**2. Emerging roles of Ca2+ in emesis**

#### **2.1. Emetic receptor stimulation increases intracellular Ca2+ concentration**

Excitatory receptor activation by corresponding agonists can increase cytosolic Ca2+ levels via both mobilization of intracellular Ca2+ stores (e.g., endoplasmic reticulum = ER) and influx from extracellular fluid [17]. The evoked cytoplasmic Ca2+ increase may result from direct activation of ion channels, or indirectly via signal transduction pathways following G protein-coupled receptor activation. The neurokinin NK1 receptor (NK<sup>1</sup> R) is a member of the tachykinin family of G-protein-coupled receptors. NK<sup>1</sup> R stimulation by substance P or corresponding selective agonists such as GR73632, can increase cytosolic Ca2+ concentration. In fact GR73632-induced activation of NK<sup>1</sup> Rs can evoke intracellular Ca2+ release from the sarco/ endoplasmic reticulum stores via Gα/q-mediated phospholipase C pathway, which subsequently evokes extracellular Ca2+ influx through L-type Ca2+ channels (LTCCs) [17–19]. The serotonergic 5-HT<sup>3</sup> receptor (5-HT<sup>3</sup> R) is a Ca2+-permeable ligand-gated ion channel [20]. Cell lines studies have demonstrated that activation of 5-HT<sup>3</sup> Rs by 5-HT or its analogs can evoke extracellular Ca2+ influx into cells in a manner sensitive to both 5-HT<sup>3</sup> R antagonists (tropisetron, MDL7222, metoclopramide) and LTCC blockers (verapamil, nimodipine, nitrendipine) [20–24]. These studies suggest that both L-type- and 5-HT<sup>3</sup> -receptor Ca2+-permeable ion channels are involved in extracellular Ca2+ influx evoked by 5-HT<sup>3</sup> R agonists. Moreover, 5-HT<sup>3</sup> R activation indirectly causes release of Ca2+ from ryanodine-sensitive intracellular Ca2+ stores subsequent to the evoked extracellular Ca2+ influx which greatly amplifies the cytoplasmic concentration of Ca2+ [23]. In fact, our findings from behavioral studies in the least shrew emesis model [25] further support the notion of Ca2+-induced Ca2+ release following 5-HT<sup>3</sup> R stimulation, which will be discussed in more detail in Section 3.4. Other emetogens such as agonists of dopamine D<sup>2</sup> - [26, 27], cholinergic M<sup>1</sup> - [28, 29], histaminergic H<sup>1</sup> - [30, 31], and opiate μ- [32, 33] receptors, as well as cisplatin [34], prostaglandins [35, 36], rotavirus NSP4 protein [37, 38] and bacterial toxins [39, 40] also possess the potential to mobilize Ca2+ which involve extracellular Ca2+ influx and/or Ca2+ release from intracellular Ca2+ pools. Much of the discussed evidence has been acquired from isolated cells.

The least shrew is an emesis-competent mammal whose reactions to common emetogens are well-defined and correlate closely with human responses [2]. 2-Methyl-5-HT is a well-known selective emetic agonist targeting the emesis-prone 5-HT<sup>3</sup> Rs [4]. This vomit-competent species is an excellent animal model for studying the emetic activity of diverse agents [2]. In fact least shrews exhibit dose-dependent full emetic responses to intraperitoneal administration of both the peripherally-acting 5-HT, as well as to its central nervous system-penetrating analog, 2-Methyl-5-HT [4, 41, 42]. In our studies, incubation of least shrew brainstem slices containing the dorsal vagal complex emetic loci with 2-Methyl-5-HT, results in a rapid increase in intracellular Ca2+ concentration as reflected by an increase in fluo-4 AM fluorescence intensity in a palonosetron (a 5-HT<sup>3</sup> R antagonist)/nifedipine-sensitive manner [22, 25].
