The characterization, localization and regulation of endothelin in ovine pars intermedia
Introduction
The potent vasoconstrictor, endothelin-1 (ET-1), a 21-amino acid peptide, was first isolated and the gene cloned by Yanigasawa et al. [1]from cultured porcine aortic endothelial cells. Subsequent molecular studies revealed a further two distinct isoforms of endothelin, reflecting the identification of three related endothelin (ET) genes encoding ET-1, ET-2 and ET-3 2, 3, 4, 5. Thus, ET-1, -2 and -3 are products of the same gene family, and are present in both endothelial [6]and non-endothelial tissues 7, 8, 9. The three ET isoforms appear to be expressed in a tissue-specific manner [10]. For example, ET-3 is predominantly expressed in intestine, kidney, lung, adrenal, liver, pituitary and brain 11, 12, 13. ET-2 is abundant in lung and colon [11], whilst ET-1, although present in brain 11, 14, lungs, aorta and stomach 11, 13, is the only form expressed by endothelial cells (EC) [1].
In the rat brain and, in particular, the hypothalamo-pituitary axis (HPA), ET-1 and ET-3 are both present 9, 10, 15; ET-1 is present in the supraoptic nucleus and paraventricular nucleus and immunoreactive ET has been found in neurons of the posterior pituitary [16].
The bovine pituitary intermediate lobe and the rat intermediate lobe have been shown to contain endothelin-like peptides 17, 18which, it has been speculated, may be implicated in the regulation of prolactin release. In this context prolactin release-inhibiting factor has been reported in extracts of rat and bovine neurointermediate lobe (NIL) of the pituitary gland [19]. This material, when isolated by Sephadex G-25 chromatography or by C-18 high performance liquid chromatography (HPLC), co-elutes with the synthetic forms of all members of the mammalian ET family of peptides, and when preabsorbed with an ET anti-serum which recognizes all members of the ET family of peptides, its in vitro bioactivity is abolished. These results suggest that a prolactin release-inhibiting factor is present in the NIL and is an ET-like peptide.
ET-3 is the predominant form expressed in the anterior pituitary (AP) and a local paracrine role for the ET isoform ET-3 in prolactin release has a precedent in the rat anterior pituitary (AP) where ET-3 has been shown to stimulate the release of pituitary hormones, such as luteinizing hormone, in addition to inhibiting the release of prolactin from the lactotroph 20, 21, 22. Two distinct subtypes of endothelin receptors have been cloned and expressed: the ETA subtype 23, 24, 25which recognizes ET-1 preferentially; and the ETB receptor subtype 26, 27which recognizes all three isoforms equally. Anterior pituitary cells in culture respond to ET with activation of calcium mobilizing pathways through specific ETA receptors [28].
Taken together, these studies strongly suggest that ET present in the hypothalamo-pituitary axis participates in neuroendocrine regulation. In the intermediate lobe, however, very little is known about the molecular forms, cellular distribution and the processing of ET. Given the pituitary intermediate lobe (IL) contains only one cell type, the melanotroph, the IL provides an extremely useful and unique in vivo model for the study of the regulation, function and processing of ET in endocrine cells.
In studies initially designed to localize and characterize ET in the ovine hypothalamo-pituitary axis, we noted specific expression of ET in the IL of the ovine pituitary. This tissue has already proven to be of great value as a model for the investigation of the processing and expression of adrenocorticotrophin, endorphin and the melanocyte-stimulating hormone precursor pro-opiomelanocortin (POMC) 29, 30, as the IL consists of a single population of secretory cells, the melanotrophs, which synthesize and release POMC and POMC-related peptides 29, 30. The expression, biosynthesis and secretion of these peptides is largely under tonic dopaminergic and GABAergic inhibitory control [31], and are thus sensitive to manipulation by dopaminergic agonists and antagonists [30]. Given that very little is known about the processing, regulation and function of ET in non-endothelial tissues, particularly in endocrine tissues such as the pituitary, the IL provides an excellent in vivo model for the study of the regulation and function of ET in endocrine cells.
In this study, the precise molecular forms of ET in the IL have been characterized, by combining specific radioimmunoassay with reverse phase (RP)-HPLC. In addition, ET was localized in the melanotroph by immunohistochemistry. Finally, the effects of chronic treatment with both a dopamine agonist and antagonist, and surgical intervention on ET levels and processing in the ovine IL were examined.
Section snippets
Immunohistochemistry
Whole pituitaries were obtained fresh from sheep and after rapid removal, 5-mm slices were placed into Zamboni's fixative (2% formaldehyde, 0.02% picric acid in 0.1 M phosphate buffer, pH 7.0) for 24 h. The tissue was then washed (3×10 min) in dimethyl sulphoxide (DMSO; Sigma Chemical Co., USA) and (3×10 min) in phosphate-buffered saline (PBS; 9.0 g NaCl, 0.136 g KH2PO4, 1.28 g Na2HPO4, 1.0 g NaN3 made up to 1 l with distilled water, pH 7.0). The tissue was then placed in transport medium (30%
Immunohistochemistry
Sections of ovine intermediate lobe (IL) were stained consecutively for endothelin (CTAB), α-melanocyte stimulating hormone (anti-α-MSH) and Von Willebrand Factor (anti-VWF antibody) as described in Section 2.
The IL contains a single population of cells, the melanotrophs, which secrete proopiomelanocortin and related products. The anti-α-MSH antibody (specific for the melanotroph) was used to confirm the precise location to the IL melanotroph. Fig. 1a is stained with the anti-α-MSH, and shows
Discussion
Although the vasoconstrictor action of ET-1 is undisputed, radioimmunoassay (RIA) and in situ hybridization studies have shown that the three endothelin (ET) isoforms are expressed in many tissues other than endothelial cells, including endocrine cells 37, 38and also in the nervous system 11, 39, 40, 41. The demonstration of ET mRNA 13, 42and ET-like immunoreactivity 11, 42and the presence of ET binding sites in brain and pituitary gland 43, 44suggested physiological effects of the peptides in
Acknowledgements
Special thanks to Professor J. Furness, Melbourne University and to Cath Wallace and Heather Woodman for technical assistance, to Bruce Doughton at Werribee Research Station; and to Colleen Thomas and Dr. Robin Woods for the antisera CTAB. This work was supported by the National Health and Research Council of Australia and the Appel Family Bequest with the Truby and Florence Williams Charitable Trust for V.M.
References (54)
- et al.
A novel peptide, vasoactive intestinal contractor, of a new (endothelin) peptide family
J Biol Chem.
(1989) - et al.
Cloning and sequence analysis of cDNA encoding the precursor of a human endothelin-derived vasoconstrictor peptide, endothelin: identity of human and porcine endothelin
FEBS Lett.
(1988) - et al.
The endothelin ETB receptor mediates both vasodilatation and vasoconstriction in vivo
Biochem. Biophys. Res. Commun.
(1992) - et al.
Neurendocrine actions of endothelins
Trends Pharmacol. Sci.
(1992) - et al.
Regional distribution of immunoreactive endothelin in rats
Peptides
(1989) - et al.
Abundance of endothelin-3 in rat intestine, pituitary gland and brain
Biochem. Biophys. Res. Commun.
(1989) - et al.
Identification of endothelin receptors in various regions of rat brain
Neuropeptides
(1990) - et al.
Inhibitory effects of endothelin-1 and endothelin-3 on prolactin release: possible involvement of endogenous endothelin isopeptides in the rat anterior pituitary
Life Sci.
(1992) - et al.
A prolactin release inhibiting activity isolated from neurointermediate lobe extracts is an endothelin-like peptide
Regul. Pept.
(1992) - et al.
Endothelin-3 inhibits prolactin and stimulates LH, FSH and TSH secretion from pituitary cell culture
Biochem. Biophys. Res. Commun.
(1991)
Cloning and characterization of cDNA encoding human A-type endothelin receptor
Biochem. Biophys. Res. Commun.
Cloning and expression of human endothelin-1 receptor cDNA
FEBS. Lett.
Cloning and sequence analysis of a cDNA encoding human nonselective type of endothelin receptor
Biochem. Biophys. Res. Commun.
The endothelin-A receptor subtype transduces the effects of the endothelins in the anterior pituitary gland
Biochem. Biophys. Res. Comm.
Characterization of ACTH-like immunoreactive peptides in rat brain using high performance liquid chromatography
Neurosci. Lett.
The combined use of size exclusion and reversed-phase high performance liquid chromatography for characterization of β endorphin processing pathways
J. Chromatgr.
Protein measurement with the Folin phenol reagent
J. Biol. Chem.
Regional distribution of immunoreactive endothelin in rats
Peptides
Endothelin localizes in the dorsal horn and acts on the spinal neurons: possible involvement of dihydropyridine-sensitive calcium channels and substance P release
Neurosci. Lett.
Autoradiographic visualization of the binding sites for []endothelin in rat and human brain
Neurosci. Lett.
Autoradiographical localization of []-endothelin-1 binding sites in rat brain
Neurosci. Res.
Paradoxical signal transduction mechanism of endothelins and sarafotoxins in cultured pituitary cells: stimulation of phosphoinositide turnover and inhibition of prolactin release
Mol. Cell. Endocrinol.
A novel potent vasoconstictor peptide produced by vascular endothelial cells
Nature (London)
The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes
Proc. Natl. Acad. Sci. USA
Primary structure, synthesis, and biological activity of rat endothelin, an endothelium derived vasoconstrictor peptide
Proc. Natl. Acad. Sci. USA
Topographical location of endothelin mRNA and peptide immunoreactivity in neurones of the human brain
Histochemistry
Effects of endothelin-1 and inhibition of nitric oxide production with Ns-monomethyl-l-arginine on arterial pressure and regional blood flow in anaesthetized rats
J Cardiovasc Pharm.
Cited by (4)
Endothelin and dopamine release
2000, Progress in NeurobiologyProtein kinase C regulates the cell surface activity of endothelin-converting enzyme-1
2006, International Journal of Peptide Research and TherapeuticsThe role of endothelins in the regulation of pituitary function
2002, Experimental and Clinical Endocrinology and DiabetesEndothelin-1 mediates prostaglandin F<inf>2α</inf>-induced luteal regression in the ewe
2001, Biology of Reproduction