Steve Sutton

Neurotransmitters and peptides modulate the release of immunoreactive corticotropin-releasing factor from cultured human placental cells.

The substances stimulating the release of immunoreactive corticotropin-releasing factor from cultured human placental cells were investigated. Monolayer primary cultures of trophoblast cells from pregnant women at term were used. The immunoreactive corticotropin-releasing factor released in the culture medium eluted from high-performance liquid chromatography with the same retention time as human corticotropin-releasing factor. Norepinephrine and acetylcholine increased immunoreactive corticotropin-releasing factor release into the culture medium in a dose-related manner. Epinephrine was partially active, whereas dopamine and serotonin did not induce significant changes of immunoreactive corticotropin-releasing factor release from placental cultures. Angiotensin II, interleukin-1, oxytocin, and arginine-vasopressin also increased placental immunoreactive corticotropin-releasing factor release in a dose-related manner, whereas other peptides (vasoactive intestinal peptide, substance P, somatostatin, atrial natriuretic factor, interleukin-2) were ineffective. These results showed that several neurotransmitters and peptides stimulate the release of immunoreactive corticotropin-releasing factor from placental cells, suggesting their possible involvement in the physiologic regulation of placental immunoreactive corticotropin-releasing factor release during pregnancy and parturition.

Neurotransmitter modulation of corticotropin releasing factor secretion into the hypophysial-portal circulation

Secretion of corticotropin releasing factor (CRF) from the hypothalamic paraventricular nuclei into the hypophysial-portal circulation is modulated by a variety of neuronal afferents. Effects of intracerebroventricular acetylcholine (ACH), gamma-aminobutyric acid (GABA) and epinephrine (EPI) on immunoreactive (ir) CRF concentration in portal plasma were directly evaluated in urethanized male rats. ACH (0.1-10 nmole) administration was associated with a dose-dependent elevation of portal irCRF which was attenuated by pretreatment with either muscarinic or nicotinic receptor antagonists. GABA (0.1-10 nmole) also caused inhibition of irCRF concentration in the portal plasma which was prevented by bicuculline pretreatment. Finally, EPI (0.1-10 nmole) facilitated irCRF secretion via alpha 1- and beta-adrenergic mechanisms. These observations provide the first direct information on possible neurotransmitter actions on hypothalamic CRF secretion.

Ovine genomic urocortin: cloning, pharmacologic characterization, and distribution of central mRNA

Urocortin (Ucn), the newest member of the corticotropin-releasing factor (CRF) family of peptides, has been demonstrated to have significant physiologic and behavioral effects following its peripheral and central administration, respectively. In order to assess the differences in Ucn across species, an 18-kb sheep genomic DNA fragment encoding urocortin was isolated by the hybridization screening of a lambda phage library with a probe generated from rat urocortin (rUcn) cDNA. The sheep clone contains a region that is 84% and 88% homologous to the coding region of rUcn and human Ucn (hUcn), respectively and encodes an ovine Ucn (oUcn) that is predicted to be identical to the rat peptide. Competitive binding assays demonstrated oUcn to have a high affinity (Ki=0.1 nM) for the sheep CRF-binding protein (CRF-BP) and localization studies by in situ hybridization have shown that the distribution of oUcn messenger RNA in sheep brain shares with that of rUcn in rat brain a predominant locus of expression in the Edinger-Westphal nucleus of the midbrain, though some secondary sites of expression reported in rat are not conserved. These findings demonstrate that, even across diverse species, Ucn is highly conserved with respect to its structure and pharmacology unlike CRF where significant amino acid substitutions between the rat/human and sheep peptides may underlie differences in neuroendocrine regulation.

Facilitatory role of neuropeptide Y on the onset of puberty : effect of immunoneutralization of neuropeptide Y on the release of luteinizing hormone and luteinizing-hormone-releasing hormone

To examine the role of neuropeptide Y (NPY) in the first luteinizing hormone (LH) surge of puberty, the effect of passive immunoneutralization of NPY with antiserum against NPY (anti-NPY) injected centrally (third ventricle) or peripherally (jugular vein) was studied in pubertal female rats on the day of first proestrus. Both peripheral and central anti-NPY administration reduced the magnitude of the LH surge during the afternoon of first proestrus; however, the central route of administration appeared to be most effective. Centrally administered anti-NPY also reduced the magnitude of proestrous LH-releasing hormone (LHRH) release into pituitary portal blood in these rats. These results suggest that endogenous NPY plays a facilitatory role in the generation of the LHRH surge necessary for preovulatory gonadotropin release and puberty.

Characterization of a sheep brain corticotropin releasing factor binding protein.

We report here the identification, purification and cDNA cloning of a corticotropin releasing factor (CRF) binding protein(s) (CRF-BP) from sheep brain. Native sheep and rat brain CRF-BP and recombinant rat CRF-BP were shown to be N-glycosylated. Two membrane associated forms of brain CRF-BPs of 33 and 35 kDa were purified from sheep brain homogenates after solubilization in the presence of detergent. N-Terminal sequence analysis revealed that the 35 kDa protein is proteolytically cleaved near the N-terminus giving rise to an 18 amino acid peptide and a 33 kDa CRF-BP. Both the purified 33 and 35 kDa ovine CRF-BPs could be specifically cross linked to ovine [125I]CRF and human [125I]CRF. In contrast, recombinant rat CRF-BP can only be cross-linked to human [125I]CRF. A 1.7 kb cDNA clone (Basil 7) encoding an open reading frame for a 324 amino acid CRF-BP precursor was cloned from a sheep brain lambda gtlO cDNA library and was shown to have 85% and 87% amino acid homology to the rat and human proteins, respectively. Competitive binding analysis of the recombinant sheep CRF-BP (Basil 7) expressed in CHO cells revealed that it binds human and ovine CRF with high affinity. However, the recombinant sheep CRF-BP (Basil 7) had approximately 50-fold higher affinity for human CRF than for the ovine peptide. These data present the first biochemical proof that CRF-BP is in the brain and provides evidence for the existence of different forms of CRF-BP which have evolved across species to regulate CRF.

Lack of Correlation Between Immunoreactive Corticotropin- Releasing Factor Concentration Profiles in Hypophysial-Portal and Peripheral Plasma

Corticotropin‐releasing factor (CRF‐41) is the primary mediator of adrenocorticotropin secretion from the adenohypophysis. This 41‐amino‐acid peptide is synthesized in perikarya of the paraventricular nuclei, transported to nerve terminals in the external zone of the median eminence and released into the hypophysial‐portal circulation. It is also synthesized in multiple extrahypothalamic and peripheral sites. In addition, immunoreactive (ir) CRF‐41 is present in the systemic circulation, raising the possibility that systemic measurements might provide a useful index of hypothalamic irCRF‐41 secretion. This hypothesis was tested in several rat models. Neither bilateral destruction of hypothalamic irCRF‐41 producing perikarya, nor infundibular stalk transection altered peripheral plasma irCRF‐41 concentration. Furthermore, central administration of norepinephrine, an agent previously shown to evoke irCRF‐41 secretion into the portal circulation, was without effect on peripheral irCRF‐41 concentration. Finally, while increased irCRF‐41 levels in both the hypophysial‐portal and the peripheral circulation were associated with nitroprusside‐induced hypotension, bilateral paraventricular nuclei lesions blocked irCRF‐41 secretion into the hypophysial‐portal circulation without blunting the rise observed in the peripheral circulation. The source of peripheral irCRF‐41 remains undetermined; however, the adrenals may be excluded as bilateral adrenalectomy failed to alter circulating irCRF‐41 levels. Therefore, our observations do not support the concept that peripheral irCRF‐41 levels provide a useful index of hypothalamic secretion of this peptide into the hypophysial‐portal circulation under the conditions tested.

Characterization of the N-linked glycan of recombinantly-expressed corticotropin releasing factor binding protein

The N-linked glycan present on corticotropin releasing factor binding protein (CRFBP) recombinantly expressed in CHO cells has been characterized using a variety of techniques including sequential hydrolysis with specific exoglycosidases monitored with reversed-phase high performance liquid chromatography (RP-HPLC), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ES-MS). Determining the intact glycan mass and comparing possible glycan compositions with entries in the Complex Carbohydrate Database facilitated the choice of enzymes. Tandem mass spectrometry (MS/MS) was used to confirm the structure of the glycan. Using this approach, complementary information could be obtained through specific glycosidase reactions monitored with MS and MS n fragmentation of the intact or enzymatically-modified glycopeptides. No evidence was found for the presence of sites of O-linked glycosylation. In addition, to understand better the role of the glycan on CRFBP, we characterized the in vitro binding affinity of a mutant non-glycosylated [Gln180] CRFBP and an enzymatically deglycosylated form of CRFBP to human corticotropin releasing factor (hCRF).

Competitive antagonists of the corticotropin releasing factor (CRF) scanned with a i-(i+3) Glu Lys Bridge

CRF [1] is involved in a wide spectrum of central nervous system (CNS)-mediated effects, suggesting that this peptide plays an important role within the brain, especially in response to stressful stimuli [2]. Systematic SAR investigations have resulted in the development of CRF antagonists such as [3], members of the (standard) family [4] and conformationally restricted analogs [5] that are effective in the CNS. Those results, predictive methods and physicochemical measurements have suggested that CRF and its family members (urotensins and sauvagine) assume an conformation when interacting with the CRF receptors. To further test this hypothesis, we have scanned the whole rat/human sequence with an i-(i + 3) bridge consisting of the Glu-Xaa-Xbb-Lys scaffold which we and others had shown to be compatible with maintenance or enhancement of structure in at least some unpredictable cases.