ABSTRACT
Interstitial cells of Cajal (ICC) and PDGFRα+ cells regulate smooth muscle motility in the gastrointestinal (GI) tract. However, their role(s) in esophageal motility are still unclear. The mouse esophagus has traditionally been described as almost entirely skeletal muscle in nature though ICC have been identified along its entire length. The current study evaluated the distribution of skeletal and smooth muscle within the esophagus using a mouse selectively expressing eGFP in smooth muscle cells (SMCs). The relationship of SMCs to ICC and PDGFRα+ cells was also examined. SMCs declined in density in the oral direction however SMCs represented ~ 25% of the area in the distal esophagus suggesting a likeness to the transition zone observed in humans. ANO1+ intramuscular ICC (ICC-IM) were distributed along the length of the esophagus though like SMCs, declined proximally. ICC-IM were closely associated with SMCs but were also found in regions devoid of SMCs. Intramuscular and submucosal PDGFRα+ cells were densely distributed throughout the esophagus though only intramuscular PDGFRα+ cells within the LES and distal esophagus highly expressed SK3. ICC-IM and PDGFRα+ cells were closely associated with nNOS+, VIP+, VAChT+ and TH+ neurons throughout the LES and distal esophagus. GFAP+ cells resembling intramuscular enteric glia were observed within the muscle and were closely associated with ICC-IM and PDGFRα+ cells, occupying a similar location to c. These data suggest that the mouse esophagus is more similar to the human than thought previously and thus set the foundation for future functional and molecular studies using transgenic mice.
ABSTRACT
The internal anal sphincter (IAS) functions to maintain continence. Previous studies utilizing mice with cell-specific expression of GCaMP6f revealed two distinct subtypes of intramuscular interstitial cells of Cajal (ICC-IM) with differing Ca2+ activities in the IAS. The current study further examined Ca2+ activity in ICC-IM and its modulation by inhibitory neurotransmission. The spatiotemporal properties of Ca2+ transients in Type II ICC-IM mimicked those of smooth muscle cells (SMCs) indicating their joint participation in the "SIP" syncytium. Electrical field stimulation (EFS; atropine present) abolished localized and whole-cell Ca2+ transients in Type I and II ICC-IM. The purinergic antagonist MRS2500 did not abolish EFS responses in either cell type whereas the NOS inhibitor L-NNA abolished responses in Type I but not Type II ICC-IM. Combined antagonists abolished EFS responses in Type II ICC-IM. In both ICC-IM subtypes, the ability of EFS to inhibit Ca2+ release was abolished by L-NNA, but not MRS2500 suggesting that the nitrergic pathway directly inhibits ICC-IM by blocking Ca2+ release from intracellular stores. Since IRAG1 is expressed in ICC-IM it is possible that it participates in the inhibition of Ca2+ release by nitric oxide. PDGFRᵯC+ cells but not ICC-IM expressed P2Y1R and SK3 suggesting that the purinergic pathway indirectly blocks whole-cell Ca2+ transients in Type II ICC-IM via PDGFRᵯC+ cells. This study provides the first direct evidence for functional coupling between inhibitory motor neurons and ICC-IM subtypes in the IAS with contractile inhibition ultimately dependent upon electrical coupling between SMCs, ICC and PDGFRᵯC+ cells via the SIP syncytium.
ABSTRACT
BACKGROUND: The internal anal sphincter (IAS) exhibits slow waves (SWs) and tone that are dependent upon L-type Ca2+ channels (CavL ) suggesting that phasic events (ie, SWs) play a fundamental role in tone generation. The present study further examined phasic activity in the IAS by measuring the spatiotemporal properties of Ca2+ transients (CTs) in IAS smooth muscle cells (SMCs). METHODS: Ca2+ transients were recorded with spinning disk confocal microscopy from the IAS of SM-GCaMP mice. Muscles were pinned submucosal surface up at two different lengths. Drugs were applied by inclusion in the superfusate. KEY RESULTS: Ca2+ transients displayed ongoing rhythmic firings at both lengths and were abolished by nifedipine and the KATP channel activator pinacidil indicating their dependence upon CavL . Like SWs, CTs were greatest in frequency (average 70.6 cpm) and amplitude at the distal extremity and conducted proximally. Removal of the distal IAS reduced but did not abolish CTs. The time constant for clearing cytoplasmic Ca2+ averaged 0.46 seconds and basal Ca2+ levels were significantly elevated. CONCLUSIONS & INFERENCES: The similarities in spatiotemporal and pharmacological properties of CTs and SWs suggest that SW gives rise to CTs while muscle stretch is not required. Elevated relative basal Ca2+ in the IAS is likely due to the inability of cells to clear or sequester Ca2+ between rapid frequency voltage-dependent Ca2+ entry events, that is, conditions that will lead to tone development. The conduction of CTs from distal to proximal IAS will lead to orally directed contractions and likely contribute to the maintenance of fecal continence.
