Search

Enteric neurotransmission is critical for coordinating motility throughout the gastrointestinal (GI) tract. However, there is considerable controversy regarding the cells that are responsible for the transduction of these neural inputs. In the present study, utilization of a cell-specific calcium biosensor GCaMP6f, the spontaneous activity and neuroeffector responses of intramuscular ICC (ICC-IM) to motor neural inputs was examined. Simultaneous intracellular microelectrode recordings and high-speed video-imaging during nerve stimulation was used to reveal the temporal relationship between changes in intracellular Ca

Significance TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-permeable, nonselective cation channel that is localized to late endosomes and lysosomes. Here, we investigated the function of TRPML1 channels in regulating lower urinary tract (LUT) smooth muscle cell (SMC) contractility. We found that TRPML1 forms a stable signaling complex with ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR). We further showed that TRPML1 channels are important for initiating an essential Ca2+-signaling negative feedback mechanism between RyRs on SR membranes and K+ channels on the plasma membrane. Knockout of TRPML1 channels in mice impaired this pathway, resulting in LUT smooth muscle hypercontractility and symptoms of overactive bladder. Our findings demonstrate a critical role for TRPML1 in LUT function., TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-permeable, nonselective cation channel that is predominantly localized to the membranes of late endosomes and lysosomes (LELs). Intracellular release of Ca2+ through TRPML1 is thought to be pivotal for maintenance of intravesicular acidic pH as well as the maturation, fusion, and trafficking of LELs. Interestingly, genetic ablation of TRPML1 in mice (Mcoln1−/−) induces a hyperdistended/hypertrophic bladder phenotype. Here, we investigated this phenomenon further by exploring an unconventional role for TRPML1 channels in the regulation of Ca2+-signaling activity and contractility in bladder and urethral smooth muscle cells (SMCs). Four-dimensional (4D) lattice light-sheet live-cell imaging showed that the majority of LELs in freshly isolated bladder SMCs were essentially immobile. Superresolution microscopy revealed distinct nanoscale colocalization of LEL-expressing TRPML1 channels with ryanodine type 2 receptors (RyR2) in bladder SMCs. Spontaneous intracellular release of Ca2+ from the sarcoplasmic reticulum (SR) through RyR2 generates localized elevations of Ca2+ (“Ca2+ sparks”) that activate plasmalemmal large-conductance Ca2+-activated K+ (BK) channels, a critical negative feedback mechanism that regulates smooth muscle contractility. This mechanism was impaired in Mcoln1−/− mice, which showed diminished spontaneous Ca2+ sparks and BK channel activity in bladder and urethra SMCs. Additionally, ex vivo contractility experiments showed that loss of Ca2+ spark–BK channel signaling in Mcoln1−/− mice rendered both bladder and urethra smooth muscle hypercontractile. Voiding activity analyses revealed bladder overactivity in Mcoln1−/− mice. We conclude that TRPML1 is critically important for Ca2+ spark signaling, and thus regulation of contractility and function, in lower urinary tract SMCs.

Key points β-Nicotinamide adenine dinucleotide (β-NAD) is a key inhibitory neurotransmitter in the colon. The neuroeffector junction in the gut consists of enteric motor neurons and SIP syncytium, including smooth muscle cells (SMCs), interstitial cells of Cajal (ICC), and cells expressing platelet-derived growth factor receptor α (PDGFRα+ cells). Measuring metabolism of 1,N6 -etheno-NAD (eNAD) in colonic tunica muscularis and in SMCs, ICC and PDGFRα+ cells with HPLC-FLD, we report that (1) in tissues, eNAD is degraded to eADP-ribose, eAMP and e-adenosine (eADO) by CD38, ENPP1 and NT5E, (2) with SMCs and PDGFRα+ cells, eNAD is metabolized to eADO by ENPP1 and NT5E, (3) eNAD is not metabolized by ICC, (4) NT5E is expressed chiefly by SMCs and moderately by PDGFRα+ cells, (5) SIP cells are not the primary location of CD38. These data argue that the duration and strength of purinergic neurotransmission can be modulated by targeting multiple enzymes with specialized cellular distribution in the colon. Abstract Prior studies suggest that β-nicotinamide adenine dinucleotide (β-NAD) is an important inhibitory motor neurotransmitter in the enteric nervous system. Metabolism of β-NAD at the neuroeffector junction (NEJ) is likely to be necessary for terminating inhibitory neurotransmission and may also produce bioactive metabolites. The enteric NEJ consists of enteric neurons and postjunctional cells of the SIP syncytium, including smooth muscle cells (SMCs), interstitial cells of Cajal (ICC), and cells expressing platelet-derived growth factor receptor α (PDGFRα+ cells). We examined possible specialized functions of the NEJ in β-NAD metabolism by determining the degradation of 1,N6 -etheno-NAD (eNAD) in colonic tunica muscularis of wild-type, Cd38-/- , Nt5e-/- , Enpp1-/- and Cd38-/- /Nt5e-/- mice and in SIP cells from mice expressing cell-specific fluorescent reporters purified by fluorescence activated cell sorting (FACS). We measured eNAD and its metabolites eADP-ribose (eADPR), eAMP and e-adenosine (eADO) from tissues and sorted SIP cells using liquid chromatography. eNAD exposed to colonic muscularis of wild-type mice produced eADPR, eAMP and eADO. CD38 mediated the conversion of eNAD to eADPR, whereas ENPP1 mediated degradation of eNAD and eADPR to eAMP. NT5E (aka CD73) was the primary enzyme forming eADO from eAMP. PDGFRα+ cells and SMCs were involved in production of eADO from eNAD, and ICC were not involved in extracellular metabolism of eNAD. CD38 mediated the eNAD metabolism in whole tissues, but CD38 did not appear to be functionally expressed by SMCs or ICC. NT5E was expressed in SMCs > PDGFRα+ cells. Our data show that extracellular metabolism of β-NAD in the colon is mediated by multiple enzymes with cell-specific expression.

Colonic intramuscular interstitial cells of Cajal (ICC-IM) are associated with cholinergic varicosities, suggesting a role in mediating excitatory neurotransmission. Ca2+ release in ICC-IM activates Ano1, a Ca2+ -activated Cl- conductance, causing tissue depolarization and increased smooth muscle excitability. We employed Ca2+ imaging of colonic ICC-IM in situ, using mice expressing GCaMP6f in ICC to evaluate ICC-IM responses to excitatory neurotransmission. Expression of muscarinic type 2, 3 (M2 , M3 ), and NK1 receptors were enriched in ICC-IM. NK1 receptor agonists had minimal effects on ICC-IM, whereas neostigmine and carbachol increased Ca2+ transients. These effects were reversed by DAU 5884 (M3 receptor antagonist) but not AF-DX 116 (M2 receptor antagonist). Electrical field stimulation (EFS) in the presence of L-NNA and MRS 2500 enhanced ICC-IM Ca2+ transients. Responses were blocked by atropine or DAU 5884, but not AF-DX 116. ICC-IM responses to EFS were ablated by inhibiting Ca2+ stores with cyclopiazonic acid and reduced by inhibiting Ca2+ influx via Orai channels. Contractions induced by EFS were reduced by an Ano1 channel antagonist, abolished by DAU 5884, and unaffected by AF-DX 116. Colonic ICC-IM receive excitatory inputs from cholinergic neurons via M3 receptor activation. Enhancing ICC-IM Ca2+ release and Ano1 activation contributes to excitatory responses of colonic muscles.

The internal anal sphincter (IAS) generates phasic contractions and tone. Slow waves (SWs) produced by interstitial cells of Cajal (ICC) underlie phasic contractions in other gastrointestinal regions. SWs are also present in the IAS where only intramuscular ICC (ICC-IM) are found, however the evidence linking ICC-IM to SWs is limited. This study examined the possible relationship between ICC-IM and SWs by recording Ca2+ transients in mice expressing a genetically-encoded Ca2+-indicator in ICC (Kit-Cre-GCaMP6f). A role for L-type Ca2+ channels (CavL) and anoctamin 1 (ANO1) was tested since each is essential for SW and tone generation. Two distinct ICC-IM populations were identified. Type I cells (36% of total) displayed localised asynchronous Ca2+ transients not dependent on CavL or ANO1; properties typical of ICC-IM mediating neural responses in other gastrointestinal regions. A second novel sub-type, i.e., Type II cells (64% of total) generated rhythmic, global Ca2+ transients at the SW frequency that were synchronised with neighbouring Type II cells and were abolished following blockade of either CavL or ANO1. Thus, the spatiotemporal characteristics of Type II cells and their dependence upon CavL and ANO1 all suggest that these cells are viable candidates for the generation of SWs and tone in the IAS.

The process of urine removal from the kidney occurs via the renal pelvis (RP). The RP demarcates the beginning of the upper urinary tract and is endowed with smooth muscle cells. Along the RP, organized contraction of smooth muscle cells generates the force required to move urine boluses toward the ureters and bladder. This process is mediated by specialized pacemaker cells that are highly expressed in the proximal RP that generate spontaneous rhythmic electrical activity to drive smooth muscle depolarization. The mechanisms by which peristaltic contractions propagate from the proximal to distal RP are not fully understood. In this study, we utilized a transgenic mouse that expresses the genetically encoded Ca

The peristaltic reflex is a fundamental behavior of the gastrointestinal (GI) tract in which mucosal stimulation activates propulsive contractions. The reflex occurs by stimulation of intrinsic primary afferent neurons with cell bodies in the myenteric plexus and projections to the lamina propria, distribution of information by interneurons, and activation of muscle motor neurons. The current concept is that excitatory cholinergic motor neurons are activated proximal to and inhibitory neurons are activated distal to the stimulus site. We found that atropine reduced, but did not block, colonic migrating motor complexes (CMMCs) in mouse, monkey, and human colons, suggesting a mechanism other than one activated by cholinergic neurons is involved in the generation/propagation of CMMCs. CMMCs were activated after a period of nerve stimulation in colons of each species, suggesting that the propulsive contractions of CMMCs may be due to the poststimulus excitation that follows inhibitory neural responses. Blocking nitrergic neurotransmission inhibited poststimulus excitation in muscle strips and blocked CMMCs in intact colons. Our data demonstrate that poststimulus excitation is due to increased Ca2+ transients in colonic interstitial cells of Cajal (ICC) following cessation of nitrergic, cyclic guanosine monophosphate (cGMP)-dependent inhibitory responses. The increase in Ca2+ transients after nitrergic responses activates a Ca2+-activated Cl− conductance, encoded by Ano1, in ICC. Antagonists of ANO1 channels inhibit poststimulus depolarizations in colonic muscles and CMMCs in intact colons. The poststimulus excitatory responses in ICC are linked to cGMP-inhibited cyclic adenosine monophosphate (cAMP) phosphodiesterase 3a and cAMP-dependent effects. These data suggest alternative mechanisms for generation and propagation of CMMCs in the colon.

Cyclophosphamide (CYP)-induced cystitis is a rodent model that shares many features common to the cystitis occurring in patients, including detrusor overactivity (DO). Platelet-derived growth factor receptor alpha positive (PDGFRα+) cells regulate muscle excitability in murine bladders during filling. PDGFRα+ cells express small conductance Ca2+-activated K+ channels (predominantly SK3) that provide stabilization of membrane potential during filling. We hypothesized that down-regulation of the regulatory functions of PDGFRα+ cells and/or loss of PDGFRα+ cells generates the DO in CYP-treated mice. After CYP treatment, transcripts of Pdgfrα and Kcnn3 and PDGFRα and SK3 protein were reduced in detrusor muscle extracts. The distribution of PDGFRα+ cells was also reduced. Inflammatory markers were increased in CYP-treated detrusor muscles. An SK channel agonist, CyPPA, increased outward current and hyperpolarization in PDGFRα+ cells. This response was significantly depressed in PDGFRα+ cells from CYP-treated bladders. Contractile experiments and ex vivo cystometry showed increased spontaneous contractions and transient contractions, respectivley in CYP-treated bladders with a reduction of apamin sensitivity, reflecting the reduction in the SK conductance expressed by PDGFRα+ cells. In summary, PDGFRα+ cells were reduced and the SK3 conductance was downregulated in CYP-treated bladders. These changes are consistent with the development of DO after CYP treatment.

Metalloendopeptidase ADAM-Like Decysin 1 (ADAMDEC1) is an anti-inflammatory peptidase that is almost exclusively expressed in the gastrointestinal (GI) tract. We have recently found abundant and selective expression of Adamdec1 in colonic mucosal PDGFRα

Platelet-derived growth factor receptor α (PDGFRα)-positive interstitial cells (PIC) are interposed between enteric nerve fibers and smooth muscle cells (SMCs) in the tunica muscularis of the gastrointestinal tract. PIC have robust expression of small conductance CaGene expression analysis, CaAdcyap1r1 (encoding PACPIC of mouse and human colon utilize PAC

Dysfunction of embryo transport causes ectopic pregnancy which affects approximately 2% of conceptions in the US and Europe, and is the most common cause of pregnancy-related death in the first trimester. Embryo transit involves a valve-like tubal-locking phenomenon that temporarily arrests oocytes at the ampullary-isthmic junction (AIJ) where fertilisation occurs, but the mechanisms involved are unknown. Here we show that female mice lacking the orphan adhesion G-protein coupled receptor Adgrd1 are sterile because they do not relieve the AIJ restraining mechanism, inappropriately retaining embryos within the oviduct. Adgrd1 is expressed on the oviductal epithelium and the post-ovulatory attenuation of tubal fluid flow is dysregulated in Adgrd1-deficient mice. Using a large-scale extracellular protein interaction screen, we identified Plxdc2 as an activating ligand for Adgrd1 displayed on cumulus cells. Our findings demonstrate that regulating oviductal fluid flow by Adgrd1 controls embryo transit and we present a model where embryo arrest at the AIJ is due to the balance of abovarial ciliary action and the force of adovarial tubal fluid flow, and in wild-type oviducts, fluid flow is gradually attenuated through Adgrd1 activation to enable embryo release. Our findings provide important insights into the molecular mechanisms involved in embryo transport in mice., Lack of correct embryo transport can cause ectopic pregnancy. Here, the authors show that female mice lacking the adhesion G-protein coupled receptor Adgrd1 are infertile, due to embryos being trapped in the ampulla as the result of dysregulated oviductal fluid flow.

Gastroparesis is characterized by symptoms suggestive of, and objective evidence of, delayed gastric emptying in the absence of mechanical obstruction. This review addresses the normal emptying of solids and liquids from the stomach and details the myogenic and neuromuscular control mechanisms, including the specialized function of the pyloric sphincter, that result in normal emptying, based predominantly on animal research. A clear understanding of fundamental mechanisms is necessary to comprehend derangements leading to gastroparesis, and additional research on human gastric muscles is needed. The section on pathophysiology of gastroparesis considers neuromuscular diseases that affect nonsphincteric gastric muscle, disorders of the extrinsic neural control, and pyloric dysfunction that lead to gastroparesis. The potential cellular basis for gastroparesis is attributed to the effects of oxidative stress and inflammation, with increased pro-inflammatory and decreased resident macrophages, as observed in full-thickness biopsies from patients with gastroparesis. Predominant diagnostic tests involving measurements of gastric emptying, the use of a functional luminal imaging probe, and high-resolution antral duodenal manometry in characterizing the abnormal motor functions at the gastroduodenal junction are discussed. Management is based on supporting nutrition; dietary interventions, including the physical reduction in particle size of solid foods; pharmacological agents, including prokinetics and anti-emetics; and interventions such as gastric electrical stimulation and pyloromyotomy. These are discussed briefly, and comment is added on the potential for individualized treatments in the future, based on optimal gastric emptying measurement and objective documentation of the underlying pathophysiology causing the gastroparesis.

Urethral smooth muscle (USM) generates tone to prevent urine leakage from the bladder during filling. USM tone has been thought to be a voltage-dependent process, relying on Ca2+ influx via voltage-dependent Ca2+ channels in USM cells, modulated by the activation of Ca2+-activated Cl− channels encoded by Ano1. However, recent findings in the mouse have suggested that USM tone is voltage independent, relying on Ca2+ influx through Orai channels via store-operated Ca2+ entry (SOCE). We explored if this pathway also occurred in the pig using isometric tension recordings of USM tone. Pig USM strips generated myogenic tone, which was nearly abolished by the Cav1.2 channel antagonist nifedipine and the ATP-dependent K+ channel agonist pinacidil. Pig USM tone was reduced by the Orai channel blocker GSK-7975A. Electrical field stimulation (EFS) led to phentolamine-sensitive contractions of USM strips. Contractions of pig USM were also induced by phenylephrine. Phenylephrine-evoked and EFS-evoked contractions of pig USM were reduced by ~50–75% by nifedipine and ~30% by GSK-7975A. Inhibition of Ano1 channels had no effect on tone or EFS-evoked contractions of pig USM. In conclusion, unlike the mouse, pig USM exhibited voltage-dependent tone and agonist/EFS-evoked contractions. Whereas SOCE plays a role in generating tone and agonist/neural-evoked contractions in both species, this dominates in the mouse. Tone and agonist/EFS-evoked contractions of pig USM are the result of Ca2+ influx primarily through Cav1.2 channels, and no evidence was found supporting a role of Ano1 channels in modulating these mechanisms.

BACKGROUND: Alpha-type platelet-derived growth factor receptor (PDGFRα) is a cell surface tyrosine kinase receptor for members of the platelet-derived growth factor family. PDGFRα plays an important role in the regulation of several biological processes and contributes to the pathophysiology of a broad range of human cancers, including glioma. Here, we hypothesize that the genes directly or indirectly influenced by PDGFRα might be useful for prognosis in glioma. METHODS: By comparing the genome-wide gene expression pattern between PDGFRα+ and PDGFRα− cells from human oligodendrocyte progenitor, we defined the genes potentially influenced by PDGFRα. RESULTS: The PDGFRα-influenced genes are strongly associated with cancer-related pathways. We subsequently developed a prognostic gene signature derived from the PDGFRα-influenced genes. This gene signature is able to predict clinical outcome of glioma. This signature is also independent of traditional prognostic factors of glioma. Resampling tests indicate that the prognostic power of this gene signature outperforms random gene sets selected from human genome. More importantly, this signature is superior to the random gene signatures selected from glioma related genes. CONCLUSIONS: Despite the absence of clear elucidation of molecular mechanisms, this study suggests the vital role of PDGFRα in carcinogenesis. Furthermore, the PDGFRα-based gene signature provides a promising prognostic tool for glioma and validates PDGFRα as a novel and effective therapeutic target in human cancers.

Pacemaker depolarization in interstitial cells of Cajal (ICCs) is believed to be induced by Ca2+ transients and activation of anoctamin-1 (Ano1) channels in the plasma membrane. However, block of store-operated calcium entry (SOCE) or the Na-K-2Cl cotransporter (NKCC1) terminates pacemaker activity in ICC, indicating these transporters are involved in the initiation or maintenance of pacemaker activity. We hypothesized that SOCE contributes to pacemaker depolarization by maintaining [Ca2+] in the endoplasmic reticulum, which is the underlying source of Ca2+ transients for activation of Ano1. NKCC1 maintains the Cl− gradient supporting the driving force for inward current mediated by Ano1. Currently mechanisms sustaining release of Ca2+ and activation of Ano1 channels during the plateau phase of slow waves are unknown, but the reverse mode of the Na+/Ca2+ exchange may contribute. We generated a mathematical model of pacemaker activity based on current empirical observations from ICC of mouse small intestine that incorporates functions of SOCE and NKCC1. This model reproduces experimental findings, suggesting roles for SOCE and Ano1 channels: blocking of either NKCC1 or SOCE in our model terminates pacemaker activity. Direct contribution of NKCC1 to pacemaker activity in a beat-to-beat manner is not predicted by our model. Instead, NKCC1 plays a maintenance role supporting the driving force for Cl− efflux. Incorporation of SOCE allows the model to drive pacemaker activity without a diastolic depolarization, as observed in cardiac pacemaking. Further biological experiments are necessary to validate and further refine the roles of NKCC1, Na+/Ca2+ exchange, and Ano1 in the pacemaker mechanism of ICC.

Key points Colonic intramuscular interstitial cells of Cajal (ICC-IM) exhibit spontaneous Ca2+ transients manifesting as stochastic events from multiple firing sites with propagating Ca2+ waves occasionally observed. Firing of Ca2+ transients in ICC-IM is not coordinated with adjacent ICC-IM in a field of view or even with events from other firing sites within a single cell. Ca2+ transients, through activation of Ano1 channels and generation of inward current, cause net depolarization of colonic muscles. Ca2+ transients in ICC-IM rely on Ca2+ release from the endoplasmic reticulum via IP3 receptors, spatial amplification from RyRs and ongoing refilling of ER via the sarcoplasmic/endoplasmic-reticulum-Ca2+ -ATPase. ICC-IM are sustained by voltage-independent Ca2+ influx via store-operated Ca2+ entry. Some of the properties of Ca2+ in ICC-IM in the colon are similar to the behaviour of ICC located in the deep muscular plexus region of the small intestine, suggesting there are functional similarities between these classes of ICC. Abstract A component of the SIP syncytium that regulates smooth muscle excitability in the colon is the intramuscular class of interstitial cells of Cajal (ICC-IM). All classes of ICC (including ICC-IM) express Ca2+ -activated Cl- channels, encoded by Ano1, and rely upon this conductance for physiological functions. Thus, Ca2+ handling in ICC is fundamental to colonic motility. We examined Ca2+ handling mechanisms in ICC-IM of murine proximal colon expressing GCaMP6f in ICC. Several Ca2+ firing sites were detected in each cell. While individual sites displayed rhythmic Ca2+ events, the overall pattern of Ca2+ transients was stochastic. No correlation was found between discrete Ca2+ firing sites in the same cell or in adjacent cells. Ca2+ transients in some cells initiated Ca2+ waves that spread along the cell at ∼100 µm s-1 . Ca2+ transients were caused by release from intracellular stores, but depended strongly on store-operated Ca2+ entry mechanisms. ICC Ca2+ transient firing regulated the resting membrane potential of colonic tissues as a specific Ano1 antagonist hyperpolarized colonic muscles by ∼10 mV. Ca2+ transient firing was independent of membrane potential and not affected by blockade of L- or T-type Ca2+ channels. Mechanisms regulating Ca2+ transients in the proximal colon displayed both similarities to and differences from the intramuscular type of ICC in the small intestine. Similarities and differences in Ca2+ release patterns might determine how ICC respond to neurotransmission in these two regions of the gastrointestinal tract.

Transient outward, or “A-type,” currents are rapidly inactivating voltage-gated potassium currents that operate at negative membrane potentials. A-type currents have not been reported in the gastric fundus, a tonic smooth muscle. We used whole cell voltage clamp to identify and characterize A-type currents in smooth muscle cells (SMCs) isolated from murine fundus. A-type currents were robust in these cells with peak amplitudes averaging 1.5 nA at 0 mV. Inactivation was rapid with a time constant of 71 ms at 0 mV; recovery from inactivation at −80 mV was similarly rapid with a time constant of 75 ms. A-type currents in fundus were blocked by 4-aminopyridine (4-AP), flecainide, and phrixotoxin-1 (PaTX1). Remaining currents after 4-AP and PaTX1 displayed half-activation potentials that were shifted to more positive potentials and showed incomplete inactivation. Currents after tetraethylammonium (TEA) displayed half inactivation at −48.1 ± 1.0 mV. Conventional microelectrode and contractile experiments on intact fundus muscles showed that 4-AP depolarized membrane potential and increased tone under conditions in which enteric neurotransmission was blocked. These data suggest that A-type K+ channels in fundus SMCs are likely active at physiological membrane potentials, and sustained activation of A-type channels contributes to the negative membrane potentials of this tonic smooth muscle. Quantitative analysis of Kv4 expression showed that Kcnd3 was dominantly expressed in fundus SMCs. These data were confirmed by immunohistochemistry, which revealed Kv4.3-like immunoreactivity within the tunica muscularis. These observations indicate that Kv4 channels likely form the A-type current in murine fundus SMCs.

Volume accommodation occurs via a novel mechanism involving interstitial cells in detrusor muscles. The interstitial cells in the bladder are PDGFRα+ , and they restrain the excitability of smooth muscle at low levels and prevent the development of transient contractions (TCs). A common clinical manifestation of spinal cord injury (SCI)-induced bladder dysfunction is detrusor overactivity (DO). Although a myogenic origin of DO after SCI has been suggested, a mechanism for development of SCI-induced DO has not been determined. In this study we hypothesized that SCI-induced DO is related to loss of function in the regulatory mechanism provided by PDGFRα+ cells. Our results showed that transcriptional expression of Pdgfra and Kcnn3 was decreased after SCI. Proteins encoded by these genes also decreased after SCI, and a reduction in PDGFRα+ cell density was also documented. Loss of PDGFRα + cells was due to apoptosis. TCs in ex vivo bladders during filling increased dramatically after SCI, and this was related to the loss of regulation provided by SK channels, as we observed decreased sensitivity to apamin. These findings show that damage to the mechanism restraining muscle contraction during bladder filling that is provided by PDGFRα + cells is causative in the development of DO after SCI.

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, propulsive contractions. Regular RP contractions rely on pacemaker activity, which originates from the most proximal region of the RP at the pelvis-kidney junction (PKJ). Due to the difficulty in accessing and preserving intact preparations of the PKJ, most investigations on RP pacemaking have focused on single-cell electrophysiology and Ca2+ imaging experiments. Although important revelations on RP pacemaking have emerged from such work, these experiments have several intrinsic limitations, including the inability to accurately determine cellular identity in mixed suspensions and the lack of in situ imaging of RP pacemaker activity. These factors have resulted in a limited understanding of the mechanisms that underlie normal rhythmic RP contractions. In this paper, a protocol is described to prepare intact segments of mouse PKJ using a vibratome sectioning technique. By combining this approach with mice expressing cell-specific reporters and genetically encoded Ca2+ indicators, investigators may be able to more accurately study the specific cell types and mechanisms responsible for peristaltic RP contractions that are vital for normal urine transport.

Two types of voltage-dependent inward currents were evoked by depolarization in murine antral smooth muscle cells (SMCs) bathed in Ca2+-containing physiological solution: high-voltage-activated (HVA) and low-voltage-activated (LVA) inward currents. We examined whether the LVA current was due to: 1) T-type Ca2+ channels, 2) Ca2+-activated Cl−channels, 3) nonselective cation channels (NSCC), or 4) voltage-dependent K+ channels. Replacement of external Ca2+ (2 mM) with equimolar Ba2+ increased the amplitude of the HVA current but blocked the LVA current. Nicardipine blocked the HVA current, and in the presence of nicardipine, T-type Ca2+ blockers failed to block LVA current. A Cl− channel antagonist had little effect on LVA current. Cation-free external solution completely abolished both HVA and LVA currents. Addition of Ca2+ to the solution restored only HVA currents. Addition of K+ (5 mM) to otherwise cation-free solution induced LVA current that reversed at −20 mV. These data suggest that LVA current is not due to T-type Ca2+ channels, Ca2+-activated Cl− channels, or NSCC. A-type K+ (KA) currents and delayed rectifying K+ (KDR) currents can be resolved in antral SMCs dialyzed with a solution containing 140 mM K+. When cells were exposed to high K+ external solution and dialyzed with Cs+-rich solution in the presence of nicardipine, LVA current was evoked and reversed at positive potentials. LVA currents were blocked by K+ channel blockers, 4-aminopyridine, and tetraethylammonium. In conclusion, LVA inward currents can be generated by K+ influx via KA channels in murine antral SMCs when cells were dialyzed with Cs+-rich solution.

Classical concepts of peripheral neurotransmission were insufficient to explain enteric inhibitory neurotransmission. Geoffrey Burnstock and colleagues developed the idea that ATP or a related purine satisfies the criteria for a neurotransmitter and serves as an enteric inhibitory neurotransmitter in GI muscles. Cloning of purinergic receptors and development of specific drugs and transgenic mice have shown that enteric inhibitory responses depend upon P2Y(1) receptors in post-junctional cells. The post-junctional cells that transduce purinergic neurotransmitters in the GI tract are PDGFRα(+) cells and not smooth muscle cells (SMCs). PDGFRα(+) cells express P2Y(1) receptors, are activated by enteric inhibitory nerve stimulation and generate Ca(2+) oscillations, express small-conductance Ca(2+)-activated K(+) channels (SK3), and generate outward currents when exposed to P2Y(1) agonists. These properties are consistent with post-junctional purinergic responses, and similar responses and effectors are not functional in SMCs. Refinements in methodologies to measure purines in tissue superfusates, such as high-performance liquid chromatography (HPLC) coupled with etheno-derivatization of purines and fluorescence detection, revealed that multiple purines are released during stimulation of intrinsic nerves. β-NAD(+) and other purines, better satisfy criteria for the purinergic neurotransmitter than ATP. HPLC has also allowed better detection of purine metabolites, and coupled with isolation of specific types of post-junctional cells, has provided new concepts about deactivation of purine neurotransmitters. In spite of steady progress, many unknowns about purinergic neurotransmission remain and require additional investigation to understand this important regulatory mechanism in GI motility.

Obesity is a critical risk factor of several life-threatening diseases and the prevalence in adults has dramatically increased over the past ten years. In the USA the age-adjusted prevalence of obesity in adults was 42.4%, i.e., with a body mass index (BMI, weight (kg)/height (m)2) that exceeds 30 kg/m2. Obese individuals are at the higher risk of obesity-related diseases, co-morbid conditions, lower quality of life, and increased mortality more than those in the normal BMI range i.e., 18.5–24.9 kg/m2. Surgical treatment continues to be the most efficient and scientifically successful treatment for obese patients. Sleeve gastrectomy or vertical sleeve gastrectomy (VSG) is a relatively new gastric procedure to reduce body weight but is now the most popular bariatric operation. To date there have been few studies examining the changes in the cellular components and pacemaker activity that occur in the gastric wall following VSG and whether normal gastric activity recovers following VSG. In the present study we used a murine model to investigate the chronological changes of gastric excitability including electrophysiological, molecular and morphological changes in the gastric musculature following VSG. There is a significant disruption in specialized interstitial cells of Cajal in the gastric antrum following sleeve gastrectomy. This is associated with a loss of gastric pacemaker activity and post-junctional neuroeffector responses. Over a 4-month recovery period there was a gradual return in interstitial cells of Cajal networks, pacemaker activity and neural responses. These data describe for the first time the changes in gastric interstitial cells of Cajal networks, pacemaker activity and neuroeffector responses and the time-dependent recovery of ICC networks and normalization of motor activity and neural responses following VSG.

Background & Aims Interstitial cells of Cajal (ICCs) and pancreatic β cells require receptor tyrosine kinase (KIT) to develop and function properly. Degeneration of ICCs is linked to diabetic gastroparesis. The mechanisms linking diabetes and gastroparesis are unclear, but may involve microRNA (miRNA)-mediated post-transcriptional gene silencing in KIT+ cells. Methods We performed miRNA-sequencing analysis from isolated ICCs in diabetic mice and plasma from patients with idiopathic and diabetic gastroparesis. miR-10b-5p target genes were identified and validated in mouse and human cell lines. For loss-of-function studies, we used KIT+ cell-restricted mir-10b knockout mice and KIT+ cell depletion mice. For gain-of-function studies, a synthetic miR-10b-5p mimic was injected in multiple diabetic mouse models. We compared the efficacy of miR-10b-5p mimic treatment vs antidiabetic and prokinetic medicines. Results miR-10b-5p is highly expressed in ICCs from healthy mice, but drastically depleted in ICCs from diabetic mice. A conditional knockout of mir-10b in KIT+ cells or depletion of KIT+ cells in mice leads to degeneration of β cells and ICCs, resulting in diabetes and gastroparesis. miR-10b-5p targets the transcription factor Kruppel-like factor 11 (KLF11), which negatively regulates KIT expression. The miR-10b-5p mimic or Klf11 small interfering RNAs injected into mir-10b knockout mice, diet-induced diabetic mice, and TALLYHO polygenic diabetic mice rescue the diabetes and gastroparesis phenotype for an extended period of time. Furthermore, the miR-10b-5p mimic is more effective in improving glucose homoeostasis and gastrointestinal motility compared with common antidiabetic and prokinetic medications. Conclusions miR-10b-5p is a key regulator in diabetes and gastrointestinal dysmotility via the KLF11-KIT pathway. Restoration of miR-10b-5p may provide therapeutic benefits for these disorders.

Interstitial cells of Cajal (ICC) generate pacemaker activity responsible for phasic contractions in colonic segmentation and peristalsis. ICC along the submucosal border (ICC-SM) contribute to mixing and more complex patterns of colonic motility. We show the complex patterns of Ca2+ signaling in ICC-SM and the relationship between ICC-SM Ca2+ transients and activation of smooth muscle cells (SMCs) using optogenetic tools. ICC-SM displayed rhythmic firing of Ca2+transients ~ 15 cpm and paced adjacent SMCs. The majority of spontaneous activity occurred in regular Ca2+ transients clusters (CTCs) that propagated through the network. CTCs were organized and dependent upon Ca2+ entry through voltage-dependent Ca2+ conductances, L- and T-type Ca2+ channels. Removal of Ca2+ from the external solution abolished CTCs. Ca2+ release mechanisms reduced the duration and amplitude of Ca2+ transients but did not block CTCs. These data reveal how colonic pacemaker ICC-SM exhibit complex Ca2+-firing patterns and drive smooth muscle activity and overall colonic contractions.

High fidelity segmentation of both macro and microvascular structure of the retina plays a pivotal role in determining degenerative retinal diseases, yet it is a difficult problem. Due to successive resolution loss in the encoding phase combined with the inability to recover this lost information in the decoding phase, autoencoding based segmentation approaches are limited in their ability to extract retinal microvascular structure. We propose RV-GAN, a new multi-scale generative architecture for accurate retinal vessel segmentation to alleviate this. The proposed architecture uses two generators and two multi-scale autoencoding discriminators for better microvessel localization and segmentation. In order to avoid the loss of fidelity suffered by traditional GAN-based segmentation systems, we introduce a novel weighted feature matching loss. This new loss incorporates and prioritizes features from the discriminator’s decoder over the encoder. Doing so combined with the fact that the discriminator’s decoder attempts to determine real or fake images at the pixel level better preserves macro and microvascular structure. By combining reconstruction and weighted feature matching loss, the proposed architecture achieves an area under the curve (AUC) of 0.9887, 0.9914, and 0.9887 in pixel-wise segmentation of retinal vasculature from three publicly available datasets, namely DRIVE, CHASE-DB1, and STARE, respectively. Additionally, RV-GAN outperforms other architectures in two additional relevant metrics, mean intersection-over-union (Mean-IOU) and structural similarity measure (SSIM).

Interstitial cells of Cajal (ICC) generate pacemaker activity responsible for phasic contractions in colonic segmentation and peristalsis. ICC along the submucosal border (ICC-SM) contributing to mixing and more complex patterns of colonic motility. We show the complex patterns of Ca2+signaling in ICC-SM and the relationship between ICC-SM Ca2+transients and activation of SMCs using optogenetic tools. ICC-SM displayed rhythmic firing of Ca2+transients ∼15 cpm and paced adjacent SMCs. The majority of spontaneous activity occurred in regular Ca2+ transients clusters (CTCs) that propagated through the network. CTCs were organized and dependent upon Ca2+entry through voltage-dependent Ca2+conductances, L- and T-type Ca2+channels. Removal of Ca2+from the external solution abolished CTCs. Ca2+release mechanisms reduced the duration and amplitude of Ca2+transients but did not block CTCs. These data reveal how colonic pacemaker ICC-SM exhibit complex Ca2+firing patterns and drive smooth muscle activity and overall colonic contractions.SynopsisHow Ca2+signaling in colonic submucosal pacemaker cells couples to smooth muscle responses is unknown. This study shows how ICC modulate colonic motility via complex Ca2+signaling and defines Ca2+transients’ sources using optogenetic techniques.

High-resolution Ca(2+) imaging to study cellular Ca(2+) behaviors has led to the creation of large datasets with a profound need for standardized and accurate analysis. To analyze these datasets, spatio-temporal maps (STMaps) that allow for 2D visualization of Ca(2+) signals as a function of time and space are often used. Methods of STMap analysis rely on a highly arduous process of user defined segmentation and event-based data retrieval. These methods are often time consuming, lack accuracy, and extremely variable between users. We designed a novel automated machine-learning based plugin for the analysis of Ca(2+) STMaps (STMapAuto). The plugin includes optimized tools for Ca(2+) signal preprocessing, automated segmentation, and automated extraction of key Ca(2+) event information such as: duration, spatial spread, frequency, propagation angle, and intensity in a variety of cell types including the Interstitial cells of Cajal (ICC). The plugin is fully implemented in Fiji and able to accurately detect and expeditiously quantify Ca(2+) transient parameters from ICC. The plugin’s speed of analysis of large-datasets was 197-fold faster than the commonly used single pixel-line method of analysis. The automated machine-learning based plugin described dramatically reduces opportunities for user error and provides a consistent method to allow high-throughput analysis of STMap datasets.

In this issue of Science Signaling, Thakore et al. report that the Ca(2+) permeable channel TRPML1 closely associates with ryanodine receptors to induce Ca(2+) sparks in native arterial myocytes. Functional studies revealed a key role for TRPML1 channels in regulation of arterial myocyte contractility and blood pressure.

TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-permeable, nonselective cation channel localized to the membranes of endosomes and lysosomes and is not present or functional on the plasma membrane. Ca2+ released from endosomes and lysosomes into the cytosol through TRPML1 channels is vital for trafficking, acidification, and other basic functions of these organelles. Here, we investigated the function of TRPML1 channels in fully differentiated contractile vascular smooth muscle cells (SMCs). In live-cell confocal imaging studies, we found that most endosomes and lysosomes in freshly isolated SMCs from cerebral arteries were essentially immobile. Using nanoscale super-resolution microscopy, we found that TRPML1 channels present in late endosomes and lysosomes formed stable complexes with type 2 ryanodine receptors (RyR2) on the sarcoplasmic reticulum (SR). Spontaneous Ca2+ signals resulting from the release of SR Ca2+ through RyR2s ("Ca2+ sparks") and corresponding Ca2+-activated K+ channel activity are critically important for balancing vasoconstriction. We found that these signals were essentially absent in SMCs from TRPML1-knockout (Mcoln1-/- ) mice. Using ex vivo pressure myography, we found that loss of this critical signaling cascade exaggerated the vasoconstrictor responses of cerebral and mesenteric resistance arteries. In vivo radiotelemetry studies showed that Mcoln1-/- mice were spontaneously hypertensive. We conclude that TRPML1 is crucial for the initiation of Ca2+ sparks in SMCs and the regulation of vascular contractility and blood pressure.

Patients with diabetes often develop gastrointestinal motor problems, including gastroparesis. Previous studies have suggested this gastric motor disorder was a consequence of an enteric neuropathy. Disruptions in interstitial cells of Cajal (ICC) have also been reported. A thorough examination of functional changes in gastric motor activity during diabetes has not yet been performed. We comprehensively examined the gastric antrums of Lepob mice using functional, morphological, and molecular techniques to determine the pathophysiological consequences in this type 2 diabetic animal model. Video analysis and isometric force measurements revealed higher frequency and less robust antral contractions in Lepob mice compared with controls. Electrical pacemaker activity was reduced in amplitude and increased in frequency. Populations of enteric neurons, ICC, and platelet-derived growth factor receptor α+ cells were unchanged. Analysis of components of the prostaglandin pathway revealed upregulation of multiple enzymes and receptors. Prostaglandin-endoperoxide synthase-2 inhibition increased slow wave amplitudes and reduced frequency of diabetic antrums. In conclusion, gastric pacemaker and contractile activity is disordered in type 2 diabetic mice, and this appears to be a consequence of excessive prostaglandin signaling. Inhibition of prostaglandin synthesis may provide a novel treatment for diabetic gastric motility disorders.