Histamine receptors in human epithelial cells – characterization of the receptor G-protein-effector system B. L. Legnazzi, F. Monczor, E. Rivera, R. Bergoc and C. Davio Laboratorio de Radioiso ´topos, Facultad de Farmacia y Bioquı ´mica, Universidad de Buenos Aires, Junı ´n 956 PB, 1113 Buenos Aires, Argentina, Fax +54 1 962 5341, e-mail: Cardavio@huemul.ffyb.uba.ar Introduction We have previously described functional H 1 and H 2 histamine receptors in human breast epithelial cells. Both receptors were positively coupled to phospholipase C activation. Neither H 1 nor H 2 receptors were coupled to the adenyl cyclase system, although studies with prosta- glandin E 2 (PGE 2 ), cholera toxin and forskolin showed that guanine nucleotide-binding stimulatory protein (Gs) and adenyl cyclase are present in this cell line [1]. The purpose of the present investigation was to study the receptor G-protein- effector system in the normal mammary epithelial HBL-100 cell line. Materials and methods Cell culture HBL-100 cells, obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA), were plated in 24 well plastic dishes and grown to 60% confluence at 37 °C in a humidified atmosphere with 5% CO 2 in RPMI 1640 medium supplemented with 10% foetal calf serum. Binding assay [ 3 H]-tiotidine and [ 3 H]-mepyramine were employed as radioligands. Incubations were performed with 10 5 cells/well (or purified membrane fraction equivalent to this number of cells) for 40 min at 4 °C in 50 mM Tris-HCl buffer, and terminated by dilution in cold buffer, radioactivity was extracted with 0.5 ml ethanol. Non-specific binding was determined with 1 mM histamine. Membrane purified fractions were obtained by disrupting cells by sonication in 50 mM Tris-HCl buffer and then centrifuging for 15 min at 8500 g; the supernatant was further centrifuged at 30000 g for 40 min. The resulting pellet was resuspended in 50 mM Tris-HCl buffer and pre-incubated at 37 °C with 100 M GTPgS for 2 h. The binding data were evaluated with Ligand, a non-linear, weighted, least squares curve-fitting procedure [2] and Scatchard plot analysis. Phosphoinositide turnover Cells were incubated at 37 °C for 24 h with [ 3 H]-myo-inositol (2 mCi/ml). The total [ 3 H]-inositol phosphates produced after 20 min stimulation with histamine or specific H 1 and H 2 agonists, were determined as previously described [3]. Results and discussion Saturation analysis using intact cells showed two sites for [ 3 H]-tiotidine and [ 3 H]-mepyramine binding, one site with high affinity and low capacity, and the other one with low affinity and high capacity. Studies with guanosine 5 0 -O- (3-thiotriphosphate) (GTPgS), in purified membrane frac- tions, showed no modification in the total number of sites and the high affinity component was not further observed in the [ 3 H]-tiotidine assay (Table 1). In contrast, pretreatment with GTPgS in the [ 3 H]-mepyramine assay resulted in loss of the low affinity component; but as in the [ 3 H]-tiotidine assay, the total number of sites was not altered (Table 2). The presence of a double site for [ 3 H]-tiotidine and [ 3 H]- mepyramine and the modification of the binding profile after the treatment with GTPgS suggest the presence of a G- protein receptor complex similar to that described for other members of the G-protein coupled receptors superfamily [4]. The fact that an antagonist shows a different affinity for the uncoupled receptor than for the receptor G-protein complex can not be explained by any of the classical interaction models. Neither the ‘allosteric ternary complex model’ [5] nor the ‘two state model’ [6] can predict our binding results Inflamm. res. 47, Supplement 1 (1998) S40–S41  Birkha ¨user Verlag, Basel, 1998 1023-3830/98/010S40-02 $ 1.50+0.20/0 Inflammation Research Correspondence to: C. Davio Table 1. 3 H-tiotidine binding parameters. Control assay Pretreatment with GTPgS Kd (nM) Q (sites/cell) Kd (nM) Q (sites/cell) 2.4  0.6 45500  6100 – – 21  4 195000  25000 19  4 215500  32000 Data shown are the mean  SD of triplicate assays. Similar results were obtained in four independent experiments performed with different preparations of HBL-100 cells. 