Stimulation of G-protein-coupled receptors (GPCR) linked to Gα_q/11 may activate the MAP kinase pathway either directly, i.e. by intracellular mechanisms, or indirectly by increased shedding of growth factors of the epidermal growth factor (EGF) family, which then act as paracrine or autocrine mediators (Prenzel et al. 1999). The potential presence of the two mechanisms in different members of heterogeneous cell populations clearly complicates the interpretation of responses to GPCR stimulation. We have distinguished these mechanisms using flow cytometry of heterogeneous cell populations defined by expression of green fluorescent protein (GFP).

The human gastric epithelial cell line, AGS, stably transfected with the gastrin-CCK_B receptor (AGS-G_R cells) was co-cultured with ASG cells that had been stably transfected with GFP and which do not express the gastrin-CCK_B receptor (Varro et al. 2002). Activation of the MAP kinase pathway was studied by flow cytometry using antibodies to phospho p42/44 MAP kinase, or total MAP kinase, and detection by AlexaFluor647-conjugated second antibody (Chow et al. 2001), measuring fluorescence in each cell type by gating on GFP fluorescence.

There were similar increases in phospho-p42/44 MAP kinase in AGS-G_R (2.4 ± 0.5-fold, mean ± S.E.M., n = 5) and AGS-GFP (2.3 ± 0.4) cells in response to the EGF receptor ligand, transforming growth factor (TGF)-α (100 ng ml^-1, 30 min) and a smaller (1.6 ± 0.1 and 1.8 ± 0.2, respectively) increase was seen with acidic fibroblast growth factor (FGF, 25 ng ml^-1). In co-cultures, gastrin (1 nM, 30 min) increased phospho-p42/44MAP kinase in both ASG-G_R (3.1 ± 0.4-fold) and AGS-GFP cells (1.5 ± 0.1-fold). The responses in AGS-GRP cells to gastrin were fully reversed by AG1478 (3 mM), which inhibits EGF-receptor tyrosine kinase activity (P < 0.05, unpaired t test). In contrast, in the same co-cultures, AG1478 did not significantly inhibit the AGS-G_R cell responses to gastrin. Interestingly, neither the inhibitor of erb-B-2 receptor tyrosine kinase activity, AG825 (5 mM) nor the inhibitor of FGF-receptor tyrosine kinase SU5402 (25 mM) had any effect on gastrin-induced p42/44MAP kinase phosphorylation in either cell type.

We conclude that flow cytometry may be used to distinguish activation of signalling pathways in mixed populations of cells. Gastrin stimulation of MAP kinase activation can be partly direct, i.e. intracellular, and partly due to release of EGF-receptor ligands.