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

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...

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Published in:Proceedings of the National Academy of Sciences - PNAS 1993-05, Vol.90 (9), p.4231-4235
Main Authors: Wynick, David, Smith, David M., Ghatei, Mohammad, Akinsanya, Karen, Bhogal, Ranjev, Purkiss, Paul, Byfield, Peter, Yanaihara, Noboru, Bloom, Stephen R.
Format: Article
Language:English
Subjects:
Amino acids
Animals
Antagonists
Anterior pituitary
Antiserum
Binding, Competitive
Biological and medical sciences
Biology
Cell Membrane - metabolism
Cell receptors
Cell structures and functions
characterization
Female
Fundamental and applied biological sciences. Psychology
Galanin
Galanin receptors
In Vitro Techniques
Inhibitory concentration 50
Kinetics
Molecular and cellular biology
Neurology
Neuropeptide receptors
Neuropeptides - metabolism
Neuropeptides - pharmacology
Peptide Fragments - pharmacology
Peptides - metabolism
Peptides - pharmacology
pituitary (anterior)
Pituitary Gland, Anterior - drug effects
Pituitary Gland, Anterior - metabolism
Prolactin - metabolism
Rats
Rats, Wistar
Reagents
Receptors
Receptors, Galanin
Receptors, Gastrointestinal Hormone - metabolism
Rodents
Secretion
Sex hormones
Substance P - analogs & derivatives
Swine
Thalamus - metabolism
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Summary: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. Direct effects of galanin at the level of the pituitary have been defined, yet, paradoxically, a number of studies have been unable to demonstrate galanin binding to an anterior pituitary receptor. Porcine galanin stimulated prolactin release from dispersed rat anterior pituitary cells up to 180% ± 12% (mean ± SEM) of control secretion. The addition of a specific galanin antiserum caused a profound inhibition of basal prolactin release, maximal inhibition being 12% ± 0.5% of control secretion. Addition of M15 produced no effect on basal or galanin-stimulated prolactin release. Galanin fragment 3-29 was fully active when compared to galanin 1-29. Fragments 5-29 and 8-29 stimulated prolactin release to a lesser extent and galanin 1-15, 10-29, and 20-29 had no significant prolactin-releasing activity. Using [mono(125I)iodo-Tyr26]galanin or porcine125I-labeled Bolton-Hunter [mono(125I)iodo-Lys25]galanin, no anterior pituitary membrane binding was observed. In contrast,125I-labeled Bolton-Hunter N-terminally labeled galanin allowed characterization of a single high-affinity anterior pituitary galanin receptor with a Kdof 4.4 ± 0.34 nM and a Bmaxof 79 ± 8.3 fmol/mg of protein. The IC50for porcine galanin was 0.51 ± 0.04 nM but for M15 was in excess of 10 μ M. Galanin 3-29 fully displaced the label with an IC50of 0.96 ± 0.7 nM. The IC50for galanin 5-29 was 200 nM, whereas 8-29 and 1-15 were >1 μ M. Galanin 10-29 and 20-29 failed to displace the label. These data suggest the presence of a high-affinity pituitary galanin receptor, designated GAL-R2, in which region 3-10 and amino acid 25 are crucial for membrane binding and biological activity, in contrast to the known gut/brain galanin receptor (designated GAL-R1). A number of tissues known to bind or respond to galanin were screened. GAL-R2would appear to be expressed only in the anterior pituitary and hypothalamus.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.90.9.4231