Nitrergic neuromuscular transmission in the mouse internal anal sphincter is accomplished by multiple pathways and postjunctional effector cells

Abstract

The effector cells and second messengers participating in nitrergic neuromuscular transmission (NMT) were investigated in the mouse internal anal sphincter (IAS). Protein expression of guanylate cyclase (GCα, GCβ) and cyclic GMP-dependent protein kinase I (cGKI) were examined in cryostat sections with dual-labeling immunohistochemical techniques in PDGFRα(+) cells, interstitial cells of Cajal (ICC), and smooth muscle cells (SMC). Gene expression levels were determined with quantitative PCR of dispersed cells from Pdgfrα(egfp/+), Kit(copGFP/+), and smMHC(Cre-egfp) mice sorted with FACS. The relative gene and protein expression levels of GCα and GCβ were PDGFRα(+) cells > ICC ≫ SMC. In contrast, cGKI gene expression sequence was SMC = ICC > PDGFRα(+) cells whereas cGKI protein expression sequence was neurons > SMC ≫ ICC = PDGFRα(+) cells. The functional role of cGKI was investigated in cGKI(-/-) mice. Relaxation with 8-bromo (8-Br)-cGMP was greatly reduced in cGKI(-/-) mice whereas responses to sodium nitroprusside (SNP) were partially reduced and forskolin responses were unchanged. A nitrergic relaxation occurred with nerve stimulation (NS, 5 Hz, 60 s) in cGKI(+/+) and cGKI(-/-) mice although there was a small reduction in the cGKI(-/-) mouse. N(ω)-nitro-l-arginine (l-NNA) abolished responses during the first 20-30 s of NS in both animals. The GC inhibitor ODQ greatly reduced or abolished SNP and nitrergic NS responses in both animals. These data confirm an essential role for GC in NO-induced relaxation in the IAS. However, the expression of GC and cGKI by all three cell types suggests that each may participate in coordinating muscular responses to NO. The persistence of nitrergic NMT in the cGKI(-/-) mouse suggests the presence of a significant GC-dependent, cGKI-independent pathway.

Interstitial cell network volume is reduced in the terminal bowel of ageing mice

“…This region is of interest because the frequency and amplitude of slow waves has been reported to be greatest in the most distal region of the IAS 12. In the mouse IAS, ICC‐MY have been reported to be few in number, not form a network and to decline distally 12, 13, 14. In keeping with these previous studies, only a very small number of ICC‐MYs were observed in the ASR.…”

“…The results obtained here for the mouse terminal bowel are therefore in line with previous reports on other parts of the GI tract in two other species, indicating that a reduction in ICC network volume during ageing may be a general phenomenon. Evidence that ICC play a critical role in regulation of smooth muscle contractility is now widely accepted15 including in the terminal bowel 13. These results therefore suggest that changes in ICCs during ageing described here may be an important factor in the physiological changes observed in this region.…”

Cell‐specific effects of nitric oxide on the efficiency and frequency of long distance contractions in murine colon

“…However, the detailed regulation of nitrergic neurotransmission is still incompletely understood. Previous studies already showed an intricate modulation of spontaneous contractions via NO‐GC in smooth muscle cells (SMC), interstitial cells of Cajal (ICC) as well as fibroblast‐like cells (FLC) . The nitrergic impact on long distance contractions (LDCs), the main propulsive motor pattern in mice, was still unclear.…”

Changes in murine anorectum signaling across the life course

“…Previous work has shown that the young murine IAS contracts following stimulation of excitatory cholinergic neurons, the release of acetylcholine (ACh) and activation of both M2 and M3 receptors which increase intracellular Ca 2+ and reduce cAMP, respectively, prolonging the activity of myosin light chain kinase . For appropriate defecation, the young IAS is relaxed by a combination of inhibitory neurotransmitters, including nitric oxide, ATP (or another purine) and vasoactive intestinal peptide (VIP) . There is some evidence for the co‐localization of the inhibitory neurotransmitters in specific motor neurons in both mouse and human gastrointestinal tissue .…”

“…There is some evidence for the co‐localization of the inhibitory neurotransmitters in specific motor neurons in both mouse and human gastrointestinal tissue . The release of these inhibitory transmitters is frequency dependent with purinergic transmitters released at the lowest firing frequency, followed by NO and then VIP as the neuronal firing frequencies increase . In the mouse, release of these neurotransmitters typically hyperpolarizes IAS circular smooth muscle cells, closing voltage gated calcium channels and reducing intracellular calcium .…”