Serotonin 5-HT3 receptor-mediated vomiting occurs via the activation of Ca2+/CaMKII-dependent ERK1/2 signaling in the least shrew (Cryptotis parva)

Abstract

Stimulation of 5-HT3 receptors (5-HT3Rs) by 2-methylserotonin (2-Me-5-HT), a selective 5-HT3 receptor agonist, can induce vomiting. However, downstream signaling pathways for the induced emesis remain unknown. The 5-HT3R channel has high permeability to extracellular calcium (Ca(2+)) and upon stimulation allows increased Ca(2+) influx. We examined the contribution of Ca(2+)/calmodulin-dependent protein kinase IIα (Ca(2+)/CaMKIIα), interaction of 5-HT3R with calmodulin, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling to 2-Me-5-HT-induced emesis in the least shrew. Using fluo-4 AM dye, we found that 2-Me-5-HT augments intracellular Ca(2+) levels in brainstem slices and that the selective 5-HT3R antagonist palonosetron, can abolish the induced Ca(2+) signaling. Pre-treatment of shrews with either: i) amlodipine, an antagonist of L-type Ca(2+) channels present on the cell membrane; ii) dantrolene, an inhibitor of ryanodine receptors (RyRs) Ca2+-release channels located on the endoplasmic reticulum (ER); iii) a combination of their less-effective doses; or iv) inhibitors of CaMKII (KN93) and ERK1/2 (PD98059); dose-dependently suppressed emesis caused by 2-Me-5-HT. Administration of 2-Me-5-HT also significantly: i) enhanced the interaction of 5-HT3R with calmodulin in the brainstem as revealed by immunoprecipitation, as well as their colocalization in the area postrema (brainstem) and small intestine by immunohistochemistry; and ii) activated CaMKIIα in brainstem and in isolated enterochromaffin cells of the small intestine as shown by Western blot and immunocytochemistry. These effects were suppressed by palonosetron. 2-Me-5-HT also activated ERK1/2 in brainstem, which was abrogated by palonosetron, KN93, PD98059, amlodipine, dantrolene, or a combination of amlodipine plus dantrolene. However, blockade of ER inositol-1, 4, 5-triphosphate receptors by 2-APB, had no significant effect on the discussed behavioral and biochemical parameters. This study demonstrates that Ca(2+) mobilization via extracellular Ca(2+) influx through 5-HT3Rs/L-type Ca(2+) channels, and intracellular Ca(2+) release via RyRs on ER, initiate Ca(2+)-dependent sequential activation of CaMKIIα and ERK1/2, which contribute to the 5-HT3R-mediated, 2-Me-5-HT-evoked emesis.

Figure 1. Effects of prior administration of extracellular and intracellular Ca²⁺ antagonists on emesis induced by the 5-HT₃R agonist 2-Me-5-HT, which evokes Ca²⁺ responses.

Graph A) Increased intracellular Ca²⁺ levels (as demonstrated by fluo-4 AM) caused by the selective 5-HT₃R agonist, 2-Me-5-HT (1 µM), in the least shrew brainstem area postrema (AP) region in the absence (vehicle, left panel) and presence of the selective 5-HT₃R antagonist, palonosetron (1 µM) (right panel). Graphs B–E) Effects of Ca²⁺ modulators on the frequency and percentage of shrews vomiting in response to 2-Me-5-HT administration (5 mg/kg, i.p.). Different groups of least shrews were given an injection of either the corresponding vehicle, or varying doses of: 1) the L-type Ca²⁺ channel blocker, amlodipine (s.c.) (B); 2) the ryanodine receptor antagonist, dantrolene (i.p.) (C); 3) lower but combined doses of amlodipine (Aml, 5 mg/kg, s.c.) plus dantrolene (Dan, 10 mg/kg, i.p.) (D); or 4) the inositol-1, 4, 5-triphosphate receptor blocker, 2-APB (i.p.) (E); which were administered 30 min prior to 2-Me-5-HT injection. For each case, the vomiting responses were recorded for 30 min post 2-Me-5-HT administration. The frequency data is presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 compared with corresponding vehicle-pretreated controls.

Figure 2. 2-Me-5-HT enhances 5-HT₃R-calmodulin (CaM) colocalization in a palonosetron-sensitive manner in least shrew brainstem and intestine.

Graphs A and B) Effects of the 5-HT₃R agonist 2-Me-5-HT and the 5-HT₃R antagonist palonosetron on 5-HT₃R-CaM interaction in the least shrew brainstem as revealed by co-immunoprecipitation (IP). Palonosetron (Palo, 5 mg/kg, s.c) or its vehicle (Veh) was administered 30 min prior to 2-Me-5-HT (or its vehicle) in different groups of shrews. Twenty minutes following 2-Me-5-HT administration (5 mg/kg, i.p.), brainstems were collected from the Control (Ctl) group (Veh + Veh), 2-Me-5-HT group (Veh + 2-Me-5-HT), Palonosetron group (Palo + Veh) and Palonosetron + 2-Me-5-HT group (Palo + 2-Me-5-HT). Proteins were immunoprecipitated by rabbit anti-5-HT₃R antibody and Western blots were developed on 5-HT₃R immunoprecipitates using goat anti-5-HT₃R antibody and mouse anti-CaM antibody. The ratio of optical density for CaM (17 kD) to 5-HT₃R (55 kD) was acquired and expressed as fold change of control. A) The representative Western blot, and B) Summarized data. *P<0.05 vs. the Control. Graphs C and D show the immunohistochemical analysis of 5-HT₃R-CaM colocalization in brainstem (C) and intestinal slices (D) from shrews treated as described for A and B. 10 µm thick cryo-sections of brainstem and intestine were co-labeled with rabbit anti-5-HT₃R and mouse anti-CaM antibodies. Representative high magnification fluorescence images (200×) show colocalization of 5-HT₃R and CaM in the area postrema (AP) region of brainstem (C) and jejual segment of intestine (D) which were increased following 5-HT₃R stimulation by 2-Me-5HT (5 mg/kg, i.p.). A 30 min prior exposure to the 5-HT₃R antagonist palonosetron (5 mg/kg, s.c.) abolished the 2-Me-5-HT-induced enhancement of the 5-HT₃R-CaM colocalization. Scale bar, 10 µm.

Figure 3. Palonosetron suppresses the ability of 2-Me-5-HT to increase CaMKIIα phosphorylation in the least shrew brainstem.

A) The time-course of 2-Me-5-HT-induced CaMKIIα activation in the least shrew brainstem. Shrews were injected with the 5-HT₃R agonist 2-Me-5-HT (5 mg/kg, i.p.) and brainstems were collected at 5, 10, 20, 30 and 60 min. Phosphorylated CaMKIIα at Thr286 (pCaMKIIα) and total CaMKIIα of samples from individual animals were determined by immunoblot with rabbit anti-pCaMKIIα and mouse anti-CaMKIIα antibodies. The ratios of pCaMKIIα (∼50 kD) to CaMKIIα were calculated and expressed as fold change of vehicle-treated controls (0 min). n = 3 per group. *P<0.05 vs. 0 min. Graph A shows the summarized data and the insets exhibit the representative Western blot. B) Palonosetron (5 mg/kg, s.c.) or its vehicle was given 30 min before 2-Me-5-HT. Immunoblots were performed on the brainstems of the least shrews sacrificed 20 min after 2-Me-5-HT administration using anti-pCaMKIIα and CaMKIIα antibodies. n = 3 per treatment group. *P<0.05 vs. vehicle/vehicle control. ^#P<0.05 vs. vehicle + 2-Me-5-HT. Graph B displays the summarized data and the insets show the representative Western blot. C) Representative low magnification (20×) images for the brainstem dorsal vagal complex (DVC) emetic nuclei including the area postrema (AP), the nucleus tractus solitarius (NTS) and the dorsal motor nucleus of the vagus (DMNX) from sections co-labeled with rabbit anti-CaMKIIα (red) and mouse anti-pCaMKIIα (green) antibodies. Shrews were sacrificed 20 min after vehicle or 2-Me-5-HT administration. Scale bar, 100 µm. D) Representative images of high magnification (100×) showed 5-HT₃R-mediated CaMKIIα activation in brainstem AP area. Scale bar, 10 µm.

Figure 4. Palonosetron suppresses the ability of 2-Me-5-HT to upregulate CaMKIIα phosphorylation in enterochromaffin (EC) cells.

The isolated EC cells from the least shrew intestine were incubated with the 5-HT₃R antagonist palonosetron (1 µM) or its vehicle for 30 min and then the 5-HT₃R agonist 2-Me-5-HT (1 µM) was added for the next 30 min. The corresponding antagonist and agonist vehicles were also incubated with EC cells and were used as control. A) The control and treated EC cells were harvested to analyze CaMKIIα phosphorylation (Thr286) using Western blot. n = 3 experiments per treatment group. *P<0.05 vs. vehicle/vehicle control. ^#P<0.05 vs. vehicle + 2-Me-5-HT. Graph A shows the summarized data and the insets represent the representative Western blot. B) Representative fluorescence images show the immunoreactivity for CaMKIIα (red) and pCaMKIIα (green) in EC cells treated as described in (A) and subjected to immunocytochemistry to determine 5HT₃R-mediated CaMKIIα activation in isolated EC cells in vitro. Nuclei of EC cells were shown with DAPI stains. Scale bar, 4 µm.

Figure 5. 2-Me-5-HT-induced CaMKIIα activation is dependent upon Ca²⁺ mobilization mediated by L-type Ca²⁺ channels (LTCCs) and ryanodine receptors (RyRs).

Different groups of shrews were administrated with either vehicle (Veh), or one the following agents, the LTCC blocker amlodipine (Aml, 10 mg/kg, s.c.), the RyR blocker dantrolene (Dan, 20 mg/kg, i.p.), a combination of less effective doses of amlodipine (5 mg/kg, s.c.) and dantrolene (10 mg/kg, i.p.) (Aml+Dan), or inositol-1, 4, 5-triphosphate receptor blocker 2-APB (10 mg/kg, i.p.), and 30 min later injected with 2-Me-5-HT (5 mg/kg, i.p.). Immunoblots were performed on brainstems of least shrews sacrificed 20 min after 2-Me-5-HT injection using anti-pCaMKIIα and CaMKIIα antibodies. n = 3 per group. The inset (A) shows the representative Western blot, and the graph (B) shows the fold change from individual experimental results. *P<0.05 vs. Veh/Veh control (Ctl). ^#P<0.05 vs. Veh + 2-Me-5-HT. ^aP<0.05 vs. 2-APB + 2-Me-5-HT).

Figure 6. Effects of CaMKII inhibition on 5-HT₃R-mediated emesis.

A) The CaMKII inhibitor KN93 (i.p.) or its vehicle was administered to different groups of shrews 30 min prior to 2-Me-5-HT (5 mg/kg, i.p.) injection. The emetic responses were recorded for 30 min following 2-Me-5-HT injection. *P<0.05, **P<0.01 and ***P<0.001 vs. vehicle-pretreated control group. B) Immunoblot analyses of CaMKIIα phosphorylation were performed on brainstems collected from the experimental shrews 20 min after 2-Me-5-HT injection in the absence or presence of KN93 (10 mg/kg, i.p.). n = 3 per group. Graph B shows the fold change from individual experimental results and the insets demonstrate the representative Western blot. *P<0.05 vs. vehicle/vehicle control. ^#P<0.05 vs. vehicle + 2-Me-5-HT.

Figure 7. Involvement of Ca²⁺/CaMKIIα in 5-HT₃R-mediated ERK activation.

A) Time-course of 2-Me-5-HT-induced ERK1/2 activation in the least shrew brainstem. Least shrews were injected with 5 mg/kg (i.p.) 2-Me-5-HT and their brainstems were collected at 5, 10, 20 and 30 min (n = 3 per group). Phosphorylated (pERK1/2) and total ERK1/2 of the same sample from different shrews were determined by immunoblot with the antibodies to pERK1/2 and to total ERK1/2. The ratios of pERK1/2 (42 kD/44 kD) to ERK1/2 were calculated and expressed as fold change of vehicle-treated control (0 min). Graph A represents the summarized data and the insets show the representative Western blot. *P<0.05 vs. 0 min. Graphs B–D) Immunoblot analyses of ERK1/2 phosphorylation were performed on brainstems collected from the experimental shrews 10 min after 2-Me-5-HT treatment (5 mg/kg, i.p.) in the absence (vehicle) or presence of antagonists. B) Selective blockade of 5-HT₃Rs with palonosetron (5 mg/kg, s.c.) 30 min prior to 2-Me-5-HT injection. *P<0.05 vs. vehicle/vehicle control and ^#P<0.05 vs. vehicle + 2-Me-5-HT. C) Either vehicle (Veh, i.p.), the inositol-1, 4, 5-triphosphate receptor blocker 2-APB (10 mg/kg. i.p.), L-type Ca²⁺ channel blocker amlodipine (Aml, 10 mg/kg, s.c.), ryanodine receptor blocker dantrolene (Dan, 20 mg/kg, i.p.) or a combination (Aml+Dan) of less effective doses of amlodipine (5 mg/kg, s.c.) and dantrolene (10 mg/kg, i.p.) were administered to different groups of shrews 30 min prior to 2-Me-5-HT injection. *P<0.05 vs. Veh/Veh control (Ctl). ^#P<0.05 vs. Veh + 2-Me-5-HT. ^aP<0.05 vs. 2-APB + 2-Me-5-HT. D) Inhibition of CaMKII with KN93 (10 mg/kg, i.p.) blocked 2-Me-5-HT-evoked ERK1/2 phosphorylation in brainstem. n = 3 per group. Graphs show the summarized data and insets show representative Western blots. *P<0.05 vs. vehicle/vehicle control. ^#P<0.05, vs. vehicle + 2-Me-5-HT.

Figure 8. Suppressive effects of ERK inhibition on 5-HT₃R-mediated emesis.

A) The cited doses of the ERK inhibitor PD98509 were administered to different groups of shrews 30 min prior to 2-Me-5-HT (5 mg/kg, i.p.) injection. The vomit parameters were recorded for 30 min post 2-Me-5-HT injection. The vomit frequency data are presented as mean ± SEM. **P<0.01 and ***P<0.001 vs. vehicle-pretreated control. B) PD98059 (5 mg/kg, i.p.) or its vehicle (i.p.) was administered to different groups of shrews 30 min prior to 2-Me-5-HT (5 mg/kg, i.p.) injection and immunoblot analyses of ERK1/2 phosphorylation were performed on shrew brainstems collected 10 min after 2-Me-5-HT treatment. n = 3 per group. Graph B shows the summarized data and the insets show the representative Western blot. *P<0.05 vs. control vehicle/vehicle, ^#P<0.05 vs. Vehicle + 2-Me-5-HT.

Figure 9. 5-HT_2ARs antagonism has no significant effect on 2-Me-5-HT-evoked vomiting and CaMKIIα activation in the least shrew brainstem.

A) Shrews were pretreated with the 5-HT2_AR antagonist SR34649B (5, 10 mg/kg, s,c.) or vehicle 30 min prior to 2-Me-5-HT (5 mg/kg, i.p.) administration. The vomit parameters were recorded for 30 min post 2-Me-5-HT injection. B) Immunoblot analyses of CaMKIIα phosphorylation were performed on brainstems collected from the experimental shrews 20 min after 2-Me-5-HT treatment in the absence or presence of SR34649B (10 mg/kg, s.c.). n = 3 per group. Graph B shows the summarized data and the insets show the representative Western blot. *P<0.05 vs. control (vehicle/vehicle treated).

Figure 10. Summary of the proposed 5-HT₃R-mediated downstream signaling pathway underlying 2-Me-5-HT-induced emesis in the least shrew.

5-HT₃R stimulation by the selective agonist 2-Me-5-HT causes an influx of extracellular Ca²⁺ through 5-HT₃Rs/L-type Ca²⁺ ion channels which increases the free cytoplasmic concentration of Ca²⁺, thereby promoting Ca²⁺ release via calcium-induced calcium release (CICR) from the endoplasmic reticulum stores through ryanodine receptors (RyRs). This elevation in cellular Ca²⁺ level initiates attachment of calmodulin (CaM) with the 5-HT₃R, and leads to CaMKIIα activation and subsequent ERK1/2 signaling. The 5-HT₃R antagonist palonosetron⁽¹⁾, the L-type Ca²⁺ channel blocker amlodipine⁽²⁾, the RyR blocker dantrolene⁽³⁾, the CaMKII inhibitor KN93⁽⁴⁾, and the ERK inhibitor PD98059⁽⁵⁾, respectively exhibit anti-emetic efficacy against 2-Me-5-HT-induced vomiting. These findings demonstrate that the 2-Me-5-HT-induced emesis is regulated by 5-HT₃R-mediated Ca²⁺/CaMKII-dependent ERK signaling pathway.