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

  • Louiza Belkacemi
    Affiliations
    Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, Pomona, CA 91766, USA
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  • Weixia Zhong
    Affiliations
    Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, Pomona, CA 91766, USA
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  • Nissar A. Darmani
    Correspondence
    Corresponding author at: Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, 309 E. Second Street, Pomona, CA 91766-1854, USA.
    Affiliations
    Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, Pomona, CA 91766, USA
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Open AccessPublished:April 10, 2021DOI:https://doi.org/10.1016/j.autneu.2021.102807

      Highlights

      • Dopamine D2 receptor agonist quinpirole induces emesis in shrews.
      • Quinpirole phosphorylates PI3K/mTOR/Akt and PI3K/PKCαβII/ERK1/2/Akt pathways in brainstems.
      • Sulpiride prevents emesis in shrews.
      • Sulpiride inhibits PI3K/mTOR/Akt and PI3K/PKCαβII/ERK1/2/Akt pathways phosphorylation in brainstems.
      • The above signaling pathways function possibly through synergetic/parallel actions.

      Abstract

      With its five receptor subtypes (D15), dopamine is implicated in a myriad of neurological illnesses. Dopamine D2 receptor-based agonist therapy evokes nausea and vomiting. The signaling mechanisms by which dopamine D2 receptors evoke vomiting remains unknown. Phosphatidylinositol 3-kinases (PI3K)- and protein kinase C (PKC)-related signaling cascades stimulate vomiting post-injection of various emetogens in emetically competent animals. This study investigated potential mechanisms involved in dopamine D2 receptor-mediated vomiting using least shrews. We found that vomiting evoked by the selective dopamine D2 receptor agonist quinpirole (2 mg/kg, i.p.) was significantly suppressed by: i) a dopamine D2 preferring antagonist, sulpiride (s.c.); ii) a selective PI3K inhibitor, LY294002 (i.p.); iii) a PKCαβII inhibitor, GF109203X (i.p.); and iv) a selective inhibitor of extracellular signal-regulated protein kinase1/2 (ERK1/2), U0126 (i.p.). Quinpirole-evoked c-fos immunofluorescence in the nucleus tractus solitarius (NTS) was suppressed by pretreatment with sulpiride (8 mg/kg, s.c.). Western blot analysis of shrew brainstem emetic loci protein lysates revealed a significant and time-dependent increase in phosphorylation of Akt (protein kinase B (PKB)) at Ser473 following a 30-min exposure to quinpirole (2 mg/kg, i.p.). Pretreatment with effective antiemetic doses of sulpiride, LY294002, GF109203X, or U0126 significantly reduced quinpirole-stimulated phosphorylation of emesis-associated proteins including p-85PI3K, mTOR (Ser2448/2481), PKCαβII (Thr638/641), ERK1/2 (Thr202/204), and Akt (Ser473). Our results substantiate the implication of PI3K/mTOR/Akt and PI3K/PKCαβII/ERK1/2/Akt signaling pathways in dopamine D2 receptor-mediated vomiting. Potential novel antiemetics targeting emetic proteins associated with these signaling cascades may offer enhanced potency and/or efficacy against emesis.

      Graphical abstract

      Keywords

      1. Introduction

      Dopamine is a neurotransmitter involved in physiological functions both in the central nervous system (CNS) as well as in peripheral nervous systems and tissues (
      • Girault J.A.
      • Greengard P.
      The neurobiology of dopamine signaling.
      ). Aberrations in dopamine signaling, due to either over-activation or dysfunction, produce CNS disorders including Parkinson's disease (PD), mania/psychosis, restless leg syndrome (RLS), and addiction to name a few (
      • Beaulieu J.M.
      • Del’guidice T.
      • Sotnikova T.D.
      • Lemasson M.
      • Gainetdinov R.R.
      Beyond cAMP: the regulation of Akt and GSK3 by dopamine receptors.
      ;
      • Iversen S.D.
      • Iversen L.L.
      Dopamine: 50 years in perspective.
      ). Dopamine effects are implemented through activation of two classes of dopamine membrane-bound receptors, namely dopamine D1-like (D1 and D5) and dopamine D2-like (D2, D3, and D4) receptors (
      • De Mei C.
      • Ramos M.
      • Iitaka C.
      • Borrelli E.
      Getting specialized: presynaptic and postsynaptic dopamine D2 receptors.
      ). Both classes of dopamine receptors belong to the G-protein-coupled receptor family (
      • Gurevich E.V.
      • Gainetdinov R.R.
      • Gurevich V.V.
      G protein-coupled receptor kinases as regulators of dopamine receptor functions.
      ;
      • Usiello A.
      • et al.
      Distinct functions of the two isoforms of dopamine D2 receptors.
      ). Compounds that activate the dopamine D2-like receptors have been used clinically to improve symptoms of some of the dopamine-dependent neurological disorders (
      • Oboshi Y.
      • et al.
      In vivo mesolimbic D2/3 receptor binding predicts posttherapeutic clinical responses in restless legs syndrome: a positron emission tomography study.
      ). For instance, apomorphine has been used for both PD (
      • Yarnall A.J.
      • et al.
      Apomorphine: a potential modifier of amyloid deposition in Parkinson’s disease?.
      ) and Alzheimer's disease (
      • Ribaric S.
      The pharmacological properties and therapeutic use of apomorphine.
      ), and pramipexole with ropinirole are approved PD and RLS patients (
      • Gerlach M.
      • Double K.
      • Arzberger T.
      • Leblhuber F.
      • Tatschner T.
      • Riederer P.
      Dopamine receptor agonists in current clinical use: comparative dopamine receptor binding profiles defined in the human striatum.
      ). Unfortunately, these and related drugs evoke emesis (vomiting) as an incapacitating side-effect (
      • Kurin M.
      • Bielefeldt K.
      • Levinthal D.J.
      Prevalence of nausea and vomiting in adults using ropinirole: a systematic review and meta-analysis.
      ;
      • Lang D.G.
      • Correia M.J.
      Studies of solitary semicircular canal hair cells in the adult pigeon. II. Voltage-dependent ionic conductances.
      ).
      Functional pathophysiology of nausea and vomiting indicates that these processes are regulated by a balance between the gastrointestinal enteric nervous system and the CNS (
      • Darmani N.A.
      • Ray A.P.
      Evidence for a re-evaluation of the neurochemical and anatomical bases of chemotherapy-induced vomiting.
      ). The central emetic nuclei of the CNS are localized to the brainstem dorsal vagal complex (DVC) and consist of the chemoreceptor trigger zone containing the area postrema (AP), the nucleus tractus solitarius (NTS), and the dorsal motor nucleus of the vagus (DMNX) (
      • Darmani N.A.
      • Ray A.P.
      Evidence for a re-evaluation of the neurochemical and anatomical bases of chemotherapy-induced vomiting.
      ). The peripheral emetic loci include neurons of the enteric nerves system, vagal afferents and enterochromaffin cells (EC cells) lining the gastrointestinal tract. The vagal afferents input emetic stimuli to the brainstem DVC to evoke vomiting (
      • Babic T.
      • Browning K.N.
      The role of vagal neurocircuits in the regulation of nausea and vomiting.
      ;
      • Darmani N.A.
      • Ray A.P.
      Evidence for a re-evaluation of the neurochemical and anatomical bases of chemotherapy-induced vomiting.
      ). Regarding dopamine evoked emesis, evidence from ferrets (
      • Yoshikawa T.
      • Yoshida N.
      • Hosoki K.
      Involvement of dopamine D3 receptors in the area postrema in R(+)-7-OH-DPAT-induced emesis in the ferret.
      ), dogs, and least shrews (Cryptotis parva) implicate dopamine D2 and D3 receptors in the emetic response (
      • Darmani N.A.
      • Zhao W.
      • Ahmad B.
      The role of D2 and D3 dopamine receptors in the mediation of emesis in Cryptotis parva (the least shrew).
      ;
      • Yoshikawa T.
      • Yoshida N.
      • Hosoki K.
      Involvement of dopamine D3 receptors in the area postrema in R(+)-7-OH-DPAT-induced emesis in the ferret.
      ). This is expected since both dopamine and emetic dopamine D2-like receptors (dopamine D2 and D3) are present in the AP, NTS, and DMNX.
      Signaling cascades driven by the stimulation of the dopamine D2-like receptors are either scantly defined or remain unknown in emesis (
      • Belkacemi L.
      • Darmani N.A.
      Dopamine receptors in emesis: molecular mechanisms and potential therapeutic function.
      ). Recent emesis related signaling studies from our group have demonstrated that: i) dopamine D2 receptor-evoked vomiting via administration of quinpirole is paralleled by an increase in phosphorylated glycogen synthase kinase (phospho-GSK-3) expression, and pharmacological inhibition of phospho-GSK-3 decrease the evoked emesis (
      • Zhong W.
      • Darmani N.A.
      The pivotal role of glycogen synthase kinase 3 (GSK-3) in vomiting evoked by specific emetogens in the least shrew (Cryptotis parva).
      ); ii) vomiting mediated by activation of the SP neurokinin NK1 receptor by its selective agonist GR73632, involves phospholipase C with subsequent activation of diverse interplaying signaling pathways including extracellular regulated protein kinase 1/2 (ERK1/2), as well as the α and βII isoforms of protein kinase C (PKCαβII) at (Thr638/641) (
      • Zhong W.
      • Chebolu S.
      • Darmani N.A.
      Intracellular emetic signaling cascades by which the selective neurokinin type 1 receptor (NK1R) agonist GR73632 evokes vomiting in the least shrew (Cryptotis parva).
      ); and finally, iii) the L-type Ca2+ channel (LTCC)/inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)-dependent PI3K (phosphoinositide 3-kinase)/Akt and PKCαβII/ERK1/2 signaling pathways are also involved in NK1R-mediated vomiting (
      • Zhong W.
      • Chebolu S.
      • Darmani N.A.
      Intracellular emetic signaling cascades by which the selective neurokinin type 1 receptor (NK1R) agonist GR73632 evokes vomiting in the least shrew (Cryptotis parva).
      ). Collectively, these data illustrate the importance of PI3K/Akt and PKCαβII/ERK1/2 signaling mechanisms in emesis. mTOR (mammalian target of rapamycin) is part of the PI3K-associated kinase protein family that includes mTOR complex 1 (mTORC1) phosphorylated by Akt and mTOR complex 2 (mTORC2), which phosphorylates Akt at its Ser473 (
      • Khan M.A.
      • Jain V.K.
      • Rizwanullah M.
      • Ahmad J.
      • Jain K.
      PI3K/AKT/mTOR pathway inhibitors in triple-negative breast cancer: a review on drug discovery and future challenges.
      ). The regulatory mechanisms of the PI3K/mTOR/Akt signaling pathway essential in neurodegenerative diseases (
      • Xu F.
      • Na L.
      • Li Y.
      • Chen L.
      Roles of the PI3K/AKT/mTOR signalling pathways in neurodegenerative diseases and tumours.
      ), may also be important during vomiting.
      Thus, in the current study, we focused on signal transduction pathways of dopamine D2 receptor-evoked emesis. Specifically, we determined whether activation of PI3K/mTOR/Akt and PI3K/PKCαβII/ERK1/2 pathways is implicated in dopamine D2 receptor-mediated emesis. Overall, we provide evidence for PI3K/mTOR/Akt and PI3K//PKCαβII/ERK1/2/Akt pathways association with quinpirole-evoked vomiting in the least shrew (Cryptotis parva).

      2. Material and methods

      2.1 Animals and drugs

      Adult male and female least shrews (4–6 g, 35–60 days old) from Western University Health Sciences Animal Facility were housed in groups of 5–10 on a 14:10 light: dark cycle, at a humidity-controlled room temperature of 21 ± 1 °C, with an ad libitum supply of food and water (
      • Darmani N.A.
      Cannabinoids of diverse structure inhibit two DOI-induced 5-HT(2A) receptor-mediated behaviors in mice.
      ;
      • Darmani N.A.
      • Belkacemi L.
      • Zhong W.
      Delta(9)-THC and related cannabinoids suppress substance P- induced neurokinin NK1-receptor-mediated vomiting via activation of cannabinoid CB1 receptor.
      ). Shrews used in this investigation received care according to the “Guide for the Care and Use of Laboratory Animals, 8th edition” DHSS Publication, revised, 2011. All the procedures were approved by the Institutional Animal Care and Use Committee of the Western University of Health Sciences, CA. Maximal efforts were made to minimize both animals' suffering and the number of animals used in the experiments.
      Drugs used in this study include: (−) quinpirole 2 HCL and sulpiride were purchased from Millipore Sigma (St. Louis); LY294002 and U0126 were obtained from Tocris (Minneapolis); GF109203X was purchased from Calbiochem (Burlington). All other reagents were acquired from Millipore Sigma (St. Louis) or Fisher Scientific (Pittsburgh). Antibody manufacturers and dilutions are summarized in Table 1. Quinpirole was dissolved in autoclaved distilled deionized water, whereas sulpiride was dissolved in water with the addition of a 2 μl volume of 1/3 concentrated HCL and back titrated to pH 5 with NaOH. LY294002, GF109203X, and U0126 were dissolved in 25% DMSO in water. The drugs were injected at a volume of 0.1 ml/10 g of body weight.
      Table 1Antibodies dilutions and manufacturers.
      Antibody nameAntibody dilutionManufacturer
      Total Akt1:2000Cell Signaling Tech
      p-Akt (Ser473)1:2000Cell Signaling Tech
      Total ERK1/21:3000Cell Signaling Tech.
      p-ERK1/2 (Th202/204)1:1000Cell Signaling Tech.
      Total mTOR1:2000Cell Signaling Tech.
      p-mTOR Ser24481:2000Cell Signaling Tech.
      p-mTOR Ser28411:2000Cell Signaling Tech.
      p-85PI3K1:1000Santa Cruz Biotech.
      p-PKCαβII (Thr638/641)1:2000Cell Signaling Tech.
      GAPDH1:10000Millipore Sigma

      2.2 Behavioral studies

      On the day of the experiment, shrews were brought into the experimental room from the animal facility, weighed, and transferred to 20 × 18 × 21 cm clean clear plastic individual cages. The drug-naïve male and female shrews were randomly allocated to the control and the experimental groups regardless of their cage of origin and permitted to acclimate for a 1-h time during which daily food was withdrawn. The shrews were given four mealworms (Tenebrio sp.) each 30 min before the administration of emetogens to help identify wet vomits. Experiments were performed between 8:00 am and 5:00 pm.
      In the first set of experiments, we confirmed previously published data showing that a 2 mg/kg i.p.- injection of quinpirole caused a robust frequency of vomits in all tested animals (
      • Darmani N.A.
      • Zhao W.
      • Ahmad B.
      The role of D2 and D3 dopamine receptors in the mediation of emesis in Cryptotis parva (the least shrew).
      ). Based upon our published studies, we assessed drug interaction with potential inhibitors. Thus, different groups of shrews were pretreated at zero minute with an injection of either corresponding vehicle [(intraperitoneal (i.p.) or subcutaneous (s.c.)], or varying doses of one of following antagonists/inhibitors including: i) sulpiride, a dopamine D2 receptor-selective antagonist (8 mg/kg, s.c., N = 14–15 within each group) (
      • Darmani N.A.
      • Zhao W.
      • Ahmad B.
      The role of D2 and D3 dopamine receptors in the mediation of emesis in Cryptotis parva (the least shrew).
      ); ii) LY294002, a potent PI3K inhibitor (0, 1, 2.5, and 10 mg/kg, i.p., N = 6–8 within each group) (
      • Xiao M.
      • et al.
      S100A11 promotes human pancreatic cancer PANC-1 cell proliferation and is involved in the PI3K/AKT signaling pathway.
      ); iii) GF109203X, a PKC blocker selectively targeting PKCαβII (0, 5, 10, and 20 mg/kg, i.p., N = 12 within each group) (
      • Asehnoune K.
      • Strassheim D.
      • Mitra S.
      • Yeol Kim J.
      • Abraham E.
      Involvement of PKCalpha/beta in TLR4 and TLR2 dependent activation of NF-kappaB.
      ); iv) U0126, a highly selective inhibitor of both ERK1 and ERK2 (0, 5, and 10 mg/kg, i.p., N = 9 within each group) (
      • Zhong W.
      • Chebolu S.
      • Darmani N.A.
      Intracellular emetic signaling cascades by which the selective neurokinin type 1 receptor (NK1R) agonist GR73632 evokes vomiting in the least shrew (Cryptotis parva).
      ); and v) a combination of GF109203X + U0126 (0, 1.25, 2.5, and 5 mg/kg, i.p., N = 8–11 within each group). After 30 min following each pretreatment, quinpirole (2 mg/kg, i.p.) was administered to each shrew and the vomit frequency (oral ejections of food or liquid) was recorded for 30 min. Each shrew was euthanized with an overdose of isoflurane following the termination of each experiment.

      2.3 c-fos immunostaining of emetic cell nuclei

      Different shrew groups (N = 3 within each group) were treated with either sulpiride (8 mg/kg. s.c.) or the corresponding vehicle, 30 min before quinpirole (2 mg/kg, i.p.) injection. Shrews injected with the vehicle of sulpiride (s.c.) followed by the vehicle of quinpirole (i.p.) were used as controls. After 30 min observation, each animal was anesthetized with isoflurane and perfused with ice-cold 4% paraformaldehyde in pH 7.4, 0.1 M phosphate-buffered saline (PBS) for 10 min. Brainstems were dissected according to Ray et al. (
      • Ray A.P.
      • Chebolu S.
      • Darmani N.A.
      Receptor-selective agonists induce emesis and Fos expression in the brain and enteric nervous system of the least shrew (Cryptotis parva).
      ), cryoprotected with 30% sucrose in 0.01 M PBS overnight and sectioned on a freezing microtome (Leica, Bannockburn) into 20-μm sections. The sections were observed with a light microscope and those harboring the whole DVC were subjected to immunostaining. The sections were blocked with 0.1 M PBS containing 10% donkey serum and 0.3% Triton X-100 after which they were incubated overnight at 4 °C in rabbit anti-c-fos polyclonal antibody (1:4000, Abcam, San Diego) in 0.1 M PBS containing 5% donkey serum and 0.3% Triton X-100. The next day, the sections were washed 3 times (10 min each) in PBS and incubated in Alexa Fluor 594 donkey anti-rabbit IgG (1:500, Invitrogen, Eugene) secondary antibody. Nuclei of cells were stained with DAPI (Vector Laboratories, Burlingame). Images were acquired under a confocal microscope (Zeiss) with Zen software using 20× objective. We quantified the fluorescence seen from the binding of a fluorophore bound to a secondary antibody. c-fos-immunofluorescent cell nuclei for a given region (AP, NTS, or DMNX) were counted using ImageJ. For each region, 2 sections at 90-μm intervals were counted for each animal. The means of each region per animal were further compared. The circumventricular organ AP and the different cytoarchitectonic detail including cell size and packing distinguishing DMNX from NTS within the DVC were explained by our lab as part of a stereotaxic atlas of the least shrew brainstem (
      • Ray A.P.
      • Darmani N.A.
      A histologically derived stereotaxic atlas and substance P immunohistochemistry in the brain of the least shrew (Cryptotis parva) support its role as a model organism for behavioral and pharmacological research.
      ).

      2.4 Western blot analysis

      The time-dependent profile of Akt phosphorylation was determined using different groups of shrews (N = 4–6 within each group), sacrificed at 0, 15, and 30 min following administration of quinpirole (2 mg/kg, i.p.) or vehicle (autoclaved distilled deionized water, i.p.). To identify the relationship between dopamine D2 receptor and kinases of the PI3K/mTOR/Akt and PI3K/PKCαβII/ERK1/2/Akt pathways, different groups of shrews (N = 4–6 per group) were pretreated with: i) sulpiride (8 mg/kg, s.c.); ii) LY294002 (10 mg/kg, i.p.); iii) GF109203X, (20 mg/kg, i.p.); iv) U0126 (10 mg/kg, i.p); or v) a combination of GF109203X + U0126 (5 mg/kg each, i.p). For each experiment, a corresponding vehicle was included. After 30 min following each pretreatment, an emetic dose of quinpirole (2 mg/kg, i.p.) was administered, and animals were observed for 15 min.
      Shrew brainstems were collected after 0, 15, and 30 min for the time course study and after 15 min for the antagonist interaction experiments. Brainstem medullary structures were isolated as described elsewhere (
      • Zhong W.
      • Darmani N.A.
      The pivotal role of glycogen synthase kinase 3 (GSK-3) in vomiting evoked by specific emetogens in the least shrew (Cryptotis parva).
      ;
      • Zhong W.
      • Chebolu S.
      • Darmani N.A.
      Intracellular emetic signaling cascades by which the selective neurokinin type 1 receptor (NK1R) agonist GR73632 evokes vomiting in the least shrew (Cryptotis parva).
      ;
      • Zhong W.
      • Hutchinson T.E.
      • Chebolu S.
      • Darmani N.A.
      Serotonin 5-HT3 receptor-mediated vomiting occurs via the activation of Ca2+/CaMKII-dependent ERK1/2 signaling in the least shrew (Cryptotis parva).
      ) and homogenized in lysis buffer. Protein extracts from brainstem lysates were subjected to Western blot. The protein extracts from brainstem lysates were subjected to Western blot analysis. Information regarding the primary antibodies is summarized in Table 1. The following secondary antibodies were used for western blot: goat anti-rabbit IRDye 680RD and goat anti-mouse IRDye 800CW (1:10000; LI-COR, Nebraska). Bound antibodies were visualized correspondingly using the Odyssey imaging system (Lincoln). The ratios of phosphorylated forms of mTOR (Ser2448/2481), ERK1/2 (Thr202/204), and Akt (Ser473) to their respective total protein forms were calculated. The ratios of phosphorylated forms of p-85PI3K, and PKCα/βII (Thr638/641) were reported to GAPDH. For normalization, all values were divided by the average value at time point 0 min for the quinpirole time course study and by the values of the respective controls for the antagonist studies and presented as fold-change of respective controls.

      2.5 Statistical analysis

      The frequency of emesis data was analyzed by Kruskal–Wallis (KW) nonparametric one-way analysis of variance (ANOVA) and post hoc analysis by Dunn's multiple comparisons test and reported as the mean ± SEM. The incidence of emesis (percentage of animals vomiting) was analyzed by the Chi-square test to determine whether there were differences between groups. When appropriate, pairwise comparisons were also made by this method. For Western blotting, ordinary one-way ANOVA was performed followed by Dunnett's post hoc test to determine statistical significance between experimental groups and corresponding controls. A P-value of <0.05 was necessary to achieve statistical significance.

      3. Results

      3.1 Antiemetic effects of dopamine D2 receptor antagonist sulpiride and inhibitors of PI3K, PKCαβII, ERK1/2, and Akt on quinpirole - induced emesis

      Current results confirmed our previous findings (
      • Darmani N.A.
      • Zhao W.
      • Ahmad B.
      The role of D2 and D3 dopamine receptors in the mediation of emesis in Cryptotis parva (the least shrew).
      ) that relative to the vehicle-pretreated group, the administration of the selective dopamine D2 receptor antagonist sulpiride (8 mg/kg, s.c.) significantly decreases the frequency of vomits induced by the selective dopamine D2 receptor agonist quinpirole (2 mg/kg, i.p) [(KW (2, 41) = 35.8; P < 0.0001)] during the 30-min observation period (Fig. 1A ). A significant decrease in the percentage of animals vomiting (χ2 (2, 41) = 36.8; P < 0.0001) was also seen with the 8 mg/kg sulpiride (85.71%; (P < 0.0001) (Fig. 1B). Since sulpiride (8 mg/kg, s.c.) significantly attenuated both the mean frequency of vomiting and the percentage of shrews vomiting, this dose was used in our mechanistic studies.
      Fig. 1
      Fig. 1Antiemetic effects of dopamine D2 receptor antagonist sulpiride and selective inhibitors of PI3K, PKCαβII, and ERK1/2 against vomiting evoked by dopamine D2 receptor agonist quinpirole (2 mg/kg, i.p.).
      (A–B) Effect of dopamine D2 receptor antagonist sulpiride: Different groups of shrews received either sulpiride vehicle (0 mg/kg, s.c.) or a single dose of sulpiride (8 mg/kg, s.c.), 30 min before an emetic dose of quinpirole (2 mg/kg, i.p.). Emetic parameters were recorded for the next 30 min. (A) The frequency (mean ± SEM) of emesis. N = 9 per group. **** P < 0.01 vs. Vehicle + Quinpirole (2 mg/kg), Kruskal–Wallis non-parametric one-way ANOVA and followed by Dunn's post hoc test. B. The percentage of shrews vomiting. ****P < 0.0001 vs. Vehicle + Quinpirole (2 mg/kg), Chi-square test.
      (C–D) Effect of the PI3K potent inhibitor LY20094: Different groups of shrews received either 25% DMSO vehicle + water or pretreated with varying doses of LY20094 (0, 1, 2.5, or 10 mg/kg, i.p.) prior to the injection of a fully effective emetic dose of quinpirole (2 mg/kg, i.p.). Emetic parameters were recorded for the next 30 min. (C) Frequency of emesis presented as mean ± SEM of emesis. N = 6–8 per group. *P < 0.05 vs. Vehicle + Quinpirole (2 mg/kg), Kruskal–Wallis non-parametric one-way ANOVA followed by Dunn's post hoc test. (D) Percentage of shrews vomiting. N = 6–8 per group. *P < 0.05 and **P = 0.001 vs. 0 mg/kg, Chi-square test.
      (E–F) Effect of the selective PKCαβII inhibitor GF109203X: Different groups of shrews received either 25% DMSO vehicle + water or pretreated with varying doses of GF109203X (0, 5, 10, 20 mg/kg, i.p.) prior to the injection of a fully effective emetic dose of quinpirole (2 mg/kg, i.p.). Emetic parameters were recorded for the next 30 min. (E) Frequency of emesis presented as mean ± SEM of emesis. N = 12 per group *P < 0.05 and ***P < 0.001 vs. Vehicle + Quinpirole (2 mg/kg), Kruskal–Wallis non-parametric one-way ANOVA followed by Dunn's post hoc test. (F) Percentage of shrews vomiting. **P < 0.005 and ****P < 0.0001 vs. 0 mg/kg, Chi-square test.
      (G–H) Effect of ERK1/2 inhibitor U0126: Different groups of shrews received either 25% DMSO vehicle + water or pretreated with varying doses of U0126 (0, 5, or 10 mg/kg, i.p.) prior to the injection of a fully effective emetic dose of quinpirole (2 mg/kg, i.p.). Emetic parameters were recorded for the next 30 min. (G) Frequency of emesis presented as mean ± SEM of emesis.
      N = 9. **P < 0.01 vs. Vehicle + Quinpirole (2 mg/kg), Kruskal–Wallis non-parametric one-way ANOVA followed by Dunn's post hoc test. (H) Percentage of shrews vomiting. N = 9 per group. **P < 0.01 vs. 0 mg/kg, Chi-square test.
      (I–J) Additive effect of non-effective combined doses of GF109203X + U0126: Different groups of shrews received either 25% DMSO vehicle + water or pretreated with varying doses of GF109203X + U0126 (0, 1.25, or 5 mg/kg, i.p. each) prior to the injection of a fully effective emetic dose of quinpirole. Emetic parameters were recorded for the next 30 min. (I) Frequency of emesis presented as mean ± SEM of emesis. ***P < 0.001 vs. Vehicle + Quinpirole (2 mg/kg), Kruskal–Wallis non-parametric one-way ANOVA followed by Dunn's post hoc test. (J) Percentage of shrews vomiting. N = 8–11 per group. ****P < 0.0001 vs. 0 mg/kg, Chi-square test.
      Next, we tested the antiemetic potential of varying doses of the potent PI3K potent inhibitor LY294002 (0, 1, 2.5, and 10 mg/kg, i.p.) against vomiting caused by quinpirole (2 mg/kg, i.p.). Relative to the vehicle-pretreated control group, i.p.-administered LY294002 significantly and dose dependently attenuated the mean frequency of emesis during the 30-min observation period [(KW (3, 22) = 9.32; P < 0.05)] with a significant reduction occurring at its 10 mg/kg (P < 0.05) (Fig. 1C). Significant decreases in the percentage of shrews vomiting (χ2 (3, 22) = 12.01; P < 0.01) occurred at its 2.5 and 10 mg/kg dose (50.0%; P < 0.05 and 83.3%; P = 0.001, respectively) (Fig. 1D).
      We then evaluated the antiemetic potential of varying doses of the selective PKCαβII inhibitor GF109203X (0, 5, 10, and 20 mg/kg, i.p.), against vomiting evoked by quinpirole (2 mg/kg, i.p.). Relative to the vehicle-pretreated control group, GF109203X significantly reduced the mean frequency of quinpirole-induced vomiting during the 30-min observation period [(KW (3, 44) = 19.1; P = 0.0003)] with significant reductions in the frequency of quinpirole-induced vomits occurring at its 10 and 20 mg/kg (i.p.) doses (P = 0.05 and < 0.0001, respectively) (Fig. 1E). Likewise, GF109203X significantly protected shrews from quinpirole-evoked vomiting (χ2 (3, 44) = 19.1; P = 0.0003) with significant reductions in the percentage of shrews vomiting observed at its 10 and 20 mg/kg doses (50%; P = 0.005 and 83.3%; P < 0.0001, respectively) (Fig. 1F).
      Varying doses of the ERK1/2 inhibitor U0126 (0, 5, and 10 mg/kg, i.p.) were tested against quinpirole (2 mg/kg, i.p.)-induced vomiting. Relative to vehicle-pretreated control group, i.p.-administered U0126 significantly and dose-dependently reduced the mean frequency of quinpirole-induced vomiting during the 30-min observation period [(KW (2, 24) = 9.5; P < 0.01)] and substantial reduction in the frequency of vomits was observed at its 10 mg/kg dose (P < 0.01) (Fig. 1G). Moreover, U0126 significantly protected shrews from quinpirole-evoked vomiting (χ2 (2, 24) = 9.0; P < 0.01) with a significant reduction in the percentage of shrews vomiting occurring at its 10 mg/kg dose (66.7%; P < 0.01) (Fig. 1H).
      We then tested the efficacy of mixtures of noneffective doses of GF109203X and U0126 (0, 1.25, 2.5, or 5 mg/kg each, i.p.) against quinpirole (2 mg/kg, i.p.). The combination of the two agents significantly and dose-dependently reduced the mean frequency of quinpirole-induced vomiting during the 30-min observation period [(KW (3,31) = 14.70; P = 0.002)] with a significant decrease in the frequency of quinpirole-induced vomits occurring at their 5 mg/kg combined dose (P < 0.001) (Fig. 1I). Likewise, GF109203X + U0126 significantly protected shrews from quinpirole-evoked vomiting (χ2 (3, 31) = 18.4; P < 0.0001) with a significant reduction in the percentage of shrews vomiting at their combined 5 mg/kg dose (87.50%; P < 0.0001) (Fig. 1J).

      3.2 Dopamine D2 receptor selective antagonist prevents quinpirole - evoked c-fos expression in the DVC emetic nuclei of the shrew brainstem

      To determine central responsiveness to dopamine D2 preferring agonist quinpirole (2 mg/kg, i.p.), we performed c-fos immunostaining and quantified c-fos immnofluorescent positive nuclei in the DVC containing emetic nuclei AP, NTS, and DMNX. As summarized in Fig. 2, no significant difference was found in the AP c-fos counts between the quinpirole treated (Fig. 2C – D, G) and control group (P = 0.55) (Fig. 2A - B, G). In contrast, quinpirole caused significant c-fos induction in the NTS (P = 0.0023) as reflected by the increased mean number of c-fos immnofluorescent positive nuclei in this zone (Fig. 2C – D, G), relative to the non-vomiting vehicle control group (Fig. 2A - B, G). In the DMNX, there was a trend toward an increase in the number of c-fos immnofluorescent positive nuclei (Fig. 2C – D, G) but without significance versus that of the control group (P = 0.22) (Fig. 2A - B, G).
      Fig. 2
      Fig. 2Quinpirole mediated c-fos induction in brainstem emetic nuclei is reversed by pretreatment with sulpiride.
      (A–F) Immunolabeling was performed on brainstem sections as described in the section. The area postrema (AP), nucleus of the solitary tract (NTS), and dorsal motor nucleus of the vagus (DMNX) form the brainstem dorsal vagal complex (DVC) emetic nuclei.
      (A–B) Relatively few c-fos-immunoreactive nuclei were found in the DVC of control shrews injected with sulpiride (s.c.) vehicle 30 min prior to the administration of quinpirole vehicle (i.p.).
      (C–D) Quinpirole (2 mg/kg., i.p.)-injected shrews that were pretreated with sulpiride vehicle (s.c.) showed increased c-fos expression throughout the NTS, less in the DMNX, and very infrequently in the AP.
      (E–F) In the presence of sulpiride (8 mg/kg, s.c.), the ability of quinpirole (2 mg/kg., i.p.) to induce c-fos expression was abrogated. Scale Bar, 50 μm.
      (G) Summary of c-fos-immunoreactive nuclei counts in the AP, NTS and DMNX zones. N = 3 shrews per group. Two sections from each shrew were used for analysis. c-fos positive nuclei (white arrows) were quantified. Data for each region was expressed as mean number of c-fos-immunoreactive cell nuclei ± SEM. One-way ANOVA followed by Dunnett's post hoc test. **P < 0.01 vs. control (i.e. Vehicle sulpiride + Vehicle quinpirole).
      To study the role of the dopamine D2 receptor in quinpirole - evoked c-fos induction, we used an effective antiemetic dose of sulpiride, which significantly prevented shrews from vomiting in response to an emetic dose of quinpirole. Sulpiride (8 mg/kg, s.c.) was injected 30 min prior to quinpirole (2 mg/kg, i.p.) administration and immunostaining was performed to determine c-fos expression on brainstem sections. In line with the behavioral data, Fig. 2 shows that the higher c-fos immunofluorescent counts observed in the NTS post quinpirole injection (Fig. 2C – D, G) was reversed by the pretreatment with sulpiride to basal control levels (P = 0.98) (Fig. 2E – F, G).

      3.3 An emetic dose of quinpirole evokes time-dependent increases in Akt (Ser473) phosphorylation

      We used Western blotting to quantify the level of phosphorylation of Akt (Ser473) in shrew brainstems injected with quinpirole (2 mg/kg, i.p.) for varying periods (0, 5, 15, and 30 min). Akt phosphorylation significantly peaked at 15 min post quinpirole injection and remained at peak level for up to 30 min (P = 0.02 and 0.01 vs. 0 min, respectively) (Fig. 3A – B and Supplementary Western Table I S1), whereas the under same conditions, total Akt levels remained similar all conditions (Fig. 3A).
      Fig. 3
      Fig. 3Time-course of an emetic dose of quinpirole on Akt phosphorylation at Ser473 in the least shrew brainstem.
      (A) Representative Western blot for Akt phosphorylation at Ser473 in the least shrew brainstems harvested from either vehicle controls (0 min) or at the indicated time-points post quinpirole (2 mg/kg, i.p.) administration. (B) The ratios of phosphorylated Akt at Ser473 to total Akt were each compared to their respective total protein expression. All ratios were normalized to the vehicle-treated control (0 min) values before analysis and expressed as fold change of controls. N = 4 per group. *P < 0.05 vs. 0 min, one-way ANOVA followed by Dunnett's post hoc test.
      Since Akt is a key downstream protein in several signaling pathways including PI3K and PKCαβII/ERK1/2, and exposure to an emetic dose of quinpirole significantly increased phosphorylated Akt at Ser473 at 15 min, we choose the latter time-point for subsequent biochemical analyses.

      3.4 Sulpiride significantly decreases quinpirole-evoked phosphorylation of emesis-associated proteins from PI3K/mTOR/Akt and PI3K/PKC/ERK/Akt pathways

      Quinpirole's ability to trigger phosphorylation of PI3K (p85), mTOR (Ser2448/2481), PKCαβII (Thr638/641), ERK1/2 (Thr202/204), and Akt (Ser473) was assessed using Western blotting. Shrews (N = 4–5 within each group) were pretreated for 30 min with either sulpiride (8 mg/kg, s.c.) or its vehicle prior to 15 min exposure to quinpirole (2 mg/kg, i.p.) injection. To determine levels of protein phosphorylation, protein lysates from brainstems of the treated shrews were subjected to Western blotting followed by densitometric analysis. As shown in Fig. 4A – B and Supplementary Western Table II S1, sulpiride significantly prevented quinpirole-evoked: i) PI3K (p85) phosphorylation [P = 0.05, Vehicle + Quinpirole (2 mg/kg) vs. Vehicle + Vehicle; P = 0.007, Sulpiride (8 mg/kg) + Quinpirole (2 mg/kg) vs. Vehicle + Quinpirole (2 mg/kg)]; ii) mTOR (Ser2448/2481) phosphorylation [P = 0.02 and 0.001, Vehicle + Quinpirole (2 mg/kg) vs. respective Vehicle + Vehicle; P = 0.002 and 0.01, Sulpiride (8 mg/kg) + Quinpirole (2 mg/kg) vs. respective Vehicle + Quinpirole (2 mg/kg)]; iii) PKCαβII (Thr638/641) phosphorylation [P = 0.003, Vehicle + Quinpirole (2 mg/kg) vs. Vehicle + Vehicle; P = 0.04, Sulpiride (8 mg/kg) + Quinpirole (2 mg/kg) vs. Vehicle + Quinpirole (2 mg/kg)]; iv) ERK1/2 (Thr202/204) phosphorylation [P = 0.008 and 0.03, Vehicle + Quinpirole (2 mg/kg) vs. respective Vehicle + Vehicle; P = 0.007 and 0.03, Sulpiride (8 mg/kg) + Quinpirole (2 mg/kg) mg/kg vs. respective Vehicle + Quinpirole (2 mg/kg)]; and v) Akt (Ser473) phosphorylation [P = 0.009, Vehicle + Quinpirole (2 mg/kg) vs. Vehicle + Vehicle; P = 0.005, Sulpiride (8 mg/kg) + Quinpirole (2 mg/kg) vs. Vehicle + Quinpirole (2 mg/kg)]. Levels of GAPDH, and total mTOR, ERK1/2, and Akt proteins remained similar under all conditions (Fig. 4A).
      Fig. 4
      Fig. 4Inhibitory effects of the dopamine D2 receptor antagonist sulpiride on quinpirole-evoked phosphorylation of p-85PI3K, mTOR (Ser2448/2481), PKCαβII (Thr638/641), ERK1/2 (Thr202/204), and Akt (Ser473) proteins in shrew brainstems.
      (A) Representative Western blots of p-85PI3K, mTOR (Ser2448/2481), PKCαβII (Thr638/641), ERK1/2 (Thr202/204), and Akt (Ser473) protein phosphorylation in shrew brainstems. Different groups of shrews were pretreated for 30 min with either sulpiride (8 mg/kg, s.c.) or its vehicle, followed by a 15 min exposure to quinpirole (2 mg/kg, i.p.). Thereafter, shrew brainstem tissue levels of protein phosphorylation were quantified. (B) The levels of phosphorylation of p85-PI3K, mTOR (Ser244/2481), PKCαβII (Thr638/641), ERK1/2 (Thr202/204), and Akt (Ser473) were compared each to their respective total protein or GAPDH and ratios obtained. All ratios were normalized to vehicle-treated controls values before analysis and expressed as fold change of controls. N = 4–5 per group. *P < 0.05 and **P < 0.01 vs. either Vehicle + Vehicle or Sulpiride + Quinpirole, one-way ANOVA followed by Dunnett's post hoc test.
      Abbreviations: VV: Pretreatment with the vehicle of sulpiride (s.c.) for 30 min followed by the vehicle of quinpirole (i.p.) (sterile distilled deionized water) for 15 min. VQ: pretreatment with the vehicle of sulpiride (s.c.) for 30 min followed by treatment with quinpirole for 15 min. SQ: Pretreatment with sulpiride for 30 min followed by treatment with quinpirole (2 mg/kg, i.p.) for 15 min.

      3.5 LY294002 suppresses quinpirole-evoked intracellular emetic protein phosphorylation

      To provide additional evidence for the involvement of phosphorylation of proteins from the PI3K/mTOR/Akt and PI3K/PKCαβII/ERK1/2/Akt pathways in quinpirole mediated emesis, shrews (N = 4–6 within each group) were pretreated (i.p.) with either the PI3K pathway inhibitor LY294002 (10 mg/kg, i.p.) or its corresponding vehicle (0 mg/kg, i.p.) for 30 min prior to quinpirole (2 mg/kg, i.p.) challenge for 15 min (Fig. 5A – B and Supplementary Western Table II S2). Densitometric analysis showed that LY294002 significantly blocked quinpirole-evoked: i) mTOR (Ser2448/2481) phosphorylation [P < 0.05 and 0.02, Vehicle + Quinpirole (2 mg/kg) vs. respective Vehicle + Vehicle; P = 0.002 and 0.02, LY294002 (10 mg/kg) + Quinpirole (2 mg/kg) vs. respective Vehicle + Quinpirole (2 mg/kg)]; ii) PKCαβII (Thr638/641) phosphorylation [P = 0.003, Vehicle + Quinpirole (2 mg/kg) vs. Vehicle + Vehicle; P = 0.003, LY294002 (10 mg/kg) + Quinpirole (2 mg/kg) vs. Vehicle + Quinpirole (2 mg/kg)]; iii) ERK1/2 (Thr202/204) phosphorylation [P = 0.005 and = 0.008, Vehicle + Quinpirole (2 mg/kg) vs. respective Vehicle + Vehicle; P < 0.001 and < 0.001, LY294002 (10 mg/kg) + Quinpirole (2 mg/kg) vs. respective Vehicle + Quinpirole (2 mg/kg)]; and iv) Akt phosphorylation [P = 0.0007, Vehicle + Quinpirole (2 mg/kg) vs. Vehicle + Vehicle; P = 0.02, LY294002 (10 mg/kg) + Quinpirole (2 mg/kg) vs. Vehicle + Quinpirole (2 mg/kg)]. Levels of GAPDH, and total mTOR, ERK1/2, and Akt remained unchanged under all conditions (Fig. 5A).
      Fig. 5
      Fig. 5Suppressive effects of the PI3K potent inhibitor LY292004 on quinpirole-evoked phosphorylation of mTOR (Ser2448/2481), PKCαβII (Thr638/641), ERK1/2 (Thr202/204), and Akt (Ser473) proteins in shrew brainstems.
      (A) Representative Western blots of mTOR (Ser2448/2481), PKCαβII (Thr638/641), ERK1/2 (Thr202/204), and Akt (Ser473) protein phosphorylation in shrew brainstems. Different groups of shrews were pretreated for 30 min with either LY292004 (10 mg/kg, i.p.) or its vehicle, followed by a 15 min exposure to quinpirole (2 mg/kg, i.p.). Thereafter, shrew brainstem tissue levels of protein phosphorylation were quantified. (B) The levels of phosphorylation of mTOR (Ser2448/2481), PKCαβII (Thr638/641), ERK1/2 (Thr202/204), and Akt (Ser473) were compared each to their respective total protein or GAPDH and ratios obtained. N = 4–6 per group. All ratios were normalized to vehicle-treated control before analysis and expressed as fold change of controls. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs. either Vehicle + Vehicle or LY292004 + Quinpirole, one-way ANOVA followed by Dunnett's post hoc test.
      Abbreviations: VV: Pretreatment with 25% DMSO vehicle of LY294002 (i.p.) for 30 min followed by quinpirole vehicle (i.p.) for 15 min. VQ: pretreatment with the vehicle of 25% DMSO vehicle of LY294002 (i.p.) for 30 min followed by treatment with quinpirole (2 mg/kg, i.p.) for 15 min. LQ: Pretreatment with LY294002 (i.p.) for 30 min followed by treatment with quinpirole (2 mg/kg, i.p.) for 15 min.

      3.6 Inhibitors of PKCαβII and ERK1/2 suppress downstream quinpirole-induced intracellular emetic protein phosphorylation

      We first evaluated PKCαβII phosphorylation status following the injection of an emetic dose of quinpirole. Shrews (N = 4 within each group) were pretreated (i.p.) with either the PKCαβII inhibitor GF109203X (20 mg/kg, i.p.) or its corresponding vehicle (0 mg/kg, i.p.) for 30 min prior to a 15-min challenge with quinpirole (2 mg/kg, i.p.) after which the phosphorylation states of ERK1/2 and Akt were determined (Fig. 6A, D and Supplementary Western Table II S3). GF109203X significantly blocked: i) quinpirole-mediated ERK1/2 (Thr202/204) phosphorylation [P = 0.04 and = 0.024, Vehicle + Quinpirole (2 mg/kg) vs. respective Vehicle + Vehicle; P = 0.0006 and = 0.0002, GF109203X (20 mg/kg) + Quinpirole (2 mg/kg) vs. respective Vehicle + Quinpirole (2 mg/kg)]; and ii) Akt (Ser473) phosphorylation [P = 0.02, Vehicle + Quinpirole (2 mg/kg) vs. Vehicle + Vehicle; P = 0.02, GF109203X (20 mg/kg) + Quinpirole (2 mg/kg) vs. Vehicle + Quinpirole (2 mg/kg); P = 0.0003, GF109203X (20 mg/kg) + Vehicle vs. Vehicle + Vehicle)]. Levels of total ERK1/2 and Akt remained unchanged under all conditions (Fig. 6A).
      Fig. 6
      Fig. 6Suppressive effects of the selective PKCαβII (GF109203X) and ERK1/2 (U0126) inhibitors either alone or in combination on quinpirole-evoked phosphorylation of PKCαβII (Thr638/641), ERK1/2 (Thr202/204), and Akt (Ser473) proteins in shrew brainstems.
      (A–C) Representative Western blots for PKCαβII (Thr638/641), ERK1/2 (Thr202/204), and Akt (Ser473) in shrew brainstems. Shrews were pretreated for 30 min with (A) GF109203X (20 mg/kg, i.p.), (B) U0126 (10 mg/kg, i.p.), or (C) GF109203X + U0126 (5 mg/kg, i.p. each) or their corresponding vehicles, their brainstems were isolated 15 min after quinpirole (2 mg/kg, i.p.) injection, and levels of protein phosphorylation were quantified. The levels of phosphorylation of PKCαβII (Thr638/641), ERK1/2 (Thr202/204), and Akt (Ser473) were compared each to their respective total protein or GAPDH and ratios obtained. (D) All ratios were normalized to vehicle-treated control values before analysis and expressed as fold change of corresponding control. N = 4 per group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Vehicle + Vehicle or Specific inhibitors + Quinpirole, one-way ANOVA followed by Dunnett's post hoc test.
      Abbreviations: VV: Pretreatment with 25% DMSO vehicle (i.p.) for 30 min followed by sterile distilled deionized water (quinpirole vehicle, i.p.) for 15 min. VQ: pretreatment with 25% DMSO vehicle i.p. for 30 min followed by treatment with quinpirole (2 mg/kg, i.p.) for 15 min. PQ: pretreatment with (GF109203X (20 mg/kg, i.p.) for 30 min followed by treatment with quinpirole (2 mg/kg, i.p.) for 15 min, UQ: Pretreatment with U0126 (10 mg/kg i.p.) for 30 min followed by treatment with quinpirole (2 mg/kg, i.p.) for 15 min, or PUQ: pretreatment with GF109203X + U0126 (5 mg/kg each, i.p.) for 30 min followed by quinpirole (2 mg/kg, i.p.) treatment for 15 min.
      Second, we assessed the contribution of ERK1/2 activation to quinpirole (2 mg/kg) stimulated phosphorylation. Shrews were pretreated (i.p.) 30 min prior to quinpirole (2 mg/kg, i.p.) injection with either the ERK1/2 inhibitor U0126 (10 mg/kg, i.p.) or its corresponding vehicle (0 mg/kg, i.p.). U0126 significantly prevented quinpirole-stimulated Akt (Ser473) phosphorylation [P = 0.02, Vehicle + Quinpirole (2 mg/kg) vs. Vehicle + Vehicle; P = 0.03, U0126 (10 mg/kg) + Quinpirole (2 mg/kg) vs. Vehicle + Quinpirole (2 mg/kg)] (Fig. 6B, D and Supplementary Western Table II S4). Levels of total Akt remained unchanged under all conditions (Fig. 6B).
      Finally, we determined whether combined non-effective doses of GF109203X + U0126 (5 mg/kg each, i.p.) prior to the administration of quinpirole (2 mg/kg, i.p.) can prevent Akt (Ser473) phosphorylation. GF109203X + U0126 (5 mg/kg each) significantly blocked quinpirole-mediated Akt (Ser473) phosphorylation [P = 0.006, Vehicle + Quinpirole (2 mg/kg) vs. Vehicle + Vehicle; P = 0.003, GF109203X + U0126 (5 mg/kg each) + Quinpirole (2 mg/kg) vs. Vehicle + Quinpirole (2 mg/kg)] (Fig. 6C, D and Supplementary Western Table II S5). Levels of total Akt remained unaffected under all conditions (Fig. 6C).

      4. Discussion

      In the current study we have demonstrated that the dopamine D2 receptor preferring agonist quinpirole-evoked emesis was accompanied by a significant increase in c-fos expression in the NTS, and a trend toward higher expression in the DMNX. This was paralleled by the activation of kinases from PI3K/mTOR/Akt and PKCαβII/ERK1/2/Akt pathways with no gender differences observed among the tested animals. On the other hand, the more selective dopamine D2 antagonist sulpiride, and inhibitors of PI3K/mTOR/Akt and PKCαβII/ERK1/2/Akt pathways, prevented both quinpirole - stimulated emesis and the corresponding increases in c-fos expression as discussed below:

      4.1 Activation of DVC emetic loci is required for dopamine D2 receptor - evoked emesis

      The contribution of central emetic neurons in quinpirole-evoked vomiting is demonstrated by increased c-fos expression in the NTS, a main site for the integration of various emetic signals (
      • Ray A.P.
      • Griggs L.
      • Darmani N.A.
      Delta 9-tetrahydrocannabinol suppresses vomiting behavior and Fos expression in both acute and delayed phases of cisplatin-induced emesis in the least shrew.
      ;
      • Zhong W.
      • Chebolu S.
      • Darmani N.A.
      Thapsigargin-induced activation of Ca(2+)-CaMKII-ERK in brainstem contributes to substance P release and induction of emesis in the least shrew.
      ). In contrast, the dopaminergic agonist did not cause significant c-fos activation in the AP despite the presence of dopamine D2 receptors (
      • Hyde T.M.
      • Knable M.B.
      • Murray A.M.
      Distribution of dopamine D1-D4 receptor subtypes in human dorsal vagal complex.
      ;
      • Yoshikawa T.
      • Yoshida N.
      • Hosoki K.
      Involvement of dopamine D3 receptors in the area postrema in R(+)-7-OH-DPAT-induced emesis in the ferret.
      ). The absence of a significant increase in c-fos-immunoreactivity in the AP may be supported by prior findings indicating that systemic administration of some emetics may not involve the AP (
      • Miller A.D.
      • Leslie R.A.
      The area postrema and vomiting.
      ;
      • Ray A.P.
      • Chebolu S.
      • Darmani N.A.
      Receptor-selective agonists induce emesis and Fos expression in the brain and enteric nervous system of the least shrew (Cryptotis parva).
      ). Another potential reason may be that dopamine D2 receptors in the AP could be located on dendrites extending into the AP from NTS neurons and would therefore not increase c-fos- immunoreactivity in the AP (
      • Ray A.P.
      • Chebolu S.
      • Darmani N.A.
      Receptor-selective agonists induce emesis and Fos expression in the brain and enteric nervous system of the least shrew (Cryptotis parva).
      ). In addition, loperamide stimulated c-fos expression has been detected in the dorsomedial region of the NTS, but little or no c-fos-like immunoreactivity was found in the AP (
      • Leslie R.
      • Reynolds D.
      Functional anatomy of the emetic circuitry.
      ). Likewise,
      • Zaman S.
      • Woods A.J.
      • Watson J.W.
      • Reynolds D.J.
      • Andrews P.L.
      The effect of the NK1 receptor antagonist CP-99,994 on emesis and c-fos protein induction by loperamide in the ferret.
      found low c-fos in the AP. They observed c-fos expression the DMNX along the nuclei border. We also saw a trend toward an increase of the number of positive red nuclei in the DMNX of quinpirole-treated animals versus vehicle, which is indicative of a potential cooperation between NTS and DMNX during emesis. The pretreatment with the selective dopamine D2 receptor antagonist sulpiride, concomitantly abrogated both quinpirole - evoked emetic behavior and c-fos expression in the NTS and the DMNX, suggesting that dopamine D2 receptor may play a crucial role in quinpirole-induced responses.

      4.2 Dopamine D2 receptor-evoked emesis requires the contribution of various phosphorylated proteins

      Pretreatment with the dopamine D2 receptor antagonist sulpiride blocked quinpirole-evoked increases in the levels of phosphorylated p85PI3K, mTOR (Ser2448/2481), PKCαβII (Thr638/641), ERK1/2(Thr202/204), and Akt (Ser473) emesis-associated proteins in the least shrew brainstem. These results suggest that quinpirole-induced vomiting is a dopamine D2 receptor-dependent phenomenon and supports the potential involvement of several kinases from PI3K/mTOR/Akt and PI3K/PKCαβII/ERK1/2/Akt pathways in the dopamine D2 receptor-evoked emesis. Interestingly, these emesis-associated proteins have long been linked with various neurological diseases. Indeed, proteins of the PI3K/mTOR/Akt pathways were deemed to be vital in the onset and evolution of neurodegenerative disorders (
      • Chong Z.Z.
      • Shang Y.C.
      • Wang S.
      • Maiese K.
      A critical kinase cascade in neurological disorders: PI 3-K, Akt, and mTOR.
      ). For example, upstream of mTOR, the PI3K/AKT pathway, which controls mTOR activity is altered in PD (
      • Heras-Sandoval D.
      • Perez-Rojas J.M.
      • Hernandez-Damian J.
      • Pedraza-Chaverri J.
      The role of PI3K/AKT/mTOR pathway in the modulation of autophagy and the clearance of protein aggregates in neurodegeneration.
      ). Moreover, the disruption of PKC and ERK-mediated signal transduction significantly contributes to the pathogenesis of PD, Alzheimer's, and Huntington's disease (
      • Zeni A.L.
      • Zomkowski A.D.
      • Maraschin M.
      • Rodrigues A.L.
      • Tasca C.I.
      Involvement of PKA, CaMKII, PKC, MAPK/ERK and PI3K in the acute antidepressant-like effect of ferulic acid in the tail suspension test.
      ). When taken together, these and current findings indicate that the proteins of the PI3K/mTOR/Akt and PI3K/PKCαβII/ERK1/2/Akt pathways could be potential pharmacological targets for the dual treatment of dopamine-related neurological disorders and emesis. However, effective translation of protein kinases from these pathways into robust and safe clinical strategies will depend on the further elucidation of the complex roles that these kinases hold in the CNS.

      4.3 PI3K/mTOR/Akt signaling

      The PI3K/mTOR/Akt signaling pathway regulatory mechanisms and biological functions are crucial not only in many human neurodegenerative diseases, but also in tumors, making this system attractive for clinical use across multiple therapeutic areas. The Akt protein represents a central point of the PI3K/mTOR/Akt pathway (
      • Manning B.D.
      • Toker A.
      AKT/PKB signaling: navigating the network.
      ;
      • Testa J.R.
      • Tsichlis P.N.
      AKT signaling in normal and malignant cells.
      ;
      • Wan X.
      • Harkavy B.
      • Shen N.
      • Grohar P.
      • Helman L.J.
      Rapamycin induces feedback activation of Akt signaling through an IGF-1R-dependent mechanism.
      ) and PI3K lipid products are generally considered to be obligate and rate-limiting for appropriate Akt phosphorylation (
      • Manning B.D.
      • Toker A.
      AKT/PKB signaling: navigating the network.
      ). Akt is phosphorylated at several sites. Although the significance of most the phosphorylations remains to be fully understood (
      • Mahajan K.
      • Mahajan N.P.
      PI3K-independent AKT activation in cancers: a treasure trove for novel therapeutics.
      ), it is well-recognized 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 Ca2+ Channel (LTCC) agonist FPL64176 with vomiting (
      • Zhong W.
      • Chebolu S.
      • Darmani N.A.
      Intracellular emetic signaling evoked by the L-type Ca(2+) channel agonist FPL64176 in the least shrew (Cryptotis parva).
      ). In the current study, administration of 2 mg/kg of the dopamine D2 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. These results are in line with previous findings where i.p. injection of the selective NK1R agonist GR73632 (5 mg/kg) time-dependently upregulated phosphorylation of emesis-associated protein kinases including Akt in shrew brainstem (
      • Zhong W.
      • Chebolu S.
      • Darmani N.A.
      Intracellular emetic signaling cascades by which the selective neurokinin type 1 receptor (NK1R) agonist GR73632 evokes vomiting in the least shrew (Cryptotis parva).
      ). Our study is also consistent with diverse models of cells in culture, showing that stimulation of dopamine D2 receptor and/or D3 receptor rapidly activates Akt signaling (
      • Brami-Cherrier K.
      • Valjent E.
      • Garcia M.
      • Pages C.
      • Hipskind R.A.
      • Caboche J.
      Dopamine induces a PI3-kinase-independent activation of Akt in striatal neurons: a new route to cAMP response element-binding protein phosphorylation.
      ;
      • Mannoury la Cour C.
      • Salles M.J.
      • Pasteau V.
      • Millan M.J.
      Signaling pathways leading to phosphorylation of Akt and GSK-3beta by activation of cloned human and rat cerebral D(2)and D(3) receptors.
      ). Furthermore, in vivo administration of selective dopamine D2/D3 receptor agonists or psychostimulants (which increase dopamine extracellular levels), quickly evoke the phosphorylation of Akt in the nucleus accumbens or the dorsal striatum of mice soon after their injection (
      • Brami-Cherrier K.
      • Valjent E.
      • Garcia M.
      • Pages C.
      • Hipskind R.A.
      • Caboche J.
      Dopamine induces a PI3-kinase-independent activation of Akt in striatal neurons: a new route to cAMP response element-binding protein phosphorylation.
      ;
      • Mannoury la Cour C.
      • Salles M.J.
      • Pasteau V.
      • Millan M.J.
      Signaling pathways leading to phosphorylation of Akt and GSK-3beta by activation of cloned human and rat cerebral D(2)and D(3) receptors.
      ;
      • Svenningsson P.
      • et al.
      Diverse psychotomimetics act through a common signaling pathway.
      ).
      The potent PI3K/Akt pathway inhibitor LY294002 has often been used to study the role of the PI3K/Akt pathway in the regulation of various intracellular signaling pathways (
      • Sun J.
      • Ramnath R.D.
      • Tamizhselvi R.
      • Bhatia M.
      Role of protein kinase C and phosphoinositide 3-kinase-Akt in substance P-induced proinflammatory pathways in mouse macrophages.
      ). Inhibition of PI3K via LY294002 has been shown to lead to the dephosphorylation of Akt at Ser473 (
      • Jiang H.
      • Fan D.
      • Zhou G.
      • Li X.
      • Deng H.
      Phosphatidylinositol 3-kinase inhibitor(LY294002) induces apoptosis of human nasopharyngeal carcinoma in vitro and in vivo.
      ). In the current study we also found that quinpirole-evoked Akt (Ser473) phosphorylation can be significantly suppressed by pretreatment of shrews with LY294002 (10 mg/kg, i.p.) for 30 min. This finding agrees well with previous data demonstrating that LY294002 pretreatment for 30 min prior to the administration of a fully emetic dose of the substance P neurokinin NK1 receptor selective agonist GR73632 (5 mg/kg, i.p.), markedly attenuates the evoked Akt phosphorylation at Ser473 15 min post-GR73632 treatment. More importantly, the latter findings are indicative of a potential role for Akt and its up and/or downstream signaling proteins in the process of induction of emesis.
      One such protein is the mammalian target of rapamycin (mTOR), which is located upstream of Akt protein. As a member of the PI3K/mTOR/Akt signaling pathway (
      • Lee H.
      Phosphorylated mTOR expression profiles in human normal and carcinoma.
      ), mTOR activity can be inhibited by LY294002 and other PI3K inhibitors (
      • Ballou L.M.
      • Selinger E.S.
      • Choi J.Y.
      • Drueckhammer D.G.
      • Lin R.Z.
      Inhibition of mammalian target of rapamycin signaling by 2-(morpholin-1-yl)pyrimido[2,1-alpha]isoquinolin-4-one.
      ). We found that increased phosphorylation of mTOR (Ser2448/2481) evoked by the injection of an emetic dose of quinpirole was markedly blocked at both Ser2448/2481 phosphorylation sites by prior administration of LY294002 (10 mg/kg, i.p.), implying that mTOR phosphorylation was dependent on PI3K activation in quinpirole-evoked emesis. Mechanistically, mTOR operates through two complexes: mTORC1 (mTOR-Raptor) containing mTOR phosphorylated on Ser2448, and mTORC2 (mTOR-Rictor) comprising mTOR phosphorylated predominantly on Ser2481 (
      • Copp J.
      • Manning G.
      • Hunter T.
      TORC-specific phosphorylation of mammalian target of rapamycin (mTOR): phospho-Ser2481 is a marker for intact mTOR signaling complex 2.
      ). The exact mechanism through which the mTORC1/2 may contribute to emesis is still unclear, however, studies have shown that activation of PI3K can lead to the recruitment and activation of Akt via phosphorylation mTORC2 at Akt (Ser473) (
      • Guertin D.A.
      • Sabatini D.M.
      Defining the role of mTOR in cancer.
      ;
      • Sarbassov D.D.
      • Guertin D.A.
      • Ali S.M.
      • Sabatini D.M.
      Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex.
      ), raising the possibility that the inhibition of PI3K signaling by LY294002 may cause decreased phosphorylation of both mTORC2 and Akt as a prerequisite to the observed reduction in vomiting behavior. It is interesting to note that in the present study, mTORC1 phosphorylation at Ser2448 is significantly decreased by LY294002 as well, which is not unexpected since several reports have shown that mTORC1 is phosphorylated at Ser2448 by PI3K/Akt signaling (

      Nave BT, Ouwens M, Withers DJ, Alessi DR, Shepherd PR (1999) Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation Biochem. J. 344 Pt 2:427–431.

      ;
      • Peterson R.T.
      • Beal P.A.
      • Comb M.J.
      • Schreiber S.L.
      FKBP12-rapamycin-associated protein (FRAP) autophosphorylates at serine 2481 under translationally repressive conditions.
      ) and various Akt pathways can lead to the activation of mTORC1 (
      • Chong Z.Z.
      • Shang Y.C.
      • Maiese K.
      Cardiovascular disease and mTOR signaling.
      ). In sum, our results support the critical involvement of LY294002 in the suppression of both quinpirole-evoked emesis in least shrews and phosphorylation of mTOR (2481/2448) and Akt (Ser473) in their brainstems. However, additional work would be of interest to further define the exact role of mTOR in this system. Collectively, these findings support a pivotal role for the PI3K/mTOR/Akt signaling pathways in quinpirole-mediated emesis.

      4.4 PI3K/PKC/ERK/Akt signaling

      To increase our understanding of mechanisms implicated in dopamine D2 receptor stimulation of PI3K associated pathways, we determined the level of phosphorylation of PKCαβII at Thr638/641 and ERK1/2 at Thr202/204 in shrew brainstems pretreated with either LY294002 (10 mg/kg, i.p.) or its vehicle before exposure to an emetic dose of quinpirole for 15 min. We found that LY294002-evoked decrease in quinpirole-stimulated emesis was accompanied by significantly lower levels of PKCαβII (Thr638/641), and ERK1/2 (Thr202/204) phosphorylation, which imply PI3K activation (phosphorylation) is potentially an essential component of the PKCαβII/ERK1/2 pathway during dopamine D2 receptor-mediated emesis. Interestingly, previous work from our laboratory suggested that although LY294002 prevented GR73632-evoked ERK1/2 phosphorylation, it failed to prevent to the same extent PKCαβII phosphorylation in NK1R-mediated vomiting. The latter and current findings allude to a drug-specific effect on proteins of PI3K/PKCαβII/ERK1/2 post-receptor stimulation. Indeed, according to Mitsos et al. (
      • Mitsos A.
      • Melas I.N.
      • Siminelakis P.
      • Chairakaki A.D.
      • Saez-Rodriguez J.
      • Alexopoulos L.G.
      Identifying drug effects via pathway alterations using an integer linear programming optimization formulation on phosphoproteomic data.
      ) the drug effects are governed by the intrinsic properties of the drug (i.e., selectivity and potency) and the specific signaling transduction network of the host.
      The specific role of PKCαβII and ERK1/2 signaling in dopamine D2 receptor signaling system were further investigated. Classical PKCs include PKCα, βI, βII, and γ (
      • Lallemend F.
      • Lefebvre P.P.
      • Hans G.
      • Rigo J.M.
      • Van de Water T.R.
      • Moonen G.
      • Malgrange B.
      Substance P protects spiral ganglion neurons from apoptosis via PKC-Ca2+-MAPK/ERK pathways.
      ). GF109203X inhibits especially PKCα, β, δ, and ε with a ranked order of potency (α > βI > ε > δ) (
      • Martiny-Baron G.
      • et al.
      Selective inhibition of protein kinase C isozymes by the indolocarbazole Go 6976.
      ). Our behavioral findings showed that PKCαβII (GF109203X) and ERK1/2 (U0126) inhibitors block vomiting induced by an emetic dose of quinpirole which is in agreement with previous studies correlating both PKCαβII and ERK1/2 to emesis (
      • Hutchinson T.E.
      • Zhong W.
      • Chebolu S.
      • Wilson S.M.
      • Darmani N.A.
      L-type calcium channels contribute to 5-HT3-receptor-evoked CaMKIIalpha and ERK activation and induction of emesis in the least shrew (Cryptotis parva).
      ;
      • Zhong W.
      • Chebolu S.
      • Darmani N.A.
      Thapsigargin-induced activation of Ca(2+)-CaMKII-ERK in brainstem contributes to substance P release and induction of emesis in the least shrew.
      ). To explain the mechanistic role of PKCαβII and ERK1/2 in the PI3K signaling, firstly, we tested an antiemetic dose of GF109203X (20 mg/kg) against quinpirole (2 mg/kg) and quantified changes in ERK1/2 (Thr202/204) and Akt (Ser473) phosphorylation. We found that GF109203X markedly inhibited ERK1/2 (Thr202/204) phosphorylation evoked by quinpirole to baseline control, indicating that ERK1/2 phosphorylation occurred downstream of PKC and was dependent upon PKCαβII (Thr638/641) phosphorylation. Furthermore, pretreatment of shrews with GF109203X effectively eliminated quinpirole-induced phosphorylation of Akt in the shrew brainstems, which implies that the phosphorylation of PKCαβII (Thr638/641), ERK1/2 (Thre202/204), and Akt (Ser473) may represent essential events in signal transduction cascades underlying dopamine D2 receptor-mediated emesis. We observed that GF109203X blocked Akt phosphorylation to a significantly lower level than baseline. We speculate that there may be a second PKC-dependent signaling mechanism contributing to the process, which is in line with Kawasaki and co-workers findings (
      • Kawakami Y.
      • et al.
      Protein kinase C betaII regulates Akt phosphorylation on Ser-473 in a cell type- and stimulus-specific fashion.
      ) demonstrating that PKCβ can regulate Akt activity by directly phosphorylating Akt at Ser473. Secondly, we blocked ERK1/2 with U0126 and observed significantly decreased phosphorylation of Akt at Ser473, indicating that Akt phosphorylation may be, at least partially, dependent on ERK1/2 phosphorylation. Finally, we conducted complementary experiments to evaluate the effect of a non-effective dose of GF109203X and U0126 (5 mg/kg, each) on vomiting and phosphorylation of Akt. The combination of low doses of both inhibitors produced significantly lower vomit frequency and percentage of shrews vomiting and prevented Akt phosphorylation in the brainstem, supporting a possible synergetic effect between PKCαβII and ERK1/2 actions during vomiting caused by an emetic dose of quinpirole. It is worth noting that LTCC agonist FPL64176 enhances phosphorylation of proteins PKCαβII, ERK1/2, and Akt in the shrew brainstem, and pretreatment with corresponding LTCC antagonists amlodipine (0.5–10 mg/kg) or nifedipine (2.5–10 mg/kg), attenuates in a dose-dependent and potent manner FPL64176-stimulated emesis (
      • Zhong W.
      • Chebolu S.
      • Darmani N.A.
      Intracellular emetic signaling cascades by which the selective neurokinin type 1 receptor (NK1R) agonist GR73632 evokes vomiting in the least shrew (Cryptotis parva).
      ), and suppresses vomiting caused by a variety of other emetogens including those produced by i.p. administration of dopamine D2/3 receptor agonists such as apomorphine (2 mg/kg) or quinpirole (2 mg/kg) (
      • Darmani N.A.
      • Zhong W.
      • Chebolu S.
      • Vaezi M.
      • Alkam T.
      Broad-spectrum antiemetic potential of the L-type calcium channel antagonist nifedipine and evidence for its additive antiemetic interaction with the 5-HT(3) receptor antagonist palonosetron in the least shrew (Cryptotis parva).
      ;
      • Zhong W.
      • Chebolu S.
      • Darmani N.A.
      Broad-spectrum antiemetic efficacy of the L-type calcium channel blocker amlodipine in the least shrew (Cryptotis parva).
      ). We postulate that quinpirole administration may also cause increased Ca2+ mobilization through LTCCs leading to PKCαβII and ERK1/2 emetic effector phosphorylation and subsequent vomiting. However, this will require further testing.
      Overall, dopamine D2 receptor stimulation triggers phosphorylation of kinases from the PI3K/PKCαβII/ERK1/2/Akt pathway in shrew brainstems after 15 min, supporting a contribution of this pathway in quinpirole-evoked vomiting.

      5. Conclusion

      The role of dopamine related signaling cascades is well studied in aberrant neurodegeneration (
      • Giacoppo S.
      • Bramanti P.
      • Mazzon E.
      Triggering of inflammasome by impaired autophagy in response to acute experimental Parkinson’s disease: involvement of the PI3K/Akt/mTOR pathway.
      ;
      • Zhao Y.
      • Wang Q.
      • Wang Y.
      • Li J.
      • Lu G.
      • Liu Z.
      Glutamine protects against oxidative stress injury through inhibiting the activation of PI3K/Akt signaling pathway in parkinsonian cell model.
      ;
      • Zhu T.
      • et al.
      HDAC6 alleviates prion peptide-mediated neuronal death via modulating PI3K-Akt-mTOR pathway.
      ) but poorly explored in emesis. This investigation is the first study to decipher the signaling mechanisms associated with dopamine D2 receptor-evoked emesis as depicted in Fig. 7 and graphical abstract. The dopamine D2 receptor preferring agonist quinpirole-evoked vomiting was significantly blocked by: i) the dopamine D2 receptor selective antagonist sulpiride, and ii) pharmacological signaling blockers such as LY294002, GF109203X, and U0126 with a concomitant and significant reductions in phosphorylation of emesis-associated kinases including PI3K, mTOR, PKCαβII, ERK1/2, and Akt. Overall, the current data suggest that the dopamine D2 receptor is required for quinpirole-evoked phosphorylation of proteins from PI3K/mTOR/Akt and PI3K/PKCαβII/ERK1/2/Akt pathways during emesis. Our results also indicate that it may be possible to inhibit diverse causes of emesis by targeting common intracellular signals downstream of their corresponding receptors/effectors.
      Fig. 7
      Fig. 7Proposed schematic diagram representing potential intracellular pathways involved in dopamine D2 receptor-mediated emesis in the least shrew.
      Dopamine D2 receptors are activated through G-protein coupled receptors (GPCR). The dopamine D2 preferring receptor agonist quinpirole induces both vomiting and increased phosphorylation of emesis-associated signaling proteins including p85PI3K, mTOR (Ser2448/2481), PKCαβII (Thr638/641), ERK1/2 (Thr202/204), and Akt (Ser473) in brainstems of least shrews. In contrast, the dopamine D2 receptor specific antagonist (sulpiride), and inhibitors of PI3K (LY294002), PKCαβII (GF109203X), and ERK1/2 (U0126) suppress quinpirole-evoked vomiting and prevent phosphorylation of the corresponding proteins in shrew brainstem. Stimulation of dopamine D2 receptors cause emesis via: i) phosphorylation of PI3K, which subsequently phosphorylates mTORC2 at Ser2841, followed by activation (phosphorylation) of Akt at Ser473 resulting in vomiting, and ii) sequential activation of PI3K, PKCαβII, and ERK1/2 as primary mediators to promote phosphorylation of Akt as prerequisite of emesis.
      (P) indicate phosphorylation sites.
      The broken arrow indicates potential mTORC2 phosphorylation of Akt
      → The solid arrows show activation (phosphorylation) of PI3K/mTOR/Akt/PKCαβII/ERK1/2 pathways.
      Red T lines indicate inhibition. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

      CRediT authorship contribution statement

      Conceived and designed the experiments: LB, NAD. Performed the experiments: LB, WZ, NAD. Analyzed the data: LB, WZ, NAD. Contributed reagents/materials/analysis tools: NAD. Contributed to the writing of the manuscript: LB WZ NAD.

      Declaration of competing interest

      We have no conflict of interest to declare.

      Acknowledgements

      This work was supported in part by the NIH - NCI grant ( CA207287 ) and WesternU intramural startup fund ( 1395 ) to NAD.

      Appendix A. Supplementary data

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