Prevention of motoneuron death by adenovirus-mediated neurotrophic factors.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motoneurons, and has no effective treatment. Experimental studies in rodents have shown that motoneurons respond to a variety of molecules including brain-derived neurotrophic factor (BDNF). and the glial-cell line-derived neurotrophic factor (GDNF). Here we investigated the neuroprotective effect of these growth factors, encoded by an adenovirus, on the death of axotomized facial motoneurons in newborn rats. We used a new gene therapy strategy that involves gene transfer to motoneurons by intramuscular injection of an adenoviral vector, which is retrogradely transported from injected target muscle (Finiels et al.,: NeuroReport 7:373-378, 1995). A significant increased survival of motoneurons was observed in animals pretreated with adenovirus encoding BDNF (34.5%, P < 0.05) ou GDNF (41.9%, P < 0.05) 1 week after axotomy. These results indicate that pretreatment with BDNF or GDNF, using this therapeutic strategy, is able to prevent the massive death of motoneurons that normally follows axotomy in the neonatal period, opening new perspectives to limit neuronal death in degenerative disorders.

An adenovirus encoding CuZnSOD protects cultured striatal neurones against glutamate toxicity.

Superoxide dismutase (SOD), a key enzyme in the detoxification of free radicals, catalyses the dismutation of superoxide O2.- to oxygen and hydrogen peroxide (H2O2). It is therefore a promising candidate for gene transfer therapy of neurological diseases in which free radicals are thought to be involved. We have constructed a recombinant adenoviral vector containing the human copper-zinc SOD cDNA. Using this vector we were able to drive the production of an active human copper-zinc SOD protein (hCuZnSOD) in various cell lines and primary cultures. Infection of striatal cells with a recombinant adenovirus expressing hCuZnSOD protected these cells from glutamate-induced cell death.

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.

[Endothelin receptors and endothelin-1 immunoreactivity in the human lung]. / Récepteurs de l'endothéline et immunoréactivité de l'endothéline-1 au niveau des poumons humains.

We investigated in human lung preparations the characteristics of endothelin-1 (ET-1) binding and the amount of ET-1-like immunoreactivity. Saturation experiments revealed the presence of a large number of high affinity specific ET-1 binding sites with a dissociation constant (Kd) of 1.35 nM and a binding capacity (Bmax) of 9.74 pmol/mg of protein. The binding was time- and temperature-dependent and dissociated by only 10% by the addition of 1 microM unlabeled ET-1. In competition experiments, [125I]ET-1 binding was totally inhibited by unlabeled ET-1 and ET-2 with inhibition constant (Ki) values of 0.20 and 0.21 nM respectively, and 80% inhibited by ET-3 with Ki value of 0.50 nM. The binding was not affected by 1 microM structurally unrelated compounds. Moreover a high level of ET-1-like immunoreactivity (2.3 pg/mg wet weight) was found in human lung by using a specific radioimmunoassay of ET-1 after extraction. HPLC analysis revealed the presence of both ET-1 and Big-ET. These results suggest that the lung may be an important target organ for ET-1 action and/or metabolism in human.

Comparison of the bronchopulmonary and pressor activities of endothelin isoforms ET-1, ET-2, and ET-3 and characterization of their binding sites in guinea pig lung.

In anesthetized and ventilated guinea pigs intravenous injection of ET-1, ET-2, or ET-3 (1 or 2 nmol/kg) induced similar dose-dependent increases in pulmonary inflation pressure (PIP) associated with increases in mean arterial blood pressure (MBP). Pretreatment of the guinea pigs with 1 mg/kg intravenous indomethacin significantly inhibited the increase in PIP evoked by 2 nmol/kg of ET-1, ET-2, or ET-3. In contrast, the increase in MBP following injection of the various ET isotypes was not significantly affected by indomethacin. Injection of ET-1, ET-2, or ET-3 (40, 120, and 400 pmol) via the pulmonary artery of isolated and perfused guinea pig lungs induced dose-dependent increases in PIP and pulmonary perfusion pressure (PPP), thromboxane B2 (TXB2) release, and formation of lung edema. In keeping with the in vivo results, no marked differences were observed between the activities of ET-1, ET-2, and ET-3 on isolated and perfused guinea pig lungs. Indomethacin (5 microM) added to the perfusion medium significantly inhibited the alterations of PIP and PPP, TXB2 release, and edema formation evoked by 400 pmol ET-1, ET-2, or ET-3. High-affinity binding sites for ET-1, ET-2, and ET-3 exhibiting similar characteristics were identified on guinea pig lung membrane. Therefore ET-1, ET-2, and ET-3 exert comparable bronchopulmonary and pressor activities in the guinea pig and probably act via interaction with the same binding site. In addition, the ET-induced increase in PIP and pulmonary vasoconstriction are primarily mediated via the production of cyclooxygenase metabolites.