Introduction

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Corticotropin-releasing hormone (CRH) is a 41 amino acid peptide hormone first isolated by Vale et al. (1981). It is a member of a group of homologous peptide hormones that includes the telostian peptide urotensin, the amphibian peptide sauvagine and the recently identified mammalian peptide urocortin (Vaughan et al., 1995; Danaldson et al., 1996). CRH plays a prominent role in regulating the physiological response to stress by stimulating pituitary corticotrophs which release ACTH which in turn leads to the secretion of adrenal glucocorticoids (Vale et al., 1981). Apart from its action to stimulate the HPA axis, CRH has autonomic, electrophysiological and behavioral effects that point to a central nervous system transmitter role for the peptide (De Souza and Grigoriadis 1995). In line with this suggestion is the widespread, but discrete distribution of CRH receptors in brain (Aguilera et al., 1987; De Souza 1987). The presence of CRH in brain regions such as the locus ceruleus, amygdala, cerebral cortex and hippocampus suggests the importance of the peptide in mood and cognition. An imbalance of the central CRH system has been implicated in anxiety disorders and depression (Dunn and Berridge 1990; Owens and Nemeroff 1991; Raadsheer et al., 1994; Ladd et al., 1996).

The effects of CRH are mediated through activation of specific adenylate cyclase linked receptors. Recently, two distinct CRH receptor subtypes, designated CRH₁ and CRH₂, were identified through molecular cloning (Kishimoto et al., 1995; Lovenberg et al., 1995; Perrin et al., 1993). Three splice variants of the human CRH₂ receptor, CRH_2, CRH₂ and CRH2 have also recently been identified (Kostich et al., 1996). Studies into the functional significance of the CRH receptors in brain have provided evidence that the CRH₁ receptors mediate the central anxiogenic effects of the peptide while CRH₂ receptors may be involved in reduction of feeding (Schulz et al., 1996; Lundkvist et al., 1996; Liebsch et al., 1995; Spina et al., 1996). However, it should be noted that due to its high affinity for and its anatomic colocalization with the CRH₂ receptor, urocortin, rather than CRH, has been suggested to be the endogenous ligand for this receptor subtype (Spina et al., 1996; Vaughan et al., 1995).

Although previous studies have demonstrated specific, saturable binding of [¹²⁵I] CRH to sites in brain and peripheral tissues (Aguilera et al., 1987), [¹²⁵I] CRH is an inadequate ligand for CRH₂ receptors (D.H. Rominger and C.M. Rominger, unpublished observation), the predominant splice variant of CRH₂ receptors found in brain. Sauvagine, the amphibian peptide analog, has been reported to possess a much higher affinity for CRH₂ receptors than CRH itself (Grigoriadis et al., 1996). This observation led us to test sauvagine as an alternative ligand for CRH₂ receptors. Support for this strategy was provided by Grigoriadis and coworkers (1996) who demonstrated specific, saturable binding of [¹²⁵I]tyr^osauvagine to cloned human CRH₂ receptors. The present study confirms and extends this prior work by characterizing the interaction of [¹²⁵I] tyr^osauvagine to cloned human CRH₂ receptors using an in vitro filtration binding technique. In contrast to earlier studies (Grigoriadis et al., 1996; Primus et al., 1997), our work characterizes human CRH₁ and CRH₂ receptors expressed in the same cell background. In addition, [¹²⁵I]tyr^osauvagine in combination with a selective CRH₁ receptor antagonist was used to measure CRH₂ receptors in rat brain using a similar in vitro method.

The availability of sequence information for the CRH receptors has made possible the anatomic localization of RNA message of the receptors by in situ hybridization techniques (Chalmers et al., 1995). However, only recently have ligands specific for either of the receptors been available (Duggan et al., 1997; Rominger et al., 1996 and Fitzgerald et al., 1996), thus differentiation of the anatomic localization of CRH₁ and CRH₂ receptor binding sites has only been possible by taking advantage of the differential affinity of the peptide agonist ovine-CRF for CRH₁ and CRH₂ receptors (Primus et al., 1997). We describe the use of [¹²⁵I] tyr^osauvagine in combination with a selective small molecule CRH₁ receptor antagonist in autoradiographic studies to compare localization of receptor binding sites to the previously published distribution of CRH receptor mRNA mapped by in situ hybridization. Part of the work described was previously published in abstract form (Rominger et al., 1996).

	Materials and Methods

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In Vitro Binding

Cell line. Stable cell lines expressing human CRH₁ or CRH₂ receptors were established with HEK293EBNA cells (Invitrogen) using LipofectAMINE (GibocoBRL) (Horlick et al., 1997). The transfected plasmids contained the receptor genes under the control of the (cytomegalovirus) immediate early gene promoter, the EBV oriP for the maintenance of the plasmid as an extrachromosomal element in nonrodent mammalian cells expressing the Epstein Barr nuclear antigen, and the hph gene from Escherichia coli to yield resistance to hygromycin B. The resulting cell lines, designated 293ECRH₁ and 293ECRH₂, were grown in Dulbecco's modified Eagle medium containing 10% fetal bovine serum at 37°C in a humid environment with 5% CO₂. Upon reaching a stable transfected state (approximately 10 days), the cells were adapted to spinner culture with cells harvested and stored at -80°C.

Animals

Male Sprague-Dawley rats, Charles River Breeding Laboratories (Wilmington, MA) weighing 250 to 300 g were housed in a light and temperature controlled room, with food and water available ad libitum. Studies were performed in accordance with the declaration of Helsinki and with the "Guide for the Care and Use of Laboratory Animals" as adopted and promulgated by the National Institutes of Health.

Materials

Tyr^osauvagine was synthesized and provided by Rose Wilk, Chemical and Physical Sciences Department of Du Pont Merck Research Laboratories (Wilmington, DE). [¹²⁵I] tyr^osauvagine, and [¹²⁵I] tyr^o ovine CRH (specific activity 2200 Ci/mmol) were obtained from New England Nuclear (Boston, MA). Unlabeled rat/human, ovine,  helical CRH, sauvagine and urotensin I were purchased from American Peptide Co. (Sunnyvale, CA). XQ041(N-((2-Bromo-4-(1-methylethyl)phenyl)-N-ethyl-4-methyl-6-(4 morpholinyl)-2-pyrimidinamine), SA631 (N-(2-Bromo-4,6-dimethoxyphenyl)-N-ethyl-4-diethylamino-6-methyl-2-triazineamine) and SC241 (N-Bis(2-methoxyethyl)-3-[2-bromo-4(1-methylethyl)phenyl]-5-methyl-³H-1,2,3-triazolo[4,5-d]pyrimidin-7-amine) were synthesized and provided by Argyrios Arvanitis, Thomas E. Christos and Rajagopal Bakthavatchalam, respectively, from the Chemical and Physical Sciences Department of DuPont Merck Research Laboratories (Wilmington, DE). 10X Phosphate buffered saline and 1 M HEPES buffer were purchased from Gibco BRL(Grand Island, NY). All other standard reagents were purchased from Sigma (St. Louis, MO).

Preparation of Membrane Homogenates

Membrane homogenates from CRH receptor expressing cells were prepared as follows. Frozen pellets of approximately 1 × 10⁸ 293E_CRH1 or 293E_CRH2 cells prepared as described above were each thawed on ice and homogenized in 10 ml of buffer (50 mM HEPES, 10 mM MgCl₂, 15 mM EGTA and 1 µg/ml each of the peptidase inhibitors aprotinin, leupeptin and pepstatin, pH 7.2 at 4°C), using a Brinkman Polytron (PT-10) setting 6 for 10 sec. The homogenate was centrifuged at 48,000 × g for 12 min and the resulting pellet was washed by repeating the homogenization and centrifugation steps. The final cell pellet was resuspended in buffer to yield a protein concentration of approximately 0.3 to 0.4 mg/ml as assayed by the method of Bradford (1976) using bovine serum albumin as standard.

To prepare brain membranes for binding assays, rats were decapitated and brains were quickly dissected on ice-chilled glass plates. Frontal cortex and cerebellum were isolated and kept on ice. To isolate the septal-hypothalamic-thalamic (SHT) block, two transverse cuts were made: a rostral cut half-way between the optic chiasm and the olfactory tubercle just rostral to the genu of the corpus callosum, and a second cut immediately caudal to the mammillary nucleus. Using the lateral ventricles as landmarks, two parasagittal cuts were made separating the SHT block from the striatum and the tissue lateral to the internal capsule. The remainder of the overlying cortex and hippocampus was then removed leaving a 0.1 g tissue block containing the septum, basal forebrain, hypothalamus and thalamus. Tissue samples weighing between 0.1 and 0.25 g were homogenized in 10 ml buffer and prepared by the method described above for the cell line homogenates. The final pellet was resuspended in buffer to yield a protein concentration of 16 mg/ml original wet weight.

[¹²⁵I] Tyr^oSauvagine and [¹²⁵I] tyr^oCRH Receptor Binding: Homogenate Assays

Conditions for all binding assays were identical independent of tissue or radioligands used. The assays were initiated by the addition of 100 µl membrane suspension (~0.15 mg protein/ml) to 200 µl of assay buffer containing .096 µCi of radioligand and various concentrations of inhibitors. Binding assays were performed in triplicate in disposable polypropylene 96-well plates (Costar Corp., Cambridge, MA) in a final volume of 0.3 ml. The assay buffer was the same as described above for membrane preparation with the addition of ovalbumin and bacitracin to final concentrations of 0.1% and 0.15 mM, respectively. Nonspecific binding was defined in the presence of 500 nM -helical CRH_9-41 and 3 µM SC241, a selective CRH₁ receptor antagonist (Fitzgerald et al., 1996) for the CRH₂ and CRH₁ receptor binding assays, respectively. The optimum incubation time at 23°C was 2 hr (fig. 2). The separation of bound from free radioligand was accomplished by rapid vacuum filtration of the incubation mixture over GFF glass fiber filters (Inotech Biosystems International, Lansing, MI) presoaked for 2 hr in 0.3% polyethylenamine (pH 13) using an Inotech cell harvester. Filters were washed three times with 0.3 ml of ice-cold phosphate buffered saline pH 7.0 containing 0.01% Triton × 100. Filters were assessed for radioactivity in a gamma counter (ICN, Huntsville, AL) at approximately 80% efficiency. Saturation studies were performed over the range of 0.005 to 60 nM using a fixed concentration of [¹²⁵I] tyr^osauvagine in the presence of differing amounts of cold tyr^o sauvagine. Nonspecific binding was defined with 500 nM -helical CRH_9-41.

DATA Analysis

The apparent equilibrium dissociation constants (K_d) and the maximum number of binding sites (B_max) from saturation binding experiments were calculated using the nonlinear iterative curve-fitting computer program (LIGAND) of Munson and Rodbard (1980) Rate constants for dissociation were calculated as previously described (Donner et al., 1980).

IC₅₀ values of competitors were calculated using the program DeltaGraph by DeltaPoint, (Monterey, CA) with the following equation used for one-site competition non-linear analysis and "pseudo" Hill model (Graeser and Neubig, 1992): B = min + [(max-min)* IC₅₀^nH]/[ I^nH + IC₅₀^nH].

Autoradiography

Tissue preparation. Rats were anesthetized with pentobarbital (Abbott Labs., North Chicago, IL) and perfused transcardially with approximately 800 ml equal parts phosphate-buffered saline and 0.32 M sucrose (pH 7.4). After rapid removal of brain, pituitary and heart, the tissues were embedded in O.C.T. compound (Miles Inc., Elkhart, IL) and frozen on powdered dry ice. Twenty micron sections were cut on a cryostat at -19°C, thaw-mounted onto chrome alum/gelatin-subbed microscope slides and stored at -70°C until used. Sections from at least nine animals were subsequently used to generate autoradiograms.

Receptor labeling. Slide mounted tissue sections were brought to room temperature and preincubated in buffer (50 mM HEPES, 10 mM MgCl₂, 15 mM EGTA, 0.1% ovalbumin, 0.15 mM bacitracin and 100 KIU/ml aprotinin, pH 7.2) for 5 min. Sections were then transferred to buffer containing 0.15 nM [¹²⁵I] tyr°sauvagine and incubated 2 hr at room temperature, in the presence or absence of 3 µM XQ041, a selective small molecule CRH₁ receptor antagonist (Fitzgerald et al., 1996). Nonspecific binding was defined by incubation with 0.5 µM -helical CRH_9-41. After the 2-hr incubation, the slide-mounted sections were washed in ice-cold PBS containing 0.01% Triton X-100 for 10 min, dipped in deionized water and dried under a stream of cold air. Slides were then exposed to Hyperfilm [³H] (Amersham Corp., Arlington Heights, IL) for 2 to 3 days. Adjacent slide-mounted sections were stained with cresyl violet to identify anatomic regions of interest.

Data analysis. Autoradiograms were digitized on a Perseptics (Knoxville, TN) system for Macintosh and optical density readings and quantitation of the data were performed using the Macintosh-based computer analysis software package Image (National Institutes of Health, Bethesda, MD). Optical density values were compared against standard curves generated using 20 µm [¹²⁵I] Micro-scales, (Amersham Corp.) and were converted to fmol/mg tissue equivalent.

	Results

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Characterization of [¹²⁵I]tyr^oSauvagine Binding to 293E_CRH2 Cell Membranes

Temperature, time and tissue concentration. Studies of [¹²⁵I]tyr^osauvagine (150 pM) binding to membranes derived from 293E_CRH2 cells conducted at 4, 23 and 37°C indicated optimum specific binding at 23°C. Varying the time of the incubation revealed that equilibrium was reached in 2 hr. Nonspecific binding, defined in the presence of 500 nM -helical CRH_9-41, remained constant over the entire 5-hr incubation period. Incubation of various amounts of 293E_CRH2 derived membrane homogenate with [¹²⁵I]tyr^osauvagine indicated that binding of the radioligand was linear with protein concentrations up to 100 µg/well (fig. 1). Nonspecific binding was independent of the amount of cellular protein added suggesting that the bulk of nonspecific binding was due to the adsorption of the iodinated ligand to the glass fiber filters. Based on the protein curve, a final protein concentration of 250 µg/ml was used in subsequent assays.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   [125I]-tyrosauvagine binding to CRH2 receptors as a function of protein concentration. Increasing amounts of 293ECRH2 cell membranes were incubated with 150 pM [125I]tyrosauvagine for 2 hr at 23°C. Nonspecific binding was determined in the presence of 500 nM -helical CRH9-41. The data are the average of triplicate determinations.

Kinetics of [¹²⁵I]tyr^oSauvagine Binding: Association/Dissociation

The time course of [¹²⁵I]tyr^osauvagine association to CRH₂ receptors in 293E_CRH2 cell membranes exhibited pseudo-first-order kinetics (fig. 2). At 23°C, equilibrium was reached within 2 hr and remained stable up to 5 hr (fig. 2). Although there was a progressive increase in the level of nonspecific binding at early time points, at times later than 20 min, nonspecific binding, defined in the presence of 500 nM -helical CRH_9-41, remained constant. Calculations based on the k_obs derived from the linear regression of the pseudo-first-order plot (inset fig. 2) yielded an association rate constant of k₊₁ = 4.36 × 10⁷ M¹min¹.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Rate of association of [125I]-tyrosauvagine to 293ECRH2 cell membranes. Aliquots of membranes were incubated with 150 pM [125I]-tyrosauvagine at 23°C in the absence or presence of 500 nM -helical CRH9-41 (nonspecific) for varying lengths of time. Linear conversion of the specific association data is included as an inset, where Beq represents specific binding at the steady state level and Bt represents the specific binding at time t. Linear regression yielded the slope, kobs = 1.53 × 10 min. Each value is an average of triplicates from an individual experiment. The experiment was repeated twice with identical results.

Dissociation of specifically bound [¹²⁵I]tyr^osauvagine was observed after the addition of 1 µM -helical CRH_9-41. As seen in figure 3, the dissociation of specific binding was time dependent. The dissociation was not monophasic, but nonlinear analysis did not produce an accurate measure of the low affinity dissociation rate. Linear regression analysis of the data (inset fig. 3) resulted in a calculated k_-1 = 6.00 × 10³ min¹ corresponding to an apparent half-time of 122 min. The equilibrium dissociation constant (K_d = k_-1/k₊₁) calculated from the kinetic data was 137.0 pM.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Time course of dissociation of [125I]-tyrosauvagine from membranes derived from 293ECRH2 cells. Aliquots of membranes (50-100 µg/well) were incubated with 150 pM [125I]-tyrosauvagine at 23°C in the absence (total) or presence of 500 nM -helical CRH9-41 (non-specific) for 2.5 hr to attain equilibrium; dissociation was initiated by the addition of 1 µM -helical CRH9-41. Linear conversion of the specific association data is included as an inset, where B represents specific binding at time t and Bo represents binding at equilibrium. The calculated T1/2 of the specific binding was 122 min. Each value is an average of triplicates from an individual experiment. The experiment was repeated twice with identical results.

Saturation of [¹²⁵I]tyr^oSauvagine Binding to 293E_CRH2 Cell Membranes

Saturation of [¹²⁵I]tyr^osauvagine binding to 293E_CRH2 cell membranes is demonstrated in figure 4. Scatchard transformation of the data (inset fig. 4) indicated a two-site model of high (K_d = 44 ± 9 pM; B_max = 2.81 ± 0.64 fmol/mg protein) and low (K_d = 4.1 ± 0.25 nM; B_max = 160 ± 24 fmol/mg protein) affinity sites (table 3). In the range of ligand concentrations (0.005-60 nM) used, iterative curve fitting analysis (LIGAND) of the data also best fit a two site/state model consistent with the Scatchard transformation analysis.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Saturation binding of [125I]-tyrosauvagine binding to 293EBNACRH2 cell membranes. A direct plot of the data showing the total amount of [125I]-tyrosauvagine bound, the amount of [125I]-tyrosauvagine bound in the presence of 500 nM -helical CRH9-41 (nonspecific) and the specific (total-nonspecific) binding. The inset shows a Scatchard transformation of the specific binding. LIGAND analysis significantly fit a two-state/site model better than a one site fit (n = 5, P < .05) Aliquots of 293 EBNA cell line expressing CRH2 receptors (50-80 µg/well) were incubated for 2 hr at 23°C in binding buffer with increasing concentrations (0.005-60 nM) of [125I]-tyro-sauvagine. Each value is an average of triplicates for an individual experiment. The experiment was repeated four more times with identical results.

Effect of Gpp(NH)p on [¹²⁵I]Tyr^oSauvagine Binding

To assess whether [¹²⁵I]tyr^osauvagine binds to functionally coupled CRH₂ receptors in the 293E_CRH2 cell line, saturation studies in the presence of 200 µM 5'-guanylylimidodiphosphate Gpp(NH)p, a nonhydrolizable GTP analogue, were performed. Analysis of saturation data in the absence of Gpp(NH)p showed a biphasic Scatchard plot and best fit a two site model. The addition of 200 µM Gpp(NH)p led to a reduction in total specific binding and a shift to the low affinity state was observed (fig. 5). Saturation data in the presence of 200 µM Gpp(NH)p best fit a linear model and indicated a K_d of 3.9 ± .98 nM and a B_max of 101 ± 37 fmol/mg protein (mean ± S.E.M.; n = 3), comparable to the low affinity state of the receptor.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Scatchard transformation of [125I]-tyrosauvagine binding to membranes of 293ECRH2 cells in the absence and presence of Gpp(NH)p. Data from binding experiments performed in the absence of Gpp(NH)p. best fit a two-site model indicating affinity of [125I]-tyrosauvagine for two states of the receptor. A parallel experiment in which membranes were incubated with 200 µM Gpp(NH)p showed a loss of the high affinity binding resulting in a one site fit that corresponded with the low affinity state of the receptor. Each value is an average of triplicates from an individual experiment. The experiment was repeated twice with identical results.

Pharmacology of [¹²⁵I]tyr^oSauvagine Binding to Human CRH₁ and CRH₂ Receptors.

The pharmacology of [¹²⁵I]tyr^osauvagine binding to membranes derived from 293E_CRH1 and 293E_CRH2 cells was examined in competition experiments using CRH-related peptide agonists, the peptide antagonist -helical CRH_9-41, and the small molecule antagonists SA631 and SC241 (table 1).

View this table: [in this window] [in a new window]   TABLE 1 Inhibition of [125I]-tyrosauvagine binding to CRH receptors in 293ECRH1 and 293ECRH2

Urotensin was the most potent peptide agonist at inhibiting [¹²⁵I]tyr^osauvagine binding to CRH₁ receptors. Urocortin was similarly potent followed by rhCRH, oCRH and sauvagine. The peptide antagonist -helical CRH_9-41 as well as the two small molecule antagonists SC241 and SA631 also exhibited potent affinity for the CRH₁ receptor.

Urotensin was the most potent peptide agonist at the CRH₂ receptor followed by urocortin and sauvagine. Sauvagine showed a 3-fold preference for CRH₂ over CRH₁, although urocortin and urotensin showed equal affinity to both receptors. rhCRH showed a 3-fold preference for the CRH₁ receptor over CRH₂ and oCRH showed a 12-fold preference for CRH₁ over CRH₂. -Helical CRH_9-41 showed a slightly greater affinity for CRH₂ over CRH₁. The two small molecule antagonists, SC241 and did not inhibit [¹²⁵I]tyr^osauvagine binding in 293E_CRH2 cells at concentrations up to 10 µM.

Binding of [¹²⁵I]-tyr^oSauvagine to Rat Brain Homogenates

To assess regional differences in CRH₁ and CRH₂ receptor density in rat brain, [¹²⁵I]tyr^osauvagine binding was examined in several brain regions in the presence and absence of either urotensin, which inhibits the binding to both CRH₁ and CRH₂ receptors, or SC241, which inhibits the binding to the CRH₁ receptor alone. The difference, at saturating concentrations, between the amount of [¹²⁵I]-tyr^osauvagine bound in the presence of urotensin and SC241 represents labeling of CRH₂ receptors. Representative curves for each compound in several brain regions are shown in figure 6. Rat cerebellum showed a ratio of about 0.2 CRH₂/CRH₁ although the ratio in frontal cortex was approximately 0.12. The area that showed the greatest level of CRH₂ binding was the rat septal-hypothalamic-thalamic region. In this area the ratio of CRH₂/CRH₁ binding was 0.55.

View larger version (36K): [in this window] [in a new window]   Fig. 6.   Regional pharmacological identification of CRH2 binding in rat membranes. Membranes prepared as described in "Materials and Methods," were incubated with 100-150 pM [125I]-tyrosauvagine and either SC241 or urotensin. A, Inhibition profiles of SC241 and urotensin in rat frontal cortex (A) septal-hypothalamic-thalamic region (SHT; B)and cerebellum (C) are shown. The data shown are from representative experiments that were replicated three times with similar results. D, Ratio of CRH2 receptor to CRH1 receptor in rat frontal cortex, SHT, and cerebellum. Values represent the mean % ± S.E.M. of three experiments conducted in duplicate, of residual [125I]-tyrosauvagine binding which represents CRH2 specific binding.

Anatomical Distribution of CRH₂ Receptors

Because we found that [¹²⁵I]-tyr^osauvagine in combination with SC241 can be used to specifically measure CRH₂ receptors, we extended the use of these agents to determine the distribution of the CRH receptor subtypes using receptor autoradiography.

The most intense signal was seen in the choroid plexus at all levels of the ventricular system (figs. 7 and 8; table 2). No receptor binding was observed in the ependyma or the meningeal layers of the brain. Only a few regions of the brain displayed high densities of CRH₂ receptors. The lateral septum contained the highest CRH₂ receptors density. This CRH₂ receptor rich area, particularly the dorsal aspect, was sharply delineated from the remainder of the septum (fig. 7). Similarly, the bed nucleus of the stria terminalis was distinctly delineated in the autoradiographs. This region contained a moderate to high density of CRH₂ receptors (fig. 7). Discrete localization of CRH₂ receptors was also observed in the amygdala, particularly in the posteromedial cortical nucleus of the amygdala. However, CRH₂ receptors were not detected in the area stretching from the bed nucleus through the substantia innominata to the amygdala, a region containing numerous CRH positive neurons. Moderate levels of CRH₂ receptors were detected in the entorhinal cortex and hippocampus (fig. 8) although neocortical areas displayed low levels (table 2).

View larger version (75K): [in this window] [in a new window]   Fig. 7.   Autoradiographic localization of CRH receptors in rat at the level of the rostral forebrain. Abbreviations.: dorso- lateral septum (LSd), Bed nu. stria terminalis (BST), suprachiasmatic nu. (Sch), supraoptic nu (SO).

View larger version (72K): [in this window] [in a new window]   Fig. 8.   Autoradiographic localization of CRH receptors in rat brain at the level of the hypothalamus. Arrows indicate choroid plexus. Abbreviations: Hippocampal fields (CA1 and CA3), dentate gyrus (DG), paraventricular nu.thalamus (PVT), thalamus (Tha), dorsal medial nu. hypothalamus (DMH), ventral medial nu. hypothalamus (VMH), amygdala complex (Amyg).

View this table: [in this window] [in a new window]   TABLE 3 Equilibrium binding of [125I]-tyro-sauvagine-labeled CRH2 receptors

In the diencephalon, the most striking CRH₂ signal was detected in the ventromedial nucleus of the hypothalamus outlining the entire nucleus. Moderate levels of CRH₂ receptors were found in the supraoptic and suprachiasmatic nuclei. CRH₂ receptors were not observable in the thalamus. Despite the presence of numerous CRH-positive axon terminals in the paraventricular nucleus of the hypothalamus, no CRH₂ signal could be identified in this hypothalamic cell group. In the brainstem only a few regions displayed modest levels of CRH₂ receptors. These include the superior and inferior colliculi, the superficial layers of the spinal trigeminal nucleus and the nucleus of the solitary tract.

	Discussion

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Our work describes the binding of [¹²⁵I]tyr^osauvagine to membranes of CRH₂ receptor expressing HEK293/EBNA (293E_CRH2) cells. This study in large part confirms and extends the earlier report of Grigoriadis and coworkers (1996). However, our study used a rapid filtration assay which should prove technically superior to the centrifugation assay as used in the earlier report.

The binding of [¹²⁵I]tyr^osauvagine to CRH₂ receptors was temperature, time and tissue dependent. Optimum temperature for the binding assay was 23°C and equilibrium was reached in 2 hr. Although a low level of [¹²⁵I]tyr^osauvagine bound specifically to glass fiber filters soaked in 0.3% PEI, it did not interfere with the observation of a robust signal when membranes from hCRH₂ receptor containing cells were added. At optimum conditions, an 80% specific signal was obtained using the technique described.

Two pieces of evidence indicate that [¹²⁵I]tyr^osauvagine recognizes heterogeneous binding sites. First, the dissociation of specific binding was not monophasic. Second, saturation isotherm data best fit a two-site model with apparent affinity constants of 44 pM and 4.1 nM for high and low affinity states, respectively. The indication of two affinity states for CRH₂ receptor recognized by [¹²⁵I]tyr^osauvagine is at variance with the findings by Grigoriadis et al. (1996), whose saturation data indicated the existence of only one site. Grigoriadis et al. (1996) were, however, able to demonstrate that GTP and its nonhydrolyzable analogs partially inhibit [¹²⁵I]tyr^osauvagine binding to CRH₂ binding site, suggesting two states of the receptor. Our study shows that the high affinity [¹²⁵I]tyr^osauvagine binding sites are eliminated with 200 µM Gpp (NH) p.

The potency of peptide analogs of CRH to inhibit the [¹²⁵I]tyr^osauvagine binding to membranes prepared from 293E_CRH2 was examined. The rank order potency of several CRH peptide analogs was: urotensin>sauvagine=urocortin >-helical CRH_9-41 >rh-CRH o-CRH. We also tested the potency of these peptides to inhibit [¹²⁵I]tyr^ooCRH binding to membranes prepared from HEK293/EBNA cells expressing CRH₁ receptors. Differences were observed in the rank order inhibition potencies of the peptides at the two receptors. In contrast to that at the CRH₂ receptor, the order of potency at the CRH₁ receptor was: urotensin>urocortin>r/h-CRH>o-CRH=sauvagine>  helical CRH_9-41. We also demonstrated that the small molecule CRH antagonists SA631 and SC241 while having nanomolar potency at the CRH₁ receptor, had little or no affinity for CRH₂ receptors as labeled by [¹²⁵I]tyr^osauvagine.

The CRH₁ receptor selective small molecule antagonists enabled us to examine comparative densities of CRH₁ and CRH₂ receptors in several brain areas. The amount of CRH₂ specifically labeled by [¹²⁵I]tyr^osauvagine in the presence of a CRH₁ masking concentration of the small molecule antagonist SC241 correlated well with the amount of CRH₂ mRNA observed by Chalmers and coworkers (1995) in that a greater abundance of CRH₂ receptors was found in a tissue block containing septum and hypothalamus compared to those from the neocortex and cerebellum. The use of [¹²⁵I]tyr^osauvagine in combination with specific CRH₁ receptor antagonists should be helpful in elucidating the anatomic localization of CRH₂ receptors and the response of these receptors to specific neuronal lesions and drug manipulations.

We took advantage of the utility of [¹²⁵I]tyr^osauvagine in combination with a specific CRH₁ antagonist to examine the distribution of CRH₁ and CRH₂ receptors in autoradiographic studies. For the most part, our data confirm the results of a previous study on the localization of CRH₂ mRNA in the rat brain obtained by in situ hybridization (Chalmers et al., 1995). However, a few notable differences were observed. For example, while our receptor binding study parallels the high levels of CRH₂ mRNA in the choroid plexus, we did not observe CRH₂ binding in ependymal cells lining the ventricles in contrast to the report of CRH₂ mRNA in these cells. Conversely, previous in situ hybridization studies reported low to no detectable levels of CRH₂ mRNA in the superficial layers of the spinal trigeminal nucleus and the solitary nucleus (Chalmers et al., 1995), whereas our study detected moderate levels of CRH₂ receptors. Apart from these few exceptions, the CRH₂ receptors demonstrated in our study by receptor autoradiography match the data obtained by in situ hybridization studies on the localization of CRH₂ mRNA. The anatomic overlap of the autoradiographic and in situ hybridization data suggest that CRH₂ receptors are postsynaptic. In the choroid plexus, we found that total [¹²⁵I]-tyr^osauvagine receptor binding was not different from that measured in the presence of XQ041, confirming the conclusion of Chalmers et al. (1995) that the choroid plexus is devoid of CRH₁ receptors. Also in agreement with the earlier report, the high levels of CRH receptors in the lateral septum appear to be almost exclusively of the CRH₂ type. Close inspection of our autoradiograms of sections through the lateral septum reveals a notable discrepancy between the density of CRH terminals detected by immunohistochemistry (Sakanaka et al., 1987) and by receptor autoradiography. Both our autoradiographic receptor binding data and the results of in situ hybridization experiments indicate the presence of CRH₂ receptors in the dorsal sector of the lateral septum, yet CRH terminals in the septum appear to be concentrated most heavily in ventral sector of the septum (Sakanaka et al., 1987). Physiological studies by Siggins et al. (1985) suggest that CRH afferents exert an inhibitory influence on GABAergic septal projection neurons that receive a major excitatory input from the hippocampus and amygdala and in turn project to widespread regions in the hypothalamus. The extent to which CRH₂ receptors in the lateral septum contribute to the inhibitory projection system through which the hippocampus and the amygdala influences autonomic centers in the hypothalamus remains to be explored.

The discrete distribution of high levels of CRH₂ receptors in the posterior medial cortical nucleus of the amygdala matches the high levels of CRH₂ mRNA in this region. The posterior medial cortical nucleus receives a prominent input from the main and accessory olfactory tract. CRH neurons have been described in olfactory regions and it is conceivable that CRH₂ receptors in the posterior medial cortical nucleus mediate at least in part olfactory cues to the amygdala. Lesion studies have shown that the corticomedial complex of the amygdala also facilitates the expression of maternal behavior in rats (Fleming et al., 1983) This present data suggest a possible role of CRH₂ receptors in olfaction and the expression of maternal behavior.

The high density of CRH₂ receptors in the VMH correlates well with the presence of high CRH mRNA levels in this nucleus. This cell group also contains a high density of CRH terminals (Sakanaka et al., 1987). There is evidence that at least a subset of these terminals originate from cells in the amygdala that contains a large number of CRH neurons that provide a dense projection to the VMH (Canteras et al., 1992). The VMH shares strong connections with brain regions involved in appetite and reproductive behaviors. While suggestions have been made relating CRH₂ receptors to appetite (Spina et al., 1996), additional studies are needed to make these suggestions convincing. It remains to be determined to what extent CRH₂ receptors influence reproduction.

The presence of CRH₂ receptors in the NTS and the superficial layers of the spinal trigeminal nucleus has not previously been reported. The NTS serves as the sole relay of all visceral afferents reaching the brainstem via the VIIth, IXth and Xth cranial nerves. CRH afferents to the solitary nucleus originate in CRH cells in the central nucleus of the amygdala and the bed nucleus of the stria terminalis. Whether primary afferents from cranial nerve ganglia of the VIIth, IXth and Xth nerve contain CRH is not known. High levels of CRH receptor binding of the CRH₁ type in cranial nerve nuclei have been reported by DeSouza and Insel (1990). CRH immunoreactivity has been localized to C-fibers terminating in the superficial layers of the dorsal horn suggesting that a subpopulation of primary sensory neurons contains CRH. Thus, there is circumstantial evidence that cranial nerve afferents including those terminating in the nucleus of the solitary tract utilize CRH as transmitter. Our findings imply that CRH₂ receptors in the NTS may play a role in the transmission of inputs from the viscera to the brainstem. It should be noted, however, that the identity of the peptide stained with ovine CRH has been called into question after the demonstration of cross-reactivity between antisera to ovine CRH and substance P (Berkenbosch et al., 1986).

Recently Primus et al. (1997) studied the distribution of CRH₁ and CRH₂ receptors in rat brain using[¹²⁵I]-sauvagine autoradiography. The authors used ovine-CRF to mask CRH₁ receptors to study the distribution of CRH₂ receptors. Although our report confirms their results that the CRH₁ receptors predominate in cerebellum and that high levels of CRH₂ receptors are found in the lateral septum and amygdala, there are significant differences between the two studies. In particular, Primus et al. (1997) found high levels of CRH₂ receptors in neocortex. We find, in agreement with earlier reports on mRNA localization (Chalmers et al., 1995) that the level of CRH₂ receptors in neocortex is very low. This discrepancy might be explained by the fact that at 100 nM ovine-CRF, the concentration used by Primius et al. (1997), a significant amount of specific [¹²⁵I]-sauvagine binding to CRH₁ receptors remains. This points to the importance (as Primus et al. points out) of using an agent highly specific for one receptor when two different receptors are being labeled in differential autoradiographic studies.

As with CRH₁ receptors, the widespread distribution of CRH₂ receptors in functionally diverse regions of the central nervous system indicates their association with several different neuronal systems. The extensive connections between the amygdala, septum and bed nucleus of the stria terminalis, regions containing high levels of CRH₂ point to a role of these receptors in a variety of behavioral and regulatory functions.

We confirm and extend the work of Grigoriadis et al. (1996). Our data indicate that [¹²⁵I]tyr^osauvagine is an excellent ligand to characterize human and rat CRH₂ receptors. The filtration assay used in the current work offers advantages over the centrifugation technique described earlier, both in terms of technical ease and the clear ability to distinguish high and low affinity [¹²⁵I]tyr^osauvagine binding sites associated with CRH₂ receptors. The combination of [¹²⁵I]tyr^osauvagine with a small molecule ligand specific for CRH₁ receptors described here offers a novel approach for studying CRH₂ receptors in tissues containing CRH₁ and CRH₂ receptors. Finally, this work describes the successful use of a combination of ligands to localize CRH₂ receptors in brain and in several peripheral regions using receptor autoradiography.