Characterization of a high-affinity galanin receptor in the rat anterior pituitary: absence of biological effect and reduced membrane binding of the antagonist M15 differentiate it from the brain/gut receptor.

Abstract

Structure-activity studies demonstrate that galanin fragments 1-15 and 2-29 are fully active, whereas fragment 3-29 has been reported to be inactive, in a number of different in vivo models. M15, a chimeric peptide comprising galanin 1-13 and substance P 5-11, has recently been found to be a potent galanin antagonist. Galanin, a 29-amino acid peptide, was originally isolated from porcine intestine (1) and subsequently reported to be widely distributed in gut, pancreas, and the peripheral and central nervous systems (2, 3). Highest levels of galanin synthesis and storage occur within the hypothalamus in the median eminence (4), but it is also abundantly expressed in the anterior pituitary, where it has been shown to be estrogen inducible (5).Various studies have demonstrated effects of galanin on basal and stimulated release of prolactin (6, 7), growth hormone (8-11), and luteinizing hormone (12, 13) either from dispersed pituitary cells or at the hypothalamic level modulating dopamine, somatostatin (SRIF; somatotropin releaseinhibiting factor), and gonadotropin-releasing hormone (GnRH) release into the portal circulation. Recently galanin has been shown to be episodically released into the hypothalamo-pituitary portal circulation at concentrations in the nanomolar range (12), providing further evidence for its putative role as a modulator of pituitary function.In view of the high concentrations of galanin reaching or secreted by the anterior pituitary it is puzzling that a number of studies have failed to demonstrate anterior pituitary binding of [mono(125I)iodo-Tyr26]galanin (1251-Tyr-galanin) by membrane assay or by autoradiography in the rat or pig (14,15), although these studies demonstrated good thalamicand/or hypothalamic-specific galanin binding.Bartfai et al. (16) have recently synthesized a high-affinity galanin antagonist by fusing galanin 1-13 with substance P 5-11, which they termed M-15. This antagonist has been shown to be a potent inhibitor, at doses of 1 nM or less (with an IC50 of 0.1 nM), of the actions of galanin on the pancreas, smooth muscle, and hippocampus.The following studies demonstrate that galanin stimulates the release of prolactin from cultured anterior pituitary cells and that a specific galanin antiserum blocks basal prolactin secretion. The galanin antagonist M15 has no effect on basal or galanin-stimulated prolactin release. Using galanin 1251-labeled with the Bolton-Hunter (1251-BH) reagent at the N terminus, a single high-affinity galanin receptor in the pituitary is characterized. Unlike the brain/gut galanin receptor, the C-terminal part of the peptide is crucial for pituitary membrane binding. MATERIALS AND METHODSPituitary Dispersion. Anterior pituitaries were removed from randomly cycling female Wistar rats (Interfauna, Huntingdon, U.K.) immediately after death by decapitation and dispersed as described (17). Tissue was incubated while shaking at 37°C for 1 hr in Hanks' balanced salt solution (HBSS, GIBCO; containing 0.1% bovine serum albumin, pH 7.4) containing 0...

Expression Cloning of a Rat Hypothalamic Galanin Receptor Coupled to Phosphoinositide Turnover

“…To clone additional members of the galanin receptor family, we used an expression cloning strategy based on the hypothesis that there were multiple galanin receptors present in hypothalamus (17,29). A randomly primed cDNA expression library was constructed from rat hypothalamus and screened in pools by radioligand binding/photoemulsion detection using 125 IpGAL.…”

“…Multiple receptor subtypes are proposed to underlie the physiological effects of galanin (15)(16)(17)(18), but until recently only one cloned galanin receptor subtype (GALR1) had been described (19 -21). We report here the isolation by expression cloning and the pharmacological characterization of a novel galanin receptor from a rat hypothalamic cDNA library.…”

Molecular Characterization and Expression of Cloned Human Galanin Receptors GALR2 and GALR3

“…Because many diverse biological effects can be attributable to galanin, it was postulated early on that galanin receptor (GALR) subtypes exist (Bartfai et al, 1991;Hedlund et al, 1992;Wynick et al, 1993).…”

Galanin-receptor ligand M40 peptide distinguishes between putative galanin-receptor subtypes.

“…Galanin hyperpolarizes noradrenergic cell bodies in the locus coeruleus (11) Accordingly, a galanin-receptor subtype has been suggested, which is composed of nervous tissue and pancreatic galanin receptors that recognize the N-terminal 1-to 15-aa or 1-to 16-aa fragment of galanin as high-affinity agonists, whereas another putative galanin-receptor subtype in smooth muscle requires both the N and C terminus of galanin for binding and biological action (25). On the basis of the differential affinity ofgalanin (3-29) and ofa galanin-receptor antagonist M15 (17), existence of an additional galaninreceptor subtype has been suggested in the rat anterior pituitary (26), which differs from other CNS galanin receptors. Finally, galanin-(1-15)-binding sites have been demonstrated in the dorsal hippocampus, neocortex, and neostriatum, areas that seem to lack galanin-(1-29)-binding sites (27).…”