Propulsive colonic contractions are mediated by inhibition-driven poststimulus responses that originate in interstitial cells of Cajal.

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.

Clinical analyses of ultrasound-guided nerve block in lower-extremity surgery: A retrospective study.

PURPOSE: The purpose of this study is to examine the clinical effects and results of lower-extremity surgery under ultrasound-guided nerve block; time required for nerve block, anesthesia onset time, duration of anesthesia, duration of analgesia, tolerable tourniquet time, visual analog scale (VAS) satisfaction score, and anesthetic-related complications. METHODS: A total of 3312 cases (2597 patients) from January 2010 to April 2015 were analyzed retrospectively. A senior author performed ultrasound-guided nerve block of the lateral femoral cutaneous nerve (LFCN, 630 cases), femoral nerve (FN, 2503 cases), obturator nerve (ON, 366 cases), sciatic nerve (SN, 3271 cases), or posterior femoral cutaneous nerve (PFCN, 222 cases) depending on the type of surgery. Time required for nerve block, anesthesia onset time, duration of anesthesia, duration of analgesia, tolerable tourniquet time, VAS satisfaction score, and anesthetic-related complications were analyzed. RESULTS: The mean times required were 1.1 min for SN block, 2.5 min for FN/SN block (1762 cases), and 4.8 min for FN/SN/LFCN/ON block. The mean anesthesia onset time was 48 min. The mean durations of anesthesia were 4.5 h for FN dermatome and 5.6 h for SN dermatome. The mean duration of analgesia was 11.5 h. The mean tolerable tourniquet times after were 35, 51, and 84 min after SN block, FN/SN block, and FN/SN/LFCN/ON block, respectively. The mean VAS satisfaction score was 9.3. There were no anesthetic-related complications, such as infection, hematoma, paralysis, or nerve irritation. CONCLUSION: Selective block of the LFCN, FN, ON, SN, and PFCN based on the locations of lesions and type of surgery showed favorable clinical results with high efficacy. Ultrasound-guided nerve block may be a good option for anesthesia and analgesia in lower-extremity surgery.

Myosalpinx Contractions Are Essential for Egg Transport Along the Oviduct and Are Disrupted in Reproductive Tract Diseases.

Oviducts (also called fallopian tubes) are smooth muscle-lined tubular organs that at one end extend in a trumpet bell-like fashion to surround the ovary, and at the other connect to the uterus. Contractions of the oviduct smooth muscle (myosalpinx) and the wafting motion of the ciliated epithelium that lines these tubes facilitate bidirectional transport of gametes so that newly released ovum(s) are transported in one direction (pro-uterus) while spermatozoa are transported in the opposite direction (pro-ovary). These transport processes must be temporally coordinated so that the ovum and spermatozoa meet in the ampulla, the site of fertilization. Once fertilized, the early embryo begins another precisely timed journey towards the uterus for implantation. Myosalpinx contractions facilitate this journey too, while luminal secretions from secretory epithelial cells aid early embryo maturation.The previous paradigm was that oviduct transport processes were primarily controlled by fluid currents generated by the incessant beat of the ciliated epithelium towards the uterus. More recently, video imaging and spatiotemporal mapping have suggested a novel paradigm in which ovum/embryo transport is highly dependent upon phasic and propulsive contractions of the myosalpinx. A specialized population of pacemaker cells, termed oviduct interstitial cells of Cajal (ICC-OVI), generate the electrical activity that drives these contractions. The ionic mechanisms underlying this pacemaker activity are dependent upon the calcium-activated chloride conductance, Ano1.This chapter discusses the basis of oviduct pacemaker activity, its hormonal regulation, and the underlying mechanisms and repercussions when this activity becomes disrupted during inflammatory responses to bacterial infections, such as Chlamydia.

Femoral Footprint for Anatomical Single-Bundle Anterior Cruciate Ligament Reconstruction: A Cadaveric Study.

PURPOSE: To identify the femoral footprint of the anterior cruciate ligament (ACL) in Koreans. MATERIALS AND METHODS: Eighteen embalmed cadaveric knees (mean age, 70 years) were examined. First, the shape of the ACL was determined macroscopically. After the ACL femoral footprint was defined, the ACL was cut from the femur and a Kirschner wire was inserted into the center of the ACL, and the position was verified with a C-arm. The position was quantified on the C-arm field using the quadrant method. The length and width of the ACL were measured. RESULTS: Macroscopically, the ACL is a flat single bundle with an average length of 34 mm and an average width of 9 mm. On average, the center of the ACL insertion site measured with the quadrant method was positioned at 29.5%±2.8% in an anterior direction (from posterior), and at 38.5%±3.2% in a distal direction (from Blumensaat's line). The anterior and posterior margins of the ACL femoral footprint were the resident's ridge and the cartilage margin of the lateral femoral condyle, respectively. CONCLUSIONS: The center of the ACL femoral footprint is positioned more anteriorly and distally than the positions identified in previous studies.

Extraforaminal approach of biportal endoscopic spinal surgery: a new endoscopic technique for transforaminal decompression and discectomy.

This study was performed to describe the extraforaminal approach of biportal endoscopic spinal surgery (BESS) as a new endoscopic technique for transforaminal decompression and discectomy and to demonstrate the clinical outcomes of this new procedure for the first time. Twenty-one patients (27 segments) who underwent the extraforaminal approach of BESS between March 2015 and April 2016 were enrolled according to the inclusion and exclusion criteria. The operative time (minutes/level) and complications after the procedure were recorded. The visual analog scale (VAS) score was checked to assess the degree of radicular leg pain preoperatively and at the time of the last follow-up. The modified Macnab criteria were used to examine the clinical outcomes at the time of the last follow-up. The mean duration of the follow-up period was 14.8 months (minimum duration 12 months). The mean operative time was 96.7 minutes for one level. The mean VAS score for radicular leg pain dropped from a preoperative score of 7.5 ± 0.9 to a final follow-up score of 2.5 ± 1.2 (p < 0.001). The final outcome according to the modified Macnab criteria was excellent in 5 patients (23.8%), good in 12 (57.2%), fair in 4 (19.0%), and poor in 0. Therefore, excellent or good results (a satisfied outcome) were obtained in 80.9% of the patients. Complications were limited to one dural tear (4.8%). The authors found that the extraforaminal approach of BESS was a feasible and advantageous endoscopic technique for the treatment of foraminal lesions, including stenosis and disc herniation. They suggest that this technique represents a useful, alternative, minimally invasive method that can be used to treat lumbar foraminal stenosis and disc herniation.

Temporary ipsilateral stiff shoulder after operative fixation of distal radial fractures.

BACKGROUND: This study was conducted to identify variables affecting the development of temporary stiff shoulder after operative fixation for distal radial fractures (DRF). MATERIALS AND METHODS: The study retrospectively analyzed 167 patients who had undergone internal fixation using volar locking plate for DRF between 2010 and 2013. Group 1 was denoted as the "normal group," and group 2 was denoted as the "stiff shoulder group." Basic demographic factors evaluated included age, sex, bone mineral density (BMD), and the dominancy. Also investigated were radiologic variables, including concurrent fractures of the styloid process, positive ulnar variances, classification of DRF, and morphologic type of the distal radioulnar joint. Finally, the type of plate, methods used for postoperative protection, and time of union were analyzed. RESULTS: Group 1 consisted of 114 patients, and group 2 consisted of 53 patients. On overall univariate analysis, BMD, hand dominancy, and the protective methods after plating were significantly different between the 2 groups. On multivariate analysis, a lower BMD and injury on the nondominant side were significant factors for shoulder stiffness. Stiffness was significantly higher in patients with a mean BMD < -2.6 than in patients with a mean BMD ≥ -2.6. At the final follow-up, all of the 53 patients in group 2 were relieved of the symptoms of a stiff shoulder. CONCLUSIONS: A lower BMD and injury on the nondominant distal radius were distinct factors for the development of a stiff shoulder after operative fixation in DRF. Fortunately, nonoperative treatments, such as stretching exercises/injections, were useful for the relief of these symptoms in the short-term follow-up.

Complete sequence analysis of the mitochondrial genome of Auriculastra duplicata (Mollusca, Gastropoda, Ellobiidae).

The mitochondrial genome of the gastropod Auriculastra duplicata was completely sequenced. It was 13,920 bp in length and comprised 37 genes; two rrn genes and 22 trn genes. Phylogenetic analyses based on the concatenated protein-coding genes depicted the polyphyly of all species belonging to the family Ellobiidae; however, monophyly was observed among all species belonging to the subfamily Ellobiinae, in which A. duplicata clustered consistently with Auriculinella bidentata.

Convergence of inhibitory neural inputs regulate motor activity in the murine and monkey stomach.

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

Development of biomarker positivity analysis system for cancer diagnosis based on clinical data.

In Korea, there were 224,000 new cases of cancer and 75,334 deaths caused by cancer in 2013, which was three times more than the number of death caused by heart disease, the second leading cause of death. This study proposes a biomarker positivity analysis system based on clinical data, for personalized diagnosis and therapy of cancer. Data of 78,912 cases were obtained from immunopathology and surgical pathology reports. Data on sex, age, organ, diagnosis, and biomarkers were entered into a database. To verify the reliability of the clinical data, an additional 50,450 cases from positivity-related research papers were added. The proposed biomarker positivity analysis system makes it possible to extract and combine information for searching. The positivity values are in graphical and tabular format for ease of use. With a link to the internal network of the hospital, real-time pathology reports are available. Twenty-five pathology specialists are chosen as subjects to further confirm the reliability of this system; primary assessment results demonstrate a satisfaction level of 4.7 out of 5 and a concordance rate of 79% with positive data under the same conditions as reported in the literature. In the present study, analysis methods and platforms using large volumes of clinical and literature data are developed for cancer prognoses. It is expected that these tools will benefit both healthcare professionals and non-professionals involved in cancer diagnosis and treatment.

A novel class of interstitial cells in the mouse and monkey female reproductive tracts.

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

Uridine adenosine tetraphosphate is a novel neurogenic P2Y1 receptor activator in the gut.

Enteric purinergic motor neurotransmission, acting through P2Y1 receptors (P2Y1R), mediates inhibitory neural control of the intestines. Recent studies have shown that NAD(+) and ADP ribose better meet criteria for enteric inhibitory neurotransmitters in colon than ATP or ADP. Here we report that human and murine colon muscles also release uridine adenosine tetraphosphate (Up4A) spontaneously and upon stimulation of enteric neurons. Release of Up4A was reduced by tetrodotoxin, suggesting that at least a portion of Up4A is of neural origin. Up4A caused relaxation (human and murine colons) and hyperpolarization (murine colon) that was blocked by the P2Y1R antagonist, MRS 2500, and by apamin, an inhibitor of Ca(2+)-activated small-conductance K(+) (SK) channels. Up4A responses were greatly reduced or absent in colons of P2ry1(-/-) mice. Up4A induced P2Y1R-SK-channel-mediated hyperpolarization in isolated PDGFRα(+) cells, which are postjunctional targets for purinergic neurotransmission. Up4A caused MRS 2500-sensitive Ca(2+) transients in human 1321N1 astrocytoma cells expressing human P2Y1R. Up4A was more potent than ATP, ADP, NAD(+), or ADP ribose in colonic muscles. In murine distal colon Up4A elicited transient P2Y1R-mediated relaxation followed by a suramin-sensitive contraction. HPLC analysis of Up4A degradation suggests that exogenous Up4A first forms UMP and ATP in the human colon and UDP and ADP in the murine colon. Adenosine then is generated by extracellular catabolism of ATP and ADP. However, the relaxation and hyperpolarization responses to Up4A are not mediated by its metabolites. This study shows that Up4A is a potent native agonist for P2Y1R and SK-channel activation in human and mouse colon.

Adenosine 5-diphosphate-ribose is a neural regulator in primate and murine large intestine along with ß-NAD(+).

Adenosine 5'-triphosphate (ATP) has long been considered to be the purine inhibitory neurotransmitter in gastrointestinal (GI) muscles, but recent studies indicate that another purine nucleotide, ß-nicotinamide adenine dinucleotide (ß-NAD(+)), meets pre- and postsynaptic criteria for a neurotransmitter better than ATP in primate and murine colons. Using a small-volume superfusion assay and HPLC with fluorescence detection and intracellular microelectrode techniques we compared ß-NAD(+) and ATP metabolism and postjunctional effects of the primary extracellular metabolites of ß-NAD(+) and ATP, namely ADP-ribose (ADPR) and ADP in colonic muscles from cynomolgus monkeys and wild-type (CD38(+/+)) and CD38(−/−) mice. ADPR and ADP caused membrane hyperpolarization that, like nerve-evoked inhibitory junctional potentials (IJPs), were inhibited by apamin. IJPs and hyperpolarization responses to ADPR, but not ADP, were inhibited by the P2Y1 receptor antagonist (1R,2S,4S,5S)-4-[2-iodo-6-(methylamino)-9H-purin-9-yl]-2-(phosphonooxy)bicyclo[3.1.0]hexane-1-methanol dihydrogen phosphate ester tetraammonium salt (MRS2500). Degradation of ß-NAD(+) and ADPR was greater per unit mass in muscles containing only nerve processes than in muscles also containing myenteric ganglia. Thus, mechanisms for generation of ADPR from ß-NAD(+) and for termination of the action of ADPR are likely to be present near sites of neurotransmitter release. Degradation of ß-NAD(+) to ADPR and other metabolites appears to be mediated by pathways besides CD38, the main NAD-glycohydrolase in mammals. Degradation of ß-NAD(+) and ATP were equal in colon. ADPR like its precursor, ß-NAD(+), mimicked the effects of the endogenous purine neurotransmitter in primate and murine colons. Taken together, our observations support a novel hypothesis in which multiple purines contribute to enteric inhibitory regulation of gastrointestinal motility.

Analysis of pacemaker activity in the human stomach.

Extracellular electrical recording and studies using animal models have helped establish important concepts of human gastric physiology. Accepted standards include electrical quiescence in the fundus, 3 cycles per minute (cpm) pacemaker activity in corpus and antrum, and a proximal-to-distal slow wave frequency gradient. We investigated slow wave pacemaker activity, contractions and distribution of interstitial cells of Cajal (ICC) in human gastric muscles. Muscles were obtained from patients undergoing gastric resection for cancer, and the anatomical locations of each specimen were mapped by the operating surgeon to 16 standardized regions of the stomach. Electrical slow waves were recorded with intracellular microelectrodes and contractions were recorded by isometric force techniques. Slow waves were routinely recorded from gastric fundus muscles. These events had similar waveforms as slow waves in more distal regions and were coupled to phasic contractions. Gastric slow wave frequency was significantly greater than 3 cpm in all regions of the stomach. Antral slow wave frequency often exceeded the highest frequency of pacemaker activity in the corpus. Chronotropic mechanisms such as muscarinic and prostaglandin receptor binding, stretch, extracelluar Ca(2+) and temperature were unable to explain the observed slow wave frequency that exceeded accepted normal levels. Muscles from all regions through the thickness of the muscularis demonstrated intrinsic pacemaker activity, and this corresponded with the widespread distribution in ICC we mapped throughout the tunica muscularis. Our findings suggest that extracellular electrical recording has underestimated human slow wave frequency and mechanisms of human gastric function may differ from standard laboratory animal models.

ß-nicotinamide adenine dinucleotide is an enteric inhibitory neurotransmitter in human and nonhuman primate colons.

BACKGROUND & AIMS: An important component of enteric inhibitory neurotransmission is mediated by a purine neurotransmitter, such as adenosine 5'-triphosphate (ATP), binding to P2Y1 receptors and activating small conductance K(+) channels. In murine colon ß-nicotinamide adenine dinucleotide (ß-NAD) is released with ATP and mimics the pharmacology of inhibitory neurotransmission better than ATP. Here ß-NAD and ATP were compared as possible inhibitory neurotransmitters in human and monkey colons. METHODS: A small-volume superfusion assay and high-pressure liquid chromatography with fluorescence detection were used to evaluate spontaneous and nerve-evoked overflow of ß-NAD, ATP, and metabolites. Postjunctional responses to nerve stimulation, ß-NAD and ATP were compared using intracellular membrane potential and force measurements. Effects of ß-NAD on smooth muscle cells (SMCs) were recorded by patch clamp. P2Y receptor transcripts were assayed by reverse transcription polymerase chain reaction. RESULTS: In contrast to ATP, overflow of ß-NAD evoked by electrical field stimulation correlated with stimulation frequency and was diminished by the neurotoxins, tetrodotoxin, and ω-conotoxin GVIA. Inhibitory junction potentials and responses to exogenous ß-NAD, but not ATP, were blocked by P2Y receptor antagonists suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS 2179), and (1R,2S,4S,5S)-4-[2-Iodo-6-(methylamino)-9H-purin-9-yl]-2-(phosphonooxy)bicyclo[3.1.0]hexane-1-methanol dihydrogen phosphate ester tetraammonium salt (MRS 2500). ß-NAD activated nonselective cation currents in SMCs, but failed to activate outward currents. CONCLUSIONS: ß-NAD meets the criteria for a neurotransmitter better than ATP in human and monkey colons and therefore may contribute to neural regulation of colonic motility. SMCs are unlikely targets for inhibitory purine neurotransmitters because dominant responses of SMCs were activation of net inward, rather than outward, current.

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 Ca(2+)-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 (Tmem16a(tm1Bdh)(/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.

Changes in the structure and function of ICC networks in ICC hyperplasia and gastrointestinal stromal tumors.

BACKGROUND & AIMS: Gastrointestinal stromal tumors (GISTs) express the receptor tyrosine kinase c-kit. Approximately 90% of GISTs have gain-of-function mutations in the Kit gene, which leads to its constitutive activation and drives malignant behavior of GISTs. Interstitial cells of Cajal (ICC) express c-kit; however, it is unknown whether uncontrolled hyperplasia of ICC is responsible for GISTs. Here, we sought to determine whether gain-of-function mutations in Kit lead to hyperplasia of all classes of ICC, whether ICC hyperplasia begins before birth, and whether functional defects occur in ICC hyperplasia or the development of GISTs. METHODS: Heterozygous mutant Kit(V558Delta)/+ mice that develop symptoms of human familial GISTs and prematurely die from pathology of the gastrointestinal tract were utilized and compared with wild-type controls. C-kit-immunohistochemistry and intracellular electrical recording of spontaneous and nerve-evoked activity were applied to examine the density and functionality of ICC in these mutants. RESULTS: There was considerable hyperplasia in all classes of ICC throughout the GI tract of Kit(V558Delta)/+ mice, except for ICC in the deep muscular plexus of the intestine. Spontaneous electrical activity and postjunctional neural responses in hyperplastic ICC tissues appeared normal but were up-regulated in the cecum, where GISTs were commonly found. CONCLUSIONS: Kit gain-of-function leads to hyperplasia of most classes of ICC throughout the GI tract. ICC retain normal pacemaker function and enteric neural responses well after development of hyperplasia.

Block of inhibitory junction potentials and TREK-1 channels in murine colon by Ca2+ store-active drugs.

Post-junctional enteric inhibitory responses are composed of at least two components attributed to the release of a purine and nitric oxide (NO). The nitrergic component is characterized by membrane potential hyperpolarization; however, the conductances involved and the role of Ca(2+) stores in regulating these conductances are controversial. Conventional microelectrode recordings were performed in intact muscle strips and whole-cell voltage clamp experiments were performed on freshly dispersed cells and COS7 cells stably transfected with TREK-1 channels. Here we show that several Ca(2+) store-active compounds, including caffeine, ryanodine, and cyclopiazonic acid, reduce inhibitory junction potentials and responses to sodium nitroprusside in murine colonic muscles. We previously proposed that two-pore K(+) channels of the TREK family mediate a portion of the hyperpolarization response to NO in colonic muscles. We tested the effects of Ca(2+) store-active drugs in COS cells expressing murine TREK-1 channels and found these compounds block TREK-1 currents. These effects were greatly attenuated by dialysing cells with protein kinase A inhibitory peptide (PKAI). Caffeine also blocked stretch-dependent K(+) (SDK) channels, thought to be due to expression of TREK channels, in colonic myocytes, but these effects were not apparent in excised patches. Taken together our data show that Ca(2+) store-active compounds inhibit TREK-1 channels, native SDK channels, and nitrergic inhibitory junction potentials. These effects appear to be due, in part, to the cAMP/PKA stimulatory actions of these drugs and inhibitory effects of TREK channels.

Beta-nicotinamide adenine dinucleotide is an inhibitory neurotransmitter in visceral smooth muscle.

Peripheral inhibitory nerves are physiological regulators of the contractile behavior of visceral smooth muscles. One of the transmitters responsible for inhibitory neurotransmission has been reputed to be a purine, possibly ATP. However, the exact identity of this substance has never been verified. Here we show that beta-nicotinamide adenine dinucleotide (beta-NAD), an inhibitory neurotransmitter candidate, is released by stimulation of enteric nerves in gastrointestinal muscles, and the pharmacological profile of beta-NAD mimics the endogenous neurotransmitter better than ATP. Levels of beta-NAD in superfusates of muscles after nerve stimulation exceed ATP by at least 30-fold; unlike ATP, the release of beta-NAD depends on the frequency of nerve stimulation. beta-NAD is released from enteric neurons, and release was blocked by tetrodotoxin or omega-conotoxin GVIA. beta-NAD is an agonist for P2Y1 receptors, as demonstrated by receptor-mediated responses in HEK293 cells expressing P2Y1 receptors. Exogenous beta-NAD mimics the effects of the enteric inhibitory neurotransmitter. Responses to beta-NAD and inhibitory junction potentials are blocked by the P2Y1-selective antagonist, MRS2179, and the nonselective P2 receptor antagonists, pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid and suramin. Responses to ATP are not blocked by these P2Y receptor inhibitors. The expression of CD38 in gastrointestinal muscles, and specifically in interstitial cells of Cajal, provides a means of transmitter disposal after stimulation. beta-NAD meets the traditional criteria for a neurotransmitter that contributes to enteric inhibitory regulation of visceral smooth muscles.

Diol- and triol-type ginseng saponins potentiate the apoptosis of NIH3T3 cells exposed to methyl methanesulfonate.

In this study we investigated the effect of ginseng saponins on the p53-dependent apoptosis in NIH3T3 cells exposed to methyl methanesulfonate (MMS), an alkylating agent. Trypan blue exclusion assay, cell morphology studies, and apoptotic index determined by acridine orange staining showed that the postincubation of MMS-exposed cells in medium containing diol- (PD) or triol-type (PT) ginseng saponins potentiate the apoptotic cell death. FACS analysis indicated that the increased apoptotic cell population in the saponin-postincubation group was accompanied by the accumulation of cells in G0/G1 phase. By Western blot analyses it was demonstrated that postincubation of saponins increases the expression of p53 and p21 in MMS-exposed cells but decreased that of CDK2, cyclin E and D1, and PCNA. The upregulation of p53 and p21 and downregulation of CDK2 was shown to be p53-dependent in experiments using the p53 antisense oligonucleotide. These results suggest that ginseng saponins contain components potentiating the apoptosis of MMS-exposed NIH3T3 cells via p53 and p21 activation, accompanied with by downregulation of cell cycle-related protein expression.