Intracellular emetic signaling cascades by which the selective neurokinin type 1 receptor (NK1R) agonist GR73632 evokes vomiting in the least shrew (Cryptotis parva)

Abstract

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.

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

“…These results are in line with previous findings where i.p. injection of the selective NK 1 R agonist GR73632 (5 mg/kg) time-dependently upregulated phosphorylation of emesis-associated protein kinases including Akt in shrew brainstem (Zhong et al, 2019). Our study is also consistent with diverse models of cells in culture, showing that stimulation of dopamine D 2 receptor and/ or D 3 receptor rapidly activates Akt signaling (Brami-Cherrier et al, 2002;Mannoury la Cour et al, 2011).…”

“…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 D 2 receptorselective antagonist (8 mg/kg, s.c., N = 14-15 within each group) (Darmani et al, 1999); ii) LY294002, a potent PI3K inhibitor (0, 1, 2.5, and 10 mg/kg, i.p., N = 6-8 within each group) (Xiao et al, 2018); iii) GF109203X, a PKC blocker selectively targeting PKCαβII (0, 5, 10, and 20 mg/kg, i.p., N = 12 within each group) (Asehnoune et al, 2005); 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 et al, 2019); 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.)…”

“…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 and Darmani, 2020;Zhong et al, 2019;Zhong et al, 2014b) and homogenized in lysis buffer. Protein extracts from brainstem lysates were subjected to Western blot.…”

“…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 FPL64176stimulated emesis (Zhong et al, 2019), and suppresses vomiting caused by a variety of other emetogens including those produced by i.p. administration of dopamine D 2/3 receptor agonists such as apomorphine (2 mg/kg) or quinpirole (2 mg/kg) (Darmani et al, 2014;Zhong et al, 2014a).…”

“…Signaling cascades driven by the stimulation of the dopamine D 2 -like receptors are either scantly defined or remain unknown in emesis (Belkacemi and Darmani, 2020). Recent emesis related signaling studies from our group have demonstrated that: i) dopamine D 2 receptorevoked 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 and Darmani, 2020); ii) vomiting mediated by activation of the SP neurokinin NK 1 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 et al, 2019); and finally, iii) the L-type Ca 2+ channel (LTCC)/inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP 3 R)-dependent PI3K (phosphoinositide 3-kinase)/Akt and PKCαβII/ERK1/2 signaling pathways are also involved in NK 1 Rmediated vomiting (Zhong et al, 2019). Collectively, these data illustrate the importance of PI3K/Akt and PKCαβII/ERK1/2 signaling mechanisms in emesis.…”