The actions of tuberoinfundibular peptide on the hypothalamo-pituitary axes.

Tuberoinfundibular peptide is a recently discovered agonist for the PTH receptor-2; the latter has a wide distribution including the external zone of the median eminence of the hypothalamus, suggesting a role in neuroendocrine function. We have investigated the effects of tuberoinfundibular peptide on the hypothalamo-pituitary axes in vitro and in vivo. Tuberoinfundibular peptide had effects on the hypothalamo-pituitary-adrenal axis with increased release of ACTH-releasing factor (tuberoinfundibular peptide 100 nM 4.4 +/- 0.6 pmol/explant vs. control 2.9 +/- 0.4 pmol/explant, P < 0.001) and increased release of arginine vasopressin (tuberoinfundibular peptide 100 nM 563.5 +/- 55.5 fmol/explant vs. control 73.4 +/- 9.6 fmol/explant, P < 0.01) from in vitro hypothalamic explants. Intracerebroventricular administration of tuberoinfundibular peptide and PTH((1-34)) resulted in elevated plasma ACTH at 10 min post injection (saline 13.5 +/- 2.1 pg/ml, tuberoinfundibular peptide 3 nmol 32.3 +/- 4.0 pg/ml; P < 0.01 to saline: PTH((1-34)) 10 nmol 28.9 +/- 3.2 pg/ml: P < 0.05 to saline). Tuberoinfundibular peptide also had both in vitro and in vivo effects on the hypothalamo-pituitary-gonadal axis with increased release of LH-releasing hormone (tuberoinfundibular peptide 100 nM 28.5 +/- 5.1 fmol/explant vs. control 19.3 +/- 2.5 fmol/explant, P < 0.05) from in vitro hypothalamic explants. Both intracerebroventricular and peripheral administration of tuberoinfundibular peptide had effects on the hypothalamo-pituitary-gonadal axis. Intracerebroventricular injection of tuberoinfundibular peptide increased plasma LH (tuberoinfundibular peptide 10 nmol 0.70 +/- 0.09 ng/ml vs. saline 0.42 +/- 0.04 ng/ml at 10 min, P < 0.05). Intraperitoneal administration of tuberoinfundibular peptide also increased plasma LH (tuberoinfundibular peptide 30 nmol 0.53 +/- 0.09 ng/ml vs. saline 0.21 +/- 0.04 ng/ml at 10 min, P < 0.05). In addition to these actions on the hypothalamo-pituitary-adrenal and hypothalamo-pituitary-gonadal axes, an increased release of GH-releasing factor (GRF) from hypothalamic explants (tuberoinfundibular peptide 100 nM 770.9 +/- 90.7 pg/explant vs. control 657.8 +/- 77.7 pg/explant, P < 0.01) was observed. Overall, these data show the actions of tuberoinfundibular peptide on the hypothalamo-pituitary axes and suggest that it may play a role in the control of the hypothalamo-pituitary-adrenal and hypothalamo-pituitary-gonadal axes.

The novel hypothalamic peptide ghrelin stimulates food intake and growth hormone secretion.

Ghrelin, a novel 28 amino acid peptide found in hypothalamus and stomach, was recently identified as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R). We have now found that both intracerebroventricular (i.c.v.) and intraperitoneal (i.p.) administration of ghrelin in freely feeding rats stimulated food intake. The onset of increased feeding was rapid and after i.c.v. administration was sustained for 24 hours. Following i.c.v. administration of 3 nmol ghrelin, the duration and magnitude of the feeding stimulation was similar to that following 5 nmol neuropeptide Y (NPY). Plasma growth hormone (GH) concentration increased following both i.c.v. and i.p. administration of ghrelin. Release of adrenocorticotrophic hormone (ACTH) was stimulated and thyroid stimulating hormone (TSH) inhibited following i.c.v. administration of ghrelin. These data suggest a possible role for the newly identified endogenous hypothalamic peptide, ghrelin, in stimulation of feeding and growth hormone secretion.

Rat tumour necrosis factor-alpha: expression in recombinant Pichia pastoris, purification, characterization and development of a novel ELISA.

Tumour necrosis factor-alpha is a pro-inflammatory cytokine involved in many aspects of acute phase and immune responses. Species specificity in the biological action and receptor binding of TNF-alpha make it desirable to use homologous reagents in experimental models, both in vivo and in vitro. As the rat is the model of choice in many investigations on fever, trauma and pathology, there is a need for specific rat reagents. In this paper, we describe the production of recombinant rat TNF-alpha in milligram quantities, using a methylotrophic yeast expression system, Pichia pastoris. Recombinant TNF-alpha was produced intracellularly in a soluble form, cells were lysed and the protein purified by ammonium sulphate precipitation, Sephadex G75 fractionation and finally, ion-exchange chromatography. The purified recombinant rat TNF-alpha had a molecular mass of 17401.38 +/- 0.38 Da, which is within 1 Da of the value predicted by the sequence data, taking into account N-acetylation of the initial methionine residue and a single disulphide bridge between amino acids 70 and 101. Recombinant rat TNF-alpha was shown to be 20 x fold more biologically active in the WEHI cytotoxicity assay, than the human standard preparation. Polyclonal antibodies were raised against purified recombinant rat TNF-alpha, these reagents were used to develop a novel enzyme-linked immunosorbant assay (ELISA). The ELISA was sensitive to 10 pg.ml- 1 rat TNF-alpha and was specific for TNF-alpha, showing no cross-reactivity with rat IL-1alpha, rat IL-1beta, rat IL-1Ra or rat IL-6. The ELISA was used to measure TNF-alpha in the plasma of rats injected with bacterial endotoxin and in cultures of rat white blood cells. The ELISA was shown to be a robust method suitable for use in assaying samples generated in both in vivo or in vitro experiments.

Rat interleukin 6: expression in recombinant Escherichia coli, purification and development of a novel ELISA.

Interleukin 6 (IL-6) is a cytokine involved in many aspects of the acute phase and immune responses. Cloning of rat IL-6 cDNA into the pET-21d expression plasmid under control of a bacteriophage T7 RNA polymerase promoter system allowed isopropylthio-galactopyranoside (IPTG)-inducible production of recombinant rat IL-6 in Escherichia coli. The cloning, expression and purification of rat IL-6 is described. In this expression system, rat IL-6 was produced in insoluble inclusion bodies. The protein was solubilized in 6 M guanidine hydrochloride and refolded in a glutathione redox system. Refolded rat IL-6 was purified to homogeneity using anion-exchange chromatography on SP-Trisacryl. The purified recombinant rat IL-6 had a molecular mass of 21 756.38+/-0.25 Da, which is within 0.01% of the predicted value, taking into account cleavage of the N-terminal methionine residue and the formation of two disulfide bridges. Recombinant rat IL-6 was 2-3-fold more bioactive than the human standard preparation in the B9 hybridoma bioassay. Purified rat IL-6 was used to raise polyclonal antibodies in sheep and these reagents were used to develop a novel rat IL-6 enzyme-linked immunosorbent assay (ELISA). The ELISA is sensitive to 10 pg/ml and has been shown to detect IL-6 in plasma from rats injected with lipopolysaccharide (LPS).

Differences in carbohydrate composition of FSH preparations detected with lectin-ELISA systems.

FSH is a glycoprotein containing N-linked carbohydrates which exhibit a variety of forms ranging from mono- to multibranched structures. Variation in glycosylation, particularly the degree of terminal sialylation, determines the half-life of the hormone and hence its in vivo bioactivity. The glycoform content of FSH preparations can differ according to the source (e.g. pituitary, urine), cell line (for rDNA-derived material) and selectivity of purification procedures, and may create difficulties in the preparation and characterization of standards and therapeutic products. In order to develop a simple method to detect changes in glycocomposition, an FSH ELISA was modified by the incorporation of lectins of recognized sugar specificity, and used to examine the terminal sugar composition of ampouled preparations of pituitary, urinary and rDNA-derived FSH. FSH was captured with a specific monoclonal antibody (MAb) and detected with either biotinylated anti-FSH MAb (ELISA) or the sugar-specific lectins (L-ELISA) from Triticum vulgaris (sialic acid, SA), Sambucus nigra (alpha 2,6-linked SA), Maackia amurensis (alpha 2,3-linked SA) or Ricinus communis (free terminal galactose; GAL). Relative estimates of the amounts of terminal SA, its different forms and GAL were derived from the L-ELISA/ELISA data compared with the highly sialylated 1st International Standard for pituitary FSH (IS) 83/575. All the FSH preparations had less SA than the IS with the ratio of alpha 2,3- and alpha 2,6-linked SA varying between preparations. The amounts of alpha 2,6-linked SA relative to the IS were not significantly different in the urinary and pituitary preparations whereas alpha 2,3-linked SA in all preparations was generally less than that of the standard.(ABSTRACT TRUNCATED AT 250 WORDS)