Grant W. Hennig

Expression of anoctamin 1/TMEM16A by interstitial cells of Cajal is fundamental for slow wave activity in gastrointestinal muscles

Interstitial cells of Cajal (ICC) generate pacemaker activity (slow waves) in gastrointestinal (GI) smooth muscles, but the mechanism(s) of pacemaker activity are controversial. Several conductances, such as Ca2+‐activated Cl− channels (CaCC) and non‐selective cation channels (NSCC) have been suggested to be involved in slow wave depolarization. We investigated the expression and function of a new class of CaCC, anoctamin 1 (ANO1), encoded by Tmem16a, which was discovered to be highly expressed in ICC in a microarray screen. GI muscles express splice variants of the Tmem16a transcript in addition to other paralogues of the Tmem16a family. ANO1 protein is expressed abundantly and specifically in ICC in all regions of the murine, non‐human primate (Macaca fascicularis) and human GI tracts. CaCC blocking drugs, niflumic acid and 4,4′‐diisothiocyano‐2,2′‐stillbene‐disulfonic acid (DIDS) reduced the frequency and blocked slow waves in murine, primate, human small intestine and stomach in a concentration‐dependent manner. Unitary potentials, small stochastic membrane depolarizations thought to underlie slow waves, were insensitive to CaCC blockers. Slow waves failed to develop by birth in mice homozygous for a null allele of Tmem16a (Tmem16atm1Bdh/tm1Bdh) and did not develop subsequent to birth in organ culture, as in wildtype and heterozygous muscles. Loss of function of ANO1 did not inhibit the development of ICC networks that appeared structurally normal as indicated by Kit antibodies. These data demonstrate the fundamental role of ANO1 in the generation of slow waves in GI ICC.

Quantitative analysis of peristalsis in the guinea-pig small intestine using spatio-temporal maps.

1 Peristalsis was evoked in guinea‐pig small intestine by slow fluid infusion and recorded onto video and digitized. Spatio‐temporal maps of diameter and longitudinal movement were constructed and parameters of motion were calculated. 2 During the filling of the isolated segments of intestine, rhythmic local longitudinal movements were observed at several points along the preparation. These phasic longitudinal muscle contractions were associated with small but significant local increases in diameter and probably reflect a passive mechanical coupling by connective tissue in the gut wall. In addition, occasional synchronized longitudinal muscle contractions caused net shortening of the preparation and always preceded the onset of peristaltic emptying. 3 Peristaltic emptying was characterized by a contraction of the circular muscle which usually started at the oral end of the preparation, that propagated aborally, propelling the contents. However, in 19% of trials, the first circular muscle contraction occurred in the aboral half of the preparation. 4 The propagation of peristalsis consisted of separate sequential circular muscle contractions several centimetres long, particularly in the oral half of the preparation, giving a ‘step‐like’ appearance to the spatio‐temporal map. The gut was transiently distended aboral to the propagating circular muscle contraction due to the propulsion of contents. 5 At each point in the preparation, the longitudinal muscle remained contracted during the propulsive part of the circular muscle contraction. Only when the circular muscle contraction became lumen occlusive did lengthening of the longitudinal muscle take place. 6 Spatio‐temporal maps are a powerful tool to visualize and analyse the complexity of gastrointestinal motility patterns.

Male germ cells express abundant endogenous siRNAs.

In mammals, endogenous siRNAs (endo-siRNAs) have only been reported in murine oocytes and embryonic stem cells. Here, we show that murine spermatogenic cells express numerous endo-siRNAs, which are likely to be derived from naturally occurring double-stranded RNA (dsRNA) precursors. The biogenesis of these testicular endo-siRNAs is DROSHA independent, but DICER dependent. These male germ cell endo-siRNAs can potentially target hundreds of transcripts or thousands of DNA regions in the genome. Overall, our work has unveiled another hidden layer of regulation imposed by small noncoding RNAs during male germ cell development.

Analysis of motor patterns in the isolated guinea‐pig large intestine by spatio‐temporal maps

We investigated and quantified the spontaneous patterns of motility in the isolated guinea‐pig proximal and distal colon taken from adult animals. During spontaneous emptying, profiles of proximal and distal colon were recorded with a video camera, and image analysis was used to construct spatio‐temporal maps of the motions of the intestinal wall. Four patterns of motility were recorded. In the proximal colon there were neurally mediated contractions that propagated in the aboral direction at 4.1 mm s–1, gently pushing the soft contents aborally; these are likely to represent spontaneous peristaltic behaviour. A second pattern, insensitive to tetrodotoxin (TTX; 0.6 μM), consisted, in both oral and aboral propagation, of shallow contractions of the circular muscle (ripples). These contractions propagated aborally at 2.8 ± 0.45 mm s–1 and orally at 2.03 ± 0.31 mm s–1 (n=10). Of these TTX‐resistant contractions, 22.5% propagated both orally and aborally from a common origin. The orally propagated component of these myogenic contractions is likely to correspond to the antiperistalsis widely described in the proximal colon. In the distal colon, two patterns of motor activity were observed. One, induced by natural or artificial pellets, consisted of peristaltic contractions that pushed the pellets aborally at 0.8 mm s–1 and expelled a pellet every 108 s. In the interval between pellet propulsion and after the distal colon had emptied all of its pellets a second, nerve‐mediated pattern of motor activity, consisting of clusters of annular circular muscle contractions separated by short dilated regions, slowly propagated aborally at 0.3 mm s–1. Both of these motor patterns were abolished by TTX (0.6 μM). A latex balloon, inserted at the oral end of the empty isolated distal colon and inflated to a size similar to faecal pellets, was propelled at 1.4 mm s–1. Epoxy resin‐covered natural pellets were propelled at a similar speed of 1.6 mm s−1.Our data revealed that myogenic and neurogenic patterns of propagated contractions in the colon occur in isolated preparations and are involved in emptying the colon.

Electrical Slow Waves in the Mouse Oviduct Are Dependent upon a Calcium Activated Chloride Conductance Encoded by Tmem16a

ABSTRACT Myosalpinx contractions are critical for oocyte transport along the oviduct. A specialized population of pacemaker cells—oviduct interstitial cells of Cajal—generate slow waves, the electrical events underlying myosalpinx contractions. The ionic basis of oviduct pacemaker activity is unknown. We examined the role of a new class of Ca2+-activated Cl− channels (CaCCs)—anoctamin 1, encoded by Tmem16a—in oviduct slow wave generation. RT-PCR revealed the transcriptional expression of Tmem16a-encoded CaCCs in the myosalpinx. Intracellular microelectrode recordings were performed in the presence of two pharmacologically distinct Cl− channel antagonists, anthracene-9-carboxylic acid and niflumic acid. Both of these inhibitors caused membrane hyperpolarization, reduced the duration of slow waves, and ultimately inhibited pacemaker activity. Niflumic acid also inhibited propagating calcium waves within the myosalpinx. Slow waves were present at birth in wild-type and heterozygous oviducts but failed to develop by birth in mice homozygous for a null allele of Tmem16a (Tmem16atm1Bdh/tm1Bdh). These data suggest that Tmem16a-encoded CaCCs contribute to membrane potential and are responsible for the upstroke and plateau phases of oviduct slow waves.

Localized Release of Serotonin (5-Hydroxytryptamine) by a Fecal Pellet Regulates Migrating Motor Complexes in Murine Colon

BACKGROUND & AIMS The colonic migrating motor complex (CMMC) is a motor pattern that regulates the movement of fecal matter through a rhythmic sequence of electrical activity and/or contractions along the large bowel. CMMCs have largely been studied in empty preparations; we investigated whether local reflexes generated by a fecal pellet modify the CMMC to initiate propulsive activity. METHODS Recordings of CMMCs were made from the isolated murine large bowel, with or without a fecal pellet. Transducers were placed along the colon to record muscle tension and propulsive force on the pellet and microelectrodes were used to record electrical activity from either side of a fecal pellet, circular muscle cells oral and anal of a pellet, and in colons without the mucosa. RESULTS Spontaneous CMMCs propagated in both an oral or anal direction. When a pellet was inserted, CMMCs increased in frequency and propagated anally, exerting propulsive force on the pellet. The amplitude of slow waves increased during the CMMC. Localized mucosal stimulation/circumferential stretch evoked a CMMC, regardless of stimulus strength. The serotonin (5-hydroxytryptamine-3) receptor antagonist ondansetron reduced the amplitude of the CMMC, the propulsive force on the pellet, and the response to mucosal stroking, but increased the apparent conduction velocity of the CMMC. Removing the mucosa abolished spontaneous CMMCs, which still could be evoked by electrical stimulation. CONCLUSIONS The fecal pellet activates local mucosal reflexes, which release serotonin (5-hydroxytryptamine) from enterochromaffin cells, and stretch reflexes that determine the site of origin and propagation of the CMMC, facilitating propulsion.

Rapid anterograde and retrograde tracing from mesenteric nerve trunks to the guinea-pig small intestine in vitro.

Abstract A novel technique for rapid anterograde labelling of cut axons in vitro was used to visualise the peripheral branches of mesenteric nerve trunks supplying the guinea-pig small intestine. Biotinamide, dissolved in an artificial intracellular solution, was applied to the cut ends of the mesenteric nerves and the tissue was maintained in organ culture overnight. Labelled nerve fibres were visualised by fluorescein isothiocyanate (FITC)-conjugated streptavidin. Intense staining of nerve fibres and terminal varicosities in the ganglia and internodal strands of the myenteric plexus was achieved up to 15 mm from the application site. Filled fibres formed baskets around some myenteric nerve cell bodies, suggesting target-specific neurotransmission. When combined with multiple-labelling immunohistochemistry for tyrosine hydroxylase (TH), calcitonin gene-related protein (CGRP) or choline acetyltransferase (ChAT), most anterogradely labelled nerve fibres, and many pericellular baskets, were found to be TH immunoreactive, indicating their postganglionic sympathetic origin. Double-labelling immunohistochemistry revealed that the postganglionic sympathetic pericellular baskets preferentially surrounded 5-hydroxytryptamine (5-HT)-handling myenteric neurons. Some biotinamide-filled fibres were CGRP immunoreactive, and are likely to originate from spinal sensory neurons. We describe for the first time many pericellular baskets labelled from the mesenteric nerves which were ChAT immunoreactive. Retrogradely filled intestinofugal nerve cell bodies were also observed, all of which had a single axon arising from a small nerve cell body with short filamentous or lamellar dendrites. Many of these cells were ChAT immunoreactive. This in vitro technique is effective in identifying the fine arrangement of nerve terminals arising from nerve trunks in the periphery.

Excitatory and inhibitory motor reflexes in the isolated guinea‐pig stomach

1 We have described and analysed the movements of the isolated stomach during distension by correlating intragastric pressure with video recordings, and investigated the presence of intrinsic inhibitory and excitatory reflexes. 2 Isolated guinea‐pig stomachs, placed in an organ bath, were slowly distended with Krebs solution using a syringe pump via a cannula through the pylorus. The changes in intragastric pressure during cycles of distension were monitored by pressure transducers connected to both oesophageal and pyloric cannulae. The resistivity of the gastric wall (change in pressure with volume, δP/δV) and the amplitude and frequency of phasic pressure events were calculated from pressure recordings. 3 The movements of the stomach were also recorded onto videotape. The motion of the gastric wall during distension cycles was analysed to establish the patterns of contractions, their propagation and the distribution of fluid in the stomach. During filling, fluid was preferentially accommodated in the fundus. Propagating (peristaltic) contractions, often starting in the fundus, moved aborally towards the pylorus. The peak of the phasic pressure event was observed when a contraction reached the orad antrum. As it reached the pylorus, intragastric pressure was at its minimum. 4 During the initial phase of distension, intragastric pressure increased steeply. Tetrodotoxin and hyoscine reduced both the resistivity and amplitude of phasic pressure events. Hexamethonium had a similar effect. Thus distension appears to activate an excitatory reflex pathway, involving nicotinic ganglionic transmission. This reflex increases wall tension and enhances myogenic peristaltic contractions. 5 In control preparations, with larger distension volumes, the intragastric pressure decreased, despite the continued infusion of Krebs solution. l‐NAME and apamin abolished this drop in pressure, indicating that gastric enteric inhibitory mechanisms prevail at larger distension volumes. After blockade of the excitatory reflex, hexamethonium antagonized the inhibitory response, indicating that activation of inhibitory mechanisms involves nicotinic transmission, probably on enteric inhibitory motoneurons. 6 Both the excitatory and inhibitory reflexes in the isolated stomach operate within a physiological range of gastric volumes. The excitatory reflex predominates at small distension volumes, leading to large phasic propagated contractions that mix the contents and may lead to emptying of the stomach. The inhibitory reflex, described previously as adaptive relaxation, can maximally relax the stomach and is activated preferentially at higher distension volumes to accommodate the contents. The interplay of these reflex pathways in the isolated stomach produces a rich repertoire of gastric movements. 7 The isolated stomach preparation, used with a combination of kinematic, kinetic and pharmacological methods, provides a highly suitable means of investigating the mechanisms of gastric motility.

The mitochondrial genome encodes abundant small noncoding RNAs

Small noncoding RNAs identified thus far are all encoded by the nuclear genome. Here, we report that the murine and human mitochondrial genomes encode thousands of small noncoding RNAs, which are predominantly derived from the sense transcripts of the mitochondrial genes (host genes), and we termed these small RNAs mitochondrial genome-encoded small RNAs (mitosRNAs). DICER inactivation affected, but did not completely abolish mitosRNA production. MitosRNAs appear to be products of currently unidentified mitochondrial ribonucleases. Overexpression of mitosRNAs enhanced expression levels of their host genes in vitro, and dysregulated mitosRNA expression was generally associated with aberrant mitochondrial gene expression in vivo. Our data demonstrate that in addition to 37 known mitochondrial genes, the mammalian mitochondrial genome also encodes abundant mitosRNAs, which may play an important regulatory role in the control of mitochondrial gene expression in the cell.

TTX-sensitive and TTX-insensitive control of spontaneous gut motility in the developing zebrafish (Danio rerio) larvae.

SUMMARY Spontaneous regular gut motility in zebrafish begins around 4 days post fertilisation (d.p.f.) and is modulated by release of acetylcholine and nitric oxide. The role of intrinsic or extrinsic innervation for initiating and propagating the spontaneous contractions, however, is not well understood. By creating spatiotemporal maps, we could examine spontaneous motility patterns in zebrafish larvae in vivo at 4 and 7 d.p.f. in more detail. Tetrodotoxin (TTX) was added to elucidate the importance of nervous control. Anterograde and retrograde contraction waves originated in the same region, just posterior to the intestinal bulb. This area correlates well with the distribution of Hu (human neuronal protein C/D)-immunoreactive nerve cell bodies. Whereas numerous immunoreactive nerve cells were present in the mid and distal intestine at both 4 and 7 d.p.f., fewer cells were seen anterior to the origin of contractions. The overall frequency of contractions (1.16±0.15 cycles min–1, N=14 at 4 d.p.f.; 1.05±0.09 cycles min–1, N=13 at 7 d.p.f.) and the interval between individual anterograde contraction waves (54.8±7.9 s at 4 d.p.f., N=14; 56.9±4.4 s, N=13 at 7 d.p.f.) did not differ between the two stages but the properties of the contractions were altered. The distance travelled by each wave increased from 591.0±43.8 μm at 4 d.p.f. (N=14) to 719.9±33.2 μm at 7 d.p.f. (N=13). By contrast, the velocity decreased from 4 d.p.f. (49.5±5.5 μm s–1, N=12) to 7 d.p.f. (27.8±3.6 μm s–1, N=13). At 4 d.p.f., TTX did not affect any of the parameters whereas at 7 d.p.f. anterograde frequency (control 1.07±0.12 cycles min–1, N=8; TTX 0.55±0.13 cycles min–1, N=8) and distance travelled (control 685.1±45.9 μm, N=8; TTX 318.7±88.7 μm, N=6) were decreased. In conclusion, enteric or extrinsic innervation does not seem to be necessary to initiate spontaneous contractions of the gut in zebrafish larvae. However, later in development, nerves have an increasingly important role as modulators of intestinal activity.)