Rapid development of vasopressin-induced hydroosmosis in kidney collecting tubules measured by a new fluorescence technique.
AuthorsKuwahara M, Berry CA, Verkman AS
JournalBiophys J
PubMed ID3224145
'The pre-steady-state kinetics of the vasopressin-induced increase in collecting tubule osmotic water permeability (Pf) has been measured by a new fluorescence technique. Isolated cortical collecting tubules (CCT) from rabbit kidney were perfused with physiological buffers containing the impermeant fluorophores fluorescein sulfonate (FS) and pyrenetetrasulfonic acid (PTSA). Tubules were subject to ... More
The delta-isoform of protein kinase C causes inducible nitric-oxide synthase and nitric oxide up-regulation: key mechanism for oxidant-induced carbonylation, nitration, and disassembly of the microtubule cytoskeleton and hyperpermeability of barrier of intestinal epithelia.
AuthorsBanan A, Farhadi A, Fields JZ, Zhang LJ, Shaikh M, Keshavarzian A
JournalJ Pharmacol Exp Ther
PubMed ID12606598
'Using intestinal (Caco-2) cells, we found that oxidant-induced disruption of barrier integrity requires microtubule disassembly. Protein kinase C (PKC)-delta isoform seems to be essential for disruption, but the mechanism is unknown. Because inducible nitric-oxide synthase (iNOS) is key to oxidant stress, we hypothesized that PKC-delta activation is essential in oxidant-induced ... More
Phospholipase C-gamma inhibition prevents EGF protection of intestinal cytoskeleton and barrier against oxidants.
AuthorsBanan A, Fields JZ, Zhang Y, Keshavarzian A
JournalAm J Physiol Gastrointest Liver Physiol
PubMed ID11447022
'Loss of intestinal barrier integrity is associated with oxidative inflammatory GI disorders including inflammatory bowel disease. Using monolayers of human intestinal epithelial (Caco-2) cells, we recently reported that epidermal growth factor (EGF) protects barrier integrity against oxidants by stabilizing the microtubule cytoskeleton, but the mechanism downstream of the EGF receptor ... More
Activation of delta-isoform of protein kinase C is required for oxidant-induced disruption of both the microtubule cytoskeleton and permeability barrier of intestinal epithelia.
AuthorsBanan A, Fields JZ, Farhadi A, Talmage DA, Zhang L, Keshavarzian A
JournalJ Pharmacol Exp Ther
PubMed ID12235228
'Using monolayers of intestinal (Caco-2) cells, we showed that oxidants disassemble the microtubule cytoskeleton and disrupt barrier integrity (permeability) (Banan et al., 2000a). Because exposure of our parental cells to oxidants causes protein kinase C (PKC)-delta to be translocated to particulate fractions, we hypothesized that PKC-delta activation is required for ... More
Transepithelial water permeability in microperfused distal airways. Evidence for channel-mediated water transport.
AuthorsFolkesson HG, Matthay MA, Frigeri A, Verkman AS
JournalJ Clin Invest
PubMed ID8609221
'Water movement across the airway epithelium is important for regulation of the volume and composition of airspace fluid. A novel approach is reported here to measure osmotic and diffusional water permeability in intact airways. Small airways (100-200 microns diameter, 1-2 mm length) from guinea pig lung were microdissected and perfused ... More
Rotavirus alters paracellular permeability and energy metabolism in Caco-2 cells.
'Rotaviruses infect epithelial cells of the small intestine, but the pathophysiology of the resulting severe diarrhea is incompletely understood. Histological damage to intestinal epithelium is not a consistent feature, and in vitro studies showed that intestinal cells did not undergo rapid death and lysis during viral replication. We show that ... More
PKC-beta1 isoform activation is required for EGF-induced NF-kappaB inactivation and IkappaBalpha stabilization and protection of F-actin assembly and barrier function in enterocyte monolayers.
AuthorsBanan A, Zhang LJ, Farhadi A, Fields JZ, Shaikh M, Keshavarzian A
JournalAm J Physiol Cell Physiol
PubMed ID14602581
'Using monolayers of intestinal Caco-2 cells, we reported that activation of NF-kappaB is required for oxidative disruption and that EGF protects against this injury but the mechanism remains unclear. Activation of the PKC-beta1 isoform is key to monolayer barrier integrity. We hypothesized that EGF-induced activation of PKC-beta1 prevents oxidant-induced activation ... More
Multiple innervation of tonic endplates revealed by activity-dependent uptake of fluorescent probes.
AuthorsLichtman JW, Wilkinson RS, Rich MM
JournalNature
PubMed ID3982503
'During development of the vertebrate nervous system, there is a widespread reduction in the number of axons innervating target cells. This phenomenon, often called synapse elimination, has been particularly well studied at the neuromuscular junction of developing twitch muscle fibres: following a period of polyneuronal innervation, axonal branches are retracted, ... More
Theta-isoform of PKC is required for alterations in cytoskeletal dynamics and barrier permeability in intestinal epithelium: a novel function for PKC-theta.
AuthorsBanan A, Zhang LJ, Shaikh M, Fields JZ, Farhadi A, Keshavarzian A
JournalAm J Physiol Cell Physiol
PubMed ID14985240
'Using intestinal Caco-2 cells, we previously showed that assembly of cytoskeleton is required for monolayer barrier function, but the underlying mechanisms remain poorly understood. Because the theta-isoform of PKC is present in wild-type (WT) intestinal cells, we hypothesized that PKC-theta is crucial for changes in cytoskeletal and barrier dynamics. We ... More
Modulation of K+ conductance by intracellular pH in pancreatic beta-cells.
AuthorsRosario LM, Rojas E
JournalFEBS Lett
PubMed ID2422060
Measurements of the effects of NH3/NH4+ on glucose-induced electrical activity in beta-cells from microdissected mouse islets of Langerhans and on intracellular pH in single collagenase-isolated islets pre-loaded with a fluorescent pH probe were performed and are reported here. Application of NH3/NH4+ (15 mM) in the presence of glucose (11 mM) ... More
Vita Blue: a new 633-nm excitable fluorescent dye for cell analysis.
AuthorsLee LG, Berry GM, Chen CH
JournalCytometry
PubMed ID2714106
Several new derivatives of fluorescein were synthesized. The dyes were characterized by NMR; and the absorbance, excitation, and emission spectra were measured. The fluorescence quantum yields of the dyes were determined. The pKa3 values of the dyes were measured by fluorescence titration. The characteristics of the fluorescein and sulfonefluorescein derivatives ... More
Nitric oxide dilates tight junctions and depletes ATP in cultured Caco-2BBe intestinal epithelial monolayers.
We tested the hypothesis that nitric oxide (NO) modulates the permeability of tight junctions in a model intestinal epithelium (Caco-2BBe monolayers). Incubation with sodium nitroprusside (SNP) resulted in time- and concentration-dependent decreases in transepithelial resistance. Permeability to fluorescein sulfonic acid increased during incubation for 24 h in the presence of ... More
iNOS upregulation mediates oxidant-induced disruption of F-actin and barrier of intestinal monolayers.
AuthorsBanan A, Fields JZ, Zhang Y, Keshavarzian A
JournalAm J Physiol Gastrointest Liver Physiol
PubMed ID11352817
Using oxidant-induced hyperpermeability of monolayers of intestinal (Caco-2) cells as a model for the pathophysiology of inflammatory bowel disease (IBD), we previously showed that oxidative injury to the F-actin cytoskeleton is necessary for the disruption of monolayer barrier integrity. We hypothesized that this cytoskeletal damage is caused by upregulation of ... More
Direct measurement of trans-Golgi pH in living cells and regulation by second messengers.
AuthorsSeksek O, Biwersi J, Verkman AS
JournalJ Biol Chem
PubMed ID7890600
In the endocytic compartment, an acidic pH plays a key role in receptor and ligand sorting, vesicular transport, and protein degradation. In the secretory compartment, indirect estimates of trans-Golgi pH based on partitioning of weak bases and following viral infection suggest a mildly acidic pH of > 6.0. We developed ... More
Frequent occurrence of identical heavy and light chain Ig rearrangements.
AuthorsSeidl KJ, MacKenzie JD, Wang D, Kantor AB, Kabat EA, Herzenberg LA, Herzenberg LA
JournalInt Immunol
PubMed ID9184914
Single-cell PCR analyses of expressed Ig H and L chain sequences presented here show that certain rearrangements occur repeatedly and account for a major segment of the well-studied repertoire of B-1 cell autoantibodies that mediate the lysis of bromelain-treated mouse erythrocytes, i.e. antibodies reactive with phosphatldyicholine (PtC). We repeatedly isolated ... More
PKC-beta1 mediates EGF protection of microtubules and barrier of intestinal monolayers against oxidants.
AuthorsBanan A, Fields JZ, Talmage DA, Zhang Y, Keshavarzian A
JournalAm J Physiol Gastrointest Liver Physiol
PubMed ID11518696
Using monolayers of human intestinal (Caco-2) cells, we found that oxidants and ethanol damage the cytoskeleton and disrupt barrier integrity; epidermal growth factor (EGF) prevents damage by enhancement of protein kinase C (PKC) activity and translocation of the PKC-beta1 isoform. To see if PKC-beta1 mediates EGF protection, cells were transfected ... More
Hyperpermeability of intestinal epithelial monolayers is induced by NO: effect of low extracellular pH.
AuthorsUnno N, Menconi MJ, Smith M, Aguirre DE, Fink MP
JournalAm J Physiol
PubMed ID9176198
Nitric oxide (NO.) increases the permeability of Caco-2BBe enterocytic monolayers. Many of the toxic effects of NO. are thought to be mediated by the peroxynitrite anion (ONOO-), which, under mildly acidic conditions, can rearrange to yield an intermediate with reactivity similar to toxic OH.. Accordingly, we assessed the permeability of ... More
Key role of PKC and Ca2+ in EGF protection of microtubules and intestinal barrier against oxidants.
AuthorsBanan A, Fields JZ, Zhang Y, Keshavarzian A
JournalAm J Physiol Gastrointest Liver Physiol
PubMed ID11292590
Using monolayers of human intestinal (Caco-2) cells, we showed that growth factors (GFs) protect microtubules and barrier integrity against oxidative injury. Studies in nongastrointestinal cell models suggest that protein kinase C (PKC) signaling is key in GF-induced effects and that cytosolic calcium concentration ([Ca2+](i)) is essential in cell integrity. We ... More
Cytosolic ionized Ca2+ modulates chemical hypoxia-induced hyperpermeability in intestinal epithelial monolayers.
AuthorsUnno N, Baba S, Fink MP
JournalAm J Physiol
PubMed ID9575852
We reported previously that ATP depletion induced by glycolytic inhibition or cellular hypoxia increases the permeability of intestinal epithelial monolayers [N. Unno, M. J. Menconi, A. L. Salzman, M. Smith, S. Hagen, Y. Ge, R. M. Ezzell, and M. P. Fink. Am. J. Physiol. 270 (Gastrointest. Liver Physiol. 33): G1010-G1021, ... More
Rapid fluorescence assay of glucose and neutral solute transport using an entrapped volume indicator.
AuthorsKim YK, Illsley NP, Verkman AS
JournalAnal Biochem
PubMed ID3189785
A rapid fluorescence method is described for quantitative measurement of glucose and neutral solute transport in cells and sealed membrane vesicles based on volume changes accompanying transport. Membrane vesicles are loaded with the volume-sensitive indicator fluorescein sulfonate (FS) which undergoes concentration-dependent fluorescence self-quenching. In response to an inward solute gradient, ... More
Membrane water and solute permeability determined quantitatively by self-quenching of an entrapped fluorophore.
AuthorsChen PY, Pearce D, Verkman AS
JournalBiochemistry
PubMed ID3179272
Quantitative determination of rapid water and solute transport and solute reflection coefficients by light-scattering methods is complicated by dependence of vesicle or cell light scattering on nonvolume factors including solution refractive index, cell motion, and membrane aggregation. To overcome these difficulties, a fluorescence technique has been developed to measure accurately ... More
Flow rate measurements in isolated perfused kidney tubules by fluorescence photobleaching recovery.
AuthorsFlamion B, Bungay PM, Gibson CC, Spring KR
JournalBiophys J
PubMed ID1760509
We have developed a new application of the fluorescence photobleaching recovery (FPR) technique for instantaneous measurement of volume flow rates at any axial position along isolated perfused kidney tubules. The method requires fast data acquisition of emitted fluorescence through a photomultiplier (time resolution, 0.5 ms) coupled with differential interference contrast ... More
Phosphatidic acid-phosphatidylethanolamine interaction and apocytochrome c translocation across model membranes.
AuthorsMiao Q, Han X, Yang F
JournalBiochem J
PubMed ID11237873
The translocation of apocytochrome c (apocyt.c) across large unilamellar vesicles (LUVs) constructed from mixtures of anionic and zwitterionic phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), has been studied. It was shown that the import ratio of horse heart apocyt.c in LUVs composed of phosphatidic acid (PA) combined with PE and PC ... More
Nitric oxide and its metabolites mediate ethanol-induced microtubule disruption and intestinal barrier dysfunction.
AuthorsBanan A, Fields JZ, Decker H, Zhang Y, Keshavarzian A
JournalJ Pharmacol Exp Ther
PubMed ID10945852
Loss of gastrointestinal (GI) barrier integrity has been implicated in a wide range of inflammatory illnesses, including alcoholic cirrhosis. Using monolayers of Caco-2 (intestinal) cells as a model, we showed that the ability of ethanol (EtOH) to disrupt intestinal barrier integrity depends on damage to the microtubule (MT) cytoskeleton, especially ... More
Inhibition of oxidant-induced nuclear factor-kappaB activation and inhibitory-kappaBalpha degradation and instability of F-actin cytoskeletal dynamics and barrier function by epidermal growth factor: key role of phospholipase-gamma isoform.
AuthorsBanan A, Zhang LJ, Shaikh M, Fields JZ, Farhadi A, Keshavarzian A
JournalJ Pharmacol Exp Ther
PubMed ID14724221
Using monolayers of intestinal (Caco-2) cells as a model for studying inflammatory bowel disease (IBD), we previously showed that nuclear factor-kappaB (NF-kappaB) activation is required for oxidant-induced disruption of cytoskeletal and barrier integrity. Epidermal growth factor (EGF) stabilizes the F-actin cytoskeleton and protects against oxidant damage, but the mechanism remains ... More
Ethanol-induced barrier dysfunction and its prevention by growth factors in human intestinal monolayers: evidence for oxidative and cytoskeletal mechanisms.
AuthorsBanan A, Choudhary S, Zhang Y, Fields JZ, Keshavarzian A
JournalJ Pharmacol Exp Ther
PubMed ID10565827
Exposure of intestinal mucosa to ethanol (EtOH) disrupts barrier function and growth factors [epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha)] are protective, but the mechanisms remain obscure. Accordingly, we sought to determine whether the molecular mechanism of EtOH-induced intestinal barrier dysfunction involves oxidative stress and disassembly of microtubules ... More
Evidence that nuclear factor-kappa B activation is critical in oxidant-induced disruption of the microtubule cytoskeleton and barrier integrity and that its inactivation is essential in epidermal growth factor-mediated protection of the monolayers of intestinal epithelia.
AuthorsBanan A, Farhadi A, Fields JZ, Mutlu E, Zhang L, Keshavarzian A
JournalJ Pharmacol Exp Ther
PubMed ID12815011
Using monolayers of intestinal (Caco-2) cells, we showed that oxidants disrupt the microtubule cytoskeleton and barrier integrity; epidermal growth factor (EGF) was protective via stabilization of the microtubules. Because proinflammatory conditions activate nuclear factor-kappaB (NF-kappaB), we hypothesized that oxidants disrupt barrier integrity through activation of NF-kappaB and that EGF protects ... More
Zeta isoform of protein kinase C prevents oxidant-induced nuclear factor-kappaB activation and I-kappaBalpha degradation: a fundamental mechanism for epidermal growth factor protection of the microtubule cytoskeleton and intestinal barrier integrity.
AuthorsBanan A, Fields JZ, Zhang LJ, Shaikh M, Farhadi A, Keshavarzian A
JournalJ Pharmacol Exp Ther
PubMed ID12893839
Oxidant damage and gut barrier disruption contribute to the pathogenesis of a variety of inflammatory gastrointestinal disorders, including inflammatory bowel disease (IBD). In our studies using a model of the gastrointestinal (GI) epithelial barrier, monolayers of intestinal (Caco-2) cells, we investigated damage to and protection of the monolayer barrier. We ... More
Modulation of intestinal permeability by nitric oxide donors: implications in intestinal delivery of poorly absorbable drugs.
AuthorsYamamoto A, Tatsumi H, Maruyama M, Uchiyama T, Okada N, Fujita T
JournalJ Pharmacol Exp Ther
PubMed ID11123366
The effects of nitric oxide (NO) donors NOC5 [3-(2-hydroxy-1-(methylethyl)-2-nitrosohydrazino)-1-propanamine ] and NOC12 [N-ethyl-2-(1-ethyl-hydroxy-2-nitrosohydrazino)-ethanamine] on the permeability of 5(6)-carboxyfluorescein (CF) across the intestinal membrane were examined by an in vitro Ussing chamber method. The NO donors significantly increased the intestinal permeability of CF and their absorption-enhancing effects were concentration-dependent over the ... More
Regulation of aquaporin-2 trafficking by vasopressin in the renal collecting duct. Roles of ryanodine-sensitive Ca2+ stores and calmodulin.
In the renal collecting duct, vasopressin increases osmotic water permeability (P(f)) by triggering trafficking of aquaporin-2 vesicles to the apical plasma membrane. We investigated the role of vasopressin-induced intracellular Ca(2+) mobilization in this process. In isolated inner medullary collecting ducts (IMCDs), vasopressin (0.1 nm) and 8-(4-chlorophenylthio)-cAMP (0.1 mm) elicited marked ... More
Hyperpermeability and ATP depletion induced by chronic hypoxia or glycolytic inhibition in Caco-2BBe monolayers.
AuthorsUnno N, Menconi MJ, Salzman AL, Smith M, Hagen S, Ge Y, Ezzell RM, Fink MP
JournalAm J Physiol
PubMed ID8764209
Previous studies have documented that the barrier function of epithelial or endothelial monolayers is deranged when cellular ATP levels are rapidly decreased to very low levels by inhibitors of mitochondrial and glycolytic function. We hypothesized that lesser degrees of ATP depletion also might affect epithelial permeability, particularly if the perturbation ... More
PKC-zeta prevents oxidant-induced iNOS upregulation and protects the microtubules and gut barrier integrity.
AuthorsBanan A, Zhang L, Fields JZ, Farhadi A, Talmage DA, Keshavarzian A
JournalAm J Physiol Gastrointest Liver Physiol
PubMed ID12223351
Using intestinal (Caco-2) monolayers, we reported that inducible nitric oxide synthase (iNOS) activation is key to oxidant-induced barrier disruption and that EGF protects against this injury. PKC-zeta was required for protection. We thus hypothesized that PKC-zeta activation and iNOS inactivation are key in EGF protection. Wild-type (WT) Caco-2 cells were ... More
The beta 1 isoform of protein kinase C mediates the protective effects of epidermal growth factor on the dynamic assembly of F-actin cytoskeleton and normalization of calcium homeostasis in human colonic cells.
AuthorsBanan A, Fields JZ, Farhadi A, Talmage DA, Zhang L, Keshavarzian A
JournalJ Pharmacol Exp Ther
PubMed ID12023512
Using intestinal monolayers, we showed that F-actin cytoskeletal stabilization and Ca(2+) normalization contribute to epidermal growth factor (EGF)-mediated protection against oxidant injury. However, the intracellular mediator responsible for these protective effects remains unknown. Since the protein kinase C-beta1 (PKC-beta1) isoform is abundant in our naive (N) cells, we hypothesized that ... More
Key role of PLC-gamma in EGF protection of epithelial barrier against iNOS upregulation and F-actin nitration and disassembly.
AuthorsBanan A, Zhang LJ, Shaikh M, Fields JZ, Farhadi A, Keshavarzian A
JournalAm J Physiol Cell Physiol
PubMed ID12788694
Upregulation of inducible nitric oxide synthase (iNOS) is key to oxidant-induced disruption of intestinal (Caco-2) monolayer barrier, and EGF protects against this disruption by stabilizing the cytoskeleton. PLC-gamma appears to be essential for monolayer integrity. We thus hypothesized that PLC-gamma activation is essential in EGF protection against iNOS upregulation and ... More
Acidosis induces hyperpermeability in Caco-2BBe cultured intestinal epithelial monolayers.
AuthorsMenconi MJ, Salzman AL, Unno N, Ezzell RM, Casey DM, Brown DA, Tsuji Y, Fink MP
JournalAm J Physiol
PubMed ID9176208
We previously demonstrated that ileal mucosal acidosis in pigs reversibly increases intestinal permeability to hydrophilic macromolecules, even in the absence of tissue hypoxia [A.L. Salzman, H. Wang, P.S. Wollert, T.J. Vandermeer, C.C. Compton, A.G. Denenberg, and M.P. Fink. Am. J. Physiol. 266 (Gastrointest, Liver Physiol, 29): G633-G646, 1994]. In an ... More
Late-Stage Glioma Is Associated with Deleterious Alteration of Gut Bacterial Metabolites in Mice.