Modulation of intracellular calcium activity in interstitial cells of Cajal by inhibitory neural pathways within the internal anal sphincter.

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 present 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 nitric oxide synthase (NOS) inhibitor N^G-nitro-l-arginine (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 inositol (1,4,5)-trisphosphate receptor-associated cGMP kinase substrate I (IRAG1) is expressed in ICC-IM, it is possible that it participates in the inhibition of Ca²⁺ release by nitric oxide. Platelet-derived growth factor receptor α (PDGFRα)⁺ cells but not ICC-IM expressed P2Y₁ receptors (P2Y₁R) and small-conductance Ca²⁺-activated K⁺ channels (SK3), suggesting that the purinergic pathway indirectly blocks whole cell Ca²⁺ transients in Type II ICC-IM via PDGFRα⁺ 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 PDGFRα⁺ cells via the SIP syncytium.NEW & NOTEWORTHY Two intramuscular interstitial cells of Cajal (ICC-IM) subtypes exist within the internal anal sphincter (IAS). This study provides the first evidence for direct coupling between nitrergic motor neurons and both ICC-IM subtypes as well as indirect coupling between purinergic inputs and Type II ICC-IM. The spatiotemporal properties of whole cell Ca²⁺ transients in Type II ICC-IM mimic those of smooth muscle cells (SMCs), suggesting that ICC-IM modulate the activity of SMCs via their joint participation in a SIP syncytium (SMCs, ICC, and PDGFRα⁺ cells).