited not only CRF-stimulated, but also Ucn-stimulated, ACTH secretion. The sequence similarity between CRF and Ucn, particularly in the N-terminal domain, also means that in the absence of cross-reactivity data to the contrary, antiserum or antibodies raised against CRF (in particular N-terminally directed antisera/antibodies) may well cross-react with other CRF-like peptides such as Ucn. These considerations led us to test the hypothesis that Ucn is an important endogenous ligand for the best characterized of CRF receptor functions, namely regulation of pituitary ACTH secretion.

Ucn is clearly a more potent ACTH secretagogue than CRF itself, as demonstrated here by the greater plasma ACTH concentrations observed after the administration of equimolar doses of CRF or Ucn and by previous work showing that stimulation of ACTH secretion from rat anterior pituitary cells in primary cell culture by Ucn is apparent with an EC50 approximately 7-fold lower than that observed with CRF (23, 24). The present dose-response studies in vivo showed that at equimolar doses, Ucn elicited a more sustained elevation in plasma ACTH concentrations than that observed with CRF. This did not appear to be due to differences in activity between the two ACTH secretagogues, however, as CRF and Ucn doses that produced similar peak plasma ACTH concentrations produced very similar plasma ACTH profiles (Fig. 2Go). The present work also shows that the stimulatory effect of Ucn on ACTH secretion occurs via CRF receptor-dependent mechanisms, as evidenced by the inhibition of Ucn-induced ACTH secretion produced by pretreatment with the potent CRF receptor antagonist, astressin (43).

We also found that the availability of endogenous AVP was required for full expression of the ACTH-releasing capacity of both Ucn and CRF. It is well recognized that CRF and AVP interact synergistically to stimulate ACTH secretion (47, 48, 49, 50, 51), and studies in either AVP-deficient (Brattleboro) or normal rats treated with an anti-AVP serum or AVP receptor antagonists have shown that in the absence of AVP signaling, pituitary-adrenal responses to stressful stimuli are blunted (52, 53, 54, 55, 56, 57). The present studies are the first of which we are aware that show that the acute removal of AVP availability for interaction with its receptors in vivo blunts the pituitary ACTH response to CRF itself and indicate that endogenous AVP potentiates the response to elevated levels of CRF. Anti-AVP also inhibited the plasma ACTH response to Ucn, indicating a similar potentiating effect of AVP on Ucn-induced ACTH secretion.

In the present study we determined whether Ucn plays a significant role in the regulation of ACTH secretion by comparing the effects of passive immunization with antiserum raised to either CRF or Ucn. At the doses used, these antisera were clearly specific for their respective peptide immunogens. We found that although anti-CRF sera reduced basal plasma ACTH concentrations, anti-Ucn sera had no discernible impact. Similarly, anti-CRF virtually abolished electrofootshock-induced ACTH secretion. In contrast, the data obtained with anti-Ucn sera demonstrate that Ucn plays little, if any, endogenous role in the regulation of electrofootshock-induced ACTH secretion in the intact male rat.

It should be noted, however, that we investigated plasma ACTH concentrations in rats only at one time of day (midday), which corresponds to the diurnal trough of HPA axis activity in nocturnal animals. We cannot discount the possibility that Ucn may play a role in regulating pituitary ACTH secretion at other times of the day. Furthermore, we used only a single stress paradigm (intermittent electrofootshock), and there may be other stressful situations in which Ucn is an important mediator of ACTH secretion. In this regard, recent reports indicating that chronic salt loading (38) and dehydration (39) result in increased expression of Ucn-like immunoreactivity within the supraoptic nucleus, paraventricular nucleus, and median eminence suggest that under these circumstances Ucn may play some role in regulating pituitary ACTH secretion.

In conclusion, the present work shows that Ucn is a potent ACTH secretagogue in the intact male rat, and that it stimulates ACTH secretion via mechanisms similar to those employed by CRF (CRF receptors and interaction with AVP). However, it is endogenous CRF that is the primary mediator of basal and electrofootshock-induced ACTH secretion, and under these conditions Ucn does not play a significant role in regulating plasma ACTH concentrations.


   Footnotes
 
1 This work was supported by NIH Grant DK-26741 and The Foundation for Research. Back

2 Present address: North Western Injury Research Center, Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom. Back

Received May 5, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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