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.

Keywords: ERK; FPL64176; L-type Ca(2+) channel; Ryanodine receptor; Serotonin.

Figure 1. The L-type Ca²⁺ channel antagonist nifedipine blocks FPL64176-induced emesis

Least shrews were pretreated subcutaneously (s.c.) (n = 6 per group) with either the LTCC antagonist nifedipine (10 mg/kg) or its vehicle (0 mg/kg) for 30 min, followed by an intraperitoneal (i.p.) FPL64176 injection at its fully effective emetic dose, 10 mg/kg. Each shrew was placed in the observation cage and frequencies of vomiting were recorded separately for the next 30 min. A) The vomiting frequency data between groups were analyzed using unpaired t-test and expressed as mean ± S.E.M. **P < 0.01 vs. 0 mg/kg. B) The percentage of animals vomiting at different doses was compared using Chi-square test. ***P < 0.001 vs. 0 mg/kg.

Figure 2. FPL64176 triggers c-Fos induction in brainstem emetic nuclei in a LTCC antagonist (nifedipine)-sensitive manner

A–C): Examples of c-Fos immunoreactivity (Fos-IR) in the brainstem. Immunolabeling was performed on free-floating coronal tissue sections using avidin–biotin–peroxidase amplification and nickel-enhanced diaminobenzidine visualization. The area postrema (AP), nucleus of the solitary tract (NTS) and dorsal motor nucleus of the vagus (DMNX) constitute the brainstem dorsal vagal complex (DVC) emetic nuclei. Note the different cytoarchitectonic details such as cell size and packing, which differentiate each nucleus from each other. A) Relatively few Fos-IR nuclei are found in the DVC of control shrew injected with nifedipine vehicle 30 min prior to the vehicle of FPL64176. B) In FPL64176 (10 mg/kg., i.p.)-injected shrew pretreated with nifedipine vehicle (Veh), shows increased Fos-IR throughout the NTS and DMNX, but only infrequently in AP and none in hypoglossal nucleus (XII). C) In the presence of LTCC antagonist nifedipine (10 mg/kg., s.c.), the ability of FPL64176 (10 mg/kg., i.p.) to induce Fos-IR was abrogated. Scale Bar, 100 μm. D) Summary data of groups as demonstrated in A, B and C. n = 3 shrews per group. 3 sections from each shrew were used for analysis. Fos-IR positive nuclei (dark and filled) but not weakly labeled nuclei were quantified. Data for each region was expressed as mean number of Fos-IR nuclei ± S.E.M. One-way ANOVA followed by Dunn’s post hoc test. **P < 0.01 vs. control (i.e. Veh+Veh).

Figure 3. Effect of Ca²⁺ modulators on FPL64176-induced emesis

Varying doses of the intracellular Ca²⁺ release channel RyR inhibitor dantrolene (A–B) (n = 6), the IP₃R inhibitor 2-APB (C–D) (n = 6) or corresponding vehicles (i.e. 0 mg/kg; n = 6) were injected (i.p.) to different groups of shrews. Following 30 min exposure animals were injected with FPL 64176 (10 mg/kg, i.p.) and observed for next 30 min. The vomiting frequency data were analyzed using the Kruskal-Wallis non-parametric one-way ANOVA followed by Dunn’s post hoc test and expressed as mean ± S.E.M. The percentage of animals vomiting within groups at different doses was compared using Chi-square test. A–B) Dantrolene dose-dependently reduced both emetic indices in response to FPL64176. *P < 0.05, **P < 0.01 vs. 0 mg/kg. C–D) 2-APB at all tested dosages failed to affect FPL64176-induced emesis. E–F) Combination of nifedipine with dantrolene at ineffective doses exerted additive anti-emetic efficacy against FPL64176. Nifedipine (0.25 mg/kg, s.c.) plus dantrolene (0.5 m/kg., i.p.) or corresponding vehicles (i.e. 0 mg/kg) were administered to different groups (n = 7) of shrews 30 min prior to FPL 64176 (10 mg/kg., i.p.) challenge. **P < 0.01 vs. Nifedipine 0.25 mg/kg + Dantrolene 0.5 mg/kg.

Figure 4. FPL64176 upregulates PKCα/βII, ERK1/2 and Akt phosphorylation in brainstem in a LTCC antagonist nifedipine-sensitive manner

A–B) Time-courses of FPL64176-induced PKCα/βII, ERK1/2 and Akt phosphorylation the least shrew brainstem. Shrews (n =3 per group) were injected with either vehicle (0 min) or FPL 64176 (10 mg/kg., i.p.). Brainstems were collected at 0, 5, 10, 15, 20, 25, 30 and 60 min. Phospho-PKCα/βII at Thr638/641 (pPKCα/βII), GAPDH, phospho-ERK1/2 at Thr202/204 (pERK1/2), ERK1/2, phospho-Akt at Ser473 (pAkt) and Akt of protein samples extracted from individual brainstems were determined by Western blots. Panel A shows representative blots. Panel B shows summarized data. Bands were quantified using ImageJ software. Ratios of pPKCα/βII (~ 80 kD) to GAPDH (~ 37 kD), pERK1/2 (42/44 kD) to ERK1/2 and pAkt (~ 60 kD) to Akt were calculated and expressed as fold change (mean ± S.E.M.) of control (0 min). *P < 0.05, ***P < 0.001 vs. 0 min, One-way ANOVA followed by Dunnett’s test. C–D) Nifedipine abolished PKCα/βII, ERK1/2 and Akt phosphorylation evoked by FPL64176. Following a 30 min prior treatment with either vehicle (Veh) or nifedipine (10 mg/kg., s.c.), shrews were given FPL64176 (10 mg/kg., i.p.). Shrews injected with vehicle of nifedipine followed by vehicle of FPL64176 were used as controls (Ctl). Following 20 min after the second injection, collected brainstems were subjected to Western blots. Panel C shows representative blots. Panel D shows summarized data. N = 3 shrews per group. **P < 0.01, ***P < 0.001 vs. Ctl. One-way ANOVA followed by Dunnett’s test.

Figure 5. Effects of inhibitors of PKC, ERK1/2 and PI3K-Akt on FPL64176-induced emesis

Varying doses of inhibitors of PKC (GF109203X), MEK-ERK1/2 cascade (PD98059), PI3K-Akt (LY294002) or corresponding vehicles (i.e. 0 mg/kg) were administered (i.p.) to different groups of shrews 30 min before to FPL64176 (10 mg/kg., i.p.) injection (n = 6 shrews/group). Vomiting was recorded for the next 30 min post FPL64176 treatment. The vomiting frequency data were analyzed using the Kruskal-Wallis non-parametric one-way ANOVA followed by Dunn’s post hoc test and expressed as mean ± S.E.M. The percentage of animals vomiting within groups at different doses was compared using Chi-square test. GF109203X (10 mg/kg) (A–B), PD98059 (2.5 and 5 mg/kg) (C–D) and LY294002 (10 mg/kg) (E–F) significantly decreased both emetic indices (i.e. Vomit frequency & Percentage of shrews vomiting) in response to FPL64176. *P < 0.05, **P < 0.01, ***P < 0.001 vs. 0 mg/kg.

Figure 6. Effect of the NK₁ receptor antagonist netupitant on FPL64176-induced emesis

A–B) Netupitant dose-dependently reduced both emetic parameters in response to FPL64176. Varying doses of netupitant or its vehicle (i.e. 0 mg/kg) were injected (i.p.) to different groups of shrews (n = 6). Following 30 min exposure, animals were challenged with FPL 64176 (10 mg/kg, i.p.) and were observed for the next 30 min. The vomiting frequency data were analyzed using the Kruskal-Wallis non-parametric one-way ANOVA followed by Dunn’s post hoc test and expressed as mean ± S.E.M. The percentage of animals vomiting within groups at different doses was compared using Chi-square test. **P < 0.01, ***P < 0.001 vs. 0 mg/kg. C–D) A combination of ineffective dose of serotonin type 3 receptor antagonist palonosetron with netupitant exerted additive antiemetic efficacy against FPL64176. Palonosetron (0.1 mg/kg, s.c.) plus netupitant (1 m/kg., i.p.) or corresponding vehicles (i.e. 0 mg/kg) were administered to different groups (n = 8) of shrews 30 min prior to FPL 64176 (10 mg/kg., i.p.) challenge. *P < 0.05, **P < 0.01 vs. Palonosetron 0.1 mg/kg + Netupitant 1 mg/kg.

Figure 7. Example of images (20×) of serotonin and substance P immunolabeling following injection of the emetic agent, FPL64176

Shrews (n = 3 per group) were euthanized at 15 min post vehicle control or FPL64176 (10 mg/kg, i.p.) injection, and immunohistochemistry were conducted as described in methods section “5-HT and SP Immunohistochemistry”. Coronal brain sections (20 μm) containing the brainstem DVC emetic nuclei, area postrema (AP), nucleus of the solitary tract (NTS) and dorsal motor nucleus of the vagus (DMNX) were used. Sections were immunolabeled with rabbit anti-serotonin (5-HT) antibody and rat anti-substance P (SP) antibody overnight followed by FITC-conjugated donkey anti-rabbit secondary antibody incubation and Cy3^TM3-conjugated donkey anti-rat secondary antibody incubation. After counterstaining with DAPI, images were acquired using a confocal microscope. Left panels A, C, and E show representative images of sections from vehicle controls labelled with anti-5-HT (green), anti-SP antibodies (red) and DAPI (blue) respectively. Right panels B and D show representative images of sections from FPL64176-treated shrews labelled with anti-5-HT and anti-SP antibodies respectively. Image from DAPI-stained sections of FPL64176-treated shrews is not shown. Scale bar, 100 μm. Boxed areas (the dorsomedial NTS subnucleus) in panels A and B were further magnified and displayed as Figure 8. Panel F. Average Fluorescence Intensities of 5-HT in boxed area in A and B and SP staining in the field as boarded by the dashed lines in C and D. **P < 0.01. n.s., non-significant. Unpaired t-test.

Figure 8. Example of images (100×) of serotonin-immunoreactivity in the dorsomedial NTS related to administration of the emetic agent, FPL64176

Shrews were treated and immunohistochemistry were performed as described in Figure 7. Panel A and B, high magnification (100×) of boxed area (the dorsomedial NTS) of figures 7A and 7B, show more evidence of density of serotonin (5-HT)-immunoreactive varicosities (green) at 15 min post FPL64176 (10 mg/kg., i.p.) administration (panel B), in contrast with vehicle control (panel A). C–D) Cell nuclei counterstained with DAPI (blue). E–F) Merged version demonstrates co-labeled neurons. Scale bar, 10 μm.