α/Sulfono-γ-AA peptide hybrids agonist of GLP-1R with prolonged action both in vitro and in vivo.

Peptides are increasingly important resources for biological and therapeutic development, however, their intrinsic susceptibility to proteolytic degradation represents a big hurdle. As a natural agonist for GLP-1R, glucagon-like peptide 1 (GLP-1) is of significant clinical interest for the treatment of type-2 diabetes mellitus, but its in vivo instability and short half-life have largely prevented its therapeutic application. Here, we describe the rational design of a series of α/sulfono-γ-AA peptide hybrid analogues of GLP-1 as the GLP-1R agonists. Certain GLP-1 hybrid analogues exhibited enhanced stability (t 1/2 > 14 days) compared to t 1/2 (<1 day) of GLP-1 in the blood plasma and in vivo. These newly developed peptide hybrids may be viable alternative of semaglutide for type-2 diabetes treatment. Additionally, our findings suggest that sulfono-γ-AA residues could be adopted to substitute canonical amino acids residues to improve the pharmacological activity of peptide-based drugs.

RNA sequencing least shrew (Cryptotis parva) brainstem and gut transcripts following administration of a selective substance P neurokinin NK1 receptor agonist and antagonist expands genomics resources for emesis research.

The least shrew is among the subset of animals that are capable of vomiting and therefore serves as a valuable research model for investigating the biochemistry, molecular biology, pharmacology, and genomics of emesis. Both nausea and vomiting are associated with a variety of illnesses (bacterial/viral infections, bulimia, exposure to toxins, gall bladder disease), conditions (pregnancy, motion sickness, emotional stress, overeating) and reactions to drugs (chemotherapeutics, opiates). The severe discomfort and intense fear associated with the stressful symptoms of nausea and emesis are the major reason for patient non-compliance when being treated with cancer chemotherapeutics. Increased understanding of the physiology, pharmacology and pathophysiology underlying vomiting and nausea can accelerate progress for developing new antiemetics. As a major animal model for emesis, expanding genomic knowledge associated with emesis in the least shrew will further enhance the laboratory utility of this model. A key question is which genes mediate emesis, and are they expressed in response to emetics/antiemetics. To elucidate the mediators of emesis, in particular emetic receptors, their downstream signaling pathways, as well as the shared emetic signals, we carried out an RNA sequencing study focused on the central and peripheral emetic loci, the brainstem and gut. Thus, we sequenced RNA extracted from brainstem and gut tissues from different groups of least shrews treated with either a neurokinin NK1 receptor selective emetic agonist, GR73632 (5 mg/kg, i.p.), its corresponding selective antagonist netupitant (5 mg/kg, i.p.), a combination of these two agents, versus their corresponding vehicle-pretreated controls and drug naïve animals. The resulting sequences were processed using a de novo transcriptome assembly and used it to identify orthologs within human, dog, mouse, and ferret gene sets. We compared the least shrew to human and a veterinary species (dog) that may be treated with vomit-inducing chemotherapeutics, and the ferret, another well-established model organism for emesis research. The mouse was included because it does not vomit. In total, we identified a final set of 16,720 least shrew orthologs. We employed comparative genomics analyses as well as gene ontology enrichment, KEGG pathway enrichment and phenotype enrichment to better understand the molecular biology of genes implicated in vomiting.

Design of Potent and Proteolytically Stable Biaryl-Stapled GLP-1R/GIPR Peptide Dual Agonists.

Recent clinical trials have revealed that the chimeric peptide hormones simultaneously activating glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) demonstrate superior efficacy in glycemic control and body weight reduction, better than those activating the GLP-1R alone. However, the linear peptide-based GLP-1R/GIPR dual agonists are susceptible to proteolytic cleavage by common digestive enzymes present in the gastrointestinal tract and thus not suitable for oral administration. Here, we report the design and synthesis of biaryl-stapled peptides, with and without fatty diacid attachment, that showed potent GLP-1R/GIPR dual agonist activities. Compared to a linear peptide dual agonist and semaglutide, the biaryl-stapled peptides displayed drastically improved proteolytic stability against the common digestive enzymes. Furthermore, two stapled peptides showed excellent efficacy in an oral glucose tolerance test in mice, owing to their potent receptor activity in vitro and good pharmacokinetics exposure upon subcutaneous injection. By exploring a more comprehensive set of biaryl staplers, we expect that this stapling method could facilitate the design of the stapled peptide-based dual agonists suitable for oral administration.

The Contribution of Phospholipase C in Vomiting in the Least Shrew (Cryptotis Parva) Model of Emesis.

Gq and Gßγ protein-dependent phospholipase C (PLC) activation is extensively involved in G protein-coupled receptor (GPCR)-mediated signaling pathways which are implicated in a wide range of physiological and pathological events. Stimulation of several GPCRs, such as substance P neurokinin 1-, dopamine D2/3-, histamine H1- and mu-opioid receptors, can lead to vomiting. The aim of this study was to investigate the role of PLC in vomiting through assessment of the emetic potential of a PLC activator (m-3M3FBS), and the antiemetic efficacy of a PLC inhibitor (U73122), in the least shrew model of vomiting. We find that a 50 mg/kg (i.p.) dose of m-3M3FBS induces vomiting in ∼90% of tested least shrews, which was accompanied by significant increases in c-Fos expression and ERK1/2 phosphorylation in the shrew brainstem dorsal vagal complex, indicating activation of brainstem emetic nuclei in m-3M3FBS-evoked emesis. The m-3M3FBS-evoked vomiting was reduced by pretreatment with diverse antiemetics including the antagonists/inhibitors of: PLC (U73122), L-type Ca2+ channel (nifedipine), IP3R (2-APB), RyR receptor (dantrolene), ERK1/2 (U0126), PKC (GF109203X), the serotoninergic type 3 receptor (palonosetron), and neurokinin 1 receptor (netupitant). In addition, the PLC inhibitor U73122 displayed broad-spectrum antiemetic effects against diverse emetogens, including the selective agonists of serotonin type 3 (2-Methyl-5-HT)-, neurokinin 1 receptor (GR73632), dopamine D2/3 (quinpirole)-, and muscarinic M1 (McN-A-343) receptors, the L-type Ca2+ channel (FPL64176), and the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin. In sum, PLC activation contributes to emesis, whereas PLC inhibition suppresses vomiting evoked by diverse emetogens.

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

Nausea and vomiting are common gastrointestinal complaints that can be triggered by diverse emetic stimuli through central and/or peripheral nervous systems. Both nausea and vomiting are considered as defense mechanisms when threatening toxins/drugs/bacteria/viruses/fungi enter the body either via the enteral (e.g., the gastrointestinal tract) or parenteral routes, including the blood, skin, and respiratory systems. While vomiting is the act of forceful removal of gastrointestinal contents, nausea is believed to be a subjective sensation that is more difficult to study in nonhuman species. In this review, the authors discuss the anatomical structures, neurotransmitters/mediators, and corresponding receptors, as well as intracellular emetic signaling pathways involved in the processes of nausea and vomiting in diverse animal models as well as humans. While blockade of emetic receptors in the prevention of vomiting is fairly well understood, the potential of new classes of antiemetics altering postreceptor signal transduction mechanisms is currently evolving, which is also reviewed. Finally, future directions within the field will be discussed in terms of important questions that remain to be resolved and advances in technology that may help provide potential answers.

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

With its five receptor subtypes (D1-5), 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 PKCαßII 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), PKCαßII (Thr638/641), ERK1/2 (Thr202/204), and Akt (Ser473). Our results substantiate the implication of PI3K/mTOR/Akt and PI3K/PKCαßII/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.

Central and peripheral emetic loci contribute to vomiting evoked by the Akt inhibitor MK-2206 in the least shrew model of emesis.

Akt (protein kinase B) signaling is frequently activated in diverse cancers. Akt inhibitors such as perifosine and MK-2206 have been evaluated as potential cancer chemotherapeutics. Although both drugs are generally well tolerated, among their most common side-effects vomiting is a major concern. Here we investigated whether these Akt inhibitors evoke emesis in the least shrew model of vomiting. Indeed, both perifosine and MK-2206 induced vomiting with maximal efficacies of 90% at 50 mg/kg (i.p.) and 100% at 10 mg/kg (i.p.), respectively. MK-2206 (10 mg/kg, i.p.) increased c-Fos immunoreactivity both centrally in the shrew brainstem dorsal vagal complex (DVC) emetic nuclei, and peripherally in the jejunum. MK-2206 also evoked phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in both the DVC emetic nuclei and the enteric nervous system in the jejunum. The ERK1/2 inhibitor U0126 suppressed MK-2206-induced emesis dose-dependently. We then evaluated the suppressive efficacy of diverse antiemetics against MK-2206-evoked vomiting including antagonists/inhibitors of the: L-type Ca2+ channel (nifedipine at 2.5 mg/kg, subcutaneously (s.c.)); glycogen synthase kinase 3 (GSK-3) (AR-A014418 at 10 mg/kg and SB216763 at 0.25 mg/kg, i.p.); 5-hydroxytryptamine 5-HT3 receptor (palonosetron at 0.5 mg/kg, s.c.); substance P neurokinin NK1 receptor (netupitant at 10 mg/kg, i.p.) and dopamine D2/3 receptor (sulpride at 8 mg/kg, s.c.). All tested antagonists/blockers attenuated emetic parameters to varying degrees. In sum, this is the first study to demonstrate how pharmacological inhibition of Akt evokes vomiting via both central and peripheral mechanisms, a process which involves multiple emetic receptors.

Ultra-low doses of the transient receptor potential vanilloid 1 agonist, resiniferatoxin, prevents vomiting evoked by diverse emetogens in the least shrew (Cryptotis parva).

Published studies have shown that the transient receptor potential vanilloid 1 (TRPV1) receptor agonist, resiniferatoxin (RTX), has pro and antiemetic effects. RTX can suppress vomiting evoked by a variety of nonselective emetogens such as copper sulfate and cisplatin in several vomit-competent species. In the least shrew, we have already demonstrated that combinations of ultra-low doses of RTX and low doses of the cannabinoid CB1/2 receptor agonist delta-9-tetrahydrocannabinol (Δ-THC) produce additive antiemetic effects against cisplatin-evoked vomiting. In the current study, we investigated the broad-spectrum antiemetic potential of very low nonemetic doses of RTX against a diverse group of specific emetogens including selective and nonselective agonists of serotonergic 5-hydroxytrptamine (5-HT3) receptor (5-HT and 2-Me-5-HT), dopaminergic D2 receptor (apomorphine and quinpirole), cholinergic M1 receptor (pilocarpine and McN-A-343), as well as the selective substance P neurokinin NK1 receptor agonist GR73632, the selective L-Type calcium channel agonist FPL64176, and the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA) inhibitor thapsigargin. When administered subcutaneously, ultra-low (0.01 µg/kg) to low (5.0 µg/kg) doses of RTX suppressed vomiting induced by the aforementioned emetogens in a dose-dependent fashion with 50% inhibitory dose values ranging from 0.01 to 1.26 µg/kg. This study is the first to demonstrate that low nanomolar nonemetic doses of RTX have the capacity to completely abolish vomiting caused by diverse receptor specific emetogens in the least shrew model of emesis.

Δ9-THC and related cannabinoids suppress substance P- induced neurokinin NK1-receptor-mediated vomiting via activation of cannabinoid CB1 receptor.

Δ9-THC suppresses cisplatin-induced vomiting through activation of cannabinoid CB1 receptors. Cisplatin-evoked emesis is predominantly due to release of serotonin and substance P (SP) in the gut and the brainstem which subsequently stimulate their corresponding 5-HT3-and neurokinin NK1-receptors to induce vomiting. Δ9-THC can inhibit vomiting caused either by the serotonin precursor 5-HTP, or the 5-HT3 receptor selective agonist, 2-methyserotonin. In the current study, we explored whether Δ9-THC and related CB1/CB2 receptor agonists (WIN55,212-2 and CP55,940) inhibit vomiting evoked by SP (50 mg/kg, i.p.) or the NK1 receptor selective agonist GR73632 (5 mg/kg, i.p.). Behavioral methods were employed to determine the antiemetic efficacy of cannabinoids in least shrews. Our results showed that administration of varying doses of Δ9-THC (i.p. or s.c.), WIN55,212-2 (i.p.), or CP55,940 (i.p.) caused significant suppression of SP-evoked vomiting in a dose-dependent manner. When tested against GR73632, Δ9-THC also dose-dependently reduced the evoked emesis. The antiemetic effect of Δ9-THC against SP-induced vomiting was prevented by low non-emetic doses of the CB1 receptor inverse-agonist/antagonist SR141716A (<10 mg/kg). We also found that the NK1 receptor antagonist netupitant can significantly suppress vomiting caused by a large emetic dose of SR141716A (20 mg/kg). In sum, Δ9-THC and related cannabinoids suppress vomiting evoked by the nonselective (SP) and selective (GR73632) neurokinin NK1 receptor agonists via stimulation of cannabinoid CB1 receptors.

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

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

The anti-asthmatic drug pranlukast suppresses the delayed-phase vomiting and reverses intracellular indices of emesis evoked by cisplatin in the least shrew (Cryptotis parva).

The introduction of second generation serotonin 5-HT3 receptor (5-HT3) antagonist palonosetron combined with long-acting substance P neurokinin NK1 receptor (NK1) antagonists (e.g. netupitant) has substantially improved antiemetic therapy against early- and delayed-phases of emesis caused by highly emetogenic chemotherapeutics such as cisplatin. However, the improved efficacy comes at a cost that many patients cannot afford. We introduce a new class of antiemetic, the antiasthmatic leukotriene CysLT1 receptor antagonist pranlukast for the suppression of cisplatin-evoked vomiting. Pranlukast (10mg/kg) by itself significantly reduced the mean frequency of vomits (70%) and fully protected least shrews from vomiting (46%) during the delayed-phase of cisplatin (10mg/kg)-evoked vomiting. Although, pranlukast tended to substantially reduce both the mean frequency of vomits and the number of shrews vomiting during the early-phase, these reductions failed to attain significance. When combined with a first (tropisetron)- or a second (palonosetron)-generation 5-HT3 receptor antagonist, pranlukast potentiated their antiemetic efficacy during both phases of vomiting. In addition, pranlukast by itself prevented several intracellular signal markers of cisplatin-evoked delayed-vomiting such as phosphorylation of ERK1/2 and PKA. When pranlukast was combined with either palonosetron or tropisetron, these combinations suppressed the evoked phosphorylation of: i) ERK1/2 during both acute- and delayed-phase, ii) PKCα/ß at the peak acute-phase, and iii) PKA at the peak delayed-phase. The current and our published findings suggest that overall behavioral and intracellular signaling effects of pranlukast via blockade of CysLT1 receptors generally appear to be similar to the NK1 receptor antagonist netupitant with some differences.

Ca2+ signaling and emesis: Recent progress and new perspectives.

Cisplatin-like chemotherapeutics cause vomiting via calcium (Ca2+)-dependent release of multiple neurotransmitters (dopamine, serotonin, substance P, etc.) from the gastrointestinal enterochromaffin cells and/or the brainstem. Intracellular Ca2+ signaling is triggered by activation of diverse emetic receptors (including tachykininergic NK1, serotonergic 5-HT3, dopaminergic D2, cholinergic M1, or histaminergic H1), whose activation in vomit-competent species can evoke emesis. Other emetogens such as cisplatin, rotavirus NSP4 protein and bacterial toxins can also induce intracellular Ca2+ elevation. Netupitant is a highly selective neurokinin NK1 receptor (NK1R) antagonist and palonosetron is a selective second-generation serotonin 5-HT3 receptor (5-HT3R) antagonist with a distinct pharmacological profile. An oral fixed combination of netupitant/palonosetron (NEPA; Akynzeo(®)) with >85% antiemetic efficacy is available for use in the prevention of acute and delayed chemotherapy-induced nausea and vomiting (CINV). Cannabinoid CB1 receptor agonists possess broad-spectrum antiemetic activity since they prevent vomiting caused by a variety of emetic stimuli including the chemotherapeutic agent cisplatin, 5-HT3R agonists, and D2R agonists. Our findings demonstrate that application of the L-type Ca2+ channel (LTCC) agonist FPL 64176 and the intracellular Ca2+ mobilizing agent thapsigargin (a sarco/endoplasmic reticulum Ca2+-ATPase inhibitor) cause vomiting in the least shrew. On the other hand, blockade of LTCCs by corresponding antagonists (nifedipine or amlodipine) not only provide broad-spectrum antiemetic efficacy against diverse agents that specifically activate emetogenic receptors such as 5-HT3, NK1, D2, and M1 receptors, but can also potentiate the antiemetic efficacy of palonosetron against the non-specific emetogen, cisplatin. In this review, we will provide an overview of Ca2+ involvement in the emetic process; discuss the relationship between Ca2+ signaling and the prevailing therapeutics in control of vomiting; highlight the evidence for Ca2+-signaling blockers/inhibitors in suppressing emetic behavior in the least shrew model of emesis as well as in the clinical setting; and also draw attention to the clinical benefits of Ca2+-signaling blockers/inhibitors in the treatment of nausea and vomiting.

Intracellular vomit signals and cascades downstream of emetic receptors: Evidence from the least shrew (Cryptotis parva) model of vomiting.

Nausea and vomiting are often considered as stressful symptoms of many diseases and drugs. In fact they are the most feared and debilitating side-effects of many cancer chemotherapeutics and the main cause of patient noncompliance. Despite years of substantial research, the intracellular emetic signals are at best poorly understood or remain unknown. Among different receptor-mediated emetic signaling cascades, one potential converging signal appears to be changes in the cytosolic concentration of Ca2+. In this editorial, we focus on Ca2+-related intracellular signals underlying emesis mediated by various emetogens. This strategy will help us understand common signaling mechanisms downstream of diverse emetogens and should therefore promote development of new antiemetics for the treatment nausea and vomiting caused by diverse diseases, drugs, as well as viruses and bacterial infections.

Thapsigargin-induced activation of Ca(2+)-CaMKII-ERK in brainstem contributes to substance P release and induction of emesis in the least shrew.

Cytoplasmic calcium (Ca(2+)) mobilization has been proposed to be an important factor in the induction of emesis. The selective sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin, is known to deplete intracellular Ca(2+) stores, which consequently evokes extracellular Ca(2+) entry through cell membrane-associated channels, accompanied by a prominent rise in cytosolic Ca(2+). A pro-drug form of thapsigargin is currently under clinical trial as a targeted cancer chemotherapeutic. We envisioned that the intracellular effects of thapsigargin could cause emesis and planned to investigate its mechanisms of emetic action. Indeed, thapsigargin did induce vomiting in the least shrew in a dose-dependent and bell-shaped manner, with maximal efficacy (100%) at 0.5 mg/kg (i.p.). Thapsigargin (0.5 mg/kg) also caused increases in c-Fos immunoreactivity in the brainstem emetic nuclei including the area postrema (AP), nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus (DMNX), as well as enhancement of substance P (SP) immunoreactivity in DMNX. In addition, thapsigargin (0.5 mg/kg, i.p.) led to vomit-associated and time-dependent increases in phosphorylation of Ca(2+)/calmodulin kinase IIα (CaMKIIα) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) in the brainstem. We then explored the suppressive potential of diverse chemicals against thapsigargin-evoked emesis including antagonists of: i) neurokinin-1 receptors (netupitant), ii) the type 3 serotonin receptors (palonosetron), iii) store-operated Ca(2+) entry (YM-58483), iv) L-type Ca(2+) channels (nifedipine), and v) SER Ca(2+)-release channels inositol trisphosphate (IP3Rs) (2-APB)-, and ryanodine (RyRs) (dantrolene)-receptors. In addition, the antiemetic potential of inhibitors of CaMKII (KN93) and ERK1/2 (PD98059) were investigated. All tested antagonists/blockers attenuated emetic parameters to varying degrees except palonosetron, however a combination of non-effective doses of netupitant and palonosetron exhibited additive antiemetic efficacy. A low-dose combination of nifedipine and 2-APB plus dantrolene mixture completely abolished thapsigargin-evoked vomiting, CaMKII-ERK1/2 activation and SP elevation. In addition, pretreatment with KN93 or PD98059 suppressed thapsigargin-induced increases in SP and ERK1/2 activation. Intracerebroventricular injection of netupitant suppressed vomiting caused by thapsigargin which suggests that the principal site of evoked emesis is the brainstem. In sum, this is the first study to demonstrate that thapsigargin causes vomiting via the activation of the Ca(2+)-CaMKII-ERK1/2 cascade, which is associated with an increase in the brainstem tissue content of SP, and the evoked emesis occurs through SP-induced activation of neurokinin-1 receptors.

Epidemiological Investigation of Asymptomatic Dogs with Leishmania Infection in Southwestern China Where Visceral Leishmaniasis is Intractable.

Heishui county, located in northwest Sichuan province, southwestern China, is an endemic area of zoonotic visceral leishmaniasis (VL) and is the most intractable area. VL is never destroyed in it. Asymptomatic dogs (Leishmania parasites have been diagnosed but clinically healthy) are considered to be a potential reservoir host in zoonotic VL area, and most can lead to infection of individuals, that is a new challenge for controlling VL in humans. The present study aimed to assess the Leishmania infection rate of asymptomatic dogs in Heishui county. Total 105 asymptomatic domestic dogs were gathered from 4 districts in Heishui county to investigate the infection rate with serological and molecular methods based on ELISA and kinetoplast minicircle DNA(kDNA) PCR, respectively. Out of 105 dogs, 44 (41.9%) were positive by more than 1 method; 21 (20.0%) were positive by ELISA, and 30 (28.6%) were positive by kDNA-PCR. Our study showed that Leishmania infection of domestic dogs which is clinically healthy is prevalent in the studied district, and the asymptomatic dogs infected by Leishmania may be the primary reason for the prevalence of visceral leishmaniasis in the area.

[Development and application of rapid molecular method for detection of asymptomatic infection of Leishmania].

OBJECTIVE: To develop a rapid molecular biological method for detection of the asymptomatic infection of Leishmania. METHODS: Two pairs of primers named RV1-RV2 and K13A-K13B were selected to be the fast diagnosis primers since they were designed according to the conserved region of Leishmania kinetoplast DNA (kDNA) minicircles. The PCR amplification products of Leishmania donovani promastigote from Shandong Province were sequenced to compare their conservatism. The method was applied to detect 105 venous blood samples from healthy home canine and 7 venous blood samples from home canine suffered from Kala-azar in Heishui County of Sichuan Province, and 75 venous blood samples from susceptible population (no leishmaniasis symptoms) and 7 venous blood samples from patients in Xinjiang Kashi area in order to verify the feasibility and accuracy of the method. RESULTS: The size of PCR products was consistent with the expected fragments with high conservative among Leishmania species. The positive rates of 105 home canine samples and 75 susceptible population samples were 37.14% (39/105) and 82.67% (62/75) rspectively, and the positive rates of home canine suffered from Kala-azar and patients were all 100%(7/7). CONCLUSION: This rapid diagnosis method is suitable for detection of asymptomatic infection of Leishmania in Kalaazar endemic areas of China with high sensitive and specific, thus it has bright perspective to be used.

L-type calcium channels contribute to 5-HT3-receptor-evoked CaMKIIα and ERK activation and induction of emesis in the least shrew (Cryptotis parva).

Activation of serotonergic 5-HT3 receptors by its selective agonist 2-methyl serotonin (2-Me-5-HT) induces vomiting, which is sensitive to selective antagonists of both 5-HT3 receptors (palonosetron) and L-type calcium channels (LTCC) (amlodipine or nifedipine). Previously we demonstrated that 5-HT3 receptor activation also causes increases in a palonosetron-sensitive manner in: i) intracellular Ca(2+) concentration, ii) attachment of calmodulin (CaM) to 5-HT3 receptor, and iii) phosphorylation of Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) and extracellular-signal-regulated kinase 1/2 (ERK1/2). Here, we investigate the role of the short-acting LTCC blocker nifedipine on 2-Me-5-HT-evoked intracellular Ca(2+) increase and on downstream intracellular emetic signaling, which have been shown to be coupled with 2-Me-5-HT׳s emetic effects in the least shrew. Using the cell-permeant Ca(2+) indicator fluo-4 AM, here we present evidence for the contribution of Ca(2+) influx through LTCCs (sensitive to nifedipine) in 2-Me-5-HT (1µM) -evoked rise in cytosolic Ca(2+) levels in least shrew brainstem slices. Nifedipine pretreatment (10mg/kg, s.c.) also suppressed 2-Me-5-HT-evoked interaction of 5-HT3 receptors with CaM as well as phosphorylation of CaMKIIα and ERK1/2 in the least shrew brainstem, and 5-HT3 receptors -CaM colocalization in jejunum of the small intestine. In vitro exposure of isolated enterochromaffin cells of the small intestine to 2-Me-5-HT (1µM) caused CaMKIIα phosphorylation, which was also abrogated by nifedipine pretreatment (0.1µM). In addition, pretreatment with the CaMKII inhibitor KN62 (10mg/kg, i.p.) suppressed emesis and also the activation of CaMKIIα, and ERK in brainstem caused by 2-Me-5-HT (5mg/kg, i.p.). This study provides further mechanistic explanation for our published findings that nifedipine can dose-dependently protect shrews from 2-Me-5-HT-induced vomiting.

Differential and additive suppressive effects of 5-HT3 (palonosetron)- and NK1 (netupitant)-receptor antagonists on cisplatin-induced vomiting and ERK1/2, PKA and PKC activation.

To better understand the anti-emetic profile of the 5-HT3 (palonosetron)- and the tachykinin NK1 (netupitant) -receptor antagonists, either alone or in combination, we evaluated the effects of palonosetron and/or netupitant pretreatment on cisplatin-evoked vomiting and changes in the phosphorylation of brainstem kinases such as the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), protein kinase C alpha/beta (PKCα/ß), and protein kinase A (PKA) in the least shrew. Our results demonstrate that cisplatin (10mg/kg, i.p.) causes emesis in the least shrew over 40h with respective peak early- and delayed-phases occurring at 1 - 2 and 32 - 34h post-injection. During the early phase (0 - 16h post cisplatin), palonosetron (0.1mg/kg, s.c.) significantly protected shrews from vomiting with a near complete suppression of vomit frequency. Palonosetron also significantly protected shrews from vomiting during the delayed phase (27 - 40h post cisplatin), but the reduction in mean vomit frequency failed to achieve significance. On the other hand, netupitant (5mg/kg, i.p.) totally abolished vomiting during the delayed phase, and tended to suppress the mean vomit frequency during the acute phase. The combined treatment protected shrews almost completely from vomiting during both phases. Brainstem pERK1/2 levels were significantly elevated at all time-points except at 40h post-cisplatin administration. PKA phosphorylation tended to be elevated throughout the delayed phase, but a significant increase only occurred at 33h. Brainstem pPKCα/ß levels were enhanced during acute-phase with a significant elevation at 2h. Palonosetron, netupitant or their combination had no effect on elevated pERK1/2 levels during acute phase, but the combination reversed ERK1/2 phosphorylation at 33h post-cisplatin treatment. In addition, only the combined regimen prevented the cisplatin-induced PKCα/ß phosphorylation observed at the acute phase. On the other hand, palonosetron and netupitant, either alone or in combination, were effective in reducing the induced elevated pPKA levels during the delayed phase. These effects on cisplatin-related emetic signals downstream of 5-HT3- and NK1-receptors help us to better understand the intracellular basis of cisplatin-induced vomiting.

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

[Construction and identification of pcDNA3-HBsAg-p30-ROP2 expression vector].

OBJECTIVE: To construct a multi-gene recombinant pcDNA3-HBsAg-p30-ROP2 expression vector and identify it preliminarily. METHODS: According to recombinant pcDNA3-p30-ROP2 restriction sites, HBV HBsAg gene sequences of primers were designed and synthesized to amplify target fragment, and then cloned into pcDNA3-HbsAg-p30-ROP2 expression vector. After sequencing, it was identified finally by restriction enzyme digestion and other molecular biology techniques. RESULTS: HBV HBsAg gene segment was amplified by PCR and the multi-gene recombinant pcDNA3-HBsAg-p30-ROP2 expression vector was constructed and identified to be correct as theoretical values. The PCR and restriction enzyme digestion results showed that HBsAg and p30-ROP2 gene in recombinant plasmid were confirmed by DNA sequencing. CONCLUSION: The multi-gene recombinant pcD-NA3-HBsAg-p30-ROP2 expression vector is successfully constructed.