Submitted on November 9, 2007
Accepted on February 22, 2008
Stimulation of the neurokinin 3 receptor activates protein kinase C
and protein kinase D in enteric neurons
Daniel Philip Poole1*, Silvia Amadesi2, Enrique Rozengurt3, Michelle Thacker4, Nigel W. Bunnett2, and John B Furness4
1 Surgery and Physiology, University of California-San Francisco, San Francisco, California, United States; Anatomy & Cell Biology and Centre for Neuroscience, University of Melbourne, Parkville, Victoria, Australia
2 Surgery and Physiology, University of California-San Francisco, San Francisco, California, United States
3 Medicine, UCLA School of Medicine, Los Angeles, California, United States
4 Anatomy & Cell Biology and Centre for Neuroscience, University of Melbourne, Parkville, Victoria, Australia
* To whom correspondence should be addressed. E-mail: PooleD{at}surgery.ucsf.edu.
Tachykinins, acting through NK3 receptors (NK3R), contribute to excitatory transmission to intrinsic primary afferent neurons (IPANs) of the small intestine. Although this transmission is protein kinase C (PKC)-dependent, its maintenance could depend on protein kinase D (PKD), a downstream target of PKC. Here we show that PKD1/2-immunoreactivity occurred exclusively in intrinsic primary afferent neurons (IPANs) of the guinea-pig ileum. PKC
was colocalized with PKD1/2 in IPANs. PKC and PKD1/2 trafficking was studied in myenteric neurons. In untreated preparations, PKC and PKD1/2 were cytosolic and no signal for activated (phosphorylated) PKD was detected. The NK3R-agonist senktide evoked a transient translocation of PKC and PKD1/2 from the cytosol to the plasma membrane, and induced PKD1/2 phosphorylation at the plasma membrane. PKC translocation was maximal at 10s and returned to the cytosol within 2min. Phosphorylated-PKD1/2 was detected at the plasma membrane within 15s, and translocated to the cytosol by 2min, where it remained active up to 30min after NK3R stimulation. PKD1/2 activation was reduced by a PKC inhibitor and prevented by NK3R inhibition. NK3R-mediated PKC and PKD activation was confirmed in HEK293 cells transiently expressing NK3R and GFP-tagged PKC, PKD1, PKD2 or PKD3. Senktide caused membrane translocation and activation of kinases within 30s. After 15min, phosphorylated PKD had returned to the cytosol. PKD activation was confirmed through Western blotting. Thus, stimulation of NK3R activates PKC and PKD in sequence, and sequential activation of these kinases may account for rapid and prolonged modulation of IPAN function.
