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.

Keywords: HCN channel; ZD7288; calcium; emesis; emetic nuclei; least shrew.

FIGURE 1

Expression of HCN channel subtypes 1 and 2. HCN channel immunostaining with rabbit anti-HCN1 and mouse anti- HCN2 primary antibodies followed by Alexa Fluor 594 donkey anti-rabbit and 488 donkey anti-mouse secondary antibodies were performed on coronal brainstem sections (20 μm) prepared from naïve least shrews (n = 3 shrews). Nuclei were stained with DAPI in blue. (A-C) Representative images (20x) show expression of HCN1 but not HCN2 observed in the least shrew brainstem dorsal vagal complex (DVC) area including the three emetic nuclei, the area postrema (AP), the nucleus tractus solitarius (NTS) and the dorsal motor nucleus of the vagus (DMNX). Scale bar, 100 µm. (D–O) Representative images (60x) show differential expression of HCN1 observed in the area postrema (AP), the nucleus tractus solitarius (NTS), the dorsal motor nucleus of the vagus (DMNX) and the hypoglossal nuclei (XII). Scale bar, 50 µm.

FIGURE 2

Expression of HCN channel subtypes 3 and 4. HCN channel immunostaining with rabbit anti-HCN4 and mouse anti-HCN3 primary antibodies followed by Alexa Fluor 594 donkey anti-rabbit and 488 donkey anti-mouse secondary antibodies were performed on coronal brainstem sections (20 μm) prepared from naïve least shrews. (A-B) Representative images (20x) show expression of HCN4 but not HCN3 observed in the least shrew brainstem dorsal vagal complex (DVC) area including three emetic nuclei, the area postrema (AP), the nucleus tractus solitarius (NTS) and the dorsal motor nucleus of the vagus (DMNX). Scale bar, 100 µm. (C–F) Representative images (60x) show differential expression of HCN4 observed in the area postrema (AP), the nucleus tractus solitarius (NTS), the dorsal motor nucleus of the vagus (DMNX) and the hypoglossal nuclei (XII). Scale bar, 50 µm.

FIGURE 3

The dose-response emetic effect of the HCN blockers in the lease shrew. Different groups of least shrews were given varying doses of ZD7288 (i.p., n = 8 shrews per group or i. c.v., n = 6) or ivabradine hydrochloride (i.p., n = 6 shrews per group), and were observed for the next 30 min (A, C, E) The frequency of emesis was analyzed with Kruskal-Wallis non-parametric one-way ANOVA followed by Dunnett’s post hoc test and presented as mean ± SEM. (B, D, F) Percentage of shrews vomiting was analyzed with chi-square test and presented as mean. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. 0 mg/kg.

FIGURE 4

Immunohistochemical analysis of c-Fos following emesis induced by systemic administration of the HCN channel blocker ZD7288. Least shrews were sacrificed 90 min post vehicle treatment, or after the first vomiting occurred post systemic administration (1 mg/kg, i. p.) of ZD7288 (n = 4 shrews per group). Shrew brainstem section (20 μm) and intestinal jejunum sections (25 μm) were stained with rabbit c-Fos antibody and Alexa Fluor 594 donkey anti-rabbit secondary antibody. Nuclei were stained with DAPI in blue. (A, C) Representative tile-scanned images show a robust c-Fos induction in the brainstem dorsal vagal complex (DVC) in response to ZD7288 (1 mg/kg, i. p.). Scale bar, 100 μm. (B, D) Representative single filed images (20x) show c-Fos expression evoked by ZD7288 observed in the DVC emetic nuclei, the area postrema (AP), the nucleus tractus solitarius (NTS) and the dorsal motor nucleus of the vagus (DMNX), within the brainstem. Scale bar, 200 μm. (E–H) Representative images (20x) show low c-Fos expression induced by ZD7288 in the enteric nervous system (ENS) of the intestinal jejunum. Scale bar, 100 μm.

FIGURE 5

Quantified data for ZD7288-induced c-Fos expression in the least shrew brainstem dorsal vagal complex, containing the area postrema (AP), the nucleus tractus solitarius (NTS) and the dorsal motor nucleus of the vagus (DMNX), as well as the enteric nervous system (ENS) embedded in the wall of jejunum. Values represent the mean number of c-Fos positive cells in each region of interest per section and are presented as mean ± SEM (n = 4 shrews per group). *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control (treated with vehicle of ZD7288), Unpaired t-test.

FIGURE 6

Immunohistochemical analysis of c-Fos expression in NeuN-positive neurons of ENS. Co-staining of jejunum sections from ZD7288 (1 mg/kg, i. p.)-treated shrews with rabbit c-Fos and mouse anti-NeuN antibodies followed by Alexa Fluor 594 donkey anti-rabbit and 488 donkey anti-mouse secondary antibodies. NeuN, a neuronal marker of intrinsic primary afferent neurons of the enteric nervous system. Nuclei were stained with DAPI in blue. (A–D) Representative images (60x) show c-Fos expression seen in neurons of the enteric nervous system (ENS), which is embedded in the lining of the intestine. Scale bar, 100 μm.

FIGURE 7

ZD7288 increases c-Fos expression of serotonin neurons in the least shrew brainstem dorsal vagal complex. (A–F) Tryptophan hydroxylase 2 (TPH2) is the rate-limiting enzyme in the synthesis of neuronal serotonin. Representative images (20x) showing anti- TPH2, anti-c-Fos and merged immunofluorescence staining in vehicle controls and shrews administered with ZD7288 (1 mg/kg, i. p.) (n = 3 shrews per group). Scale bars = 100 μm. (G–R) Representative higher magnification images (×60) showing c-Fos expressing evoked by ZD7288 (1 mg/kg, i. p.) localized in TPH2 positive serotonin neurons of the brainstem dorsal vagal complex, composed of the area postrema (AP), the nucleus tractus solitarius (NTS) and the dorsal motor nucleus of the vagus (DMNX). Scale bars = 10 μm.

FIGURE 8

Immunohistochemical analysis of ERK1/2 phosphorylation following the HCN channel blocker ZD7288-induced emesis. Least shrews were sacrificed 15 min after vehicle or ZD7288 injection (1 mg/kg, i. p.) (n = 3 shrews per group). Brainstem sections (20 μm) and intestinal jejunum sections (25 μm) were stained with rabbit anti-phospho-ERK1/2 antibody and Alexa Fluor 594 donkey anti-rabbit secondary antibody. Nuclei were stained with DAPI in blue. (A, C) Representative tile scan images show a strong upregulation of ERK1/2 phosphorylation (pERK) in the brainstem dorsal vagal complex (DVC) in response to ZD7288. Scale bar, 200 μm. (B, D) Representative images show ERK1/2 phosphorylation evoked by ZD7288 seen in the DVC throughout three emetic nuclei, the area postrema (AP), the nucleus tractus solitarius (NTS) and the dorsal motor nucleus of the vagus (DMNX). Scale bar, 100 μm. (E) Statistical analysis of integrated density of ERK1/2 phosphorylation evoked by ZD7288 (1 mg/kg, i. p.) in the brainstem dorsal vagal complex. **p < 0.01 vs. Control, Unpaired t-test.

FIGURE 9

Effect of ERK1/2 inhibitor U0126 on the HCN channel blocker ZD7288-induced emesis. Different groups of shrews were given vehicle or varying doses of the ERK1/2 inhibitor U0126 (i.p.) (n = 7 shrews per group), 30 min prior to ZD7288 (1 mg/kg, i. p.) administration. Shrews were observed the next 30 min. (A) The frequency of emesis was analyzed with Kruskal-Wallis non-parametric one-way ANOVA followed by Dunnett’s post hoc test and presented as mean ± SEM. (B) Percentage of shrews vomiting was analyzed with chi-square test and presented as mean. ***p < 0.001 vs. 0 mg/kg.

FIGURE 10

Effects of modulators of cell membrane Ca²⁺ channels on the HCN channel blocker ZD7288-induced emesis. Different groups of least shrews were given an injection of either the corresponding vehicle, or varying doses of: 1) The L-type Ca²⁺ channel (LTCC) inhibitor nifedipine (s.c.) (n = 6 shews per group); 2) the TRPV1R agonist resiniferatoxin (RTX) (s.c.) (n = 8); 3) store-operated Ca²⁺ entry blocker MRS 1845 (i.p.) (n = 8); 4) T-type Ca²⁺ channel inhibitor Z944 (i.p.) (n = 9), 30 min prior to ZD7288 injection (1 mg/kg, i. p.). Emetic parameters were recorded for the next 30 min (A, C, E, G) The frequency of emesis was analyzed with Kruskal-Wallis non-parametric one-way ANOVA followed by Dunnett’s post hoc test and presented as mean ± SEM. (B, D, F, H) Percentage of shrews vomiting was analyzed with chi-square test and presented as mean. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. 0 mg/kg.

FIGURE 11

Effects of intracellular Ca²⁺ channel modulators on the HCN channel blocker ZD7288-induced emesis. Thirty minutes prior to an injection of ZD7288 (1 mg/kg, i. p.), different groups of least shrews were given an injection (i.p.) of either the corresponding vehicle, or varying doses of: 1) the ryanodine receptor (RyR) antagonist dantrolene (n = 8 shrews per group) (A, B), and 2) the inositol-1, 4, 5-triphosphate receptor IP₃R antagonist 2-APB (n = 6) (C, D). Emetic parameters were recorded for the next 30 min post ZD8288 injection. (A, C) The frequency of emesis was analyzed with Kruskal-Wallis non-parametric one-way ANOVA followed by Dunnett’s post hoc test and presented as mean ± SEM. (B, D) Percentage of shrews vomiting was analyzed with chi-square test and presented as mean. *p < 0.05, **p < 0.01, ****p < 0.0001 vs. 0 mg/kg.

FIGURE 12

Efficacy of receptor-selective antiemetics against the HCN channel blocker ZD7288-induced emesis. Different groups of least shrews were given an injection of either the corresponding vehicles (0 mg/kg), or varying doses of 5-HT₃R antagonist palonosetron (s.c.) (n = 6 shrews per group) (A and B), NK₁R antagonist netupitant (i.p.) (n = 6) (C and D), or the D_2/3R antagonist sulpride (s.c.) (n = 6) (E and F), 30 min prior to ZD7288 administration (1 mg/kg, i. p.). Emetic parameters were recorded for the next 30 min (A, C) The frequency of emesis was analyzed with Kruskal-Wallis non-parametric one-way ANOVA followed by Dunnett’s post hoc test and presented as mean ± SEM. (E) The frequency of emesis was analyzed with Unpaired t-test and presented as mean ± SEM. (B, D, F) Percentage of shrews vomiting was analyzed with chi-square test and presented as mean. *p < 0.05, **p < 0.01, ***p < 0.001 vs. 0 mg/kg.