Phospholipase C (PLC), catalyses the formation of inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG), which release Ca²⁺ from intracellular stores and activate protein kinase C (PKC), respectively. Here, we assess agonist-induced IP₃ and DAG production, PKC activation and Ca²⁺ release at the single cell level using fluorescent 'biosensors'. The aim of this study was to utilize these biosensors to assess the pharmacological relationships between IP₃/Ca²⁺ responses and DAG/PKC activation in single CHO cells co-expressing M₃ muscarinic and _1B adrenoceptors.

IP₃, DAG production, and PKC activation were determined by transient transfection of cells with
eGFP-PH, eGFP-C1₂-PKC (Nahorski et al., 2003) and eGFP-MARCKS (Arbuzova et al., 2002), respectively, and agonist-induced responses were measured as changes in cytosolic fluorescence. Ca²⁺ mobilization was detected using epifluorescence microscopy of fura 2-loaded cells (Young et al., 2003).

IP3, DAG and PKC activity responses to methacholine (MCh) and noradrenaline (NA) were observed as initial peaks, followed by sustained plateau phases. Peak-and-plateau Ca²⁺ responses were also seen except at low [NA]. Peak response pEC₅₀ and R_max values for IP₃ and DAG production, Ca²⁺ mobilization and PKC activity are shown in Table 1. An approx. 2 fold higher R_max for IP₃- and DAG-stimulated by MCh indicates that the M₃ is more efficiently coupled to the PLC pathway than the _1B. While the data reveal modest signal amplification between IP₃ and Ca²⁺ mobilisation (~2.5-4 fold shift), a much more marked amplification of the DAG signal with respect to PKC activity (16 and 19 fold shift for MCh and NA, respectively) was observed. DAG responses to MCh or NA were unaffected by inclusion of 30 mM butan-1-ol to eliminate phospholipase D-derived DAG generation.

Table 1. Potency (pEC₅₀) and maximal response (R_max) estimates for different readouts of MCh- and NA-stimulated PLC activation measured in single CHO-m3_1B cells. Data are shown as means ± s.e. mean for
n 12 cells over 3 experiments. IP₃ and PKC values represent fold increase and DAG relative decline in biosensor cytosolic fluorescence. Ca²⁺ values represent 340/380 changes for fura 2-fluorescence.

These data allow quantitative assessment of GPCR signal amplification for the first time in single cells, and highlight how poorly coupled receptors (e.g. _1B adrenoceptor) can nevertheless elicit robust downstream responses.

Arbuzova, A. et al. (2002) Biochem. J. 362, 1-12.
Young, K.W. et al. (2003) J. Biol. Chem. 278, 20753-20760.
Nahorski, S.R. et al. (2003) Trends Neurosci. 26, 444-452.

We gratefully acknowledge the support of the Wellcome Trust.