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The HCN Channel Blocker ZD7288 Induces Emesis in the Least Shrew (Cryptotis parva).

ABSTRACT: Subtypes (1-4) of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are widely expressed in the central and peripheral nervous systems, as well as the cells of smooth muscles in many organs. They mainly serve to regulate cellular excitability in these tissues. The HCN channel blocker ZD7288 has been shown to reduce apomorphine-induced conditioned taste aversion on saccharin preference in rats suggesting potential antinausea/antiemetic effects. Currently, in the least shew model of emesis we find that ZD7288 induces vomiting in a dose-dependent manner, with maximal efficacies of 100% at 1 mg/kg (i.p.) and 83.3% at 10 µg (i.c.v.). HCN channel subtype (1-4) expression was assessed using immunohistochemistry in the least shrew brainstem dorsal vagal complex (DVC) containing the emetic nuclei (area postrema (AP), nucleus tractus solitarius and dorsal motor nucleus of the vagus). Highly enriched HCN1 and HCN4 subtypes are present in the AP. A 1 mg/kg (i.p.) dose of ZD7288 strongly evoked c-Fos expression and ERK1/2 phosphorylation in the shrew brainstem DVC, but not in the in the enteric nervous system in the jejunum, suggesting a central contribution to the evoked vomiting. The ZD7288-evoked c-Fos expression exclusively occurred in tryptophan hydroxylase 2-positive serotonin neurons of the dorsal vagal complex, indicating activation of serotonin neurons may contribute to ZD7288-induced vomiting. To reveal its mechanism(s) of emetic action, we evaluated the efficacy of diverse antiemetics against ZD7288-evoked vomiting including the antagonists/inhibitors of: ERK1/2 (U0126), L-type Ca²⁺ channel (nifedipine); store-operated Ca²⁺ entry (MRS 1845); T-type Ca²⁺ channel (Z944), IP₃R (2-APB), RyR receptor (dantrolene); the serotoninergic type 3 receptor (palonosetron); neurokinin 1 receptor (netupitant), dopamine type 2 receptor (sulpride), and the transient receptor potential vanilloid 1 receptor agonist, resiniferatoxin. All tested antiemetics except sulpride attenuated ZD7288-evoked vomiting to varying degrees. In sum, ZD7288 has emetic potential mainly via central mechanisms, a process which involves Ca²⁺ signaling and several emetic receptors. HCN channel blockers have been reported to have emetic potential in the clinic since they are currently used/investigated as therapeutic candidates for cancer therapy related- or unrelated-heart failure, pain, and cognitive impairment.

SUBMITTER: Zhong W

PROVIDER: S-EPMC8117105 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:Glycogen synthase kinase 3 (GSK-3) is a constitutively active multifunctional serine-threonine kinase which is involved in diverse physiological processes. GSK-3 has been implicated in a wide range of diseases including neurodegeneration, inflammation, diabetes and cancer. GSK-3 is a downstream target for protein kinase B (Akt) which phosphorylates GSK-3 and suppresses its activity. Based upon our preliminary findings, we postulated Akt's involvement in emesis. The aim of this study was to investigate the participation of GSK-3 and the antiemetic potential of two GSK-3 inhibitors (AR-A014418 and SB216763) in the least shrew model of vomiting against fully-effective emetic doses of diverse emetogens, including the nonselective and/or selective agonists of serotonin type 3 (e.g. 5-HT or 2-Methyl-5-HT)-, neurokinin type 1 receptor (e.g. GR73632), dopamine D2 (e.g. apomorphine or quinpirole)-, and muscarinic 1 (e.g. pilocarpine or McN-A-343) receptors, as well as the L-type Ca2+ channel agonist (FPL64176), the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin, and the chemotherapeutic agent, cisplatin. We first determined if these emetogens could regulate the phosphorylation level of GSK-3 in the brainstem emetic loci of least shrews and then investigated whether AR-A014418 and SB216763 could protect against the evoked emesis. Phospho-GSK-3α/β Ser21/9 levels in the brainstem and the enteric nerves of jejunum in the small intestine were upregulated following intraperitoneal (i.p.) administration of all the tested emetogens. Furthermore, administration of AR-A014418 (2.5-20 mg/kg, i.p.) dose-dependently attenuated both the frequency and percentage of shrews vomiting in response to i.p. administration of 5-HT (5 mg/kg), 2-Methyl-5-HT (5 mg/kg), GR73632 (5 mg/kg), apomorphine (2 mg/kg), quinpirole (2 mg/kg), pilocarpine (2 mg/kg), McN-A-343 (2 mg/kg), FPL64176 (10 mg/kg), or thapsigargin (0.5 mg/kg). Relatively lower doses of SB216763 exerted antiemetic efficacy, but both inhibitors barely affected cisplatin (10 mg/kg)-induced vomiting. Collectively, these results support the notion that vomiting is accompanied by a downregulation of GSK-3 activity and pharmacological inhibition of GSK-3 protects against pharmacologically evoked vomiting.

Project description: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 II? (Ca(2+)/CaMKII?), 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 CaMKII? 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 CaMKII? and ERK1/2, which contribute to the 5-HT3R-mediated, 2-Me-5-HT-evoked emesis.

Project description:To characterize mechanisms involved in neurokinin type 1 receptor (NK1R)-mediated emesis, we investigated the brainstem emetic signaling pathways following treating least shrews with the selective NK1R agonist GR73632. In addition to episodes of vomiting over a 30-min observation period, a significant increase in substance P-immunoreactivity in the emetic brainstem dorsal motor nucleus of the vagus (DMNX) occurred at 15 min post an intraperitoneal (i.p.) injection GR73632 (5 mg/kg). In addition, time-dependent upregulation of phosphorylation of several emesis -associated protein kinases occurred in the brainstem. In fact, Western blots demonstrated significant phosphorylations of Ca2+/calmodulin kinase IIα (CaMKIIα), extracellular signal-regulated protein kinase1/2 (ERK1/2), protein kinase B (Akt) as well as α and βII isoforms of protein kinase C (PKCα/βII). Moreover, enhanced phospho-ERK1/2 immunoreactivity was also observed in both brainstem slices containing the dorsal vagal complex emetic nuclei as well as in jejunal sections from the shrew small intestine. Furthermore, our behavioral findings demonstrated that the following agents suppressed vomiting evoked by GR73632 in a dose-dependent manner: i) the NK1R antagonist netupitant (i.p.); ii) the L-type Ca2+ channel (LTCC) antagonist nifedipine (subcutaneous, s.c.); iii) the inositol trisphosphate receptor (IP3R) antagonist 2-APB (i.p.); iv) store-operated Ca2+ entry inhibitors YM-58483 and MRS-1845, (i.p.); v) the ERK1/2 pathway inhibitor U0126 (i.p.); vi) the PKC inhibitor GF109203X (i.p.); and vii) the inhibitor of phosphatidylinositol 3-kinase (PI3K)-Akt pathway LY294002 (i.p.). Moreover, NK1R, LTCC, and IP3R are required for GR73632-evoked CaMKIIα, ERK1/2, Akt and PKCα/βII phosphorylation. In addition, evoked ERK1/2 phosphorylation was sensitive to inhibitors of PKC and PI3K. These findings indicate that the LTCC/IP3R-dependent PI3K/PKCα/βII-ERK1/2 signaling pathways are involved in NK1R-mediated vomiting.

Project description:Temsirolimus is a prodrug form of sirolimus (rapamycin). With its analogs (everolimus, ridaforolimus, and rapamycin), it forms a group of anticancer agents that block the activity of one of the two mammalian targets of rapamycin (mTOR) complexes, mTORC1. We investigated the emetic potential of varying doses (0, 0.5, 1, 2.5, 5, 10, 20, and 40 mg/kg, i.p.) of temsirolimus in the least shrew. Temsirolimus caused a bell-shaped and dose-dependent increase in both the mean vomit frequency and the number of shrews vomiting with maximal efficacy at 10 mg/kg (p < 0.05 and p < 0.02, respectively). Its larger doses (20 or 40 mg/kg) had no significant emetic effect. We also evaluated the emetic potential of its analogs (5, 10, and 20 mg/kg, i.p.), all of which exhibited a similar emetic profile. Our observational studies indicated that temsirolimus can reduce the shrew motor activity at 40 mg/kg, and subsequently, we examined the motor effects of its lower doses. At 10 and 20 mg/kg, it did not affect the spontaneous locomotor activity (distance moved) but attenuated the mean rearing frequency in a U-shaped manner at 10 mg/kg (p < 0.05). We then determined the broad-spectrum antiemetic potential of a 20 mg/kg (i.p.) dose of temsirolimus against diverse emetogens, including selective and nonselective agonists of 1) dopaminergic D2/3 receptors (apomorphine and quinpirole); 2) serotonergic 5-HT3 receptors [5-HT (serotonin) and 2-methyl-5-HT]; 3) cholinergic M1 receptors (pilocarpine and McN-A-343); 4) substance P neurokinin NK1 receptors (GR73632); 5) the L-type calcium (Ca2+) channel (LTCC) (FPL64176); 6) the sarcoplasmic endoplasmic reticulum Ca2+ ATPase inhibitor, thapsigargin; 7) the CB1 receptor inverse agonist/antagonist, SR141716A; and 8) the chemotherapeutic cisplatin. Temsirolimus prevented vomiting evoked by the aforementioned emetogens with varying degrees. The mechanisms underlying the pro- and antiemetic effects of temsirolimus evaluated by immunochemistry for c-fos expression demonstrated a c-fos induction in the AP and NTS, but not DMNX with the 10 mg/kg emetic dose of temsirolimus, whereas its larger antiemetic dose (20 mg/kg) had no significant effect. Our study is the first to provide preclinical evidence demonstrating the promising antiemetic potential of high doses of temsirolimus and possibly its analogs in least shrews.

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The HCN Channel Blocker ZD7288 Induces Emesis in the Least Shrew (Cryptotis parva).

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