**5. Conclusion**

Note in conclusion that the specific features of agonist responses, including kinetics and magnitude, all-or-nothing behavior and gradual dose-response delay were correctly reproduced by Ca2+ signals elicited by Ca2+ uncaging (**Figure 5**). This supports the idea that agonist-evoked Ca2+ signaling in MSCs includes two different but coupled stages. Initially, agonists stimulate coupling of suitable GPCRs via appropriate G-proteins to PLC, thus triggering IP<sup>3</sup> production, activation of IP<sup>3</sup> receptors (IP<sup>3</sup> Rgrad) followed by the release of Ca2+ ions from Ca2+ store. This machinery generates an initial, presumably local and gradual Ca2+ signal (**Figure 11**). When exceeding the threshold, this local Ca2+ signal stimulates CICR that is mediated by IP<sup>3</sup> receptors (IP<sup>3</sup> RCICR) presumably located in another, spatially separated Ca2+ store. By involving the trigger-like mechanism CICR, a cell generates Ca2+ responses of virtually universal shape and magnitude at different agonist concentrations above the

**Figure 11.** Working model of agonist transduction in MSCs. The binding of agonists to GPCRs stimulates PLC-dependent hydrolysis of PIP<sup>2</sup> to DAG and IP<sup>3</sup> . The consequent activation of IP<sup>3</sup> receptors (IP<sup>3</sup> Rgrad) mediates Ca2+ release from related Ca2+ store, producing an initial Ca2+ signal that gradually rises with agonist concentration (red curve). As soon as this signal reaches the threshold level (dotted line), the process determining agonist-dependent delay of a cellular response, one stimulates IP<sup>3</sup> receptors of another type (IP<sup>3</sup> RCICR) in separated Ca2+ store and triggers CICR. This provides a significant amplification mechanism that finalizes transduction with a large and global Ca2+ signal (blue curve).

cut-off dose. Of course, the presented model is a simplification of the actual transduction process, and roles for other common contributors to intracellular Ca2+ signaling, including Ca2+ pumps, mitochondria, Ca2+ buffer as well as Ca2+-dependent enzymes and ion channels, remain to be elucidated.
