Minho Kang

The role of β-arrestin2 in the mechanism of morphine tolerance in the mouse and guinea pig gastrointestinal tract

β-Arrestin2 has been reported to play an essential role in analgesic tolerance. Analgesic tolerance without concomitant tolerance to constipation is a limiting side effect of chronic morphine treatment. Because tolerance to morphine develops in the mouse ileum but not the colon, we therefore examined whether the role of β-arrestin2 in the mechanism of morphine tolerance differs in the ileum and colon. In both guinea pig and mouse, chronic in vitro exposure (2 h, 10 μM) to morphine resulted in tolerance development in the isolated ileum but not the colon. The IC50 values for morphine-induced inhibition of electrical field stimulation contraction of guinea pig longitudinal muscle myenteric plexus shifted rightward in the ileum from 5.7 ± 0.08 (n = 9) to 5.45 ± 0.09 (n = 6) (p < 0.001) after morphine exposure. A significant shift was not observed in the colon. Similar differential tolerance was seen between the mouse ileum and the colon. However, tolerance developed in the colon from β-arrestin2 knockout mice. β-Arrestin2 and extracellular signal-regulated kinase 1/2 expression levels were determined further by Western blot analyses in guinea pig longitudinal muscle myenteric plexus. A time-dependent decrease in the expression of β-arrestin2 and extracellular signal-regulated kinase 1/2 occurred in the ileum but not the colon after 2 h of morphine (10 μM) exposure. Naloxone prevented the decrease in β-arrestin2. In the isolated ileum from guinea pigs chronically treated in vivo with morphine for 7 days, neither additional tolerance to in vitro exposure of morphine nor a decrease in β-arrestin2 occurred. We conclude that a decrease in β-arrestin2 is associated with tolerance development to morphine in the gastrointestinal tract.

Hydrogen Sulfide as an Allosteric Modulator of ATP- Sensitive Potassium Channels in Colonic Inflammation.

The ATP-sensitive potassium channel (KATP) 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 KATP channels in smooth muscle cells in experimental colitis. Colonic inflammation was induced in BALB/C mice after intracolonic administration of trinitrobenzene sulfonic acid. KATP currents were measured at a holding potential of −60 mV in high K+ external solution. The concentration response to levcromakalim (LEVC), a KATP 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 EC50 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 KATP currents were significantly greater in inflamed colon compared with controls. In control cells, pretreatment with 100 µM NaHS shifted the EC50 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 KATP currents in colonic inflammation.

Protein and gene expression of Ca2+ channel isoforms in murine colon: effect of inflammation

L-Type voltage-dependent Ca2+ channels (L-VDCC) mediate calcium influx in response to membrane depolarization and regulate intracellular processes such as contraction, secretion, neurotransmission, and gene expression. Colonic inflammation significantly attenuates calcium currents in smooth muscle; however, the basis for this remains unclear. In this study we examined the protein and mRNA expression of two isoforms of Cav1.2, encoded by either exon 1a or 1b. Both isoforms were detected by Western blots, immunohistochemistry and RT-PCR in smooth muscle cells. Neither the protein nor mRNA expression measured by real-time PCR of either isoforms was affected in colonic myocytes from dextran sulfate sodium-treated mice. In whole-cell voltage-clamp experiments, the amplitude of the calcium currents were decreased by almost 70% by inflammation. The calcium channel currents were attenuated by 50±3% by the c-src kinase specific inhibitor, PP2, in control cells but only 19±7% in cells from inflamed mice. These studies suggest that decreased calcium channel currents following colonic inflammation are not due to decreased expression but may result from altered regulation by the non-receptor cellular tyrosine kinase, c-src kinase.

Nitrotyrosylation of Ca2+ Channels Prevents c-Src Kinase Regulation of Colonic Smooth Muscle Contractility in Experimental Colitis

Basal levels of c-Src kinase are known to regulate smooth muscle Ca2+ channels. Colonic inflammation results in attenuated Ca2+ currents and muscle contraction. Here, we examined the regulation of calcium influx-dependent contractility by c-Src kinase in experimental colitis. Ca2+-influx induced contractions were measured by isometric tension recordings of mouse colonic longitudinal muscle strips depolarized by high K+. The Emax to CaCl2 was significantly less in inflamed tissues (38.4 ± 7.6%) than controls, indicative of reduced Ca2+ influx. PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine], a selective Src kinase inhibitor, significantly reduced the contractile amplitude and shifted the pD2 from 3.88 to 2.44 in controls, whereas it was ineffective in inflamed tissues (3.66 versus 3.43). After pretreatment with a SIN-1 (3-morpholinosydnonimine)/peroxynitrite combination, the maximal contraction to CaCl2 was reduced by 46 ± 7% in controls but unaffected in inflamed tissues (13 ± 11%). Peroxynitrite also prevented the inhibitory effect of PP2 in control tissues. In colonic single smooth muscle cells, PP2 inhibited Ca2+ currents by 84.1 ± 3.9% in normal but only 36.2 ± 13% in inflamed tissues. Neither the Ca2+ channel Cav1.2b, gene expression, nor the c-Src kinase activity was altered by inflammation. Western blot analysis showed no change in the Ca2+ channel protein expression but increased nitrotyrosylated-Ca2+ channel proteins during inflammation. These data suggest that post-translational modification of Ca2+ channels during inflammation, possibly nitrotyrosylation, prevents c-Src kinase regulation resulting in decreased Ca2+ influx.

Ion channel remodeling in gastrointestinal inflammation

Background Gastrointestinal inflammation significantly affects the electrical excitability of smooth muscle cells. Considerable progress over the last few years have been made to establish the mechanisms by which ion channel function is altered in the setting of gastrointestinal inflammation. Details have begun to emerge on the molecular basis by which ion channel function may be regulated in smooth muscle following inflammation. These include changes in protein and gene expression of the smooth muscle isoform of L‐type Ca2+ channels and ATP‐sensitive K+ channels. Recent attention has also focused on post‐translational modifications as a primary means of altering ion channel function in the absence of changes in protein/gene expression. Protein phosphorylation of serine/theronine or tyrosine residues, cysteine thiol modifications, and tyrosine nitration are potential mechanisms affected by oxidative/nitrosative stress that alter the gating kinetics of ion channels. Collectively, these findings suggest that inflammation results in electrical remodeling of smooth muscle cells in addition to structural remodeling. Purpose The purpose of this review is to synthesize our current understanding regarding molecular mechanisms that result in altered ion channel function during gastrointestinal inflammation and to address potential areas that can lead to targeted new therapies.

Denitration of L-type calcium channel

Tyrosine nitration results in altered function of selective proteins, including human smooth muscle L‐type calcium channel, hCav1.2b. We report here that Cav1.2 is also subject to “denitration”. Cell lysates from activated macrophage‐like cell line, RAW264.7 cells, reversed peroxynitrite‐induced nitration of the carboxy terminus of Cav1.2 in a 1D gel assay. Tyrosine phosphorylation of the calcium channel by c‐src kinase was blocked by nitration but reversed by pretreatment with RAW264.7 cell lysates. These findings indicate that denitration may be a physiological mechanism to restore cellular excitability during inflammation.

The effect of tyrosine nitration of L-type Ca2+ channels on excitation-transcription coupling in colonic inflammation.

Background and purpose:  Excitation–transcriptional coupling involves communication between plasma membrane ion channels and gene expression in the nucleus. Calcium influx through L‐type Ca2+ channels induces phosphorylation of the transcription factor, cyclic‐AMP response element binding protein (CREB) and downstream activation of the cyclic‐AMP response element (CRE) promoter regions. Tyrosine nitration of Ca2+ channels attenuates interactions with c‐Src kinase, decreasing Ca2+ channel currents and smooth muscle contraction during colonic inflammation. In this study we examined the effect of tyrosine nitration and colonic inflammation on Ca2+ channel mediated phosphorylation of CREB and CRE activation.

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-1β, 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.

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.

α7-nAChR-mediated suppression of hyperexcitability of colonic dorsal root ganglia neurons in experimental colitis

Controlled clinical trials of nicotine transdermal patch for treatment of ulcerative colitis have been shown to improve histological and global clinical scores of colitis. Here we report that nicotine (1 microM) suppresses in vitro hyperexcitability of colonic dorsal root ganglia (DRG) (L(1)-L(2)) neurons in the dextran sodium sulfate (DSS)-induced mouse model of acute colonic inflammation. Nicotine gradually reduced regenerative multiple-spike action potentials in colitis mice to a single action potential. Nicotines effect on hyperexcitability of inflamed neurons was blocked in the presence of an alpha(7)-nicotinic acetylcholine receptor (nAChR) antagonist, methyllicaconitine, while choline, the alpha(7)-nAChR agonist, induced a similar effect to that of nicotine. Consistent with these findings, nicotine failed to suppress hyperexcitability in colonic DRG neurons from DSS-treated alpha(7) knockout mice. Furthermore, colonic DRG neurons from DSS-treated alpha(7) knockout mice were characterized by lower rheobase (10 +/- 5 vs. 77 +/- 13 pA, respectively) and current threshold (28 +/- 4 vs. 103 +/- 8 pA, respectively) levels than DSS-treated C57BL/J6 mice. An interesting observation of this study is that 8 of 12 colonic DRG (L(1)-L(2)) neurons from control alpha(7) knockout mice exhibited multiple-spike action potential firing while no wild-type neurons did. Overall, our findings suggest that nicotine at low 1 microM concentration suppresses in vitro hyperexcitability of inflamed colonic DRG neurons in a mouse model of acute colonic inflammation via activation of alpha(7)-nAChRs.