Uncoupling of Ca2+ sparks from BK channels in cerebral arteries underlies hypoperfusion in hypertension-induced vascular dementia.

The deficit in cerebral blood flow (CBF) seen in patients with hypertension-induced vascular dementia is increasingly viewed as a therapeutic target for disease-modifying therapy. Progress is limited, however, due to uncertainty surrounding the mechanisms through which elevated blood pressure reduces CBF. To investigate this, we used the BPH/2 mouse, a polygenic model of hypertension. At 8 mo of age, hypertensive mice exhibited reduced CBF and cognitive impairment, mimicking the human presentation of vascular dementia. Small cerebral resistance arteries that run across the surface of the brain (pial arteries) showed enhanced pressure-induced constriction due to diminished activity of large-conductance Ca2+-activated K+ (BK) channels-key vasodilatory ion channels of cerebral vascular smooth muscle cells. Activation of BK channels by transient intracellular Ca2+ signals from the sarcoplasmic reticulum (SR), termed Ca2+ sparks, leads to hyperpolarization and vasodilation. Combining patch-clamp electrophysiology, high-speed confocal imaging, and proximity ligation assays, we demonstrated that this vasodilatory mechanism is uncoupled in hypertensive mice, an effect attributable to physical separation of the plasma membrane from the SR rather than altered properties of BK channels or Ca2+ sparks, which remained intact. This pathogenic mechanism is responsible for the observed increase in constriction and can now be targeted as a possible avenue for restoring healthy CBF in vascular dementia.

Functionally linked potassium channel activity in cerebral endothelial and smooth muscle cells is compromised in Alzheimer's disease.

The brain microcirculation is increasingly viewed as a potential target for disease-modifying drugs in the treatment of Alzheimer's disease patients, reflecting a growing appreciation of evidence that cerebral blood flow is compromised in such patients. However, the pathogenic mechanisms in brain resistance arteries underlying blood flow defects have not yet been elucidated. Here we probed the roles of principal vasodilatory pathways in cerebral arteries using the APP23 mouse model of Alzheimer's disease, in which amyloid precursor protein is increased approximately sevenfold, leading to neuritic plaques and cerebrovascular accumulation of amyloid-ß similar to those in patients with Alzheimer's disease. Pial arteries from APP23 mice (18 mo old) exhibited enhanced pressure-induced (myogenic) constriction because of a profound reduction in ryanodine receptor-mediated, local calcium-release events ("Ca2+ sparks") in arterial smooth muscle cells and a consequent decrease in the activity of large-conductance Ca2+-activated K+ (BK) channels. The ability of the endothelial cell inward rectifier K+ (Kir2.1) channel to cause dilation was also compromised. Acute application of amyloid-ß 1-40 peptide to cerebral arteries from wild-type mice partially recapitulated the BK dysfunction seen in APP23 mice but had no effect on Kir2.1 function. If mirrored in human Alzheimer's disease, these tandem defects in K+ channel-mediated vasodilation could account for the clinical cerebrovascular presentation seen in patients: reduced blood flow and crippled functional hyperemia. These data direct future research toward approaches that reverse this dual vascular channel dysfunction, with the ultimate aim of restoring healthy cerebral blood flow and improving clinical outcomes.

Urothelium-derived prostanoids enhance contractility of urinary bladder smooth muscle and stimulate bladder afferent nerve activity in the mouse.

The transitional epithelial cells (urothelium) that line the lumen of the urinary bladder form a barrier between potentially harmful pathogens, toxins, and other bladder contents and the inner layers of the bladder wall. The urothelium, however, is not simply a passive barrier, as it can produce signaling factors, such as ATP, nitric oxide, prostaglandins, and other prostanoids, that can modulate bladder function. We investigated whether substances produced by the urothelium could directly modulate the contractility of the underlying urinary bladder smooth muscle. Force was measured in isolated strips of mouse urinary bladder with the urothelium intact or denuded. Bladder strips developed spontaneous tone and phasic contractions. In urothelium-intact strips, basal tone, as well as the frequency and amplitude of phasic contractions, were 25%, 32%, and 338% higher than in urothelium-denuded strips, respectively. Basal tone and phasic contractility in urothelium-intact bladder strips were abolished by the cyclooxygenase (COX) inhibitor indomethacin (10 µM) or the voltage-dependent Ca2+ channel blocker diltiazem (50 µM), whereas blocking neuronal sodium channels with tetrodotoxin (1 µM) had no effect. These results suggest that prostanoids produced in the urothelium enhance smooth muscle tone and phasic contractions by activating voltage-dependent Ca2+ channels in the underlying bladder smooth muscle. We went on to demonstrate that blocking COX inhibits the generation of transient pressure events in isolated pressurized bladders and greatly attenuates the afferent nerve activity during bladder filling, suggesting that urothelial prostanoids may also play a role in sensory nerve signaling.NEW & NOTEWORTHY This paper provides evidence for the role of urothelial-derived prostanoids in maintaining tone in the urinary bladder during bladder filling, not only underscoring the role of the urothelium as more than a barrier but also contributing to active regulation of the urinary bladder. Furthermore, cyclooxygenase products greatly augment sensory nerve activity generated by bladder afferents during bladder filling and thus may play a role in perception of bladder fullness.

Oviductal motile cilia are essential for oocyte pickup but dispensable for sperm and embryo transport.

Mammalian oviducts play an essential role in female fertility by picking up ovulated oocytes and transporting and nurturing gametes (sperm/oocytes) and early embryos. However, the relative contributions to these functions from various cell types within the oviduct remain controversial. The oviduct in mice deficient in two microRNA (miRNA) clusters (miR-34b/c and miR-449) lacks cilia, thus allowing us to define the physiological role of oviductal motile cilia. Here, we report that the infundibulum without functional motile cilia failed to pick up the ovulated oocytes. In the absence of functional motile cilia, sperm could still reach the ampulla region, and early embryos managed to migrate to the uterus, but the efficiency was reduced. Further transcriptomic analyses revealed that the five messenger ribonucleic acids (mRNAs) encoded by miR-34b/c and miR-449 function to stabilize a large number of mRNAs involved in cilium organization and assembly and that Tubb4b was one of their target genes. Our data demonstrate that motile cilia in the infundibulum are essential for oocyte pickup and thus, female fertility, whereas motile cilia in other parts of the oviduct facilitate gamete and embryo transport but are not absolutely required for female fertility.

Afferent nerve activity in a mouse model increases with faster bladder filling rates in vitro, but voiding behavior remains unaltered in vivo.

Storage and voiding functions in urinary bladder are well-known, yet fundamental physiological events coordinating these behaviors remain elusive. We sought to understand how voiding function is influenced by the rate at which the bladder fills. We hypothesized that faster filling rates would increase afferent sensory activity and increase micturition rate. In vivo, this would mean animals experiencing faster bladder filling would void more frequently with smaller void volumes. To test this hypothesis, we measured afferent nerve activity during different filling rates using an ex vivo mouse bladder preparation and assessed voiding frequency in normally behaving mice noninvasively (UroVoid). Bladder afferent nerve activity depended on the filling rate, with faster filling increasing afferent nerve activity at a given volume. Voiding behavior in vivo was measured in UroVoid cages. Male and female mice were given access to tap water or, to induce faster bladder filling rates, water containing 5% sucrose. Fluid intake increased dramatically in mice consuming 5% sucrose. As expected, micturition frequency was elevated in the sucrose group. However, even with the greatly increased rate of urine production, void volumes were unchanged in both genders. Although faster filling rates generated higher afferent nerve rates ex vivo, this did not translate into more frequent, smaller-volume voids in vivo. This suggests afferent nerve activity is only one factor contributing to the switch from bladder filling to micturition. Together with afferent nerve activity, higher centers in the central nervous system and the state of arousal are likely critical to coordinating the micturition reflex.

Epithelial 5-HT4 Receptors as a Target for Treating Constipation and Intestinal Inflammation.

Because of their importance in the regulation of gut functions, several therapeutic targets involving serotonin-related proteins have been developed or repurposed to treat motility disorders, including serotonin transporter inhibitors, tryptophan hydroxylase blockers, 5-HT3 antagonists, and 5-HT4 agonists. This chapter focuses on our discovery of 5-HT4 receptors in the epithelial cells of the colon and our efforts to evaluate the effects of stimulating these receptors. 5-HT4 receptors appear to be expressed by all epithelial cells in the mouse colon, based on expression of a reporter gene driven by the 5-HT4 receptor promoter. Application of 5-HT4 agonists to the mucosal surface causes serotonin release from enterochromaffin cells, mucus secretion from goblet cells, and chloride secretion from enterocytes. Luminal administration of 5-HT4 agonists speeds up colonic motility and suppresses distention-induced nociceptive responses. Luminal administration of 5-HT4 agonists also decreases the development of, and improves recovery from, experimental colitis. Recent studies determined that the prokinetic actions of minimally absorbable 5-HT4 agonists are just as effective as absorbable compounds. Collectively, these findings indicate that targeting epithelial receptors with non-absorbable 5-HT4 agonists could offer a safe and effective strategy for treating constipation and colitis.

Motile cilia of the male reproductive system require miR-34/miR-449 for development and function to generate luminal turbulence.

Cilia are cell-surface, microtubule-based organelles that project into extracellular space. Motile cilia are conserved throughout eukaryotes, and their beat induces the flow of fluid, relative to cell surfaces. In mammals, the coordinated beat of motile cilia provides highly specialized functions associated with the movement of luminal contents, as seen with metachronal waves transporting mucus in the respiratory tract. Motile cilia are also present in the male and female reproductive tracts. In the female, wave-like motions of oviductal cilia transport oocytes and embryos toward the uterus. A similar function has been assumed for motile cilia in efferent ductules of the male-i.e., to transport immotile sperm from rete testis into the epididymis. However, we report here that efferent ductal cilia in the male do not display a uniform wave-like beat to transport sperm solely in one direction, but rather exert a centripetal force on luminal fluids through whip-like beating with continual changes in direction, generating turbulence, which maintains immotile spermatozoa in suspension within the lumen. Genetic ablation of two miRNA clusters (miR-34b/c and -449a/b/c) led to failure in multiciliogenesis in murine efferent ductules due to dysregulation of numerous genes, and this mouse model allowed us to demonstrate that loss of efferent duct motile cilia causes sperm aggregation and agglutination, luminal obstruction, and sperm granulomas, which, in turn, induce back-pressure atrophy of the testis and ultimately male infertility.

Mechanisms of Connexin-Related Lymphedema.

RATIONALE: Mutations in GJC2 and GJA1, encoding Cxs (connexins) 47 and 43, respectively, are linked to lymphedema, but the underlying mechanisms are unknown. Because efficient lymph transport relies on the coordinated contractions of lymphatic muscle cells (LMCs) and their electrical coupling through Cxs, Cx-related lymphedema is proposed to result from dyssynchronous contractions of lymphatic vessels. OBJECTIVE: To determine which Cx isoforms in LMCs and lymphatic endothelial cells are required for the entrainment of lymphatic contraction waves and efficient lymph transport. METHODS AND RESULTS: We developed novel methods to quantify the spatiotemporal entrainment of lymphatic contraction waves and used optogenetic techniques to analyze calcium signaling within and between the LMC and the lymphatic endothelial cell layers. Genetic deletion of the major lymphatic endothelial cell Cxs (Cx43, Cx47, or Cx37) revealed that none were necessary for the synchronization of the global calcium events that triggered propagating contraction waves. We identified Cx45 in human and mouse LMCs as the critical Cx mediating the conduction of pacemaking signals and entrained contractions. Smooth muscle-specific Cx45 deficiency resulted in 10- to 18-fold reduction in conduction speed, partial-to-severe loss of contractile coordination, and impaired lymph pump function ex vivo and in vivo. Cx45 deficiency resulted in profound inhibition of lymph transport in vivo, but only under an imposed gravitational load. CONCLUSIONS: Our results (1) identify Cx45 as the Cx isoform mediating the entrainment of the contraction waves in LMCs; (2) show that major endothelial Cxs are dispensable for the entrainment of contractions; (3) reveal a lack of coupling between lymphatic endothelial cells and LMCs, in contrast to arterioles; (4) point to lymphatic valve defects, rather than contraction dyssynchrony, as the mechanism underlying GJC2- or GJA1-related lymphedema; and (5) show that a gravitational load exacerbates lymphatic contractile defects in the intact mouse hindlimb, which is likely critical for the development of lymphedema in the adult mouse.

Postnatal Restriction of Activity-Induced Ca2+ Responses to Schwann Cells at the Neuromuscular Junction Are Caused by the Proximo-Distal Loss of Axonal Synaptic Vesicles during Development.

Terminal or perisynaptic Schwann cells (TPSCs) are nonmyelinating, perisynaptic glial cells at the neuromuscular junction (NMJ) that respond to neural activity by increasing intracellular calcium (Ca2+) and regulate synaptic function. The onset of activity-induced TPSC Ca2+ responses, as well as whether axonal Schwann cells (ASCs) along the nerve respond to nerve stimulation during development, is unknown. Here, we show that phrenic nerve stimulation in developing male and female mice elicited Ca2+ responses in both ASCs and TPSCs at embryonic day 14. ASC responses were lost in a proximo-distal gradient over time, but could continue to be elicited by bath application of neurotransmitter, suggesting that a loss of release rather than a change in ASC competence accounted for this response gradient. Similar to those of early postnatal TPSCs, developing ASC/TPSC responses were mediated by purinergic P2Y1 receptors. The loss of ASC Ca2+ responses was correlated to the proximo-distal disappearance of synaptophysin immunoreactivity and synaptic vesicles in phrenic axons. Accordingly, developing ASC Ca2+ responses were blocked by botulinum toxin. Interestingly, the loss of ASC Ca2+ responses was also correlated to the proximo-distal development of myelination. Finally, compared with postnatal TPSCs, neonatal TPSCs and ASCs displayed Ca2+ signals in response to lower frequencies and shorter durations of nerve stimulation. Together, these results with GCaMP3-expressing Schwann cells provide ex vivo evidence that both axons and presynaptic terminals initially exhibit activity-induced vesicular release of neurotransmitter, but that the subsequent loss of axonal synaptic vesicles accounts for the postnatal restriction of vesicular release to the NMJ.SIGNIFICANCE STATEMENT Neural activity regulates multiple aspects of development, including myelination. Whether the excitation of developing neurons in vivo results in the release of neurotransmitter from both axons and presynaptic terminals is unclear. Here, using mice expressing the genetically encoded calcium indicator GCaMP3 in Schwann cells, we show that both terminal/perisynaptic Schwann cells at the diaphragm neuromuscular junction and axonal Schwann cells along the phrenic nerve exhibit activity-induced calcium responses early in development, mediated by the vesicular release of ATP from the axons of motor neurons acting on P2Y1 receptors. These ex vivo findings corroborate classic in vitro studies demonstrating transmitter release by developing axons, and thus represent a tool to study the mechanisms and significance of this process during embryonic development.

Differential sensitivity of gastric and small intestinal muscles to inducible knockdown of anoctamin 1 and the effects on gastrointestinal motility.

KEY POINTS: Electrical pacemaking in gastrointestinal muscles is generated by specialized interstitial cells of Cajal that produce the patterns of contractions required for peristalsis and segmentation in the gut. The calcium-activated chloride conductance anoctamin-1 (Ano1) has been shown to be responsible for the generation of pacemaker activity in GI muscles, but this conclusion is established from studies of juvenile animals in which effects of reduced Ano1 on gastric emptying and motor patterns could not be evaluated. Knocking down Ano1 expression using Cre/LoxP technology caused dramatic changes in in gastric motor activity, with disrupted slow waves, abnormal phasic contractions and delayed gastric emptying; modest changes were noted in the small intestine. Comparison of the effects of Ano1 antagonists on muscles from juvenile and adult small intestinal muscles suggests that conductances in addition to Ano1 may develop with age and contribute to pacemaker activity. ABSTRACT: Interstitial cells of Cajal (ICC) generate slow waves and transduce neurotransmitter signals in the gastrointestinal (GI) tract, facilitating normal motility patterns. ICC express a Ca2+ -activated Cl- conductance (CaCC), and constitutive knockout of the channel protein anoctamin-1 leads to loss of slow waves in gastric and intestinal muscles. These knockout experiments were performed on juvenile mice. However, additional experiments demonstrated significant differences in the sensitivity of gastric and intestinal muscles to antagonists of anoctamin-1 channels. Furthermore, the significance of anoctamin-1 and the electrical and mechanical behaviours facilitated by this conductance have not been evaluated on the motor behaviours of adult animals. Cre/loxP technology was used to generate cell-specific knockdowns of anoctamin-1 in ICC (KitCreERT2/+ ;Ano1tm2jrr/+ ) in GI muscles. The recombination efficiency of KitCreERT was evaluated with an eGFP reporter, molecular techniques and immunohistochemistry. Electrical and contractile experiments were used to examine the consequences of anoctamin-1 knockdown on pacemaker activity, mechanical responses, gastric motility patterns, gastric emptying and GI transit. Reduced anoctamin-1 caused loss of gastric, but not intestinal slow waves. Irregular spike complexes developed in gastric muscles, leading to uncoordinated antral contractions, delayed gastric emptying and increased total GI transit time. Slow waves in intestinal muscles of juvenile mice were more sensitive to anoctamin-1 antagonists than slow waves in adult muscles. The low susceptibility to anoctamin-1 knockdown and weak efficacy of anoctamin-1 antagonists in inhibiting slow waves in adult small intestinal muscles suggest that a conductance in addition to anoctamin-1 may develop in small intestinal ICC with ageing and contribute to pacemaker activity.

TRPV4 blockade reduces voiding frequency, ATP release, and pelvic sensitivity in mice with chronic urothelial overexpression of NGF.

Transient receptor potential vanilloid family member 4 (TRPV4) transcript and protein expression increased in the urinary bladder and lumbosacral dorsal root ganglia of transgenic mice with chronic urothelial overexpression of nerve growth factor (NGF-OE). We evaluated the functional role of TRPV4 in bladder function with open-outlet cystometry, void spot assays, and natural voiding (Urovoid) assays with the TRPV4 antagonist HC-067047 (1 µM) or vehicle in NGF-OE and littermate wild-type (WT) mice. Blockade of TRPV4 at the level of the urinary bladder significantly (P ≤ 0.01) increased the intercontraction interval (2.2-fold) and void volume (2.6-fold) and decreased nonvoiding contractions (3.0-fold) in NGF-OE mice, with lesser effects (1.3-fold increase in the intercontraction interval and 1.3-fold increase in the void volume) in WT mice. Similar effects of TRPV4 blockade on bladder function in NGF-OE mice were demonstrated with natural voiding assays. Intravesical administration of HC-067047 (1 µM) significantly (P ≤ 0.01) reduced pelvic sensitivity in NGF-OE mice but was without effect in littermate WT mice. Blockade of urinary bladder TRPV4 or intravesical infusion of brefeldin A significantly (P ≤ 0.01) reduced (2-fold) luminal ATP release from the urinary bladder in NGF-OE and littermate WT mice. The results of the present study suggest that TRPV4 contributes to luminal ATP release from the urinary bladder and increased voiding frequency and pelvic sensitivity in NGF-OE mice.

BDNF downregulates ß-adrenergic receptor-mediated hypotensive mechanisms in the paraventricular nucleus of the hypothalamus.

Brain-derived neurotrophic factor (BDNF) is upregulated in the paraventricular nucleus of the hypothalamus (PVN) in response to hypertensive stimuli such as stress and hyperosmolality, and BDNF acting in the PVN plays a key role in elevating sympathetic activity and blood pressure. However, downstream mechanisms mediating these effects remain unclear. We tested the hypothesis that BDNF increases blood pressure, in part by diminishing inhibitory hypotensive input from nucleus of the solitary tract (NTS) catecholaminergic neurons projecting to the PVN. Male Sprague-Dawley rats received bilateral PVN injections of viral vectors expressing either green fluorescent protein (GFP) or BDNF and bilateral NTS injections of vehicle or anti-dopamine-ß-hydroxylase-conjugated saporin (DSAP), a neurotoxin that selectively lesions noradrenergic and adrenergic neurons. BDNF overexpression in the PVN without NTS lesioning significantly increased mean arterial pressure (MAP) in awake animals by 18.7 ± 1.8 mmHg. DSAP treatment also increased MAP in the GFP group, by 9.8 ± 3.2 mmHg, but failed to affect MAP in the BDNF group, indicating a BDNF-induced loss of NTS catecholaminergic hypotensive effects. In addition, in α-chloralose-urethane-anesthetized rats, hypotensive responses to PVN injections of the ß-adrenergic agonist isoprenaline were significantly attenuated by BDNF overexpression, whereas PVN injections of phenylephrine had no effect on blood pressure. BDNF treatment was also found to significantly reduce ß1-adrenergic receptor mRNA expression in the PVN, whereas expression of other adrenergic receptors was unaffected. In summary, increased BDNF expression in the PVN elevates blood pressure, in part by downregulating ß-receptor signaling and diminishing hypotensive catecholaminergic input from the NTS to the PVN.NEW & NOTEWORTHY We have shown that BDNF, a key hypothalamic regulator of blood pressure, disrupts catecholaminergic signaling between the NTS and the PVN by reducing the responsiveness of PVN neurons to inhibitory hypotensive ß-adrenergic input from the NTS. This may be occurring partly via BDNF-mediated downregulation of ß1-adrenergic receptor expression in the PVN and results in an increase in blood pressure.

SU9516 Increases α7ß1 Integrin and Ameliorates Disease Progression in the mdx Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by mutations in the dystrophin gene, resulting in a complete loss of the dystrophin protein. Dystrophin is a critical component of the dystrophin glycoprotein complex (DGC), which links laminin in the extracellular matrix to the actin cytoskeleton within myofibers and provides resistance to shear stresses during muscle activity. Loss of dystrophin in DMD patients results in a fragile sarcolemma prone to contraction-induced muscle damage. The α7ß1 integrin is a laminin receptor protein complex in skeletal and cardiac muscle and a major modifier of disease progression in DMD. In a muscle cell-based screen for α7 integrin transcriptional enhancers, we identified a small molecule, SU9516, that promoted increased α7ß1 integrin expression. Here we show that SU9516 leads to increased α7B integrin in murine C2C12 and human DMD patient myogenic cell lines. Oral administration of SU9516 in the mdx mouse model of DMD increased α7ß1 integrin in skeletal muscle, ameliorated pathology, and improved muscle function. We show that these improvements are mediated through SU9516 inhibitory actions on the p65-NF-κB pro-inflammatory and Ste20-related proline alanine rich kinase (SPAK)/OSR1 signaling pathways. This study identifies a first in-class α7 integrin-enhancing small-molecule compound with potential for the treatment of DMD.

Exposure to seawater increases intestinal motility in euryhaline rainbow trout (Oncorhynchus mykiss).

Upon exposure to seawater, euryhaline teleosts need to imbibe and desalinate seawater to allow for intestinal ion and water absorption, as this is essential for maintaining osmotic homeostasis. Despite the potential benefits of increased mixing and transport of imbibed water for increasing the efficiency of absorptive processes, the effect of water salinity on intestinal motility in teleosts remains unexplored. By qualitatively and quantitatively describing in vivo intestinal motility of euryhaline rainbow trout (Oncorhynchus mykiss), this study demonstrates that, in freshwater, the most common motility pattern consisted of clusters of rhythmic, posteriorly propagating contractions that lasted ∼1-2 min followed by a period of quiescence lasting ∼4-5 min. This pattern closely resembles mammalian migrating motor complexes (MMCs). Following a transition to seawater, imbibed seawater resulted in a significant distension of the intestine and the frequency of MMCs increased twofold to threefold with a concomitant reduction in the periods of quiescence. The increased frequency of MMCs was also accompanied by ripple-type contractions occurring every 12-60 s. These findings demonstrate that intestinal contractile activity of euryhaline teleosts is dramatically increased upon exposure to seawater, which is likely part of the overall response for maintaining osmotic homeostasis as increased drinking and mechanical perturbation of fluids is necessary to optimise intestinal ion and water absorption. Finally, the temporal response of intestinal motility in rainbow trout transitioning from freshwater to seawater coincides with previously documented physiological modifications associated with osmoregulation and may provide further insight into the underlying reasons shaping the migration patterns of salmonids.

The presence and role of interstitial cells of Cajal in the proximal intestine of shorthorn sculpin (Myoxocephalus scorpius).

Rhythmic contractions of the mammalian gastrointestinal tract can occur in the absence of neuronal or hormonal stimulation owing to the generation of spontaneous electrical activity by interstitial cells of Cajal (ICC) that are electrically coupled to smooth muscle cells. The myogenically driven component of gastrointestinal motility patterns in fish probably also involves ICC; however, little is known of their presence, distribution and function in any fish species. In the present study, we combined immunohistochemistry and in vivo recordings of intestinal motility to investigate the involvement of ICC in the motility of the proximal intestine in adult shorthorn sculpin (Myoxocephalus scorpius). Antibodies against anoctamin 1 (Ano1, a Ca2+-activated Cl- channel), revealed a dense network of multipolar, repeatedly branching cells in the myenteric region of the proximal intestine, similar in many regards to the mammalian ICC-MY network. The addition of benzbromarone, a potent blocker of Ano1, altered the motility patterns seen in vivo after neural blockade with TTX. The results indicate that ICC are integral for the generation and propagation of the majority of rhythmic contractile patterns in fish, although their frequency and amplitude can be modulated via neural activity.

Spontaneous Ca(2+) transients in interstitial cells of Cajal located within the deep muscular plexus of the murine small intestine.

KEY POINTS: Interstitial cells of Cajal at the level of the deep muscular plexus (ICC-DMP) in the small intestine generate spontaneous Ca(2+) transients that consist of localized Ca(2+) events and limited propagating Ca(2+) waves. Ca(2+) transients in ICC-DMP display variable characteristics: from discrete, highly localized Ca(2+) transients to regionalized Ca(2+) waves with variable rates of occurrence, amplitude, duration and spatial spread. Ca(2+) transients fired stochastically, with no cellular or multicellular rhythmic activity being observed. No correlation was found between the firing sites in adjacent cells. Ca(2+) transients in ICC-DMP are suppressed by the ongoing release of inhibitory neurotransmitter(s). Functional intracellular Ca(2+) stores are essential for spontaneous Ca(2+) transients, and the sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA) pump is necessary for maintenance of spontaneity. Ca(2+) release mechanisms involve both ryanodine receptors (RyRs) and inositol triphosphate receptors (InsP3 Rs). Release from these channels is interdependent. ICC express transcripts of multiple RyRs and InsP3 Rs, with Itpr1 and Ryr2 subtypes displaying the highest expression. ABSTRACT: Interstitial cells of Cajal in the deep muscular plexus of the small intestine (ICC-DMP) are closely associated with varicosities of enteric motor neurons and generate responses contributing to neural regulation of intestinal motility. Responses of ICC-DMP are mediated by activation of Ca(2+) -activated Cl(-) channels; thus, Ca(2+) signalling is central to the behaviours of these cells. Confocal imaging was used to characterize the nature and mechanisms of Ca(2+) transients in ICC-DMP within intact jejunal muscles expressing a genetically encoded Ca(2+) indicator (GCaMP3) selectively in ICC. ICC-DMP displayed spontaneous Ca(2+) transients that ranged from discrete, localized events to waves that propagated over variable distances. The occurrence of Ca(2+) transients was highly variable, and it was determined that firing was stochastic in nature. Ca(2+) transients were tabulated in multiple cells within fields of view, and no correlation was found between the events in adjacent cells. TTX (1 µm) significantly increased the occurrence of Ca(2+) transients, suggesting that ICC-DMP contributes to the tonic inhibition conveyed by ongoing activity of inhibitory motor neurons. Ca(2+) transients were minimally affected after 12 min in Ca(2+) free solution, indicating these events do not depend immediately upon Ca(2+) influx. However, inhibitors of sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA) pump and blockers of inositol triphosphate receptor (InsP3 R) and ryanodine receptor (RyR) channels blocked ICC Ca(2+) transients. These data suggest an interdependence between RyR and InsP3 R in the generation of Ca(2+) transients. Itpr1 and Ryr2 were the dominant transcripts expressed by ICC. These findings provide the first high-resolution recording of the subcellular Ca(2+) dynamics that control the behaviour of ICC-DMP in situ.

Spatio-Temporal Mapping and the Enteric Nervous System.

Study of the enteric nervous system (ENS) is somewhat less glamorous than other body systems but offers a unique opportunity to study the sensory, interneuronal and motor outputs of a highly developed neural network in the same tissue. This has not been a trivial task, and even after a century, we still struggle to understand both the simple (e.g. reflexes) and complex (e.g. MMCs) behaviors the gut produces. On top of that, other control networks (such as ICC) that are integrated with ENS at varying levels, can modify ENS activity directly or indirectly. While many of the methods used to study the ENS were originally developed in other systems (e.g. brain/heart), a few were spawned "in the offal" so to speak, due to the unique characteristics of the gut. The brief perspective below outlines how spatio-temporal maps (ST Maps) originated and continue to flourish in GI research as a tool to describe and analyze the complexity of GI movements.I apologize that I am not able to specifically mention all the people involved in the development and use of ST Maps in enteric/motility research due to space constraints (GWH, July 2014).

Temporal sequence of activation of cells involved in purinergic neurotransmission in the colon.

KEY POINTS: Platelet derived growth factor receptor α (PDGFRα(+) ) cells in colonic muscles are innervated by enteric inhibitory motor neurons. PDGFRα(+) cells generate Ca(2+) transients in response to exogenous purines and these responses were blocked by MRS-2500. Stimulation of enteric neurons, with cholinergic and nitrergic components blocked, evoked Ca(2+) transients in PDGFRα(+) and smooth muscle cells (SMCs). Responses to nerve stimulation were abolished by MRS-2500 and not observed in muscles with genetic deactivation of P2Y1 receptors. Ca(2+) transients evoked by nerve stimulation in PDGFRα(+) cells showed the same temporal characteristics as electrophysiological responses. PDGFRα(+) cells express gap junction genes, and drugs that inhibit gap junctions blocked neural responses in SMCs, but not in nerve processes or PDGFRα(+) cells. PDGFRα(+) cells are directly innervated by inhibitory motor neurons and purinergic responses are conducted to SMCs via gap junctions. ABSTRACT: Interstitial cells, known as platelet derived growth factor receptor α (PDGFRα(+) ) cells, are closely associated with varicosities of enteric motor neurons and suggested to mediate purinergic hyperpolarization responses in smooth muscles of the gastrointestinal tract (GI), but this concept has not been demonstrated directly in intact muscles. We used confocal microscopy to monitor Ca(2+) transients in neurons and post-junctional cells of the murine colon evoked by exogenous purines or electrical field stimulation (EFS) of enteric neurons. EFS (1-20 Hz) caused Ca(2+) transients in enteric motor nerve processes and then in PDGFRα(+) cells shortly after the onset of stimulation (latency from EFS was 280 ms at 10 Hz). Responses in smooth muscle cells (SMCs) were typically a small decrease in Ca(2+) fluorescence just after the initiation of Ca(2+) transients in PDGFRα(+) cells. Upon cessation of EFS, several fast Ca(2+) transients were noted in SMCs (rebound excitation). Strong correlation was noted in the temporal characteristics of Ca(2+) transients evoked in PDGFRα(+) cells by EFS and inhibitory junction potentials (IJPs) recorded with intracellular microelectrodes. Ca(2+) transients and IJPs elicited by EFS were blocked by MRS-2500, a P2Y1 antagonist, and absent in P2ry1((-/-)) mice. PDGFRα(+) cells expressed gap junction genes, and gap junction uncouplers, 18ß-glycyrrhetinic acid (18ß-GA) and octanol blocked Ca(2+) transients in SMCs but not in neurons or PDGFRα(+) cells. IJPs recorded from SMCs were also blocked. These findings demonstrate direct innervation of PDGFRα(+) cells by motor neurons. PDGFRα(+) cells are primary targets for purinergic neurotransmitter(s) in enteric inhibitory neurotransmission. Hyperpolarization responses are conducted to SMCs via gap junctions.

A novel class of somatic small RNAs similar to germ cell pachytene PIWI-interacting small RNAs.

PIWI-interacting RNAs (piRNAs) are small noncoding RNAs that bind PIWI family proteins exclusively expressed in the germ cells of mammalian gonads. MIWI2-associated piRNAs are essential for silencing transposons during primordial germ cell development, and MIWI-bound piRNAs are required for normal spermatogenesis during adulthood in mice. Although piRNAs have long been regarded as germ cell-specific, increasing lines of evidence suggest that somatic cells also express piRNA-like RNAs (pilRNAs). Here, we report the detection of abundant pilRNAs in somatic cells, which are similar to MIWI-associated piRNAs mainly expressed in pachytene spermatocytes and round spermatids in the testis. Based on small RNA deep sequencing and quantitative PCR analyses, pilRNA expression is dynamic and displays tissue specificity. Although pilRNAs are similar to pachytene piRNAs in both size and genomic origins, they have a distinct ping-pong signature. Furthermore, pilRNA biogenesis appears to utilize a yet to be identified pathway, which is different from all currently known small RNA biogenetic pathways. In addition, pilRNAs appear to preferentially target the 3'-UTRs of mRNAs in a partially complementary manner. Our data suggest that pilRNAs, as an integral component of the small RNA transcriptome in somatic cell lineages, represent a distinct population of small RNAs that may have functions similar to germ cell piRNAs.

Ano1, a Ca2+-activated Cl- channel, coordinates contractility in mouse intestine by Ca2+ transient coordination between interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) are pacemaker cells that generate electrical activity to drive contractility in the gastrointestinal tract via ion channels. Ano1 (Tmem16a), a Ca(2+)-activated Cl(-) channel, is an ion channel expressed in ICC. Genetic deletion of Ano1 in mice resulted in loss of slow waves in smooth muscle of small intestine. In this study, we show that Ano1 is required to maintain coordinated Ca(2+) transients between myenteric ICC (ICC-MY) of small intestine. First, we found spontaneous Ca(2+) transients in ICC-MY in both Ano1 WT and knockout (KO) mice. However, Ca(2+) transients within the ICC-MY network in Ano1 KO mice were uncoordinated, while ICC-MY Ca(2+) transients in Ano1 WT mice were rhythmic and coordinated. To confirm the role of Ano1 in the loss of Ca(2+) transient coordination, we used pharmacological inhibitors of Ano1 activity and shRNA-mediated knock down of Ano1 expression in organotypic cultures of Ano1 WT small intestine. Coordinated Ca(2+) transients became uncoordinated using both these approaches, supporting the conclusion that Ano1 is required to maintain coordination/rhythmicity of Ca(2+) transients. We next determined the effect on smooth muscle contractility using spatiotemporal maps of contractile activity in Ano1 KO and WT tissues. Significantly decreased contractility that appeared to be non-rhythmic and uncoordinated was observed in Ano1 KO jejunum. In conclusion, Ano1 has a previously unidentified role in the regulation of coordinated gastrointestinal smooth muscle function through coordination of Ca(2+) transients in ICC-MY.