Can colonic migrating motor complexes occur in mice lacking the endothelin-3 gene?

In mammals, colonic migrating motor complexes (CMMC) are a major propulsive contraction responsible for the expulsion of faecal content. Mice with a mutation of the endothelin-3 gene raised on a 129SL background strain have ~70% colonic aganglionosis, lack CMMC, and are lethal within 12 days postpartum. In contrast, endothelin-3 mutant mice raised and maintained on a C57BL6 background strain (lethal-spotted (ls/ls) mice) can live for much longer, but it is unclear whether CMMC generation is preserved in these mice also lacking the endothelin-3 gene. The aim of this study was to determine whether CMMC exist in ls/ls mouse colon and, if so, whether their existence and frequency are related to the length of aganglionosis. Spatiotemporal mapping and mechanical recordings of colonic wall movements were made from isolated whole colons obtained from wild-type and ls/ls mice. Although ls/ls mice had a megacolon, they still generated CMMC in the ganglionic segment, which on some occasions could propagate short distances into the aganglionic region. There was large variability in aganglionosis length, which showed a weak correlation with the existence or frequency of CMMC. Interestingly, CMMC propagation velocity was slower in ls/ls mice when evoked by intraluminal fluid. A myogenic motor pattern was identified in the aganglionic region that was maintained under tonic inhibition. We show that despite megacolon, ls/ls mice still generate CMMC in the ganglionic region. These offspring have sufficient propulsive motility in the ganglionic segment to live a normal murine lifespan and rarely die of bowel obstruction.

Loss of visceral pain following colorectal distension in an endothelin-3 deficient mouse model of Hirschsprung's disease.

Endothelin peptides and their endogenous receptors play a major role in nociception in a variety of different organs. They also play an essential role in the development of the enteric nervous system. Mice with deletions of the endothelin-3 gene (lethal spotted mice, ls/ls) develop congenital aganglionosis. However, little is known about how nociception might be affected in the aganglionic rectum of mice deficient in endothelin-3. In this study we investigated changes in spinal afferent innervation and visceral pain transmission from the aganglionic rectum in ls/ls mice. Electromyogram recordings from anaesthetized ls/ls mice revealed a deficit in visceromotor responses arising from the aganglionic colorectum in response to noxious colorectal distension. Loss of visceromotor responses (VMRs) in ls/ls mice was selective, as no reduction in VMRs was detected after stimulation of the bladder or somatic organs. Calcitonin gene related peptide (CGRP) immunoreactivity, retrograde neuronal tracing and extracellular afferent recordings from the aganglionic rectum revealed decreased colorectal spinal innervation, combined with a reduction in mechanosensitivity of rectal afferents. The sensory defect in ls/ls mice is primarily associated with changes in low threshold wide dynamic range rectal afferents. In conclusion, disruption of endothelin 3 gene expression not only affects development and function of the enteric nervous system, but also specific classes of spinal rectal mechanoreceptors, which are required for visceral nociception from the colorectum.

Benzalkonium chloride-treated anorectums mimicked endothelin-3-deficient aganglionic anorectums on manometry.

BACKGROUND/PURPOSE: The anorectal spasticity in Hirschsprung disease may be caused by the absence of enteric ganglia and/or the presence of hypertrophic nerves. Anorectal manometry of chemically denervated rectums was compared with that of congenital aganglionic rectums that also possessed hypertrophic nerves. METHODS: Aganglionic and ganglionic littermates were produced from breeding heterozygous lethal-spotted mice. Benzalkonium chloride was endorectally injected into ganglionic rectums to ablate the neural elements. Anorectal manometry was performed before the injection and on day 14 postinjection. The anorectal resting pressure was calculated based on the manometric tracing. Rectums were retrieved on day 14 for histologic evaluations. RESULTS: Benzalkonium chloride injection successfully ablated the rectal ganglia. Although ganglionic littermates exhibited regular slow waves on anorectal manometry, aganglionic lethal-spotted mice showed irregular waves. Similar to lethal spotted mice, benzalkonium chloride-treated mice exhibited significantly higher anorectal resting pressure than that of ganglionic mice. The slow waves were absent in benzalkonium chloride-treated mice. CONCLUSION: Benzalkonium chloride treatment produced aganglionic rectums that had higher resting pressure similar to the congenital aganglionic rectums. This suggests that hypertrophic nerves in congenital aganglionosis are not necessary to produce the anorectal spasticity.

Cooperative and indispensable roles of endothelin 3 and KIT signalings in melanocyte development.

The development of melanocytes from neural crest-derived precursor cells depends on signaling by the receptor tyrosine kinase KIT and the G protein-coupled endothelin receptor B (EDNRB) pathways. Loss-of-function mutations in either of these two signaling receptor molecules cause a loss or a marked reduction in the number of melanocyte precursors in the embryo and finally lead to loss of the coat color. Using cultures of embryonic stem (ES) cells to induce melanocyte differentiation in vitro, we investigated the requirement for EDNRB signaling during the entire developmental process of the melanocyte, in association with that for KIT signaling. During the 21-day period necessary for the induction of mature melanocytes from undifferentiated ES cells, endothelin 3 (EDN3), a ligand for EDNRB, increased the number of melanocytes in proportion to the period during which it was present. We tested the compensatory effect of EDNRB signaling on KIT signaling in vivo by using Kit(W-LacZ)/Kit(W-LacZ) ES cells and confirmed that the ectopic expression of EDN3 in the skin reduced the white spotting of Kit(W57)/Kit(W57)mice. KIT ligand (KITL) and EDN3 worked synergistically to induce melanocyte differentiation in vitro; however, the complete lack of EDNRB signaling attained by the use of EDN3-/- ES cells and an EDNRB antagonist, BQ788, revealed that the resulting failure of melanocyte development was not compensated by the further activation of KIT signaling by adding KITL. Simultaneous blockade of EDNRB and KIT signalings eliminated melanocyte precursors completely, suggesting that the maintenance or survival of early melanocyte precursors at least required the existence of either EDNRB or KIT signalings.

Normal ventilation and ventilatory responses to chemical stimuli in juvenile mutant mice deficient in endothelin-3.

Congenital central hypoventilation syndrome (CCHS) and Hirschsprung's disease (HSCR) are often classified as neurocristopathies and are thought to share a common molecular pathogenesis related to the genes that control the development of neural crest cells. We examined whether endothelin-3 (ET-3), one of the developmental regulators of neural crest cells and of which null mutation results in aganglionic megacolon in mice, fulfills the requirements for such a common molecule. To investigate the possible involvement of ET-3 in central ventilatory control, we measured ventilation in mutant mice deficient in ET-3 by whole body plethysmography. Tidal volume and breathing frequency were measured during breathing of room air, hypoxic, hyperoxic, or hypercapnic gas mixtures in awake and anesthetized mice. There were no significant differences in resting ventilation as well as ventilatory responses to hypoxia and hypercapnia between ET-3-knockout mice and wild-type mice. Our results indicate that ET-3 can not be considered as a common pathogenic mechanism for CCHS and HSCR at least in mice.