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Biomedical subjects

J M Mullin

Publications and source records attributed to J M Mullin.

At least 19 recordsLinked to original sources
PubMed2006

Transepithelial leak in Barrett's esophagus.

Using orally administered sucrose as a probe of gastrointestinal permeability, this study focused on determining whether Barrett's metaplasia exhibits a paracellular transepithelial leak to small nonelectrolytes. Subjects in five separate classes (nonendoscoped, asymptomatic controls; endoscoped, asymptomatic controls; gastroesophageal reflux disease without mucosal complications; grossly visible esophagitis; and Barrett's esophagus) consumed a sucrose solution at bedtime and collected all overnight urine. Urine volume was measured and sucrose concentration was determined by high-performance liquid chromatography. Patients with Barrett's were observed to exhibit a transepithelial leak to sucrose whose mean value was threefold greater than that seen in healthy control subjects or patients with reflux but without any mucosal defect. A parallel study of claudin tight junction proteins in endoscopy biopsy samples showed that whereas Barrett's metaplasia contains dramatically more claudin-2 and claudin-3 than is found in normal esophageal mucosa, it is markedly lower in claudins 1 and 5, indicating very different tight junction barriers.

Amylases↗
PubMed2004

Epithelial barriers, compartmentation, and cancer.

Epithelial cells, and the tight junctions between them, form a polarized barrier between luminal and serosal fluid compartments and segregate luminal growth factors from their basal-lateral receptors. Breakdown of this barrier should allow access of growth factors in the luminal fluid to their receptors on the basal-lateral cell membranes, as recently demonstrated for heregulin and erbB receptors in airway epithelia. It should also allow luminal growth factors to access the stroma. This property may have adaptive value for epithelial tissues in general, as an elegant response to injury, but may also promote cancer formation in premalignant epithelial tissues in which the tight junctions have become chronically leaky to growth factors.

Animals↗
PubMed2002

Age- and diet-related increase in transepithelial colon permeability of Fischer 344 rats.

When transepithelial permeability of rat distal colon is evaluated on the basis of transepithelial electrical resistance, age does not have an effect. Age likewise did not affect the decrease in resistance brought about by phorbol ester exposure. However, age was shown to correlate with increased transepithelial permeability when diffusion of the nonelectrolyte, D-mannitol, was used as an indicator. A phorbol ester-induced increase in transepithelial permeability to D-mannitol was observed to increase with age. Basal permeability to D-mannitol was significantly higher in older rats when the animals were allowed to age on a high-fat diet. Distance from the rectum was shown to be a potential complicating factor in these studies, since distal colon closer to the rectum was observed to have lower transepithelial permeability. The potential effect of such increased leakiness on the increased frequency of colon cancer in older individuals is discussed.

Aging↗
PubMed2001

Phorbol ester treatment increases paracellular permeability across IEC-18 gastrointestinal epithelium in vitro.

The phorbol ester, TPA, transiently increases the transepithelial permeability across the gastrointestinal epithelium formed by IEC-18. There was a significant decrease in transepithelial resistance (R(T)) between 0 and 1.5 hr, accompanied by increased flux of polyethylene glycol (4000 MW), suggesting that the increase was across the tight junction. By 2 hr, the decrease in R(T) reversed and maintained control level. The transepithelial permeability increase was prevented by coincubation with the protein kinase C (PKC) inhibitor bisindolylmaleimide. There was a rapid (within 15 min) translocation of PKC-alpha from the cytosolic to the "membrane-associated" compartment, followed by a down-regulation that was detectable within 60 min of TPA treatment. The down-regulation of PKC-alpha from the membrane was prevented by either calpain inhibitor I or MG-132 and resulted in a sustained permeability increase. The permeability changes were not accompanied by significant effects on the amount or localization of the tight junctional proteins, occludin and ZO-1. However, occludin did show a reversible increase in phosphorylation with TPA treatment. Together these data support a role for PKC-alpha-mediated regulation of barrier permeability in an in vitro model of small intestinal epithelium, perhaps through modulation of the phosphorylation state of the tight junctional protein, occludin.

Animals↗
PubMed2000

The transient increase of tight junction permeability induced by bryostatin 1 correlates with rapid downregulation of protein kinase C-alpha.

The role of PKC-alpha in altered epithelial barrier permeability following the activation of PKC by TPA (12-O-tetradecanoyl phorbol 13-acetate) and bryostatin 1 in LLC-PK1 cells was investigated in this study. Like TPA, bryostatin 1 binds to and activates PKC but unlike TPA, it is not a tumor promoter. TPA at 10(-7) M induced a sustained 95% decrease in transepithelial electrical resistance (R(t)) across LLC-PK1 epithelial cell sheets, while 10(-7) M bryostatin 1 caused only a 30% decrease in R(t), which spontaneously reversed after 5 h. Simultaneous exposure of cell sheets to 10(-7) M TPA and 10(-7) M bryostatin 1 blunted the increase in epithelial permeability observed with 10(-7) M TPA alone. Co-incubation of cell sheets with bryostatin 1 and MG-132, a proteasomal inhibitor, caused a further decrease in R(t) at the 6-h time point and inhibited the recovery in R(t) seen with bryostatin 1 alone at this time point. TPA caused a rapid translocation of PKC-alpha from the cytosol to the membrane of the cell where it remained elevated. Bryostatin 1 treatment resulted in a slower translocation of PKC-alpha from the cytosol to the membrane and a much more rapid downregulation of PKC-alpha, with disappearance from this compartment after only 6 h. The classical PKC inhibitor Go6976 prevented the decrease in R(t) seen with TPA. Treatment of cells with TPA and bryostatin 1 resulted in a PKC-alpha translocation and downregulation profile which more closely resembled that seen with bryostatin 1 alone. Co-incubation of cells with MG-132 and bryostatin 1 caused a slower downregulation of PKC-alpha from the membrane fraction. Bryostatin 1 treatment of cells expressing a dominant/negative form of PKC-alpha resulted in a slower and less extensive decrease in R(t) compared to the corresponding control cells. For both TPA and bryostatin 1, the level of PKC-alpha in the membrane-associated fraction of the treated cells correlated closely with increased transepithelial permeability. Due to its transient effect on tight junction permeability, bryostatin 1 offers a novel pharmacological tool to investigate junctional physiology.

Animals↗
PubMed2000

Modification of tight junction function by protein kinase C isoforms.

The regulation of tight junction permeability by a variety of signal transduction pathways is summarized. An emphasis is placed on regulation of paracellular permeability by the protein kinase C family of isoforms, which involves the reporting of a large number of studies using the phorbol ester family of protein kinase C activators. The ability of protein kinase C activation to open epithelial barriers to a very wide range of solutes is emphasized, but then countered with discussion of the role of phorbol esters and protein kinase C activation in epithelial carcinogenesis. The ability of protein kinase C activation to enable growth factors to leak from luminal fluid compartments of epithelial tissues into lateral intercellular and interstitial fluid spaces may play a role in this carcinogenic action. An examination of protein kinase C effects on the phosphorylation states of tight junctional proteins suggests that downstream kinases and/or phosphatases mediate protein kinase C's effect on tight junction permeability. A role for protein kinase C in transepithelial drug delivery is questioned herein. The tight junctional leakiness associated with protein kinase C activation and apparently intrinsic to transformed epithelia suggests a potentially useful role for tight junction leakiness as a marker for early cancer diagnosis.

Animals↗
PubMed2000

Plasma concentrations of soluble tumor necrosis factor receptor I and tumor necrosis factor during cardiopulmonary bypass.

BACKGROUND: Tumor necrosis factor-alpha (TNF) has been implicated in the development of postoperative morbidity after cardiopulmonary bypass for myocardial revascularization. Despite their postulated roles as modulators of TNF bioavailability, soluble TNF receptors have not been characterized in patients undergoing this procedure and is the focus of this study. METHODS: Soluble tumor necrosis factor receptor I (sTNFRI) and TNF were measured by immunoassay in plasma samples collected from 36 patients at events before, during, and after cardiopulmonary bypass. RESULTS: Plasma concentrations of sTNFRI averaged 1.39 ng/mL at the start of the operation. Preoperative sTNFRI concentrations were found to significantly correlate with a preoperative morbidity assessment score, age, duration of bypass, duration of supplemental oxygen, and length of hospital stay. Plasma sTNFRI increased in all of the patients during the procedure. Plasma concentrations of sTNFRI and TNF did not correlate at any time. CONCLUSIONS: Preoperative measurement of sTNFRI could potentially serve as a reliable indicator for prophylactic treatment with an anti-TNF therapy. Such a therapeutic approach might help attenuate inflammatory processes thought to underlie postoperative morbidity associated with cardiopulmonary bypass.

Aged↗
PubMed2000

Regulation of protein kinase C-delta and -epsilon isoforms by phorbol ester treatment of LLC-PK1 renal epithelia.

BACKGROUND: LLC-PK1 renal epithelia are a widely used model for proximal tubular physiology and differentiation. Protein kinase C (PKC) has been observed to play a role in both processes. This study examines the subcellular distribution and down-regulation of PKC-delta and PKC-epsilon isoforms in phorbol ester-treated LLC-PK1 epithelia. METHODS: Cells were treated with 10-7 mol/L 12-O-tetradecanoyl phorbol 13-acetate (TPA) for up to seven days and were extracted as total cell lysates as well as cytosolic, membrane-associated (Triton-X soluble) and a third (Triton-X insoluble) fraction. The expression and cellular localization of PKC-delta and PKC-epsilon isoforms were then detected using Western immunoblot and immunofluorescence. RESULTS: Based on the use of an anti-PKC-delta monoclonal antibody, TPA was observed to cause a rapid decrease in total PKC-delta content, which then returned to near control levels by seven days of treatment. Immunofluorescence indicated that PKC-delta had a cytoskeletal localization within the cells, and a subtle cytoskeletal rearrangement occurred upon exposure to TPA. Western immunoblots showed that PKC-delta did not undergo the expected membrane translocation upon activation by TPA, but simply disappeared immediately from the cytosolic compartment. Conventional cell fractionation procedures such as homogenization and Triton extraction prior to Western immunoblot will, however, fail to evaluate completely PKC-delta in LLC-PK1 epithelia because of the highly stringent measures necessary to extract PKC-delta from the cytoskeletal compartment of these cells. Furthermore, we observed that a second (polyclonal) PKC-delta antibody may recognize phosphorylated forms of PKC-delta, which went unrecognized by the other antibody. PKC-epsilon was present in the cytosol, membrane, and Triton-X-insoluble fractions of the cells. TPA treatment resulted in a partial translocation of PKC-epsilon to both the membrane and Triton-X-insoluble fractions of the cell, but total PKC-epsilon remained essentially unchanged. CONCLUSIONS: The present data indicate that the localization of PKC-delta and subsequent redistribution within the LLC-PK1 cells in response to TPA treatment is highly unique and distinct from that of PKC-epsilon and PKC-alpha. An important methodological finding is that one given antibody may not recognize all phosphoproteins of a given PKC isoform.

Animals↗
PubMed2000

Increased tight junction permeability can result from protein kinase C activation/translocation and act as a tumor promotional event in epithelial cancers.

Exposure of LLC-PK1 epithelial cell cultures to phorbol ester tumor promoters causes immediate translocation of protein kinase C-alpha (PKC-alpha) from cytosolic to membrane-associated compartments. With a very similar time course, a dramatic and sustained increase in tight junctional (paracellular) permeability occurs. This increased permeability extends not only to salts and sugars but macromolecules as well. Fortyfold increases of transepithelial fluxes of biologically active EGF and insulin occur. Recovery of tight junction barrier function coincides with proteasomal downregulation of PKC-alpha. The failure to downregulate activated membrane-associated PKC-alpha has correlated with the appearance of multilayered cell growth and persistent leakiness of tight junctions. Accelerated downregulation of PKC-alpha results in only a partial and transient increase in tight junction permeability. Transfection of a dominant/negative PKC-alpha results in a slower increase in tight junction permeability in response to phorbol esters. In a separate study using rat colon, dimethylhydrazine (DMH)-induced colon carcinogenesis has been preceded by linear increases in both the number of aberrant crypts and transepithelial permeability, as a function of weeks of DMH treatment. Adenocarcinomas of both rat and human colon have been found to have uniformly leaky tight junctions. Whereas most human colon hyperplastic and adenomatous polyps contain nonleaky tight junctions, adenomatous polyps with dysplastic changes did possess leaky tight junctions. Our overall hypothesis is that tight junctional leakiness is a late event in epithelial carcinogenesis but will allow for growth factors in luminal fluid compartments to enter the intercellular and interstitial fluid spaces for the first time, binding to receptors that are located on only the basal-lateral cell surface, and causing changes in epithelial cell kinetics. Tight junctional leakiness is therefore a promotional event that would be unique to epithelial cancers.

Adenocarcinoma↗
PubMed2000

Protein kinase C activation leads to dephosphorylation of occludin and tight junction permeability increase in LLC-PK1 epithelial cell sheets.

Activation of protein kinase C by exposure of LLC-PK1 renal epithelial cells to 10(-7) M TPA, a tumor promoting phorbol ester, results in a rapid and sustained increase in paracellular permeability as evidenced by a decrease in transepithelial electrical resistance. Occludin, the first identified transmembrane protein to be localized to the tight junction of both epithelial and endothelial cells is thought play an important role in tight junction barriers. Although transepithelial electrical resistance fell to less than 20% of initial values within 1 hour of TPA exposure, transmission electron microscopy showed no change in the gross morphology of the tight junction of cells treated with 10(-7) M TPA for up to 2 hours. Immunofluorescence microscopy revealed a more rapid change in the membrane distribution of ZO-1 compared to occludin in the TPA-treated cells. Immunoblot analysis indicated that occludin levels in total cell lysates as well as cytosolic, membrane (Triton-X soluble) and cytoskeletal (Triton-X insoluble) fractions remained unchanged for at least 2 hours in cells treated with 10(-7) M TPA compared to their corresponding control cells. As the phosphorylation state of occludin is thought to be important in both tight junction assembly and regulation, the effect of phorbol ester treatment on the phosphorylation of occludin was investigated. Surprisingly, activation of protein kinase C with 10(-7) M TPA resulted in a time-dependent decrease in threonine phosphorylation of occludin which correlated closely with the rapid decrease in transepithelial electrical resistance. This dephosphorylation of occludin, occurring after activation of a serine/threonine kinase by TPA, suggested that protein kinase C was not acting directly on this tight junction target protein. If occludin dephosphorylation is involved in increasing tight junction permeability, then protein kinase C is apparently further upstream in the signaling pathway regulating epithelial barrier function, with a downstream serine/threonine phosphatase acting upon occludin.

Animals↗
PubMed1999

Activation of NF-kappaB is necessary for the restoration of the barrier function of an epithelium undergoing TNF-alpha-induced apoptosis.

Tumor necrosis factor-alpha (TNF) induces apoptosis in confluent LLC-PK1 epithelial cells, but also activates NF-kappaB, a negative regulator of apoptosis. The presence of increased TNF-induced apoptosis causes a transient increase in epithelial permeability, but the epithelial barrier function recovers, as assessed by measuring the transepithelial electrical resistance, the paracellular flux of mannitol and by the electron microscopic evaluation of the penetration of the electron-dense dye ruthenium red across the tight junctions. The integrity of the epithelial cell layer is maintained by rearrangement of non-apoptotic cells in the monolayer and by the phagocytosis of apoptotic fragments. To study the role of NF-kappaB in an epithelium exposed to TNF, NF-kappaB was inhibited in LLC-PK1 epithelial cells with either the dietary compound, curcumin, or by transfection with a dominant negative mutant inhibitor I kappaB alpha. Replacement of serine 32 and 36 by alanine has been shown to prevent its phosphorylation and degradation, blocking NF-kappaB activation. Inhibition of NF-kappaB altered the morphology of TNF-induced apoptotic cells, which showed lack of fragmentation and membrane blebbings, and absence of phagocytosis by neighboring cells. TNF treatment of NF-kappaB-inhibited cells also caused altered distribution of the tight junction-associated protein ZO-1, increased epithelial leakiness, and impaired the recovery of the epithelial barrier function, which normally occurs 6 hours after TNF treatment of LLC-PK1 cells. These data demonstrate that NF-kappaB activation is required for the maintenance of the barrier function of an epithelium undergoing TNF-induced apoptosis.

Animals↗
PubMed1999

Increased tight junctional permeability is associated with the development of colon cancer.

Epithelial tissues act as barriers between two fluid compartments, and the epithelial barrier function is provided by the epithelial cells and the tight junctions (TJs) that connect them. We have shown previously that chronic treatment of a cultured epithelial monolayer with phorbol ester tumor promoters induces an increase in transepithelial paracellular permeability and produces tumor-like polyps, suggesting an association between TJ permeability and tumor formation. In this study, we analyzed the association between TJ permeability and formation of tumors in vivo. The permeability of the TJs was assessed in normal human and rat colon epithelia and in colon tumors by measuring the transepithelial electrical resistance, the paracellular flux rate of D-[(14)C]mannitol and the electron microscopic evaluation of the penetration of the electron dense dye ruthenium red across the TJs. By these criteria, the TJs of human colon tumors, including carcinomas and adenomatous polyps, and the TJs of 1,2-dimethylhydrazine (DMH)-induced rat colon tumors were leakier than the TJs of normal colon. Treatment of rats with the carcinogen DMH induced a progressive increase in the number of aberrant colonic crypts, considered the putative pre-neoplastic colonic phenotype while increasing TJ permeability of the colon epithelium prior to the development of tumors. These results showed that increased TJ permeability of the colon epithelium and consequently a decrease in epithelial barrier function precede the development of colon tumors.

1,2-Dimethylhydrazine↗
PubMed1998

Protein kinase C activation increases transepithelial transport of biologically active insulin.

Protein kinase C activation leads to tight junctional leakiness and, consequently, to increased transepithelial (paracellular) solute flux across epithelial barriers. This leakiness is shown here to result in as much as a 20-fold increase in the transepithelial flux of insulin. Using an epithelial/fibroblast coculture model, this transepithelially transported insulin is shown to be biologically active. The 3T3 fibroblasts situated on one side of the epithelial barrier exhibited increased insulin binding and resulting DNA synthesis when the epithelial junctions were made leaky to insulin on the opposite side of the epithelial barrier. The dramatically enhanced permeability of macromolecules across epithelial cell layers undergoing protein kinase C activation may play a significant role in epithelial cancer, immunology, and drug delivery.

3T3 Cells↗
PubMed1998

Tumor necrosis factor-alpha increases sodium and chloride conductance across the tight junction of CACO-2 BBE, a human intestinal epithelial cell line.

CACO-2 BBE was used to determine the response of a gastrointestinal epithelium to tumor necrosis factor-alpha (TNF). Incubation of CACO-2 BBE with TNF did not produce any effect on transepithelial resistance (TER) within the first 6 hr but resulted in a 40-50% reduction in TER and a 30% decrease in 1SC (short circuit current) relative to time-matched control at 24 hr. The decrease in TER was sustained up to 1 week following treatment with TNF and was not associated with a significant increase in the transepithelial flux of [14C]-D-mannitol or the penetration of ruthenium red into the lateral intercellular space. Dilution potential and transepithelial 22Na+ flux studies demonstrated that TNF-treatment of CACO-2 BBE cell sheets increased the paracellular permeability of the epithelium to Na+ and Cl-. The increased transepithelial permeability did not associate with an increase in the incidence of apoptosis. However, there was a TNF-dependent increase in [3H]-thymidine labeling that was not accompanied by a change in DNA content of the cell sheet. The increase in transepithelial permeability was concluded to be across the tight junction because: (i) 1 mM apical amiloride reduced the basolateral to apical flux of 22Na+, and (ii) dilution potential studies revealed a bidirectionally increased permeability to both Na+ and Cl-. These data suggest that the increase in transepithelial permeability across TNF-treated CACO-2 BBE cell sheets arises from an alteration in the charge selectivity of the paracellular conductive pathway that is not accompanied by a change in its size selectivity.

Caco-2 Cells↗
PubMed1998

Pentobarbital affects transepithelial electrophysiological parameters regulated by protein kinase C in rat distal colon.

For rat distal colon, the transepithelial electrical parameters, short circuit current (Iscc) and transepithelial electrical resistance (TER), respectively, measure net transepithelial electrolyte transport activity and the barrier function of the epithelium. Studies with a variety of epithelial cell cultures have shown greater than 90% decreases of TER within minutes of exposure of in vitro cell sheets to phorbol esters. The phorbol ester and protein kinase C (PKC) activator, phorbol dibutyrate (PDBU), was observed to produce an over 100% elevation of Iscc but only a small yet significant 20-30% decrease of TER across rat distal colon. Inhibition of the above effects of PDBU by the PKC inhibitor bisindolylmaleimide (GFX) is further evidence that in rat distal colon, Iscc and TER are under regulatory control by PKC. When animals received anesthesia with intraperitoneal pentobarbital prior to removal of the colon, the effect of PDBU on Iscc was significantly reduced, and the effect of PDBU on TER was almost completely inhibited. This effect of pentobarbital on PKC-mediated transepithelial permeability parameters is consistent with the known ability of anesthetics to alter protein kinase C activity. Exposure of rat colon to pentobarbital produced as much as a 90% inhibition of calcium-dependent PKC activity, whereas calcium-independent activity was stimulated by as much as 35%. Prior anesthetic use may be therefore a complicating factor in observing PKC-mediated effects on epithelial barrier function using epithelial tissue models.

Adjuvants, Anesthesia↗
PubMed1998

Overexpression of protein kinase C-delta increases tight junction permeability in LLC-PK1 epithelia.

The Ca2+-independent delta-isoform of protein kinase C (PKC-delta) was overexpressed in LLC-PK1 epithelia and placed under control of a tetracycline-responsive expression system. In the absence of tetracycline, the exogenous PKC-delta is expressed. Western immunoblots show that the overexpressed PKC-delta is found in the cytosolic, membrane-associated, and Triton-insoluble fractions. Overexpression of PKC-delta produced subconfluent and confluent epithelial morphologies similar to that observed on exposure of wild-type cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. Transepithelial electrical resistance (RT) in cell sheets overexpressing PKC-delta was only 20% of that in cell sheets incubated in the presence of tetracycline, in which the amount of PKC-delta and RT were similar to those in LLC-PK1 parental cell sheets. Overexpression of PKC-delta also elicited a significant increase in transepithelial flux of D-[14C]mannitol and a radiolabeled 2 x 10(6)-molecular-weight dextran, suggesting with the RT decrease that overexpression increased paracellular, tight junctional permeability. Electron microscopy showed that PKC-delta overexpression results in a multilayered cell sheet, the tight junctions of which are almost uniformly permeable to ruthenium red. Freeze-fracture electron microscopy indicates that overexpression of PKC-delta results in a more disorganized arrangement of tight junctional strands. As with LLC-PK1 cell sheets treated with 12-O-tetradecanoylphorbol-13-acetate, the reduced RT, increased D-mannitol flux, and tight junctional leakiness to ruthenium red that are seen with PKC-delta overexpression suggest the involvement of PKC-delta in regulation of tight junctional permeability.

Animals↗