**2.2. Emetic potential of Ca2+ channel activators: behavioral and immunohistochemical evidence**

manner in specific vomit-associated neuroanatomical structures. In both the periphery and the brainstem, emetic neurotransmitters/mediators—such as acetylcholine, dopamine, 5-HT, substance P, prostaglandins, leukotrienes, and/or histamine—may act independently or in combination to evoke vomiting after cisplatin administration [16] (**Figure 1**). In this review, we focus on the current evidence supporting the significance of Ca2+ signaling in emesis generation and its relationship to antiemetic efficacy, as well as the corresponding development

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

responding selective agonists such as GR73632, can increase cytosolic Ca2+ concentration. In

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

tron, MDL7222, metoclopramide) and LTCC blockers (verapamil, nimodipine, nitrendipine)

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>

stimulation, which will be discussed in more detail in Section 3.4. Other emetogens such as

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

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

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

R) is a member of the

R antagonists (tropise-

R

R


R stimulation by substance P or cor-

Rs by 5-HT or its analogs can evoke


Rs [4]. This vomit-competent species

R agonists. Moreover, 5-HT<sup>3</sup>

Rs can evoke intracellular Ca2+ release from the sarco/

R) is a Ca2+-permeable ligand-gated ion channel [20]. Cell


of potential novel antiemetic medications, as shown in brief in **Figure 2**.

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

protein-coupled receptor activation. The neurokinin NK1 receptor (NK<sup>1</sup>

extracellular Ca2+ influx into cells in a manner sensitive to both 5-HT<sup>3</sup>


tachykinin family of G-protein-coupled receptors. NK<sup>1</sup>

receptor (5-HT<sup>3</sup>

lines studies have demonstrated that activation of 5-HT<sup>3</sup>

[20–24]. These studies suggest that both L-type- and 5-HT<sup>3</sup>

discussed evidence has been acquired from isolated cells.

selective emetic agonist targeting the emesis-prone 5-HT<sup>3</sup>

nels are involved in extracellular Ca2+ influx evoked by 5-HT<sup>3</sup>

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

fact GR73632-induced activation of NK<sup>1</sup>

serotonergic 5-HT<sup>3</sup>

112 Calcium and Signal Transduction

agonists of dopamine D<sup>2</sup>

A variety of Ca2+-permeable ion-channels mediating extracellular Ca2+ influx are present in the plasma membrane. Among them are voltage-gated LTCCs, which can be activated by membrane depolarization, and serve as the principal route of Ca2+ entry in electrically excitable cells such as neurons and muscle [43, 44]. Recently we have acquired direct evidence for the proposal that Ca2+ mobilization is an important facet in the mediation of emesis. In fact we have identified the novel emetogen FPL64176 (**Figure 2**), a selective agonist of LTCCs, which causes vomiting in the least shrew in a dose-dependent manner [45, 46]. All tested shrews vomited at a 10 mg/kg dose of FPL64176 administered intraperitoneally (i.p.). LTCCs have been shown to be present in enterochromaffin cells of guinea pig and human small intestinal crypts [47]. Furthermore, in these cells FPL64176 not only can enhance cytosolic Ca2+ concentration, but also increases 5-HT release from enterochromaffin cells [47]. The latter findings may have underpinnings for the mechanisms underlying FPL64176-evoked vomiting observed in least shrew model of emesis. FPL64176 (10 mg/kg., i.p.) can cause Ca2+-dependent 5-HT release from shrew intestinal enterochromaffin cells which in turn could increase vagal afferent activity via stimulation of 5-HT<sup>3</sup> receptors, thereby indirectly triggering emetic signals in the brainstem [2, 48].

Our most recent work has focused on the Ca2+-mobilizing agent thapsigargin (**Figure 3**), a specific and potent inhibitor of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump which transports the free cytosolic Ca2+ into the lumen of the sarco/endoplasmic reticulum to

**Figure 3.** A schematic representation of extracellular Ca2+ influx and intracellular Ca2+ release contributing to thapsigargin-elicited Ca2+ mobilization. Intracellular Ca2+ release from the sarco/endoplasmic reticulum (SER) Ca2+ stores through the inositol triphosphate receptors (IP<sup>3</sup> Rs) and ryanodine receptors (RyRs) is counter-balanced by continuous Ca2+ uptake from the cytoplasm into SER stores by the SER Ca2+-ATPase pump (SERCA). Thapsigargin is a specific inhibitor of SERCA and thus enhances cytosolic levels of Ca2+, a process involving SER Ca2+ release via IP<sup>3</sup> Rs and RyRs as well as extracellular Ca2+ entry through Ca2+ channels located in the plasma membrane including store-operated Ca2+ channels (SOCE) and L-type Ca2+ channels (LTCCs) [49–60].

counter-balance the cytosolic intracellular Ca2+ release from the sarco/endoplasmic reticulum into the cytoplasm via the inositol trisphosphate receptors (IP<sup>3</sup> Rs) and ryanodine receptors (RyRs) [49–51]. Thapsigargin also causes intracellular release of stored Ca2+ from the sarco/ endoplasmic reticulum into the cytosol which subsequently evokes extracellular Ca2+ influx predominantly through store-operated Ca2+ entry (SOCE) in non-excitable cells [52–54]. In total, these events lead to a significant rise in the free concentration of cytosolic Ca2+ [55–57]. In addition, a partial involvement of LTCCs in thapsigargin-evoked contraction has also been demonstrated in rat stomach smooth muscle cells [58], rat gastric smooth muscle [59], and cat gastric smooth muscle [60]. On the other hand, the potential of thapsigargin as a Ca2+ modulating cancer chemotherapeutic agent has been evaluated in both cells and animal models [61]. Thapsigargin-evoked increases in cytosolic Ca2+ concentration can lead to cell apoptosis, which can result in eradication of cancer cells of the breast [62], prostate [63], colon [64] and kidneys [65]. Clinically, a prodrug form of thapsigargin, mipsagargin, is currently under clinical trial as a targeted cancer chemotherapeutic agent with selective toxicity against cancer cells in tumor sites with minimal side-effects to the host [66–69]. In our studies, intraperitoneal administration of thapsigargin (0.1–10 mg/kg) caused vomiting in least shrews in a dose-dependent, but bell-shaped manner, with maximal efficacy at 0.5 mg/kg. An important consideration for the emetic potential of thapsigargin is that it augments the cytosolic levels of free Ca2+ in emetic loci as a result of SERCA inhibition as indicated in our latest discussed finding [70], which is the first study to reflect emesis as a major side-effect of thapsigargin when delivered systemically.

of NK1

of 5-HT<sup>3</sup>

NK1

expressing both 5-HT<sup>3</sup>

**3.2. Cannabinoid CB1**

tors (CB1

CB1

CB1

Rs subsequent to release of substance P in the brainstem [77, 78]. However, our more recent findings suggest that 5-HT and substance P are concomitantly involved in both emetic phases in the gastrointestinal tract as well as in the brainstem [2, 16]. While netupitant is

humans [79, 80] and the least shrew model of emesis [45]. A successful regimen of an oral fixed combined dose of netupitant/palonosetron (NEPA) (**Figure 2**) has been formulated with over 85% clinical efficacy, good tolerability, and high central nervous system penetrance for the prophylactic treatment of acute and delayed chemotherapy-induced nausea and vomiting

suppression of Ca2+ signaling is involved in antiemetic efficacy of both palonosetron and netupitant. Indeed, Rojas et al. [83, 84] have shown that palonosetron causes a persistent inhibition

lar Ca2+ influx. They have further demonstrated that palonosetron can prevent enhancement of substance P-induced intracellular Ca2+ release in response to serotonin in NG108–15 cells

prepared least shrew brainstem slices also demonstrate that palonosetron can abolish enhance-

2-Methyl-5-HT [25]. The latter finding provides more relevant ex-vivo evidence for the Ca2+ modulating antiemetic effect of palonosetron in a vomit-competent species. The role of netupitant in suppression of substance P-evoked enhancement of intracellular Ca2+ levels has also been demonstrated via Ca2+ mobilization assays in vitro in CHO cells expressing the human

Rs. Moreover, pronetupitant, an intravenous alternative to the oral netupitant, appears to be more potent than netupitant in both in vitro Ca2+ measurement studies and in vivo animal behavioral evaluations of substance P in rats [86]. In addition, another clinically approved NK<sup>1</sup>

antagonist antiemetic rolapitant, has been shown to suppress the ability of the selective NK<sup>1</sup>

agonist GR73632 to evoke intracellular Ca2+ release [9, 87–89]. The discussed findings clearly suggest that Ca2+ is a major player in the initiation of vomiting evoked by diverse emetogens.

levonantradol and nabilone, were evaluated in cancer patients for suppression of chemotherapy-induced nausea and vomiting that were not effectively controlled by other available antiemetics [2, 90]. Cannabinoids are increasingly being tested as antiemetics against cisplatin-induced emesis in animal experiments using house musk shrews [91], ferrets [92], or least shrews [73, 93]; nausea-related behavior in rats [91]; radiation-induced emesis in the least shrew [94]; as well as both phases of chemotherapy-induced nausea and vomiting in the clinic

[95–97]. Cannabinoid agonists exert their antiemetic efficacy via direct activation of CB<sup>1</sup>

Rs in the brainstem nuclei involved in emesis has been confirmed, with a high density of

R immunoreactivity in the dorsal motor nucleus of the vagus and the medial subnucleus

ment of intracellular Ca2+ levels in brainstem slices evoked by the selective 5-HT<sup>3</sup>

R function as reflected by a near complete suppression of 5-HT-evoked extracellu-

Recent evidence accumulated from HEK293 cells stably transfected with 5-HT<sup>3</sup>

R antagonist, palonosetron is

http://dx.doi.org/10.5772/intechopen.78370

Role of Calcium in Vomiting

115

Rs suggest that

R agonist

R

R


R antagonists [92, 94, 98–100].

recep-

R antagonist with a unique antiemetic profile in both

Rs [85]. Our Ca2+ monitoring studies performed on acutely-

R antagonists, several phyto- and synthetic

Rs in emesis is currently lacking [101]. The presence of

a highly selective and a longer-acting second generation NK<sup>1</sup>

in cancer patients receiving chemotherapy [9, 81, 82].

Rs and NK1

 **receptor agonists**

cannabinoids including dronabinol (delta-9-tetrahydrocannabinol, Δ<sup>9</sup>

R) since their antiemetic effects were reversed by CB<sup>1</sup>

Before the introduction of first generation 5-HT<sup>3</sup>

Significant evidence for a role for CB<sup>2</sup>

considered as a second generation 5-HT<sup>3</sup>

c-Fos induction has been used to evaluate differential neuronal activation [71]. Our lab has applied immunostaining and detected c-Fos induction in the brainstem emetic nuclei to demonstrate central responsiveness to peripheral administration of a variety of emetogens [4, 70, 72, 73]. The participation of the central emetic neurons in FPL64176-induced vomiting is further indicated by evoked c-Fos expression in brainstem emetic nuclei, the nucleus tractus solitarius and the dorsal motor nucleus of the vagus (unpublished data). Thus, the bloodbrain barrier permeable agent FPL64176 [74–76] could excite emetic neurons directly in the nucleus tractus solitarius and the dorsal motor nucleus of the vagus. Thapsigargin (0.5 mg/kg) also causes increases in c-Fos immunoreactivity in the brainstem emetic nuclei including the area postrema, the nucleus tractus solitarius and the dorsal motor nucleus of the vagus [70].
