Corticotropin-releasing factor (CRF) is a major hypophysiotropic peptide regulating pituitary-adrenal response to stress, and it Is also widely expressed in the central nervous system. (2), with some major areas of mismatch (14). One potential basis for this discrepancy derives from the recent identification of a distinct CRF-binding protein (CRF-BP), which is prominently expressed in brain, including subsets of CRF-containing pathways and/or their targets (15). The identification of this distinct high-affinity CRF-binding moiety, which is in a position to modify central and pituitary actions of CRF by autocrine or paracrine mechanisms, could limit the ability to draw inferences concerning CRF-R distributions on the basis of ligand-binding patterns alone. The present study employed hybridization histochemical methods to provide an initial overview ofthe cellular sites ofexpression ofthe CRF-R in rat brain and pituitary. Dual staining approaches were used to directly compare loci of CRF-R mRNA expression with those of ACTH in the pituitary and of CRF-BP in brain. MATERIALS AND METHODSIn Situ Hybridization. Male (n = 7) and female (n = 3) adult Sprague-Dawley albino rats (200-300 g) were perfused transcardially with 4% paraformaldehyde in 0.1 M pH 9.5 sodium borate buffer, and regularly spaced series of 20-to 30-,umthick frozen sections through brain and pituitary were taken as described (16). Radiolabeled antisense and sense (control) complementary RNA (cRNA) copies were synthesized from a full-length or N-terminal 344 bp fragment of the rat CRF cDNA subcloned in pBluescript KS vector (Stratagene). (a-33P]UTP was used for probe synthesis, and in situ hybridization was performed as previously described (16,17). cRNA probes to full-length rat CRF-R coding region were labeled to 40-60% total incorporation, and hybridization was carried out under high stringency [50%6 (vol/vol) formamide with final washes carried out in 0.2x SSC at 700C (lx SSC is 0.15 M NaCl/0.015 M sodium citrate, pH 7.0)].Combined in Situ Hybridization and Immun emis try. Concurrent localization of ACTH immunoreactivity -ir) and CRF-R mRNA in pituitaries was performed by using a modification of a procedure described by Watts and Swanson (18). This involved first applying a conventional biotinavidin-immunoperoxidase protocol, to localize primary antibodies raised in rabbit against the rat corticotropin fragment ACTH-(23-39). Rat ACTH-(23-39) was coupled to human a-globulins with bisdiazotized benzidine, and antisera were

In recent studies to clone and characterize genes coding for the corticotropin-releasing factor-binding protein (CRF-BP), analysis of the tissue distribution of the CRF-BP gene indicated a high level of expression in the rat brain. We have now characterized by immunohistochemical and hybridization histochemical means the cellular localization of CRF-BP protein and mRNA expression, respectively. Results from both approaches converged to indicate that CRF-BP is expressed predominantly in the cerebral cortex, including all major archi-, paleo-, and neocortical fields. Other prominent sites of mRNA and protein expression include subcortical limbic system structures (amygdala, bed nucleus of the stria terminalis), sensory relays associated with the auditory, olfactory, vestibular, and trigeminal systems, several raphe nuclei, and a number of cell groups in the brainstem reticular core. Expression in the hypothalamus appears largely limited to the ventral premammillary and dorsomedial nuclei; only isolated CRF-BP-stained cells are apparent in neurosecretory cell groups. Dual immunstaining for CRF and CRF-BP revealed a partial colocalization in some of these regions. In addition, prominent CRF-BP-stained terminal fields have been identified in association with CRF-expressing cell groups in circumscribed hypothalamic and limbic structures. In the anterior pituitary, CRF-BP mRNA and immunoreactivity were colocalized with corticotropin-immunoreactivity in a majority of corticotropes. Thus, CRF-BP could serve to modify the actions of CRF by intra-and intercellular mechanisms, in CRF-related pathways in the central nervous system and pituitary.

Corticotropin-releasing factor (CRF), is a potent stimulator of synthesis and secretion of preopiomelanocortin-derived peptides. Although CRF concentrations in the human peripheral circulation are normally low, they increase throughout pregnancy and fall rapidly after parturition. Maternal plasma CRF probably originates from the placenta, which responds to the bioactive peptide and produces the peptide and its messenger RNA. Even though CRF concentrations in late gestational maternal plasma are similar to those in rat hypothalamic portal blood and to those that can stimulate release of adrenocorticotropic hormone (ACTH) in vitro, maternal plasma ACTH concentrations increase only slightly with advancing gestation and remain within the normal range. Several groups have now reported the existence of a CRF-binding protein in human plasma which inactivates CRF and which has been proposed to prevent inappropriate pituitary-adrenal stimulation in pregnancy. The binding protein was recently purified from human plasma. We have now isolated and partially sequenced the binding protein, allowing us to clone and characterize its complementary DNA from human liver and rat brain. Expression of the cDNAs for human and rat binding protein in COS7 cells showed that these proteins bind CRF with the same affinity as the native human protein. Both rat and human recombinant binding proteins inhibit CRF binding to a CRF antibody and inhibit CRF-induced ACTH release by pituitary cells in vitro.

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