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Corticotropin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays an important role in the stress response in the hypothalamus. We describe the development of an immortalized hypothalamic cell line which expresses CRH. We hypothesized that this cell line would possess the relevant characteristics of parvocellular CRH-expressing neurones such as glucocorticoid receptor (GR) expression and vasopressin (VP) coexpression. For production of hypothalamic cells, embryonic day 19 rat pup hypothalami were dissected and dissociated into tissue culture dishes. They were immortalized by retrovirus-mediated transfer of the SV40 large T antigen gene at 3 days of culture and then screened for expression of CRH following dilution cloning. One cell line was chosen (IVB) which exhibited CRH-like immunoreactivity (CRH-LI) and expressed CRH, VP and CRH 1 receptor RNA via the reverse transcriptase-polymerase chain reaction. In addition, the cell line expressed the neuronal marker, microtubule-associated protein-2. We veri®ed that the CRH-LI from IVB cell lysates coeluted with CRH standard via reversed-phase high-performance liquid chromatography (HPLC). Furthermore, oxidation of the lysate converted its HPLC pro®le to that identical with oxidized CRH standard. In addition, IVB cells exhibited high af®nity binding to CRH. Incubation of IVB cells with CRH lead to increases in cAMP levels and protein kinase A activity in a concentration-dependent manner. Incubation of IVB cells with CRH also resulted in increases in phospho-cyclic-AMP response element binding protein (CREB) imunostaining as detected by immunocytochemical analysis. Finally, CRH treatment of IVB cell lines has been linked to CREB-mediated gene expression as determined via the PathDetect CREB trans-reporting system. The characteristics of IVB cells, such as CRH and VP coexpression, GR expression and a biologically active CRH-R1-mediated signalling pathway, suggest that this neuronal cell line may serve as model of parvocellular CRH neurones.Corticotropin-releasing hormone (CRH) is a 41 amino acid containing neuropeptide which coordinates the neuroendocrine, behavioural, autonomic and immune responses to stress (1, 2). It is secreted from the hypothalamus and acts on pituitary corticotrophs to enhance secretion of adrenocorticotropic hormone (ACTH) and other pro-opiomelanocortin products. ACTH, in turn, stimulates glucocorticoid synthesis and secretion in the adrenal cortex. CRH containing parvocellular neurones coexpress vasopressin (VP) and are subject to negative feedback by glucocorticoids following stressful conditions (3).The cellular effects of CRH are mediated by high-af®nity receptors (4±7). In addition to the anterior and intermediate lobes of the pituitary (8), CRH receptors are located in a wide variety of locations in the central nervous system (CNS), including the parvocellular neurones of the paraventricular nucleus where they are induced by stress (9). There are two CRH receptor types.

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A molecular recognition code has been hypothesized to exist in which ligands and their binding sites are encoded on complementary segments of genomic DNA. We have tested this hypothesis by generating a rabbit antibody to a synthetic decapeptide (complementary peptide) encoded by an RNA complementary to the mRNA for luteinizing hormonereleasing hormone (LHRH) and determining whether this antibody recognizes the LHRH receptor. When the antibody was used for immunoperoxidase staining of enzymatically dispersed rat anterior pituitary cells, only those that contained and secreted luteinizing hormone (i.e., the gonadotropes) were recognized. This staining could be abolished by preincubation with the complementary peptide or with an LHRH agonist, suggesting that the antibody is specific to the complementary peptide and is directed at the binding site of the receptor. Further evidence that the antibody recognizes the LHRH receptor was obtained in immunoblot experiments on solubilized receptors from pituitary glands. Immunoperoxidase staining with the antibody revealed two bands at 60 kDa and 51 kDa, which are values similar to those previously obtained for the LHRH receptor in photoaffimity-labeling experiments. The staining of these bands was inhibited by preincubation with the complementary peptide or an LHRH agonist. The antibody as well as the complementary peptide to LHRH also suppressed LHRH-stimulated luteinizing hormone release in a quantitative reverse hemolytic plaque assay, presumably by binding to the LHRH receptor and by binding LHRH, respectively. These findings suggest that the synthetic decapeptide whose sequence is specified by the complementary RNA to LHRH mRNA is sufficiently similar to an LHRH binding site that the peptide not only binds LHRH but was also recognized by the immune system as such a site. These findings provide strong support for the hypothesis that recognition molecules are encoded by complementary segments of genomic DNA.A hypothesis has been developed recently that the binding sites of interacting proteins are encoded by complementary strands ofgenomic DNA (1). This molecular recognition code hypothesis arose from the observations that codons for hydrophobic amino acids are complemented by those for hydrophilic amino acids and vice versa and that the average tendency of codons for uncharged (slightly hydrophilic) amino acids is to be complemented by codons for other uncharged amino acids (2). Based on this pattern it was suggested that two peptides represented by complementary RNAs would assume conformations that resulted in specific and high-affinity binding of the pair. Consistent with this suggestion was the finding that the mRNA sequences complementary to the mRNA sequences for the receptors to epidermal growth factor, interleukin 2, and transferrin bore regions of high amino acid and nucleotide homology with their respective ligands (3). This hypothesis was tested by preparing synthetic peptide counterparts of corticotropin (ACTH) and y-endorphin that were specified by R...

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The structural similarities between human PRL (hPRL) and human GH (hGH) suggest a common evolutionary origin of the two hormones. The existence of these similarities also suggests the existence of a developmental intermediate cell type which has the potential to produce and secrete both hPRL and hGH. We have applied reverse hemolytic plaque assays to monitor the secretions of individual fetal human pituitary cells to determine if any of these cells secrete both hPRL and hGH. When hPRL and hGH assays were performed sequentially on cells obtained from five fetuses of 18-22 weeks gestational age, we consistently found a subpopulation of cells that formed plaques in both assays, i.e. they secreted both hPRL and hGH. The majority of fetal cells secreting hPRL also secreted hGH at this stage of development. These data were corroborated with light and electron microscopic immunocytochemical localization of hPRL and hGH in a subpopulation of previously identified fetal somatotrophs. These findings suggest that in addition to the classical somatotrophs and lactotrophs, the fetal human pituitary contains an additional cell type which secretes both hPRL and hGH. The dual hormone-secreting cell may represent a common progenitor of these classical cell types. It also may be the cell of origin of those pituitary tumors that secrete both PRL and GH.