Phosphoinositide and Ca2+ signalling in Chinese hamster ovary cells expressing recombinant muscarinic M3 receptors.

It is well established that M3 muscarinic receptors couple, via the Gq/11 family of G-proteins, to the activation of phosphoinositidase C (PIC) leading to Ins(l,4,5)P3 formation and intracellular Ca2+ mobilisation (Caulfield, 1993). The ability of many muscarinic agonists to initiate a response via the phosphoinositide pathway is known to be tissue dependent. Using recombinant human muscarinic M3 receptors, expressed as a homogeneous population in a host CHO-Kl cell line, the significance of receptor-G-protein stoichiometry, in agonist mediated inositol phosphate and Ca2+ responses, was investigated. Agonist-stimulated PIC and Ca2+ responses in cell lines expressing different densities of the M3 receptor, or cells treated with an alkylating agent, were compared in an attempt to determine the relationship between M3 muscarinic occupancy by full and partial agonists and the efficiency of coupling to the effector PIC. The transfected cell lines appeared to have a classical 'reserve' for PIC activation, which was dependent on receptor density and partly dependent on extracellular Ca2+ concentration. This reserve appeared to be greater when PIC activity was assessed by total [3H]inositol phosphate accumulation rather than Ins(l,4,5)P3 mass accumulation, indicating that the method of assessment must be taken into account when determining the characteristics of phosphoinositide signalling. Agonists appeared to be more efficient at mobilising intracellular Ca2+ than activating PIC, as dose response curves for agonist-stimulated Ca2+ mobilisation lay to the left of those for PIC activation. The ability of agonists to mobilise intracellular Ca2+ also appeared to be dependent on receptor density and extracellular Ca2+ concentration. However, there did not appear to be a receptor reserve for this response. In conclusion, the results obtained for the PIC response appear to follow the predictions of classical receptor theory and Ca2+ mobilisation, as a response downstream of PIC activation, is more sensitive to agonists, as would be expected following an amplification of the signal. However, following amplification, an increase in receptor reserve would be expected. Clearly this is not the case and the agonist induced increases in intracellular Ca2+ in this study cannot be adequately described by classical receptor theory.