Andre Crespo

The human galanin receptor: ligand-binding and functional characteristics in the Bowes melanoma cell line

The human galanin receptor has been characterized pharmacologically from the Bowes melanoma cell line. Using porcine [125I]galanin as the radioligand, a single population of non-interacting high-affinity binding sites (KD = 0.05 +/- 0.01 nM; Bmax = 135 +/- 7 fmol/mg protein) was demonstrated. Human galanin peptide competitively inhibited the specific binding of [125I]galanin (IC50 = 0.35 +/- 0.13 nM) and decreased the forskolin-stimulated cAMP production (EC50 = 0.46 +/- 0.05 nM) with a maximal inhibition of 63 +/- 2% at 10(-7) M. Rat and porcine galanin peptides and the chimeric peptides M15, M35, M32, M40 and C7 also dose-dependently inhibited the forskolin-stimulated cAMP production, while the fragment porcine galanin-(3-29) and [D-Trp2]galanin were found to be inactive. The specific binding of [125I]galanin was decreased in a dose-dependent manner by GTP and the cAMP response was inhibited by the pertussis toxin, suggesting the activation of a G-protein dependent process. The Bowes cell line thus appears to be a relevant tool for the study of human galanin receptor.

Characterization of a Human NK1 Tachykinin Receptor in the Astrocytoma Cell Line U 373 MG

Abstract: The human NK1 tachykinin receptor in the astrocytoma cell line U 373 MG was characterized using selective agonists and antagonists described for this receptor in the rat. Specific [3H]substance P binding sites were present on cell homogenates, whereas [3H]neurokinin A or [3H]‐senktide binding sites were absent. The binding was saturable and reversible. The binding of [3H]substance P was inhibited by very low concentrations of [L‐Pro9]substance P and [Sar9,Met(O2)11]substance P; septide was ∼ 1,000‐fold less potent. The most potent peptide antagonist was trans‐4‐hydroxy‐1‐(1 H‐indol‐3‐ylcarbonyl)‐L‐prolyl‐N‐methyl‐N‐(phenylmethyl)‐L‐tyrosineamide. The rank order of potency for the nonpeptide antagonists was (S,S)‐CP 96,345 > (±)‐CP 96,345 > (±)‐2‐chlorobenzylquinuclidinone > (R,R)‐CP 96,345 > RP 67580 > RP 68651. In [3H]‐inositol‐labeled cells, substance P stimulated phosphatidylinositol turnover. A good correlation was found when the abilities of NK1 receptor agonists for stimulating inositol phosphate production and for inhibiting [3H]substance P binding were compared. Similarly, the binding and functional assays were well correlated for the antagonists. As a result of its high sensitivity and selectivity, the U 373 MG cell line thus appears an excellent tool for investigating the pharmacology of the human NK1 receptor.

Monoclonal antibodies against synovial collagenase: use for immunopurification and characterization of the latent and active enzyme.

Monoclonal antibodies have been produced against porcine synovial collagenase which recognize both the active enzyme and its inactive precursor. These antibodies inhibited the collagenolytic activity of collagenase, but not its activity with a synthetic peptide substrate. The antibodies were also able to recognize human synovial, human skin fibroblast and human chondrocyte collagenase but not the enzyme from human granulocytes. One of the monoclonal antibodies was successfully used for the immunopurification of the porcine enzyme and these experiments led to the demonstration of an endogenous activator of procollagenase in the synovial cell culture medium.