Expression of GalR1 and GalR2 galanin receptor messenger ribonucleic acid in proopiomelanocortin neurons of the rat arcuate nucleus: effect of testosterone.

Previous studies have shown that galanin-containing fibers make synaptic contacts with POMC neurons in the arcuate nucleus. However, the ability of POMC neurons to express galanin receptors has never been assessed. The present study was designed to investigate whether POMC neurons express galanin receptor messenger RNA (mRNA) and whether testosterone could modulate galanin receptor gene expression. A dual-labeling in situ hybridization histochemistry, using 35S-labeled (galanin receptors GalR1 or GalR2) and digoxigenin-labeled (POMC) riboprobes, was performed on brain sections from intact, castrated, and testosterone-replaced adult male rats. For analysis, the arcuate nucleus was divided into four rostro-caudal areas. The results revealed that both GalR1 and GalR2 mRNAs were expressed in POMC neurons. Most POMC neurons expressing galanin receptor mRNAs were found in the rostral parts of the nucleus. Castration reduced the labeling density of galanin receptor mRNAs in POMC neurons, and testosterone prevented the effects of castration in all rostro-caudal subdivisions of the arcuate nucleus. Taken together, these data indicate that galanin can directly modulate the activity of POMC neurons, via an action on GalR1 or GalR2 receptors, particularly in the rostral-arcuate nucleus. In addition, testosterone can modulate the expression of GalR1 and GalR2. Because POMC neurons located in the rostral part of the nucleus are known to project preferentially to the preoptic area, POMC neurons expressing the galanin receptor genes may play an important role in the regulation of the GnRH neuroendocrine axis.

Semiquantitative distribution of galanin-receptor (GAL-R1) mRNA-containing cells in the male rat hypothalamus.

The semiquantitative distribution of mRNA encoding for rat galanin receptor (GAL-R1) was examined by in situ hybridization in the rat hypothalamus using a 35S-riboprobe. Most hypothalamic nuclei expressed GAL-R1 mRNA. In the anterior hypothalamus, high levels of expression were found in the medial preoptic area, paraventricular and supraoptic nuclei. Numerous cells also expressed the GAL-R1 mRNA with a moderate level of expression in the periventricular region. Very few GAL-R1-expressing cells were present in the suprachiasmatic nucleus. In the medial hypothalamus, numerous expressing cells were detected in the dorsomedial and ventromedial nuclei. The arcuate nucleus was moderately labeled throughout its rostrocaudal extent; labeled cell bodies were visible in the ventromedial and ventrolateral subdivisions as well. These results indicate that the GAL-R1 mRNA is not only expressed in anterior hypothalamic nuclei but also in the mediobasal hypothalamus and periventricular region. This hypothalamic distribution correlates well with that of 125I-GAL-binding sites and GAL-immunoreactive fibers. This distribution represents the morphological substrate for GAL roles in the hypothalamic regulation of neuroendocrine, behavioral and autonomic functions.

Molecular determinants of the species selectivity of neurokinin type 1 receptor antagonists.

Most nonpeptide neurokinin (NK)1 antagonists display a marked difference in affinity for rat versus human NK1 receptors. The molecular basis for the species selectivity of RP67580 and CP96,345 has been previously addressed [J. Biol. Chem. 267:25668-25671 (1992); J. Biol. Chem. 268:2319-2323 (1993)]. We are extending these previous results to additional NK1 antagonists, which are members of different chemical families. Included is a new perhydroisoindolol, RPR100893, which unlike its parent compound (RP67580) is human receptor selective. Chimeric rat/human NK1 receptors, as well as rat and human mutant NK1 receptors, were constructed and expressed in COS-1 cells, and affinities for substance P and the various antagonists were determined in binding studies. With human receptor-selective antagonists, the rat R290(S-->I) mutation was the most effective in increasing antagonist affinity (from 7- to 23-fold). Combination with the R116(L-->V) mutation led to an additional increase in affinity for trans-4-hydroxy-1-(1H-indol-3-ylcarbonyl)-L-prolyl-N- methyl-N-(phenylmethyl)-L-tyrosineamide (a derivative of FK888) and to nearly full human receptor affinity for RPR100893 and (+/-)-CP99,994. Based on the gains in affinities, these results confirm and extend the role of residues 116 and 290 of the NK1 receptor in the species selectivity of these three new human receptor-selective NK1 antagonists. In comparison, the affinity of RP67580, the least selective molecule, was most affected by changes at position 116, and combination with mutations at either position 97 (V-->E) or position 290 led to the human receptor phenotype. For the heterosteroid KAN610857, modifications of the rat receptor at positions 97 and 290, and to a lesser degree position 116, were the most effective in reducing affinity. Two double-mutants [R(97,290) and R(116,290)], although different from those identified for RP67580, also displayed human receptor-like affinity. Therefore, the molecular determinants of the species selectivity appear to be different, in part, between rat and human receptor-selective compounds, even between closely related chemical families.

Cloning, pharmacological characterization, and anatomical distribution of a rat cDNA encoding for a galanin receptor.

We have cloned and expressed a rat cDNA, designated GALR1-rat, that encodes a galanin receptor based on homology, pharmacology, and anatomical criteria. This cDNA was isolated from a rat brain cDNA library. The nucleotide sequence of the cloned receptor revealed an open reading frame encoding a 346-amino-acid protein, showing 90.8% identity with the previously cloned human galanin receptor. Membranes prepared from COS cells transiently expressing GALR1-rat specifically bind 125I-galanin with high affinity (Kd = 0.12 +/- 0.01 nM). Rat, porcine, and human galanin were able to displace 125I-galanin with nanomolar Ki (0.08 +/- 0.03, 0.10 +/- 0.01, and 0.14 +/- 0.03 nM, respectively), whereas the Ki values for the porcine galanin fragments galanin-(1-16), galanin-(2-29), and galanin-(3-29) were 0.95 +/- 0.21 nM, 7.14 +/- 0.51 nM, and > 1 microM, respectively. The rank order potency of these ligands is consistent with that reported for the native galanin receptor. The distribution of the mRNA corresponding to the galanin receptor encoded by GALR1-rat was determined by in situ hybridization to rat brain sections. High levels of galanin receptor mRNA were detected in the ventral hippocampal formation, thalamic, amygdala, and medulla oblongata nuclei, and in the dorsal horn of the spinal cord.

Molecular cloning of a functional human galanin receptor.

The ubiquitous neuropeptide galanin controls numerous functions such as endocrine secretions, intestinal motility, and behavioral activities. These regulatory effects of galanin are mediated through the interaction with specific membrane receptors and involve the pertussis toxin-sensitive guanine nucleotide binding proteins Gi/Go as transducing elements. We report here the isolation of a cDNA coding for a human galanin receptor from a Bowes melanoma cell line cDNA expression library, by using a radioligand binding strategy. The nucleotide sequence of the cloned receptor reveals an open reading frame encoding a 349-amino acid protein with seven putative hydrophobic transmembrane domains and significant homology with members of the guanine nucleotide binding protein-coupled neuropeptide receptor family. The cloned receptor expressed in COS cells specifically binds human, porcine, and rat galanin with high affinity (Kd in the nanomolar range) and mediates the galanin inhibition of adenylate cyclase. A 2.8-kb galanin receptor transcript was identified in several human tissues. Cloning of this galanin receptor should enhance our knowledge of its distribution, structure, and function in human physiology and pathophysiology.