AJP - Gastrointestinal and Liver Physiology

Harmer, A. R.; Smith, P. M.; Gallacher, D. V.

doi: 10.1152/ajpgi.00096.2004pmid: 15308468

Polarized Ca 2+ signals that originate at and spread from the apical pole have been shown to occur in acinar cells from lacrimal, parotid, and pancreatic glands. However, "local" Ca 2+ signals, that are restricted to the apical pole of the cell, have been previously demonstrated only in pancreatic acinar cells in which the primary function of the Ca 2+ signal is to regulate exocytosis. We show that submandibular acinar cells, in which the primary function of the Ca 2+ signal is to drive fluid and electrolyte secretion, are capable of both Ca 2+ waves and local Ca 2+ signals. The generally accepted model for fluid and electrolyte secretion requires simultaneous Ca 2+ -activation of basally located K + channels and apically located Cl – channels. Whereas a propagated cell-wide Ca 2+ signal is clearly consistent with this model, a local Ca 2+ signal is not, because there is no increase in intracellular Ca 2+ concentration at the basal pole of the cell. Our data provide the first direct demonstration, in submandibular acinar cells, of the apical and basal location of the Cl – and K + channels, respectively, and confirm that local Ca 2+ signals do not Ca 2+ -activate K + channels. We reevaluate the model for fluid and electrolyte secretion and demonstrate that Ca 2+ -activation of the Cl – channels is sufficient to voltage-activate the K + channels and thus demonstrate that local Ca 2+ signals are sufficient to support fluid secretion. Ca 2+ mobilization; patch-clamp; K + channel; Cl – channel Address for reprint requests and other correspondence: P. M. Smith, Clinical Dental Sciences, The Univ. of Liverpool, The Edwards Bldg., Daulby St. Liverpool L69 3GN (E-mail: [email protected] )

Khan, W. I.; Motomura, Y.; Blennerhassett, P. A.; Kanbayashi, H.; Varghese, A. K.; El-Sharkawy, R. T.; Gauldie, J.; Collins, S. M.

doi: 10.1152/ajpgi.00159.2004pmid: 15308470

In our previous studies, we demonstrated that during Trichinella spiralis infection, T helper (Th) 2 cells contribute to the development of intestinal muscle hypercontractility and worm expulsion from the gut via STAT6. In addition, we have linked the altered muscle contractility to the eviction of the parasite and thereby to the host defense. However, the initial events linking infection to the development of muscle hypercontractility are poorly understood. In this study, we examined the contribution of CD40-CD40 ligand (CD40L) interaction in the development of intestinal muscle hypercontractility, in monocyte chemoattractant protein-1 (MCP-1) production, and in the Th2 response in CD40 ligand-deficient (CD40L –/–) mice infected with T. spiralis . Expulsion of intestinal worms was substantially delayed in CD40L –/– mice compared with the wild-type mice after T. spiralis infection. Consistent with delayed worm expulsion, there was a significant attenuation of intestinal muscle contractility in CD40L –/– mice. Infected CD40L –/– mice also exhibited marked impairment in the production of MCP-1, IL-4, IL-13, IgG1, IgE, and mouse mucosal MCP 1 (MMCP-1), and in goblet cell response. These results demonstrate that CD40-CD40 ligand interaction plays an important role in MCP-1 production, Th2 response, intestinal muscle hypercontractility, and worm expulsion in nematode infection. The present data suggest that the early events leading to the generation of Th2 response include CD40-CD40 ligand interaction, which subsequently influences the production of Th2 cytokines, most likely via upregulation of MCP-1. CD40 ligand; monocyte chemoattractant protein-1; T helper 2 cells; muscle; Trichinella spiralis ; host defense Address for reprint requests and other correspondence: S. M. Collins, Room 4W8, HSC, McMaster Univ. Medical Centre, Hamilton, Ontario L8N 3Z5, Canada (E-mail: [email protected] )

Mammen, Joshua M. V.; Song, J. Cecilia; Yoo, James; Kim, Peter S.; Davis, Harold W.; Calvo, M. Isabel; Worrell, Roger T.; Matlin, Karl S.; Matthews, Jeffrey B.

doi: 10.1152/ajpgi.00139.2004pmid: 15358594

Ischemia is the central pathogenic factor underlying a spectrum of intestinal disorders. The study of the cellular signaling responses to ischemic stress in nonepithelial cells has progressed substantially in the previous several years, but little is known about the response in epithelial cells. Unique features of the epithelial response to ischemic stress suggest differential regulation with regards to signaling. The PKC family of proteins has been implicated in ischemic stress in nonepithelial systems. The role of PKC isoforms in chemical ischemia in intestinal epithelial cells is evaluated in this study. Additionally, the phosphorylation of the F-actin cross-linking protein myristoylated alanine-rich C kinase substrate (MARCKS) is also studied. Chemical ischemia resulted in the transient activation of only the isoform PKC-ε as detected by translocation employing the subcellular fractionation technique. The pharmacological agonists phorbol 12-myristate 13-acetate and carbachol also led to the translocation of PKC-ε. By immunofluoresence, MARCKS is noted to be located at the lateral membrane under control conditions. In response to carbachol, MARCKS translocates to the cytosol, indicating its phosphorylation, which is additionally confirmed biochemically. Consistent with this observation, carbachol induces the translocation of PKC-ε to proximity with MARCKS at the lateral membrane. In response to chemical ischemia, MARCKS fails to translocate and phosphorylation does not increase. Additionally, the translocation of PKC-ε is not to the lateral membrane but rather basally. The data suggest that the differential translocation of PKC-ε in response to pharmacological agonists versus ischemic stress may lead to different effects on downstream targets. F-actin; hypoxia; ischemia; myristolated alanine-rich C kinase substrate; phorbol ester Address for reprint requests and other correspondence: J. B. Matthews, Dept. of Surgery, Univ. of Cincinnati College of Medicine, 231 Albert Sabin Way, PO Box 670558, Cincinnati, OH 45267–0558 (E-mail: [email protected] )

Lim, Inja; Gibbons, Simon J; Lyford, Gregory L.; Miller, Steven M.; Strege, Peter R.; Sarr, Michael G.; Chatterjee, Suvro; Szurszewski, Joseph H.; Shah, Vijay H.; Farrugia, Gianrico

doi: 10.1152/ajpgi.00205.2004pmid: 15319183

Carbon monoxide (CO) is increasingly recognized as a physiological messenger. CO is produced in the gastrointestinal tract with diverse functions, including regulation of gastrointestinal motility, interacting with nitric oxide (NO) to mediate neurotransmission. The aim of this study was to determine the effect of CO on the human intestinal L-type Ca 2+ channel expressed in HEK cells and in native cells using the patch-clamp technique. Extracellular solution contained 10 mM Ba 2+ as the charge carrier. Maximal peak Ba 2+ current ( I Ba ) was significantly increased by bath application of 0.2% CO to transfected HEK cells (18 ± 3%). The NO donor S -nitroso- N -acetylpenicillamine also increased I Ba , and CO (0.2%) increased NO production in transfected HEK cells. The CO-induced increase in I Ba was blocked when cells were pretreated with 1H-1,2,4-oxadiazolo4,3-aquinoxalin-1-one (10 µM) or inhibitors of NO synthase (NOS). The PKA inhibitor KT-5720 (0.5 µM) and milrinone (3 µM), a phosphodiesterase (PDE) III inhibitor, blocked the effect of CO on I Ba . Similar effects were seen in freshly dissociated human intestinal smooth muscle cells. The data suggest that exogenous CO can activate native and heterologously expressed intestinal L-type Ca 2+ channels through a pathway that involves activation of NOS, increased NO, and cGMP levels, but not PKG. Rather, the pathway appears to involve PKA, partly by reducing cAMP breakdown through inhibition of PDE III. CO-induced NO production may explain the apparent discrepancy between the low affinity of guanylyl cyclase for CO and the robust cGMP production evoked by CO. ion channels; gases; human studies; patch clamp Address for reprint requests and other correspondence: G. Farrugia, 8 Guggenheim Bldg. Mayo Clinic, 200 First St. SW, Rochester, MN 55905 (E-mail: [email protected] )

Diehl, Anna Mae

doi: 10.1152/ajpgi.00376.2004pmid: 15591584

Liver regeneration is necessary to recover from alcoholic liver injury. Herein, we review evidence that ethanol interferes with liver regeneration. Briefly, alcoholic fatty livers demonstrate increased rates of hepatocyte death. The latter provides a regenerative stimulus. However, unlike mature hepatocytes in healthy adult livers, most surviving mature hepatocytes in alcoholic fatty livers cannot replicate. Therefore, less mature cells (progenitors) must differentiate to replace dead hepatocytes. Little is known about the general mechanisms that modulate the differentiation of liver progenitors in adults. Delineation of these mechanisms and clarification of how ethanol influences them might suggest new therapies for alcoholic liver disease. cell senescence; progenitor cells; hepatocyte differentiation Address for reprint requests and other correspondence: A. M. Diehl, Gastroenterology Division, Genome Science Research Bldg. 1, Suite 1073, Box 3256, Durham, NC 27710 (E-mail: [email protected] )

Hung, Daniel Y.; Siebert, Gerhard A.; Chang, Ping; Burczynski, Frank J.; Roberts, Michael S.

doi: 10.1152/ajpgi.00196.2004pmid: 15345470

Nonalcoholic fatty liver disease is the most common of all liver diseases. The hepatic disposition 3 Hpalmitate and its low-molecular-weight metabolites in perfused normal and steatotic rat liver were studied using the multiple indicator dilution technique and a physiologically based slow diffusion/bound pharmacokinetic model. The steatotic rat model was established by administration of 17α-ethynylestradiol to female Wistar rats. Serum biochemistry markers and histology of treated and normal animals were assessed and indicated the presence of steatosis in the treatment group. The steatotic group showed a significantly higher alanine aminotransferase-to-aspartate aminotransferase ratio, lower levels of liver fatty acid binding protein and cytochrome P -450, as well as microvesicular steatosis with an enlargement of sinusoidal space. Hepatic extraction for unchanged 3 Hpalmitate and production of low-molecular-weight metabolites were found to be significantly decreased in steatotic animals. Pharmacokinetic analysis suggested that the reduced extraction and sequestration for palmitate and its metabolites was mainly attributed to a reduction in liver fatty acid binding protein in steatosis. 17α-ethynylestradiol; hepatic palmitate disposition Address for reprint requests and other correspondence: M. S. Roberts, Dept. of Medicine, Univ. of Queensland, Princess Alexandra Hospital, Woollongabba, Qld 4102, Australia (E-mail: [email protected] )

Li, Tiangang; Chiang, John Y. L.

doi: 10.1152/ajpgi.00258.2004pmid: 15331348

Bile acids, steroids, and drugs activate steroid and xenobiotic receptor pregnane X receptor (PXR; NR1I2), which induces human cytochrome P4503A4 (CYP3A4) in drug metabolism and cholesterol 7α-hydroxylase (CYP7A1) in bile acid synthesis in the liver. Rifampicin, a human PXR agonist, inhibits bile acid synthesis and has been used to treat cholestatic diseases. The objective of this study is to elucidate the mechanism by which PXR inhibits CYP7A1 gene transcription. The mRNA expression levels of CYP7A1 and several nuclear receptors known to regulate the CYP7A1 gene were assayed in human primary hepatocytes by quantitative real-time PCR (Q-PCR). Rifampicin reduced CYP7A1 and small heterodimer partner (SHP; NR02B) mRNA expression suggesting that SHP was not involved in PXR inhibition of CYP7A1. Rifampicin inhibited CYP7A1 reporter activity and a PXR binding site was localized to the bile acid response element-I. Mammalian two-hybrid assays revealed that PXR interacted with hepatic nuclear factor 4α (HNF4α, NR2A1) and rifampicin was required. Coimmunoprecipitation assay confirmed PXR interaction with HNF4α. PXR also interacted with peroxisome proliferator-activated receptor γ coactivator (PGC-1α), which interacted with HNF4α and induced CYP7A1 gene transcription. Rifampicin enhanced PXR interaction with HNF4α and reduced PGC-1α interaction with HNF4α. Chromatin immunoprecipitation assay showed that PXR, HNF4α, and PGC-1α bound to CYP7A1 chromatin, and rifampicin dissociated PGC-1α from chromatin. These results suggest that activation of PXR by rifampicin promotes PXR interaction with HNF4α and blocks PGC-1α activation with HNF4α and results in inhibition of CYP7A1 gene transcription. Rifampicin inhibition of bile acid synthesis may be a protective mechanism against drug and bile acid-induced cholestasis. bile acid synthesis; gene regulation; nuclear receptors; peroxisome proliferator-activated receptor γ coactivator Address correspondence: J. Y. L. Chiang, Dept. of Biochemistry and Molecular Pathology, Northeastern Ohio Univ. College of Medicine, 4209 State Route 44, P.O. Box 95, Rootstown, OH 44272 (E-mail: [email protected] )

Shimamoto, Chikao; Fujiwara, Shoko; Kato, Masumi; Ito, Shigenori; Katsu, Ken-ichi; Mori, Hiroshi; Nakahari, Takashi

doi: 10.1152/ajpgi.00060.2004pmid: 15345468

The effects of indomethacin (IDM) and aspirin (ASA) on ACh (10 µM) -stimulated exocytotic events were studied in guinea pig antral mucous cells by using video optical microscopy. IDM or ASA, which inhibits cyclooxygenase (COX), decreased the frequency of ACh-stimulated exocytotic events by 30% or 60%, respectively. The extent of inhibition induced by ASA (60%) decreased by 30% when IDM or arachidonic acid (AA, the substrate of COX) was added. IDM, unlike ASA, appears to induce the accumulation of AA, which enhances the frequency of ACh-stimulated exocytotic events in ASA-treated cells. ONO-8713 (100 µM; an inhibitor of the EP1–EP4 prostaglandin receptors) and N -2-(( p -bromocinnamyl)amino)ethyl-5-isoquinolinesulfonamide, HCl (H-89, 20 µM; an inhibitor of PKA) also decreased the frequency of ACh-stimulated exocytotic events by 60%. However, the supplementation of PGE 2 (1 µM) prevented the IDM-induced decrease in the frequency of ACh-stimulated exocytotic events. SC-560 (an inhibitor of COX-1) decreased the frequency of ACh-stimulated exocytotic events by 30%, but NS-398 (an inhibitor of COX-2) did not. Moreover, IDM decreased the frequency of exocytotic events stimulated by ionomycin, suggesting that COX-1 activity is stimulated by an increase in intracellular Ca 2+ concentration (Ca 2+ i ). ACh and ionomycin increased PGE 2 release in antral mucosal cells. In conclusion, in ACh-stimulated antral mucous cells, an increase in Ca 2+ i activates Ca 2+ -regulated exocytotic events and PGE 2 release mediated by COX-1. The released PGE 2 induces the accumulation of cAMP, which enhances the Ca 2+ -regulated exocytosis. The autocrine mechanism mediated by PGE 2 maintains the high-level mucin release from antral mucous cells during ACh stimulation. gastric mucin secretion; cyclic adenosine 5'-monophosphate; cyclooxygenase-1; indomethacin; aspirin Address for reprint requests and other correspondence: T. Nakahari, Dept. of Physiology, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki 569-8686, Japan (E-mail: [email protected] )

Motomura, Y.; Kanbayashi, H.; Khan, W. I.; Deng, Y.; Blennerhassett, P. A.; Margetts, P. J.; Gauldie, J.; Egashira, K.; Collins, S. M.

doi: 10.1152/ajpgi.00186.2004pmid: 15297261

Peritoneal fibrosis formation is a consequence of inflammation/injury and a significant medical problem to be solved. The effects of soluble VEGF receptor type I (sFlt-1) gene transfer on experimental peritoneal fibrosis were examined and compared with soluble transforming growth factor- (TGF- ) receptor type II (sTGF RII) gene transfer. Male C57BL/6 mice were injected with 1.5 x 10 8 plaque-forming unit of adenovirus encoding active TGF- (AdTGF ) intraperitoneally. Some mice had been treated with sTGF RII or sFlt-1 plasmid injection into skeletal muscle with electroporation 4 days before virus administration. Mice were euthanized at day 14 after virus administration. AdTGF induced significant elevation of serum active TGF- , caused significant inflammatory response weight loss, elevation of serum amyloid-P (SAP) and IL-12, increased expression of monocyte chemoattractant protein-1 (MCP-1) mRNA, and induced marked thickening of the peritoneum and collagen deposition. Gene transfer of sFlt-1 reduced the collagen deposition ∼81% in mesenteric tissue. Treatment with sFlt-1 decreased ICAM-1 and MCP-1 mRNA expression significantly. Significant negative correlation between serum sFlt-1 and placental growth factor level was observed, whereas there was no significant negative correlation between sFlt-1 and VEGF. On the other hand, sTGF RII treatment enhanced the AdTGF -induced inflammation (significant elevation of SAP, TNF-α, and IL-12 levels and upregulation of ICAM-1 and MCP-1 mRNA expressions) and failed to prevent collagen deposition. These observations indicate that sFlt-1 gene transfer might be of therapeutic benefit in peritoneal fibrosis. inflammation; adhesion; placental growth factor; monocyte chemoattractant protein-1; anti-inflammatory cytokine Address for reprint requests and other correspondence: S. M. Collins, Rm. 4W8, McMaster Univ. Medical Center, 1200 Main St. West, Hamilton, Ontario L8N 3Z5, Canada (E-mail: [email protected] )

Nanthakumar, N. Nanda; Young, Cheryl; Ko, Jae Sung; Meng, Di; Chen, Ji; Buie, Timothy; Walker, W. Allan

doi: 10.1152/ajpgi.00169.2004pmid: 15591589

Necrotizing enterocolitis (NEC) is a major inflammatory disease of the premature human intestine that can be prevented by glucocorticoids if given prenatally before the 34th wk of gestation. This observation suggests that a finite period of steroid responsiveness exists as has been demonstrated in animal models. Human intestinal xenografts were used to determine whether a glucocorticoid responsive period exists in the developing human intestine. Developmental responsiveness was measured by lactase activity and inflammatory responsiveness by IL-8, IL-6, and monocyte chemotactic protein-1 (MCP-1) induction after an endogenous (IL-1 ) or exogenous (LPS) proinflammatory stimulus, respectively. Functional development of ileal xenografts were monitored for 30 wk posttransplantation, and the lactase activity recapitulated that predicted by in utero development. Cortisone acetate accelerated the ontogeny of lactase at 20 wk (immature) but the effect was lost by 30 wk (mature) posttransplant. Concomitant with accelerated maturation, the IL-8 response to both IL-1 and LPS was significantly dampened (from 6- to 3-fold) by glucocorticoid pretreatment in the immature but not mature xenografts. The induction of IL-8 was reflected at the level of IL-8 mRNA, suggesting transcriptional regulation. The excessive activation of IL-8 in the immature gut was mediated by a prolonged activation of ERK and p38 kinases and nuclear translocation of NF-κB due to low levels of IκB. Steroid pretreatment in immature intestine dampens activation of all three signaling pathways in response to proinflammatory stimuli. Therefore, accelerating intestinal maturation by glucocorticoids within the responsive period by accelerating functional and inflammatory maturation may provide an effective preventive therapy for NEC. cortisone acetate; interleukin-8; human ileum; intestinal inflammation; signal transduction Address for reprint requests and other correspondence: N. N. Nanthakumar, Developmental Gastroenterology Laboratory, Massachusetts General Hospital-East, 114, 16th St., Rm. 3650, Charlestown, MA 02129 (E-mail: [email protected] )