Serotonin 5-HT3 Receptor-Mediated Vomiting Occurs via the Activation of Ca2+/CaMKII-Dependent ERK1/2 Signaling in the Least Shrew (Cryptotis parva)

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 (Ca2+) and upon stimulation allows increased Ca2+ influx. We examined the contribution of Ca2+/calmodulin-dependent protein kinase IIα (Ca2+/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 Ca2+ levels in brainstem slices and that the selective 5-HT3R antagonist palonosetron, can abolish the induced Ca2+ signaling. Pre-treatment of shrews with either: i) amlodipine, an antagonist of L-type Ca2+ 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 Ca2+ mobilization via extracellular Ca2+ influx through 5-HT3Rs/L-type Ca2+ channels, and intracellular Ca2+ release via RyRs on ER, initiate Ca2+-dependent sequential activation of CaMKIIα and ERK1/2, which contribute to the 5-HT3R-mediated, 2-Me-5-HT-evoked emesis.


Introduction
Chemotherapy (e.g. cisplatin)-induced nausea and vomiting (CINV) is mediated via neurochemical circuits that involve braingut interactions [1]. The critical sites for CINV includes the medullary emetic nuclei of the dorsal vagal complex (DVC) in the brainstem, as well as the enteric nervous system (ENS) and enterochromaffin cells (EC cells) in the gastrointestinal tract (GIT) [2,3]. The DVC emetic nuclei consists of the nucleus tractus solitarius (NTS), the dorsal motor nucleus of the vagus (DMNX) and the area postrema (AP) [1]. These brainstem emetic loci can be activated by emetogens, such as serotonin, either directly or indirectly through gastrointestinal signaling [4]. Among several, serotonin (5-hydroxytryptamine = 5-HT) is one important emetic neurotransmitter in both the brainstem and the gastrointestinal tract (GIT) that contributes to induction of CINV. In the GIT 5-HT is mainly produced and stored in the enterochromaffin (EC) cells and its release is regulated by the ENS as well as by multiple receptors present on EC cells including serotonergic 5-HT 3 receptors (5-HT 3 Rs) [3,5,6]. The diverse functions associated with 5-HT are due to the existence of a large family of serotonergic receptors, 5-HT 1 to 5-HT 7 , in which each class consist of further subtypes [7]. Unlike most serotonergic receptors which are Gprotein-coupled, the 5-HT 3 R belongs to the ligand-gated ion channel receptor superfamily and is associated with vomiting. 5-HT 3 Rs are found throughout the brainstem DVC and GIT [1,8]. In fact, cisplatin-like drugs cause vomiting via release of 5-HT from the gastrointestinal EC cells which subsequently activates local 5-HT 3 Rs present on the GIT vagal afferents [1,9,10]. This activation results in vagal nerve depolarization which subsequently triggers the brainstem DVC emetic nuclei to initiate the vomiting reflex.
The central/peripheral-acting agent 2-Methyl serotonin (2-Me-5-HT) is considered a ''more selective'' 5-HT 3 R agonist, which causes vomiting in several species including the least shrew [11,12,13]. In fact 2-Me-5-HT-induced emesis has been shown to be associated with enhanced Fos-immunoreactivity in both the DVC emetic nuclei and in the ENS of the least shrew [14]. Moreover, 5-HT 3 R-selective antagonists such as tropisetron [10] or palonosetron [15], can suppress vomiting caused by 2-Me-5-HT. However, to date, the downstream signaling pathways for the 5-HT 3 R-mediated vomiting remain unknown. Recently, it has been demonstrated that increased luminal glucose levels result in 5-HT release from EC cells, which subsequently activates vagal afferent 5-HT 3 Rs, leading to activation of the Ca 2+ /calmodulindependent kinase II (CaMKII) signaling pathway in the brainstem DVC-gut circuit in rats [16]. Activation of the extracellular signalregulated kinase 1/2 (ERK1/2) also appears to be involved in some downstream functions of 5-HT 3 Rs including pain [17] and cisplatin-induced immediate and delayed emesis [18].
In the present study we sought to evaluate the potential involvement of the above-discussed transduction signals downstream of 5-HT 3 Rs in the process of vomiting via the use of in vivo pharmacology, ex-vivo and/or in vitro immunoprecipitation, immunohistochemistry, immunocytochemistry and Western blot on isolated EC cells and/or tissues of both small intestine and brainstem in the least shrew.

Animals and Ethics statement
Adult least shrews were bred in the animal facility of Western University of Health Sciences. Previous studies had demonstrated no gender differences, so both males and females were used. Shrews were housed in groups of 5-10 on a 14:10 light:dark cycle, fed with food and water ad libitum as described previously [19]. All the shrews used were 45-60 days old and weighed between 4-5 g. This study was carried out in strict accordance with the recommendations in the guide for the Care and Use of Laboratory Animals of the National Institutes of Health (Department of Health and Human Services Publication, revised, 1985). The protocol was approved by the Western University of Health Sciences IACUC. To minimize the suffering of laboratory animals, the number of pharmacological tests was limited to the necessary minimum and the animals were observed regularly for any signs of unnecessary suffering from drug treatment. Any animal showing at least one of the following symptoms: weight loss greater than 20% of the initial weight, not eating or drinking, rough appearance of fur and/or absence of activity, were euthanized via exposure to 32% isoflurane. All experiments were conducted between 9:00 and 15:00 h.  MN), respectively. The CaMKII inhibitor KN93 and its inactive analog KN92 as well as ERK1/2 inhibitor PD98059 were obtained from Calbiochem (San Diego, CA). The L-type Ca 2+ channel antagonist amlodipine besylate was purchased from Tocris (Minneapolis, MN). The ryanodine receptor antagonist dantrolene (sodium salt) and the inositol-1, 4, 5-triphosphate receptor antagonist 2-APB, were obtained from Santa Cruz Biotechnology (Dallas, TX). Unless otherwise stated, the above drugs were dissolved in water. KN92, dantrolene sodium and 2-APB were dissolved in 25% DMSO in water. PD98059 was dissolved in 0.5% ethanol, 0.5% Tween-80 in saline.
All drugs were administered at a volume of 0.1 ml/10 g of body weight.

Ca 2+ imaging
Least shrew brainstem slice preparation and treatment. Adult least shrews were anesthetized in lethal isoflurane chamber and subsequently decapitated. Brainstems were quickly removed and transferred to ice-cold artificial cerebrospinal fluid (aCSF, pH 7.37) buffer, containing (in mM): 124 NaCl; 5 KCl; 1.3 MgCl 2 ; 2 CaCl 2 ; 10 glucose; and 26.2 NaHCO 3 , and aerated with 95% O 2 /5% CO 2 . Transverse brainstem slices (200 mm-thick) containing the DVC emetic nuclei identified as previously reported in our lab [14] were prepared using a Leica vibratome (Model-VT 100 A), maintained in aCSF buffer, and incubated with Ca 2+ indicator fluo-4 AM (5 mM; Invitrogen) for 30 min in dark at room temperature. The fluo-4 AM loaded slices were pinned to sylgard blocks (Ellsworth Adhesives, Germantown, WI) and pre-treated with either the selective 5-HT 3 R antagonist palonosetron (1 mM) or its vehicle (control) for 30 min. The pretreated slices were simultaneously placed in an open bath imaging chamber (Warner Instruments, Hamden, CT) containing aCSF and mounted on the confocal imaging stage assembled with model 710 NLO (Carl Zeiss Microscopy, Thornwood, NY) laser scanning confocal imaging workstation with inverted microscope (Olympus IX81 or Zeiss Axio Observer Z1). Since only 1 section (200 mm-thick) containing the emetic nuclei could be prepared from each shrew brainstem, one slice from 4 different shrews were used to investigate the effect of palonosetron on 2-Me-5-HT-elicited Ca 2+ increase in the AP region among the brainstem DVC emetic nuclei. 2-Me-5-HT (1 mM) was added to aCSF containing palonosetron or vehicle at the end of pretreatment using a hand pipette, exactly at the 400 th sec during the whole 1200-sec Ca 2+ image-acquisition period.
Measurement of intracellular Ca 2+ . Slices were illuminated at 488 nm with a krypton argon laser and the emitted light was collected using a photomultiplier tube. Line scans were imaged at rates from 422 to 822 lines generated every 1 s, depending on line length. To ensure that sparks within the region of interest (ROI), the AP region of the brainstem, were imaged, global Ca 2+ responses were acquired at roughly one image per second with an imaging depth of 10 mm, which is equivalent to two or three cells thick. The sampling depth was 16-bit (Zeiss 710). Ca 2+ spark recordings were made using Zeiss C-Apochromat 63x/1.20 water immersion objective. ROIs were examined post hoc and analyzed with ImageJ. Analysis of time series recordings was achieved by hand using the time series analyzer plugin for ImageJ. For presentation purposes, the fractional fluorescence intensity was calculated as F/F0. After Ca 2+ image acquisition, the data was analyzed by NIH-approved Fiji ImageJ software using the time series analyzer plugin for ImageJ. The captured images were visualized and cells with different level (500-60000) of fluorescence intensities were identified. Regions of interest were selected from the initial frame captured at 0 sec with cells showing initial fluorescence intensities between 5000-25000 and the values of fluorescence intensities at different time points were identified by time series analyzer to plot the graphs of selected regions of interest. To show the changes in Ca 2+ levels before and after 2-Me-5-HT treatment, the average fluorescence intensities were calculated for at least 12 regions of interest in each acquisition for all time points. The data is represented in a graph as the ratio (F/ F0) of final fluorescence intensity (F) for each time point to the initial fluorescence intensities (F0) at 0 sec for ROIs and is the mean value of 4 individual experiments.

Behavioral emesis studies
On the day of the experiment shrews were brought from the animal facility, separated into individual cages and allowed to adapt for at least two hours (h). Daily food was withheld 2 h prior to the start of the experiment but shrews were given 4 mealworms each prior to emetogen injection, to aid in identifying wet vomits as described previously [20].

Tissue studies
Tissue collection. Adult least shrews treated with 2-Me-5-HT (5 mg/kg, i.p.) were rapidly anesthetized with isoflurane and decapitated at the indicated time points post-treatment (see Figures). Brainstem and small intestine were quickly removed. Further division of the small intestine to recognize the jejunal segment was performed according to Ray et al [7]. Brainstem and jejunum were transferred into cold fixative 4% paraformaldehyde (PF) in phosphate-buffered saline (PBS) for cryo-sectioning and immunohistochemical staining. For biochemical assays, the lower half of brainstem, mostly medullary structures, was isolated and immediately frozen on dry ice.
Immunohistochemistry. The optimal-cutting-temperature compound-embedded brainstems (n = 3 animals per group) were cut into 20 mm sections using a cryostat and mounted onto slides. Sections from brainstem were observed with a light microscope and those containing the whole DVC subjected to immunohistochemistry. Slides were washed in PBS three times, fixed with 4% PF for 2 h at 4uC, then washed 3 times with PBS, permeabilized with 0.1% Triton X-100 for 30 min at 4uC, and washed again 3 times with PBS. After blockade for 1 h with the blocking buffer containing 5% bovine serum albumin (BSA) in PBS, histological sections of brainstem and intestine were co-incubated overnight at 4uC with goat anti-CaMKIIa (1:100, ab87597, Abcam) and rabbit anti-phospho-CaMKIIa (Thr286) antibodies (1:100, ab5683, Abcam) to analyze CaMKIIa phosphorylation at Thr286 site. Sections were then washed 3 times (10 min each) in PBS and incubated in Alexa Fluor 594 donkey anti-goat IgG and Alexa Fluor 488 donkey anti-rabbbit IgG (1:400, Abcam) for 2 h at room temperature. Images for the whole DVC region and for the individual areas (AP/NTS/DMNX) were acquired under a confocal microscope (Nikon) with Metamorph software using 206and 1006objectives, respectively. Nuclei of cells were stained with DAPI. DAPI is excited at 345 nm and emits at 458 nm, producing blue fluorescence.
Immunoprecipitation and Western blot. To assess the 2-Me-5-HT-induced interaction of 5-HT 3 R and CaM in the brainstem of least shrews, the animals (n = 3 per group) were treated either with vehicle, the 5-HT 3 R agonist 2-Me-5-HT (5 mg/kg, i.p.), the 5-HT 3 R antagonist palonosetron (5 mg/kg, s.c.), or a combination of both agents. The time to first vomit was generally within 15 minutes of 2-Me-5-HT injection. Thus, each shrew brainstem was isolated 20 min after 2-Me-5-HT treatment, homogenized in cold lysis buffer (50 mM Tris-HCL, pH 8, 150 mM NaCl and 1% NP-40) containing protease-and phosphatase-inhibitors cocktail (Pierce, Rockford, IL), and centrifuged at 100006g for 20 min at 4uC. Total protein concentrations in supernatants were confirmed using BCA protein assay kit (Pierce, Rockford, IL). A 1 mg protein extract from each brain lysate was immunoprecipitated overnight at 4uC with 10 mg rabbit anti-5-HT 3 R antibody (sc-28958) or rabbit IgG (sc-2027, Santa Cruz) and then incubated with 50% Protein A/G agarose slurry (20421, Thermo) for 1 h with occasional mixing at 4uC. After washing 3 times with lysis buffer by centrifuging at 7006g for 1 min at 4uC, supernatant was discarded, and 50 ml of 1.56 SDS-PAGE sample buffer was added to the saved pellets, heated at 100uC for 10 min, and centrifuged at 7006g for 1 min. Supernatants containing 5-HT 3 R immunoprecipitates were subjected to Western blot for the detection of 5-HT 3 R and CaM. Inputs from various groups were used to confirm the expression of 5-HT 3 R and CaM. GAPDH served as an internal standard. All samples were subjected to 12% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene difluoride membrane for 90 min at 90 V. After blocking with TBST solution (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) containing 5% BSA for 1 h at room temperature, membranes were incubated overnight at 4uC with mouse anti-CaM antibody (1:1000, 05-173, Millipore), goat anti-5-HT 3 R antibody (1:500, sc-19152, Santa Cruz) or mouse anti-GAPDH antibody (1:10000, MAB374, Chemicon). Infrared fluorescent-labeled anti-goat and anti-mouse secondary antibodies (1:10000, LI-COR Biosciences) were then used. Bound antibodies were visualized using Odyssey imaging system and analyzed semi-quantitatively based on densitometric values using Quantity-One 1D software (Bio-Rad). The ratios of CaM (,17 kD) to 5-HT 3 R (,55 kD) precipitated by 5-HT 3 R antibody were calculated and are shown as fold change of control.

Cellular studies
Isolation of enterochromaffin cells. The enterochromaffin (EC) cell isolation from naïve shrews was performed via a slight modification of the method described by Schä fermeyer and coworkers [21]. Buffers A, B and C were prepared according to Schä fermeyer and co-workers [21]. Shrew intestines were surgically removed and enzymatic digestion and alternative switching off and on exposure to EDTA-calcium salt was performed for isolation of intestinal mucosal cells. Each intestine (approximately 12 cm in length and 3mm in diameter) was fastened by a small metal binder clip at its anal end, and was filled with the buffer B (containing a mixture of 0.64 mg/ml pronase E and 0.5 mg/ml collagenase) by injecting and filling it with 1-1.5 ml from the proximal end which was then closed by a small metal binder clip to make sacs. The filled intestines were partially immersed in 100 mm plastic dishes containing 2 ml buffer B and incubated at 37uC for 15 min. The intestines were hung vertically from the distal metal binder and the proximal metal binder was then removed by cutting the intestine from its edge to release the digested, detached mucosal lining from muscularis propria. In addition, the mucosal lining was stripped from the distal to the proximal end of intestine by tweezing and running forceps along the intestinal length. The mucosal lining was collected into a petri dish containing buffer A (25 ml) for 20 min, then centrifuged at 1200 rpm for 10 min. Buffer B was added to the pellet, gently vortexed and stirred for 10 min. The EC cells were collected by pouring the mixture through a nylon filter mesh (pore size , 200 mm) and buffer B (25 ml) was added and centrifuged at 1200 rpm for 10 min. Enriched EC cells were obtained by step density gradient centrifugation using nycodenz gradient with adjusted density of 1.1 g/ml at the bottom of tube, followed by adjusted density of 1.07 g/ml as intermediate layer. The cell suspension was layered on top of the two layers, centrifuged at 1100 rpm for 8 min with slow deceleration. The EC cell-enriched layer was collected at the 1.070 interface, and then washed in buffer C.

Cell
Treatment and CaMKIIa phosphorylation analyses. The EC cells (n = 3 expeiments per group) were pre-incubated for 30 min with either 1 mM palonosetron or its vehicle at 37uC, followed by 5-HT 3 R stimulation with 1 mM 2-Me-5-HT (or its vehicle as control) for 20 min at 37uC. After treatment, the collected cell pellets were re-suspended in lysis buffer, sonicated and centrifuged (100006g, 10 min at 4uC). Supernatants containing total protein were quantified and used to analyze CaMKIIa phosphorylation at Thr286 by Western blot, as described above. For immunocytochemistry, control and treated EC cells (n = 3 experiments per group) were fixed with 4% PF in PBS for 10 min followed by treatment with cold methanol for 10 min at 4uC. The cells were also co-stained with rabbit anti-CaMKIIa (1:100, sc-13082, Santa Cruz) and mouse anti-phospho-CaMKIIa (Thr286) (1:100, sc-32289, Santa Cruz). The immunoreactivities were visualized by incubation with rhodamine red antirabbit and FITC-conjugated anti-mouse antibodies.

Statistical analysis
The vomit frequency data were analyzed using the Kruskal-Wallis non-parametric one-way analysis of variance (ANOVA) and followed by Dunn's post hoc test. The percentage of animals vomiting across groups at different doses was compared using the chi square test. Statistical significance for differences between two groups was tested by unpaired t-test, among groups ($3) was tested by one-way ANOVA followed by Tukey's multiple comparison tests. For time-course analyses of CaMKIIa and ERK activation, one-way ANOVA followed by Dunn's post hoc test was used. All results are presented as mean 6 SEM. P,0.05 was considered significant.

5-HT 3 R stimulation increases intracellular Ca 2+ concentration and Ca 2+ mobilization regulates 2-Me-5-HT-induced emesis
Activation of 5-HT 3 Rs regulates neuronal function by directly gating its corresponding ion channel to produce an increase in Ca 2+ influx which rapidly induces neuronal depolarization [22]. In addition, the increase in the magnitude of the intracellular Ca 2+ signal can be partly due to subsequent extracellular Ca 2+ influx via enhancement of voltage-operated Ca 2+ channels [23] due to mobilization of intracellular Ca 2+ from ER stores through the process of Ca 2+ -induced Ca 2+ release (CICR) [24].
2-Me-5-HT enhances interaction of 5-HT 3 R with CaM in the brainstem of least shrews 5-HT 3 R stimulation induces extracellular Ca 2+ influx which may secondarily affect the cytosolic Ca 2+ sensor protein, calmodulin (CaM), since an increase in free cytoplasmic Ca 2+ concentration can lead to activation of CaM and CaMKIIa [25]. CaM can bind a number of other targets including enzymes, ion channels, transcription factors and several plasma membrane receptors [26]. CaM not only can modulate G-protein-coupled receptor signaling including serotonergic 5-HT 1A -, 5-HT 2A -and 5-HT 2C -recptors [27,28,29], but may also regulate the actions of diverse ion channels such as voltage-gated L-type Ca 2+ channels, voltagegated sodium channels and voltage-gated potassium channels [30,31,32].
We further investigated the colocalization of 5-HT 3 R with CaM in brainstem in response to 2-Me-5-HT treatment by immunohistochemistry. Brainstems from the above-discussed experimental shrews were isolated, sections were prepared and immunolabeled for 5-HT 3 R and CaM. The colocalization between 5-HT 3 R and CaM in different DVC emetic loci in the brainstem (NTS, DMNX, and AP) were then evaluated. Brainstem sections obtained from the 2-Me-5-HT-treated shrews exhibited significantly enhanced 5-HT 3 R-CaM colocalization in AP area relative to vehicle control, whereas the brainstem sections obtained from least shrews pretreated with palonosetron followed by 2-Me-5-HT (i.e. palonosetron + 2-Me-5-HT) did not show significant alteration in 5-HT 3 R-CaM colocalization, which was similar to control ( Figure 2C). However, 5-HT 3 R activation with 2-Me-5-HT had no major effect on 5-HT 3 R-CaM colocalization in NTS and DMNX ( Figure S1). The above results indicate that activation of 5-HT 3 Rs can lead to the close physical connection between 5-HT 3 R and CaM in AP emetic region of the brainstem.

2-Me-5-HT enhances colocalization of 5-HT 3 R with CaM in the GIT of least shrews
Since the GIT plays a major role in vomiting and Darmani et al. [1] have previously demonstrated that largest increases in jejunal 5-HT tissue levels were closely associated with cisplatininduced peak early and delayed vomit frequency, the colocalization between 5-HT 3 R and CaM in the shrew jejunum after administration of 2-Me-5-HT was also analyzed by immunohistochemistry ( Figure 2D). After a 20-min exposure to 2-Me-5-HT, the least shrew intestines (excluding colon and stomach) were dissected from vehicle/vehicle-treated control, vehicle/2-Me-5-HT, and palonosetron + 2-Me-5HT treatment groups. Transverse sections were prepared from jejunum. The cryosections were immunolabeled for 5-HT 3 R and CaM and intestinal mucosal cells from jejunal regions were analyzed for interaction of 5-HT 3 R with CaM. As shown in Figure 2D, relative to the control group, the jejunal section from least shrews treated with 2-Me-5-HT exhibited significantly enhanced 5-HT 3 R-CaM colocalization. On the other hand, the jejunal sections obtained from least 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 Ca 2+ 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 6 SEM. *P,0.05, **P,0.01, ***P,0.001 and ****P,0.0001 compared with corresponding vehicle-pretreated controls. doi:10.1371/journal.pone.0104718.g001 shrews pretreated with palonosetron followed by 2-Me-5-HT injection, showed no significant change in 5-HT 3 R-CaM colocalization and were essentially identical to the vehicle control. These results demonstrate that 2-Me-5-HT induces a 5-HT 3 R-mediated increase in 5-HT 3 R-CaM colocalization in the jejunum of the least shrew intestine.

Activation of CaMKIIa by 2-Me-5-HT in brainstem of least shrews occurs via 5-HT 3 Rs
CaMKIIa is a downstream kinase which is activated by Ca 2+ / CaM signaling, and integrates transient, localized changes in intracellular Ca 2+ levels to induce diverse downstream responses [25,33]. To determine the involvement of CaMKIIa in 2-Me-5-HT-induced emesis, we performed Western blots to analyze the degree of activation by CaMKIIa autophosphorylation at Thr286 We further confirmed our Western blot results with immunohistochemistry ( Figure 3C). Brainstem sections from vehicle control, 2-Me-5-HT and palonosetron + 2-Me-5-HT groups were prepared, and co-stained with anti-CaMKIIa and anti-phospho-CaMKIIa Thr286 antibodies. In figure 3C, immunolabeling for CaMKIIa in the control brainstem section indicated the cytoarchitectonic differences among the AP, NTS and DMNX under low magnification (206). Immunoreactive brainstem sections showed that systemic administration of 2-Me-5-HT induced a significant increase in CaMKIIa phosphorylation at Thr286 (pCaMKIIa) throughout the DVC including AP, NTS and DMNX, but especially in AP region of least shrew brainstem ( Figure 3C; Figure S2). Pre-treatment with palonosetron significantly suppressed the pCaMKIIa increase in the AP region of shrew brainstem in response to 2-Me-5-HT ( Figure 3D).

2-Me-5-HT induces CaMKIIa activation via 5-HT 3 Rs in the EC cells in vitro
The 5-HT-releasing EC cells present in the GIT are involved in the induction of emesis (see introduction). Furthermore, 2-Me-5-HT can activate 5-HT 3 Rs present on EC cells to promote release of endogenous serotonin from these cells and the induced release is sensitive to selective corresponding antagonists [3,5,6]. To investigate the direct actions of 2-Me-5-HT on EC cells, in this study we isolated EC cells from the least shrew GIT mucosa. The EC cells were incubated with either vehicle or palonosetron (1 mM) 30 min prior to addition of 2-Me-5-HT (1 mM), and cells were harvested at 20 min. The control group was exposed to vehicles of both palonosetron and 2-Me-5-HT in accord with the above described procedure. Western blots were performed on total proteins extracted from cell lysates to analyze the phosphorylation level of CaMKIIa at Thr286. The results showed significant increases in pCaMKIIa levels after 2-Me-5-HT exposure (P,0.05 vs. vehicle/vehicle control) ( Figure 4A). Palonosetron pretreatment prevented the induced increase in CaMKIIa phosphorylation in response to 2-Me-5-HT (P,0.05 vs. vehicle + 2-Me-5-HT) ( Figure 4A). Moreover, results obtained from immunoblots were confirmed using immunocytochemistry. The immunofluorescence of control EC cells showed weak immunoreactivity to CaMKIIa phophorylation at Thr286, which was elevated by 2-Me-5-HT incubation ( Figure 4B). Pretreatment with palonosetron reversed the observed CaMKIIa activation evoked by 2-Me-5-HT ( Figure 4B). These results provide evidence that 2-Me-5-HT directly increases CaMKIIa activation in vitro in EC cells via 5-HT 3 Rs.

and intestine. Graphs A and B) Effects of the 5-HT 3 R agonist 2-Me-5-HT and the 5-HT 3 R antagonist palonosetron on 5-HT 3 R-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-HT 3 R antibody and Western blots were developed on 5-HT 3 R immunoprecipitates using goat anti-5-HT 3 R antibody and mouse anti-CaM antibody. The ratio of optical density for CaM (17 kD) to 5-HT 3 R (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-HT 3 R-CaM colocalization in brainstem (C) and intestinal slices (D) from shrews treated as described for A and B. 10 mm thick cryo-sections of brainstem and intestine were co-labeled with rabbit anti-5-HT 3 Figure 5B). These results are in concordance with our described behavioral findings which suggest that elevation of intracellular Ca 2+ via extracellular influx through L-type Ca 2+ channels and intracellular Ca 2+ release from ER Ca 2+ stores through RyRs, but not IP 3 Figure 6B). These observations suggest that the Ca 2+ -modulated CaMKIIa activation in the brainstem is involved in 5-HT 3 R-mediated emesis.

2-Me-5-HT-induced vomiting is independent of 5HT 2Aand 5-HT 6 -receptor activity
It has been suggested that functional interaction exists between 5-HT 2A Rs and 5-HT 3 Rs [35]. To rule out the possibility that 5-HT 2A Rs may be involved in emetic response evoked by 2-Me-5-HT, we evaluated the effect of 5-HT 2A/C R antagonist, SR46349B [36,37]. Thus, SR46349B (5 and 10 mg/kg, s.c.) or its vehicle were administered to different groups of least shrews 30 min prior to 2-Me-5-HT. The vomiting response was recorded for the following 30 min. SR46349B (5 or 10 mg/kg) failed to significantly suppress either the frequency or the percentage of shrews vomiting in response to 2-Me-5-HT ( Figure 9A). Western blots were further performed on brainstem protein extracts from least shrew pretreated with either SR46349B (10 mg/kg) or its vehicle 30 min prior to 2-Me-5-HT (5 mg/kg) injection. Tested animals were sacrificed at 20 min after 2-Me-5-HT injection. Consistent  with the behavioral results, SR46349B had no significant effect (P.0.05) on the ability of 2-Me-5-HT to increase pCaMKIIa ( Figure 9B). These findings strongly suggest that the 5-HT 3 R, and not the 5-HT 2A R subtype, is specifically involved in 2-Me-5-HTinduced emesis-related responses.

Discussion
The concept and laboratory testing of antiemetic efficacy of 5-HT 3 R antagonists against CINV began in the early 1980s. To date, understanding of emetic signals downstream of 5-HT 3 R has remained elusive. Since chemotherapeutics such as cisplatin induce vomiting via concomitant release of several different emetogenic neurotransmitters [1], deciphering the downstream signal transduction mechanism(s) of a particular emetic transmitter in CINV becomes challenging, to say the least. Thus, in the current study we chose to investigate the post-receptor emetic signaling pathway of the more selective 5-HT 3 R ''preferring'' agonist 2-Me-5-HT in the least shrew. The advantage of this model over the long-standing and well-established ferret model is that unlike ferrets, shrews vomit consistently and in a dosedependent manner in response to systemic administration of serotonin [11,20]. Although serotonin cannot penetrate the bloodbrain-barrier, its methylated analog, 2-Me-5-HT, does. We utilized pharmacological, behavioral, immunohistochemical, and Western blot techniques to reveal the central and peripheral Our findings support the hypothesis that, following 5-HT 3 R activation, 2-Me-5-HT causes an influx of extracellular Ca 2+ through 5-HT 3 Rs/Ltype Ca 2+ channels, which subsequently evokes Ca 2+ -induced Ca 2+ release (CICR) from intracellular ER Ca 2+ stores via activation of RyRs Ca 2+ channels present on the ER membrane. The enhanced Ca 2+ mobilization is also sequentially linked to the intracellular activation of the CaMKIIa-ERK pathway in the brainstem, which plays an important role in 2-Me-5-HT-induced vomiting. (See our proposed signaling pathway in Figure 10).

Involvement of extracellular Ca 2+ influx and CICR in 5-HT 3 R-mediated emesis
Stimulation of 5-HT 3 Rs can increase intracellular Ca 2+ levels via extracellular influx through both 5-HT 3 R-and voltage-dependent L-type Ca 2+ -channels present in the cell membrane [23,41,42,43,44]. In fact, the observed in vitro increase in Ca 2+ influx into isolated cell lines is sensitive to both 5-HT 3 R-and Ltype Ca 2+ channel-selective antagonists [42,43]. In the current ex vivo study we confirm that the selective 5-HT 3 R antagonist palonosetron can suppress the 5-HT 3 R-mediated, 2-Me-5-HTevoked enhancements of intracellular Ca 2+ concentration in the least shrew brainstem slices. Likewise, we have recently demonstrated that vomiting caused by specific stimulation of 5-HT 3 Rs in the least shrew is sensitive to selective antagonists of both 5-HT 3 Rs (e.g. palonosetron) and L-type Ca 2+ channels (e.g. nifedipine) [15]. Moreover, the newly identified and novel emetogen FPL64176, a selective agonist of the L-type Ca 2+ channels, causes vomiting in the least shrew in a dose-dependent manner. Not only palonosetron and nifedipine on their own can suppress FPL 64176-induced vomiting in a dose-dependent and potent manner, their ineffective but combined doses demonstrate significantly greater antiemetic  efficacy against vomiting caused by several emetogens including FPL64176, 2-Me-5-HT and cisplatin [15]. These in vivo findings support the proposed cross-talk that occurs between 5-HT 3 Rs and L-type Ca 2+ channels in vitro [45]. Consistent with these observations, in the current study we have demonstrated that vomiting triggered by 2-Me-5-HT is dose-dependently inhibited by another L-type Ca 2+ channel blocker, amlodipine. Furthermore, both nifedipine and amlodipine are effective antiemetics against vomiting caused by diverse emetogens [15,46]. Intracellular Ca 2+ release from the endoplasmic reticulum (ER) can occur via at least two classes of receptors present in ER membrane termed IP 3 Rs and RyRs [47]. In addition, a functional linkage between L-type Ca 2+ channels and RyRs appear to exist which plays an important role in intracellular Ca 2+ release following voltage-dependent Ca 2+ entry through L-type Ca 2+channels [48,49]. In the current study, we first determined whether 2-Me-5-HT-induced vomiting can be differentially modulated via manipulation of IP 3 Rs and RyRs. We found that the 5-HT 3 R-mediated vomiting was insensitive to the IP 3 R antagonist, 2-APB, but in contrast, was dose-dependently suppressed by the RyR antagonist, dantrolene. Furthermore, a combination of the semi-effective doses of amlodipine and dantrolene, was more potent than each antagonist being tested alone. These behavioral findings suggest that 5-HT 3 R stimulation drives extracellular Ca 2+ through both 5-HT 3 Rs and L-type Ca 2+ channels, which subsequently trigger Ca 2+ release via RyRs from intracellular ER stores (i.e. CICR), which greatly amplifies free Ca 2+ levels in the cytoplasm. Our in vivo findings are consistent with a previous in vitro cellular study which demonstrated that 5-HT 3 R activation evokes extracellular Ca 2+ entry which then triggers such Ca 2+ release from intracellular stores in a RyRssensitive manner (i.e. CICR) [42].

Participation of CaM in 5-HT 3 R-mediated emesis
An increase in free cytoplasmic Ca 2+ concentration can lead to activation of CaM and subsequent CaMKIIa [25]. The Ca 2+ sensor CaM can regulate diverse functions by binding to hundreds of target proteins [50]. Our co-immunoprecipitation and immunohistochemistry findings provide the first evidence for an enhanced specific activity-dependent physical interaction between 5-HT 3 R and CaM in both the shrew brainstem and their colocalization in the jejunum following 2-Me-5-HT administration, since the observed association is sensitive to the 5-HT 3 R antagonist palonosetron. Indeed, it is already known that CaM can interact with several G-protein-coupled receptors including serotonergic 5-HT 1A [25]-, 5-HT 2A [26]-, and 5-HT 2C [27]-, as well as muscarinic M 1 -receptors [51], and alters their function via various means including desensitization, receptor internalization and trafficking. Therefore, our findings raise the possibility that in response to 5-HT 3 R activation by 2-Me-5-HT, CaM might influence the localization, clustering, and trafficking of 5HT 3 R as well as 5HT 3 R-mediated signal transduction via direct or indirect binding to 5-HT 3 R. Moreover, not only does CaM bind to L-type Ca 2+ channels (LTCC) [52,53] but our preliminary unpublished findings suggest that the discussed CaM-5-HT 3 R interaction can be also suppressed by the L-type Ca 2+ channel antagonist, amlodipine. Thus, our findings suggest that 5-HT 3 R-CaM interaction appears to be regulated by 5-HT 3 R and LTCC activities which support the proposed crosstalk between 5-HT 3 R and L-type Ca 2+ channels [45]. Based on our current report, the full role for CaM in the regulation of 5-HT 3 R signaling in general and in emesis in particular remains to be fully characterized, and more systematic experiments remain to be conducted, especially, (1) investigation of the consequences of 5-HT 3 R-CaM interaction on 5-HT 3 R function as an ion channel; and (2) cellular studies investigating the specific interruption of the 5-HT 3 R-CaM Figure 10. Summary of the proposed 5-HT 3 R-mediated downstream signaling pathway underlying 2-Me-5-HT-induced emesis in the least shrew. 5-HT 3 R stimulation by the selective agonist 2-Me-5-HT causes an influx of extracellular Ca 2+ through 5-HT 3 Rs/L-type Ca 2+ ion channels which increases the free cytoplasmic concentration of Ca 2+ , thereby promoting Ca 2+ release via calcium-induced calcium release (CICR) from the endoplasmic reticulum stores through ryanodine receptors (RyRs). This elevation in cellular Ca 2+ level initiates attachment of calmodulin (CaM) with the 5-HT 3 R, and leads to CaMKIIa activation and subsequent ERK1/2 signaling. The 5-HT 3 R antagonist palonosetron (1) , the L-type Ca 2+ 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-HT 3 R-mediated Ca 2+ / CaMKII-dependent ERK signaling pathway. doi:10.1371/journal.pone.0104718.g010 interaction with specific small molecules or peptides directly targeting the protein complexes as well as the influence of this specific blockade on 5-HT 3 R-mediated signaling pathway and emesis.

5-HT 3 R-mediated emesis occurs via Ca 2+ -dependent activation of CaMKIIa
CaMKII is a protein kinase that is widely expressed in a variety of tissues [54]. It autophosphorylates in response to elevated intracellular Ca 2+ and functions as an intracellular signaling protein. Phosphorylated CaMKII (pCaMKII) has a relatively unique property that allows prolonged phosphorylation in response to transient Ca 2+ signals making it an excellent marker for cellular activation. Furthermore, enhanced current through Ltype voltage-gated Ca 2+ channels can stimulate CaMKII activity which is required for various effects including induction of longterm potentiation [55], and cocaine-induced sensitization-specific adaptation of trafficking of GluA1 subunit of AMPA receptor [56]. Thus, a third novel aspect of this study was to determine whether Ca 2+ /CaMKII signaling is involved in the 5-HT 3 R-mediated 2-Me-5-HT-induced vomiting. In fact both vomit frequency and the degree of CaMKIIa activation appear to have a temporal relationship, since within 20 min of systemic injection, 2-Me-5-HT not only caused maximal number of vomits, but also induced maximal increase in CaMKIIa phosphorylation at Thr286 in brainstem as revealed by Western blots and immunohistochemistry. Similar to the reported differential increases in c-Fos immunoreactivity in the AP, NTS and DMNX of the least shrew in response to 2-Me-5-HT administration [14], CaMKIIa was also activated by 2-Me-5-HT in all of these brainstem DVC emetic nuclei, but the AP region exhibited higher activation. In addition, in the current study an identical pattern of results was obtained from isolated intestinal EC cells exposed to 2-Me-5-HT in vitro. Both Western blots of total protein extracted from least shrew EC cells and immunocytochemistry of EC cells exhibited substantial increases in pCaMKIIa levels. Moreover, pretreatment with the 5-HT 3 R antagonist palonosetron reversed the 2-Me-5-HT-induced increases in pCaMKIIa in the above-discussed in vivo and in vitro experiments. Since 5-HT 3 Rs are expressed in distinct cells in the GIT including functionally discrete classes of neurons as well as EC cells, 5-HT 3 R stimulation may involve the activation of both neuronal and nonneuronal pathways [5,8]. In fact activation of 5-HT 3 Rs present on the surface of EC cells by 2-Me-5-HT can induce release of endogenous serotonin which can be prevented by prior exposure to selective 5-HT 3 R antagonists [5]. The released endogenous serotonin may then activate 5-HT 3 Rs on vagal nerve endings to initiate the vomiting reflex [6]. Thus, our current findings also appear to suggest the potential involvement of intracellular signaling mechanisms within EC cells in response to emetogens (2-Me-5-HT and possibly cisplatin or bacterial and viral toxins) for the release of endogenous serotonin in the mediation of emesis. In line with our above discussed findings, 5-HT release following perfusion of gut with glucose in rats has been shown to increase CaMKII phosphorylation in the EC cells, NTS and DMNX via activation of 5-HT 3 Rs [16]. Furthermore, 2-Me-5-HT-induced activation of CaMKIIa was abolished by prior treatment of least shrews with either the L-type Ca 2+ channel antagonist amlodipine, the RyR antagonist dantrolene, or a combination of their less effective doses, but not by the IP 3 R antagonist 2-APB, which is consistent with the earlier discussed effects of these Ca 2+ modulators on 2-Me-5-HT-induced vomiting presented in this study. In addition, the CaMKII inhibitor KN93 (but not its inactive analog KN92) [57] not only suppressed CaMKIIa phosphorylation in the shrew brainstem in response to 2-Me-5-HT, but also decreased the induced vomiting in a dose-dependent and potent manner. These results demonstrate that CaMKIIa activation contributes to 5-HT 3 R-mediated vomiting and is under regulation of extracellular Ca 2+ influx through 5-HT 3 R/L-type Ca 2+ channels as well as intracellular Ca 2+ release from the ER stores via the RyRs.

ERK signaling is necessary for 5-HT 3 R-induced emesis
We have recently demonstrated that significant activation of ERK1/2 is associated with peak vomit frequency during both the immediate and delayed phases of emesis caused by cisplatin in the least shrew [18]. In addition, serotonin plays an important role in both emetic phases in the brainstem and the GIT [9]. The final innovative finding of this study is that ERK1/2 activation in the brainstem occurs during 2-Me-5-HT-induced vomiting in the least shrew. This is also the first evidence that 5-HT 3 R stimulation is directly coupled to ERK1/2 phosphorylation. This upregulation of ERK1/2 was abolished by prior treatment with either palonosetron, amlodipine, dantrolene, KN93, or the ERK inhibitor PD98059, suggesting that extracellular Ca 2+ influx, CICR from ER stores via RyRs, and CaMKII activation are sequential prior components of the ERK1/2 cascade involved in 5-HT 3 R-mediated signaling pathway. Our behavioral evidence that inhibition of ERK1/2 activation with PD98059 attenuated 2-Me-5-HT-induced emesis provides further credibility for the involvement of ERK1/2 in the induction of 5-HT 3 R-mediated emesis.

2-Me-5-HT-induced vomiting is independent of 5-HT 2A R and 5-HT 6 R activation
Although 2-Me-5-HT is generally considered a 5-HT 3 R selective agonist, it does possess affinity for 5-HT 2A Rs and 5-HT 6 Rs [58]. In fact 2-Me-5-HT administration in the least shrew can induce the protypical 5-HT 2A receptor-mediated head-twitch behavior [13]. Furthermore, 5-HT 2A R stimulation can increase intracellular Ca 2+ levels and affect L-type Ca 2+ currents [59,60]. In addition, functional interaction can occur between these two receptors where activation of 5-HT 2A R potentiates 5-HT 3 R function [35]. In the current study we have demonstrated that the 5-HT 2A R antagonist SR46349B, does not reduce the ability of 2-Me-5-HT to either induce vomiting or activate CaMKIIa in the shrew brainstem. Moreover, i.p. administration of the selective 5-HT 2A R agonist, DOI, produces the head-twitch response in the least shrew [61] but not emesis [Darmani, unpublished observation]. Likewise, at diverse doses, we tested the antiemetic potential of two selective 5-HT 6 R antagonists (Ro-046790 and Ro-04368554). Both antagonists failed to suppress 2-Me-5-HT-evoked vomiting in the least shrew. Since we have recently demonstrated cAMP/PKA signaling is involved in mediation of cyclophosphamide-induced emesis [62], and activation of 5-HT 6 Rs can activate the cAMP/PKA cascade [63], we investigated the effect of 2-Me-5-HT on PKA phosphorylation at Thr197. 2-Me-5-HT had no significant effect on the latter parameter indicating that neither 5-HT 6 R nor its downstream signaling is involved in the induced vomiting (data not shown). Thus, the discussed findings strongly demonstrate that 5-HT 3 Rs (but not 5-HT 2A Rs or 5-HT 6 Rs) are specifically involved in the mediation of 2-Me-5-HT-induced emesis and related downstream signaling.

Conclusions
In summary, we postulate the following signaling pathway underlying 5-HT 3 R-mediated emesis: 2-Me-5-HT acts in both the brainstem DVC and the GIT emetic loci to increase extracellular influx of Ca 2+ through both 5-HT 3 Rs and the L-type Ca 2+ channels, which leads to CICR from intracellular ER calcium stores via RyRs. This 5-HT 3 R activation-induced increase in intracellular Ca 2+ concentration initiates attachment of CaM to the 5-HT 3 R, and causes Ca 2+ -dependent activation of CaMKIIa which further results in ERK1/2 activation and vomiting (see Figure 10). The latter schematic provides new targets for the development of more novel antiemetics against diverse emetogens in general, and for those emetic agents (chemotherapeutics, bacterial and viral toxins) that employ 5-HT to induce vomiting, in particular. Figure S1 Effects of 2-Me-5-HT treatment on 5-HT 3 Rcalmodulin (CaM) colocalization in the least shrew brainstem nucleus tractus solitaries (NTS) and dorsal motor nucleus of the vagus (DMNX). Shrews were treated with 2-Me-5-HT (5 mg/kg, i.p.) or vehicle for 20 min. 5-HT 3 R-CaM colocalization was determined through co-stained brainstem slices with 5-HT 3 R (red) and CaM (green). Graphs A and B are representative images (2006) of NTS (A) and DMNX (B). Nuclei were shown with DAPI stains. Scale bar, 10 mm. (TIF) Figure S2 Effects of 2-Me-5-HT treatment on pCaMKIIa in the least shrew brainstem nucleus tractus solitaries (NTS) and dorsal motor nucleus of the vagus (DMNX). Shrews were treated with 2-Me-5-HT (5 mg/kg, i.p.) or vehicle for 20 min. CaMKIIa activation was determined through co-stained brainstem slices with CaMKIIa (red) and pCaMKIIa (green). Graphs A and B are representative images (1006) of NTS (A) and DMNX (B). Nuclei were shown with DAPI stains. Scale bar, 10 mm. (TIF)