thioperamide and Stomach-Neoplasms

The histamine H3 receptor agonist (R)alpha-methylhistamine (MeHA) inhibited, in a nanomolar range, basal and carbachol-stimulated inositol phosphate formation in the human gastric tumoral cell line HGT1-clone 6. The inhibition was reversed by micromolar concentrations of the histamine H3 receptor antagonist thioperamide and was sensitive to cholera or pertussis toxin treatment. Using [3H]N alpha-MeHA as specific tracer, high affinity binding sites were demonstrated with a Bmax of 54 +/- 3 fmol/mg of protein and a KD of either 0.61 +/- 0.04 or 2.2 +/- 0.4 nM, in the absence or presence of 50 microM GTP[gamma]S, respectively. The binding sites were solubilized by Triton X-100 and prepurified by gel chromatography. They were separated from the histamine H2 receptor sites by filtration through Sepharose-famotidine and finally retained on Sepharose-thioperamide. The purified sites concentrated in one single silver-stained protein band of 70 kDa in SDS-polyacrylamide gel electrophoresis. They specifically bound [3H]N alpha-MeHA with a KD of 1.6 +/- 0.1 nM and a Bmax of 12,000 +/- 750 pmol/mg of protein. This corresponds to a 90,225-fold purification over cell lysate and a purity degree of 84%. Binding was competitively displaced by N alpha-MeHA (IC50 = 5.8 +/- 0.7 nM), (R) alpha-MeHA (IC50 = 9 +/- 1 nM), and thioperamide (IC50 = 85 +/- 10 nM), but not by famotidine (H2 antagonist) or by mepyramine (H1 antagonist). These findings provide the first evidence for solubilization, purification, and molecular mass characterization of the histamine H3 receptor protein and for the negative coupling of this receptor phosphatidylinositol turnover through a so far unidentified G protein.

Topics: Binding Sites; Carbachol; Cholera Toxin; Chromatography, Affinity; Chromatography, Gel; Guanosine 5'-O-(3-Thiotriphosphate); Histamine Antagonists; Humans; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Methylhistamines; Molecular Weight; Phosphatidylinositols; Piperidines; Receptors, Histamine; Receptors, Histamine H3; Stomach Neoplasms; Tumor Cells, Cultured

Histamine 0.1 microM-0.1 mM increased adenylate cyclase activity five- to ten-fold in human fundic membranes, with a potency Ka = 3 microM. The histamine dose-response curve was mimicked by the H3 receptor agonist (R) alpha-MeHA, but at 100 times lower potency, Ka = 0.3 mM. Histamine-induced adenylate cyclase activation was abolished by H2, H1 and H3 receptor antagonists, according to the following order of potency IC50: famotidine (0.3 microM) greater than triprolidine (0.1 mM) thioperamide (2 mM), respectively. Famotidine has no action on membrane components activating the adenylate cyclase system, including the Gs subunit of the enzyme stimulated by forskolin and cell surface receptors sensitive to isoproterenol (beta 2-type), PGE2 and VIP. The Schild plot was linear for famotidine (P less than 0.01) with a regression coefficient r = 0.678. The slope of the regression line was 0.64 and differs from unity. Accordingly, famotidine showed a slow onset of inhibition and dissociation from the H2 receptor in human cancerous HGT-1 cells. The results demonstrate that famotidine is a potent and selective H2 receptor antagonist with uncompetitive actions in human gastric mucosa. Consequently, famotidine might be a suitable drug with long-lasting actions in the treatment of Zollinger-Ellison syndrome. The results also confirm and extend the previous observations that (R) alpha-MeHA and thioperamide are two selective ligands at histamine H3 receptor sites. In the human gastric mucosa, these drugs are respectively 330 and 6700 times less potent than histamine and famotidine on the adenylate cyclase system. The possible involvement of histamine H3 receptors in the regulation of gastric secretion is proposed.

Topics: Adenylyl Cyclases; Cyclic AMP; Famotidine; Gastric Fundus; Histamine; Histamine H2 Antagonists; Humans; In Vitro Techniques; Methylhistamines; Piperidines; Ranitidine; Receptors, Histamine; Stomach Neoplasms; Thiazoles; Triprolidine