Validation of mitochondrial biomarkers and immune dynamics in polycystic ovary syndrome.

PROBLEM: Polycystic ovary syndrome (PCOS), a prevalent endocrine-metabolic disorder, presents considerable therapeutic challenges due to its complex and elusive pathophysiology. METHOD OF STUDY: We employed three machine learning algorithms to identify potential biomarkers within a training dataset, comprising GSE138518, GSE155489, and GSE193123. The diagnostic accuracy of these biomarkers was rigorously evaluated using a validation dataset using area under the curve (AUC) metrics. Further validation in clinical samples was conducted using PCR and immunofluorescence techniques. Additionally, we investigate the complex interplay among immune cells in PCOS using CIBERSORT to uncover the relationships between the identified biomarkers and various immune cell types. RESULTS: Our analysis identified ACSS2, LPIN1, and NR4A1 as key mitochondria-related biomarkers associated with PCOS. A notable difference was observed in the immune microenvironment between PCOS patients and healthy controls. In particular, LPIN1 exhibited a positive correlation with resting mast cells, whereas NR4A1 demonstrated a negative correlation with monocytes in PCOS patients. CONCLUSION: ACSS2, LPIN1, and NR4A1 emerge as PCOS-related diagnostic biomarkers and potential intervention targets, opening new avenues for the diagnosis and management of PCOS.

Potential Causal Association between Plasma Metabolites, Immunophenotypes, and Female Reproductive Disorders: A Two-Sample Mendelian Randomization Analysis.

BACKGROUND: While extensive research highlighted the involvement of metabolism and immune cells in female reproductive diseases, causality remains unestablished. METHODS: Instrumental variables for 486 circulating metabolites (N = 7824) and 731 immunophenotypes (N = 3757) were derived from a genome-wide association study (GWAS) meta-analysis. FinnGen contributed data on 14 female reproductive disorders. A bidirectional two-sample Mendelian randomization study was performed to determine the relationships between exposures and outcomes. The robustness of results, potential heterogeneity, and horizontal pleiotropy were examined through sensitivity analysis. RESULTS: High levels of mannose were found to be causally associated with increased risks of gestational diabetes (GDM) (OR [95% CI], 6.02 [2.85-12.73], p = 2.55 × 10-6). A genetically predicted elevation in the relative count of circulating CD28-CD25++CD8+ T cells was causally related to increased female infertility risk (OR [95% CI], 1.26 [1.14-1.40], p = 1.07 × 10-5), whereas a high absolute count of NKT cells reduced the risk of ectopic pregnancy (OR [95% CI], 0.87 [0.82-0.93], p = 5.94 × 10-6). These results remained consistent in sensitivity analyses. CONCLUSIONS: Our study supports mannose as a promising GDM biomarker and intervention target by integrating metabolomics and genomics.

Azo-PROTAC: Novel Light-Controlled Small-Molecule Tool for Protein Knockdown.

Reversibly altering endogenous protein levels are persistent issues. Herein, we designed photoswitchable azobenzene-proteolysis targeting chimeras (Azo-PROTACs) by including azobenzene moieties between ligands for the E3 ligase and the protein of interest. Azo-PROTACs are light-controlled small-molecule tools for protein knockdown in cells. The light-induced configuration change can switch the active state to induce protein degradation activity, which can be reversely controlled by light exposure in intact cells. We compared the protein degradation abilities of Azo-PROTACs with different configurations and linker lengths. Using the stable form with the best degradation ability against the BCR-ABL fusion and ABL proteins in myelogenous leukemia K562 cells, we showed that Azo-PROTAC combines the potent protein knockdown and facile cell uptake properties of the small-molecule PROTAC with a reversible photoswitchability, offering a promising chemical knockdown strategy based on the light-induced reversible on/off properties.

Automatic Identification of Breast Ultrasound Image Based on Supervised Block-Based Region Segmentation Algorithm and Features Combination Migration Deep Learning Model.

Breast cancer is a high-incidence type of cancer for women. Early diagnosis plays a crucial role in the successful treatment of the disease and the effective reduction of deaths. In this paper, deep learning technology combined with ultrasound imaging diagnosis was used to identify and determine whether the tumors were benign or malignant. First, the tumor regions were segmented from the breast ultrasound (BUS) images using the supervised block-based region segmentation algorithm. Then, a VGG-19 network pretrained on the ImageNet dataset was applied to the segmented BUS images to predict whether the breast tumor was benign or malignant. The benchmark data for bio-validation were obtained from 141 patients with 199 breast tumors, including 69 cases of malignancy and 130 cases of benign tumors. The experiment showed that the accuracy of the supervised block-based region segmentation algorithm was almost the same as that of manual segmentation; therefore, it can replace manual work. The diagnostic effect of the combination feature model established based on the depth feature of the B-mode ultrasonic imaging and strain elastography was better than that of the model established based on these two images alone. The correct recognition rate was 92.95%, and the AUC was 0.98 for the combination feature model.

Pectic polysaccharides extracted from Rauvolfia verticillata (Lour.) Baill. var. hainanensis Tsiang ameliorate ulcerative colitis via regulating the MAPKs and NF-κB pathways in dendritic cells.

This study was to investigate the effects and mechanisms of pectic polysaccharides (PP) extracted from Rauvolfia verticillata (Lour.) Baill. var. hainanensis Tsiang on dextran sulphate sodium (DSS)-induced ulcerative colitis (UC). Eighty female BALB/c mice were randomly divided into four groups: Control, DSS, DSS + salicylazosulfapyridine (SASP), and DSS+ PP. The disease activity index (DAI), overall physical activity, and blood stool were monitored daily to evaluate severity of UC. Histological scores of the colon were observed. The expression of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPKs) pathways in colon tissues and bone marrow-derived dendritic cells (DCs) was assessed by western blot, immunohistochemistry, electrophoretic mobility shift assay (EMSA) and real time polymerase chain reaction (RT-PCR). Cytokines were measured by enzyme-linked immunosorbent assay (ELISA). The overall physical activity, DAI and histological scores decreased in DSS+SASP and DSS+PP groups, compared with the DSS-alone group. Also, tumour necrosis factor α (TNF-α) and interleukin 6 (IL-6) reduced significantly while the expression of IκBα was up-regulated, extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK) and p38 were activated, in DSS+SASP and DSS+PP groups. PP inhibited activation of MAPKs and NF-κB pathways in the bone-marrow-derived DCs. In conclusion, PP significantly ameliorated murine DSS-induced UC model, via regulation of MAPKs and NF-κB pathways in DCs.

KangFuXin Liquid in the Treatment of Diabetic Foot Ulcer: A Systematic Review and Meta-Analysis.

BACKGROUND: Diabetic foot ulcer (DFU) is one of the most common complications of diabetes mellitus, with the wound not healing as expected and healing slowly. Poor control can develop into gangrene and even amputation. Currently, the existing treatments are not satisfactory enough. In China, KangFuXin liquid (KFXL) has been clinically used to treat DFU and has shown good clinical efficacy. In order to provide more reference to clinicians and experts, evidence of efficacy for it needs to be further rigorously evaluated. METHODS: Eight electronic databases were searched to identify eligible randomized clinical trials (RCTs) published from construction of the library to April 2019. There is no language or data restriction; 11 trials involving 889 participants met the inclusion criteria. These RCTs compared the total effective rate, cure rate, cure time, and adverse events associated with KFXL. The Cochrane Handbook guidelines were used to assess the risk of bias and to evaluate the methodological quality of eligible studies. The methodological quality of included studies was generally low. Dichotomous and continuous data were presented using risk ratios (RRs) and mean differences (MDs), respectively. RESULTS: Compared with the basic treatment, meta-analyses showed that KFXL combined with basic treatment can improve the total effective rate (RR = 1.38; 95% CI = 1.23-1.54; P < 0.00001; fixed effect model: I 2 = 32%) and cure rate (RR = 1.67; 95% CI = 1.17-2.38; P=0.005; random effect model: I 2 = 65%), and shorten the healing time (MD = -5.73; 95% CI = -6.95 to -4.52; P < 0.00001; random effect model). Moreover, under the same basic treatment, KFXL had a better effect than external use of pharmaceutical medications (RR = 1.95; 95% CI = 1.30-2.93; P=0.001), but the cure rate was not significantly different. Also, KFXL had nothing to do with adverse reactions. CONCLUSION: The evidence confirms that KFXL is an effective treatment for DFU. However, further large-scale, rigorously designed trials and high-quality studies are needed to confirm the role of KFXL in the treatment of DFU.

Commensal microbiota induces colonic barrier structure and functions that contribute to homeostasis.

The intestinal barrier encompasses structural, permeability and immune aspects of the gut mucosa that, when disrupted, may contribute to chronic inflammation. Although gnotobiotic studies have demonstrated the effects of microbiota on mucosal and systemic immunity, as well as intestinal barrier architecture and innate immune characteristics, its impact on barrier function remains unclear. We compared germ-free and conventional mice, as well as mice colonized with human fecal microbiota that were followed for 21 days post-colonization. Colonic barrier structure was investigated by immunohistochemistry, molecular and electron microscopy techniques. Permeability was assessed in colon tissue by Ussing chambers, and by serum LPS and MDP detection using TLR4- and NOD2-NFκB reporter assays. Microbiota profile was determined by Illumina 16S rRNA gene sequencing. Low dose dextran sodium sulfate was administered to assess microbiota-induced barrier changes on resistance to colonic injury. Permeability to paracellular probes and mucus layer structure resembled that of conventional mice by day 7 post-colonization, coinciding with reduced claudin-1 expression and transient IL-18 production by intestinal epithelial cells. These post-colonization adaptations were associated with decreased systemic bacterial antigen exposure and reduced susceptibility to intestinal injury. In conclusion, commensal colonization promotes physiological barrier structural and functional adaptations that contribute to intestinal homeostasis.

Abnormal absorptive colonic motor activity in germ-free mice is rectified by butyrate, an effect possibly mediated by mucosal serotonin.

The role of short-chain fatty acids (SCFAs) in the control of colonic motility is controversial. Germ-free (GF) mice are unable to produce these metabolites and serve as a model to study how their absence affects colonic motility. GF transit is slower than controls, and colonization of these mice improves transit and serotonin [5-hydroxytryptamine (5-HT)] levels. Our aim was to determine the role SCFAs play in improving transit and whether this is dependent on mucosal 5-HT signaling. Motility was assessed in GF mice via spatiotemporal mapping. First, motor patterns in the whole colon were measured ex vivo with or without luminal SCFA, and outflow from the colon was recorded to quantify outflow caused by individual propulsive contractions. Second, artificial fecal pellet propulsion was measured. Motility was then assessed in tryptophan hydroxylase-1 (TPH1) knockout (KO) mice, devoid of mucosal 5-HT, with phosphate buffer, butyrate, or propionate intraluminal perfusion. GF mice exhibited a lower proportion of propulsive contractions, lower volume of outflow/contraction, slower velocity of contractions, and slower propulsion of fecal pellets compared with controls. SCFAs changed motility patterns to that of controls in all parameters. Butyrate administration increased the proportion of propulsive contractions in controls yet failed to in TPH1 KO mice. Propionate inhibited propulsive contractions in all mice. Our results reveal significant abnormalities in the propulsive nature of colonic motor patterns in GF mice, explaining the decreased transit time in in vivo studies. We show that butyrate but not propionate activates propulsive motility and that this may require mucosal 5-HT. NEW & NOTEWORTHY Understanding the role that the microbiota play in governing the physiology of colonic motility is lacking. Here, we offer for the first time, to our knowledge, a detailed analysis of colonic motor patterns and pellet propulsion using spatiotemporal mapping in the absence of microbiota. We show a striking difference in germ-free and control phenotypes and attribute this to a lack of fermentation-produced short-chain fatty acid. We then show that butyrate but not propionate can restore motility and that the butyrate effect likely requires mucosal 5-hydroxytryptamine.

Altered Esophageal Mucosal Structure in Patients with Celiac Disease.

Background/Aim. Reflux symptoms (RS) are common in patients with celiac disease (CD), a chronic enteropathy that affects primarily the small intestine. We evaluated mucosal integrity and motility of the lower esophagus as mechanisms contributing to RS generation in patients with CD. Methods. We enrolled newly diagnosed CD patients with and without RS, nonceliac patients with classical reflux disease (GERD), and controls (without RS). Endoscopic biopsies from the distal esophagus were assessed for dilated intercellular space (DIS) by light microscopy and electron microscopy. Tight junction (TJ) mRNA proteins expression for zonula occludens-1 (ZO-1) and claudin-2 and claudin-3 (CLDN-2; CLDN-3) was determined using qRT-PCR. Results. DIS scores were higher in patients with active CD than in controls, but similar to GERD patients. The altered DIS was found even in CD patients without RS and normalized after one year of a gluten-free diet. CD patients with and without RS had lower expression of ZO-1 than controls. The expression of CLDN-2 and CLDN-3 was similar in CD and GERD patients. Conclusions. Our study shows that patients with active CD have altered esophageal mucosal integrity, independently of the presence of RS. The altered expression of ZO-1 may underlie loss of TJ integrity in the esophageal mucosa and may contribute to RS generation.

Vagal Fibers Form Associations With Interstitial Cells of Cajal During Fetal Development.

Vagal intramuscular arrays (IMAs) have been shown to form complexes with intramuscular interstitial cells of Cajal (ICC). We tested the hypothesis that associations between vagal nerve endings and ICC arise in fetal development. Intraganglionic laminar endings (IGLEs) and IMAs were identified by applying 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanineperchlorate (DiI) to vagal nerve trunks and myenteric plexus (MP) and intramuscular (IM) ICC were immunolabeled with antibodies to c-Kit in fetal and early postnatal mice (E16-P7). At E16, c-Kit immunoreactive cells were abundant in the primordial smooth muscle, with early ICC networks discernable by E18 and ongoing organization at P1 and P7. The distribution of vagal endings was found to change during the course of development, with significantly more putative IGLEs in the prenatal compared to the postnatal period and less IMAs in the prenatal compared to postnatal period. Associations of ICC with both IGLEs and IMAs were detected as early as E16 and were maintained into postnatal life. These findings suggest that vagal fibers begin to associate with ICC during prenatal development. Future studies will be needed to determine the mechanisms through which vagal endings and ICC interact.

Ano1 is a better marker than c-Kit for transcript analysis of single interstitial cells of Cajal in culture.

The interstitial cells of Cajal (ICC) drive the slow wave-associated contractions in the small intestine. A commonly used marker for these cells is c-Kit, but another marker named Ano1 was recently described. This study uses single-cell RT-PCR, qPCR and immunohistochemistry to determine if Ano1 could be reliably used as a molecular marker for ICC in single-cell mRNA analysis. Here, we report on the relationship between the expression of c-Kit and Ano1 in single ICC in culture. We observed that Ano1 is expressed in more than 60% of the collected cells, whereas c-Kit is found only in 22% of the cells (n = 18). When we stained ICC primary cultures for c-KIT and ANO1 protein, we found complete co-localization in all the preparations. We propose that this difference is due to the regulation of c-Kit mRNA in culture. This regulation gives rise to low levels of its transcript, while Ano1 is expressed more prominently in culture on day 4. We also propose that Ano1 is more suitable for single-cell expression analysis as a marker for cell identity than c-Kit at the mRNA level. We hope this evidence will help to validate and increase the success of future studies characterizing single ICC expression patterns.

Discrepancies between c-Kit positive and Ano1 positive ICC-SMP in the W/Wv and wild-type mouse colon; relationships with motor patterns and calcium transients.

BACKGROUND: Interstitial cells of Cajal associated with the submuscular plexus (ICC-SMP) generate omnipresent slow-wave activity in the colon and are associated with prominent motor patterns. Our aim was to investigate colon motor dysfunction in W/W(v) mice in which the ICC are reportedly reduced. METHODS: Whole organ colon motility was studied using spatio-temporal mapping; immunohistochemical staining was carried out for c-Kit and Ano1; calcium imaging was applied to ICC-SMP. KEY RESULTS: Discrepancies between Ano1 and c-Kit staining were found in both wild-type and W/W(v) colon. ICC-SMP were reduced to ~50% in the W/W(v) mouse colon according to c-Kit immunohistochemistry, but Ano1 staining indicated a normal network of ICC-SMP. The latter was consistent with rhythmic calcium transients occurring at the submucosal border of the colon in W/W(v) mice, similar to the rhythmic transients in wild-type ICC-SMP. Furthermore, the motor pattern associated with ICC-SMP pacemaking, the so-called 'ripples' were normal in the W/W(v) colon. CONCLUSIONS & INFERENCES: c-Kit is not a reliable marker for quantifying ICC-SMP in the mouse colon. Ano1 staining revealed a normal network of ICC-SMP consistent with the presence of a normal 'ripples' motor pattern. We detected a class of Ano1 positive c-Kit negative cells that do not depend on Kit expression for maintenance, a feature shared with ICC progenitors.

Cholinergic signalling-regulated KV7.5 currents are expressed in colonic ICC-IM but not ICC-MP.

Interstitial cells of Cajal (ICC) and the enteric nervous system orchestrate the various rhythmic motor patterns of the colon. Excitation of ICC may evoke stimulus-dependent pacemaker activity and will therefore have a profound effect on colonic motility. The objective of the present study was to evaluate the potential role of K(+) channels in the regulation of ICC excitability. We employed the cell-attached patch clamp technique to assess single channel activity from mouse colon ICC, immunohistochemistry to determine ICC K(+) channel expression and single cell RT-PCR to identify K(+) channel RNA. Single channel activity revealed voltage-sensitive K(+) channels, which were blocked by the KV7 blocker XE991 (n = 8), which also evoked inward maxi channel activity. Muscarinic acetylcholine receptor stimulation with carbachol inhibited K(+) channel activity (n = 8). The single channel conductance was 3.4 ± 0.1 pS (n = 8), but with high extracellular K(+), it was 18.1 ± 0.6 pS (n = 22). Single cell RT-PCR revealed Ano1-positive ICC that were positive for KV7.5. Double immunohistochemical staining of colons for c-Kit and KV7.5 in situ revealed that intramuscular ICC (ICC-IM), but not ICC associated with the myenteric plexus (ICC-MP), were positive for KV7.5. It also revealed dense cholinergic innervation of ICC-IM. ICC-IM and ICC-MP networks were found to be connected. We propose that the pacemaker network in the colon consists of both ICC-MP and ICC-IM and that one way of exciting this network is via cholinergic KV7.5 channel inhibition in ICC-IM.

Enteric sensory neurons communicate with interstitial cells of Cajal to affect pacemaker activity in the small intestine.

Enteric sensory neurons (the AH neurons) play a role in control of gastrointestinal motor activity; AH neuron activation has been proposed to change propulsion into segmentation. We sought to find a mechanism underlying this phenomenon. We formulated the hypothesis that AH neurons increase local ICC-MP (interstitial cells of Cajal associated with the myenteric plexus) pacemaker frequency to disrupt peristalsis and promote absorption. To that end, we sought structural and physiological evidence for communication between ICC-MP and AH neurons. We designed experiments that allowed us to simultaneously activate AH neurons and observe changes in ICC calcium transients that underlie its pacemaker activity. Neurobiotin injection in AH neurons together with ICC immunohistochemistry proved the presence of multiple contacts between AH neuron varicosities and the cell bodies and processes of ICC-MP. Generating action potential activity in AH neurons led to increase in the frequency and amplitude of calcium transients underlying pacemaker activity in ICC. When no rhythmicity was seen, rhythmic calcium transients were evoked in ICC. As a control, we stimulated nitrergic S neurons, which led to reduction in ICC calcium transients. Hence, we report here the first demonstration of communication between AH neurons and ICC. The following hypothesis can now be formulated: AH neuron activation can disrupt peristalsis directed by ICC-MP slow wave activity, through initiation of a local pacemaker by increasing ICC pacemaker frequency through increasing the frequency of ICC calcium transients. Evoking new pacemakers distal to the proximal lead pacemaker will initiate both retrograde and antegrade propulsion causing back and forth movements that may disrupt peristalsis.

On the origin of rhythmic contractile activity of the esophagus in early achalasia, a clinical case study.

A patient with early achalasia presented spontaneous strong rhythmic non-propulsive contractions at ~7/min, independent of swallows. Our aim was to evaluate characteristics of the rhythmic contractions, provide data on the structure of pacemaker cells in the esophagus and discuss a potential role for interstitial cells of Cajal (ICC) in the origin of rhythmicity. We hypothesize that intramuscular ICC (ICC-IM) are the primary pacemaker cells. The frequency but not the amplitude of the rhythmic contractions was inhibited by the phosphodiesterase inhibitor drotaverine consistent with cAMP inhibiting pacemaker currents in ICC-IM. The frequency increased by wet swallows but not dry swallows, consistent with stretch causing increase in slow wave frequency in ICC-IM. New studies on archival material showed that ICC-IM were present throughout the human esophageal musculature and were not diminished in early achalasia. Although ICC-IM exhibited a low density, they were connected to PDGFRα-positive fibroblast-like cells with whom they formed a dense gap junction coupled network. Nitrergic innervation of ICC was strongly diminished in early achalasia because of the loss of nitrergic nerves. It therefore appears possibly that ICC-IM function as pacemaker cells in the esophagus and that the network of ICC and PDGFRα-positive cells allows for coupling and propagation of the pacemaker activity. Loss of nitrergic innervation to ICC in achalasia may render them more excitable such that its pacemaker activity is more easily expressed. Loss of propagation in achalasia may be due to loss of contraction-induced aboral nitrergic inhibition.

Interstitial cell of Cajal loss correlates with the degree of inflammation in the human appendix and reverses after inflammation.

BACKGROUND: Normal gut motility relies on the complex interaction between the interstitial cell of Cajal (ICC) and the enteric nerve networks. Inflammation of the gastrointestinal tract adversely affects both ICC and enteric nerves. We aimed to determine the distribution of ICC and nerve networks in patients with appendicitis. METHODS: Specimens from controls and patients with appendicitis were examined with immunohistochemistry (c-Kit for ICC, beta III tubulin [Tuj-1] and neuronal nitric oxide synthase [histochemical diaphorase] for nitrergic neurons) and electron microscopy (EM). Data were quantified using image analysis. RESULTS: We found a profound decrease in c-Kit immunoreactivity (c-Kit IR) in the advanced inflammatory stages of appendicitis, which correlated with the severity of inflammation. Electron microscopy confirmed ultrastructural injury in both ICC and nerve fiber networks during acute inflammation. After the inflammation resolved, interval appendices displayed a recovery in ICC c-Kit IR to control levels and normal ultrastructure. The neuronal network also displayed ultrastructural recovery; however, neuronal nitric oxide synthase activity did not recover. CONCLUSIONS: Severe inflammation results in significant ultrastructural damage of nerves and ICC networks in appendicitis. The loss of c-Kit IR is likely due to impaired ICC cytophysiology because ICC was still present under EM. After resolution of acute inflammation, ICC recovers their normal ultrastructure and c-Kit IR.

Damage-free top-down processes for fabricating two-dimensional arrays of 7 nm GaAs nanodiscs using bio-templates and neutral beam etching.

The first damage-free top-down fabrication processes for a two-dimensional array of 7 nm GaAs nanodiscs was developed by using ferritin (a protein which includes a 7 nm diameter iron core) bio-templates and neutral beam etching. The photoluminescence of GaAs etched with a neutral beam clearly revealed that the processes could accomplish defect-free etching for GaAs. In the bio-template process, to remove the ferritin protein shell without thermal damage to the GaAs, we firstly developed an oxygen-radical treatment method with a low temperature of 280 °C. Then, the neutral beam etched the defect-free nanodisc structure of the GaAs using the iron core as an etching mask. As a result, a two-dimensional array of GaAs quantum dots with a diameter of ∼ 7 nm, a height of ∼ 10 nm, a high taper angle of 88° and a quantum dot density of more than 7 × 10(11) cm(-2) was successfully fabricated without causing any damage to the GaAs.

Two independent networks of interstitial cells of cajal work cooperatively with the enteric nervous system to create colonic motor patterns.

Normal motility of the colon is critical for quality of life and efforts to normalize abnormal colon function have had limited success. A better understanding of control systems of colonic motility is therefore essential. We report here a hypothesis with supporting experimental data to explain the origin of rhythmic propulsive colonic motor activity induced by general distention. The theory holds that both networks of interstitial cells of Cajal (ICC), those associated with the submuscular plexus (ICC-SMP) and those associated with the myenteric plexus (ICC-MP), orchestrate propagating contractions as pacemaker cells in concert with the enteric nervous system (ENS). ICC-SMP generate an omnipresent slow wave activity that causes propagating but non-propulsive contractions ("rhythmic propagating ripples") enhancing absorption. The ICC-MP generate stimulus-dependent cyclic depolarizations propagating anally and directing propulsive activity ("rhythmic propulsive motor complexes"). The ENS is not essential for both rhythmic motor patterns since distention and pharmacological means can produce the motor patterns after blocking neural activity, but it supplies the primary stimulus in vivo. Supporting data come from studies on segments of the rat colon, simultaneously measuring motility through spatiotemporal mapping of video recordings, intraluminal pressure, and outflow measurements.

On the origin of rhythmic calcium transients in the ICC-MP of the mouse small intestine.

Interstitial cells of Cajal associated with the myenteric plexus (ICC-MP) are pacemaker cells of the small intestine, producing the characteristic omnipresent electrical slow waves, which orchestrate peristaltic motor activity and are associated with rhythmic intracellular calcium oscillations. Our objective was to elucidate the origins of the calcium transients. We hypothesized that calcium oscillations in the ICC-MP are primarily regulated by the sarcoplasmic reticulum (SR) calcium release system. With the use of calcium imaging, study of the effect of T-type calcium channel blocker mibefradil revealed that T-type channels did not play a major role in generating the calcium transients. 2-Aminoethoxydiphenyl borate, an inositol 1,4,5 trisphosphate receptor (IP(3)R) inhibitor, and U73122, a phospholipase C inhibitor, both drastically decreased the frequency of calcium oscillations, suggesting a major role of IP(3) and IP(3)-induced calcium release from the SR. Immunohistochemistry proved the expression of IP(3)R type I (IP(3)R-I), but not type II (IP(3)R-II) and type III (IP(3)R-III) in ICC-MP, indicating the involvement of the IP(3)R-I subtype in calcium release from the SR. Cyclopiazonic acid, a SR/endoplasmic reticulum calcium ATPase pump inhibitor, strongly reduced or abolished calcium oscillations. The Na-Ca exchanger (NCX) in reverse mode is likely involved in refilling the SR because the NCX inhibitor KB-R7943 markedly reduced the frequency of calcium oscillations. Immunohistochemistry revealed 100% colocalization of NCX and c-Kit in ICC-MP. Testing a mitochondrial NCX inhibitor, we were unable to show an essential role for mitochondria in regulating calcium oscillations in the ICC-MP. In summary, ongoing IP(3) synthesis and IP(3)-induced calcium release from the SR, via the IP(3)R-I, are the major drivers of the calcium transients associated with ICC pacemaker activity. This suggests that a biochemical clock intrinsic to ICC determines the pacemaker frequency, which is likely directly linked to kinetics of the IP(3)-activated SR calcium channel and IP(3) metabolism.