The characterization, localization and regulation of endothelin in ovine pars intermedia

Abstract

The pituitary intermediate lobe (IL) contains a single population of cells and has recently been shown to express endothelin (ET)-like peptides. The IL thus provides an excellent in vivo model to study regulation, function and processing of ET in an endocrine cell. The primary aims of the present study were to locate and characterize the precise molecular forms of ET in the ovine IL and determine if levels and/or processing of ET is under dopaminergic or other influences. We have developed a radioimmunoassay (RIA) that detects each form of ET and, when combined with reverse phase-HPLC (RP-HPLC), shows the ovine IL to contain predominantly the ET-1 isoform. In addition, using a specific anti-endothelin antiserum for immunohistochemistry (IHC), we localized ET-1 with α-melanocyte stimulating hormone (α-MSH) within the melanotroph. The effects of dopamine agonists, antagonists and hypothalamo-pituitary disconnection (HPD) on both tissue levels and processing of ET in the ovine IL were also examined. Normal sheep were treated chronically with haloperidol or bromocriptine to investigate the possibility of dopaminergic regulation of ET in the IL. In the haloperidol-treated group, plasma prolactin levels did not vary significantly from day 0 to day 8, but the bromocriptine treatment reduced prolactin levels (t=9.4 P<0.01). Neither bromocriptine nor haloperidol, however, affected tissue ET peptide levels or forms. After HPD, the HPLC profile of pooled IL showed that ET-1 levels in the IL are slightly increased with no change in molecular forms.

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 ET_A subtype 23, 24, 25which recognizes ET-1 preferentially; and the ET_B 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 ET_A 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.