Endocannabinoid biosynthetic enzymes regulate pain response via LKB1-AMPK signaling.

Diacylglycerol lipase-beta (DAGLß) serves as a principal 2-arachidonoylglycerol (2-AG) biosynthetic enzyme regulating endocannabinoid and eicosanoid metabolism in immune cells including macrophages and dendritic cells. Genetic or pharmacological inactivation of DAGLß ameliorates inflammation and hyper-nociception in preclinical models of pathogenic pain. These beneficial effects have been assigned principally to reductions in downstream proinflammatory lipid signaling, leaving alternative mechanisms of regulation largely underexplored. Here, we apply quantitative chemical- and phospho-proteomics to find that disruption of DAGLß in primary macrophages leads to LKB1-AMPK signaling activation, resulting in reprogramming of the phosphoproteome and bioenergetics. Notably, AMPK inhibition reversed the antinociceptive effects of DAGLß blockade, thereby directly supporting DAGLß-AMPK crosstalk in vivo. Our findings uncover signaling between endocannabinoid biosynthetic enzymes and ancient energy-sensing kinases to mediate cell biological and pain responses.

The role of morphine- and fentanyl-induced impairment of intestinal epithelial antibacterial activity in dysbiosis and its impact on the microbiota-gut-brain axis.

Recent evidence suggests that chronic exposure to opioid analgesics such as morphine disrupts the intestinal epithelial layer and causes intestinal dysbiosis. Depleting gut bacteria can preclude the development of tolerance to opioid-induced antinociception, suggesting an important role of the gut-brain axis in mediating opioid effects. The mechanism underlying opioid-induced dysbiosis, however, remains unclear. Host-produced antimicrobial peptides (AMPs) are critical for the integrity of the intestinal epithelial barrier as they prevent the pathogenesis of the enteric microbiota. Here, we report that chronic morphine or fentanyl exposure reduces the antimicrobial activity in the ileum, resulting in changes in the composition of bacteria. Fecal samples from morphine-treated mice had increased levels of Akkermansia muciniphila with a shift in the abundance ratio of Firmicutes and Bacteroidetes. Fecal microbial transplant (FMT) from morphine-naïve mice or oral supplementation with butyrate restored (a) the antimicrobial activity, (b) the expression of the antimicrobial peptide, Reg3γ, (c) prevented the increase in intestinal permeability and (d) prevented the development of antinociceptive tolerance in morphine-dependent mice. Improved epithelial barrier function with FMT or butyrate prevented the enrichment of the mucin-degrading A. muciniphila in morphine-dependent mice. These data implicate impairment of the antimicrobial activity of the intestinal epithelium as a mechanism by which opioids disrupt the microbiota-gut-brain axis.

Low-Efficacy Mu Opioid Agonists as Candidate Analgesics: Effects of Novel C-9 Substituted Phenylmorphans on Pain-Depressed Behavior in Mice.

Low efficacy mu opioid receptor (MOR) agonists may serve as novel candidate analgesics with improved safety relative to high-efficacy opioids. This study used a recently validated assay of pain-depressed behavior in mice to evaluate a novel series of MOR-selective C9-substituted phenylmorphan opioids with graded MOR efficacies. Intraperitoneal injection of dilute lactic acid (IP acid) served as a noxious stimulus to depress locomotor activity by mice in an activity chamber composed of two compartments connected by an obstructed door. Behavioral measures included (1) crosses between compartments (vertical activity over the obstruction) and (2) movement counts quantified as photobeam breaks summed across compartments (horizontal activity). Each drug was tested alone and as a pretreatment to IP acid. A charcoal-meal test and whole-body-plethysmography assessment of breathing in 5% CO2 were also used to assess gastrointestinal (GI) inhibition and respiratory depression, respectively. IP acid produced a concentration-dependent depression in crosses and movement that was optimally alleviated by intermediate- to low-efficacy phenylmorphans with sufficient efficacy to produce analgesia with minimal locomotor disruption. Follow-up studies with two low-efficacy phenylmorphans (JL-2-39 and DC-1-76.1) indicated that both drugs produced naltrexone-reversible antinociception with a rapid onset and a duration of ~1hr. Potency of both drugs increased when behavior was depressed by a lower IP-acid concentration, and neither drug alleviated behavioral depression by a non-pain stimulus (IP lithium chloride). Both drugs produced weaker GI inhibition and respiratory depression than fentanyl and attenuated fentanyl-induced GI inhibition and respiratory depression. Results support further consideration of selective, low-efficacy MOR agonists as candidate analgesics. Significance Statement This study used a novel set of mu opioid receptor (MOR)-selective opioids with graded MOR efficacies to examine the lower boundary of MOR efficacy sufficient to relieve pain-related behavioral depression in mice. Two novel low-efficacy opioids (JL-2-39, DC-1-76.1) produced effective antinociception with improved safety relative to higher- or lower-efficacy opioids, and results support further consideration of these and other low-efficacy opioids as candidate analgesics.

The Guts of the Opioid Crisis.

Bidirectional interactions of the gut epithelium with commensal bacteria are critical for maintaining homeostasis within the gut. Chronic opioid exposure perturbs gut homeostasis through a multitude of neuro-immune-epithelial mechanisms, resulting in the development of analgesic tolerance, a major underpinning of the current opioid crisis. Differences in molecular mechanisms of opioid tolerance between the enteric and central pain pathways pose a significant challenge for managing chronic pain without untoward gastrointestinal effects.

The Cannabinoid Receptor Type 1 Positive Allosteric Modulator ZCZ011 Attenuates Naloxone-Precipitated Diarrhea and Weight Loss in Oxycodone-Dependent Mice.

Opioid use disorder reflects a major public health crisis of morbidity and mortality in which opioid withdrawal often contributes to continued use. However, current medications that treat opioid withdrawal symptoms are limited by their abuse liability or lack of efficacy. Although cannabinoid 1 (CB1) receptor agonists, including Δ9-tetrahydrocannabinol, ameliorate opioid withdrawal in both clinical and preclinical studies of opioid dependence, this strategy elicits cannabimimetic side effects as well as tolerance and dependence after repeated administration. Alternatively, CB1 receptor positive allosteric modulators (PAMs) enhance CB1 receptor signaling and show efficacy in rodent models of pain and cannabinoid dependence but lack cannabimimetic side effects. We hypothesize that the CB1 receptor PAM ZCZ011 attenuates naloxone-precipitated withdrawal signs in opioid-dependent mice. Accordingly, male and female mice given an escalating dosing regimen of oxycodone, a widely prescribed opioid, and challenged with naloxone displayed withdrawal signs that included diarrhea, weight loss, jumping, paw flutters, and head shakes. ZCZ011 fully attenuated naloxone-precipitated withdrawal-induced diarrhea and weight loss and reduced paw flutters by approximately half, but its effects on head shakes were unreliable, and it did not affect jumping behavior. The antidiarrheal and anti-weight loss effects of ZCZ0111 were reversed by a CB1 not a cannabinoid receptor type 2 receptor antagonist and were absent in CB1 (-/-) mice, suggesting a necessary role of CB1 receptors. Collectively, these results indicate that ZCZ011 completely blocked naloxone-precipitated diarrhea and weight loss in oxycodone-dependent mice and suggest that CB1 receptor PAMs may offer a novel strategy to treat opioid dependence. SIGNIFICANCE STATEMENT: Opioid use disorder represents a serious public health crisis in which current medications used to treat withdrawal symptoms are limited by abuse liability and side effects. The CB1 receptor positive allosteric modulator (PAM) ZCZ011, which lacks overt cannabimimetic behavioral effects, ameliorated naloxone-precipitated withdrawal signs through a CB1 receptor mechanism of action in a mouse model of oxycodone dependence. These results suggest that CB1 receptor PAMs may represent a viable strategy to treat opioid withdrawal.

Experimental Colitis Enhances the Rate of Antinociceptive Tolerance to Morphine via Peripheral Opioid Receptors.

Opioids are highly effective analgesics, however, their therapeutic use is limited by adverse effects that include respiratory depression, dependence, and tolerance. Inflammation has been implicated as a significant driver for the development of tolerance to opioids. Recent studies show that chronic morphine in mice results in gut microbial dysbiosis and inflammation in the colon. In the present study, we examined whether colonic inflammation results in tolerance to the antinociceptive effects of morphine. Colonic inflammation was induced in mice by intrarectal administration of 2,4,6-trinitro-benzene sulfonic acid. The development of antinociceptive tolerance was determined by warm-water tail-immersion assay in mice implanted with 25-, 50-, or 75-mg morphine pellet. Colonic inflammation significantly enhanced the rate at which tolerance developed in each cohort of chronic morphine-treated mice. At the lowest dose of morphine pellet (25 mg), antinociceptive tolerance only developed in the presence of colonic inflammation, whereas in 50- and 75-mg pelleted mice, tolerance developed faster in the inflamed animals than in the noninflamed mice. The enhanced antinociceptive tolerance was attenuated with daily administration of peripheral opioid receptor antagonist, 6ß-N-heterocyclic-substituted naltrexamine derivative [17-cyclopropylmethyl-3,14ß-dihydroxy-4,5α-epoxy-6ß-[(4'pyridyl)acetamido]morphinan (NAP)], irrespective of colonic inflammation. Collectively, these findings show that the rate of tolerance to morphine antinociception is exaggerated in the presence of colonic inflammation, and tolerance is prevented by a peripheral µ-opioid receptor antagonist. These studies suggest a peripheral component to the development of antinociceptive tolerance to opioids. Furthermore, peripherally selective opioid antagonists may be useful adjuncts in opioid-based pain management. SIGNIFICANCE STATEMENT: This study supports the notion that inflammation influences the development of antinociceptive tolerance to chronic morphine exposure. We found that, in the presence of colonic inflammation, the rate of development of tolerance to the antinociceptive effects of morphine increased. We also found that treatment with a peripheral opioid receptor antagonist prevented morphine antinociceptive tolerance. Increasing opioid intake during an inflammatory state would result in decreased analgesia and enhanced analgesic tolerance, which puts patients with inflammatory bowel diseases, inflammatory joint diseases, and sickle cell anemia at risk for heavy opioid use.

Analysis of carbenoxolone by ultra-high-performance liquid chromatography tandem mass spectrometry in mouse brain and blood after systemic administration.

Carbenoxolone is a derivative of glycyrrhetinic acid found in the root of Glycyrrhiza glabra, colloquially known as licorice. It has been used as a treatment for peptic and oral ulcers. In recent years, carbenoxolone has been utilized in basic research for its ability to block gap junctional communication. Better understanding the distribution of carbenoxolone after systemic administration can lead to a better understanding of its potential sites of action. Presented is an ultra high-performance liquid chromatography tandem mass spectrometer (UHPLC-MS/MS) method for the identification and quantification of carbenoxolone in mouse blood and brain tissue. Twenty mice were injected intraperitoneally with 25 mg/kg carbenoxolone and brain tissue and blood were collected for analysis. Blood concentrations (mean ± SD) at 15, 30, 60 and 120 min were determined to be (n = 5) 5394 ± 778, 2636 ± 836, 1564 ± 541 and 846 ± 252 ng/mL, respectively. Brain concentrations (mean ± SD) at 15, 30, 60 and 120 mins were determined to be (n = 5) 171 ± 62, 102 ± 35, 55 ± 10 and 27 ± 9 ng/g, respectively. The analysis of these specimens at the four different time points resulted in blood and brain half-lives in mice of ~43 and 41 min, respectively. The UHPLC-MS/MS method was determined to be sensitive and robust for quantification of carbenoxolone.

Ethanol Reversal of Oxycodone Tolerance in Dorsal Root Ganglia Neurons.

Oxycodone is a semisynthetic opioid compound that is widely prescribed, used, and abused today, and has a well-established role in shaping the current opioid epidemic. Previously, we have shown that tolerance develops to the antinociceptive and respiratory depressive effects of oxycodone in mice, and that a moderate dose of acute ethanol or a protein kinase C (PKC) inhibitor reversed that tolerance. To investigate further if tolerance was occurring through neuronal mechanisms, our aims for this study were to assess the effects of acute and prolonged oxycodone in isolated dorsal root ganglia (DRG) neurons and to determine if this tolerance was reversed by either ethanol or a PKC inhibitor. We found that an acute exposure to 3 µM oxycodone reduced neuronal excitability, as measured by increased threshold potentials and reduced action potential amplitude, without eliciting measurable changes in resting membrane potential. Exposure to 10 µM oxycodone for 18-24 hours prevented oxycodone's effect on neuronal excitability, indicative of tolerance development. The development of opioid tolerance was mitigated in DRG neurons from ß-arrestin 2 knockout mice. Oxycodone tolerance was reversed in isolated DRG neurons by the acute application of either ethanol (20 mM) or the PKC inhibitor, bisindolylmaleimide XI hydrochloride (Bis XI), when a challenge of 3 µM oxycodone significantly reduced neuronal excitability following prolonged exposure. Through these studies, we concluded that oxycodone acutely reduced neuronal excitability, tolerance developed to this effect, and reversal of that tolerance occurred at the level of a single neuron, suggesting that reversal of oxycodone tolerance by either ethanol or Bis XI involves cellular mechanisms.

Connexin-purinergic signaling in enteric glia mediates the prolonged effect of morphine on constipation.

Morphine is one of the most widely used drugs for the treatment of pain. However, side effects, including persistent constipation and antinociceptive tolerance, limit its clinical efficacy. Prolonged morphine treatment results in a "leaky" gut, predisposing to colonic inflammation that is facilitated by microbial dysbiosis and associated bacterial translocation. In this study, we examined the role of enteric glia in mediating this secondary inflammatory response to prolonged treatment with morphine. We found that purinergic P2X receptor activity was significantly enhanced in enteric glia that were isolated from mice with long-term morphine treatment (in vivo) but not upon direct exposure of glia to morphine (in vitro). LPS, a major bacterial product, also increased ATP-induced currents, as well as expression of P2X4, P2X7, IL6, IL-1ß mRNA in enteric glia. LPS increased connexin43 (Cx43) expression and enhanced ATP release from enteric glia cells. LPS-induced P2X currents and proinflammatory cytokine mRNA expression were blocked by the Cx43 blockers Gap26 and carbenoxolone. Likewise, colonic inflammation related to prolonged exposure to morphine was significantly attenuated by carbenoxolone (25 mg/kg). Carbenoxolone also prevented gut wall disruption and significantly reduced morphine-induced constipation. These findings imply that enteric glia activation is a significant modulator of morphine-related inflammation and constipation.-Bhave, S., Gade, A., Kang, M., Hauser, K. F., Dewey, W. L., Akbarali, H. I. Connexin-purinergic signaling in enteric glia mediates the prolonged effect of morphine on constipation.

CCR5 mediates HIV-1 Tat-induced neuroinflammation and influences morphine tolerance, dependence, and reward.

The HIV-1 regulatory protein, trans-activator of transcription (Tat), interacts with opioids to potentiate neuroinflammation and neurodegeneration within the CNS. These effects may involve the C-C chemokine receptor type 5 (CCR5); however, the behavioral contribution of CCR5 on Tat/opioid interactions is not known. Using a transgenic murine model that expresses HIV-1 Tat protein in a GFAP-regulated, doxycycline-inducible manner, we assessed morphine tolerance, dependence, and reward. To assess the influence of CCR5 on these effects, mice were pretreated with oral vehicle or the CCR5 antagonist, maraviroc, prior to morphine administration. We found that HIV-1 Tat expression significantly attenuated the antinociceptive potency of acute morphine (2-64 mg/kg, i.p.) in non-tolerant mice. Consistent with this, Tat attenuated withdrawal symptoms among morphine-tolerant mice. Pretreatment with maraviroc blocked the effects of Tat, reinstating morphine potency in non-tolerant mice and restoring withdrawal symptomology in morphine-tolerant mice. Twenty-four hours following morphine administration, HIV-1 Tat significantly potentiated (∼3.5-fold) morphine-conditioned place preference and maraviroc further potentiated these effects (∼5.7-fold). Maraviroc exerted no measurable behavioral effects on its own. Protein array analyses revealed only minor changes to cytokine profiles when morphine was administered acutely or repeatedly; however, 24 h post morphine administration, the expression of several cytokines was greatly increased, including endogenous CCR5 chemokine ligands (CCL3, CCL4, and CCL5), as well as CCL2. Tat further elevated levels of several cytokines and maraviroc pretreatment attenuated these effects. These data demonstrate that CCR5 mediates key aspects of HIV-1 Tat-induced alterations in the antinociceptive potency and rewarding properties of opioids.

Ethanol Reversal of Tolerance to the Antinociceptive Effects of Oxycodone and Hydrocodone.

This study compared the development of tolerance to two orally bioavailable prescription opioids, oxycodone and hydrocodone, to that of morphine, and the reversal of this tolerance by ethanol. Oxycodone (s.c.) was significantly more potent in the mouse tail-withdrawal assay than either morphine or hydrocodone. Oxycodone was also significantly more potent in this assay than hydrocodone when administered orally. Tolerance was seen following chronic subcutaneous administration of each of the three drugs and by the chronic administration of oral oxycodone, but not following the chronic oral administration of hydrocodone. Ethanol (1 g/kg i.p.) significantly reversed the tolerance to the subcutaneous administration of each of the three opioids that developed when given 30 minutes prior to challenge doses. It took twice as much ethanol, when given orally, to reverse the tolerance to oxycodone. We investigated whether the observed tolerance to oxycodone and its reversal by ethanol were due to biodispositional changes or reflected a true neuronal tolerance. As expected, a relationship between brain oxycodone concentrations and activity in the tail-immersion test existed following administration of acute oral oxycodone. Following chronic treatment, brain oxycodone concentrations were significantly lower than acute concentrations. Oral ethanol (2 g/kg) reversed the tolerance to chronic oxycodone, but did not alter brain concentrations of either acute or chronic oxycodone. These studies show that there is a metabolic component of tolerance to oxycodone; however, the reversal of that tolerance by ethanol is not due to an alteration of the biodisposition of oxycodone, but rather is neuronal in nature.

Sex Differences and Drug Dose Influence the Role of the α7 Nicotinic Acetylcholine Receptor in the Mouse Dextran Sodium Sulfate-Induced Colitis Model.

INTRODUCTION: α7 nicotinic acetylcholine receptors (nAChRs) play an important role in vagus nerve-based cholinergic anti-inflammatory effects. This study was designed to assess the role of α7 nAChRs in dextran sodium sulfate (DSS)-induced colitis in male and female mouse. We first compared disease activity and pathogenesis of colitis in α7 knockout and wild-type mice. We then evaluated the effect of several α7 direct and indirect agonists on the severity of disease in the DSS-induced colitis. METHODS: Male and female adult mice were administered 2.5% DSS solution freely in the drinking water for 7 consecutive days and the colitis severity (disease activity index) was evaluated as well as colon length, colon histology, and levels of tumor necrosis factor-alpha colonic levels. RESULTS: Male, but not female, α7 knockout mice displayed a significantly increased colitis severity and higher tumor necrosis factor-alpha levels as compared with their littermate wild-type mice. Moreover, pretreatment with selective α7 ligands PHA-543613, choline, and PNU-120596 decreased colitis severity in male but not female mice. The anti-colitis effects of these α7 compounds dissipated when administered at higher doses. CONCLUSIONS: Our results suggest the presence of a α7-dependent anti-colitis endogenous tone in male mice. Finally, our results show for the first time that female mice are less sensitive to the anti-colitis activity of α7 agonists. Ovarian hormones may play a key role in the sex difference effect of α7 nAChRs modulation of colitis in the mouse. IMPLICATIONS: Our collective results suggest that targeting α7 nAChRs could represent a viable therapeutic approach for intestinal inflammation diseases such as ulcerative colitis with the consideration of sex differences.

Enhanced Sensitivity of α3ß4 Nicotinic Receptors in Enteric Neurons after Long-Term Morphine: Implication for Opioid-Induced Constipation.

Opioid-induced constipation is a major side effect that persists with long-term opioid use. Previous studies demonstrated that nicotine-induced contractions are enhanced after long-term morphine exposure in guinea pig ileum. In the present study, we examined whether the increased sensitivity to nicotine could be observed in single enteric neurons after long-term morphine exposure, determined the subunits in mouse enteric neurons, and examined the effect of nicotine in reversing opioid-induced constipation. Nicotine (0.03-1 mM) dose-dependently induced inward currents from a holding potential of -60 mV in isolated single enteric neurons from the mouse ileum. The amplitude of the currents, but not the potency to nicotine, was significantly increased in neurons receiving long-term (16-24 h) but not short-term (10 min) exposure to morphine. Quantitative mRNA analysis showed that nicotinic acetylcholine receptor (nAChR) subunit expression in the mouse ileum was α3 ≥ ß2 > ß4 > α5 > α4 > ß3 > α6. Nicotine-induced currents were obtained in neurons from α7, ß2, α5, and α6 knockout mice. The currents were, however, inhibited by mecamylamine (10 µM) and the α3ß4 blocker α-conotoxin AuIB (3 µM), suggesting that nicotine-induced currents were mediated by the α3ß4 subtype of nAChRs on enteric neurons. Conversely, NS3861, a partial agonist at α3ß4 nAChR, enhanced fecal pellet expulsion in a dose-dependent manner in mice that received long-term, but not short-term, morphine treatment. Overall, our findings suggest that the efficacy of nAChR agonists on enteric neurons is enhanced after long-term morphine exposure, and activation of the α3ß4 subtype of nAChR reverses chronic, but not acute, morphine-induced constipation.

Effects of HIV-1 Tat on enteric neuropathogenesis.

The gastrointestinal (GI) tract presents a major site of immune modulation by HIV, resulting in significant morbidity. Most GI processes affected during HIV infection are regulated by the enteric nervous system. HIV has been identified in GI histologic specimens in up to 40% of patients, and the presence of viral proteins, including the trans-activator of transcription (Tat), has been reported in the gut indicating that HIV itself may be an indirect gut pathogen. Little is known of how Tat affects the enteric nervous system. Here we investigated the effects of the Tat protein on enteric neuronal excitability, proinflammatory cytokine release, and its overall effect on GI motility. Direct application of Tat (100 nm) increased the number of action potentials and reduced the threshold for action potential initiation in isolated myenteric neurons. This effect persisted in neurons pretreated with Tat for 3 d (19 of 20) and in neurons isolated from Tat(+) (Tat-expressing) transgenic mice. Tat increased sodium channel isoforms Nav1.7 and Nav1.8 levels. This increase was accompanied by an increase in sodium current density and a leftward shift in the sodium channel activation voltage. RANTES, IL-6, and IL-1ß, but not TNF-α, were enhanced by Tat. Intestinal transit and cecal water content were also significantly higher in Tat(+) transgenic mice than Tat(-) littermates (controls). Together, these findings show that Tat has a direct and persistent effect on enteric neuronal excitability, and together with its effect on proinflammatory cytokines, regulates gut motility, thereby contributing to GI dysmotilities reported in HIV patients.

Interactive HIV-1 Tat and morphine-induced synaptodendritic injury is triggered through focal disruptions in Na⁺ influx, mitochondrial instability, and Ca²âº overload.

Synaptodendritic injury is thought to underlie HIV-associated neurocognitive disorders and contributes to exaggerated inflammation and cognitive impairment seen in opioid abusers with HIV-1. To examine events triggering combined transactivator of transcription (Tat)- and morphine-induced synaptodendritic injury systematically, striatal neuron imaging studies were conducted in vitro. These studies demonstrated nearly identical pathologic increases in dendritic varicosities as seen in Tat transgenic mice in vivo. Tat caused significant focal increases in intracellular sodium ([Na(+)]i) and calcium ([Ca(2+)]i) in dendrites that were accompanied by the emergence of dendritic varicosities. These effects were largely, but not entirely, attenuated by the NMDA and AMPA receptor antagonists MK-801 and CNQX, respectively. Concurrent morphine treatment accelerated Tat-induced focal varicosities, which were accompanied by localized increases in [Ca(2+)]i and exaggerated instability in mitochondrial inner membrane potential. Importantly, morphine's effects were prevented by the µ-opioid receptor antagonist CTAP and were not observed in neurons cultured from µ-opioid receptor knock-out mice. Combined Tat- and morphine-induced initial losses in ion homeostasis and increases in [Ca(2+)]i were attenuated by the ryanodine receptor inhibitor ryanodine, as well as pyruvate. In summary, Tat induced increases in [Na(+)]i, mitochondrial instability, excessive Ca(2+) influx through glutamatergic receptors, and swelling along dendrites. Morphine, acting via µ-opioid receptors, exacerbates these excitotoxic Tat effects at the same subcellular locations by mobilizing additional [Ca(2+)]i and by further disrupting [Ca(2+)]i homeostasis. We hypothesize that the spatiotemporal relationship of µ-opioid and aberrant AMPA/NMDA glutamate receptor signaling is critical in defining the location and degree to which opiates exacerbate the synaptodendritic injury commonly observed in neuroAIDS.

Interaction between hydrogen sulfide-induced sulfhydration and tyrosine nitration in the KATP channel complex.

Hydrogen sulfide (H2S) is an endogenous gaseous mediator affecting many physiological and pathophysiological conditions. Enhanced expression of H2S and reactive nitrogen/oxygen species (RNS/ROS) during inflammation alters cellular excitability via modulation of ion channel function. Sulfhydration of cysteine residues and tyrosine nitration are the posttranslational modifications induced by H2S and RNS, respectively. The objective of this study was to define the interaction between tyrosine nitration and cysteine sulfhydration within the ATP-sensitive K(+) (KATP) channel complex, a significant target in experimental colitis. A modified biotin switch assay was performed to determine sulfhydration of the KATP channel subunits, Kir6.1, sulphonylurea 2B (SUR2B), and nitrotyrosine measured by immunoblot. NaHS (a donor of H2S) significantly enhanced sulfhydration of SUR2B but not Kir6.1 subunit. 3-Morpholinosydnonimine (SIN-1) (a donor of peroxynitrite) induced nitration of Kir6.1 subunit but not SUR2B. Pretreatment with NaHS reduced the nitration of Kir6.1 by SIN-1 in Chinese hamster ovary cells cotransfected with the two subunits, as well as in enteric glia. Two specific mutations within SUR2B, C24S, and C1455S prevented sulfhydration by NaHS, and these mutations prevented NaHS-induced reduction in tyrosine nitration of Kir6.1. NaHS also reversed peroxynitrite-induced inhibition of smooth muscle contraction. These studies suggest that posttranslational modifications of the two subunits of the KATP channel interact to alter channel function. The studies described herein demonstrate a unique mechanism by which sulfhydration of one subunit modifies tyrosine nitration of another subunit within the same channel complex. This interaction provides a mechanistic insight on the protective effects of H2S in inflammation.

Hydrogen sulfide as an allosteric modulator of ATP-sensitive potassium channels in colonic inflammation.

The ATP-sensitive potassium channel (K(ATP)) in mouse colonic smooth muscle cell is a complex containing a pore-forming subunit (Kir6.1) and a sulfonylurea receptor subunit (SUR2B). These channels contribute to the cellular excitability of smooth muscle cells and hence regulate the motility patterns in the colon. Whole-cell voltage-clamp techniques were used to study the alterations in K(ATP) channels in smooth muscle cells in experimental colitis. Colonic inflammation was induced in BALB/C mice after intracolonic administration of trinitrobenzene sulfonic acid. K(ATP) currents were measured at a holding potential of -60 mV in high K(+) external solution. The concentration response to levcromakalim (LEVC), a K(ATP) channel opener, was significantly shifted to the left in the inflamed smooth-muscle cells. Both the potency and maximal currents induced by LEVC were enhanced in inflammation. The EC(50) values in control were 6259 nM (n = 10) and 422 nM (n = 8) in inflamed colon, and the maximal currents were 9.9 ± 0.71 pA/pF (60 µM) in control and 39.7 ± 8.8 pA/pF (3 µM) after inflammation. As was seen with LEVC, the potency and efficacy of sodium hydrogen sulfide (NaHS) (10-1000 µM) on K(ATP) currents were significantly greater in inflamed colon compared with controls. In control cells, pretreatment with 100 µM NaHS shifted the EC(50) for LEV-induced currents from 2838 (n = 6) to 154 (n = 8) nM. Sulfhydration of sulfonylurea receptor 2B (SUR2B) was induced by NaHS and colonic inflammation. These data suggest that sulfhydration of SUR2B induces allosteric modulation of K(ATP) currents in colonic inflammation.

Increased PDE5 activity and decreased Rho kinase and PKC activities in colonic muscle from caveolin-1-/- mice impair the peristaltic reflex and propulsion.

Caveolae are specialized regions of the plasma membrane that concentrate receptors and associated signaling molecules critical in regulation of cellular response to transmitters and hormones. We have determined the effects of caveolin-1 (Cav-1) deletion, caveolin-1 siRNA, and caveolar disruption in mice on the signaling pathways that mediate contraction and relaxation in colonic smooth muscle and on the components of the peristaltic reflex in isolated tissue and propulsion in intact colonic segments. In Cav-1-/- mice, both relaxation and contraction were decreased in smooth muscle cells and muscle strips, as well as during both phases of the peristaltic reflex and colonic propulsion. The decrease in relaxation in response to the nitric oxide (NO) donor was accompanied by a decrease in cGMP levels and an increase in phosphodiesterase 5 (PDE5) activity. Relaxation by a PDE5-resistant cGMP analog was not affected in smooth muscle of Cav-1-/- mice, suggesting that inhibition of relaxation was due to augmentation of PDE5 activity. Similar effects on relaxation, PDE5 and cGMP were obtained in muscle cells upon disruption of caveolae by methyl-ß-cyclodextrin or suppression of Cav-1. Sustained contraction mediated via inhibition of myosin light chain phosphatase (MLCP) activity is regulated by Rho kinase and PKC via phosphorylation of two endogenous inhibitors of MLCP: myosin phosphatase-targeting subunit (MYPT1) and 17-kDa PKC-potentiated protein phosphatase 1 inhibitor protein (CPI-17), respectively. The activity of both enzymes and phosphorylation of MYPT1 and CPI-17 were decreased in smooth muscle from Cav-1-/- mice. We conclude that the integrity of caveolae is essential for contractile and relaxant activity in colonic smooth muscle and the maintenance of neuromuscular function at organ level.

Novel insights on the effect of nicotine in a murine colitis model.

Studies showed that nicotine has a positive influence on symptoms of ulcerative colitis. In the present study, we explored the effect of nicotine treatment using different routes of administration in the dextran sodium sulfate (DSS) colitis mouse model. We also investigated the effects of cotinine, a major metabolite of nicotine, in the model. C57BL6 adult male mice were given DSS solution freely in the drinking water for seven consecutive days, and tap water was given thereafter. Disease severity, length of the colon, colon tissue histology, and inflammatory markers, including colonic myeloperoxidase activity and colonic tumor necrosis factor-α levels, were evaluated. The effect of nicotine and cotinine treatments via various different routes of administration were examined the DSS model. In addition, we measured the plasma levels of nicotine and cotinine in our treatment protocols. Administration of low, but not high, doses of oral nicotine in DSS-treated mice resulted in a significant decrease in disease severity, histologic damage scores, as well as colonic level of tumor necrosis factor-α. However, the anti-inflammatory effect of nicotine was not seen after chronic s.c. or minipump infusion of the drug. Differences in plasma levels of nicotine and cotinine do not seem to account for this lack of effect. Finally, oral cotinine alone failed to show a significant effect in the DSS model of colitis. These results highlight that dose and route of administration play a critical role in the protective effect of nicotine in the DSS mouse colitis model.

Sepiapterin ameliorates chemically induced murine colitis and azoxymethane-induced colon cancer.

The effects of modulating tetrahydrobiopterin (BH4) levels with a metabolic precursor, sepiapterin (SP), on dextran sodium sulfate (DSS)-induced colitis and azoxymethane (AOM)-induced colorectal cancer were studied. SP in the drinking water blocks DSS-induced colitis measured as decreased disease activity index (DAI), morphologic criteria, and recovery of Ca(2+)-induced contractility responses lost as a consequence of DSS treatment. SP reduces inflammatory responses measured as the decreased number of infiltrating inflammatory macrophages and neutrophils and decreased expression of proinflammatory cytokines interleukin 1ß (IL-1ß), IL-6, and IL-17A. High-performance liquid chromatography analyses of colonic BH4 and its oxidized derivative 7,8-dihydrobiopterin (BH2) are inconclusive although there was a trend for lower BH4:BH2 with DSS treatment that was reversed with SP. Reduction of colonic cGMP levels by DSS was reversed with SP by a mechanism sensitive to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a specific inhibitor of the NO-sensitive soluble guanylate cyclase (sGC). ODQ abrogates the protective effects of SP on colitis. This plus the finding that SP reduces DSS-enhanced protein Tyr nitration are consistent with DSS-induced uncoupling of NOS. The results agree with previous studies that demonstrated inactivation of sGC in DSS-treated animals as being important in recruitment of inflammatory cells and in altered cholinergic signaling and colon motility. SP also reduces the number of colon tumors in AOM/DSS-treated mice from 7 to 1 per unit colon length. Thus, pharmacologic modulation of BH4 with currently available drugs may provide a mechanism for alleviating some forms of colitis and potentially minimizing the potential for colorectal cancer in patients with colitis.