*4.5.2. Activation and inhibition of CaMKII, ERK1/2, PKC, and Akt are correspondingly linked to emesis induction and prevention*

Vomit-associated Ca2+ mobilization as well as time-dependent Ca2+/calmodulin kinase IIα (CaMKIIα) and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphorylation in the least shrew brainstem occurs: (i) following 5-HT<sup>3</sup> R-evoked vomiting caused by its selective agonist 2-Methyl-5-HT [46], (ii) thapsigargin-induced emesis in the least shrew [70], as well as (iii) NK1 R-mediated vomiting evoked by the selective NK<sup>1</sup> R agonist GR73632 in the least shrew [167] (**Figure 5**). Our additional behavioral evidence that inhibitors of CaMKII or ERK1/2 attenuate the evoked emesis provides further credence for involvement of CaMKII and ERK1/2 downstream of the discussed emetic receptors/effectors. Furthermore, other published evidence support phosphorylation of protein kinase Cα/βII (PKCα/βII) and ERK1/2 in least shrew brainstem are associated with cisplatin-induced emesis [144, 149] (**Figure 5**). In fact significant upregulation of ERK1/2 phosphorylation occurs with peak vomit frequency during both the immediate and delayed phases of emesis caused by cisplatin in the least shrew [144, 149].

It has been suggested that glucocorticoids' antiemetic efficacy could be due to their antiinflammatory effects [174] probably via a reduction in the synthesis of prostaglandins and leukotrienes [175], both of which can be increased during chemotherapy [6]. Although not all, but several prostaglandins (e.g., PGE2 and PGF2a) and cysteinyl leukotrienes (e.g., LTC4

and LTD4 ), appear to be potent emetogens [72, 149, 176, 177]. Our findings demonstrate that unlike other leukotrienes (e.g., LTA4 , LTB4 and LTF4 ), the above discussed leukotrienes are effective emetogens with the following potency order: LTC4 = LTD4 > LTE4 . Regarding LTC4, the evoked vomiting was shown to be suppressed in a dose-dependent manner in the least shrew by the antiasthmatic drug pranlukast, the corresponding cysteinyl leukotrienes receptor 1 (CysLT1R) antagonist [72]. Although not available in the USA, the cost of other members of this class of drugs (montelukast and Zafirlukast) that are sold in the USA is less than one dollar per pill. Based on pranlukast's efficacy against LTC4 -induced vomiting [72], we envisaged it may have potential utility against cisplatin-evoked emesis. Our most recent publication [178] shows the potential of pranlukast (currently used for the treatment of various respiratory disorders including asthma), as a new class of antiemetic for the suppression of the acute- and delayed- phases of cisplatin-evoked vomiting in the least shrew. An intraperitoneal (i.p.) dose of 10 mg/kg pranlukast by itself significantly reduced the mean frequency of vomits by 70% and fully protected 46% of least shrews during the delayed-phase of cisplatin (10 mg/kg, i.p.)-evoked vomiting. Although pranlukast tended to substantially reduce both the mean frequency of vomits and the number of shrews vomiting during the early-phase, these reductions failed to attain significance. When pranlukast was combined with a first (tropisetron)- or a second (palonosetron)-generation 5-HT<sup>3</sup> R antagonist, it potentiated their antiemetic efficacy during both acute- and delayed-phases of cisplatin-evoked vomiting. Moreover, pranlukast potentiated the antiemetic efficacy of serotonin 5-HT<sup>3</sup> receptor antagonists, tropisetron and palonosetron, against chemotherapyinduced nausea and vomiting. In fact per hour efficacy antiemetic profile of pranlukast combined with palonosetron or tropisetron during both phases of chemotherapy-induced nausea and vomiting in the least shrew resembles those of: (i) the NK<sup>1</sup> receptor antagonist netupitant (5 mg/kg) plus palonosetron (0.1 mg/kg) in the same species [144]; (ii) netupitant plus ondansetron in ferrets [179]; and (iii) ondansetron plus aprepitant in combination with dexamethasone in ferrets [179]; and (iv) palonosetron plus netupitant in combination with dexamethasone in ferrets [179]. If analogs of pranlukast such as montelukast and zafirlukast can also provide similar antiemetic potential, then clinical trials should be initiated since this class of drugs are relatively inexpensive than available effective antiemetic regimens against chemotherapy-induced nausea and vomiting. Our related biochemical data indicates the mechanisms of antiemetic action of pranlukast are linked to suppression of cisplatin-elicited PKCα/βII, ERK1/2 and PKA activation (phosphorylation) in the least shrew brainstem [178]. Moreover, suppression of these signaling molecules may be shared in the anti-inflammatory signaling pathway of pranlukast.

**Figure 5.** Summarized behavioral and biochemical evidence for intracellular signaling molecules (cAMP, PKA, CaMKII, ERK1/2, PKC) related to emesis based on the least shrew emesis model. First, cyclic AMP (cAMP) is synthesized by adenylate cyclase and cAMP activates protein kinase A (PKA) [110, 168]. The adenylyl cyclase/cAMP/PKA signaling pathway can mediate vomiting. Indeed, increased levels of endogenous cAMP can evoke vomiting in animal models [109, 170] as well as humans [171–173], which can be prevented by adenylate cyclase inhibitor SQ22536 [109]. Evoked PKA-phosphorylation is associated with peak vomit frequency during both immediate- and delayed-phases of vomiting caused by cancer chemotherapeutics including cisplatin and cyclophosphamide in the least shrew [109, 144, 149]. In addition, Ca2+/calmodulin kinase IIα (CaMKIIα) and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2)

R agonist 2-Methyl-5-HT

R-evoked vomiting caused by its

R agonist GR73632 in the least

R agonist GR73632 [167]. Phosphorylation of protein kinase Cα/βII (PKCα/

phosphorylation in the least shrew brainstem were elevated in vomiting evoked by the 5-HT<sup>3</sup>

tion in the least shrew brainstem occurs: (i) following 5-HT<sup>3</sup>

βII) and ERK1/2 in least shrew brainstem were also upregulated in the vomiting induced by cisplatin [144, 149].

*4.5.2. Activation and inhibition of CaMKII, ERK1/2, PKC, and Akt are correspondingly linked* 

demonstrated that increased brain cAMP levels evoke vomiting which can be prevented by SQ22536 (**Figure 5**), an inhibitor of adenylyl cyclase [109]. Moreover, PKA-phosphorylation is associated with peak vomit frequency during both immediate- and delayed-phases of vomiting caused by either cisplatin or cyclophosphamide in the least shrew [109, 144, 149] (**Figure 5**).

Vomit-associated Ca2+ mobilization as well as time-dependent Ca2+/calmodulin kinase IIα (CaMKIIα) and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphoryla-

selective agonist 2-Methyl-5-HT [46], (ii) thapsigargin-induced emesis in the least shrew [70], as

shrew [167] (**Figure 5**). Our additional behavioral evidence that inhibitors of CaMKII or ERK1/2 attenuate the evoked emesis provides further credence for involvement of CaMKII and ERK1/2 downstream of the discussed emetic receptors/effectors. Furthermore, other published evidence support phosphorylation of protein kinase Cα/βII (PKCα/βII) and ERK1/2 in least shrew brainstem are associated with cisplatin-induced emesis [144, 149] (**Figure 5**). In fact significant upregulation of ERK1/2 phosphorylation occurs with peak vomit frequency during both the immediate and delayed phases of emesis caused by cisplatin in the least shrew [144, 149].

It has been suggested that glucocorticoids' antiemetic efficacy could be due to their antiinflammatory effects [174] probably via a reduction in the synthesis of prostaglandins and leukotrienes [175], both of which can be increased during chemotherapy [6]. Although not all, but several prostaglandins (e.g., PGE2 and PGF2a) and cysteinyl leukotrienes (e.g., LTC4

R-mediated vomiting evoked by the selective NK<sup>1</sup>

[46], thapsigargin [70], or the selective NK<sup>1</sup>

122 Calcium and Signal Transduction

*to emesis induction and prevention*

well as (iii) NK1

When antiemetic mechanism of action of pranlukast against LTC4-induced vomiting or cisplatin-induced responses is discussed, Ca2+ is also an essential element. Montelukast and pranlukast were found to inhibit nucleotide-induced Ca2+ mobilization in a human monocytemacrophage-like cell line, DMSO-differentiated U937 [180]. CysLT1 receptors belonging to the rhodopsin family of the G protein-coupled receptor genes respond to LTD4 with a strong increase in cytosolic Ca2+ concentration partially sensitive to pertussis toxin, and with the activation of the Ras-MAPK cascade totally dependent upon Gi/o [144]. These signaling effects were totally inhibited by various specific CysLT1-receptor antagonists, and CysLT1 antagonists inhibit both the P2Y agonist-induced activation of phospholipase C and intracellular Ca2+ mobilization [144].
