79 results on '"J. Gregory Fitz"'
Search Results
2. Hepatic Thermal Injury Promotes Colorectal Cancer Engraftment in C57/black 6 Mice
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Karim C. El Kasmi, Yuki Fujiwara, Richard D. Schulick, Aimee L. Anderson, J. Gregory Fitz, Yuwen Zhu, Alison L. Halpern, Jeniann A. Yi, and Carlton C. Barnett
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Vascular Endothelial Growth Factor A ,0301 basic medicine ,Physiology ,Colorectal cancer ,Adenocarcinoma ,medicine.disease_cause ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Immune system ,Cell Line, Tumor ,Tumor Microenvironment ,medicine ,Animals ,Arginase ,Thermal injury ,business.industry ,Liver Neoplasms ,Burns, Electric ,Cell Biology ,Macrophage Activation ,Hypoxia-Inducible Factor 1, alpha Subunit ,medicine.disease ,Mice, Inbred C57BL ,Vascular endothelial growth factor ,Disease Models, Animal ,030104 developmental biology ,Liver ,chemistry ,Tumor progression ,030220 oncology & carcinogenesis ,Hemostasis ,Concomitant ,Colonic Neoplasms ,Disease Progression ,Cancer research ,Carcinogenesis ,business ,Neoplasm Transplantation ,Research Article ,Signal Transduction - Abstract
Treatment options for liver metastases (primarily colorectal cancer) are limited by high recurrence rates and persistent tumor progression. Surgical approaches to management of these metastases typically use heat energy including electrocautery, argon beam coagulation, thermal ablation of surgical margins for hemostasis, and preemptive thermal ablation to prevent bleeding or to effect tumor destruction. Based on high rates of local recurrence, these studies assess whether local effects of hepatic thermal injury (HTI) might contribute to poor outcomes by promoting a hepatic microenvironment favorable for tumor engraftment or progression due to induction of procancer cytokines and deleterious immune infiltrates at the site of thermal injury. To test this hypothesis, an immunocompetent mouse model was developed wherein HTI was combined with concomitant intrasplenic injection of cells from a well-characterized MC38 colon carcinoma cell line. In this model, HTI resulted in a significant increase in engraftment and progression of MC38 tumors at the site of thermal injury. Furthermore, there were local increases in expression of messenger ribonucleic acid (mRNA) for hypoxia-inducible factor-1α (HIF1α), arginase-1, and vascular endothelial growth factor α and activation changes in recruited macrophages at the HTI site but not in untreated liver tissue. Inhibition of HIF1α following HTI significantly reduced discreet hepatic tumor development (P = 0.03). Taken together, these findings demonstrate that HTI creates a favorable local environment that is associated with protumorigenic activation of macrophages and implantation of circulating tumors. Discrete targeting of HIF1α signaling or inhibiting macrophages offers potential strategies for improving the outcome of surgical management of hepatic metastases where HTI is used.
- Published
- 2020
3. Cholangiocyte Ion Channels
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J. Gregory Fitz
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Chemistry ,Biophysics ,Cholangiocyte ,Ion channel - Published
- 2020
4. Hepatology after Hepatitis C
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J. Gregory Fitz
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Liver Cirrhosis ,0301 basic medicine ,medicine.medical_specialty ,Carcinoma, Hepatocellular ,medicine.medical_treatment ,Population ,Hepacivirus ,Liver transplantation ,Gastroenterology ,03 medical and health sciences ,Liver disease ,0302 clinical medicine ,Internal medicine ,Prevalence ,medicine ,Humans ,Disease Eradication ,Intensive care medicine ,education ,Hepatitis ,education.field_of_study ,business.industry ,Liver Diseases ,Liver Neoplasms ,General Medicine ,Hepatitis C ,Hepatitis B ,Hepatology ,medicine.disease ,Liver Transplantation ,030104 developmental biology ,030211 gastroenterology & hepatology ,business ,Viral hepatitis - Abstract
The ∼90% probability of curing individual patients with hepatitis C virus (HCV)using direct-acting antivirals represents one of the most dramatic medical success stories of the modern era, and the journey from viral discovery to treatment occurred over just ∼25 years. The realities of the global burden of disease (2-3% of the world's population is infected), limited access to care and cost of treatment mean that HCV will continue to be a major problem for the next 25 years. But what if HCV (and hepatitis B) could be eradicated? Since liver transplantation and HCV management have been the mainstays of academic hepatology practice, where do we go from here? Unfortunately, we are in an era where the incidence and prevalence of liver diseases around the globe is increasing, and death from complications of cirrhosis is now among the top 10 causes in most countries; so hepatologists are expected to play a major role in the future. Despite remarkable progress, success at the population level is limited by the resource-intensive nature of caring for patients with end-stage disease. Accordingly, the major advances in the next decade are likely to focus on (i) the earlier identification of individuals and populations at higher risk for liver diseases, and (ii) initiation in high-risk populations of specific strategies for early detection and treatment of fibrosis, cancer and cirrhosis. The answers will lie in large part in the further exploration of the human genome in carefully phenotyped patients. Risk variants in the PNPLA3 gene represent the best example to date. The risk variants are common and are enriched in certain populations around the globe; and individuals that possess risk variants are more likely to have liver injury from fatty liver disease (even as children), alcohol and viral hepatitis. Further, those with liver injury are more likely to progress to cirrhosis and hepatoma. Similarly, in those with established liver disease, use of biomarkers and other strategies for early detection of fibrosis and hepatoma will pay dividends as the next generation of treatments focusing on (i) anti-fibrotic strategies and (ii) liver regeneration move to the forefront. There remains an important need to invest in hepatology as a growth industry even after the (unlikely) eradication of HCV.
- Published
- 2016
5. Yamada's Atlas of Gastroenterology
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Daniel K. Podolsky, Michael Camilleri, J. Gregory Fitz, Anthony N. Kalloo, Fergus Shanahan, Timothy C. Wang, Daniel K. Podolsky, Michael Camilleri, J. Gregory Fitz, Anthony N. Kalloo, Fergus Shanahan, and Timothy C. Wang
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- Gastrointestinal system--Diseases, Gastroenterology
- Abstract
Access to accurate, high-quality images is vital for ensuring effective management of patients with GI complaints. The fifth edition of Yamada's Atlas of Gastroenterology sees the return of the gold-standard multi-media atlas that provides gastroenterologists with an outstanding visual tool covering all facets of the field. Every GI disorder from liver abscesses, endocrine neoplasms of the pancreas, to motility disorders of the esophagus are fully illustrated through the use of endoscopic ultrasonographs, computed tomography scans, magnetic resonance images, radionuclide images, and angiograms.
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- 2016
6. Yamada's Textbook of Gastroenterology
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Daniel K. Podolsky, Michael Camilleri, J. Gregory Fitz, Anthony N. Kalloo, Fergus Shanahan, Timothy C. Wang, Daniel K. Podolsky, Michael Camilleri, J. Gregory Fitz, Anthony N. Kalloo, Fergus Shanahan, and Timothy C. Wang
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- Gastrointestinal system--Diseases, Gastroenterology
- Abstract
Yamada's Textbook of Gastroenterology has for 20 years been the most comprehensive gastroenterology reference book, combining an encyclopaedic basic science approach to GI and liver disease with the latest clinical thinking, especially in diagnostic and therapeutic developments. It is universally respected across the globe.The original outstanding editorial team was led by Tadataka Yamada, MD, one of the world's leading figures in GI research. As in previous editions, the new textbook reflects the collective efforts of the editors and a hugely impressive team of contributors, who are each experts in their specific areas. Now with another world leader in gastroenterology as Editor-in-Chief, Daniel K. Podolsky MD, President and Professor of Internal Medicine at the University of Texas Southwestern Medical Center, together with a stellar group of associate editors, the 6th edition of this iconic textbook has been expanded and enhanced in many ways with new content and technology.
- Published
- 2016
7. Andreoli and Carpenter's Cecil Essentials of Medicine
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Ivor Benjamin, Robert C. Griggs, J. Gregory Fitz, Edward J. Wing, Ivor Benjamin, Robert C. Griggs, J. Gregory Fitz, and Edward J. Wing
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- Internal medicine
- Abstract
Students, residents, and instructors swear by Andreoli and Carpenter's Cecil Essentials of Medicine because it presents just the right amount of information, just the right way. This updated edition has been revised to provide the most current, easy-to-digest review of internal medicine. Comprehensive yet concise, it focuses on the high-yield core knowledge important to those established in or just entering the field. - Excellent images and photographs vividly illustrate the appearance and clinical features of disease. - Full-color design makes absorbing and retaining information as effortless as possible. - Highlights the core principles of medicine and how they apply to patient care. - Focused revision reduces the number of pages from the previous edition, providing more high-yield core information in an accessible format. - Clear, concise writing style facilitates comprehension, while new figures, tables, and end-of-chapter references enhance readability and retention. - Consistent format provides clarity. Each section describes key physiology and biochemistry, followed by comprehensive accounts of the diseases of the organ system or field covered in the chapters. - Brand-new chapters on Thrombosis and Head and Neck Infections ensure coverage of the topics most relevant to each reader's needs. - Student Consult eBook version included with purchase. This enhanced eBook experience includes web-only extras, additional figures and tables, clinical photos, radiologic images, video procedures, imaging studies, and audio recordings, in addition to the fully searchable text and all of the images from the book.
- Published
- 2015
8. Yamada's Atlas of Gastroenterology
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Timothy C. Wang, Michael Camilleri, J. Gregory Fitz, Daniel K. Podolsky, Fergus Shanahan, and Anthony N. Kalloo
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medicine.anatomical_structure ,Atlas (anatomy) ,business.industry ,medicine ,Anatomy ,business - Published
- 2016
9. Increased Phosphoenolpyruvate Carboxykinase Gene Expression and Steatosis during Hepatitis C Virus Subgenome Replication
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J. Gregory Fitz, Ishtiaq Qadri, Jerome Schaack, Shaikh M. Rahman, Jacob E. Friedman, Francis R. Simon, Gordan Kilic, Mahua Choudhury, Mieko Iwahashi, Livia Puljak, Trina A. Knotts, and Rachel C. Janssen
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Ccaat-enhancer-binding proteins ,FOXO1 ,Cell Biology ,Biology ,CREB ,medicine.disease ,Biochemistry ,Molecular biology ,digestive system diseases ,IRS1 ,Insulin receptor ,Insulin resistance ,biology.protein ,medicine ,NS5A ,Molecular Biology ,Protein kinase B - Abstract
Chronic hepatitis C virus (HCV) infection greatly increases the risk for type 2 diabetes and nonalcoholic steatohepatitis; however, the pathogenic mechanisms remain incompletely understood. Here we report gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) transcription and associated transcription factors are dramatically up-regulated in Huh.8 cells, which stably express an HCV subgenome replicon. HCV increased activation of cAMP response element-binding protein (CREB), CCAAT/enhancer-binding protein (C/EBPβ), forkhead box protein O1 (FOXO1), and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and involved activation of the cAMP response element in the PEPCK promoter. Infection with dominant-negative CREB or C/EBPβ-shRNA significantly reduced or normalized PEPCK expression, with no change in PGC-1α or FOXO1 levels. Notably, expression of HCV nonstructural component NS5A in Huh7 or primary hepatocytes stimulated PEPCK gene expression and glucose output in HepG2 cells, whereas a deletion in NS5A reduced PEPCK expression and lowered cellular lipids but was without effect on insulin resistance, as demonstrated by the inability of insulin to stimulate mobilization of a pool of insulin-responsive vesicles to the plasma membrane. HCV-replicating cells demonstrated increases in cellular lipids with insulin resistance at the level of the insulin receptor, increased insulin receptor substrate 1 (Ser-312), and decreased Akt (Ser-473) activation in response to insulin. C/EBPβ-RNAi normalized lipogenic genes sterol regulatory element-binding protein-1c, peroxisome proliferator-activated receptor γ, and liver X receptor α but was unable to reduce accumulation of triglycerides in Huh.8 cells or reverse the increase in ApoB expression, suggesting a role for increased lipid retention in steatotic hepatocytes. Collectively, these data reveal an important role of NS5A, C/EBPβ, and pCREB in promoting HCV-induced gluconeogenic gene expression and suggest that increased C/EBPβ and NS5A may be essential components leading to increased gluconeogenesis associated with HCV infection.
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- 2012
10. Approach to the Patient with Abnormal Liver Chemistries or Jaundice
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J. Gregory Fitz
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medicine.medical_specialty ,business.industry ,Jaundice ,medicine.disease ,Gastroenterology ,Serum bilirubin ,Liver disease ,Internal medicine ,Nonalcoholic fatty liver disease ,Medicine ,Abnormal liver ,Hepatocellular injury ,medicine.symptom ,business - Published
- 2015
11. Initiation of Purinergic Signaling by Exocytosis of ATP-containing Vesicles in Liver Epithelium
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Katherine Luby-Phelps, Charles Kresge, Meghana Sathe, Matthew A. Lewis, Andrew P. Feranchak, Abhijit Bugde, Peter P. Antich, and J. Gregory Fitz
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Vacuolar Proton-Translocating ATPases ,Carcinoma, Hepatocellular ,Biology ,Biochemistry ,Exocytosis ,Adenosine Triphosphate ,Cell Line, Tumor ,Paracrine Communication ,Animals ,Enzyme Inhibitors ,Transport Vesicles ,Autocrine signalling ,Molecular Biology ,Protein Kinase C ,Cell Size ,Microscopy, Confocal ,Liver cell ,Vesicle ,Liver Neoplasms ,Purinergic receptor ,Receptors, Purinergic ,Epithelial Cells ,Cell Biology ,Purinergic signalling ,Rats ,Cell biology ,Autocrine Communication ,Endocytic vesicle ,Hepatocytes ,Macrolides ,Intracellular ,Signal Transduction - Abstract
Extracellular ATP represents an important autocrine/paracrine signaling molecule within the liver. The mechanisms responsible for ATP release are unknown, and alternative pathways have been proposed, including either conductive ATP movement through channels or exocytosis of ATP-enriched vesicles, although direct evidence from liver cells has been lacking. Utilizing dynamic imaging modalities (confocal and total internal reflection fluorescence microscopy and luminescence detection utilizing a high sensitivity CCD camera) at different scales, including confluent cell populations, single cells, and the intracellular submembrane space, we have demonstrated in a model liver cell line that (i) ATP release is not uniform but reflects point source release by a defined subset of cells; (ii) ATP within cells is localized to discrete zones of high intensity that are approximately 1 mum in diameter, suggesting a vesicular localization; (iii) these vesicles originate from a bafilomycin A(1)-sensitive pool, are depleted by hypotonic exposure, and are not rapidly replenished from recycling of endocytic vesicles; and (iv) exocytosis of vesicles in response to cell volume changes depends upon a complex series of signaling events that requires intact microtubules as well as phosphoinositide 3-kinase and protein kinase C. Collectively, these findings are most consistent with an essential role for exocytosis in regulated release of ATP and initiation of purinergic signaling in liver cells.
- Published
- 2010
12. 5-Nitro-2-(3-phenylpropylamino)benzoic Acid (NPPB) Stimulates Cellular ATP Release through Exocytosis of ATP-enriched Vesicles
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Gordan Kilic, J. Gregory Fitz, Svjetlana Dolovcak, and Shar L. Waldrop
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Cell Membrane Permeability ,Angiogenesis Inhibitors ,Stimulation ,Biology ,Biochemistry ,Exocytosis ,Cell membrane ,chemistry.chemical_compound ,Adenosine Triphosphate ,Cell Line, Tumor ,Extracellular ,medicine ,Animals ,Humans ,Receptor ,Molecular Biology ,Neurons ,Receptors, Purinergic P2 ,Vesicle ,Cell Membrane ,Purinergic receptor ,Cell Biology ,Calcium Channel Blockers ,Rats ,Cell biology ,Membrane Transport, Structure, Function, and Biogenesis ,Kinetics ,Spectrometry, Fluorescence ,medicine.anatomical_structure ,chemistry ,Nitrobenzoates ,Receptors, Purinergic P2X7 ,Adenosine triphosphate - Abstract
Cells release ATP in response to physiologic stimuli. Extracellular ATP regulates a broad range of important cellular functions by activation of the purinergic receptors in the plasma membrane. The purpose of these studies was to assess the role of vesicular exocytosis in cellular ATP release. FM1-43 fluorescence was used to measure exocytosis and bioluminescence to measure ATP release in HTC rat hepatoma and Mz-Cha-1 human cholangiocarcinoma cells. Exposure to a Cl(-) channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (50-300 microM) stimulated a 5-100-fold increase in extracellular ATP levels within minutes of the exposure. This rapid response was not a result of changes in cell viability or Cl(-) channel activity. NPPB also potently stimulated ATP release in HEK293 cells and HEK293 cells expressing a rat P2X7 receptor indicating that P2X7 receptors are not involved in stimulation of ATP release by NPPB. In all cells studied, NPPB rapidly stimulated vesicular exocytosis that persisted many minutes after the exposure. The kinetics of NPPB-evoked exocytosis and ATP release were similar. Furthermore, the magnitudes of NPPB-evoked exocytosis and ATP release were correlated (correlation coefficient 0.77), indicating that NPPB may stimulate exocytosis of a pool of ATP-enriched vesicles. These findings provide further support for the concept that vesicular exocytosis plays an important role in cellular ATP release and suggest that NPPB can be used as a biochemical tool to specifically stimulate ATP release through exocytic mechanisms.
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- 2009
13. Report of the Multisociety Task Force on GI Training
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Philip O. Katz, Lawrence S. Friedman, Michael H. Stolar, John F. Kuemmerle, John J. Vargo, Grace H. Elta, Gregory J. Gores, Lawrence J. Brandt, Darrell S. Pardi, and J. Gregory Fitz
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medicine.medical_specialty ,Certification ,Process (engineering) ,Advisory Committees ,education ,Guidelines as Topic ,Medical Oncology ,Training (civil) ,Endoscopy, Gastrointestinal ,Maintenance of Certification ,Internal medicine ,Humans ,Medicine ,Radiology, Nuclear Medicine and imaging ,Duration (project management) ,Curriculum ,Societies, Medical ,Medical education ,Education, Medical ,Hepatology ,Task force ,business.industry ,Gastroenterology ,Competency-Based Education ,United States ,Liver Transplantation ,Practice Guidelines as Topic ,Education, Medical, Continuing ,Psychology ,business - Abstract
In summary, the task force recommends that the 4 gastroenterology/hepatology societies work with the ABIM to develop a competency-based curriculum that incorporates the Maintenance of Certification process to accommodate the need and desire for training and subsequent practice in specific areas of gastroenterology/hepatology. Given the increasing complexity of treating digestive diseases, allowing trainees the opportunity to develop enhanced ability and experience in specific disease areas or procedures will benefit patients. By developing these training pathways, training programs will need to measure the achievements of trainees in terms of specific defined competencies rather than the duration of training alone.
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- 2009
14. Purinergic signaling microenvironments: An introduction
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Erik M. Schwiebert and J. Gregory Fitz
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0303 health sciences ,Adenosine ,Nucleotides ,Cellular differentiation ,Pharmacology toxicology ,Purinergic receptor ,Nucleosides ,Cell Biology ,Human physiology ,Purinergic signalling ,Biology ,Signaling ,ATP ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Paracrine signalling ,Editorial ,0302 clinical medicine ,Autacoid ,Molecular Biology ,Neuroscience ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
The common theme of this introductory article and the minireviews that follow in this special issue is the concept of microenvironments within tissues and surrounding cells that would be ideal signaling venues for a biologically active purinergic ligand. Collectively, the editors/authors and the other contributing authors agree that nucleotides and nucleosides would be most potent within a confined system. A talented cadre of purinergics has been solicited to discuss purinergic signaling in his or her favorite microenvironment within a given organ or tissue. We are gratified by the large number of original articles that also have successfully navigated the peer review process and are part of this special issue. These concepts are not simply purinergic, but the idea of maximal potency in a tissue microenvironment and surrounding specialized cells within a tissue pertains to any autacoid or paracrine agonist.
- Published
- 2008
15. Characterization of ionotrophic purinergic receptors in hepatocytes
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Livia Puljak, Ryan R. McWilliams, J. Gregory Fitz, Gordan Kilic, Svjetlana Dolovcak, Andrew P. Feranchak, R. Brian Doctor, Bonnie C. Miller, and Daniel S. Emmett
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Agonist ,medicine.medical_specialty ,Carcinoma, Hepatocellular ,medicine.drug_class ,Biology ,Mice ,Adenosine Triphosphate ,Cell Line, Tumor ,Internal medicine ,medicine ,Extracellular ,Animals ,Humans ,RNA, Neoplasm ,Patch clamp ,Receptor ,Calcium signaling ,Hepatology ,Receptors, Purinergic P2 ,Liver Neoplasms ,Purinergic receptor ,Rats ,Cell biology ,Mice, Inbred C57BL ,Glucose ,Endocrinology ,medicine.anatomical_structure ,Receptors, Purinergic P2X ,Hepatocyte ,Hepatocytes ,RNA ,Receptors, Purinergic P2X4 ,Intracellular - Abstract
Ionotrophic purinergic (P2X) receptors function as receptor-gated cation channels, where agonist binding leads to opening of a nonselective cation pore permeable to both Na+ and Ca2+. Based on evidence that extracellular adenosine 5′-triphosphate (ATP) stimulates glucose release from liver, these studies evaluate whether P2X receptors are expressed by hepatocytes and contribute to ATP-dependent calcium signaling and glucose release. Studies were performed in isolated hepatocytes from rats and mice and hepatoma cells from humans and rats. Transcripts and protein for both P2X4 and P2X7 were detectable, and immunohistochemistry of intact liver revealed P2X4 in the basolateral and canalicular domains. In whole cell patch clamp studies, exposure to the P2X4/P2X7 receptor agonist 2′3′-O-(4-benzoyl-benzoyl)-adenosine 5′-triphosphate (BzATP; 10 μM) caused a rapid increase in membrane Na+ conductance. Similarly, with Fluo-3 fluorescence, BzATP induced an increase in intracellular [Ca2+]. P2X4 receptors are likely involved because the calcium response to BzATP was inhibited by Cu2+, and the P2X4 modulators Zn2+ and ivermectin (0.3-3 μM) each increased intracellular [Ca2+]. Exposure to BzATP decreased cellular glycogen content; and P2X4 receptor messenger RNA increased in glycogen-rich liver samples. Conclusion: These studies provide evidence that P2X4 receptors are functionally important in hepatocyte Na+ and Ca2+ transport, are regulated by extracellular ATP and divalent cation concentrations, and may constitute a mechanism for autocrine regulation of hepatic glycogen metabolism. (HEPATOLOGY 2007.)
- Published
- 2007
16. Research in academic medical centers: Two threats to sustainable support
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J. Larry Jameson, Nancy C. Andrews, Pamela B. Davis, Arthur S. Levine, J. Gregory Fitz, Robert N. Golden, Larry Shapiro, Kenneth S. Polonsky, Allen M. Spiegel, Jeffrey R. Balser, Karen H. Antman, Vivian S. Lee, Robert J. Alpern, Jeremy M Berg, Lee Goldman, E. Albert Reece, Marsha D. Rappley, Paul B. Rothman, and Debra A. Schwinn
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Academic Medical Centers ,Biomedical Research ,business.industry ,Uncertainty ,MEDLINE ,Humans ,Revenue ,Medicine ,General Medicine ,Public relations ,business - Abstract
Reductions in federal support and clinical revenue jeopardize biomedical research and, in turn, clinical medicine.
- Published
- 2015
17. Purinergic regulation of cholangiocyte secretion: identification of a novel role for P2X receptors
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J. Gregory Fitz, Ryan R. McWilliams, Thomas Matzakos, Andrew P. Feranchak, Sylene M. Johnson, and R. Brian Doctor
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Gene isoform ,Physiology ,Biology ,Cholangiocyte ,Adenosine Triphosphate ,Chlorides ,Cell Line, Tumor ,Physiology (medical) ,medicine ,Extracellular ,Animals ,Bile ,Humans ,Protein Isoforms ,Secretion ,RNA, Messenger ,Purine metabolism ,Receptor ,Cells, Cultured ,Staining and Labeling ,Hepatology ,Receptors, Purinergic P2 ,Purinergic receptor ,Gastroenterology ,Immunohistochemistry ,Adenosine ,Rats ,Electrophysiology ,Bile Ducts, Intrahepatic ,Biochemistry ,Receptors, Purinergic P2X ,Receptors, Purinergic P2X4 ,medicine.drug - Abstract
The P2X family of ligand-gated cation channels is comprised of seven distinct isoforms activated by binding of extracellular purines. Although originally identified in neurons, there is increasing evidence for expression of P2X receptors in epithelia as well. Because ATP is released by both hepatocytes and cholangiocytes, these studies were performed to evaluate whether P2X receptors are present in cholangiocytes and contribute to local regulation of biliary secretion and bile formation. RT-PCR of cDNA from cultured normal rat cholangiocytes detected transcripts for P2X receptors 2, 3, 4, and 6; products from P2X3 and P2X4 were robust and always detectable. In cholangiocyte lysates, P2X4 protein was readily detected, and immunohistochemical staining of intact rat liver revealed P2X4 protein concentrated in intrahepatic bile ducts. To assess the functional significance of P2X4, isolated Mz-ChA-1 cells were exposed to the P2X4-preferring agonist 2′,3′-O-(4-benzoyl-benzoyl)-ATP (BzATP), which activated inward currents of −18.2 + 3.0 pA/pF. In cholangiocyte monolayers, BzATP but not P2X3 agonists elicited robust Cl−secretory responses (short-circuit current) when applied to either the apical (Δ Isc22.1 ± 3.3 μA) or basolateral (18.5 ± 1.6 μA) chamber, with half-maximal stimulation at ∼10 μM and ∼1 μM, respectively. The response to BzATP was unaffected by suramin (not significant) and was inhibited by Cu2+( P < 0.01). These studies provide molecular and biochemical evidence for the presence of P2X receptors in cholangiocytes. Functional studies indicate that P2X4 is likely the primary isoform involved, representing a novel and functionally important component of the purinergic signaling complex modulating biliary secretion.
- Published
- 2005
18. Inhibition of cellular responses to insulin in a rat liver cell line. A role for PKC in insulin resistance
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Vinay Parameswara, Yuren Wei, Michael J. Pagliassotti, Gordan Kilic, J. Gregory Fitz, Victoria Esser, Ishtiaq Qadri, and Livia Puljak
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medicine.medical_specialty ,biology ,Physiology ,Insulin ,medicine.medical_treatment ,Liver cell ,medicine.disease ,Exocytosis ,Insulin oscillation ,Insulin receptor ,chemistry.chemical_compound ,Endocrinology ,Insulin resistance ,chemistry ,Internal medicine ,Insulin receptor substrate ,medicine ,biology.protein ,Arachidonic acid - Abstract
The initial response of liver cells to insulin is mediated through exocytosis of Cl− channel-containing vesicles and a subsequent opening of plasma membrane Cl− channels. Intracellular accumulation of fatty acids leads to profound defects in metabolism, and is closely associated with insulin resistance. It is not known whether the activity of Cl− channels is altered in insulin resistance and by which mechanisms. We studied the effects of fatty acid accumulation on Cl− channel opening in a model liver cell line. Overnight treatment with amiodarone increased the fat content by ∼2-fold, and the rates of gluconeogenesis by ∼5-fold. The ability of insulin to suppress gluconeogenesis was markedly reduced indicating that amiodarone treatment induces insulin resistance. Western blot analysis showed that these cells express the same number of insulin receptors as control cells. However, insulin failed to activate exocytosis and Cl− channel opening. These inhibitory effects were mimicked in control cells by exposures to arachidonic acid (15 μm). Further studies demonstrated that fatty acids stimulate the PKC activity, and inhibition of PKC partially restored exocytosis and Cl− channel opening in insulin-resistant cells. Accordingly, activation of PKC with PMA in control cells potently inhibited the insulin responses. These results suggest that stimulation of PKC activity in insulin resistance contributes to the inhibition of cellular responses to insulin in liver cells.
- Published
- 2005
19. Calcium-dependent regulation of secretion in biliary epithelial cells: The role of apamin-sensitive SK channels
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R. Brian Doctor, J. Gregory Fitz, Kathryn Brookman, Andrew P. Feranchak, Marlyn Troetsch, and Sylene M. Johnson
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medicine.medical_specialty ,Potassium Channels ,Small-Conductance Calcium-Activated Potassium Channels ,Biology ,Apamin ,Cholangiocyte ,SK channel ,Potassium Channels, Calcium-Activated ,chemistry.chemical_compound ,Cell Line, Tumor ,Internal medicine ,medicine ,Animals ,Humans ,Secretion ,Patch clamp ,Cells, Cultured ,Ionophores ,Hepatology ,Ussing chamber ,Ionomycin ,Purinergic receptor ,Gastroenterology ,Biological Transport ,Epithelial Cells ,Rats ,Cell biology ,Bee Venoms ,Bile Ducts, Intrahepatic ,Endocrinology ,chemistry ,Potassium ,Calcium - Abstract
Background & Aims: Increases in intracellular Ca2+ are thought to complement cAMP in stimulating Cl− secretion in cholangiocytes, although the site(s) of action and channels involved are unknown. We have identified a Ca2+-activated K+ channel (SK2) in biliary epithelium that is inhibited by apamin. The purpose of the present studies was to define the role of SK channels in Ca2+-dependent cholangiocyte secretion. Methods: Studies were performed in human Mz-Cha-1 cells and normal rat cholangiocytes (NRC). Currents were measured by whole-cell patch clamp technique and transepithelial secretion by Ussing chamber. Results: Ca2+-dependent stimuli, including purinergic receptor stimulation, ionomycin, and increases in cell volume, each activated K+-selective currents with a linear IV relation and time-dependent inactivation. Currents were Ca2+ dependent and were inhibited by apamin and by Ba2+. In intact liver, immunoflourescence with an antibody to SK2 showed a prominent signal in cholangiocyte plasma membrane. To evaluate the functional significance, NRC monolayers were mounted in a Ussing chamber, and the short-circuit current (Isc) was measured. Exposure to ionomycin caused an increase in Isc 2-fold greater than that induced by cAMP. Both the basal and ionomycin-induced Isc were inhibited by basolateral Ba2+, and ∼58% of the basolateral K+ current was apamin sensitive. Conclusions: These studies demonstrate that cholangiocytes exhibit robust Ca2+-stimulated secretion significantly greater in magnitude than that stimulated by cAMP. SK2 plays an important role in mediating the increase in transepithelial secretion due to increases in intracellular Ca2+. SK2 channels, therefore, may represent a target for pharmacologic modulation of bile flow.
- Published
- 2004
20. Cholangiocytes exhibit dynamic, actin-dependent apical membrane turnover
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R. Brian Doctor, Laura Fouassier, Rolf Dahl, Gordan Kilic, and J. Gregory Fitz
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Cytochalasin D ,Physiology ,Population ,Antineoplastic Agents ,Biology ,Endocytosis ,Peptides, Cyclic ,Exocytosis ,Cholangiocyte ,Cell Line ,Depsipeptides ,Cyclic AMP ,Animals ,Transport Vesicles ,education ,Cytoskeleton ,Actin ,Fluorescent Dyes ,Nucleic Acid Synthesis Inhibitors ,education.field_of_study ,Vesicle ,Cell Membrane ,Cell Polarity ,Biological Transport ,Cell Biology ,Apical membrane ,Actins ,Rats ,Cell biology ,Bile Ducts - Abstract
The present studies of cholangiocytes used complementary histological, biochemical, and electrophysiological methods to identify a dense population of subapical vesicles, quantify the rates of vesicular trafficking, and assess the contribution of the actin cytoskeleton to membrane trafficking. FM 1–43 fluorescence measured significant basal rates of total exocytosis (1.33 ± 0.16% plasma membrane/min) in isolated cholangiocytes and apical exocytosis in cholangiocyte monolayers. Cell surface area remained unchanged, indicating that there was a concurrent, equivalent rate of endocytosis. FM 1–43 washout studies showed that 36% of the endocytosed membrane was recycled to the plasma membrane. 8-(4-Chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (CPT-cAMP; cAMP analog) increased exocytosis by 71 ± 31%, whereas the Rp diastereomer of adenosine 3′,5′-cyclic monophosphothioate (Rp-cAMPS; protein kinase A inhibitor) diminished basal exocytosis by 53 ± 11%. A dense population of 140-nm subapical vesicles arose, in part, from apical membrane endocytosis. Phalloidin staining showed that a subpopulation of the endocytosed vesicles was encapsulated by F-actin. Furthermore, membrane trafficking was inhibited by disrupting the actin cytoskeleton with cytochalasin D (51 ± 13% of control) or jasplakinolide (58 ± 9% of control). These studies indicate that there is a high rate of vesicular trafficking at the apical membrane of cholangiocytes and suggest that both cAMP and the actin cytoskeleton contribute importantly to these events.
- Published
- 2002
21. Heterotrimeric G-proteins Activate Cl− Channels through Stimulation of a Cyclooxygenase-dependent Pathway in a Model Liver Cell Line
- Author
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Gordan Kilic and J. Gregory Fitz
- Subjects
Carcinoma, Hepatocellular ,Patch-Clamp Techniques ,Time Factors ,Intracellular pH ,Indomethacin ,GTPgammaS ,Inositol 1,4,5-Trisphosphate ,Biology ,Models, Biological ,Biochemistry ,Phospholipases A ,Cell Line ,Norepinephrine ,chemistry.chemical_compound ,Adenosine Triphosphate ,Chloride Channels ,GTP-Binding Proteins ,Heterotrimeric G protein ,Animals ,Virulence Factors, Bordetella ,Patch clamp ,Molecular Biology ,Membrane potential ,Arachidonic Acid ,Phospholipase C ,Liver cell ,Cell Membrane ,Electric Conductivity ,Cell Biology ,Rats ,Cell biology ,Electrophysiology ,Liver ,Pertussis Toxin ,chemistry ,Guanosine 5'-O-(3-Thiotriphosphate) ,Prostaglandin-Endoperoxide Synthases ,Calcium ,Adrenergic alpha-Agonists ,Intracellular ,Protein Binding - Abstract
Circulating hormones produce rapid changes in the Cl(-) permeability of liver cells through activation of plasma membrane receptors coupled to heterotrimeric G-proteins. The resulting effects on intracellular pH, membrane potential, and Cl(-) content are important contributors to the overall metabolic response. Consequently, the purpose of these studies was to evaluate the mechanisms responsible for G-protein-mediated changes in membrane Cl(-) permeability using HTC hepatoma cells as a model. Using patch clamp techniques, intracellular dialysis with 0.3 mm guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) increased membrane conductance from 10 to 260 picosiemens/picofarads due to activation of Ca(2+)-dependent Cl(-) currents that were outwardly rectifying and exhibited slow activation at depolarizing potentials. These effects were mimicked by intracellular AlF(4)(-) (0.03 mm) and inhibited by pertussis toxin (PTX), consistent with current activation through Galpha(i). Studies using defined agonists and inhibitors indicate that Cl(-) channel activation by GTPgammaS occurs through an indomethacin-sensitive pathway involving sequential activation of phospholipase C, mobilization of Ca(2+) from inositol 1,4,5-trisphosphate-sensitive stores, and stimulation of phospholipase A(2) and cyclooxygenase (COX). Accordingly, the conductance responses to GTPgammaS or to intracellular Ca(2+) were inhibited by COX inhibitors. These results indicate that PTX-sensitive G-proteins regulate the Cl(-) permeability of HTC cells through Ca(2+)-dependent stimulation of COX activity. Thus, receptor-mediated activation of Galpha(i) may be essential for hormonal regulation of liver transport and metabolism through COX-dependent opening of a distinct population of plasma membrane Cl(-) channels.
- Published
- 2002
22. Regulation of Cholangiocyte Secretion
- Author
-
J. Gregory Fitz
- Subjects
Anions ,Cystic Fibrosis Transmembrane Conductance Regulator ,Cell Communication ,Biology ,Endocytosis ,Exocytosis ,Cholangiocyte ,Adenylyl cyclase ,chemistry.chemical_compound ,Chlorides ,Chloride Channels ,Cyclic AMP ,Bile ,Humans ,Cyclic adenosine monophosphate ,Secretion ,Chloride-Bicarbonate Antiporters ,Hepatology ,Cell Membrane ,Epithelial Cells ,Apical membrane ,Cell biology ,Bile Ducts, Intrahepatic ,chemistry ,Biochemistry ,Hepatocytes ,Chloride channel ,Education, Medical, Continuing - Abstract
Observations from a variety of model systems suggest that ductular bile formation is mediated in large part by transepithelial transport of Cl(-) ions and have identified Cl(-) channels in the apical membrane as important targets for hormones and other factors that modulate bile volume and composition through effects on duct cells. Signaling through secretin receptors that stimulate adenylyl cyclase and activate the cystic fibrosis transmembrane regulator (CFTR) Cl(-) channels represents a prototype for cholangiocyte secretion. However, recent observations indicate that cholangiocytes also express a variety of receptors that modulate secretory responses in the absence of effects on cyclic adenosine monophosphate (cAMP), and Cl(-) channels unrelated to CFTR have been identified. Moreover, rapid exocytosis of subapical vesicles is coupled closely to different Cl(-) secretory responses. These observations suggest that the protein composition of the apical domain is regulated by selective exocytosis and endocytosis of channel-containing vesicles, leading to rapid modulation of the transport capacity of individual cells in response to changing physiologic demands.
- Published
- 2002
23. p38 MAP kinase modulates liver cell volume through inhibition of membrane Na+ permeability
- Author
-
Andrew P. Feranchak, Tomas Berl, Juan Capasso, Paul A. Wojtaszek, Jiahuai Han, and J. Gregory Fitz
- Subjects
General Medicine - Published
- 2001
24. Insulin Stimulates Membrane Conductance in a Liver Cell Line
- Author
-
Gordan Kilic, R. Brian Doctor, and J. Gregory Fitz
- Subjects
Vesicle ,Liver cell ,Cell Biology ,Biology ,Endocytosis ,Biochemistry ,Exocytosis ,Cell biology ,Wortmannin ,chemistry.chemical_compound ,Insulin receptor ,chemistry ,biology.protein ,Patch clamp ,Molecular Biology ,Ion channel - Abstract
Activation of insulin receptors stimulates a rapid increase in the ion permeability of liver cells. To evaluate whether this response involves insertion of ion channels, plasma membrane turnover was measured in a model liver cell line using the fluorescent membrane marker FM1–43. Under basal conditions, the rate of constitutive membrane turnover was ∼2%min−1, and balanced exocytosis and endocytosis maintained the total cell membrane area constant. Exposure to insulin stimulated a transient increase in membrane turnover of up to 10-fold above constitutive rates. The response was concentration-dependent (0.001–10 μm). Insulin also caused a parallel increase in membrane conductance as measured by whole-cell patch clamp recording due to opening of Cl−- and K+-selective ion channels. The insulin-stimulated membrane turnover did not appear to involve the constitutive recycling compartments, suggesting that a distinct pool of vesicles may be involved. The effects of insulin on membrane turnover and membrane conductance were inhibited by blockers of phosphoinositide 3-kinase LY294002 and wortmannin or by disrupting microtubule assembly with nocodazole. Taken together, these findings indicate that insulin stimulates recruitment of new membranes through phosphoinositide 3-kinase-dependent mechanisms. Thus, regulated insertion of a separate population of ion channel-containing vesicles may represent one mechanism for mediating the changes in membrane conductance that are essential for the cellular response to insulin.
- Published
- 2001
25. Liver cell volume regulation: Size matters
- Author
-
Andrew P. Feranchak, Jeffrey C. Dunkelberg, and J. Gregory Fitz
- Subjects
medicine.medical_specialty ,Hepatology ,Liver cell ,Cell volume ,Biology ,Ion Channels ,Cell biology ,Adenosine Triphosphate ,Endocrinology ,medicine.anatomical_structure ,Liver ,Internal medicine ,Hepatocyte ,medicine ,Animals ,Humans ,Extracellular Space ,Cell Size ,Signal Transduction ,Volume (compression) - Published
- 2001
26. ClC-2 chloride channels contribute to HTC cell volume homeostasis
- Author
-
J. Gregory Fitz, Andrew P. Feranchak, Roderic L. Smith, Gerald H. Clayton, R. Brian Doctor, and Richard M. Roman
- Subjects
medicine.medical_specialty ,Carcinoma, Hepatocellular ,DNA, Complementary ,Patch-Clamp Techniques ,Microinjections ,Physiology ,Cell ,4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid ,Biology ,Transfection ,Antibodies ,Cell Line ,Chlorides ,Chloride Channels ,Physiology (medical) ,Internal medicine ,Gene expression ,medicine ,Animals ,Homeostasis ,Humans ,Cell Size ,Hepatology ,Reverse Transcriptase Polymerase Chain Reaction ,urogenital system ,Cell Membrane ,Purinergic receptor ,Gastroenterology ,Epithelial Cells ,Metabolism ,Rats ,Cell biology ,CLC-2 Chloride Channels ,Endocrinology ,medicine.anatomical_structure ,Hepatocyte ,Hepatocytes ,Heterologous expression ,Cell volume homeostasis - Abstract
Membrane Cl−channels play an important role in cell volume homeostasis and regulation of volume-sensitive cell transport and metabolism. Heterologous expression of ClC-2 channel cDNA leads to the appearance of swelling-activated Cl−currents, consistent with a role in cell volume regulation. Since channel properties in heterologous models are potentially modified by cellular background, we evaluated whether endogenous ClC-2 proteins are functionally important in cell volume regulation. As shown by whole cell patch clamp techniques in rat HTC hepatoma cells, cell volume increases stimulated inwardly rectifying Cl−currents when non-ClC-2 currents were blocked by DIDS (100 μM). A cDNA closely homologous with rat brain ClC-2 was isolated from HTC cells; identical sequence was demonstrated for ClC-2 cDNAs in primary rat hepatocytes and cholangiocytes. ClC-2 mRNA and membrane protein expression was demonstrated by in situ hybridization, immunocytochemistry, and Western blot. Intracellular delivery of antibodies to an essential regulatory domain of ClC-2 decreased ClC-2-dependent currents expressed in HEK-293 cells. In HTC cells, the same antibodies prevented activation of endogenous Cl−currents by cell volume increases or exposure to the purinergic receptor agonist ATP and delayed HTC cell volume recovery from swelling. These studies provide further evidence that mammalian ClC-2 channel proteins are functional and suggest that in HTC cells they contribute to physiological changes in membrane Cl−permeability and cell volume homeostasis.
- Published
- 2001
27. Regulation of biliary secretion by extracellular nucleotides
- Author
-
J. Gregory Fitz and Andrew P. Feranchak
- Subjects
Paracrine signalling ,Biochemistry ,Drug Discovery ,Purinergic receptor ,Intrahepatic bile ducts ,Secretion ,Apical membrane ,Biology ,Purinergic signalling ,Signal transduction ,Cholangiocyte ,Cell biology - Abstract
Bile formation is an essential function of the liver and depends upon complimentary actions of hepatocytes and intrahepatic bile duct cells, or cholangiocytes. Cholangiocytes contribute importantly to the volume and composition of bile secreted by the liver through absorption and secretion of fluid and electrolytes. Biophysical measurements suggest that increases in Cl - permeability at the apical membrane of cholangiocytes represent an important cellular site for regulation of secretion. Recent evidence suggests that extracellular ATP may serve as a signal modifying cholangiocyte secretion through the regulation of membrane Cl - permeability. Through activation of P 2Y2 receptors in the apical (luminal) membrane of cholangiocytes, ATP functions as a local autocrine/paracrine regulatory factor that modulates biliary secretion. Consequently, the purpose of this review is to describe the cellular mechanisms thought to contribute to ATP release and review the evidence supporting a role for ATP in the regulation of cholangiocyte Cl - -secretion and bile formation. Characterization of the factors that regulate local nucleotide concentrations and purinergic signaling may represent therapeutic targets for the pharmacologic modulation of bile composition and flow.
- Published
- 2001
28. Modified Culture Conditions Enhance Expression of Differentiated Phenotypic Properties of Normal Rat Cholangiocytes
- Author
-
R. Brian Doctor, J. Gregory Fitz, Nicholas R. Larusso, Kelli D. Salter, and Richard M. Roman
- Subjects
medicine.medical_specialty ,Cellular differentiation ,Cell Differentiation ,Cell Biology ,Biology ,Phenotype ,Cholangiocyte ,Cell Line ,Culture Media ,Rats ,Pathology and Forensic Medicine ,Cell biology ,Endocrinology ,Cell culture ,Internal medicine ,medicine ,Animals ,Secretion ,Bile Ducts ,Molecular Biology ,Biliary tract disease - Abstract
Modified Culture Conditions Enhance Expression of Differentiated Phenotypic Properties of Normal Rat Cholangiocytes
- Published
- 2000
29. Evidence for Ezrin-Radixin-Moesin-binding Phosphoprotein 50 (EBP50) Self-association through PDZ-PDZ Interactions
- Author
-
C. Chris Yun, J. Gregory Fitz, R. Brian Doctor, and Laura Fouassier
- Subjects
Sodium-Hydrogen Exchangers ,Macromolecular Substances ,Immunoprecipitation ,Recombinant Fusion Proteins ,Blotting, Western ,PDZ domain ,Biology ,Transfection ,Biochemistry ,Cell Line ,Protein–protein interaction ,Animals ,Amino Acid Sequence ,Cloning, Molecular ,Molecular Biology ,Integral membrane protein ,Binding Sites ,Epithelial Cells ,Cell Biology ,Membrane transport ,Phosphoproteins ,Fusion protein ,Cell biology ,Membrane protein ,Phosphoprotein ,Carrier Proteins ,Dimerization - Abstract
Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (EBP50) is a versatile membrane-cytoskeleton linking protein that binds to the COOH-tail of specific integral membrane proteins through its two PDZ domains. These EBP50 binding interactions have been implicated in sequestering interactive sets of proteins into common microdomains, regulating the activity of interacting proteins, and modulating membrane protein trafficking. With only two PDZ domains, it is unclear how EBP50 forms multiprotein complexes. Other PDZ proteins increase their breadth and diversity of protein interactions through oligomerization. Hypothesizing that EBP50 self-associates to amplify its functional capacity, far-Western blotting of cholangiocyte epithelial cell proteins with EBP50 fusion protein revealed that EBP50 binds to a 50-kDa protein. Far-Western blotting of EBP50 isolated by two-dimensional gel electrophoresis or immunoprecipitation demonstrates that the 50-kDa binding partner is itself EBP50. Further, co-transfection/co-precipitation studies show the self-association can occur in an intracellular environment. In vitro analysis of the EBP50-EBP50 binding interaction indicates it is both saturable and of relatively high affinity. Analysis of truncated EBP50 proteins indicates EBP50 self-association is mediated through its PDZ domains. The ability to self-associate provides a mechanism for EBP50 to expand its capacity to form multiprotein complexes and regulate membrane transport events.
- Published
- 2000
30. Functional interactions between oxidative stress, membrane Na+ permeability, and cell volume in rat hepatoma cells
- Author
-
Andrew P. Feranchak, Wolfgang Stremmel, Thorsten Schlenker, Lukas Schwake, J. Gregory Fitz, and Richard M. Roman
- Subjects
D-Amino-Acid Oxidase ,medicine.medical_specialty ,Cell Membrane Permeability ,Patch-Clamp Techniques ,Sodium ,Hypertonic Solutions ,chemistry.chemical_element ,medicine.disease_cause ,chemistry.chemical_compound ,Cytosol ,Liver Neoplasms, Experimental ,Internal medicine ,Tumor Cells, Cultured ,medicine ,Animals ,Cell Size ,chemistry.chemical_classification ,Reactive oxygen species ,Alanine ,Hepatology ,biology ,Liver cell ,Gastroenterology ,Hydrogen Peroxide ,Glutathione ,Catalase ,Glutathione synthase ,Rats ,Kinetics ,Oxidative Stress ,Endocrinology ,chemistry ,Permeability (electromagnetism) ,biology.protein ,Biophysics ,Calcium ,Reactive Oxygen Species ,Intracellular ,Oxidative stress - Abstract
Background & Aims: Oxidative stress leads to a rapid initial loss of liver cell volume, but the adaptive mechanisms that serve to restore volume have not been defined. This study aimed to evaluate the functional interactions between oxidative stress, cell volume recovery, and membrane ion permeability. Methods: In HTC rat hepatoma cells, oxidative stress was produced by exposure to H 2 O 2 or D-alanine plus D-amino acid oxidase (40 U/mL). Results: Oxidative stress resulted in a rapid decrease in relative cell volume to 0.85 ± 0.06. This was followed by an ~100-fold increase in membrane cation permeability and partial volume recovery to 0.97 ± 0.05 of original values. The volume-sensitive conductance was permeable to Na + ≃ K + >> Tris + , and whole-cell current density at −80 mV increased from −1.2 pA/pF at 10 −,5 mol/L H 2 O 2 to −95.1 pA/pF at 10 −,2 mol/L H 2 O 2 . The effects of H 2 O 2 were completely inhibited by dialysis of the cell interior with reduced glutathione, and were markedly enhanced by inhibition of glutathione synthase. Conclusions: These findings support the presence of dynamic functional interactions between cell volume, oxidative stress, and membrane Na + permeability. Stress-induced Na + influx may represent a beneficial adaptive response that partially restores cell volume over short periods, but sustained cation influx could contribute to the increase in intracellular [Na + ] and [Ca 2+ ] associated with cell injury and necrosis. GASTROENTEROLOGY 2000;118:395-403
- Published
- 2000
31. Evidence for Gd3+inhibition of membrane ATP permeability and purinergic signaling
- Author
-
Richard M. Roman, Andrew P. Feranchak, Erik M. Schwiebert, J. Gregory Fitz, and Amy K. Davison
- Subjects
Carcinoma, Hepatocellular ,Cell Membrane Permeability ,Physiology ,Cell ,Anti-Inflammatory Agents ,Gadolinium ,Biology ,Paracrine signalling ,Adenosine Triphosphate ,Chloride Channels ,Physiology (medical) ,Paracrine Communication ,Tumor Cells, Cultured ,Extracellular ,medicine ,Animals ,Autocrine signalling ,Hepatology ,Anti-Inflammatory Agents, Non-Steroidal ,Liver Neoplasms ,Purinergic receptor ,Receptors, Purinergic ,Gastroenterology ,Epithelial Cells ,Water-Electrolyte Balance ,Purinergic signalling ,Flufenamic Acid ,Rats ,Cell biology ,Autocrine Communication ,medicine.anatomical_structure ,Hypotonic Solutions ,Biochemistry ,Chloride channel ,Calcium Channels ,Isotonic Solutions ,Signal transduction ,Signal Transduction - Abstract
Extracellular ATP functions as an important autocrine and paracrine signal that modulates a broad range of cell and organ functions through activation of purinergic receptors in the plasma membrane. Because little is known of the cellular mechanisms involved in ATP release, the purpose of these studies was to evaluate the potential role of the lanthanide Gd3+as an inhibitor of ATP permeability and to assess the physiological implications of impaired purinergic signaling in liver cells. In rat hepatocytes and HTC hepatoma cells, increases in cell volume stimulate ATP release, and the localized increase in extracellular ATP increases membrane Cl−permeability and stimulates cell volume recovery through activation of P2receptors. In cells in culture, spontaneous ATP release, as measured by a luciferin-luciferase-based assay, was always detectable under control conditions, and extracellular ATP concentrations increased 2- to 14-fold after increases in cell volume. Gd3+(200 μM) inhibited volume-sensitive ATP release by >90% ( P < 0.001), inhibited cell volume recovery from swelling ( P < 0.01), and uncoupled cell volume from increases in membrane Cl−permeability ( P < 0.01). Moreover, Gd3+had similar inhibitory effects on ATP release from other liver and epithelial cell models. Together, these findings support an important physiological role for constitutive release of ATP as a signal coordinating cell volume and membrane ion permeability and suggest that Gd3+might prove to be an effective inhibitor of ATP-permeable channels once they are identified.
- Published
- 1999
32. Endogenous ATP release regulates Cl−secretion in cultured human and rat biliary epithelial cells
- Author
-
J. Gregory Fitz, Richard M. Roman, Andrew P. Feranchak, Yu Wang, and Kelli D. Salter
- Subjects
P2Y receptor ,Cell Membrane Permeability ,Physiology ,Biology ,Ion Channels ,Cholangiocyte ,Cell Line ,Adenosine Triphosphate ,Chlorides ,Chloride Channels ,Physiology (medical) ,medicine ,Extracellular ,Animals ,Homeostasis ,Humans ,Cells, Cultured ,Ion channel ,Hepatology ,Receptors, Purinergic P2 ,Purinergic receptor ,Gastroenterology ,Epithelial Cells ,Purinergic signalling ,Epithelium ,Rats ,Cell biology ,Autocrine Communication ,medicine.anatomical_structure ,Biochemistry ,Cell culture ,Bile Ducts ,Extracellular Space - Abstract
P2Y receptor stimulation increases membrane Cl−permeability in biliary epithelial cells, but the source of extracellular nucleotides and physiological relevance of purinergic signaling to biliary secretion are unknown. Our objectives were to determine whether biliary cells release ATP under physiological conditions and whether extracellular ATP contributes to cell volume regulation and transepithelial secretion. With the use of a sensitive bioluminescence assay, constitutive ATP release was detected from human Mz-ChA-1 cholangiocarcinoma cells and polarized normal rat cholangiocyte monolayers. ATP release increased rapidly during cell swelling induced by hypotonic exposure. In Mz-ChA-1 cells, removal of extracellular ATP (apyrase) and P2 receptor blockade (suramin) reversibly inhibited whole cell Cl−current activation and prevented cell volume recovery during hypotonic stress. Moreover, exposure to apyrase induced cell swelling under isotonic conditions. In intact normal rat cholangiocyte monolayers, hypotonic perfusion activated apical Cl−currents, which were inhibited by addition of apyrase and suramin to bathing media. These findings indicate that modulation of ATP release by the cellular hydration state represents a potential signal coordinating cell volume with membrane Cl−permeability and transepithelial Cl−secretion.
- Published
- 1999
33. Reorganization of cholangiocyte membrane domains represents an early event in rat liver ischemia
- Author
-
Rolf Dahl, R. Brian Doctor, J. Gregory Fitz, and Kelli D. Salter
- Subjects
Male ,medicine.medical_specialty ,Time Factors ,Biology ,Cholangiocyte ,Tight Junctions ,Rats, Sprague-Dawley ,chemistry.chemical_compound ,Adenosine Triphosphate ,Ezrin ,Cholestasis ,Ischemia ,Heterochromatin ,Internal medicine ,medicine ,Animals ,Secretion ,Viability assay ,Cytoskeleton ,Microvilli ,Hepatology ,Tight junction ,Cell Membrane ,Intracellular Membranes ,medicine.disease ,Actins ,Rats ,Bile Ducts, Intrahepatic ,Endocrinology ,chemistry ,Adenosine triphosphate ,Liver Circulation - Abstract
Cholangiocytes contribute significantly to bile formation through the vectorial secretion of water and electrolytes and are a focal site of injury in a number of diseases including liver ischemia and post-transplantation liver failure. Using ischemia in intact liver and adenosine triphosphate (ATP) depletion in cultured cells to model cholangiocyte injury, these studies examined the effects of metabolic inhibition on cholangiocyte viability and structure. During 120 minutes of ischemia or ATP depletion, cell viability and tight junctional integrity in cholangiocytes were maintained. However, both the in vivo and in vitro models displayed striking alterations in the secondary structure of the plasma membrane. After 120 minutes, the basolateral (BL) interdigitations were diminished and the apical (Ap) microvilli were significantly decreased in number. The BL and Ap membrane surface areas decreased by 42 +/- 8% and 63 +/- 2%, respectively. Despite these changes, F-actin remained predominantly localized to the membrane domains. In contrast, in a time course that paralleled the loss of microvilli, the actin-membrane linking protein ezrin progressively dissociated from the cytoskeleton. These studies indicate that cholangiocyte ATP depletion induces characteristic, domain-specific changes in the plasma membrane and implicate alterations in the membrane-cytoskeletal interactions in the initiation of the changes. Pending the re-establishment of the differentiated domains, the loss of specific secondary structures may contribute to impaired vectorial bile duct secretion and postischemic cholestasis.
- Published
- 1999
34. Thinking Outside the Cell: The Role of Extracellular Adenosine Triphosphate in Bile Formation
- Author
-
Andrew P. Feranchak and J. Gregory Fitz
- Subjects
chemistry.chemical_compound ,medicine.anatomical_structure ,Hepatology ,chemistry ,Cell ,Gastroenterology ,Extracellular ,medicine ,Bile formation ,Purinergic signalling ,Adenosine triphosphate ,Cell biology - Published
- 2007
35. Stimulation of cyclic guanosine monophosphate production by natriuretic peptide in human biliary cells
- Author
-
Marie V. St. Pierre, Douglas M. Jefferson, Jean-François J. Dufour, J. Gregory Fitz, Thorsten Schlenker, and Irwin M. Arias
- Subjects
Hepatology ,Chemistry ,medicine.drug_class ,Guanylin ,Gastroenterology ,Epithelial Cells ,Apical membrane ,NPR1 ,Molecular biology ,NPR2 ,Cell Line ,Iodine Radioisotopes ,chemistry.chemical_compound ,Chlorides ,Atrial natriuretic peptide ,Biochemistry ,Natriuretic peptide ,medicine ,Humans ,Patch clamp ,Biliary Tract ,Cyclic GMP ,Cyclic guanosine monophosphate ,Atrial Natriuretic Factor - Abstract
Background & Aims: Guanosine 3',5'-cyclic monophosphate (cGMP), whose production is stimulated by the interaction of nitric oxide, natriuretic peptides, and guanylin with their respective guanylate cyclases, activates secretion through ion channels in several epithelia. Cl − channels have been identified in the apical membrane of biliary epithelial cells. The aim of this study was to investigate the production of cGMP and its effects on Cl − permeability in biliary epithelial cells. Methods: Halide efflux measurement, whole-cell patch clamp recording, radioimmunoassay, and reverse-transcription polymerase chain reaction using two human biliary cell lines (H69 and Mz-ChA-1) were performed. Results: In cells equilibrated with 125 I, bromo-cGMP stimulated halide efflux by 22%. In whole-cell patch clamp recordings, the addition of cGMP intracellularly, or of atrial natriuretic peptide extracellularly, stimulated inward currents at negative membrane potentials, consistent with Cl − efflux through open channels. In H69 cells, atrial and C-type natriuretic peptides stimulated production of cGMP. Mz-ChA-1 responded only to atrial natriuretic peptide. Both cell lines expressed messenger RNA for the guanylate cyclase type A receptor and the guanylate cyclase free-clearance receptor. Conclusions: These data suggest that natriuretic peptide stimulates cGMP production in human biliary epithelial cells, which in turn may regulate ductular bile formation through the opening of Cl − channels. GASTROENTEROLOGY 1998;114:782-790
- Published
- 1998
36. Regulation of biliary secretion through apical purinergic receptors in cultured rat cholangiocytes
- Author
-
Stephen J. Kim, Rodger A. Liddle, Ala I. Sharara, J. Gregory Fitz, Nicholas F. LaRusso, Joelle Romac, Thorsten Schlenker, and Richard M. Roman
- Subjects
medicine.medical_specialty ,P2Y receptor ,Transcription, Genetic ,Physiology ,Cloning, Organism ,Molecular Sequence Data ,Biology ,Ion Channels ,Cholangiocyte ,Membrane Potentials ,Amiloride ,Receptors, Purinergic P2Y2 ,Mice ,Xenopus laevis ,Adenosine Triphosphate ,Chlorides ,Physiology (medical) ,Internal medicine ,medicine ,Animals ,Bile ,Humans ,Secretion ,Amino Acid Sequence ,RNA, Messenger ,Cells, Cultured ,Sequence Homology, Amino Acid ,Hepatology ,Receptors, Purinergic P2 ,Cell Membrane ,Purinergic receptor ,Gastroenterology ,Cell Polarity ,Recombinant Proteins ,Epithelium ,In vitro ,Rats ,Cell biology ,medicine.anatomical_structure ,Endocrinology ,Liver ,Biliary tract ,Oocytes ,Chloride channel ,Female ,Bile Ducts ,Sequence Alignment ,Signal Transduction - Abstract
To evaluate whether ATP in bile serves as a signaling factor regulating ductular secretion, voltage-clamp studies were performed using a novel normal rat cholangiocyte (NRC) model. In the presence of amiloride (100 μM) to block Na+channels, exposure of the apical membrane to ATP significantly increased the short-circuit current ( Isc) from 18.2 ± 5.9 to 52.8 ± 12.7 μA ( n = 18). The response to ATP is mediated by basolateral-to-apical Cl−transport because it is inhibited by 1) the Cl−channel blockers 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (1 mM), diphenylanthranilic acid (1.5 mM), or 5-nitro-2-(3-phenylpropylamino)benzoic acid (50 or 100 μM) in the apical chamber, 2) the K+channel blocker Ba2+(5 mM), or 3) the Na+-K+-2Cl−cotransport inhibitor bumetanide (200 μM) in the basolateral chamber. Other nucleotides stimulated an increase in Iscwith a rank order potency of UTP = ATP = adenosine 5′-O-(3)-thiotriphosphate, consistent with P2upurinergic receptors. ADP, AMP, 2-methylthioadenosine 5′-triphosphate, and adenosine had no effect. A cDNA encoding a rat P2ureceptor (rP2uR) was isolated from a liver cDNA library, and functional expression of the corresponding mRNA in Xenopus laevis oocytes resulted in the appearance of ATP-stimulated currents with a similar pharmacological profile. Northern analysis identified hybridizing mRNA transcripts in NRC as well as other cell types in rat liver. These findings indicate that exposure of polarized cholangiocytes to ATP results in luminal Cl−secretion through activation of P2ureceptors in the apical membrane. Release of ATP into bile may serve as an autocrine or paracrine signal regulating cholangiocyte secretory function.
- Published
- 1997
37. Metabolic Stress Opens K+ Channels in Hepatoma Cells through a Ca2+- and Protein Kinase Cα-dependent Mechanism
- Author
-
Sloan Stribling, Yusuf A. Hannun, J. Gregory Fitz, Yu Wang, John R. Raymond, Richard Roman, Ann L. Sostman, and Steve Vigna
- Subjects
Potassium Channels ,Protein Kinase C-alpha ,Molecular Sequence Data ,Deoxyglucose ,Biology ,Apamin ,Biochemistry ,chemistry.chemical_compound ,Adenosine Triphosphate ,Electric Impedance ,Tumor Cells, Cultured ,Animals ,Amino Acid Sequence ,Enzyme Inhibitors ,Protein kinase A ,Molecular Biology ,Protein Kinase C ,Protein kinase C ,Liver cell ,Electric Conductivity ,Biological Transport ,Cell Biology ,Potassium channel ,Rats ,Cell biology ,Isoenzymes ,Cytosol ,Chelerythrine ,Liver ,chemistry ,Potassium ,Calcium ,2,4-Dinitrophenol ,Ion Channel Gating ,Protein Kinases ,Dinitrophenols ,Intracellular - Abstract
These studies of a model liver cell line evaluate the mechanisms responsible for regulated release of K+ ions during metabolic stress. Metabolic inhibition of HTC hepatoma cells by exposure to 2, 4-dinitrophenol (50 microM) and 2-deoxy-D-glucose (10 mM) stimulated outward currents carried by K+ of 974 +/- 75 pA at 0 mV (n = 20, p < 0.001). Currents were inhibited by chelation of intracellular Ca2+ or exposure to apamin (50 nM), an inhibitor of SKCa channels. In cell-attached recordings from intact cells, removal of metabolic substrates (25/28 cells) or exposure to metabolic inhibitors (32/40 cells) opened K+-selective channels with a conductance of 6.5 +/- 0. 2 pS. Channels had an open probability of 0.31 +/- 0.08 and opened in bursts averaging 3.55 +/- 0.27 ms in duration (n = 6). Metabolic stress was associated with rapid translocation of the alpha isoform of protein kinase C (PKCalpha) from cytosol to membrane; and down-regulation of PKCalpha by phorbol esters or exposure to the PKC inhibitor chelerythrine (10 microM) each inhibited currents. Moreover, intracellular perfusion with purified PKCalpha activated currents in a Ca2+- and concentration-dependent manner. These findings indicate that metabolic stress leads to opening of apamin-sensitive SKCa channels in hepatoma cells through a Ca2+- and PKC-dependent mechanism and suggest that PKCalpha may be selectively involved in the response. This mechanism functionally couples the metabolic state of cells to membrane K+ permeability and represents a potential target for modification of liver injury associated with ischemia and preservation.
- Published
- 1996
38. Adenosine triphosphate activates ion permeabilities in biliary epithelial cells
- Author
-
John P. Middleton, Gayle H. Shimokura, James M. McGill, Allen W. Mangel, J. Gregory Fitz, and Srisaila Basavappa
- Subjects
Male ,Adenosine ,Cell Membrane Permeability ,Uridine Triphosphate ,Adenosine-5'-(N-ethylcarboxamide) ,Cholangiocarcinoma ,Iodine Radioisotopes ,Rats, Sprague-Dawley ,chemistry.chemical_compound ,Adenosine A1 receptor ,Adenosine Triphosphate ,Chlorides ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,Cells, Cultured ,Ion transporter ,Uridine triphosphate ,Ion Transport ,Hepatology ,Purinergic receptor ,Gastroenterology ,Purinergic signalling ,Adenosine receptor ,Rats ,Bile Duct Neoplasms ,chemistry ,Biochemistry ,Xanthines ,Potassium ,Biophysics ,Calcium ,Bile Ducts ,Rubidium Radioisotopes ,Adenosine triphosphate ,medicine.drug - Abstract
Background/Aims: The biliary epithelium contributes to bile formation through absorption and secretion of fluid and electrolytes. The effects of extracellular nucleotides on membrane ion transport were assessed in isolated bile duct cells from rats and Mz-ChA-1 cells from a human cholangiocarcinoma. Methods: The rates of efflux of 125 I and 86 Rb were used to assess membrane CI − and K + permeabilities, respectively. Patch clamp recordings of whole cell currents were used to evaluate the properties of adenosine triphosphate (ATP)-activated currents. Results: Purinergic receptor agonists ATP and uridine triphosphate stimulated 125 I and 86 Rb efflux about twofold above basal levels. The effects were reproduced by a nonhydrolyzable analogue of ATP (adenosine 5′- O -[3-thiophosphate]) and were unaffected by an adenosine receptor blocker xanthine amine congener. 125 I efflux was also stimulated by adenosine and its receptor agonists 5′- N -ethylcar-boxamidoadenosine, N 6 -(2-phenylisopropyl)adenosine; these effects were inhibited by xanthine amine congener, suggesting a separate adenosine receptor. ATP, adenosine 5′- O -(3-thiophosphate), and uridine triphosphate each stimulated release of Ca 2+ from intracellular stores, whereas adenosine had no effect. In whole cell recordings of Mz-ChA-1 cells, ATP activated an early transient outward current consistent with a K + conductance and a later, sustained inward current consistent with a CI − conductance. Conclusions: Biliary cells possess at least two classes of nucleotide receptors that modulate membrane ion permeability through Ca 2+ -dependent and -independent pathways, and ATP may be involved in the regulation of biliary secretion.
- Published
- 1994
39. Copper inhibits P2Y(2)-dependent Ca(2+) signaling through the effects on thapsigargin-sensitive Ca(2+) stores in HTC hepatoma cells
- Author
-
Shar L. Waldrop, Svjetlana Dolovcak, J. Gregory Fitz, and Gordan Kilic
- Subjects
medicine.medical_specialty ,P2Y receptor ,Thapsigargin ,Biophysics ,Biology ,Biochemistry ,Article ,Receptors, Purinergic P2Y2 ,chemistry.chemical_compound ,Inhibitory Concentration 50 ,Internal medicine ,Cell Line, Tumor ,medicine ,Extracellular ,Purinergic P2 Receptor Antagonists ,Animals ,Calcium Signaling ,Receptor ,Molecular Biology ,Receptors, Purinergic P2 ,Liver cell ,Purinergic receptor ,Cell Biology ,Purinergic signalling ,Cell biology ,Rats ,Cytosol ,Endocrinology ,chemistry ,Liver ,Calcium ,Copper - Abstract
Purinergic P2Y(2) G-protein coupled receptors play a key role in the regulation of hepatic Ca(2+) signaling by extracellular ATP. The concentration of copper in serum is about 20muM. Since copper accumulates in the liver in certain disease states, the purpose of these studies was to assess the effects of copper on P2Y(2) receptors in a model liver cell line. Exposure to a P2Y(2) agonist UTP increased [Ca(2+)](i) by stimulating Ca(2+) release from thapsigargin-sensitive Ca(2+) stores. Pretreatment of HTC cells for several minutes with copper did not affect cell viability, but potently inhibited increases in [Ca(2+)](i) evoked by UTP and thapsigargin. During this pretreatment, copper was not transported into the cytosol, and inhibited P2Y(2) receptors in a concentration-dependent manner with the IC(50) of about 15muM. These results suggest that copper inhibits P2Y(2) receptors through the effects on thapsigargin-sensitive Ca(2+) stores by acting from an extracellular side. Further experiments indicated that these effect of copper may lead to inhibition of regulatory volume decrease (RVD) evoked by hypotonic solution. Thus, copper may contribute to defective regulation of purinergic signaling and liver cell volume in diseases associated with the increased serum copper concentration.
- Published
- 2010
40. Report of the Multisociety Task Force on GI Training
- Author
-
Lawrence J. Brandt, J. Gregory Fitz, Gregory J. Gores, Philip O. Katz, Lawrence S. Friedman, John F. Kuemmerle, Michael H. Stolar, John J. Vargo, Grace H. Elta, and Darrell S. Pardi
- Subjects
Male ,medicine.medical_specialty ,Process (engineering) ,education ,Advisory Committees ,Training (civil) ,Maintenance of Certification ,Internal medicine ,medicine ,Humans ,Duration (project management) ,Curriculum ,Societies, Medical ,Medical education ,Hepatology ,business.industry ,Task force ,Gastroenterology ,Internship and Residency ,United States ,Education, Medical, Graduate ,Practice Guidelines as Topic ,Female ,Clinical Competence ,business - Abstract
In summary, the task force recommends that the 4 gastroenterology/hepatology societies work with the ABIM to develop a competency-based curriculum that incorporates the Maintenance of Certification process to accommodate the need and desire for training and subsequent practice in specific areas of gastroenterology/hepatology. Given the increasing complexity of treating digestive diseases, allowing trainees the opportunity to develop enhanced ability and experience in specific disease areas or procedures will benefit patients. By developing these training pathways, training programs will need to measure the achievements of trainees in terms of specific defined competencies rather than the duration of training alone.
- Published
- 2009
41. Modulation of colonic motility by substance P, cholecystokinin and neuropeptide Y
- Author
-
Allen W. Mangel, Ian L. Taylor, and J. Gregory Fitz
- Subjects
Atropine ,Male ,medicine.medical_specialty ,Colon ,Physiology ,Neuropeptide ,Motility ,Substance P ,Stimulation ,Tetrodotoxin ,In Vitro Techniques ,Biology ,Biochemistry ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Endocrinology ,Phentolamine ,Reference Values ,Internal medicine ,medicine ,Animals ,Neuropeptide Y ,Cholecystokinin ,Dose-Response Relationship, Drug ,Muscle, Smooth ,Neuropeptide Y receptor ,chemistry ,Rabbits ,Gastrointestinal Motility ,medicine.drug - Abstract
The effects of substance P, cholecystokinin and neuropeptide Y were examined on rabbit distal colonic motility. All three agents produced increased contractile activity but the mechanisms responsible differed depending on the agent tested. In the intact animal, peptide effects were measured under basal conditions and following exposure to atropine, tetrodotoxin and the alpha-adrenergic antagonist phentolamine. Administration of all three peptides resulted in a stimulation of colonic motility. Phentolamine did not significantly effect substance P-, cholecystokinin- or neuropeptide Y-induced activity. By contrast, the in vivo activity induced by cholecystokinin and neuropeptide Y, but not substance P, was nearly eliminated by tetrodotoxin. Only the neuropeptide Y response was partially atropine sensitive. In isolated colonic strips, cholecystokinin-induced activity, but not that produced by neuropeptide Y or substance P, was blocked by tetrodotoxin. Atropine did not significantly inhibit any of the hormone-induced contractions.
- Published
- 1991
42. HCO 3 − -coupled Na+ influx is a major determinant of Na+ turnover and Na+/K+ pump activity in rat hepatocytes
- Author
-
Steven D. Lidofsky, J. Gregory Fitz, Thomas Grotmol, Richard A. Weisiger, Bruce F. Scharschmidt, Ming Hong Xie, and Mary Cochran
- Subjects
Male ,inorganic chemicals ,Sodium-Potassium-Chloride Symporters ,Physiology ,Liver cytology ,Sodium ,Intracellular pH ,Biophysics ,Biological Transport, Active ,chemistry.chemical_element ,medicine ,Animals ,Na+/K+-ATPase ,Ouabain ,Cells, Cultured ,Ion transporter ,Membrane potential ,urogenital system ,Rats, Inbred Strains ,Cell Biology ,Hydrogen-Ion Concentration ,Rats ,Oxygen ,Bicarbonates ,medicine.anatomical_structure ,Liver ,chemistry ,Hepatocyte ,Symporter ,Carrier Proteins ,Rubidium Radioisotopes ,Nuclear chemistry - Abstract
Recent studies in hepatocytes indicate that Na(+)-coupled HCO3- transport contributes importantly to regulation of intracellular pH and membrane HCO3- transport. However, the direction of net coupled Na+ and HCO3- movement and the effect of HCO3- on Na+ turnover and Na+/K+ pump activity are not known. In these studies, the effect of HCO3- on Na+ influx and turnover were measured in primary rat hepatocyte cultures with 22Na+, and [Na+]i was measured in single hepatocytes using the Na(+)-sensitive fluorochrome SBFI. Na+/K+ pump activity was measured in intact perfused rat liver and hepatocyte monolayers as Na(+)-dependent or ouabain-suppressible 86Rb uptake, and was measured in single hepatocytes as the effect of transient pump inhibition by removal of extracellular K+ on membrane potential difference (PD) and [Na+]i. In hepatocyte monolayers, HCO3- increased 22Na+ entry and turnover rates by 50-65%, without measurably altering 22Na+ pool size or cell volume, and HCO3- also increased Na+/K+ pump activity by 70%. In single cells, exposure to HCO3- produced an abrupt and sustained rise in [Na+]i from approximately 8 to 12 mM. Na+/K+ pump activity assessed in single cells by PD excursions during transient K+ removal increased congruent to 2.5-fold in the presence of HCO3-, and the rise in [Na+]i produced by inhibition of the Na+/K+ pump was similarly increased congruent to 2.5-fold in the presence of HCO3-. In intact perfused rat liver, HCO3- increased both Na+/K+ pump activity and O2 consumption. These findings indicate that, in hepatocytes, net coupled Na+ and HCO3- movement is inward and represents a major determinant of Na+ influx and Na+/K+ pump activity. About half of hepatic Na+/K+ pump activity appears dedicated to recycling Na+ entering in conjunction with HCO3- to maintain [Na+]i within the physiologic range.
- Published
- 1991
43. Extracellular nucleotides stimulate Cl− currents in biliary epithelia through receptor-mediated IP3 and Ca2+ release
- Author
-
Andrew P. Feranchak, Kangmee Woo, R. Brian Doctor, Amal K. Dutta, and J. Gregory Fitz
- Subjects
Purinergic P2 Receptor Agonists ,P2Y receptor ,Physiology ,Inositol Phosphates ,Biology ,Adenocarcinoma ,Cholangiocyte ,Fluorescence ,Adenosine Triphosphate ,Chlorides ,Chloride Channels ,Physiology (medical) ,Extracellular ,Animals ,Humans ,Secretion ,Receptor ,Cells, Cultured ,Hepatology ,Nucleotides ,Receptors, Purinergic P2 ,Gastroenterology ,Gallbladder ,Epithelial Cells ,Receptor-mediated endocytosis ,Purinergic signalling ,Cell biology ,Rats ,Liver and Biliary Tract ,Biochemistry ,Calcium ,Gallbladder Neoplasms ,Signal transduction - Abstract
Extracellular ATP regulates bile formation by binding to P2 receptors on cholangiocytes and stimulating transepithelial Cl−secretion. However, the specific signaling pathways linking receptor binding to Cl−channel activation are not known. Consequently, the aim of these studies in human Mz-Cha-1 biliary cells and normal rat cholangiocyte monolayers was to assess the intracellular pathways responsible for ATP-stimulated increases in intracellular Ca2+concentration ([Ca2+]i) and membrane Cl−permeability. Exposure of cells to ATP resulted in a rapid increase in [Ca2+]iand activation of membrane Cl−currents; both responses were abolished by prior depletion of intracellular Ca2+. ATP-stimulated Cl−currents demonstrated mild outward rectification, reversal at ECl−, and a single-channel conductance of ∼17 pS, where E is the equilibrium potential. The conductance response to ATP was inhibited by the Cl−channel inhibitors NPPB and DIDS but not the CFTR inhibitor CFTRinh-172. Both ATP-stimulated increases in [Ca2+]iand Cl−channel activity were inhibited by the P2Y receptor antagonist suramin. The PLC inhibitor U73122 and the inositol 1,4,5-triphosphate (IP3) receptor inhibitor 2-APB both blocked the ATP-stimulated increase in [Ca2+]iand membrane Cl−currents. Intracellular dialysis with purified IP3 activated Cl−currents with identical properties to those activated by ATP. Exposure of normal rat cholangiocyte monolayers to ATP increased short-circuit currents ( Isc), reflecting transepithelial secretion. The Iscwas unaffected by CFTRinh-172 but was significantly inhibited by U73122 or 2-APB. In summary, these findings indicate that the apical P2Y-IP3 receptor signaling complex is a dominant pathway mediating biliary epithelial Cl−transport and, therefore, may represent a potential target for increasing secretion in the treatment of cholestatic liver disease.
- Published
- 2008
44. Evidence for AMPK-dependent regulation of exocytosis of lipoproteins in a model liver cell line
- Author
-
Vinay Parameswara, Svjetlana Dolovcak, Daniel S. Emmett, J. Gregory Fitz, Victoria Esser, Shar L. Waldrop, Livia Puljak, and Gordan Kilic
- Subjects
medicine.medical_specialty ,Patch-Clamp Techniques ,Apolipoprotein B ,AMPK ,cellular lipid metabolism ,Amiodarone ,AMP-Activated Protein Kinases ,Protein Serine-Threonine Kinases ,Exocytosis ,Article ,Cell Line ,AMP-activated protein kinase ,Multienzyme Complexes ,Internal medicine ,medicine ,Animals ,Humans ,Hypoglycemic Agents ,Lovastatin ,Enzyme Inhibitors ,Triglycerides ,Apolipoproteins B ,biology ,Kinase ,Liver cell ,Fatty Acids ,Lipid metabolism ,Cell Biology ,Ribonucleotides ,Aminoimidazole Carboxamide ,Lipid Metabolism ,Metformin ,Rats ,Endocrinology ,biology.protein ,Hepatocytes ,Epoxy Compounds ,lipids (amino acids, peptides, and proteins) ,Sterol Regulatory Element Binding Protein 1 ,Oxidation-Reduction ,Intracellular - Abstract
5'-AMP-activated kinase (AMPK) plays a key role in the regulation of cellular lipid metabolism. The contribution of vesicular exocytosis to this regulation is not known. Accordingly, we studied the effects of AMPK on exocytosis and intracellular lipid content in a model liver cell line. Activation of AMPK by metformin or 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) increased the rates of constitutive exocytosis by about 2-fold. Stimulation of exocytosis by AMPK occurred within minutes, and persisted after overnight exposure to metformin or AICAR. Activation of AMPK also increased the amount of triacylglycerol (TG) and apolipoprotein B (apoB) secreted from lipid-loaded cells. These effects were accompanied by a decrease in the intracellular lipid content indicating that exocytosis of lipoproteins was involved in these lipid-lowering effects. While AMPK increased the rates of fatty acid oxidation (FAO), the lipid-lowering effects were quantitatively significant even after inhibition of FAO with R-etomoxir. These results suggest that hepatic AMPK stimulates constitutive exocytosis of lipoproteins, which may function in parallel with FAO to regulate intracellular lipid content.
- Published
- 2008
45. Andreoli and Carpenter's Cecil Essentials of Medicine
- Author
-
Thomas E. Andreoli, Ivor Benjamin, Robert C. Griggs, Edward J Wing, J. Gregory Fitz, Thomas E. Andreoli, Ivor Benjamin, Robert C. Griggs, Edward J Wing, and J. Gregory Fitz
- Subjects
- Internal medicine--Textbooks, Internal medicine
- Abstract
Students, residents, and instructors swear by Andreoli and Carpenter's Cecil Essentials of Medicine because it presents just the right amount of information, just the right way. Edited by the late Thomas E. Andreoli, MD as well as Ivor Benjamin, MD, Robert C. Griggs, MD, and Edward J. Wing, MD, it focuses on core principles and how they apply to patient care, covering everything you need to know to succeed on a medical rotation or residency. Masterful editing and a user-friendly full-color design make absorbing and retaining information as effortless as possible. New chapters on'Pre- and Post-Operative Care'and'Palliative Care,'plus the integration of molecular biology and other new horizons in medicine, familiarize you with the most current clinical concepts. An expanded International Editorial Board provides increased input from respected practitioners worldwide. Excellent images and clinical photographs vividly illustrate the appearance and clinical features of disease. Masterful editing and a user-friendly full-color design make absorbing and retaining information as effortless as possible.
- Published
- 2010
46. Regulated ion transport in mouse liver cyst epithelial cells
- Author
-
Claudia R. Amura, R. Brian Doctor, J. Gregory Fitz, Sylene M. Johnson, Vincent H. Gattone, and Kelley S. Brodsky
- Subjects
Epithelial sodium channel ,medicine.medical_specialty ,Mice, Inbred Strains ,030204 cardiovascular system & hematology ,Biology ,Amiloride ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Ussing analysis ,Adenosine Triphosphate ,Internal medicine ,cAMP ,parasitic diseases ,medicine ,Extracellular ,Cyclic AMP ,Animals ,Cyst ,Secretion ,Epithelial Sodium Channels ,Molecular Biology ,Ion transporter ,030304 developmental biology ,0303 health sciences ,Ion Transport ,Cysts ,Receptors, Purinergic P2 ,Purinergic signaling ,Purinergic receptor ,Liver Neoplasms ,Sodium ,Autosomal dominant polycystic kidney disease (ADPKD) ,Epithelial Cells ,Purinergic signalling ,medicine.disease ,Cell biology ,Blot ,Endocrinology ,Molecular Medicine ,Bile Ducts ,Chlorine ,Receptors, Purinergic P2X4 - Abstract
Derived from bile duct epithelia (BDE), secretion by liver cyst-lining epithelia is positioned to drive cyst expansion but the responsible ion flux pathways have not been characterized. Cyst-lining epithelia were isolated and cultured into high resistance monolayers to assess the ion secretory pathways. Electrophysiologic studies showed a marked rate of constitutive transepithelial ion transport, including Cl − secretion and Na + absorption. Na + absorption was amiloride-sensitive, suggesting the activation of epithelial sodium channels (ENaC). Further, both cAMP i and extracellular ATP induced robust secretory responses. Western blotting and immunohistologic analysis of liver cyst epithelia demonstrated expression of P2X4, a potent purinergic receptor in normal BDE. Luminometry and bioassaying measured physiologically relevant levels of ATP in a subset of liver cyst fluid samples. Liver cyst epithelia also displayed a significant capacity to degrade extracellular ATP. In conclusion, regulated ion transport pathways are present in liver cyst epithelia and are positioned to direct fluid secretion into the lumen of liver cysts and promote increases in liver cyst expansion and growth.
- Published
- 2006
47. Extracellular ATP: Important Developments in Purinergic Signaling
- Author
-
David Gatof and J. Gregory Fitz
- Subjects
chemistry.chemical_classification ,Paracrine signalling ,Cell signaling ,chemistry ,Purinergic receptor ,Extracellular ,Nucleotide ,Purinergic signalling ,Autocrine signalling ,Intracellular ,Cell biology - Abstract
Adenosine 5’-triphosphate (ATP) is a nucleoside triphosphate composed of adenine, ribose, and three phosphate groups and is the principal carrier of chemical energy within the cell. Roughly 10 molecules of ATP are in solution throughout the intracellular space in a typical cell, providing energy for a large variety of biologic reactions that are energetically unfavorable and would otherwise not occur [4]. ATP also serves as the substrate for one of the most widely used intracellular signaling molecules, adenosine 3’,5’-cyclic monophosphate (cAMP), and is ubiquitous as a mediator of protein signaling through regulatory phosphorylation. While it has long been recognized that ATP serves as the substrate for one of the most common intracellular signaling molecules, it is more recently becoming apparent that ATP and other nucleotides also serve as potent extracellular signaling molecules. In fact, autocrine release of extracellular ATP is a ubiquitous biologic and physiologic process in numerous cell types including epithelial cells, endothelial cells, smooth muscle cells, fibroblasts, circulating lymphocytes, monocytes, red blood cells, mast cells, chondrocytes, excitatory neurons, platelets, hepatocytes, pancreatic β-cells and cholangiocytes [34]. ATP and other nucleotides exert potent autocrine and paracrine effects on cellular function through activation of purinergic receptors [12, 53]. In hepatocytes and cholangiocytes, activation of purinergic receptors through agonist binding has been linked to numerous fundamental biologic processes including cell volume regulation, glucose metabolism, bile formation, secretion and ion channel activation [40, 49, 50, 54]. Despite the recent advances in knowledge of extracellular signaling through purinergic receptors, the mechanisms of cellular ATP release remain unknown. It should be recognized that while this review focuses primarily on ATP, the mechanisms for nucleotide release presented apply to uridine nucleotides and nucleotide sugars, suggesting that these nucleotides are also capable of acting as autocrine/paracrine signaling molecules [10]. Historically, two broad models of nucleotide release have been recognized: exocytotic release of nucleotides involving the mobilization of vesicles rich in ATP, and channel-mediated release of ATP through various candidate transporters and/or exchangers. The mechanisms employed by a cell for nucleotide release will depend on the specific cell model studied and its purinergic receptor distribution. Accordingly this chapter will focus on the two principal epithelial cell types that are involved in most liver diseases: hepatocytes, which constitute the liver parenchyma, and cholangiocytes, which line the lumen of the bile ducts. The emerging role of extracellular nucleotides in the regulation of cellular physiology in these cell types was reviewed by Roman et al. [47]. This chapter builds on that work by following a similar format while focusing on more recent developments in the field.
- Published
- 2005
48. Inhibition of cellular responses to insulin in a rat liver cell line. A role for PKC in insulin resistance
- Author
-
Livia, Puljak, Michael J, Pagliassotti, Yuren, Wei, Ishtiaq, Qadri, Vinay, Parameswara, Victoria, Esser, J Gregory, Fitz, and Gordan, Kilic
- Subjects
Cell Physiology ,Fatty Acids ,Electric Conductivity ,Amiodarone ,Exocytosis ,Rats ,Isoenzymes ,Chloride Channels ,Cell Line, Tumor ,Hepatocytes ,Animals ,Insulin ,Enzyme Inhibitors ,Insulin Resistance ,Protein Kinase C - Abstract
The initial response of liver cells to insulin is mediated through exocytosis of Cl- channel-containing vesicles and a subsequent opening of plasma membrane Cl- channels. Intracellular accumulation of fatty acids leads to profound defects in metabolism, and is closely associated with insulin resistance. It is not known whether the activity of Cl- channels is altered in insulin resistance and by which mechanisms. We studied the effects of fatty acid accumulation on Cl- channel opening in a model liver cell line. Overnight treatment with amiodarone increased the fat content by approximately 2-fold, and the rates of gluconeogenesis by approximately 5-fold. The ability of insulin to suppress gluconeogenesis was markedly reduced indicating that amiodarone treatment induces insulin resistance. Western blot analysis showed that these cells express the same number of insulin receptors as control cells. However, insulin failed to activate exocytosis and Cl- channel opening. These inhibitory effects were mimicked in control cells by exposures to arachidonic acid (15 microm). Further studies demonstrated that fatty acids stimulate the PKC activity, and inhibition of PKC partially restored exocytosis and Cl- channel opening in insulin-resistant cells. Accordingly, activation of PKC with PMA in control cells potently inhibited the insulin responses. These results suggest that stimulation of PKC activity in insulin resistance contributes to the inhibition of cellular responses to insulin in liver cells.
- Published
- 2005
49. Secretion of cytokines and growth factors into autosomal dominant polycystic kidney disease liver cyst fluid
- Author
-
J. Gregory Fitz, Raj J. Shah, Matthew T. Nichols, Rolf Dahl, Tom Matzakos, Jared J. Grantham, Greg V. Stiegmann, R. Brian Doctor, and Elsa Gidey
- Subjects
Adult ,Male ,medicine.medical_specialty ,Pathology ,medicine.medical_treatment ,Autosomal dominant polycystic kidney disease ,Intrahepatic bile ducts ,Biology ,Receptors, Interleukin-8B ,Paracrine signalling ,Ischemia ,Internal medicine ,medicine ,Bile ,Humans ,Cyst ,Autocrine signalling ,Growth Substances ,Cells, Cultured ,Aged ,Hepatology ,Growth factor ,Cyst Fluid ,Epithelial Cells ,Middle Aged ,medicine.disease ,Polycystic Kidney, Autosomal Dominant ,Endocrinology ,Cytokine ,Liver ,Cytokines ,Female ,Hepatic Cyst - Abstract
The principal extrarenal manifestation of autosomal dominant polycystic kidney disease (ADPKD) involves formation of liver cysts derived from intrahepatic bile ducts. Autocrine and paracrine factors secreted into the cyst would be positioned to modulate the rate of hepatic cyst growth. The aim of this study was to identify potential growth factors present in human ADPKD liver cyst fluid. Cytokine array and enzyme-linked immunosorbent assay analysis of human ADPKD liver cyst fluid detected epithelial neutrophil attractant 78, interleukin (IL)-6 (503 ± 121 pg/mL); and IL-8 (4,488 ± 355 pg/mL); and elevated levels of vascular endothelial growth factor compared with non-ADPKD bile (849 ± 144 pg/mL vs. 270 pg/mL maximum concentration). ADPKD liver cyst cell cultures also released IL-8 and vascular endothelial growth factor, suggesting that cystic epithelial cells themselves are capable of secreting these factors. Western blotting of cultured cyst cells and immunostaining of intact cysts demonstrate that cysteine-X-cysteine receptor 2, an epithelial neutrophil attractant 78 and IL-8 receptor, is expressed at the apical domain of cyst lining epithelial cells. Suggesting the cystic epithelial cells may exist in hypoxic conditions, electron microscopy of the ADPKD liver cyst epithelium revealed morphological features similar to those observed in ischemic bile ducts. These features include elongation, altered structure, and diminished abundance of apical microvilli. In conclusion, IL-8, epithelial neutrophil attractant 78, IL-6, and vascular endothelial growth factor may serve as autocrine and paracrine factors to direct errant growth of ADPKD liver cyst epithelia. Interruption of these signaling pathways may provide therapeutic targets for inhibiting liver cyst expansion. (Hepatology 2004;40:836–846).
- Published
- 2004
50. Vesicular exocytosis contributes to volume-sensitive ATP release in biliary cells
- Author
-
J. Gregory Fitz, Gordan Kilic, and David Gatof
- Subjects
Physiology ,Enzyme Activators ,Biology ,Adenocarcinoma ,Exocytosis ,Cholangiocyte ,Fluorescence ,Paracrine signalling ,Phosphatidylinositol 3-Kinases ,Adenosine Triphosphate ,Physiology (medical) ,Cell Line, Tumor ,Extracellular ,Cyclic AMP ,Humans ,Enzyme Inhibitors ,Autocrine signalling ,Protein Kinase C ,Cell Size ,Phosphoinositide-3 Kinase Inhibitors ,Hepatology ,Purinergic receptor ,Cell Membrane ,Osmolar Concentration ,Gastroenterology ,Gallbladder ,Epithelial Cells ,Purinergic signalling ,Cell biology ,Gallbladder Neoplasms ,Signal transduction - Abstract
Extracellular ATP is a potent autocrine/paracrine signal that regulates a broad range of liver functions through activation of purinergic receptors. In biliary epithelium, increases in cell volume stimulate ATP release through a phosphoinositide 3-kinase (PI3-kinase)-dependent mechanism. Because PI3-kinase also regulates vesicular exocytosis, the purpose of these studies was to determine whether volume-stimulated vesicular exocytosis contributes to cellular ATP release. In a human cholangiocarcinoma cell line, exocytosis was measured by using the plasma membrane marker FM1–43, whereas ATP release was assessed by using a luciferase-luciferin assay. Under basal conditions, cholangiocytes exhibited constitutive exocytosis at a rate of 1.6%/min, and low levels of extracellular ATP were detected at 48.2 arbitrary light units. Increases in cholangiocyte cell volume induced by hypotonic exposure resulted in a 10-fold increase in the rate of exocytosis and a robust 35-fold increase in ATP release. Both vesicular exocytosis and ATP release were proportional to cell volume, and both exhibited similar regulatory properties including: 1) dependence on intact PI3-kinase, 2) attenuation by inhibition of PKC, and 3) potentiation by activation of PKC before hypotonic exposure. These findings demonstrate that increases in cholangiocyte cell volume stimulate ATP release and vesicular exocytosis through similar regulatory paradigms. Functional interactions among cell volume, PKC, and PI3-kinase modulate exocytosis, thereby regulating ATP release and purinergic signaling in cholangiocytes. It is hypothesized that PKC is involved in the recruitment of a volume-sensitive vesicular pool to a readily releasable state.
- Published
- 2003
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