Intracellular Emetic Signaling Cascades by Which the Selective Neurokinin Type 1 Receptor (NKR) Agonist GR73632 Evokes Vomiting in the Least Shrew (Cryptotis Parva)

To characterize mechanisms involved in neurokinin type 1 receptor (NKR)-mediated emesis, we investigated the brainstem emetic signaling pathways following treating least shrews with the selective NKR 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 Ca/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 NKR antagonist netupitant (i.p.); ii) the L-type Ca channel (LTCC) antagonist nifedipine (subcutaneous, s.c.); iii) the inositol trisphosphate receptor (IPR) antagonist 2-APB (i.p.); iv) store-operated Ca 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, NKR, LTCC, and IPR 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/IPR-dependent PI3K/PKCα/βII-ERK1/2 signaling pathways are involved in NKR-mediated vomiting.