Introduction Cultured keratinocytes can be induced to differentiate by modulating the extracellular Ca 2+ concentration. This in vitro response correlates with that observed in vivo as keratino- cytes migrate from the basement membrane toward the suprabasal differentiating layers of the epidermis [1]. The elevation of extracellular Ca 2+ increases phosphatidylinositol turnover and diacylglycerol levels and induces Protein Kinase C activity, resulting in the expression of differentia- tion markers [2]. We have previously demonstrated that stimulation of histamine receptors increases levels of inositol phosphates in mouse keratinocytes [3]. In the present work we studied the expression and functional coupling of hista- mine H 1 and H 2 receptors during Ca 2+ induced epidermal dif- ferentiation in cultured mouse keratinocytes. Materials and methods Cell culture PB cells, a papilloma derived murine cell line, were plated in 24 wells plastic dishes and incubated in MEM basic medium with 8 % FCS at 37 °C in a 5 % CO 2 humidified atmosphere. Keratinocytes were induced to differentiate by elevating the extra- cellular Ca 2+ concentration in the culture medium from 0.05 mM to 0.12 mM. Whole cell binding experiments Saturation experiments were performed on 70% confluent monolayers, using [ 3 H]-mepyramine or [ 3 H]-tiotidine as specific H 1 and H 2 radio- ligands, as described [4]. Second messenger production Intracellular cyclic AMP and total inositol phosphates (InsP) were measured in cell monolayers at 70% confluence as described previous- ly [4], after stimulation with different concentrations of histamine, H 1 or H 2 agonists (2-[3-fluoromethylphenyl]histamine and BU-E-75 re- spectively). Sodium fluoride (10 mM), forskolin (10 mM) and PGE 2 (1 mM) were used as positive controls for cyclic AMP production. Results and discussion We have previously demonstrated the expression of H 1 and H 2 receptors in cultured mouse keratinocytes [3]. In this work, using PB cells, a papilloma derived cell line which maintains the ability terminally to differentiate in response to Ca 2+ concentration [5], we demonstrated that H 2 receptor expression is regulated during keratinocyte differentiation. Treatment of cells with the higher Ca 2+ concentration (0.12 mM) resulted in a marked decrease in H 2 receptor amounts on the cell surface, as assessed by whole cell bind- ing experiments (Table 1). Similar amounts of specific H 1 antagonist binding sites were detected in PB cells grown under low or high Ca 2+ in the culture medium. Values of the dissociation constants for H 1 and H 2 specific radioligands were similar under both culture conditions (Table 1). Con- comitantly, in PB cells grown under higher Ca 2+ concentra- tions, the H 2 receptor lost its ability to increase InsP levels above basal values (Fig. 1A). In PB cells grown under low Ca 2+ conditions (0.05 mM), the InsP production induced by H 1 and H 2 receptor agonists was dose-dependent (EC 50 = 0.38 ± 0.05 mM for 2-[3- fluoromethylphenyl]histamine and EC 50 = 1.06 ± 0.24 mM for BU-E-75) and specifically blocked by the corresponding selective antagonists, indicating a receptor-mediated activa- tion of the intracellular signalling pathway (Fig. 1B). Similarly, in PB cells grown under lower Ca 2+ concentra- tion, histamine induced InsP production in a dose-dependent fashion (EC 50 = 0.234 ± 0.017 mM). This InsP production was insensitive to the H 1 antagonist mepyramine but com- pletely blocked by the H 2 antagonist tiotidine, suggesting Inflamm. res. 48, Supplement 1 (1999) S73–S74 1023-3830/99/010S73-02 $ 1.50+0.20/0 © Birkhäuser Verlag, Basel, 1999 Inflammation Research Changes in H 2 receptor expression and coupling during Ca 2+ -induced differentiation in mouse epidermal keratinocytes C. Fitzsimons 1 , H. Durán 2 , N. Engel 1 , B. Molinari 2 and E. Rivera 1 1 Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina, Fax +54 19 62 53 41, e-mail: cfitzsim@huemul.ffyb.uba.ar, erivera@huemul.ffyb.uba.ar 2 Departamento de Radiobiología, Comisión Nacional de Energía Atómica, Laboratorio Tandar, Av. General Paz y Av. de los Constituyentes, Pcia de Buenos Aires, Argentina Correspondence to: E. Rivera