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Design and synthesis of heterofunctional V1a-selective vasopressin receptor ligands with lysine at position 9

J. Howl, D. C. New, and M. Wheatley

ABSTRACT

A peptide analogue of [8-arginine]vasopressin (AVP) with Lys substituted for Gly at position 9 ([d(CH2)5Tyr(Me)2LysNH2 9]AVP; ALVP) has been synthesized as a precursor for the production of heterofunctional vasopressin receptor ligands. Three heterofunctional ligands have been prepared by attaching biotin and a photoreactive cross-linker capable of iodination, either alone or in combination, to the ε-amino group of Lys at position 9 in ALVP. The binding characteristics of these novel ligands have been determined at the V1a and V2 vasopressin receptors by employing membrane preparations of rat liver and kidney respectively. All of the analogues synthesized during the course of this study bound selectively, and with high affinity, to the V1a vasopressin receptor subtype. Our results demonstrate that the strategies described in this paper provide a convenient means of synthesizing heterofunctional vasopressin receptor ligands with preservation of subtype-specific, high affinity binding characteristics. These parameters establish the potential value of the analogues as probes for investigating V1a receptor structure and function.

DOI:
https://doi.org/10.1677/jme.0.0090123
Volume 9: Issue 2,
Pages:
123–129 (7 total)
Free access

βA- and βC-activin, follistatin, activin receptor mRNA and βC-activin peptide expression during rat liver regeneration

E J Gold, X Zhang, A M Wheatley, S L Mellor, M Cranfield, G P Risbridger, N P Groome, and J S Fleming

The mRNA expression of two activin growth factor subunits (βA- and βC-activin), activin receptor subunits (ActRIIA, ActRIIB) and the activin-binding protein follistatin, and peptide expression of βA-activin and βC-activin subunits, were examined in regenerating rat liver after partial hepatectomy (PHx). Liver samples were collected from adult, male Sprague–Dawley rats, 12–240 h (n=3–5 rats per time point) after PHx or from sham-operated controls at the same time points. Hepatocyte mitosis and apoptosis were assessed histologically and by in situ cell death detection. RT and PCR were used to assess relative gene expression. βA- and βC-activin peptide immunoreactivity was assessed in liver and serum samples by western blotting, whereas cellular expression was investigated by immunohistochemistry, using specific monoclonal antibodies. βA- and βC-activin mRNA dropped to < 50% of sham control values 12 h after PHx and remained at this level until 168 h post-PHx, when βA-activin expression increased to three times sham control values and βC-activin mRNA returned to pre-PHx levels. A peak in follistatin expression was observed 24–48 h post-PHx, coincident with an increase in hepatocyte mitosis. No changes were observed in ActRIIA mRNA, whereas ActRIIB expression paralleled that of βA-activin mRNA. βC-activin immunoreactive homo- and heterodimers were observed in regenerating liver and serum. Mitotic hepatocytes frequently contained βC-activin immunoreactivity, whereas apoptotic hepatocytes were often immunoreactive for βA-activin. We conclude that βA- and βC-activin subunit proteins are autocrine growth regulators in regenerating liver and when expressed independently lead to hepatocyte apoptosis or mitosis in a subset of hepatocytes.

DOI:
https://doi.org/10.1677/jme.1.01657
Volume 34: Issue 2,
Pages:
505–515 (11 total)