Institute of Clinical Physiology

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  ! Main idea of our research ! 

 

Tight junctions, channels and carriers  Research topics
- Tight junction proteins: claudin(s), occludin, and tricellulin
- Barrier defect in inflammatory bowel diseases (IBD)
- Cytokines like TNFa affect barrier function

- Epithelial apoptosis causes leaks
- Restitution of epithelial single cell defects
- Direct measurement of paracellular conductance
- Localization of ion channels in crypts and surface epithelium

- Epithelial Na+-channel (ENaC)

- Magnesium

- Physiology of the eye
- Glaucoma
- Retinal degeneration
cartoon Methods
- Short-circuit current (Ussing)
- Conductance scanning
- Impedance spectroscopy
- Patch-clamp
- Molecular biology
- Fluorescence microscopy
- Freeze fracture electron microscopy
cartoon Keywords
- HT-29/B6 confluent colon cell line 
- Inflammatory bowel diseases (IBD)
- Ulcerative colitis and Crohn's disease
- Leak flux diarrhea
- Apoptosis in a colonic epithelium
- Tutorial (A.H. Gitter) Electrical impedance of epithelia
Completed  projects (German text)
 

  Main idea of our research 
Physiologists always ask How does it work?
  • Physiology is no certain method, but a kind of question: We use a wide spectrum om different methods - alway aiming to answer the question "How does it work?" with respect to the investigated molecules, cells, organs or the whole body.
  • This question cannot be fully answered using classic, e.g. electrophysiological, methods alone without knowing about the molecular structures. On the other hand, molecular biology and biochemistry results usually do not offer conclusions regarding the function of the whole cell or the intact body. In our current projects we therefore combine functional (= mostly electrophysiological) and structural (biochemical, molecular biological, laser-scanning microscopical, and electron microscopical) techniques.
  • In order to understand the pathophysiology of a disease it is necessary to know the mechanisms of normal function. For most topics we study the basic mechanisms and - using the same methods - the impaired function in diseased states. Experiments are performed on isolated epithelia and cell cultures. Within this spectrum of approaches we collaborate for many years with the group of Prof. Jörg-Dieter Schulzke (Dept. of General Medicine and Dept. of Gastroenterology).

Our main research topic is the tight junction:

The intestinal barrier critically depends on the function of the tight junctions. By use of molecular biology and fluorescence microscopy techniques we study the role of the tight junction proteins of the claudin family, occludin, and tricellulin.

In inflammatory bowel diseases (ulcerative colitis and Crohn's disease) cytokines like tumor necrosis factor alpha (TNFa) induce a dramatic increase tight junction permeability. This barrier break-down is a major determinant of the disease, because it causes leak flux diarrhea and allows for unwanted uptake from the lumen. 

For measuring the barrier function we have developed two electrophysiological techniques,
... and the transporters of the cell membrane:

Tight junctions, Kanäle, Carrier
Research topics 

Tight junction proteins: claudin-1 to -24, occludin, and tricellulin.
Barrier function and genomic regulation

Epithelial tight junctions consist of the transmembranal proteins occludin, tricellulin, and 24 different claudins. Common properties are 4 transmembranal domains und 2 extrazellular loops (ECL). ECL1 is thought to determine the paracellular barrier and/or channel function. ECL2 may act as mechanical contact between opposing tight junction proteins. Also a TJ protein is JAM (junctional adhesion molecule), which forms one transmembranal domain.

   2009: 2012: parts I and II

General reviews on TJ proteins:

Coming soon:

 

There are two families of tight junction (TJ) proteins mit four transmembrane domains (tetraspan proteins):

Another protein of the TJ is JAM (Junctional Adhesion Molecule). It has one transmembrane domain. It provides adhesion between neighboring lateral cell membranes and has no barrier function.
Intracellularly located (and thus no real TJ proteins) are several scaffold proteins like ZO-1 and ZO-2 (Zonula Occludens-1 and -2). They connect many of the claudins and the TAMPs with the actin cytoskeleton.

The claudin family
General: Presently, 27 different claudins are known in mammalia. Some claudins are essential for forming the epithelial barrier while others even do the oppsosite, they form paracellular channels.

Claudin-1
Localization: Typical for epithelia with almost impermable tight junctions ("tight epithelia") like distal colon and distal kidney tubule
Function: Paracellular barrier against transepithelial diffusion
Clinical Impact: Claudin-1 is reduced in hereditary mammary cancer. A defect of claudin-1 is present in neonatal sclerotic cholangitis with ichthyosis. Expression of claudin-1 and of claudin-4 was increased in colorectal cancer and pancreatic and ovarian cancers. In human epidermis claudin-1 is the essential paracellular barrier former

Claudin-2
Localization: Typical for epithelia with rather permeable tight junctions ("leaky epithelia") like small intestine and proximal kidney tubule
Functions: (i) Claudin-2 forms paracellular channels for small cations (discovered by Amasheh et al. 2002)
                (ii) Claudin-2 also forms paracellular channels for water (discovered by Rosenthal et al. 2010)
Clinical Impact: Increased expression causes impaired barrier function 

Claudin-3
Localization: Typical for tight epithelia
Function: We have characterized claudin-3 to be a general barrier former as it reduces permeability for ions without charge preference and uncharged solutes.
Clinical Impact: Claudin-3 and -4 are receptors for the enterotoxin of Clostridium perfringens.

Claudin-4
Localization: Typical for tight epithelia
Function: Paracellular barrier
Clinical Impact: In the Williams-Beuren-Syndrome, claudin-4 is not expressed .

Claudin-5
Localization: Typical for endothelia
Function: We were able to show that claudin-5 belongs to the barrier-forming claudins and that it is expressed also in some epithelia.
Clinical Impact: Claudin-5 is deleted in patients suffering from velo-cardio-facial syndrome (DiGeorge syndrome). Claudin-5-deficient mice exhibit a barrier defect of the blood-brain barrier.

Claudin-6
Localization: Embryonic epithelia, adipose tissue
Function: Overexpression of claudin 6 results in decreased expression of other claudins and a severe barrier defect

Claudin-7
Localization: Kidney: Distal nephron
Function: Overexpression of claudin-7 decreases the paracellular Cl- conductance and increases the paracellular Na+ conductance in LLC-PK1 cells

Claudin-8
Localization: Kidney: Aldosterone-sensitive distal nephron (ASDN)
Function: Barrier for cations in tight epithelia.

Claudin-10
Localization: Claudin-10 exists in six splice variants. Two variants are of major importance, 10a, localized in kidney TJs, and variant 10b, localized in many epithelia including kidney
Function: Claudin-10a is anion selective. Claudin-10b forms a cation selective channel, which is, in contrast to claudin-2, not permeable to water.

Claudin-11 (= OSP)
Localization: CNS: oligodendrocytes, Testis: Sertoli cells, Ear: organ of Corti, Kidney: prox. Tubule, Henle's loop
Function: Barrier

Claudin-14
Localization: Ear: cochlea hair cells; Kidney: collecting duct
Function: Barrier in cochlea hair cells

Claudin-15
Localization: Intestine
Function: Cation channel

Claudin-16  (= paracellin 1)
Localization: Kidney: thick ascending limb of Henle's loop and distal tubule
Function: Claudin-16 facilitates magnesium and calcium transport
Clinical Impact: Mutations of claudin-16 (and claudin-19) are associated with familial hypomagnesemia, together with hypercalciuria and nephrocalcinosis (FHHNC)

Claudin-17
Lokalization: Kidney: abundant in proximal tubules and gradually decreasing towards distal segments. Marginal in brain.
Function: Claudin-17 forms paracellular anion channels (discovered by Krug et al. 2012, Cell. Mol. Life Sci.). As claudin-17 is predominantly expressed in proximal nephrons, which exhibit substantial though molecularly not defined paracellular chloride reabsorption, we suggest that claudin-17 has a unique physiological function in that process. Molecular: Claudin-17 anion selectivity critically depends on a positive charge at position 65.

Claudin-18
Lokalization: Lung, Stomach, Oesophagus, Ear: Cochlea, Corti organ, Stria vascularis marginal cells

Claudin-19
Lokalization: Kidney: Distal Nephron, Nerve: Schwann cells
Function: Barrier

Claudin-20
Lokalization: Eye: Retina pigment epithelium

Claudin-21

Claudin-22

Claudin-23
Lokalization: Placenta, Stomach, colon tumors

Claudin-24

Claudin-25

Claudin-26

Claudin-27

The TAMP family
General: TAMP stands for Tight junction-Associated Marvel Proteins. The TAMP family includes occludin, tricellulin, and marvelD3, which share a transmembrane domain motif called MARVEL (Myelin and lymphocyte And Related protein for VEsicle trafficking and membrane Link).

Occludin
Localization: All epithelia
Function: The function of occludin is still poorly understood. In a collaboration, the lab of Shoichiro Tsukita and our group have shown that in occludin-KO mice the tight juncion barrier is unaltered. This means that occludin either has no intrinsic barrier properties or can be replaced by other components of the tight junction.

In occludin-knockout mice the glandular structure of the stomach exhibited a complete loss of parietal cells and mucus cell hyperplasia, as a result of which acid secretion was virtually abolished. A dramatic change in gastric morphology and secretory function indicates that occludin is involved in gastric epithelial differentiation.

Little is known about the regulatory mechanisms of occludin that influence occludin gene expression. We aimed to identify the sequences essential in cis for genomic regulation of tight junction formation and to investigate their functional role in cytokine-dependent tight junction regulation.
Using genome walking cloning of occludin-specific human genomic DNA sequences, a 1853 bp DNA fragment containing the transcription start point of occludin cDNA sequences was amplified and sequenced. The proinflammatory cytokines, TNFa and interferon g diminished occludin promoter activity alone and even synergistically, suggesting a genomic regulation of alterations of the paracellular barrier. Both cytokines downregulate the expression of occludin, paralleling the barrier disturbance detected electrophysiologically. This could be an important mechanism in gastrointestinal diseases accompanied by barrier defects, for example inflammatory bowel diseases.

Tricellulin
Localization: Tricellular tight junction (tTJ), i.e. the site where three epithelial or endothelial cells meet.
Function: Tricellulin was discovered by Shoichiro Tsukita who has died in Dec. 2005, a few days before landmark paper appeared: Ikenouchi et al., 2005, J. Cell Biol. 171(6): 939-945. [PubMed] [PDF]. In cell cultures, lack of tricellulin prevents the development of the epithelial barrier. We showed that tricellulin tightens the tricellular junction against macromolecules. We propose that, at impaired tricellulin expression, the tTJ becomes a major site for the passage of macromolecules.

 

Supported by:
  • German Research Society (DFG) Research Unit (Forschergruppe) (FOR 721) "Molecular structure and function of the tight junction" (Coordinator: Fromm)
    • TP1 "Characterization of tight junction proteins as barrier or channel formers" (Fromm, S.Amasheh)
    • TP7 "Renal claudins and their interactions" (Günzel)
    • TP8 "Tight junction proteins as permeability regulators of the perineurium and of the antinociception perineurallly applied drugs" (Rittner, Brack, Fromm)
    • TPZ "Freeze fracture electron microscopy" and two-path impedance spectroscopy (Fromm, Schulzke, Krug)
    • Doctoral students of the Research Unit 721
  • DFG Collaborative Research Center (Sonderforschungsbereich) (SFB 852), "Nutrition and intestinal microbiota - host interaction in the pig" (Coordinator: Zentek)
    • Project B2 "Paracellular intestinal barrier - effects of probiotics and feed additives" (Fromm, Schulzke)
  • DFG Research Grant (AU 132/7-1 and AM 141/4-1) "Meaning of CD97 in experimental colitis and ulcerative colitis: functional characterization of the molecule in intestinal epithelial cells" (Aust, S.Amasheh, Fromm)

 
The Tight Junctions
Localization: Charité events & clubs in Berlin
Function: Live featuring classic rock
Clinical Impact: Animation of acoustically irradiated hominids

Barrier defect in inflammatory bowel diseases (IBD)

Many diseases of the intestines are caused by impaired epithelial absorption or secretion and by impaired epithelial barrier function.

The pathogenesis of the ulcerative colitis and Crohn's disease is still unknown. A typical symptom in both inflammatory bowel diseases is chronic diarrhea. We investigate the transport and barrier function of the intestine in vitro using two electrophysiological techniques, impedance spectroscopy and conductance scanning.

If the ion permeability is critically increased under pathological conditions a leak flux diarrhea occurs. This type of diarrhea is caused by massive fluxes of solutes and water from the blood into the gut lumen.

Regarding immunological mechanisms, an intact epithelial barrier keeps luminal bacteria, toxins, and antigens away from the subepithelial tissues. It is discussed, whether an impaired intestinal barrier allows for increased uptake of bacteria, toxins, and antigens which then will support the inflammation process.

Bacterial translocation through the intestinal wall has been studied under defined in vitro conditions in our lab.

 

Cytokines like TNFa affect barrier function

Cytokines like tumor necrosis factor alpha (TNFa), interleukins and interferons act as mediators of inflammation. In inflammatory bowel diseases (and in HIV infection) their local concentrations increase. We study the action of cytokines on transport and barrier function of human intestine and cell cultures originating from human colon (HT-29/B6).

Epithelial apoptosis causes leaks

Key word: Epithelial apoptosis

Current opinion assumes epithelial integrity during spontaneous apoptotic cell death. We measured, for the first time, the local conductances associated with apoptoses and show leaks of 50 nS (mean) in human intestinal epithelium. Measurements were performed employing the conductance scanning technique on confluent HT-29/B6 monolayers.

The results disprove the dogma that isolated cell apoptosis occurs without affecting the epithelial cell permeability barrier. After induction of apoptosis by tumor necrosis factor a (TNFa ) the apoptotic leaks were dramatically enhanced: not only increased the frequency 3fold, but the mean conductance increased 12fold to 600 nS. Thus apoptosis accounted for about half of the TNFa -induced permeability increase while the other half is caused by degradation of tight junctions in non-apoptotic areas.

Hence, spontaneous and, more so, induced apoptosis hollow out the intestinal barrier and may facilitate loss of solutes and uptake of noxious agents.

Restitution of epithelial single cell defects

Since the barrier function of an epithelium relies on its complete integrity, which is challenged by the loss of cells, by injury or apoptosis, rapid repair mechanisms are important. Already well known is the repair process of large epithelial wounds (restitution). The gap is closed by the immigration of intact adjacent cells within hours. Deep wounds require also cell proliferation, which takes several days. Little is known, however, about the - much faster - closure of single cell defects. Purely morphological studies cannot assess the functional leak that opens with the hiatus. Our new conductance scanning technique allows to measure the leak. Thus we investigate, for instance, the effects of inflammatory mediators on the restitution of single cell defects in epithelia of the intestine.

Localization of ion channels in crypts and surface epithelium

The conductance scanning technique in medium resolution allowed to measure in crypt and surface epithelium of the colon the conductivities for Na+, K+, Cl–, which are defined by specific apical channels.
We demonstrated that cAMP-induced Cl– secretion is localized not only - as assumed so far - in the crypts, but to a similar amount also in the surface epithelium:

The aldosterone-simulated electrogenic Na+ absorption is restricted to the surface epithelium:

  • Köckerling A, Sorgenfrei D, Fromm M (1993) Electrogenic Na+ absorption of rat distal colon is confined to surface epithelium. A voltage scanning study. Am. J. Physiol. 264: C1285-C1293. [PubMed] [PDF]

Regarding the localization of the K+ secretion it is crucial how it is stimulated: The cAMP-induced K+ secretion is localized in crypts as well as in surface epithelium, while after stimulation by aldosterone K+ secretion is induced only in the surface epithelium:

 Epithelial sodium channel (ENaC)

The amiloride- blockable epithelial Na+ channel (ENaC) is an important protein for the regulation of the Na+ balance in vertebrates. It is a limiting factor for the absorption of Na+ in several organs. ENaC is regulated in epithelia of the distal kidney tubule and the distal colon by aldosterone and other corticosteroids.

The epithelial sodium channel (ENaC) was cloned a few years ago from intestinal epithelium of the rat [Canessa et al. 1993, Nature; Lingueglia et al. 1993, FEBS Lett.; Canessa et al. 1994, Nature]. The channel-forming protein consists of two a-, one b- and one g-subunit [Kosari et al. 1998, J. Biol. Chem.; Firsov et al. 1998, EMBO J.].

ENaC is regulated by aldosterone on the genomic level. The effect of aldosterone can be divided in 3 phases of: a latent phase, a phase of increasing Na+ transport, and a phase starting after 4 hours of stimulation of the basolateral Na+/K+-ATPase [Benos et al. 1995, J. Membr. Biol.]. The main component is the regulation of the epithelial Na+ channel: a blockade of ENaC by the diuretic amiloride stops the electrogenic Na+ transport (and the effect of aldosterone) completely [Benos 1982, Am. J. Physiol.].

We investigated electrogenic Na+ transport and, in identical tissues, mRNA expression of ENaC subunits in early (EDC) and late (LDC) distal colon of the rat. In both segments 8 h in vitro incubation with 3 nM aldosterone enhanced b- and gENaC mRNA and induced Na+ transport. Na+ transport was ten times higher in LDC than in EDC.

Although it is known, that aldosterone regulates the electrogenic Na+ absorption via a transcriptional mechanism [Verrey & Beron 1996, NIPS], it was unresolved so far, whether aldosterone affects the electrogenic Na+ transport directly by de novo synthesis of channel proteins or indirectly by induction of regulatory proteins. We found that aENaC mRNA was unchanged in EDC, whereas it decreased in LDC. In LDC, b- and gENaC mRNA was induced 1 h after aldosterone addition whereas Na+ transport became apparent >1 h later. Down-regulation of aENaC does not take part in the acute regulation because it started after a lag time.

It was thought so far that the "early effect" of aldosterone is not based on genomic regulation, because ENaC transcription was reported to start later than Na+ transport. However, time correlation of b- and gENaC induction and Na+ transport stimulation suggests that the early aldosterone effect on Na+ absorption in distal colon may be due to transcriptional upregulation of b- and gENaC expression.

  • Epple HJ, Amasheh S, Mankertz J, Goltz M, Schulzke JD, Fromm M (2000) Early aldosterone effect in distal colon by transcriptional regulation of ENaC subunits. Am. J. Physiol. 278(5): G718-G724 [PubMed] [HTML] [PDF]

ENaC is induced by the mineralocorticoid rezeptor (MR) as well as by the glucocorticoid rezeptor (GR). ENaC expression is synergized by butyrate and by tumor necrosis factor-alpha (TNF-alpha).

  • Bergann T, Plöger S, Fromm A, Zeissig S, Borden SA, Fromm M, Schulzke JD (2009) A colonic mineralocorticoid receptor cell model expressing epithelial Na+ channels. Biochem. Biophys. Res. Comm. 381(2): 280-285 [PubMed] [HTML] [PDF]

  • Bergann T, Zeissig S, Fromm A, Richter JF, Fromm M, Schulzke JD (2009) Glucocorticoid and tumor necrosis factor-alpha synergize to induce absorption by the epithelial sodium channel in the colon. Gastroenterology 136(3): 933-942 [PubMed] [HTML] [PDF]

  • Ziera T, Irlbacher H, Fromm A, Latouche C, Krug SM, Fromm M, Jaisser F, Borden SA (2009) Cnksr3 is a direct mineralocorticoid receptor target gene and plays a key role in the regulation of the epithelial sodium channel. FASEB J. 23(11): 3936-3946 [PubMed] [HTML] [PDF]

In inflammatory bowel diseases (IBD) ENaC expression and thus function is reduced. This effect is mediated by pro-inflammatory cytokines like interleukin-1beta and tumor necrosis factor-alpha:

  • Zeissig S, Bergann T, Fromm A, Bojarski C, Heller F, Guenther U, Zeitz M, Fromm M, Schulzke JD (2008) Altered ENaC expression leads to impaired sodium absorption in the non-inflamed intestine in Crohn's disease. Gastroenterology 134(5):1436-1447 [PubMed] [HTML] [PDF]

  • Amasheh S, Barmeyer C, Koch CS, Tavalali S, Mankertz J, Epple HJ, Gehring MM, Florian P, Kroesen AJ, Zeitz M, Fromm M, Schulzke JD (2004) Cytokine-dependent transcriptional down-regulation of epithelial sodium channel (ENaC) in ulcerative colitis. Gastroenterology 126: 1711-1720  [PubMed] [HTML] [PDF]

  • Barmeyer C*, Amasheh S* (*shared first authorship), Tavalali S, Mankertz J, Zeitz M, Fromm M, Schulzke JD (2004) IL-1beta and TNFalpha regulate sodium absorption in rat distal colon. Biochem. Biophys. Res. Comm. 317: 500-507 [PubMed] [HTML] [PDF]

  • Bürgel N, Bojarski C, Mankertz J, Zeitz M, Fromm M, Schulzke JD (2002) Mechanisms of diarrhea in collagenous colitis. Gastroenterology 123(2): 433-443 [PubMed] [HTML] [PDF]

  • Barmeyer C, Horak I, Zeitz M, Fromm M, Schulzke JD (2003) The Interleukin-2-deficient mouse model. Pathobiology 70(3): 139-142 [PubMed] [Full text]

Supported by the Deutsche Forschungsgemeinschaft (DFG Fr 652/4-3)

Earlier papers on epithelial Na+ transport

  • Fromm M, Schulzke JD, Hegel U (1990) Aldosterone low dose, short term action in adrenalectomized glucocorticoid-substituted rats: Na, K, Cl, HCO3, osmolyte, and water transport in proximal and rectal colon. Pflügers Arch. 416: 573-579 [PubMed] [PDF]

  • Hierholzer K, Siebe H, Fromm M (1990) Inhibition of 11-b-hydroxysteroid dehydrogenase and its effect on epithelial sodium transport. Kidney Int. 38: 673-678 [Abstract+References] [PDF]

  • Fromm M, Hegel U (1987) Net ion fluxes and zero flux limiting concentrations in rat upper colon and rectum during anaesthesia-induced aldosterone liberation. Pflügers Arch. 408: 185-193 [PubMed] [PDF]

  • Fromm M, Oelkers W, Hegel U (1983) Time course of aldosterone and corticosterone plasma levels in rats during general anaesthesia and abdominal surgery. Pflügers Arch. 399: 249-254 [PubMed] [PDF]

  • Fromm M, Hegel U (1978) Segmental heterogeneity of epithelial transport in rat large intestine. Pflügers Arch. 378: 71-83 [PubMed] [PDF]

Magnesium

  • Gabriel TE, Gunzel D (2007) Quantification of Mg2+ extrusion and cytosolic Mg2+-buffering in Xenopus oocytes. Arch. Biochem. Biophys. 458(1): 3-15 [PubMed] [HTML] [PDF]

  • Gunzel D, Kucharski LM, Kehres DG, Romero MF, Maguire ME (2006) The MgtC virulence factor of Salmonella enterica serovar typhimurium activates Na+,K+-ATPase. J. Bacteriol. 188(15): 5586-5594 [PubMed] [HTML] [PDF]

  • Kausalya PJ*, Amasheh S* (*shared first authorship), Günzel D, Wurps H, Müller D, Fromm M, Hunziker W (2006) Disease-associated mutations affect intracellular traffic and paracellular Mg2+ transport function of claudin-16. J. Clin. Invest.  116(4): 878-891 [PubMed] [HTML] [PDF]

  • Ebel H, Gunther T (2006) Stimulation of choline/Mg2+ antiport in rat erythrocytes by mefloquine. Magnesium Res. 19(1): 7-11 [PubMed] [Full text]

  • Ebel H, Günther T (2005) Na+/Mg2+ antiport in erythrocytes of spontaneously hypertensive rats: role of Mg2+ in the pathogenesis of hypertension. Magnesium Res. 18(3): 175-185 [PubMed] [PDF]

  • Gunzel D, Hintz K, Durry S, Schlue WR (2005) Mg2+-malate co-transport, a mechanism for Na+-independent Mg2+ transport in neurons of the leech Hirudo medicinalis. J. Neurophysiol. 94: 441-453 [PubMed] [HTML] [PDF]

  • Gunzel D, McGuigan JAS, Schlue WR (2005) Use of Mg2+ and Ca2+ macroelectrodes to measure binding in extracellular-like physiological solutions. Front. Biosci. 10: 905-918 [PubMed] [Full text] [Request text]

  • Ebel H, Hollstein M, Gunther T (2004) Differential effect of imipramine and related compounds on Mg2+ efflux from rat erythrocytes. Biochim. Biophys. Acta - Biomembranes 1667(2):132-140 [PubMed] [HTML] [PDF]

  • Ebel H, Kreis K, Gunther T (2004) Regulation of Na+/Mg2+ antiport in rat erythrocytes. Biochim. Biophys. Acta - Biomembranes 1664(2): 150-160 [PubMed] [HTML] [PDF]

  • Ebel H, Gunther T (2003) Stimulation of Na+/Mg2+ antiport in rat erythrocytes by intracellular Cl-. FEBS Letters 543: 103-107 [PubMed] [HTML] [PDF]

  • Müller A, Günzel D, Schlue WR (2003) Activation of AMPA/kainate receptors but not acetylcholine receptors causes Mg2+ influx into Retzius neurones of the leech Hirudo medicinalis. J. Gen. Physiol. 122: 727-739 [PubMed] [HTML] [PDF]

  • Ebel H, Hollstein M, Gunther T (2002) Role of the choline exchanger in Na+-independent Mg2+ efflux from rat erythrocytes. Biochim. Biophys. Acta  - Biomembranes 1559(2): 135-144 [PubMed] [HTML] [Full paper ,PDF]
cartoon
  Methods 

Isolated but "living" gastrointestinal epithelia and epithelial cell cultures can be functionally characterized regarding their transport and barrier functions by electrophysiological methods.

Short circuit current (Ussing)

Old fashioned, but still powerful in vitro technique for determination of active ion transport (short circuit current), ion permeability (conductance or resistance, respectively) and radioisotope fluxes. Three computerized setups consisting of 6-8 chambers each. Details:

This technique is applied also in the Lab course in Physiology for 2nd year medical students. Tissues are distal colons obtained from a rural rabbit slaughtery close to Berlin. We have published this lab course:

We have developed 2 more refined techniques which allow for discrimination of local conductances within the epithelial tissue:
- Conductance scanning for the measurement of horizontal conductance distribution,
- Transmural impedance for the measurement of vertical conductance distribution.

Conductance scanning

In order to describe quantitatively the heterogenous horizontal distribution of ion permeability in an epithelium, we developed a new electophysiological method. It was named conductance scanning, because it aims at the spatial resolution of epithelial conductivity by means of a scanning microelectrode probe.

The method is based upon the measurement of local differences in current density that are recorded with microelectrodes in the electrolyte solution above the mucosal surface of the flat epithelium, while a defined clamp current is passed through the tissue. Using mathematical models the distribution of epithelial conductivity is derived from the distribution of supraepithelial current density.

  • Günzel D, Krug SM, Rosenthal R, Fromm M (2010) Biophysical methods to study tight junction permeability. Curr. Top. Membr. 65: 39-78 [Directory] [HTML] [PDF] (review / book chapter)

We have developed this method for 3 applications which differ reganding their spatial resolution and their mathematical models:

  • Günzel D, Krug SM, Rosenthal R, Fromm M (2010) Biophysical methods to study tight junction permeability. Curr. Top. Membr. 65: ### (invited Review) [12.03.10 Accepted]

  • At low resolution, local leaks can be described, which may for instance be caused by defects in the epithelial layer (erosions) 
  • At medium resolution, the ion permeability of colon crypt and surface epithelium can be distinguished
    • Köckerling et al., 1993, Am. J. Physiol. 264: C1285-C1293 [PDF]
    • Köckerling & Fromm, 1993, Am. J. Physiol. 264: C1294-C1301 [PDF]
    • Grotjohann I, Gitter AH, Köckerling A, Bertog M, Schulzke JD, Fromm M (1998) Localization of cAMP- and aldosterone-induced K+ secretion in rat distal colon by conductance scanning. J. Physiol. (Lond.) 507: 561-570 [PubMed] [HTML] [PDF]
  • Also at medium resolution, leaks can be quantified that originate from the apoptotic decomposition of individual epithelial cells 
  • At high resolution, conductance scanning allows for determination of trans- and paracellular conductivities
    • Gitter et al., 1997, Pflügers Arch. 434: 830-840 [PDF]
    • Gitter et al., 2000, Pflügers Arch. 439: 477-482 [PDF]
    • Yu ASL, Cheng MH, Angelow S, Günzel D, Kanzawa SA, Schneeberger EE, Fromm M, Coalson RD (2009) Molecular basis for cation selectivity in claudin-2-based paracellular pores: Identification of an electrostatic interaction site. J. Gen. Physiol. 133(1):111-127 [PubMed] [HTML] [PDF]

One-path impedance spectroscopy (1PI)

Key word: Electrical impedance of epithelia

Discrimination of epithelial and subepithelial ion conductance in intact gastrointestinal epithelia in vitro. The conductance of the pure epithelium is frequency-dependent, however that of the tissues underlying the intestinal epithelium is not (in the frequency range applied). This is due to the fact that the subepithelial tissues have much higher conductivity due the lack of tight junctions. This allows to locate intestinal permeability changes to the epithelium or the subepithelium. Own development. Two  setups.

Two-path impedance spectroscopy (2PI)

This is a refined technique which is employed to determine paracellular and transcellular resistance in epithelial confluent celllayers. A specific perturbation of one of the two pathways allows for recording two data sets. Para- and transcellular resistance is calculated after combinig the two data sets and fluxes of a suitable paracellular marker.
Method:

  • Krug SM, Fromm M, Günzel D (2009) Two-path impedance spectroscopy for measuring paracellular and transcellular epithelial resistance. Biophys. J. 97(8): 2202-2211 [PubMed] [HTML] [PDF] [Supplement]

  • Günzel D, Zakrzewski S, Schmid T, Pangalos M, Wiedenhoeft J, Blasse C, Ozboda C, Krug SM (2012) From TER to trans- and paracellular resistance: Lessons from impedance spectroscopy. Ann. N.Y. Acad. Sci. 1257: 142-151 [PubMed] [WebPage] [PDF] (Review)

  • Günzel D, Krug SM, Rosenthal R, Fromm M (2010) Biophysical methods to study tight junction permeability. Curr. Top. Membr. 65: 39-78 [Directory] [HTML] [PDF] (review / book chapter)

  • Schmid T, Günzel D, Bogdan M (2010) Using an artificial neural network to determine electrical properties of epithelia. In: Diamantaras K, Duch W, Iliadis LS Eds., ICANN 2010, Part I, Lect. Notes Comput. Sci. 6352: 211-216 (DOI: 10.1007/978-3-642-15819-3_28) [WebPage] [PDF]

Application:

  • Krug SM, Amasheh M, Dittmann I, Christoffel I, Fromm M, Amasheh S (2013) Sodium caprate as an enhancer of macromolecule permeation across tricellular tight junctions of intestinal cells. Biomaterials 34(1): 275-282 [PubMed] [WebPage] [PDF]

  • Krug SM, Günzel D, Conrad MP, Rosenthal R, Fromm A, Amasheh S, Schulzke JD, Fromm M (2012) Claudin-17 forms tight junction channels with distinct anion selectivity. Cell. Mol. Life Sci. 69(16): 2765-2778 [PubMed] [HTML] [PDF] [Supplement]

  • Amasheh M, Fromm A, Krug SM, Amasheh S, Andres S, Zeitz M, Fromm M, Schulzke JD (2010) TNFa-induced and berberine-antagonized tight junction barrier impairment via tyrosine kinase, pAkt, and NFkB signaling. J. Cell Sci. 123(23): 4145-4155 [PubMed] [HTML] [PDF] [Supplements]

  • Milatz S, Krug SM, Rosenthal R, Günzel D, Müller D, Schulzke JD, Amasheh S*, Fromm M* (*shared last authorship) (2010) Claudin-3 acts as a sealing component of the tight junction for ions of either charge and uncharged solutes. Biochim. Biophys. Acta Biomembr. [PubMed] [HTML] [PDF]

  • Krug SM, Amasheh S, Richter JF, Milatz S, Günzel D, Westphal JK, Huber O, Schulzke JD, Fromm M (2009) Tricellulin forms a barrier to macromolecules in tricellular tight junctions without affecting ion permeability. Mol. Biol. Cell  20: 3713-3724 [PubMed] [HTML] [PDF] [Supplement text] [Supplement video]

  • Amasheh S*, Milatz S* (*shared first authorship), Krug SM, Bergs M, Amasheh M, Schulzke JD, Fromm M (2009) Na+ absorption defends from paracellular back-leakage by claudin-8 upregulation. Biochem. Biophys. Res. Comm. 378(1): 45-50 [PubMed] [HTML] [PDF]

Patch-Clamp

Measurement of ion specificity, conductance, voltage dependency and open probability of cell membrane channels. Very common electrophysiological method, thus no detailed explanation here.

Note: The Berlin Patch Clamp Colloquium, a regular seminar of  patch clampers throughout Berlin, is organized by our former post-doc Friederike Stumpff.

Molecular biology

In most projects we alter epithelial transport or barrier properties in an electrophysiological experiment, and then use these functionally altered tissues for molecular biology.

Our current projects cover two main topics:
1.  The epithelial sodium channel (ENaC)

  • Amasheh S, Barmeyer C, Koch CS, Tavalali S, Mankertz J, Epple HJ, Gehring MM, Florian P, Kroesen AJ, Zeitz M, Fromm M, Schulzke JD (2004) Cytokine-dependent transcriptional down-regulation of epithelial sodium channel (ENaC) in ulcerative colitis. Gastroenterology 126: 1711-1720  [PubMed] [HTML] [PDF]

  • Barmeyer C*, Amasheh S* (*shared first authorship), Tavalali S, Mankertz J, Zeitz M, Fromm M, Schulzke JD (2004) IL-1beta and TNFalpha regulate sodium absorption in rat distal colon. Biochem. Biophys. Res. Comm. 317: 500-507 [PubMed] [HTML] [PDF]

  • Barmeyer C, Horak I, Zeitz M, Fromm M, Schulzke JD (2003) The Interleukin-2-deficient mouse model. Pathobiology 70(3): 139-142 [PubMed] [Full text]

  • Epple HJ, Amasheh S, Mankertz J, Goltz M, Schulzke JD, Fromm M (2000) Early aldosterone effect in distal colon by transcriptional regulation of ENaC subunits. Am. J. Physiol. 278(5): G718-G724 [PubMed] [HTML] [PDF]

2.  Tight junction proteins (occludin and claudin)

  • Zeissig S, Bürgel N, Günzel D, Richter JF, Mankertz J, Wahnschaffe U, Kroesen AJ, Zeitz M, Fromm M, Schulzke JD (2007) Changes in expression and distribution of claudin-2, -5 and -8 lead to discontinuous tight junctions and barrier dysfunction in active Crohn's disease. Gut 56(1): 61-72 [PubMed] [HTML] [PDF]

  • Zeissig S, Bojarski C, Bürgel N, Mankertz J, Zeitz M, Fromm M, Schulzke JD (2004) Downregulation of epithelial apoptosis and barrier repair in active Crohn's disease by TNFalpha antibody treatment. Gut 53: 1295-1302 [PubMed] [HTML] [PDF]

  • Barmeyer C, Harren M, Schmitz H, Heinzel-Pleines U, Mankertz J, Seidler U, Horak I, Wiedenmann B, Fromm M, Schulzke JD (2004) Mechanisms of diarrhea in the interleukin-2 deficient mouse model of colonic inflammation. Am. J. Physiol. Gastrointest. Liver Physiol. 286: G244–G252 [PubMed] [HTML] [PDF]

  • Bojarski C, Weiske J, Schöneberg T, Schröder W, Mankertz J, Schulzke JD, Florian P, Fromm M, Tauber R, Huber O (2004) The specific fate of tight junction proteins in apoptotic epithelial cells. J. Cell Sci. 117: 2097-2107 [PubMed] [HTML] [PDF]

  • Mankertz J*, Hillenbrand B* (*shared first authorship), Tavalali S, Huber O, Fromm M, Schulzke JD (2004) Functional crosstalk between Wnt signaling and Cdx-related transcriptional activation in the regulation of the claudin-2 promoter activity. Biochem. Biophys. Res. Comm. 314(4): 1001-1007 [PubMed] [HTML] [PDF]

  • Tebbe B, Mankertz J, Schwarz C, Amasheh S, Fromm M, Schultz-Ehrenburg U, Sánchez Ruderisch H, Schulzke JD, Orfanos CE (2002) Tight junction proteins: A novel class of integral membrane proteins. Expression in human epidermis and HaCaT keratinocytes. Arch. Dermatol. Res. 294: 14-18 [PubMed] [HTML] [PDF]

    •

  • Amasheh S, Meiri N, Gitter AH, Schöneberg T, Mankertz J, Schulzke JD, Fromm M (2002) Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells. J. Cell Sci. 115(24): 4969-4976 [PubMed] [HTML] [PDF]

  • Bürgel N, Bojarski C, Mankertz J, Zeitz M, Fromm M, Schulzke JD (2002) Mechanisms of diarrhea in collagenous colitis. Gastroenterology 123(2): 433-443 [PubMed] [HTML] [PDF]

  • Mankertz J, Waller JS, Hillenbrand B, Tavalali S, Florian P, Schoneberg T, Fromm M, Schulzke JD (2002) Gene expression of the tight junction protein occludin includes differential splicing and alternative promoter usage. Biochem. Biophys. Res. Comm. 298: 657-666 [PubMed] [PDF]

  • Mankertz J, Tavalali S, Schmitz H, Mankertz A, Fromm M, Schulzke JD (2000) Expression from the human occludin promoter is affected by tumor necrosis factor a and interferon g. J. Cell Sci. 113(Pt 11): 2085-2090 [PubMed] [PDF] [GenBank: Homo sapiens occludin gene, partial sequence]

For the investigation of structural and functional characteristics we expose epithelial cell layers to proinflammatory cytokines under the standardised conditions of in vitro cell culture. During the incubation period the alteration of the transepithelial resistance is monitored. Subsequently, RNA and proteins are isolated from the cells, separated electrophoretically in a gel matrix and analyzed in Northern and Western blots with gene-specific probes or antisera against tight junction proteins.

Changes in quantity of the biomolecules permit conclusion on the regulation of gene expression and the role of the individual tight junction proteins. Beside classical hybridizing procedures we apply modern molecular biology methods for a structural and functional analysis of paracellular barrier and epithelial transport. With the genome walking technique it is possible to isolate regulatory sequences structurally linked to the gene-of-interest.

After nucleic acid sequencing, reporter gene analyses are performed to characterize these sequences functionally. Motives essential for transcription factor binding can be limited by targeted mutations. Thus, conclusions on the signal transduction cascades connected with the regulation of gene expression can be drawn. This is of importance for the development of improved therapeutic approaches of inflammatory bowel diseases.

Fluorescence microscopy

Many molecules can be stained with an immunofluorescence dye and then detected or localized within the cell by confocal laser scanning microscopy (CLSM). Example: By Western blotting Claudin-1 was found in the membrane fraction of MDCK-C11 cells, which is surprising, because MDCK-C11 form a rather leaky epithelium, while claudin-1 is typical for tight epithelia. However, claudin-1 was not colocalized with occludin within the tight junction but was found below the tight junction. Therefore it does not contribute to sealing properties of the tight junction (see Fig., from Amasheh et al., 2002, J Cell Sci.):

  • Amasheh S, Meiri N, Gitter AH, Schöneberg T, Mankertz J, Schulzke JD, Fromm M (2002) Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells. J. Cell Sci. 115(24): 4969-4976 [PubMed] [HTML] [PDF]

  • Mankertz J, Waller JS, Hillenbrand B, Tavalali S, Florian P, Schoneberg T, Fromm M, Schulzke JD (2002) Gene expression of the tight junction protein occludin includes differential splicing and alternative promoter usage. Biochem. Biophys. Res. Comm. 298: 657-666 [PubMed] [PDF]

  • Florian P, Schoneberg T, Schulzke JD, Fromm M, Gitter AH (2002) Single-cell epithelial defects close rapidly by an actinomyosin purse string mechanism with functional tight junctions. J. Physiol. (Lond.) 545(2): 485-499 [PubMed] [HTML] [PDF] [Supplementary movies]

 

Freeze fracture electron microscopy of the tight junction

Permeability of tight junctions are not determined solely by their molecular composition, but also - as known for many years - by the ultrastructural arrangement, expansion, and continuity of tight junction strands. The molecular composition and the ultrastructure are related to each other. Technically, tissues are freeze fractured,  vaporized, analyzed electron microscopically. If the fracture occurs inside the lateral cell membrane often a tight junction meshwork becomme visible. In a morphometric analysis, tight junctions then are analyzed regarding the number of horizontally arranged strands, its extension, and its linearity.

  • Krug SM, Günzel D, Conrad MP, Rosenthal R, Fromm A, Amasheh S, Schulzke JD, Fromm M (2012) Claudin-17 forms tight junction channels with distinct anion selectivity. Cell. Mol. Life Sci. 69(16): 2765-2778 [PubMed] [HTML] [PDF] [Supplement]

  • Milatz S, Krug SM, Rosenthal R, Günzel D, Müller D, Schulzke JD, Amasheh S*, Fromm M* (*shared last authorship) (2010) Claudin-3 acts as a sealing component of the tight junction for ions of either charge and uncharged solutes. Biochim. Biophys. Acta Biomembr. [PubMed] [HTML] [PDF]

  • Rosenthal R, Milatz S, Krug SM, Oelrich B, Schulzke JD, Amasheh S, Günzel D, Fromm M (2010) Claudin-2, a component of the tight junction, forms a paracellular water channel. J. Cell Sci. 123(11): 1913-1921 [PubMed] [HTML] [PDF] [Supplement]

  • Krug SM, Amasheh S, Richter JF, Milatz S, Günzel D, Westphal JK, Huber O, Schulzke JD, Fromm M (2009) Tricellulin forms a barrier to macromolecules in tricellular tight junctions without affecting ion permeability. Mol. Biol. Cell 20: 3713-3724 [PubMed] [HTML] [PDF] [Supplement text] [Supplement video]

  • Zeissig S, Bürgel N, Günzel D, Richter JF, Mankertz J, Wahnschaffe U, Kroesen AJ, Zeitz M, Fromm M, Schulzke JD (2007) Changes in expression and distribution of claudin-2, -5 and -8 lead to discontinuous tight junctions and barrier dysfunction in active Crohn's disease. Gut 56(1): 61-72 [PubMed] [HTML] [PDF]

  • Schmitz H, Barmeyer C, Fromm M, Runkel N, Foss HD, Bentzel CJ, Riecken EO, Schulzke JD (1999a) Altered tight junction structure contributes to the impaired epithelial barrier function in ulcerative colitis. Gastroenterology 116: 301-309. [PubMed] [HTML] [PDF]

  • Schmitz H, Fromm M, Bentzel CJ, Scholz P, Bode H, Epple HJ, Riecken EO, Schulzke JD (1999b) Tumor necrosis factor-alpha (TNFalpha) regulates the epithelial barrier in the human intestinal cell line HT-29/B6. J. Cell Sci. 112: 137-146. [PubMed] [PDF]

  • Schulzke JD, Bentzel CJ, Schulzke I, Riecken EO, Fromm M (1998) Epithelial tight junction structure in the jejunum of children with acute and treated celiac sprue. Pediatric Res. 43: 435-441. [PubMed] [HTML]

  • Schulzke JD, Fromm M, Bentzel CJ, Zeitz M, Menge H, Riecken EO (1992) Ion transport in the experimental short bowel syndrome of the rat: Increased glucose-dependent Na-absorption is the main adaptive response. Gastroenterology 102: 497-504. [PubMed] [PDF]

  • Schulzke JD, Fromm M, Zeitz M, Menge H, Riecken EO, Bentzel CJ (1990) Tight junction regulation during impaired ion transport in blind loops of rat jejunum. Res. Exp. Med. 190: 59-68. [PubMed]

  • Schulzke JD, Fromm M, Bentzel CJ, Menge H, Riecken EO (1987) Adaptation of the jejunal mucosa in the experimental blind loop syndrome: changes in paracellular conductance and tight junction structure. Gut 28: 159-164. [PubMed] [PDF]

  • Bentzel CJ, Fromm M, Palant CE, Hegel U (1987) Protamine alters structure and conductance of Necturus gallbladder tight junctions without major electrical effects on the apical cell membrane. J. Membr. Biol. 95: 9-20. [PubMed] [PDF]

 
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The cell line HT-29/B6

The highly differentiated cell line HT-29/B6 is a sub-clone of the human colon cancer cell line HT-29 [Kreusel et al., 1991].

  • Kreusel KM, Fromm M, Schulzke JD, Hegel U (1991) Cl– secretion in epithelial monolayers of mucus-forming human colon cells (HT-29/B6). Am. J. Physiol. 261: C574-C582. [PubMed] [PDF]

HT-29/B6 cells grow on permeable supports as epithelial monolayers. Secretagoges induce chloride secretion and mucus production [Epple et al., 1997]. They form apical brush borders and complete belts of tight junctions [Schmitz et al., 1999]. Consequently, they form epithelial barriers with properties of colon crypt cells [Gitter et al., 2000] and single cell apoptosis can be induced.[Bojarski et al., 2001].

The cell line HT-29/B6 is a versatile and well characterized model epithelium suitable for studying epithelial and/or intestinal properties with electrophysiological, morphological, and molecular methods.

  • Bergann T, Plöger S, Fromm A, Zeissig S, Borden SA, Fromm M, Schulzke JD (2009) A colonic mineralocorticoid receptor cell model expressing epithelial Na+ channels. Biochem. Biophys. Res. Comm. 381(2): 280-285 [PubMed] [HTML] [PDF]

  • Bergann T, Zeissig S, Fromm A, Richter JF, Fromm M, Schulzke JD (2009) Glucocorticoid and tumor necrosis factor-alpha synergize to induce absorption by the epithelial sodium channel in the colon. Gastroenterology 136(3): 933-942 [PubMed] [HTML] [PDF]

  • Mankertz J*, Amasheh M* (*shared first authorship), Krug SM, Fromm A, Amasheh S, Hillenbrand B, Tavalali S, Fromm M, Schulzke JD (2009) Tumour necrosis factor alpha up-regulates claudin-2 expression in epithelial HT-29/B6 cells via phosphatidylinositol 3-kinase signaling. Cell Tiss. Res. 336(1): 67-77 [PubMed] [HTML] [PDF]

  • Bojarski C, Gitter AH, Bendfeldt K, Mankertz J, Schmitz H, Wagner S, Fromm M, Schulzke JD (2001) Permeability of HT-29/B6 colonic epithelium as a function of apoptosis. J. Physiol. (Lond.) 535(2): 541-552 [PubMed] [PDF]

  • Gitter AH, Bendfeldt K, Schulzke JD, Fromm M (2000) Trans-/paracellular, surface/crypt, and epithelial/subepithelial resistances of mammalian colonic epithelia. Pflügers Arch. 439(4): 477-482 [PubMed] [PDF]

  • Schmitz H, Fromm M, Bentzel CJ, Scholz P, Detjen K, Mankertz J, Bode H, Epple HJ, Riecken EO, Schulzke JD (1999) Tumor necrosis factor-alpha (TNFa) regulates the epithelial barrier in the human intestinal cell line HT-29/B6. J. Cell Sci. 112: 137-146 [PubMed] [PDF]

  • Epple HJ, Kreusel KM, Hanski C, Schulzke JD, Riecken EO, Fromm M (1997) Differential stimulation of intestinal mucin secretion by cholera toxin and carbachol. Pflügers Arch. 433: 638-647 [PubMed] [PDF]

  • ... and many more 

Inflammatory bowel diseases (IBD)

There are two inflammatory bowel diseases, ulcerative colitis and Crohn's disease. For excellent information on both diseases we refer to the German Crohn/Colitis society (DCCV) and the European Federation of Crohn's & Ulcerative Colitis Asscociations (EFCCA)

  • Bücker R, Schumann M, Amasheh S, Schulzke JD (2010) Claudins in intestinal function and disease. Curr. Top. Membr. 65: 195-227 [Directory] [HTML] [PDF] (review / book chapter)  

Leak flux diarrhea

Diarrhea can be driven by different mechanisms:

  • Altered motility:  luminal passage too fast; not enough time to absorb
  • Malabsorptive diarrhea:  unsufficient active absorption
  • Osmotic diarrhea in the narrow sense:  non-absorbable substances in the lumen (e.g. diet sugars)
  • Secretory diarrhea (traveller's diarrhea):  increased active secretion of chloride, fluid follows
  • Leak flux diarrhea:  increased passive loss into the gut lumen

Leak flux diarrhea is caused by a break-down of the epithelial barrier, mostly produced by impaired tight junctions. This allows solutes and fluid to flow back into the gut lumen (=leak flux).

  • Zeissig S, Bürgel N, Günzel D, Richter JF, Mankertz J, Wahnschaffe U, Kroesen AJ, Zeitz M, Fromm M, Schulzke JD (2007) Changes in expression and distribution of claudin-2, -5 and -8 lead to discontinuous tight junctions and barrier dysfunction in active Crohn's disease. Gut 56(1): 61-72 [PubMed] [HTML] [PDF]

  • Bürgel N, Bojarski C, Mankertz J, Zeitz M, Fromm M, Schulzke JD (2002) Mechanisms of diarrhea in collagenous colitis. Gastroenterology 123(2): 433-443 [PubMed] [HTML] [PDF]

  • Bode H, Schmidt W, Schulzke JD, Fromm M, Zippel T, Wahnschaffe U, Bendfeldt K, Riecken EO, Ullrich R (2000) The HIV protease inhibitors saquinavir, ritonavir, and nelfinavir, but not indinavir, impair the epithelial barrier in the human intestinal cell line HT-29/B6. AIDS 13(18): 2595-2597 [PubMed reference] [Related articles]

  • Schmitz H, Barmeyer C, Fromm M, Runkel N, Foss HD, Bentzel CJ, Riecken EO, Schulzke JD (1999) Altered tight junction structure contributes to the impaired epithelial barrier function in ulcerative colitis. Gastroenterology 116: 301-309.[PubMed] [HTML] [PDF] [1999 award of the DCCV]

  • Schmitz H, Fromm M, Bentzel CJ, Scholz P, Detjen K, Mankertz J, Bode H, Epple HJ, Riecken EO, Schulzke JD (1999) Tumor necrosis factor-alpha (TNFa) regulates the epithelial barrier in the human intestinal cell line HT-29/B6. J. Cell Sci. 112: 137-146 [PubMed] [PDF]

  • Stockmann M, Fromm M, Schmitz H, Schmidt W, Riecken EO, Schulzke JD (1998) Duodenal biopsies of HIV infected patients with diarrhea show epithelial barrier defects but no secretion. AIDS 12: 43-51 [PubMed]

  • Stockmann M, Fromm M, Riecken EO, Schulzke JD (1998) Non-malabsorptive mechanisms of diarrhea in HIV infection. Pathobiology 66: 165-169 [PubMed] [PDF]

Apoptosis in a colonic epithelium (HT-29/B6)

  • Schulzke JD, Bojarski C, Zeissig S, Heller F, Gitter AH, Fromm M (2006) Disrupted barrier function through epithelial cell apoptosis. Ann. N.Y. Acad. Sci. 1072: 288-299 [PubMed] [PDF]

  • Bojarski C, Gitter AH, Bendfeldt K, Mankertz J, Schmitz H, Wagner S, Fromm M, Schulzke JD (2001) Permeability of HT-29/B6 colonic epithelium as a function of apoptosis. J. Physiol. (Lond.) 535(2): 541-552 [PubMed] [HTML] [PDF]

  • Gitter AH, Bendfeldt K, Schulzke JD, Fromm M (2000) Leaks in the epithelial barrier caused by spontaneous and TNFa-induced single-cell apoptosis. FASEB J. 14(12): 1749-1753 [PubMed] [HTML] [PDF]

Electrical impedance of epithelia: Impedance spectroscopy

  • Günzel D, Zakrzewski S, Schmid T, Pangalos M, Wiedenhoeft J, Blasse C, Ozboda C, Krug SM (2012) From TER to trans- and paracellular resistance: Lessons from impedance spectroscopy. Ann. N.Y. Acad. Sci. 1257: 142-151 [PubMed] [WebPage] [PDF] (Review)

  • Günzel D, Krug SM, Rosenthal R, Fromm M (2010) Biophysical methods to study tight junction permeability. Curr. Top. Membr. 65: 39-78 [Directory] [HTML] [PDF] (review / book chapter)

  • Schmid T, Günzel D, Bogdan M (2010) Using an artificial neural network to determine electrical properties of epithelia. In: Diamantaras K, Duch W, Iliadis LS Eds., ICANN 2010, Part I, Lect. Notes Comput. Sci. 6352: 211-216 [26.07.10 In press]

  • Krug SM, Fromm M, Günzel D (2009) Two-path impedance spectroscopy for measuring paracellular and transcellular epithelial resistance. Biophys. J. 97(8): 2202-2211 [PubMed] [HTML] [PDF] [Supplement]

  • Gitter AH, Bendfeldt K, Schulzke JD, Fromm M (2000) Trans-/paracellular, surface/crypt, and epithelial/subepithelial resistances of mammalian colonic epithelia. Pflügers Arch. 439(4): 477-482 [PubMed] [PDF] [Related articles]

  • Gitter AH, Fromm M, Schulzke JD (1998) Impedance analysis for determination of epithelial and subepithelial resistance in intestinal tissues. J. Biochem. Biophys. Meth. 37: 35-46 [PubMed]

  • Fromm M, Schulzke JD, Hegel U (1985) Epithelial and subepithelial contributions to transmural electrical resistance of intact rat jejunum, in vitro. Pflügers Arch. 405: 400-402. [PubMed]