Sha, Lei

AbstractBackgroundGastroparesis is defined by delayed gastric emptying (GE) without obstruction. Studies suggest targeting heme oxygenase‐1 (HO1) may ameliorate diabetic gastroparesis. Upregulation of HO1 expression via interleukin‐10 (IL‐10) in the gastric muscularis propria is associated with reversal of delayed GE in diabetic NOD mice. IL‐10 activates the M2 cytoprotective phenotype of macrophages and induces expression of HO1 protein. Here, we assess delivery of HO1 by recombinant adeno‐associated viruses (AAVs) in diabetic mice with delayed GE.MethodsC57BL6 diabetic delayed GE mice were injected with 1 × 1012 vg scAAV9‐cre, scAAV9‐GFP, or scAAV9‐HO1 particles. Changes to GE were assessed weekly utilizing our [13C]‐octanoic acid breath test. Stomach tissue was collected to assess the effect of scAAV9 treatment on Kit, NOS1, and HO1 expression.Key ResultsDelayed GE returned to normal within 2 weeks of treatment in 7/12 mice receiving scAAV9‐cre and in 4/5 mice that received the scAAV9‐GFP, whereas mice that received scAAV9‐HO1 did not respond in the same manner and had GE that took significantly longer to return to normal (6/7 mice at 4–6 weeks). Kit, NOS1, and HO1 protein expression in scAAV9‐GFP‐treated mice with normal GE were not significantly different compared with diabetic mice with delayed GE.Conclusions and InferencesInjection of scAAV9 into diabetic C57BL6 mice produced a biological response that resulted in acceleration of GE independently of the cargo delivered by the AAV9 vector. Further research is needed to determine whether use of AAV mediated gene transduction in the gastric muscularis propria is beneficial and warranted.

Electrical slow wave activity, generated by interstitial cells of Cajal (ICC), is essential for efficient gastrointestinal (GI) transit. Electrophysiology of slow waves has been extensively studied; inclusion of bicarbonate (HCO 3 − ) in extracellular solutions is required for recording normal slow wave activity. HCO 3 − transport contributes to regulation of intracellular pH, which has multiple consequences for normal cellular function. Previous transcriptional profiling found that the Na + / HCO 3 − cotransporter (NBCe1/Slc4a4) is enriched in pacemaker myenteric ICC (ICC‐MY) compared to non pacemaker ICC in the deep muscular plexus (ICC‐DMP) of the small intestine. Our aim was to determine the distribution and identity of Slc4a4/NBCe1 variants in ICC in the GI tract. NBCe1 immunoreactivity (IR) was detected by fluorescence labeling of parafomaldehyde‐fixed cross sections and whole mounts of adult mouse small intestine, colon and stomach using primary antibodies directed against a 329 amino acid sequence in the N terminus of the NBCe1 protein. Labeling of Kit receptor tyrosine kinase with a goat anti‐Kit polyclonal antibody was used to detect ICC. Slc4a4 knockout (KO) mice were bred from heterozygous founders. Slc4a4 fl/fl mice were bred with ETV1 CreERT2/− mice to allow targeted, conditional KO of Slc4a4 in ICC in response to tamoxifen treatment (130 mg/Kg). mRNA transcripts for Slc4a4 in Kit expressing cells were isolated from Kit‐ribotag mice, which were generated by breeding Kit creERT2 with Rpl22 tm1.1Psam mice. The abundance of specific Slc4a4 transcripts was measured by quantitative real time PCR (qRT‐PCR). Green fluorescent protein (GFP)‐labeled ICC were dissociated from the small intestine of neonatal Kit copGFP mice and RNA expression levels were determined using the Single Cell‐to‐CT™ qRT‐PCR Kit from Ambion. NBCe1‐IR was absent in all cells in Slc4a4‐KO tissues but co‐localized with Kit in ICC‐MY, but not in ICC‐DMP, in small intestine of wild type mice. In the proximal and distal colon, pacemaker submucosal ICC (ICC‐SMP) but not intramuscular ICC (ICC‐IM) or ICC‐MY showed NBCe1‐IR. In the corpus and antrum of the stomach NBCe1‐IR was detected in ICC‐MY but not in ICC‐IM. NBCe1‐IR did not co‐localize with Kit in the gastric fundus. mRNA for variant C, but not variants A or B of Slc4a4, was detected in small intestinal ICC‐MY. Reduced NBCe1‐IR was restricted to small intestinal ICC‐MY of Slc4a4 conditional KO mice. NBCe1 is enriched in subtypes of ICC responsible for pacemaker activity in all regions of the GI tract indicating a role for this transporter in slow wave generation. The presence of the C variant of Slc4a4 in ICC‐MY suggests dynamic regulation of NBCe1 activity in ICC by the I P 3 R b inding protein released with i nositol 1, 4, 5‐ t risphosphate (IRBIT). Support or Funding Information Supported by NIH R01 DK57061. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

AbstractBackgroundNormal gastrointestinal motility depends on electrical slow‐wave activity generated by interstitial cells of Cajal (ICC) in the tunica muscularis of the gastrointestinal tract. A requirement for HCO3− in extracellular solutions used to record slow waves indicates a role for HCO3− transport in ICC pacemaking. The Slc4a4 gene transcript encoding the electrogenic Na+/HCO3− cotransporter, NBCe1, is enriched in mouse small intestinal myenteric region ICC (ICC‐MY) that generate slow waves. This study aimed to determine how extracellular HCO3− concentrations affect electrical activity in mouse small intestine and to determine the contribution of NBCe1 activity to these effects.MethodsImmunohistochemistry and sharp electrode electrical recordings were used.Key ResultsThe NBCe1 immunoreactivity was localized to ICC‐MY of the tunica muscularis. In sharp electrode electrical recordings, removal of HCO3‐ from extracellular solutions caused significant, reversible, depolarization of the smooth muscle and a reduction in slow‐wave amplitude and frequency. In 100 mM HCO3−, the muscle hyperpolarized and slow wave amplitude and frequency increased. The effects of replacing extracellular Na+ with Li+, an ion that does not support NBCe1 activity, were similar to, but larger than, the effects of removing HCO3‐. There were no additional changes to electrical activity when HCO3‐ was removed from Li+ containing solutions. The Na+/HCO3− cotransport inhibitor, S‐0859 (30µM) significantly reduced the effect of removing HCO3− on electrical activity.Conclusions & InferencesThese studies demonstrate a major role for Na+/HCO3− cotransport by NBCe1 in electrical activity of mouse small intestine and indicated that regulation of intracellular acid:base homeostasis contributes to generation of normal pacemaker activity in the gastrointestinal tract.

AbstractBackground and AimMuscularis macrophages (MMs) not only mediate the innate immunity, but also functionally interact with cells important for gastrointestinal motility. The aim of this study was to determine the spatial relationship and types of contacts between the MMs and neighboring cells in the muscularis propria of human and mouse stomach, small intestine, and large intestine.MethodsThe distribution and morphology of MMs and their contacts with other cells were investigated by immunohistochemistry and transmission electron microscopy.Key ResultsImmunohistochemistry showed variable shape and number of MMs according to their location in different portions of the muscle coat. By double labeling, a close association between MMs and neighboring cells, that is, neurons, smooth muscle cells, interstitial cells of Cajal (ICCs), telocytes (TCs)/PDGFRα‐positive cells, was seen. Electron microscopy demonstrated that in the muscle layers of both animal species, MMs have similar ultrastructural features and have specialized cell‐to‐cell contacts with smooth muscle cells and TCs/PDGFRα‐positive cells but not with ICCs and enteric neurons.Conclusion & InferencesThis study describes varying patterns of distribution of MMs between different regions of the gut, and reports the presence of distinct and extended cell‐to‐cell contacts between MMs and smooth muscle cells and between MMs and TCs/PDGFRα‐positive cells. In contrast, MMs, although close to ICCs and nerve elements, did not make contact with them. These findings indicate specialized and variable roles for MMs in the modulation of gastrointestinal motility whose significance should be more closely investigated in normal and pathological conditions.

In mammalian peripheral sympathetic ganglia GABA acts presynaptically to facilitate cholinergic transmission and postsynaptically to depolarize membrane potential. The GABA effect on parasympathetic pancreatic ganglia is unknown. We aimed to determine the effect of locally applied GABA on cat pancreatic ganglion neurons. Ganglia with attached nerve trunks were isolated from cat pancreata. Conventional intracellular recording techniques were used to record electrical responses from ganglion neurons. GABA pressure microejection depolarized membrane potential with an amplitude of 17.4 ± 0.7 mV. Electrically evoked fast excitatory postsynaptic potentials were significantly inhibited (5.4 ± 0.3 to 2.9 ± 0.2 mV) after GABA application. GABA-evoked depolarizations were mimicked by the GABAAreceptor agonist muscimol and abolished by the GABAAreceptor antagonist bicuculline and the Cl−channel blocker picrotoxin. GABA was taken up and stored in ganglia during preincubation with 1 mM GABA; β-aminobutyric acid application after GABA loading significantly ( P < 0.05) increased depolarizing response to GABA (15.6 ± 1.0 vs. 7.8 ± 0.8 mV without GABA preincubation). Immunolabeling with antibodies to GABA, glial cell fibrillary acidic protein, protein gene product 9.5, and glutamic acid decarboxylase (GAD) immunoreactivity showed that GABA was present in glial cells, but not in neurons, and that glial cells did not contain GAD, whereas islet cells did. The data suggest that endogenous GABA released from ganglionic glial cells acts on pancreatic ganglion neurons through GABAAreceptors.

New Findings What is the central question of this study? The aim was to investigate the roles of extracellular chloride in electrical slow waves and resting membrane potential of mouse jejunal smooth muscle by replacing chloride with the impermeant anions gluconate and isethionate. What is the main finding and its importance? The main finding was that in smooth muscle cells, the resting Cl− conductance is low, whereas transmembrane Cl− movement in interstitial cells of Cajal (ICCs) is a major contributor to the shape of electrical slow waves. Furthermore, the data confirm that ICCs set the smooth muscle membrane potential and that altering Cl− homeostasis in ICCs can alter the smooth muscle membrane potential. Intracellular Cl− homeostasis is regulated by anion‐permeable channels and transporters and contributes to excitability of many cell types, including smooth muscle and interstitial cells of Cajal (ICCs). Our aims were to investigate the effects on electrical activity in mouse jejunal muscle strips of replacing extracellular Cl− (Cl−o) with the impermeant anions gluconate and isethionate. On reducing Cl−o, effects were observed on electrical slow waves, with small effects on smooth muscle membrane voltage (Em). Restoration of Cl− hyperpolarized smooth muscle Em proportional to the change in Cl−o concentration. Replacement of 90% of Cl−o with gluconate reversibly abolished slow waves in five of nine preparations. Slow waves were maintained in isethionate. Gluconate and isethionate substitution had similar concentration‐dependent effects on peak amplitude, frequency, width at half peak amplitude, rise time and decay time of residual slow waves. Gluconate reduced free ionized Ca2+ in Krebs solutions to 0.13 mm. In Krebs solutions containing normal Cl− and 0.13 mm free Ca2+, slow wave frequency was lower, width at half peak amplitude was smaller, and decay time was faster. The transient hyperpolarization following restoration of Cl−o was not observed in W/Wv mice, which lack pacemaker ICCs in the small intestine. We conclude that in smooth muscle cells, the resting Cl− conductance is low, whereas transmembrane Cl− movement in ICCs plays a major role in generation or propagation of slow waves. Furthermore, these data support a role for ICCs in setting smooth muscle Em and that altering Cl− homeostasis in ICCs can alter smooth muscle Em.

AbstractBackgroundThe SCN5A‐encoded voltage‐gated sodium channel NaV1.5 is expressed in human jejunum and colon. Mutations in NaV1.5 are associated with gastrointestinal motility disorders. The rat gastrointestinal tract expresses voltage‐gated sodium channels, but their molecular identity and role in rat gastrointestinal electrophysiology are unknown.MethodsThe presence and distribution of Scn5a‐encoded NaV1.5 was examined by PCR, Western blotting and immunohistochemistry in rat jejunum. Freshly dissociated smooth muscle cells were examined by whole cell electrophysiology. Zinc finger nuclease was used to target Scn5a in rats. Lentiviral‐mediated transduction with shRNA was used to target Scn5a in rat jejunum smooth muscle organotypic cultures. Organotypic cultures were examined by sharp electrode electrophysiology and RT‐PCR.Key ResultsWe found NaV1.5 in rat jejunum and colon smooth muscle by Western blot. Immunohistochemistry using two other antibodies of different portions of NaV1.5 revealed the presence of the ion channel in rat jejunum. Whole cell voltage‐clamp in dissociated smooth muscle cells from rat jejunum showed fast activating and inactivating voltage‐dependent inward current that was eliminated by Na+ replacement by NMDG+. Constitutive rat Scn5a knockout resulted in death in utero. NaV1.5 shRNA delivered by lentivirus into rat jejunum smooth muscle organotypic culture resulted in 57% loss of Scn5a mRNA and several significant changes in slow waves, namely 40% decrease in peak amplitude, 30% decrease in half‐width, and 7 mV hyperpolarization of the membrane potential at peak amplitude.Conclusions & InferencesScn5a‐encoded NaV1.5 is expressed in rat gastrointestinal smooth muscle and it contributes to smooth muscle electrophysiology.

AbstractBackgroundOtilonium bromide (OB) is used as a spasmolytic drug in the treatment of the functional bowel disorder irritable bowel syndrome. Although its acute effects on colonic relaxation are well‐characterized, little is known about the effects of chronic administration ofOBon enteric neurons, neuromuscular transmission, and interstitial cells of Cajal (ICC), key regulators of the gut function.MethodsAdult Sprague–Dawley rats were treated withOBin drinking water at a dose of 2 mg/kg for 30 days. The colons ofOB‐treated and age‐matched control rats were studied by confocal immunohistochemistry to detect immunoreactivity (IR) in myenteric plexus neurons for nitrergic and tachykininergic markers, and also by microelectrode electrophysiology.Key ResultsUsing immunohistochemistry, chronicOBadministration did not change total neuron number, assessed by anti‐HuIR, but resulted in a significant increase inNK1 receptor positive neurons, a decrease in neuronal nitric oxide synthase expressing neurons, and a reduction in volume of substance P in nerve fibers in the myenteric plexus. ChronicOBadministration potentiated inhibitory and excitatory junction potentials evoked by repetitive electrical field stimulation. The various types of colonicICC, detected by KitIR, were not altered nor were slow waves or smooth muscle membrane potential.Conclusions & InferencesChronic treatment withOBcaused significant changes in the nitrergic and tachykinergic components of the myenteric plexus and in both inhibitory and excitatory neurotransmission in the rat colon.

Key points A CO‐dependent transwall gradient of resting membrane potential exists across the circular muscle layer in the gastrointestinal tract; this gradient regulates the strength of phasic muscle contraction during electrical slow wave activity. H2S, CO and NO are endogenously generated in the muscle layers of the gut wall. Inhibiting the H2S producing enzyme CSE with the CSE inhibitor PAG had no significant effect on transwall resting membrane potential gradient in control preparations but significantly shifted the entire gradient in the depolarizing direction in preparations pretreated with the NO synthase (NOS) inhibitor l‐NNA. PAG significantly shifted the gradient in the depolarizing direction in mouse preparations lacking neuronal NOS (nNOS‐KO) and the gradient was significantly shifted in the depolarizing direction in mouse preparations lacking both nNOS and CSE (CSE‐KO–nNOS‐KO). NO production was significantly higher in CSE‐KO mouse preparations compared to wild‐type mouse preparations and the amplitude of NO‐mediated slow inhibitory junction potentials (S‐IJPs) was significantly higher in CSE‐KO mouse preparations compared to the amplitude of S‐IJPs in wild‐type mouse preparations. Nearly all submucosal neurons and myenteric neurons were CSE positive and 11% of submucosal neurons and 50% of myenteric neurons were nNOS positive. Our novel findings suggested that endogenous H2S is a stealth hyperpolarizing factor on smooth muscle cells and that endogenous H2S inhibits NO production from nNOS. AbstractA transwall gradient in resting membrane potential (RMP) exists across the circular muscle layer in the mouse colon. This gradient is dependent on endogenous generation of CO. H2S is also generated in muscle layers of the mouse colon. The effect of endogenously generated H2S on the transwall gradient is not known. The aim was to investigate the role of endogenous H2S. Our results showed that the CSE inhibitor dl‐propargylglycine (PAG, 500 μm) had no effect on the transwall gradient. However, in preparations pretreated with the nitric oxide synthase inhibitor N‐nitro‐l‐arginine (l‐NNA, 200 μm) and in nNOS‐knockout (KO) mouse preparations, PAG shifted the transwall gradient in the depolarizing direction. In CSE‐KO–nNOS‐KO mice, the gradient was shifted in the depolarizing direction. Endogenous generation of NO was significantly higher in muscle preparations of CSE‐KO mice compared to wild‐type (WT) mice. The amplitude of NO‐mediated slow inhibitory junction potentials (S‐IJPs) evoked by electric field stimulation was significantly higher in CSE‐KO mouse preparations compared to the amplitude of S‐IJPs in wild‐type mouse preparations. CSE was present in all submucosal ganglion neurons and in almost all myenteric ganglion neurons. Eleven per cent of CSE positive neurons in the submucosal plexus and 50% of CSE positive neurons in the myenteric plexus also contained nNOS. Our results suggest that endogenously generated H2S acts as a stealth hyperpolarizing factor on smooth muscle cells to maintain the CO‐dependent transwall gradient and inhibits NO production from nNOS.

Hydrogen sulfide (H2S) is produced endogenously by l-cysteine metabolism. H2S modulates several ion channels with an unclear mechanism of action. A possible mechanism is through reduction-oxidation reactions attributable to the redox potential of the sulfur moiety. The aims of this study were to determine the effects of the H2S donor NaHS on NaV1.5, a voltage-dependent sodium channel expressed in the gastrointestinal tract in human jejunum smooth muscle cells and interstitial cells of Cajal, and to elucidate whether H2S acts on NaV1.5 by redox reactions. Whole cell Na+ currents were recorded in freshly dissociated human jejunum circular myocytes and NaV1.5-transfected human embryonic kidney-293 cells. RT-PCR amplified mRNA for H2S enzymes cystathionine β-synthase and cystathionine γ-lyase from the human jejunum. NaHS increased native Na+ peak currents and shifted the half-point ( V1/2) of steady-state activation and inactivation by +21 ± 2 mV and +15 ± 3 mV, respectively. Similar effects were seen on the heterologously expressed NaV1.5 α subunit with EC50s in the 10−4 to 10−3 M range. The reducing agent dithiothreitol (DTT) mimicked in part the effects of NaHS by increasing peak current and positively shifting steady-state activation. DTT together with NaHS had an additive effect on steady-state activation but not on peak current, suggesting that the latter may be altered via reduction. Pretreatment with the Hg2+-conjugated oxidizer thimerosal or the alkylating agent N-ethylmaleimide inhibited or decreased NaHS induction of NaV1.5 peak current. These studies show that H2S activates the gastrointestinal Na+ channel, and the mechanism of action of H2S is partially redox independent.