Journal of Pharmacology and Experimental Therapeutics GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

[GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL] NIM811 (N-Methyl-4-isoleucine Cyclosporine), a Mitochondrial Permeability Transition Inhibitor, Attenuates Cholestatic Liver Injury but Not Fibrosis in Mice

2008-11-12

Cholestasis causes hepatocyte death, possibly because of mitochondrial injury. This study investigated whether NIM811 (N-methyl-4-isoleucine cyclosporine), an inhibitor of the mitochondrial permeability transition (MPT), attenuates cholestatic liver injury in vivo. Cholestasis was induced in mice by bile duct ligation (BDL). NIM811 was gavaged (20 mg/kg) before BDL and daily (10 mg/kg) afterward. Mitochondrial depolarization, cell death, and MPT onset were assessed by intravital confocal/multiphoton microscopy of rhodamine 123, propidium iodide, and calcein. After BDL, serum alanine aminotransferase (ALT), hepatic necrosis, and apoptosis all increased. NIM811 decreased ALT, necrosis, and apoptosis by 60 to 86%. In vehicle-treated mice at 6 h after BDL, viable hepatocytes with depolarized mitochondria were 18/high-power field (hpf), and nonviable cells were ~1/hpf, showing that depolarization preceded necrosis. Calcein entered mitochondria after BDL, indicating MPT onset in vivo. NIM811 decreased depolarization by 72%, prevented calcein entry into mitochondria, and blocked release of cytochrome c. Hepatic tumor necrosis factor , transforming growth factor-β1, procollagen 1(I) mRNA, -smooth muscle actin, and Sirius red staining for collagen increased after BDL but were not different in vehicle- and NIM811-treated mice. Taken together, NIM811 decreased cholestatic necrosis and apoptosis but did not block fibrosis, indicating that the MPT plays an important role in cholestatic cell death in vivo.

[GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL] A Cross-Link between Protein Kinase A and Rho-Family GTPases Signaling Mediates Cell-Cell Adhesion and Actin Cytoskeleton Organization in Epithelial Cancer Cells

Leve, F., de Souza, W., Morgado-Diaz, J. A. — 2008-11-12

Disassembly of the apical junctional complex (AJC) together with actin cytoskeleton alterations are among the initial events for the development of epithelial cancer. The cell signaling pathways for these processes have been analyzed separately. However, the existence of a link between these two events has not been defined. In this study, using the extracellular calcium depletion model, we analyzed the signaling pathways regulating AJC disassembly together with actin cytoskeleton organization in colon adenocarcinoma cells (Caco-2). Changes in the location of AJC proteins were examined by immunofluorescence and immunoblotting, and tight junction (TJ) functionality was observed by measuring the transepithelial electrical resistance and permeation to ruthenium red. The actin cytoskeleton was stained with rhodamine-phalloidin and analyzed by confocal microscopy. Rho-GTPase activation was assessed by its translocation to the membrane (a hallmark of RhoA activation) and immunoblotting. Pharmacological inhibition of protein kinase A (PKA) with H-89 [N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide)] prevented AJC disassembly and actin disorganization at the apical and medial regions caused by calcium depletion. Rho inhibition using toxin A induced AJC disassembly and actin cytoskeleton reorganization. Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-ciclohexanecarboxamide], a Rho-associated kinase inhibitor, reversed redistribution of E-cadherin, but not of TJ proteins and actin disorganization caused by calcium depletion. Calcium depletion and forskolin treatment caused activation of Rho, as evidenced by their translocation to the membrane, an event concurrent to Rac and RhoGDI translocation, and this effect was also reverted by H-89. Thus, our findings demonstrate a central role of a regulatory cascade that integrates PKA and Rho-family GTPases in the AJC disassembly and actin organization in tumor epithelial cells.

[GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL] Heme Oxygenase-1 Inhibits the Proliferation of Pancreatic Stellate Cells by Repression of the Extracellular Signal-Regulated Kinase1/2 Pathway

2008-11-12

Activation of pancreatic stellate cells (PSCs) is the key process in the development of pancreatic fibrosis, a common feature of chronic pancreatitis and pancreatic cancer. In recent studies, curcumin has been shown to inhibit PSC proliferation via an extracellular signal-regulated kinase (ERK)1/2-dependent mechanism. In addition, curcumin is a potent inducer of the cytoprotective enzyme heme oxygenase-1 (HO-1) in other cell types. Therefore, the aims of this study were to 1) characterize the effect of curcumin on HO-1 gene expression in PSCs, 2) explore whether HO-1 induction contributes to the inhibitory effect of curcumin on PSC proliferation, and 3) clarify the involvement of the mitogen-activated protein kinase (MAPK) family in this context. Cultured rat PSCs were incubated with curcumin and assessed for HO-1 up-regulation by Northern blot analysis, immunoblotting, and activity assays. The effect of HO-1 on platelet-derived growth factor (PDGF)-induced PSC proliferation and MAPK activation was determined by immunoblotting, cell proliferation assays, and cell count analyses. Curcumin induced HO-1 gene expression in PSCs in a time- and dose-dependent manner and inhibited PDGF-mediated ERK1/2 phosphorylation and PSC proliferation. These effects were blocked by treatment of PSCs with tin protoporphyrin IX, an HO inhibitor, or transfection of HO-1 small interfering RNA. Our data provide evidence that HO-1 induction contributes to the inhibitory effect of curcumin on PSC proliferation. Therefore, therapeutic up-regulation of HO-1 could represent a mode for inhibition of PSC proliferation and thus may provide a novel strategy in the prevention of pancreatic fibrosis.

[GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL] Clotrimazole Ameliorates Intestinal Inflammation and Abnormal Angiogenesis by Inhibiting Interleukin-8 Expression through a Nuclear Factor-{kappa}B-Dependent Manner

2008-10-17

Increased interleukin (IL)-8 plays an important role not only in activation and recruitment of neutrophils but also in inducing exaggerated angiogenesis at the inflamed site. In the present study, we investigated the fact that clotrimazole (CLT) inhibits intestinal inflammation, and the inhibitory action is mediated through suppression of IL-8 expression. In the trinitrobenzene sulfonic acid (TNBS)-induced rat colitis model, CLT dose-dependently protected from the TNBS-induced weight loss, colon ulceration, and myeloperoxidase activity increase. In the lesion site, CLT also suppressed the TNBS-induced angiogenesis, IL-8 expression, and nuclear factor (NF)-B activation. In a cellular model of colitis using tumor necrosis factor (TNF)--stimulated HT29 colon epithelial cells, treatment with CLT significantly suppressed TNF--mediated IL-8 induction and NF-B transcriptional activity revealed by a luciferase reporter gene assay. Furthermore, cotreatment with CLT and pyrrolidine dithiocarbamate, a NF-B inhibitor, synergistically reduced the NF-B transcriptional activity as well as IL-8 expression. In an in vitro angiogenesis assay, CLT suppressed IL-8-induced proliferation, tube formation, and invasion of human umbilical vein endothelial cells. The in vivo angiogenesis assay using chick chorioallantoic membrane also showed that CLT significantly inhibited the IL-8-induced formation of new blood vessels. Taken together, these results suggest that CLT may prevent the progression of intestinal inflammation by not only down-regulating IL-8 expression but also inhibiting the action of IL-8 in both colon epithelial and vascular endothelial cells during pathogenesis of intestinal inflammation.

[GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL] Blockade of CXCL12/CXCR4 Axis Ameliorates Murine Experimental Colitis

2008-10-17

Recent studies indicate that the CXCL12/CXCR4 interaction is involved in several inflammatory conditions. However, it is unclear whether this interaction has a role in the pathophysiology of inflammatory bowel disease (IBD). We investigated the significance of this interaction in patients with IBD and in mice with dextran sulfate sodium (DSS)-induced colitis and the effect of a CXCR4 antagonist on experimental colitis. First, we measured CXCR4 expression on peripheral T cells in patients with IBD. Furthermore, we investigated CXCR4 expression on leukocytes and CXCL12 expression in the colonic tissue of mice with DSS-induced colitis, and we evaluated the effects of a CXCR4 antagonist on DSS-induced colitis and colonic inflammation of interleukin (IL)-10 knockout (KO) mice. Colonic inflammation was assessed both clinically and histologically. Cytokine production from mesenteric lymph node cells was also examined. CXCR4 expression on peripheral T cells was significantly higher in patients with active ulcerative colitis (UC) compared with normal controls, and CXCR4 expression levels of UC patients correlated with disease activity. Both CXCR4 expression on leukocytes and CXCL12 expression in colonic tissue were significantly increased in mice with DSS-induced colitis. Administration of a CXCR4 antagonist ameliorated colonic inflammation in DSS-induced colitis and IL-10 KO mice. CXCR4 antagonist reduced tumor necrosis factor- and interferon- production from mesenteric lymph node cells, whereas it did not affect IL-10 production. The percentage of mesenteric Foxp3⁺CD25⁺ T cells in DSS-induced colitis was not affected by CXCR4 antagonist. These results suggest that blockade of this chemokine axis might have potential as a therapeutic target for the treatment of IBD.

[GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL] Involvement of the Neurokinin-2 Receptor in Airway Smooth Muscle Stretch-Activated Contractions Assessed in Perfused Intact Bovine Bronchial Segments

Hernandez, J. M., Cox, G., Janssen, L. J. — 2008-10-17

The airway response to deep inspirations (DIs) in asthmatics has been shown to be ineffective in producing bronchodilation and can even cause bronchoconstriction. However, the manner by which a DI is able to cause bronchoconstriction remains ambiguous. We sought to investigate the pathway involved in this stretch-activated contraction and whether this contraction is intrinsic to airway smooth muscle (ASM). In brief, intact bovine bronchial segments were dissected, and side branches were ligated and then mounted horizontally in an organ bath. Intraluminal pressure was measured under isovolumic conditions. Instantaneously opening and then closing the tap on a column of fluid 5 to 30 cm high evoked a sudden increase in intraluminal pressure (equivalent to the height of the column of fluid) followed by a stress relaxation response of the ASM. When tissues were stimulated with carbachol (10^-8 M) or serotonin (10^-7 M) for 10 min, and the consequent agonist-evoked pressure response was dissipated manually, the response to the same transmural stretch was accompanied by a slowly developing and prolonged increase in intraluminal pressure. This stretch-activated response was significantly diminished by the stretch-activated cation channel blocker gadolinium (10^-3 M), the L-type Ca²⁺ channel blockers nifedipine (2 x 10^-6 M), diltiazem (10^-5 M), and verapamil (10^-5 M), the sensory neurotoxin capsaicin (10^-5 M), and the neurokinin (NK)₂ receptor antagonists MEN 10376 ([Tyr^I,d-Trp^6,8,9,Lys¹⁰]-NKA(4–10)) (10^-5 M) and SR48968 (N-[(2S)-4-(4-acetamido-4-phenylpiperidin-1-yl)-2-(3,4-dichlorophenyl)butyl]-N-methylbenzamide) (3 x 10^-6 M). These results show the ability of isolated airways to exhibit stretch-activated contractions and suggest a role for stretch-activated cation channels, sensory afferent neurons, the neurotransmitter NKA, and L-type Ca²⁺ channels in these isolated airway responses.

[GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL] Morphine Tolerance in the Mouse Ileum and Colon

Ross, G. R., Gabra, B. H., Dewey, W. L., Akbarali, H. I. — 2008-10-17

Repeated administration of morphine is associated with tolerance to its antinociceptive properties. However, constipation remains the major side effect of chronic exposure to morphine. In contrast, previous studies suggest that tolerance to opioids develops in the ileum of several species. In this study, we provide evidence that constipation may arise due to a lack of tolerance development to morphine in the colon. Mice received implants with either placebo or 75 mg of morphine pellets, and they were examined for morphine tolerance to antinociception, defecation, and intestinal and colonic transit after 72 h. Tissues were obtained from the ileum and distal colon, and contractile responses were measured from longitudinal and circular muscle preparations. In morphine-pelleted mice, a 5.5-fold tolerance developed to antinociception after 72 h, and a 53.2-fold tolerance developed in mice that received an additional daily morphine injection. In both models, intestinal transit but not defecation or colonic transit developed tolerance. In isolated longitudinal muscles, electrical field stimulation-induced cholinergic contractions were dose-dependently inhibited by morphine in both the ileum and colon of placebo pelleted with a pD₂ of 7.1 ± 0.4 and 7.8 ± 0.4, respectively. However, the dose response to morphine inhibition was shifted to the right for the ileum from morphine-pelleted mice (pD₂ = 5.1 ± 0.4) but not the colon (pD₂ = 6.9 ± 0.4). In circular muscle preparations, morphine induced atropine-insensitive contractions in both tissue segments. Tolerance to morphine developed in the ileum but not the colon upon repeated administration of morphine. These findings indicate that a lack of tolerance development in the colon is the basis for opioid bowel dysfunction.

[GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL] Levcromakalim and MgGDP Activate Small Conductance ATP-Sensitive K+ Channels of K+ Channel Pore 6.1/Sulfonylurea Receptor 2A in Pig Detrusor Smooth Muscle Cells: Uncoupling of cAMP Signal Pathways

Kajioka, S., Nakayama, S., Asano, H., Seki, N., Naito, S., Brading, A. F. — 2008-09-19

Pharmacological studies have suggested the existence of ATP-sensitive K⁺ (K_ATP) channel as a therapeutic target in urinary bladders; however, electrical properties have not yet been shown. Patch-clamp techniques were applied to investigate the properties of K_ATP channels in pig detrusor cells. In whole-cell configuration, levcromakalim, a K_ATP channel opener, induced a long-lasting outward current in a concentration-dependent manner. The current-voltage curve of the levcromakalim-induced membrane current intersected at approximately -80 mV. This current was abolished by glibenclamide. Intracellular application of 0.1 mM GDP significantly enhanced the levcromakalim-induced membrane current, whereas cAMP did not. Furthermore, neurotransmitters related to cAMP signaling, such as calcitonin gene-related peptide, vasointestinal peptide, adenosine, and somatostatin, had little effect on the membrane current. In cell-attached configuration, levcromakalim activated K⁺ channels with a unitary conductance of ~12 pS. When the patch configuration was changed to inside-out mode, the K⁺ channel activity ran down. Subsequent application of 1 mM GDP reactivated the channels. The openings of the ~12 pS K⁺ channels in the presence of 1 mM GDP was suppressed by ATP and glibenclamide. In reverse transcription-polymerase chain reaction, K⁺ channel pore 6.1 and sulfonylurea receptor (SUR)2A were predominant in pig detrusor cells. The 12 pS K⁺ channel activated by levcromakalim in pig detrusor smooth muscle cells is a K_ATP channel. The predominant expression of SUR2A can account for the lack of effect of neurotransmitters related to cAMP.

[GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL] Calcium Sensitization in Human Esophageal Muscle: Role for RhoA Kinase in Maintenance of Lower Esophageal Sphincter Tone

Sims, S. M., Chrones, T., Preiksaitis, H. G. — 2008-09-19

A rise in intracellular-free calcium ([Ca²⁺]_i) concentration is important for initiating contraction of smooth muscles, and Ca²⁺ sensitization involving RhoA kinase can sustain tension. We previously found that [Ca²⁺]_i was comparable in cells from the esophageal body (EB) and lower esophageal sphincter (LES) muscles, despite the fact that the LES maintains resting tone. We hypothesized that Ca²⁺ sensitization contributes to contraction in human esophageal muscle. Tension and [Ca²⁺]_i were measured simultaneously in intact human EB and LES muscles using the ratiometric Ca²⁺-sensitive dye fura-2. Spontaneous oscillations in EB muscle tension were associated with transient elevations of [Ca²⁺]_i. Carbachol caused a large increase in tension, compared with spontaneous oscillations, although the rise of [Ca²⁺]_i was similar, suggesting Ca²⁺ sensitization. The RhoA-kinase blockers (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632) and 1-(5-isoquinolinesulfonyl)-homopiperazine hydrochloride (HA-1077) reduced carbacholand nerve-evoked contraction of the EB, accompanied by smaller reduction in the rise of [Ca²⁺]_i. Protein kinase C inhibitors reduced force to a lesser extent. RhoA-kinase blockers caused concentration-dependent reduction of tension in spontaneously contracted LES muscles. Moreover, RhoA-kinase blockers reduced intrinsic nerve-evoked and carbachol-evoked contraction. However, there was no effect on nerve- or nitric oxide-mediated relaxation of LES. Ca²⁺ sensitization mediated by the RhoA-kinase pathway has an important role in contraction of human EB muscles and LES tonic contraction, a feature not previously recognized.

[GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL] E Series of Prostaglandin Receptor 2-Mediated Activation of Extracellular Signal-Regulated Kinase/Activator Protein-1 Signaling Is Required for the Mitogenic Action of Prostaglandin E2 in Esophageal Squamous-Cell Carcinoma

2008-09-19

The use of nonsteroidal anti-inflammatory drugs is associated with a lower risk for esophageal squamous cell carcinoma, in which overexpression of cyclooxygenase-2 (COX-2) is frequently reported. Prostaglandin E₂ (PGE₂), a COX-2-derived eicosanoid, is implicated in the promotion of cancer growth. However, the precise role of PGE₂ in the disease development of esophageal squamous cell carcinoma remains elusive. In this study, we investigated the effect of PGE₂ on the proliferation of cultured esophageal squamous cell carcinoma cells (HKESC-1). Results showed that HKESC-1 cells expressed all four series of prostaglandin (EP) receptors, namely, EP1 to EP4 receptors. In this regard, PGE₂ and the EP2 receptor agonist (±)-15-deoxy-16S-hydroxy-17-cyclobutyl PGE₁ methyl ester (butaprost) markedly increased HKESC-1 cell proliferation. Moreover, the mitogenic effect of PGE₂ was significantly attenuated by RNA interference-mediated knockdown of the EP2 receptor, indicating that this receptor mediated the mitogenic effect of PGE₂. In this connection, PGE₂ and butaprost induced phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2), whose down-regulation by RNA interference significantly attenuated PGE₂-induced cell proliferation. In addition, PGE₂ and butaprost increased c-Fos expression and activator protein 1 (AP-1) transcriptional activity, which were abolished by the mitogen-activated protein kinase/Erk kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)-butadiene ethanolate (U0126). AP-1-binding inhibitor curcumin also partially reversed the mitogenic effect of PGE₂. Taken together, these data demonstrate for the first time that the EP2 receptor mediates the mitogenic effect of PGE₂ in esophageal squamous cell carcinoma via activation of the Erk/AP-1 pathway. This study supports the growth-promoting action of PGE₂ in esophageal squamous cell carcinoma and the potential application of EP2 receptor antagonists in the treatment of this disease.