Sean M. Ward

A Perspective on “Contribution of postjunctional M2 muscarinic receptors to cholinergic nerve-mediated contractions of murine airway smooth muscle”.

Inhibitory motor neurons regulate several gastric motility patterns including receptive relaxation, gastric peristaltic motor patterns, and pyloric sphincter opening. Nitric oxide (NO) and purines have been identified as likely candidates that mediate inhibitory neural responses. However, the contribution from each neurotransmitter has received little attention in the distal stomach. The aims of this study were to identify the roles played by NO and purines in inhibitory motor responses in the antrums of mice and monkeys. By using wild-type mice and mutants with genetically deleted neural nitric oxide synthase ( Nos1 ^−/−) and P2Y1 receptors ( P2ry1 ^−/−) we examined the roles of NO and purines in postjunctional inhibitory responses in the distal stomach and compared these responses to those in primate stomach. Activation of inhibitory motor nerves using electrical field stimulation (EFS) produced frequency-dependent inhibitory junction potentials (IJPs) that produced muscle relaxations in both species. Stimulation of inhibitory nerves during slow waves terminated pacemaker events and associated contractions. In Nos1 ^−/− mice IJPs and relaxations persisted whereas in P2ry1 ^−/− mice IJPs were absent but relaxations persisted. In the gastric antrum of the non-human primate model Macaca fascicularis, similar NO and purine neural components contributed to inhibition of gastric motor activity. These data support a role of convergent inhibitory neural responses in the regulation of gastric motor activity across diverse species.

Background and Purpose: High‐throughput screening of compound libraries using genetically encoded fluorescent biosensors has identified several second‐generation. low MW inhibitors of the calcium‐activated chloride channel anoctamin 1 (CaCC/Ano1). Here we have (i) examined the effects of these Ano1 inhibitors on gastric and intestinal pacemaker activity; (ii) compared the effects of these inhibitors with those of the more classical CaCC inhibitor, 5‐nitro‐2‐(3‐phenylpropylalanine) benzoate (NPPB); (ii) examined the mode of action of these compounds on the waveform of pacemaker activity; and (iii) compared differences in the sensitivity between gastric and intestinal pacemaker activity to the Ano1 inhibitors.Experimental Approach: Using intracellular microelectrode recordings of gastric and intestinal muscle preparations from C57BL/6 mice, the dose‐dependent effects of Ano1 inhibitors were examined on spontaneous electrical slow waves.Key Results: The efficacy of second‐generation Ano1 inhibitors on gastric and intestinal pacemaker activity differed significantly. Antral slow waves were more sensitive to these inhibitors than intestinal slow waves. CaCC_inh‐A01 and benzbromarone were the most potent at inhibiting slow waves in both muscle preparations and more potent than NPPB. Dichlorophene and hexachlorophene were equally potent at inhibiting slow waves. Surprisingly, slow waves were relatively insensitive to T16A_inh‐A01 in both preparations.Conclusions and Implications: We have identified several second‐generation Ano1 inhibitors, blocking gastric and intestinal pacemaker activity. Different sensitivities to Ano1 inhibitors between stomach and intestine suggest the possibility of different splice variants in these two organs or the involvement of other conductances in the generation and propagation of pacemaker activity in these tissues.

Growing evidence suggests important roles for specialized platelet-derived growth factor receptor alpha-positive (PDGFRalpha⁺) cells in regulating the behaviors of visceral smooth muscle organs. Examination of the female reproductive tracts of mice and monkeys showed that PDGFRalpha⁺ cells form extensive networks in ovary, oviduct, and uterus. PDGFRalpha⁺ cells were located in discrete locations within these organs, and their distribution and density were similar in rodents and primates. PDGFRalpha⁺ cells were distinct from smooth muscle cells and interstitial cells of Cajal (ICC). This was demonstrated with immunohistochemical techniques and by performing molecular expression studies on PDGFRalpha⁺ cells from mice with enhanced green fluorescent protein driven off of the endogenous promoter for Pdgfralpha. Significant differences in gene expression were found in PDGFRalpha⁺ cells from ovary, oviduct, and uterus. Differences in gene expression were also detected in cells from different tissue regions within the same organ (e.g., uterine myometrium vs. endometrium). PDGFRalpha⁺ cells are unlikely to provide pacemaker activity because they lack significant expression of key pacemaker genes found in ICC (Kit and Ano1). Gja1 encoding connexin 43 was expressed at relatively high levels in PDGFRalpha⁺ cells (except in the ovary), suggesting these cells can form gap junctions to one another and neighboring smooth muscle cells. PDGFRalpha⁺ cells also expressed the early response transcription factor and proto-oncogene Fos, particularly in the ovary. These data demonstrate extensive distribution of PDGFRalpha⁺ cells throughout the female reproductive tract. These cells are a heterogeneous population of cells that are likely to contribute to different aspects of physiological regulation in the various anatomical niches they occupy.

Background: Loss or disruption of Kit⁺‐interstitial cells of Cajal (ICC) capable of generating pacemaker activity has been implicated in the development of numerous gastrointestinal motility disorders. We sought to develop a model where ICC could be allotransplanted into intestines naturally devoid of these cells.Methods: Enzymatically dispersed cells from the intestinal tunica muscularis of Kit^+/copGFP and Kit^V558Δ/+ gain‐of‐function mice were allotransplanted into myenteric plexus regions of W/W^V mutant intestines that lack ICC at the level of the myenteric plexus (ICC‐MY) and pacemaker activity. Immunohistochemical analysis fate mapped the development of ICC‐MY networks and intracellular microelectrode recordings provided evidence for the development of functional pacemaker activity.Key Results: Kit⁺‐ICC developed into distinct networks at the level of the myenteric plexus in organotypic cultures over 28 days and displayed robust rhythmic pacemaker activity.Conclusions & Inferences: This study demonstrates the feasibility of allotransplantation of ICC into the myenteric region of the small intestine and the establishment of functional pacemaker activity into tissues normally devoid of ICC‐MY and slow waves, thus providing a possible basis for the therapeutic treatment of patients where ICC networks have been disrupted due to a variety of pathophysiological conditions.

Background The colon undergoes distension‐induced changes in motor activity as luminal contents or feces increase wall pressure. Input from enteric motor neurons regulates this motility. Here we examined stretch‐dependent responses in circular muscle strips of murine colon.Methods Length ramps (6–31μm s⁻¹) were applied in the axis of the circular muscle layer in a controlled manner until 5 mN isometric force was reached.Key Results Length ramps produced transient membrane potential hyperpolarizations and attenuation of action potential (AP) complexes. Responses were reproducible when ramps were applied every 30 s. Stretch‐dependent hyperpolarization was blocked by TTX, suggesting AP‐dependent release of inhibitory neurotransmitter(s). Atropine did not potentiate stretch‐induced hyperpolarizations, but increased compliance of the circular layer.N^ω‐nitro‐l‐arginine (l‐NNA) inhibited stretch‐dependent hyperpolarization and decreased muscle compliance, suggesting release of NO mediates stretch‐dependent inhibition. Control membrane potential was restored by the NO donor sodium nitorprusside. Stretch‐dependent hyperpolarizations were blocked byl‐methionine, an inhibitor of stretch‐dependent K⁺(SDK) channels in colonic muscles. Loss of interstitial cells of Cajal, elicited by Kit neutralizing antibody, also inhibited responses to stretch. In presence ofl‐NNA and apamin, stretch responses became excitatory and were characterized by membrane depolarization and increased AP firing. A neurokinin‐1 receptor antagonist inhibited this stretch‐dependent increase in excitability.Conclusions and Inferences Our data show that stretch‐dependent responses in colonic muscles require tonic firing of enteric inhibitory neurons, but reflex activation of neurons does not appear to be necessary. NO causes activation of SDK channels, and stretch of muscles further activates these channels, explaining the inhibitory response to stretch in colonic muscle strips.

Kit immunohistochemistry and confocal reconstructions have provided detailed 3-dimensional images of ICC networks throughout the gastrointestinal (GI) tract. Morphological criteria have been used to establish that different classes of ICC exist within the GI tract and physiological studies have shown that these classes have distinct physiological roles in GI motility. Structural studies have focused predominately on rodent models and less information is available on whether similar classes of ICC exist within the GI tracts of humans or non-human primates. Using Kit immunohistochemistry and confocal imaging, we examined the 3-dimensional structure of ICC throughout the GI tract of cynomolgus monkeys. Whole or flat mounts and cryostat sections were used to examine ICC networks in the lower esophageal sphincter (LES), stomach, small intestine and colon. Anti-histamine antibodies were used to distinguish ICC from mast cells in the lamina propria. Kit labeling identified complex networks of ICC populations throughout the non-human primate GI tract that have structural characteristics similar to that described for ICC populations in rodent models. ICC-MY formed anastomosing networks in the myenteric plexus region. ICC-IM were interposed between smooth muscle cells in the stomach and colon and were concentrated within the deep muscular plexus (ICC-DMP) of the intestine. ICC-SEP were found in septal regions of the antrum that separated circular muscle bundles. Spindle-shaped histamine⁺ mast cells were found in the lamina propria throughout the GI tract. Since similar sub-populations of ICC exist within the GI tract of primates and rodents and the use of rodents to study the functional roles of different classes of ICC is warranted.

Key points  Normal colonic motility is regulated by excitatory and inhibitory motor neurons, and previous studies have shown that both components of neural regulation are important for normal propulsion of colonic contents. Inhibitory neural control consists of two main components, and the major neurotransmitters have been identified as nitric oxide and purines; we investigated the nature of the receptors responsible for purine inhibitory motor control of the colon using mice with P2Y1 receptors deactivated. Inhibitory control of the colon by purine neurotransmitters was dramatically decreased in these animals and transit of fecal pellets was delayed. Inhibitory responses to purine neurotransmission and exogenous β‐NAD, a neurotransmitter candidate, were completely abolished in P2Y1 receptor knockouts. These studies demonstrate the importance of purinergic neural regulation of colonic motility and suggest this form of neural regulation depends upon P2Y1 receptors to receive and transduce inhibitory neural signals.