Intracellular emetic signaling evoked by the L-type Ca2+ channel agonist FPL64176 in the least shrew (Cryptotis parva)

Abstract

Ca2+ plays a major role in maintaining cellular homeostasis and regulates processes including apoptotic cell death and side-effects of cancer chemotherapy including vomiting. Currently we explored the emetic mechanisms of FPL64176, an L-type Ca2+ channel (LTCC) agonist with maximal emetogenic effect at its 10 mg/kg dose. FPL64176 evoked c-Fos immunoreactivity in shrew brainstem sections containing the vomit-associated nuclei, nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus. FPL64176 also increased phosphorylation of proteins ERK1/2, PKCα/βII and Akt in the brainstem. Moreover, their corresponding inhibitors (PD98059, GF 109203X and LY294002, respectively) reduced FPL64176-evoked vomiting. A 30 min subcutaneous (s.c.) pretreatment with the LTCC antagonist nifedipine (10 mg/kg) abolished FPL64176-elicited vomiting, c-Fos expression, and emetic effector phosphorylation. Ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs) mediate intracellular Ca2+ release from the sarcoplasmic/endoplasmic reticulum. The RyR antagonist dantrolene (i.p.), or a combination of low doses of nifedipine and dantrolene, but not the IP3R antagonist 2-APB, significantly attenuated FPL64176-induced vomiting. The serotonin type 3 receptor (5-HT3R) antagonist palonosetron (s.c.), the neurokinin 1 receptor (NK1R) antagonist netupitant (i.p.) or a combination of non-effective doses of netupitant and palonosetron showed antiemetic potential against FPL64176-evoked vomiting. Serotonin (5-HT) and substance P immunostaining revealed FPL64176-induced emesis was accompanied by an increase in 5-HT but not SP-immunoreactivity in the dorsomedial subdivision of the NTS. These findings demonstrate that Ca2+ mobilization through LTCCs and RyRs, and subsequent emetic effector phosphorylation and 5-HT release play important roles in FPL64176-induced emesis which can be prevented by 5-HT3R and NK1R antagonists.

Mechanisms of Nausea and Vomiting: Current Knowledge and Recent Advances in Intracellular Emetic Signaling Systems

“…In addition, all tested shrews vomit at a 10 mg/kg dose of FPL64176 administered intraperitoneally (i.p.) [78,244]. These findings directly support our notion that Ca 2+ mobilization is an important facet in the mediation of emesis.…”

Signal transduction pathways involved in dopamine D2 receptor-evoked emesis in the least shrew (Cryptotis parva)

“…Although the significance of most the phosphorylations remains to be fully understood (Mahajan and Mahajan, 2012), it is wellrecognized that Akt activity depends on two phosphorylated sites one of which is Ser473. Our laboratory was the first to correlate enhanced phosphorylation of Akt at Ser473 in the DVC following the administration of the L-type Ca 2+ Channel (LTCC) agonist FPL64176 with vomiting (Zhong et al, 2018). In the current study, administration of 2 mg/kg of the dopamine D 2 receptor preferring agonist quinpirole alone caused a significant increase in phosphorylation of Akt at Ser473 in the brainstem of least shrews after 15 and 30 min.…”

The HCN Channel Blocker ZD7288 Induces Emesis in the Least Shrew (Cryptotis parva)

“…Both c-Fos and pERK1/2 have been applied as neuronal activation indicators in vivo ( Ferrini et al, 2014 ). Our published findings implicate ERK1/2 phosphorylation in the brainstem as one common event in the emetic responses elicited by i. p.-injection of distinct emetogens including the: 1) substance P neurokinin NK 1 receptor agonist GR73632, 2) LTCC activator FPL64176, 3) intracellular Ca 2+ signaling amplifier thapsigargin, 4) the serotonin 5-HT 3 receptor agonist 2-Methyl-5-HT ( Zhong et al, 2014 ; Zhong et al, 2016 ; Zhong et al, 2018 ; Zhong et al, 2019 ), and 5) chemotherapeutic agent cisplatin ( Darmani et al, 2015 ). Consistent with these findings, in the present study we find significant phosphorylation of ERK1/2 in the DVC emetic nuclei (AP, NTS and DMNX) within the least shrew brainstem at 15 min post ZD7288 treatment (1 mg/kg, i. p.).…”

“…Immunohistochemistry of the least shrew brainstem (20 µm) and jejunal (25 µm) sections was carried out as previously reported ( Zhong et al, 2016 ; Zhong et al, 2018 ; Zhong et al, 2019 ; Zhong and Darmani, 2020 ). The jejunal segment of the least shrew small intestine was dissected in accord with Ray et al (2009a) .…”

“…Rabbit anti-c-Fos polyclonal antibody (1:5000, ab190289, Abcam) and Alexa Fluor 594 donkey anti-rabbit secondary antibody (1:500, Abcam) were used. The criterion used to differentiate the AP, NTS and DMNX within the DVC has been well recognized ( Darmani et al, 2008 ; Ray et al, 2009a ; Chebolu et al, 2010 ; Zhong et al, 2016 ; Zhong et al, 2018 ; Zhong et al, 2019 ; Zhong and Darmani, 2020 ). For each animal, c-Fos positive cells in the AP, both sides of NTS and DMNX from 3 sections at 90-μm intervals were counted manually by an experimenter blind to experimental conditions.…”