Zhi S Li1, Lin Y Hung2, Kara G Margolis2, Richard T Ambron1, Ying J Sung3, Michael D Gershon1. 1. Departments of Pathology & Cell Biology, Columbia University, New York, NY, USA. 2. Departments of Pediatrics, Columbia University, New York, NY, USA. 3. Departments of Basic Science, The Commonwealth Medical College, Scranton, PA, USA.
Abstract
BACKGROUND: Intrinsic primary afferent neurons (IPANs) enable the gut to manifest reflexes in the absence of CNS input. PKG1α is selectively expressed in a subset of neurons in dorsal root ganglia (DRG) and has been linked to nociception and long-term hyperexcitability. METHODS: We used immunoblotting, immunocytochemistry, and in vitro assays of IPAN-dependent enteric functions to test hypotheses that subsets of primary neurons of the ENS and DRG share a reliance on PKG1α expression. KEY RESULTS: PKG1α immunoreactivity was demonstrated in immunoblots from isolated myenteric ganglia. PKG1α, but not PKG1β, immunoreactivity, was coincident with that of neuronal markers (HuC/D; β3-tubulin) in both enteric plexuses. PKG1α immunoreactivity also co-localized with the immunoreactivities of the IPAN markers, calbindin (100%; myenteric plexus) and cytoplasmic NeuN (98 ± 1% submucosal plexus). CGRP-immunoreactive DRG neurons, identified as visceral afferents by retrograde transport, were PKG1α-immunoreactive. We used intraluminal cholera toxin to determine whether PKG1α was necessary to enable stimulation of the mucosa to activate Fos in enteric neurons. Tetrodotoxin (1.0 µM), low Ca2+ /high Mg2+ media, and the PKG inhibitor, N46 (100 µM), all inhibited Fos activation in myenteric neurons. N46 also concentration dependently inhibited peristaltic reflexes in isolated preparations of distal colon (IC50 = 83.3 ± 1.3 µM). CONCLUSIONS & INFERENCES: These data suggest that PKG1α is present and functionally important in IPANs and visceral afferent nociceptive neurons.
BACKGROUND: Intrinsic primary afferent neurons (IPANs) enable the gut to manifest reflexes in the absence of CNS input. PKG1α is selectively expressed in a subset of neurons in dorsal root ganglia (DRG) and has been linked to nociception and long-term hyperexcitability. METHODS: We used immunoblotting, immunocytochemistry, and in vitro assays of IPAN-dependent enteric functions to test hypotheses that subsets of primary neurons of the ENS and DRG share a reliance on PKG1α expression. KEY RESULTS: PKG1α immunoreactivity was demonstrated in immunoblots from isolated myenteric ganglia. PKG1α, but not PKG1β, immunoreactivity, was coincident with that of neuronal markers (HuC/D; β3-tubulin) in both enteric plexuses. PKG1α immunoreactivity also co-localized with the immunoreactivities of the IPAN markers, calbindin (100%; myenteric plexus) and cytoplasmic NeuN (98 ± 1% submucosal plexus). CGRP-immunoreactive DRG neurons, identified as visceral afferents by retrograde transport, were PKG1α-immunoreactive. We used intraluminal cholera toxin to determine whether PKG1α was necessary to enable stimulation of the mucosa to activate Fos in enteric neurons. Tetrodotoxin (1.0 µM), low Ca2+ /high Mg2+ media, and the PKG inhibitor, N46 (100 µM), all inhibited Fos activation in myenteric neurons. N46 also concentration dependently inhibited peristaltic reflexes in isolated preparations of distal colon (IC50 = 83.3 ± 1.3 µM). CONCLUSIONS & INFERENCES: These data suggest that PKG1α is present and functionally important in IPANs and visceral afferent nociceptive neurons.