Stimulation of the neurokinin 3 receptor activates protein kinase Cε and protein kinase D in enteric neurons

Tachykinins, acting through NK₃ receptors (NK₃R), contribute to excitatory transmission to intrinsic primary afferent neurons (IPANs) of the small intestine. Although this transmission is dependent on protein kinase C (PKC), its maintenance could depend on protein kinase D (PKD), a downstream target of PKC. Here we show that PKD1/2-immunoreactivity occurred exclusively in IPANs of the guinea pig ileum, demonstrated by double staining with the IPAN marker NeuN. PKCε was also colocalized with PKD1/2 in IPANs. PKCε and PKD1/2 trafficking was studied in enteric neurons within whole mounts of the ileal wall. In untreated preparations, PKCε and PKD1/2 were cytosolic and no signal for activated (phosphorylated) PKD was detected. The NK₃R agonist senktide evoked a transient translocation of PKCε and PKD1/2 from the cytosol to the plasma membrane and induced PKD1/2 phosphorylation at the plasma membrane. PKCε translocation was maximal at 10 s and returned to the cytosol within 2 min. Phosphorylated-PKD1/2 was detected at the plasma membrane within 15 s and translocated to the cytosol by 2 min, where it remained active up to 30 min after NK₃R stimulation. PKD1/2 activation was reduced by a PKCε inhibitor and prevented by NK ₃R inhibition. NK₃R-mediated PKCε and PKD activation was confirmed in HEK293 cells transiently expressing NK₃R and green fluorescent protein-tagged PKCε, PKD1, PKD2, or PKD3. Senktide caused membrane translocation and activation of kinases within 30 s. After 15 min, phosphorylated PKD had returned to the cytosol. PKD activation was confirmed through Western blotting. Thus stimulation of NK₃R activates PKCε and PKD in sequence, and sequential activation of these kinases may account for rapid and prolonged modulation of IPAN function.