In vitro regulation of corticotropin-releasing hormone.

Studies involving regulation of corticotropin-releasing hormone (CRH) in vitro have been used to validate findings obtained in vivo and more importantly have been used as model systems to better understand signalling mechanisms responsible for the expression of the CRH gene and peptide. Many in vitro studies examining CRH have utilized hypothalamic tissue while a few have focused on the amygdala. Clonal cell lines have also been utilized as models of central nervous system CRH neurons. Stimuli that have been implicated in regulating hypothalamic CRH regulation in vitro include protein kinase A (PKA) and protein kinase C (PKC) activators, glucocorticoids, biogenic amines, cytokines and the gaseous neurotransmitters. Amygdalar CRH levels in vitro are affected by some of the same stimuli that regulate hypothalamic CRH; however there is evidence supporting differential regulation of CRH in these two brain regions by some of the same stimuli. Only a few studies in aggregate have investigated signal transduction mechanisms and these studies have focused on PKA- and glucocorticoid-mediated changes in CRH expression. Thus, much more investigative work in better understanding CRH regulation in vitro is needed.

Corticotropin-releasing hormone (CRH) expression and protein kinase A mediated CRH receptor signalling in an immortalized hypothalamic cell line.

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 CRH1 receptor RNA via the reverse transcriptase-polymerase chain reaction. In addition, the cell line expressed the neuronal marker, microtubule-associated protein-2. We verified 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 profile to that identical with oxidized CRH standard. In addition, IVB cells exhibited high affinity 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) immunostaining 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.

A molecular recognition hypothesis for nonpeptides: Na+ K+ ATPase and endogenous digitalis-like peptides.

The molecular recognition hypothesis for peptides is that binding sites of ligands and their receptors are encoded by short, complementary segments of DNA. A corollary hypothesis for nonpeptide ligands posited here is that peptide replicas may be encoded by the DNA segment complementary to the receptor binding sites for nonpeptides. This corollary was tested for digitalis. a family of cardiotonic and natriuretic steroids including ouabain. A hexapeptide (ouabain-like peptide, OLP) complementary to a ouabain binding site on sodium potassium dependent adenosine triphosphatase (Na+ K+ ATPase) exhibited activity in a digitalis bioassay. Antisera to the complementary peptide (OLP) stained the neurohypophysis in an immunocytochemical procedure. The complementary peptide was found to share an identical 4-amino acid region with the 39-amino acid glycopeptide moiety of the vasopressin-neurophysin precursor. This glycopeptide was isolated from pituitary extracts; it exhibited digitalis-like activity in the submicromolar range and cross-reacted with complementary peptide antibodies. Another digitalis-like substance with high activity also was detected in the extracts. These results demonstrate that the vasopressin-neurophysin glycopeptide has digitalis-like activity. Moreover, the findings are consistent with the hypothesis that peptide mimetics of nonpeptides are encoded in the genome.

Regulation of expression of secretogranin II mRNA in female rat pituitary and hypothalamus.

Secretogranin II (SgII) is an acidic 86-kD protein which is synthesized by most neuroendocrine cells but occurs in greatest abundance in the anterior pituitary gland where it is localized primarily in gonadotrophs. In the present studies, we investigated the regulation of SgII mRNA expression in the anterior pituitary gland by estrogens and gonadotropin-releasing hormone (GnRH) and compared the results to luteinizing hormone beta-subunit (LH beta) mRNA expression. Molecular cloning and nucleotide sequence analysis of a rat pituitary SgII cDNA revealed a derived amino acid sequence identical with that previously reported for the rat adrenal. Not previously reported were five putative nuclear localization signals, four of which coincided with dibasic residues previously thought to serve as proteolytic cleavage sites. In Northern blots, SgII mRNA was found in high abundance in the anterior pituitary gland, in moderate abundance in the brain and adrenal, and in low abundance in the ovary and testis. Measurements of pituitary SgII mRNA during the rat 4-day estrous cycle revealed an inverse relationship with LH beta mRNA: SgII mRNA decreased, whereas LH beta mRNA increased as the cycle progressed. Increases in pituitary SgII mRNA and LH beta mRNA levels occurred after ovariectomy, and decreases occurred after estrogen treatment of such animals. Likewise, pituitary SgII mRNA and LH beta mRNA levels decreased after treatment of ovariectomized animals with a GnRH antagonist. In contrast, ovariectomy significantly decreased SgII mRNA levels in the hypothalamus, and estrogen treatment increased its levels. Our studies reveal that ovarian estrogens and hypothalamic GnRH exert similar effects on SgII mRNA and LH beta mRNA expression in the pituitary. However, since their expression is inverse during the rat estrous cycle, other unidentified regulatory factors with differential effects on their expression may intervene in the regulation of SgII and LH beta mRNA levels.