Biomedical subjects

H Oberleithner

Publications and source records attributed to H Oberleithner.

At least 19 recordsLinked to original sources

PubMed2006

Bradykinin shifts endothelial fluid passage from para- to transcellular routes.

The signalling peptide bradykinin (BK) is implicated in inflammation and angiogenesis. It promotes fluid transport from blood vessels to interstitial space, and thus facilitates oedema formation. To clarify whether paracellular or transcellular pathways mediate this effect, we investigated the BK-stimulated fluid transport across endothelial monolayers in vitro by comparison of electrical and fluorescence methods. Electrical cell impedance sensing monitored a biphasic response of cell layers to BK with high time resolution: a short decrease (18%, 1 min) was followed by a more sustained increase in paracellular resistance (30%, 10 min). The two phases can be assigned to second messengers of the BK-signalling pathway: Ca(2+) for the decrease and cyclic adenosine monophosphate for the rise of resistance, respectively. Despite tightening of the intercellular clefts, BK increased the fluid permeability by 39%, indicating transcellular fluid transport. Additionally, BK stimulated both in- and outwardly directed membrane trafficking as assessed by vesicular fluid uptake (by 49%) and secretion of von Willebrandt factor (by 85%). In conclusion, the combination of electrical and fluorescence data suggests that BK induces a shift from para- to transcellular fluid transport across endothelium.

Body Fluid Compartments↗

PubMed2006

17beta-estradiol increases volume, apical surface and elasticity of human endothelium mediated by Na+/H+ exchange.

OBJECTIVE: 17beta-estradiol is known to delay the onset of atherosclerosis in women but cellular mechanisms are still unclear. Estrogens bind to specific receptors and initiate a signaling cascade that involves the activation of plasma membrane Na(+)/H(+) exchange. We hypothesized that estrogens interfere with ion transport across the plasma membrane and thus control endothelial structure and function. Therefore, we investigated the effects of the sex steroids 17beta-estradiol, progesterone, and testosterone on volume, apical surface and elasticity in human endothelium. METHODS: The atomic force microscope was used as an imaging tool and as an elasticity sensor. We applied the antiestrogen tamoxifen, the Na(+)/H(+) exchange blocker cariporide and the epithelial Na(+)channel blocker amiloride to elucidate the role of transmembrane ion transport in hormone-treated human umbilical vein endothelial cells (HUVEC). RESULTS: Incubation with 17beta-estradiol for 72 h led to a dose-dependent increase of endothelial cell volume (41%), apical cell surface (22%), and cell elasticity (53%) as compared to non-17beta-estradiol treated controls. Block of the 17beta-estradiol receptor by tamoxifen and of plasma membrane Na(+)/H(+) exchange by cariporide prevented the hormone-induced changes. Progesterone and testosterone were ineffective. CONCLUSIONS: 17beta-estradiol increases HUVEC water content and HUVEC elasticity mediated by activated estrogen receptors. The estrogen response depends on the activation of plasma membrane Na(+)/H(+) exchange. The increase in endothelial cell elasticity could be one of the vasoprotective mechanisms postulated for 17beta-estradiol.

Amiloride↗

PubMed2004

Transient permeability leak of nuclear envelope induced by aldosterone.

The mineralocorticoid hormone aldosterone controls fluid and electrolyte transport in target cells of the kidney and the cardiovascular system. Classic genomic aldosterone action involves the activation of cytosolic mineralocorticoid receptors and translocation into the cell nucleus where specific transcription processes are initiated. A key barrier of the intracellular signalling pathway is the nuclear envelope, which physically separates the nucleoplasm from the cytoplasm. It was shown recently that aldosterone changes ion conductivity of the nuclear envelope mediated by nuclear pore complexes. The latter are supramolecular nanomachines responsible for import and export of inorganic ions and macromolecules. The aim of the present study was to test whether aldosterone changes the macromolecule permeability of the nuclear envelope. Aldosterone-responsive Xenopus laevis oocytes were used as a model system. We isolated the cell nuclei at defined times after hormone injection. By means of confocal fluorescence microscopy and fluorescence-labelled dextrans we evaluated passive macromolecule import and export in isolated nuclei. 10 minutes after aldosterone injection nuclear envelope permeability of 10 kD dextran was found sharply increased. At the same time cell nuclei were found swollen by about 28%. Changes in nuclear volume and nuclear envelope permeability lasted 5 to 15 minutes and could be inhibited by the mineralocorticoid receptor blocker spironolactone. We conclude that aldosterone transiently changes the barrier function of the nuclear envelope. This short-lasting permeability change signals the start of a sustained transcription process that follows in response to steroids.

Active Transport, Cell Nucleus↗

PubMed2004

Nuclear envelope: nanoarray responsive to aldosterone.

Signalling between cytosol and nucleus is mediated by nuclear pores. These supramolecular complexes represent intelligent nanomachines regulated by a wide spectrum of factors. Among them, steroid hormones specifically interact with the pores and thus modify ion conductivity and macromolecule permeability of the nuclear envelope. In response to aldosterone the pores undergo dramatic changes in conformation, changes that depend on the nature of the transported cargo. Such changes can be imaged at the nanometer scale by using atomic force microscopy. Furthermore, steroid-induced macromolecule transport across the nuclear envelope causes osmotic water movements and nuclear swelling. Drugs that interact with intracellular steroid receptors (spironolactone) or with plasma membrane sodium channels (amiloride) inhibit swelling. Steroid hormone action is blocked when nuclear volume changes are prevented. This is shown in frog oocytes and human endothelial cells. In conclusion, nuclear pores serve as steroid-sensitive gates that determine nuclear activity.

Aldosterone↗

PubMed2003

Passive transport of macromolecules through Xenopus laevis nuclear envelope.

Although nuclear pore complexes (NPC) are considered to be key structures in gene expression, little is known about their regulatory control. In order to explore the regulatory mechanism of passive transport of small macromolecules we examined the influence of different factors on the diffusional pathway of NPCs in isolated Xenopus laevis oocyte nuclei. Diffusion of fluorescence-labeled 10-kD dextran was measured across the nuclear envelope with confocal fluorescence microscopy. Surprisingly, the filling state of the perinuclear Ca(2+) store had no influence on passive transport of 10-kD dextran. Furthermore, nuclear envelope permeability was independent of cytoplasmic pH (pH range 8.3-6.3). In contrast, nuclear swelling, induced by omission of the endogenous cytosolic macromolecules, clearly increased nuclear permeability. An antibody against the glycoprotein gp62, located at the central channel entrance, reduced macromolecule diffusion. In addition, nuclei from transcriptionally active, early developmental stages (stage II) were less permeable compared to transcriptionally inactive, late-developmental-stage (stage VI) nuclei. In stage II nuclei, atomic force microscopy disclosed NPC central channels with plugs that most likely were ribonucleoproteins exiting the nucleus. In conclusion, the difference between macromolecule permeability and previous measurements of electrical resistance strongly indicates separate routes for macromolecules and ions across the nuclear envelope.

Adenosine Triphosphate↗

PubMed2003

Intracellular calcium: a prerequisite for aldosterone action.

Transport of salt and water in various tissues is under control of the mineralocorticoid hormone aldosterone. As a liphophilic hormone, aldosterone diffuses through the plasma membrane and, then, binds to cytosolic mineralocorticoid receptors in the target cells. After binding to nuclear pore complexes, the activated receptor is translocated to the nucleus where transcription processes are initiated. After a lag period of about 20 minutes hormone-specific early mRNA transcripts leave the nucleus through nuclear pores. Some of the steps in this cascade can be followed by electrophysiology in Xenopus laevis oocyte nuclei. In addition to the genomic pathway, aldosterone exerts a rapid pre-genomic response that involves an increase in intracellular calcium. In this study, we tested for the potential role of Ca(2+) in the genomic response of the hormone. We measured the electrical resistance across the nuclear envelope in response to aldosterone, in presence and absence of intracellular Ca(2+). Nuclear envelope electrical resistance reflects receptor binding to the nuclear pore complexes ("early" resistance peak, 2 minutes after aldosterone), ongoing transcription ("transient" resistance drop, 5-15 minutes after aldosterone) and mRNA export ("late" resistance peak, 20 minutes after aldosterone). Pre-injection of the Ca(2+) chelator EGTA eliminated all electrical responses evoked by aldosterone. The transient resistance drop and the late resistance peak, induced by the hormone, were prevented by the transcription inhibitor actinomycin D, coinjected with aldosterone, while the early resistance peak remained unaffected. We conclude that (i). the presence of intracellular Ca(2+) is a prerequisite for the genomic action of aldosterone. (ii). Intracellular calcium plays a role early in the signaling cascade, either in agonist-receptor interaction, or receptor transport/docking to the nuclear pore complexes.

Aldosterone↗

PubMed2003

Endothelial cell swelling by aldosterone.

There is accumulating evidence that mineralocorticoids not only act on kidney but also on the cardiovascular system. We investigated the response of human umbilical venous endothelial cells (HUVECs) to aldosterone at a time scale of 20 minutes in absence and presence of the aldosterone antagonist spironolactone or other transport inhibitors. We applied atomic force microscopy (AFM), which measures cell volume and volume shifts between cytosol and cell nucleus. We observed an immediate cell volume increase (about 10%) approximately 1 min after addition of aldosterone (0.1 micromol/l), approaching a maximum (about 18%) 10 min after aldosterone treatment. Cell volume returned to normal 20 min after hormone exposure. Spironolactone (1 micromol/l) or amiloride (1 micromol/l) prevented the late aldosterone-induced volume changes but not the immediate change observed 1 min after hormone exposure. AFM revealed nuclear swelling 5 min after aldosterone addition, followed by nuclear shrinkage 15 min later. The Na(+)/H(+) exchange blocker cariporide (10 micromol/l) was ineffective. We conclude: (i). Aldosterone induces immediate (1 min) swelling independently of plasma membrane Na(+) channels and intracellular mineralocorticoid receptors followed by late mineralocorticoid receptor- and Na(+)-channel-dependent swelling. (ii). Intracellular macromolecule shifts cause the changes in cell volume. (iii). Both amiloride and spironolactone may be useful for medical applications to prevent aldosterone-induced vasculopathies.

Adaptation, Physiological↗

PubMed2003

Route of steroid-activated macromolecules through nuclear pores imaged with atomic force microscopy.

In eukaryotic cells, two concentric membranes, the nuclear envelope (NE), separate the nucleus from the cytoplasm. The NE is punctured by nuclear pore complexes (NPCs; molecular mass 120 MDa) that serve as regulated pathways for macromolecules entering and leaving the nuclear compartment. Transport across NPCs occurs through central channels. Such import and export of macromolecules through individual NPCs can be elicited in the Xenopus laevis oocyte by injecting the mineralocorticoid aldosterone and can be visualized with atomic force microscopy. The electrical NE resistance in intact cell nuclei can be measured in parallel. Resistance increases when macromolecules are engaged with the NPC. This article describe six observations made from these experiments and the conclusions that can be drawn from them. (i) A homogeneous population of macromolecules (approx. 100 kDa) attaches to the cytoplasmic face of the NPC 2 min after aldosterone injection. They are most likely to be aldosterone receptors. After a few minutes, they have disappeared. (ii) Large plugs (approx. molecular mass 1 MDa) appear in the central channels 20 min after hormone injection. They are most likely to be ribonucleoproteins exiting the nucleus. (iii) Electrical resistance measurements in isolated nuclei reveal transient electrical NE resistance peaks: an early (2 min) peak and a late (20 min) peak. Electrical peaks reflect macromolecule interaction with the NPC. (iv) Spironolactone blocks both the early and late peaks. This indicates that classic aldosterone receptors are involved in the pregenomic (early) and post-genomic (late) responses. (v) Actinomycin D and, independently, RNase A block the late electrical peak, confirming that plugs are genomic in nature. (vi) Intracellular calcium chelation blocks both early and late electrical peaks. Thus, the release of calcium from internal stores, which is known to be the first intracellular signal in response to aldosterone, is a prerequisite for the late genomic response.

Aldosterone↗

PubMed2002

Secretion pores in human endothelial cells during acute hypoxia.

Weibel-Palade bodies (WPB) are endothelial vesicles that store von Willebrand factor (vWF), involved in the early phase of hemostasis. In the present study we investigated the morphodynamics of single WPB plasma membrane fusion events upon hypoxic stimulation by using atomic force microscopy (AFM). Simultaneously, we measured vWF release from endothelial cells to functionally confirm WPB exocytosis. Exposing human endothelial cells to hypoxia (pO2 = 5 mm Hg) we found an acute (within minutes) release of vWF. Despite acute vWF release, potential cellular modulators of secretion, such as intracellular pH and cell volume, remained unchanged. We only detected a slight instantaneous increase of cytosolic Ca2+ concentration. Although overall cell morphology remained virtually unchanged, high resolution AFM images of hypoxic endothelial cells disclosed secretion pores, most likely the loci of WPB exocytosis on luminal plasma membrane. We conclude that short-term hypoxia barely alters overall cell morphology and intracellular milieu. However, at nanometer scale, hypoxia instantaneously switches the smooth luminal plasma membrane to a rough activated cell surface, covered with secretion pores that release vWF to the luminal cell surface.

Calcium↗

PubMed2002

Aldosterone signaling pathway across the nuclear envelope.

We describe the route by which aldosterone-triggered macromolecules enter and exit the cell nucleus of Xenopus laevis oocyte. Oocytes were microinjected with 50 fmol aldosterone and then enucleated 2-30 min after injection. After isolation, nuclear envelope electrical resistance (NEER) was measured in the intact cell nuclei by using the nuclear hourglass technique. We observed three NEER stages: an early peak 2 min after injection, a sustained depression after 5-15 min, and a final late peak 20 min after injection. Because NEER reflects the passive electrical permeability of nuclear pores, we investigated with atomic force microscopy aldosterone-induced conformational changes of individual nuclear pore complexes (NPCs). At the early peak we observed small ( congruent with 100 kDa) molecules (flags) attached to the NPC surface. At the sustained depression NPCs were found free of flags. At the late peak large ( congruent with 800 kDa) molecules (plugs) were detected inside the central channels. Ribonuclease or actinomycin D treatment prevented the late NEER peak. Coinjection of aldosterone (50 fmol) and its competitive inhibitor spironolactone (500 fmol) eliminated the electrical changes as well as flag and plug formation. We conclude: (i) The genomic response of aldosterone can be electrically measured in intact oocyte nuclei. (ii) Flags represent aldosterone receptors on their way into the cell nucleus whereas plugs represent ribonucleoproteins carrying aldosterone-induced mRNA from the nucleoplasm into the cytoplasm. (iii) Because plugs can be mechanically harvested with the atomic force microscopy stylus, oocytes could serve as a bioassay system for identifying aldosterone-induced early genes.

Aldosterone↗

PubMed2002

Stimulation of protein kinase C pathway mediates endocytosis of human nongastric H+-K+-ATPase, ATP1AL1.

The human nongastric H+-K+-ATPase, ATP1AL1, shown to reabsorb K+ in exchange for H+ or Na+, is localized in the luminal plasma membrane of renal epithelial cells. It is presumed that renal H+-K+-ATPases can be regulated by endocytosis. However, little is known about the molecular mechanisms that control plasma membrane expression of renal H+-K+-ATPases. In our study, activation of protein kinase C (PKC) using phorbol esters (phorbol 12-myristate 13-acetate) leads to clathrin-dependent internalization and intracellular accumulation of the ion pump in stably transfected Madin-Darby canine kidney cells. Functional inactivation of the H+-K+-ATPase by PKC activation is shown by intracellular pH measurements. Proton extrusion capacity of ATP1AL1-transfected cells is drastically reduced after phorbol 12-myristate 13-acetate incubation and can be prevented with the PKC blocker bisindolylmaleimide. Ion pump internalization and inactivation are specifically mediated by the PKC pathway, whereas activation of the protein kinase A pathway has no influence. Our results show that the nongastric H+-K+-ATPase is a specific target for the PKC pathway. Therefore, PKC-mediated phosphorylation is a potential regulatory mechanism for apical nongastric H+-K+-ATPase plasma membrane expression.

Animals↗

PubMed2001

Electrophoretic plugging of nuclear pores by using the nuclear hourglass technique.

The nuclear hourglass technique (NHT) was recently introduced as a novel technique that measures the electrical nuclear envelope (NE) conductance of isolated Xenopus laevis oocyte nuclei. The main conclusion drawn from NHT work so far is that nuclear pore complexes (NPCs) of oocytes are in an electrically open state under physiological conditions, with a mean conductance of 1.7 nS per NPC. Since nuclear patch-clamp data indicate that usually NPCs are electrically closed, our work has been challenged by the notion that NHT cannot assure a high resistance seal ("gigaseal") between glass wall and NE like that required for patch-clamp experiments. Thus, NHT could have dramatically underestimated NE electrical resistance. Here we demonstrate that NHT does not require a gigaseal for accurate NE conductance measurements. In addition, we present experimental conditions where mean single NPC electrical conductance is reduced 26-fold due to electrophoretic plugging by negatively charged nucleoplasmic macromolecules. In addition, data indicate that under physiological conditions (i.e., when macromolecules are offered in the cytosolic solution) the nuclear surface is heavily folded, underestimating "true" NE surface by a factor of 2.6. When "true" NE surface area is taken into consideration, modified values of mean single NPC conductances of 654 pS for electrically open conditions and 25 pS for electrically plugged conditions can be calculated. We conclude that the large overall NE conductance detected with the nuclear hourglass technique in intact Xenopus laevis oocyte nuclei can be explained by the sum of single NPC conductances in the pS range, as long as open probability is high. This confirms previous patch-clamp work concerning single NPC conductance, but disagrees with the view that mean open probability of NPC channels is usually low.

Active Transport, Cell Nucleus↗

PubMed2001

Plasma membrane protein clusters appear in CFTR-expressing Xenopus laevis oocytes after cAMP stimulation.

Membrane trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is supposed to be an important mechanism controlled by the intracellular messenger cAMP. This has been shown with fluorescence techniques, electron microscopy and membrane capacitance measurements. In order to visualize protein insertion we applied atomic force microscopy (AFM) to inside-out oriented plasma membrane patches of CFTR-expressing Xenopus laevis oocytes before and after cAMP-stimulation. In a first step, oocytes injected with CFTR-cRNA were voltage-clamped, verifying successful CFTR expression. Water-injected oocytes served as controls. Then, plasma membrane patches were excised, placed (inside out) on glass and scanned by AFM. Before cAMP-stimulation plasma membranes of both water-injected and CFTR-expressing oocytes contained about 200 proteins per micron 2. Molecular protein masses were estimated from molecular volumes measured by AFM. Before cAMP-stimulation, protein distribution showed a peak value of 11 nm protein height corresponding to 475 kDa. During cAMP-stimulation with 1 mM isobutylmethylxanthine (IBMX) plasma membrane protein density increased in water-injected oocytes to 700 proteins per micron 2 while the peak value shifted to 7 nm protein height corresponding to 95 kDa. In contrast, CFTR-expressing oocytes showed after cAMP-stimulation about 400 proteins per micron 2 while protein distribution exhibited two peak values, one peak at 10 nm protein height corresponding to 275 kDa and another one at 14 nm corresponding to 750 kDa. They could represent heteromeric protein clusters associated with CFTR. In conclusion, we visualized plasma membrane protein insertion upon cAMP-stimulation and quantified protein distribution with AFM at molecular level. We propose that CFTR causes clustering of plasma membrane proteins.

Animals↗

PubMed2001

Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli.

Many mucosal pathogens use type III secretion systems for the injection of effector proteins into target cells. The type III-secreted proteins EspB and EspD of enteropathogenic Escherichia coli (EPEC) are inserted into the target cell membrane. Together with EspA, these proteins are supposed to constitute a molecular syringe, channelling other effector proteins into the host cell. In this model, EspB and EspD would represent the tip of the needle forming a pore into target cell membranes. Although contact-dependent and Esp-mediated haemolytic activity by EPEC has already been described, the formation of a putative pore resulting in haemolysis has not been demonstrated so far. Here, we show that (i) diffusely adhering (DA)-EPEC strains exhibit a type III-dependent haemolytic activity too; (ii) this activity resides in the secreted proteins and, for DA-EPEC strains, in contrast to EPEC strains, does not require bacterial contact; and (iii) pores are introduced into the target cell membrane. Osmoprotection revealed a minimal pore size of 3-5 nm. The pores induced by type III-secreted proteins of DA-EPEC were characterized by electron microscopy techniques. Analysis by atomic force microscopy demonstrated the pores to be composed of six to eight subunits with a lateral extension of 55-65 nm and to be raised 15-20 nm above the membrane plane. We could also demonstrate an association of EspB and EspD with erythrocyte membranes and an interaction of both proteins with each other in vitro. These results, together with the homologies of EspB and EspD to proposed functional domains of other pore-forming proteins (Yop/Ipa), strongly support the idea that both proteins are directly involved in pore formation, which might represent the type III secretion system translocon.

Animals↗

PubMed2001

Evidence for Ca2+- and ATP-sensitive peripheral channels in nuclear pore complexes.

In eukaryotic cells the nuclear envelope (NE) serves as a functional barrier between cytosol and nucleoplasm perforated by nuclear pore complexes (NPCs). Both active and passive transport of ions and macromolecules are thought to be mediated by the centrally located large NPC channel. However, 3-dimensional imaging of NPCs based on electron microscopy indicates the existence of additional small channels of unknown function located in the NPC periphery. By means of the recently developed nuclear hourglass technique that measures NE electrical conductance, we evaluated passive electrically driven transport through NPCs. In isolated Xenopus laevis oocyte nuclei, we varied ambient Ca2+ and ATP in the cytosolic solution and/or chelated Ca2+ in the perinuclear stores in order to assess the role of Ca2+ in regulating passive ion transport. We noticed that NE electrical conductance is large under conditions where macromolecule permeability is known to be low. In addition, atomic force microscopy applied to native NPCs detects multiple small pores in the NPC periphery consistent with channel openings. Peripheral pores were detectable only in the presence of ATP. We conclude that NPC transport of ions and macromolecules occurs through different routes. We present a model in which NE ion flux does not occur through the central NPC channel but rather through Ca2+- and ATP-activated peripheral channels of individual NPCs.

Active Transport, Cell Nucleus↗

PubMed2001

Electrical dimension of the nuclear envelope.

Eukaryotic chromosomes are confined to the nucleus, which is separated from the rest of the cell by two concentric membranes known as the nuclear envelope (NE). The NE is punctuated by holes known as nuclear pore complexes (NPCs), which provide the main pathway for transport of cellular material across the nuclear-cytoplasmic boundary. The single NPC is a complicated octameric structure containing more than 100 proteins called nucleoporins. NPCs function as transport machineries for inorganic ions and macromolecules. The most prominent feature of an individual NPC is a large central channel, ~7 nm in width and 50 nm in length. NPCs exhibit high morphological and functional plasticity, adjusting shape to function. Macromolecules ranging from 1 to >100 kDa travel through the central channel into (and out of) the nucleoplasm. Inorganic ions have additional pathways for communication between cytosol and nucleus. NE can turn from a simple sieve that separates two compartments by a given pore size to a smart barrier that adjusts its permeabiltiy to the metabolic demands of the cell. Early microelectrode work characterizes the NE as a membrane barrier of highly variable permeability, indicating that NPCs are under regulatory control. Electrical voltage across the NE is explained as the result of electrical charge separation due to selective barrier permeability and unequal distribution of charged macromolecules across the NE. Patch-clamp work discovers NE ion channel activity associated with NPC function. From comparison of early microelectrode work with patch-clamp data and late results obtained by the nuclear hourglass technique, it is concluded that NPCs are well-controlled supramolecular structures that mediate transport of macromolecules and small ions by separate physical pathways, the large central channel and the small peripheral channels, respectively. Electrical properties of the two pathways are still unclear but could have great impact on the understanding of signal transfer across NE and gene expression.

Aldosterone↗

PubMed2000

Nuclear pore function viewed with atomic force microscopy.

In this review we focus on studies using atomic force microscopy (AFM) to describe the function of nuclear pore complexes (NPC). After a short introduction of AFM we follow the route of cargo molecules from the cytosol into the nucleus. AFM visualizes cargo before translocation into the nucleoplasm, cargo docking at the cytoplasmic NPC surface, cargo passing through the NPC and changes in NPC conformation in response to ATP, Calcium and pH. We discuss AFM experiments on nuclear envelopes on the basis of previous data obtained with more conventional techniques such as electron microscopy, confocal microscopy and other imaging techniques. Finally we draw attention to the recently developed nuclear hourglass technique that serves as a new electrophysiological approach to studying the structure-function relationship of NPC in combination with AFM at a molecular level.

Adenosine Triphosphate↗

PubMed2000

High-resistance MDCK-C7 monolayers used for measuring invasive potency of tumour cells.

We describe an electrophysiological method for evaluating the intrinsic invasive potency of tumour cells using renal cells as an in vitro assay system. A high-resistance clone of Madin-Darby canine kidney cells (MDCK-C7) was grown to confluency in a filter cup. Transepithelial electrical resistance across the MDCK-C7 monolayer was measured in a commercially available electrode chamber. After a transepithelial electrical resistance of about 4,000 omega cm2 had been reached, human melanoma or pancreatic carcinoma cells were co-cultivated with the MDCK-C7 monolayer. Both carcinoma cell lines induced resistance breakdown measured after 24 h or later depending on seeding density and cell type. Seeding carcinoma cells on the basolateral surface of MDCK-C7 cells caused a similar decrease in transepithelial resistance of the MDCK-C7 monolayer. Resistance breakdown indicates opening of tight junctions prior to tumour cell invasion. In conclusion, the high-resistance MDCK-C7 cell clone could serve as a valuable biological assay system to determine electrically the metastatic potency of tumour cells in vitro.

Animals↗