Kathleen V. Laughlin

Overexpression of protein kinase C-δ increases tight junction permeability in LLC-PK1epithelia

The Ca2+-independent δ-isoform of protein kinase C (PKC-δ) was overexpressed in LLC-PK1 epithelia and placed under control of a tetracycline-responsive expression system. In the absence of tetracycline, the exogenous PKC-δ is expressed. Western immunoblots show that the overexpressed PKC-δ is found in the cytosolic, membrane-associated, and Triton-insoluble fractions. Overexpression of PKC-δ 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 ( R T) in cell sheets overexpressing PKC-δ was only 20% of that in cell sheets incubated in the presence of tetracycline, in which the amount of PKC-δ and R Twere similar to those in LLC-PK1parental cell sheets. Overexpression of PKC-δ also elicited a significant increase in transepithelial flux ofd-[14C]mannitol and a radiolabeled 2 × 106-molecular-weight dextran, suggesting with the R T decrease that overexpression increased paracellular, tight junctional permeability. Electron microscopy showed that PKC-δ 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-δ 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 R T, increasedd-mannitol flux, and tight junctional leakiness to ruthenium red that are seen with PKC-δ overexpression suggest the involvement of PKC-δ in regulation of tight junctional permeability.

Different size limitations for increased transepithelial paracellular solute flux across phorbol ester and tumor necrosis factor-treated epithelial cell sheets.

By observing increases in the transepithelial paracellular permeability of a range of radiolabeled solutes and electron dense dyes, changes in molecular sieving caused by the cytokine, TNF (tumor necrosis factor), and the phorbol ester, TPA (12‐0‐tetra‐decanoylphorbol‐13‐acetate), were characterized. Using 14C‐labeled mannitol (mw 182), raffinose (mw 504), PEG (polyethylene glycol; mw 4000), and dextran (mw 10,000, 70,000 and 2,000,000), the transepithelial flux rates of these compounds were determined at the peak of the transepithelial electrical resistance (TER) changes caused by these two agents. TNF treatment resulted in increased permeability across LLC‐PK1 epithelial cell sheets only to relatively small solutes, with an upper limit of approximately 4,000 mw. The low molecular weight “ceiling” for the TNF‐treated epithelium is further evidence against TNF increasing transepithelial permeability by means of inducing nonspecific, microscopic “holes” in the epithelium, for which a “ceiling” would not exist. TPA treatment increases transepithelial paracellular permeability to a much broader range of solutes, extending well beyond 2 million mw. Transmission electron micrographs provide evidence that even the electron‐dense dye complex, ruthenium red, can cross tight junctions of TPA‐treated cell sheets. However, cationic ferritin cannot cross tight junctions of TPA‐treated cell sheets. This shows that there is an upper limit to solutes able to cross TPA‐treated cell sheets, but that this upper limit will include most proteins, which would then be able to cross tumor promoter‐exposed (protein kinase C‐activated) epithelial layers at accelerated rates. The biomedical implications for a high molecular weight cutoff in tumor promoter action in epithelial carcinogenesis, and for a low molecular weight cutoff in cytokine‐induced epithelial apoptosis in inflammation, are discussed. J. Cell. Physiol. 171:226–233, 1997.

Increased Tight Junction Permeability Can Result from Protein Kinase C Activation/Translocation and Act as a Tumor Promotional Event in Epithelial Cancers

Abstract: Exposure of LLC‐PK1 epithelial cell cultures to phorbol ester tumor promoters causes immediate translocation of protein kinase C‐α (PKC‐α) 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‐α. The failure to downregulate activated membrane‐associated PKC‐α has correlated with the appearance of multilayered cell growth and persistent leakiness of tight junctions. Accelerated downregulation of PKC‐α results in only a partial and transient increase in tight junction permeability. Transfection of a dominant/ negative PKC‐α 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.

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.

Pentobarbital Affects Transepithelial Electrophysiological Parameters Regulated by Protein Kinase C in Rat Distal Colon

For rat distal colon, the transepithelialelectrical parameters, short circuit current(Iscc) and transepithelial electricalresistance (TER), respectively, measure nettransepithelial electrolyte transport activity and the barrier function of theepithelium. Studies with a variety of epithelial cellcultures have shown greater than 90% decreases of TERwithin minutes of exposure of in vitro cell sheets to phorbol esters. The phorbol ester and proteinkinase C (PKC) activator, phorbol dibutyrate (PDBU), wasobserved to produce an over 100% elevation ofIscc but only a small yet significant 20-30%decrease of TER across rat distal colon. Inhibition ofthe above effects of PDBU by the PKC inhibitorbisindolylmaleimide (GFX) is further evidence that inrat distal colon, Iscc and TER are underregulatory control by PKC. When animals received anesthesia withintraperitoneal pentobarbital prior to removal of thecolon, the effect of PDBU on Iscc wassignificantly reduced, and the effect of PDBU on TER wasalmost completely inhibited. This effect ofpentobarbital on PKC-mediated transepithelialpermeability parameters is consistent with the knownability of anesthetics to alter protein kinase Cactivity. Exposure of rat colon to pentobarbital produced as muchas a 90% inhibition of calcium-dependent PKC activity,whereas calcium-independent activity was stimulated byas much as 35%. Prior anesthetic use may be therefore a complicating factor in observingPKC-mediated effects on epithelial barrier functionusing epithelial tissue models.

Sodium-independent carrier-mediated inositol transport in cultured renal epithelial (LLC-PK1) cells.

In addition to the concentrative, Na(+)-dependent inositol transport system demonstrated in many cell types, carrier-mediated, Na(+)-independent inositol transport is also shown to exist in LLC-PK1 renal epithelia. Inhibition of inositol uptake in Na(+)-free saline by 0.1 mM phloretin, and self-inhibition by net concentrations of inositol exceeding 10 mM, demonstrate the carrier-mediation of the Na(+)-independent uptake and distinguish it from flux through anion channels. The Na(+)-dependent uptake exhibits higher affinity for inositol, as seen by the stronger self-inhibition at lower inositol concentrations in Na+ saline. Kinetic analyses indicate a Km of 178 microM and a Vmax of 2447 pmol/min per microgram DNA for the Na(+)-dependent system, whereas the lower affinity, lower capacity Na(+)-independent system manifests a Km of 5.2 mM and a Vmax of 249 pmol/min per microgram DNA. the Na(+)-independent uptake further differs from the Na(+)-dependent transport by the lack of inhibitory effect of 10 microM glucose, and the greater relative inhibition of phloretin compared to that of phlorizin. Both types of uptake appear to localize predominantly to the basal-lateral cell surface. The Na(+)-independent transport is bidirectional, functioning in efflux as well as influx of inositol.

Electrophysiological differences in normal colon mucosa from diverticular disease vs cancer

To The Editor: As part of a two-year study of tight-junction permeability of human colon, we have routinely obtained colon tissue from patients who have undergone colectomy for adenocarcinoma or diverticular disease. For studies involving, eg, effects of protein kinase C activators on transepithelial permeability, we had utilized grossly normal tissue that is at least 10 cm distant from the lesion, be it a tumor or a diverticulum. Such normal-appearing tissue from the diverticular disease and cancer patients was to be our “control” group for comparisons to epithelium from inflamed mucosa (inflammatory bowel disease patients) or from tumor itself (1). This practice appears to be followed in many laboratories. In the course of these studies, we began, however, to observe a distinct trend arguing that grossly normal colon epithelium from cancer vs diverticular disease colectomies is not equivalent. The result has been that we no longer use colectomy specimens from diverticular disease patients in certain studies. After mounting any colon tissue in Ussing chambers, there is a period during which transepithelial electrical parameters come to steady state, generally 20–45 min at 37°C in a bicarbonate-buffered saline. During this time, we have observed that transepithelial electrical resistance (Rt) will rise from 20 to as high as 120 ohms 3 cm, and short circuit current (Isc) will decrease from 250 mA/cm to 50 mA/cm or lower. Whereas differences in Rt between grossly normal tissue from patients with diverticular disease vs cancer were not observed, a sharp difference in the stability of Isc was seen as shown in Figure 1. Isc values from histologically normal colon tissue of diverticular disease patients often exhibit a very steep exponential drop over the initial 60 min, frequently falling below a level (,3 mA/cm) at which accurate readings (both Isc and potential difference) can be obtained. The Isc values also initially fell steeply using grossly normal tissue from cancer patients, but generally leveled off at values above 50 mA/cm. Figure 2 shows a scatter plot of Isc values from tissue obtained from left colectomy of these two patient groups, approximately 30