Comparison of inhibitory neuromuscular transmission in the Cynomolgus monkey IAS and rectum: special emphasis on differences in purinergic transmission.

KEY POINTS: Inhibitory neuromuscular transmission (NMT) was compared in the internal anal sphincter (IAS) and rectum of the Cynomolgus monkey, an animal with high gene sequence identity to humans. Nitrergic NMT was present in both muscles while purinergic NMT was limited to the rectum and VIPergic NMT to the IAS. The profile for monkey IAS more closely resembles humans than rodents. In both muscles, SK3 channels were localized to PDGFRα+ cells that were closely associated with nNOS+ /VIP+ nerves. Gene expression levels of P2RY subtypes were the same in IAS and rectum while KCNN expression levels were very similar. SK3 channel activation and inhibition caused faster/greater changes in contractile activity in rectum than IAS. P2Y1 receptor activation inhibited contraction in rectum while increasing contraction in IAS. The absence of purinergic NMT in the IAS may be due to poor coupling between P2Y1 receptors and SK3 channels on PDGFRα+ cells. ABSTRACT: Inhibitory neuromuscular transmission (NMT) was compared in the internal anal sphincter (IAS) and rectum of the Cynomolgus monkey, an animal with a high gene sequence identity to humans. Electrical field stimulation produced nitric oxide synthase (NOS)-dependent contractile inhibition in both muscles whereas P2Y1-dependent purinergic NMT was restricted to rectum. An additional NOS-independent, α-chymotrypsin-sensitive component was identified in the IAS consistent with vasoactive intestinal peptide-ergic (VIPergic) NMT. Microelectrode recordings revealed slow NOS-dependent inhibitory junction potentials (IJPs) in both muscles and fast P2Y1-dependent IJPs in rectum. The basis for the difference in purinergic NMT was investigated. PDGFRα+ /SK3+ cells were closely aligned with nNOS+ /VIP+ neurons in both muscles. Gene expression of P2RY was the same in IAS and rectum (P2RY1>>P2RY2-14) while KCNN3 expression was 32% greater in rectum. The SK channel inhibitor apamin doubled contractile activity in rectum while having minimal effect in the IAS. Contractile inhibition elicited with the SK channel agonist CyPPA was five times faster in rectum than in the IAS. The P2Y1 receptor agonist MRS2365 inhibited contraction in rectum but increased contraction in the IAS. In conclusion, both the IAS and the rectum have nitrergic NMT whereas purinergic NMT is limited to rectum and VIPergic NMT to the IAS. The profile in monkey IAS more closely resembles that of humans than rodents. The lack of purinergic NMT in the IAS cannot be attributed to the absence of PDGFRα+ cells, P2Y1 receptors or SK3 channels. Rather, it appears to be due to poor coupling between P2Y1 receptors and SK3 channels on PDGFRα+ cells.

ANO1 in intramuscular interstitial cells of Cajal plays a key role in the generation of slow waves and tone in the internal anal sphincter.

KEY POINTS: The internal anal sphincter develops tone important for maintaining high anal pressure and continence. Controversy exists regarding the mechanisms underlying tone development. We examined the hypothesis that tone depends upon electrical slow waves (SWs) initiated in intramuscular interstitial cells of Cajal (ICC-IM) by activation of Ca2+ -activated Cl- channels (ANO1, encoded by Ano1) and voltage-dependent L-type Ca2+ channels (CavL , encoded by Cacna1c). Measurement of membrane potential and contraction indicated that ANO1 and CavL have a central role in SW generation, phasic contractions and tone, independent of stretch. ANO1 expression was examined in wildtype and Ano1/+egfp mice with immunohistochemical techniques. Ano1 and Cacna1c expression levels were examined by quantitative PCR in fluorescence-activated cell sorting. ICC-IM were the predominant cell type expressing ANO1 and the most likely candidate for SW generation. SWs in ICC-IM are proposed to conduct to smooth muscle where Ca2+ entry via CavL results in phasic activity that sums to produce tone. ABSTRACT: The mechanism underlying tone generation in the internal anal sphincter (IAS) is controversial. We examined the hypothesis that tone depends upon generation of electrical slow waves (SWs) initiated in intramuscular interstitial cells of Cajal (ICC-IM) by activation of Ca2+ -activated Cl- channels (encoded by Ano1) and voltage-dependent L-type Ca2+ channels (encoded by Cacna1c). Phasic contractions and tone in the IAS were nearly abolished by ANO1 and CavL antagonists. ANO1 antagonists also abolished SWs as well as transient depolarizations that persisted after addition of CavL antagonists. Tone development in the IAS did not require stretch of muscles, and the sensitivity of contraction to ANO1 antagonists was the same in stretched versus un-stretched muscles. ANO1 expression was examined in wildtype and Ano1/+egfp mice with immunohistochemical techniques. Dual labelling revealed that ANO1 expression could be resolved in ICC but not smooth muscle cells (SMCs) in the IAS and rectum. Ano1, Cacna1c and Kit gene expression were the same in extracts of IAS and rectum muscles. In IAS cells isolated with fluorescence-activated cell sorting, Ano1 expression was 26.5-fold greater in ICC than in SMCs while Cacna1c expression was only 2-fold greater in SMCs than in ICC. These data support a central role for ANO1 and CavL in the generation of SWs and tone in the IAS. ICC-IM are the probable cellular candidate for ANO1 currents and SW generation. We propose that ANO1 and CavL collaborate to generate SWs in ICC-IM followed by conduction to adjacent SMCs where phasic calcium entry through CavL sums to produce tone.

Spatial organization and coordination of slow waves in the mouse anorectum.

The internal anal sphincter (IAS) develops tone and is important for maintaining a high anal pressure while tone in the rectum is less. The mechanisms responsible for tone generation in the IAS are still uncertain. The present study addressed this question by comparing the electrical properties and morphology of the mouse IAS and distal rectum. The amplitude of tone and the frequency of phasic contractions was greater in the IAS than in rectum while membrane potential (Em) was less negative in the IAS than in rectum. Slow waves (SWs) were of greatest amplitude and frequency at the distal end of the IAS, declining in the oral direction. Dual microelectrode recordings revealed that SWs were coordinated over a much greater distance in the circumferential direction than in the oral direction. The circular muscle layer of the IAS was divided into five to eight 'minibundles' separated by connective tissue septa whereas few septa were present in the rectum. The limited coordination of SWs in the oral direction suggests that the activity in adjacent minibundles is not coordinated. Intramuscular interstitial cells of Cajal and platelet-derived growth factor receptor alpha-positive cells were present in each minibundle suggesting a role for one or both of these cells in SW generation. In summary, three important properties distinguish the IAS from the distal rectum: (1) a more depolarized Em; (2) larger and higher frequency SWs; and (3) the multiunit configuration of the muscle. All of these characteristics may contribute to greater tone generation in the IAS than in the distal rectum.

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

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, GCß) 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 GCß 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.

Functional role of vasoactive intestinal polypeptide in inhibitory motor innervation in the mouse internal anal sphincter.

There is evidence that vasoactive intestinal polypeptide (VIP) participates in inhibitory neuromuscular transmission (NMT) in the internal anal sphincter (IAS). However, specific details concerning VIP-ergic NMT are limited, largely because of difficulties in selectively blocking other inhibitory neural pathways. The present study used the selective P2Y1 receptor antagonist MRS2500 (1 µm) and the nitric oxide synthase inhibitor N(G)-nitro-l-arginine (l-NNA; 100 µm) to block purinergic and nitrergic NMT to characterize non-purinergic, non-nitrergic (NNNP) inhibitory NMT and the role of VIP in this response. Nerves were stimulated with electrical field stimulation (0.1-20 Hz, 4-60 s) and the associated changes in contractile and electrical activity measured in non-adrenergic, non-cholinergic conditions in the IAS of wild-type and VIP(-/-) mice. Electrical field stimulation gave rise to frequency-dependent relaxation and hyperpolarization that was blocked by tetrodotoxin. Responses during brief trains of stimuli (4 s) were mediated by purinergic and nitrergic NMT. During longer stimulus trains, an NNNP relaxation and hyperpolarization developed slowly and persisted for several minutes beyond the end of the stimulus train. The NNNP NMT was abolished by VIP6-28 (30 µm), absent in the VIP(-/-) mouse and mimicked by exogenous VIP (1-100 nm). Immunoreactivity for VIP was co-localized with neuronal nitric oxide synthase in varicose intramuscular fibres but was not detected in the VIP(-/-) mouse IAS. In conclusion, this study identified an ultraslow component of inhibitory NMT in the IAS mediated by VIP. In vivo, this pathway may be activated with larger rectal distensions, leading to a more prolonged period of anal relaxation.

Interstitial cells of Cajal in the cynomolgus monkey rectoanal region and their relationship to sympathetic and nitrergic nerves.

The morphology of interstitial cells of Cajal (ICC) in the circular muscle layer of the cynomolgus monkey internal anal sphincter (IAS) and rectum and their relationship to sympathetic and nitrergic nerves were compared by dual-labeling immunohistochemistry. Contractile studies confirmed that nitrergic nerves participate in neural inhibition in both regions whereas sympathetic nerves serve as excitatory motor nerves only in the IAS. Muscle bundles extended from myenteric to submucosal edge in rectum but in the IAS bundles were further divided into "minibundles" each surrounded by connective tissue. Dual labeling of KIT and smooth muscle myosin revealed KIT-positive stellate-shaped ICC (ICC-IAS) within each minibundle. In the rectum intramuscular ICC (ICC-IM) were spindle shaped whereas stellate-shaped ICC were located at the myenteric surface (ICC-MY). ICC were absent from both the myenteric and submucosal surfaces of the IAS. Nitrergic nerves (identified with anti-neuronal nitric oxide synthase antibodies or NADPH diaphorase activity) and sympathetic nerves (identified with anti-tyrosine hydroxylase antibody) each formed a plexus at the myenteric surface of the rectum but not the IAS. Intramuscular neuronal nitric oxide synthase- and tyrosine hydroxylase-positive fibers were present in both regions but were only closely associated with ICC-IM in rectum. Minimal association was also noted between ICC-IAS and cells expressing the nonspecific neuronal marker PGP9.5. In conclusion, the morphology of rectal ICC-IM and ICC-MY is similar to that described elsewhere in the gastrointestinal tract whereas ICC-IAS are unique. The distribution of stellate-shaped ICC-IAS throughout the musculature and their absence from both the myenteric and submucosal surfaces suggest that ICC-IAS may serve as pacemaker cells in this muscle whereas their limited relationship to nerves suggests that they are not involved in neuromuscular transmission. Additionally, the presence of numerous minibundles, each containing both ICC-IAS and nerves, suggests that this muscle functions as a multiunit type muscle.

Species dependent differences in the actions of sympathetic nerves and noradrenaline in the internal anal sphincter.

Excitatory motor innervation to the internal anal sphincter (IAS) of the monkey, the rabbit and mouse were compared. Contractile responses to electrical field stimulation of nerves (EFS, atropine 1 micromol L(-1) and N(omega)-nitro-L-arginine 100 micromol L(-1) present throughout) were examined in isolated strips of IAS. In the monkey IAS, EFS caused frequency dependent (1-30 Hz) contractions which were abolished by guanethidine (10 micromol L(-1)) or phentolamine (3 micromol L(-1)). The sympathetic neurotransmitter noradrenaline (NA) also caused concentration-dependent (10 nmol L(-1)-100 micromol L(-1)) contractions which were abolished by phentolamine revealing a small relaxation that was abolished by propranolol (3 micromol L(-1)). In contrast, EFS caused only relaxation of the mouse and rabbit IAS which was not affected by guanethidine. Furthermore, NA relaxed these muscles and relaxation was nearly abolished by combined addition of phentolamine and propranolol. In conclusion, the monkey IAS is functionally innervated by sympathetic nerves that contract the muscle via excitatory alpha-adrenergic receptors. In contrast, no significant motor function could be identified for sympathetic nerves in the rabbit or mouse IAS although adrenergic receptors linked to muscle inhibition are present. These data reveal species dependent differences in sympathetic motor innervation and suggest that some species are more appropriate than others as models for motor innervation to the human IAS.

Changes in neuromuscular transmission in the W/W(v) mouse internal anal sphincter.

BACKGROUND: Intramuscular interstitial cells of Cajal (ICC-IM) have been shown to participate in nitrergic neuromuscular transmission (NMT) in various regions of the gastrointestinal (GI) tract, but their role in the internal anal sphincter (IAS) is still uncertain. Contractile studies of the IAS in the W/W(v) mouse (a model in which ICC-IM numbers are markedly reduced) have reported that nitrergic NMT persists and that ICC-IM are not required. However, neither the changes in electrical events underlying NMT nor the contributions of other non-nitrergic neural pathways have been examined in this model. METHODS: The role of ICC-IM in NMT was examined by recording the contractile and electrical events associated with electrical field stimulation (EFS) of motor neurons in the IAS of wildtype and W/W(v) mice. Nitrergic, purinergic, and cholinergic components were identified using inhibitors of these pathways. KEY RESULTS: Under NANC conditions, purinergic and nitrergic pathways both contribute to EFS-induced inhibitory junction potentials (IJPs) and relaxation. Purinergic IJPs and relaxation were intact in the W/W(v) mouse IAS, whereas nitrergic IJPs were reduced by 50-60% while relaxation persisted. In the presence of L-NNA (NOS inhibitor) and MRS2500 (P2Y1 receptor antagonist), EFS gave rise to cholinergic depolarization and contractions that were abolished by atropine. Cholinergic depolarization was absent in the W/W(v) mouse IAS while contraction persisted. CONCLUSIONS & INFERENCES: ICC-IM significantly contributes to the electrical events underlying nitrergic and cholinergic NMT, whereas contractile events persist in the absence of ICC-IM. The purinergic inhibitory neural pathway appears to be independent of ICC-IM.