 Institut für Klinische Physiologie |
| FU Berlin / HU Berlin > Charité > Departments > Inst.Klin.Physiologie > Abstracts |
Institut für Klinische Physiologie |
| FU Berlin / HU Berlin > Charité > Departments > Inst.Klin.Physiologie > Abstracts |
J. Mater. Sci. Mater. Med. 9: 711-715 (1998)
The prerequisite for the development of a biohybrid artificial kidney is a substrate for confluent growth of renal cells forming an epithelial monolayer without any leaks. Conventional cell culture supports cannot be adapted for this purpose, because they lack adequate mechanical properties and thermal stability. From two suitable materials, polysulfone and polyacrylonitrile, two permeable polymer membranes have been produced that were, according to ISO 10933-5, not cytotoxic. Cloned Madin Darby Canine Kidney (MDCK) cells (an established renal cell line) were cultured on the surface of the plastic materials, and on conventional cell culture supports. With all materials, assays of mitochondrial and lactate dehydrogenases exhibited similar proliferation and the viability of the MDCK cells. Transmission electron microscopy showed the expression of a normal morphology of kidney tubular cells. Perfect barrier function, consequent on the formation of intercellular junctions in a confluent tight epithelium, was visualized in electron micrographs, and quantified by measurement of the transepithelial resistance. The uniformity of the cells grown was demonstrated in samples by electron microscopy and in the whole epithelium by intravital impedance analysis. It was concluded that polymeric membranes produced from polysulfone or polyacrylonitrile are appropriate substrates in the design of biohybrid kidney devices.
Z. Gastroent. 32: 30-34 (1994)
In einer Serie von Arbeiten haben Pappenheimer, Reiss und Madara 1987 die These propagiert, daß der Transport von Glukose und Aminosäuren im Dünndarm zum überwiegenden Teil passiv durch konvektive
Teilchenmitführung (solvent drag) auf dem parazellulären Weg erfolgt. Diese These ist durch Arbeiten aus den Labors von Diamond und von Fordtran angezweifelt, durch Studien aus der Arbeitsgruppe um
Madara jedoch bestätigt worden. In der vorliegenden Arbeit werden die widersprüchlichen Resultate und Interpretationen einander gegenübergestellt und abgewogen.
Physiologische Glukosekonzentrationen im Dünndarmlumen liegen mit maximal 50 mmol/l erheblich niedriger als früher angenommen; es ist jedoch nicht auszuschließen, daß im epithelnahen Mikroklima
erheblich höhere Konzentrationen auftreten. Die initialen Arbeiten von Pappenheimer sind zwar zum Teil methodisch angreifbar, jedoch ist das Konzept eines konvektiven parazellulären Transportes seit
Jahrzehnten bekannt. Es scheint so zu sein, daß Na+-gekoppelter Nährstofftransport eine parazelluläre Permeabilitätszunahme für Ionen, Monomere (z.B. Glukose, Aminosäuren) und Oligomere
verursacht, und zwar sowohl in vivo als auch in der Ussing-Kammer. Ob dem parazellulären solvent drag-vermittelten Nährstofftransport auch eine wesentliche quantitative Bedeutung für die Resorption
der Nährstoffe zukommt, kann derzeit nicht durch experimentelle Evidenz belegt werden.
Kidney Int. 38: 673-678 (1990)
SUMMARY: The effects of the steroidal compounds CHAPS and -glycyrrhetinic acid (GLY) on metabolism and biological action of corticosterone have been investigated in in vitro experiments. Using rat renal microsomes it could be demonstrated that both substances inhibited the reversible reaction corticosterone (B) <==> 11-dehydrocorticosterone (11-DHB) which is catalyzed by 11b-hydroxysteroid dehydrogenase (11-HSD, E.C. 1.1.1.146). Using stripped rat rectal epithelia the effects of both steroids on amiloride-inhibitable Na+ transport (JNa) was tested in long term (8 h) Ussing chamber experiments. B as well as 11-DHB had only weak stimulatory effects on JNa. However, when incubated together with glycyrrhetinic acid B became a potent mineralocorticoid effecting JNa qualitatively and quantitatively similar to aldosterone. It is concluded that this effect is due to a direct stimulatory effect of B on mineralocorticoid receptors which normally is masked by metabolic conversion of B mediated by 11-HSD.
Am. J. Physiol. 261: C574- C582 (1991)
SUMMARY: HT-29, an undifferentiated human colon cell line, is known to differentiate when cultured without glucose. This study aimed to characterize ion transport in the clone HT-29/B6, which was selected from HT-29 cells differentiated by glucose-free culture. HT-29/B6 cells seeded onto filter membranes grew as polarized monolayers, mainly consisting of mucus-forming cells and exhibiting high transepithelial resistance. Short-circuit current (ISC) of unstimulated HT-29/B6 monolayers in Ussing chambers was 0.1 ± 0.01 mikromol h-1cm-2, and conductance was 2.0 ± 0.2 mS/cm2. Serosal forskolin (FSK; 10-5 M) induced a sustained ISC of 1.9 ± 0.1 mikromol h-1cm-2, associated with a rise of intracellular adenosine 3',5'-cyclic monophosphate (cAMP). Isc was identified as Cl- secretion by tracer studies and by the inhibitory effects of serosal bumetanide and Ba2+. The Cl- channel blockers NPPB and DPC diminished FSK-induced Isc at respective doses of 3 x 10-4 and 10-3 M, being effective from either side of the monolayer. Cl- secretion could be triggered by vasoactive intestinal peptide (10-8 M), prostaglandin E1 (10-6 M), and dibutyryl cAMP (10-3 M) as well. In conclusion, HT-29/B6 cells grow as polarized monolayers, forming mucus and secreting Cl- in response to secretagogues. This clone may not only serve as a model for investigation of cellular mechanisms of intestinal Cl- secretion but may also be helpful to elucidate the contribution of mucus cells to this process.
Gastroenterology 102: 497-504 (1992)
SUMMARY: We studied the adaptational changes of epithelial ion transport in the short bowel syndrome. Ileal remnants 8 weeks after 70% proximal small intestinal resection in the rat were investigated. Pure epithelial resistance measured by impedance analysis decreased from 27±1 to 21±1 Ohm cm2 and PEG 4000 fluxes increased from 2.5±0.3 to 3.6±0.3 nmol h-1cm-2 indicating increased permeability of the short bowel. Unidirectional flux measurements in control ileum revealed absorptive net fluxes of Na and Cl which were assigned to electroneutral NaCl absorption, and an ISC which was accounted for by the residual flux (HCO3 secretion). Neither NaCl absorption nor HCO3 secretion were altered in short bowel. Also electrogenic Cl secretion, defined after maximal stimulation by theophylline and PGE1, was not changed in the short bowel. In contrast, electrogenic Na/glucose cotransport increased in Vmax from 2.0±0.3 in controls to 5.0±1.0 µmol h-1cm-2 in short bowel. Tight junction structure was studied by freeze fracture EM. The number of horizontal strands was unchanged, while tight junction depth was slightly increased in short bowel. Microvillus area of short bowels was increased by 20% in villus regions. Under the light microscope villus height was increased by 30%. Conclusion: The short bowel mucosa undergoes adaptive responses to reduced overall absorptive area by increasing glucose-dependent electrogenic Na absorption to 250% which is partly due to increased villus and microvillus surface area. Electrogenic Cl and HCO3 secretion and electroneutral NaCl absorption remained unchanged. The decreased epithelial resistance is due to a mucosal surface amplification.
Am. J. Physiol. 265: S10-S19 (1993)
SUMMARY: A short-circuit current experiment on epithelial ion transport is described that is suitable for student classes in human and animal physiology. Segments of late distal colon from either pig or cow are obtained from the slaughterhouse depending on the animals' daily schedule. Initial tissue preparation already in the slaughterhouse, cold storage, and proper choice of bath solutions are essential prerequisites for success. Students monitor spontaneous transepithelial voltage and short circuit current (ISC) by use of manually operated voltage clamp units. Two main transport mechanisms are studied, electrogenic Na+ absorption and Cl- secretion. Electrogenic Na+ absorption is studied by measuring the ISC drop after amiloride. Then Cl- secretion is stimulated by theophylline and subsequently inhibited by furosemide. In some experiments K+ secretion can be detected by the blocking effect of mucosal Ba++. Response of tissues from both species is similar though quantitatively different. The equipment is sturdy and inexpensive, can be provided by most departmental work shops, and has been tested for 3 years in regular lab courses. Observations made during these experiments are closely related to clinical states such as secretory diarrhea, cystic fibrosis, hyperaldosteronism, as well as to the mechanisms of clinically used diuretics.
Am. J. Physiol. 264: C1285- C1293 (1993)
SUMMARY: There is no quantitative assignment of large intestinal electrogenic Na+ absorption to surface epithelium and crypts so far. We determined the spatial distribution of
electrogenic Na+ absorption to crypts and surface epithelium of rat late distal colon using a modified voltage scanning technique. Voltage deflections resulting from external 30 Hz current
were sensed by an extracellular microelectrode stepping at 0.7 Hz above crypt openings or surface epithelium. Local conductances were calculated applying a planar model of electric field distribution
to surface epithelium and a electrostatic disc source model to the crypts. These models were confirmed by methodological experiments where the electrode position was varied in vertical and horizontal
direction. Electrogenic Na+ absorption was detected by blocking apical Na+ channels by mucosal 0.1 mM amiloride. Under control conditions surface epithelium contributed 44%
(2.0±0.2 mS/cm2) and crypts 56% (2.6±0.2 mS/cm2) to the total conductance of 4.6±0.4 mS/cm2. Electrogenic Na+ absorption was induced by 6 h in vitro
incubation in a medium containing 3 nM aldosterone. This caused a short circuit current (ISC) of 12.1±0.8 mikromol h-1cm-2 which was
paralleled by a 2.5fold increase in surface epithelial conductance to 5.1±0.4 mS/cm2 whereas crypt conductance was not significantly altered (3.0±0.2 mS/cm2). Amiloride reversed
ISC to -0.8±0.1 mikromol h-1cm-2 and decreased surface epithelium conductance to 2.3±0.3 mS/cm2 but again had no significant
effect on crypt conductance (2.5±0.3 mS/cm2). Sham incubation (no hormones added) for 6 h neither induced electrogenic transport nor altered local epithelial conductances. We conclude that
amiloride sensitive electrogenic Na+
absorption is localized in late distal colon surface epithelium and is not performed in crypt epithelia.
See also companion paper Köckerling and Fromm, 1993
Am. J. Physiol. 264: C1294-C1301 (1993)
SUMMARY: Cyclic AMP dependent Cl- secretion provides the ionic basis for secretory diarrhea. We quantified the spatial distribution of this process by measuring local ion conductance in
crypts and surface epithelium (resp. villi) of rat late distal colon and ileum. Employing an improved voltage scanning technique the tissue was clamped to a 30 Hz sine wave current and the electrical
field above the respective structures was sensed by a stepping glass microelectrode. Under control conditions, crypts and surface epithelium contributed 61% and 39%, respectively, to the total ion
conductance of distal colon. Theophylline (10 mM) increased crypt conductance (Gc) by 64% from 2.5±0.2 to 4.1±0.3 mS/cm2 and surface epithelium conductance (Gs) by 69%
from 1.6±0.1 to 2.7±0.1 mS/cm2. These changes in local conductances were completely Cl--dependent since theophylline had no effect when Cl- was replaced by gluconate.
Similar results were obtained when Cl- secretion was elicited by PGE1
(1 mikroM) or by dibutyryl-cAMP (1 mM). After stimulation, the Cl-
channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 1 mM) decreased both Gc and Gs. In rat ileum, theophylline plus dibutyryl-cAMP caused an increase in total
conductance of 19% only due to its large paracellular conductance. The ratio of scanning signals above villi and intervillous spaces was unaffected, indicating that Cl- conductance is
induced in crypts and in villi. We conclude that in distal large intestine cAMP dependent Cl- secretion is not confined to crypts, but is evenly performed also by surface cells. A similar
distribution exists in small intestine.
See also companion paper Köckerling et al., 1993
Am. J. Physiol. 264: E68-E73 (1993)
SUMMARY: It has been possible to obtain in a mammalian epithelium of dietically and surgically untreated animals a dose-response of in vitro added aldosterone (ALDO, 10-10 to 10-5 M) on electrogenic Na+ absorption (JNae). JNae was measured in the Ussing chamber on stripped rat late distal colon 8 h after in vitro addition of ALDO. Sub-maximal effects were obtained at 3 nM ALDO: after a lag time of 2 h, short circuit current increased to a maximum of 234±15 µA/cm2 and dropped after 0.1 mM amiloride to -18±3 µA/cm2, resulting in JNae 9.4±0.6 µmol h-1cm-2. Net Na+ tracer fluxes and ISC exhibited parallel time courses, so that electroneutral Na+ transport was not induced in late distal colon by acute ALDO. A plot of JNae versus GNa revealed an electromotive force (ENa) of 126±1 mV for all ALDO concentrations tested. Kinetic data were Km 1.2 nM, Vmax 10.5 µmol h-1cm-2, and Hill coefficient 2.1. In contrast to the large effect in late distal colon, 3 nM ALDO caused JNae of less than 1 µmol h-1cm-2 in early distal colon, proximal colon, and caecum. Anti-mineralocorticoid sensitivity and ENa did not vary with ALDO concentration or time of the experiment, consistent with a unique mechanism during early and late response up to 8 h, as well as at mineralocorticoid and glucocorticoid ALDO concentrations. Acute ALDO in a range of 0.1 to 10 nM fully controls electrogenic Na+ absorption between zero and Vmax in late distal colon. Under acute 3 nM ALDO electrogenic Na+ absorption is localized in late distal colon rather than in the entire distal colon as observed at very high ALDO levels.
Am. J. Physiol. 269: G42-G48 (1995)
SUMMARY: In vivo electrogenic sodium absorption (JNae) in the human rectum is controlled by acute variation of aldosterone in nanomolar concentration range. In this study we report both the induction of JNae in human rectum epithelium by nanomolar aldosterone added in vitro and the enzymatic control of glucocorticoid action on JeNa. 8 h after addition of the respective steroid JeNa was measured as amiloride sensitive short circuit current. 10 nM aldosterone caused JeNa of 5.7 ± 1.4 mikromol h-1cm-2. Cortisol in the same concentration did not induce significant JeNa. Since cortisol is readily inactivated by 11{beta}-hydroxysteroid dehydrogenase (11{beta}-HSD), the true mineralocorticoid activity of cortisol was evaluated after inhibition of 11{beta}-HSD by carbenoxolone. Carbenoxolone alone did not exhibit mineralocorticoid activity. If cortisol (10 nM) was given together with carbenoxolone (10 mikroM), the resulting JeNa (4.5 ± 0.4 mikromol h-1cm-2) was not significantly different from that after 10 nM aldosterone indicating equal intrinsic mineralocorticoid activity of cortisol and aldosterone. The same mechanisms were found in rat late distal colon. Kinetic data of carbenoxolone at 10 nM cortisol resulted in Km 0.3 mikroM, Jmax 8.4 mikromol h-1cm-2 and a Hill coefficient of 1.8. The effects of carbenoxolone and glycyrrhetinic acid did not differ. We conclude that electrogenic Na+ absorption is under complete control of mineralocorticoid action. "Spontaneous" JeNa in the beginning of the in vitro period can be explained by elevated steroid levels before tissue removal. The biological activity of 11{beta}-HSD localized in rectum epithelium suffices to completely inactivate cortisol to cortisone in vitro for several hours, thus protecting the mineralocorticoid receptor from high glucocorticoid concentrations.
Gut 37: 777-782 (1995)
SUMMARY: Epithelial barrier function and ion transport was studied in celiac sprue using a miniaturized Ussing-device for measurements on diagnostic aspiration biopsies from the jejunum of
untreated or gluten-free nourished sprue patients, or from healthy controls.
Pure epithelial resistance (Re) indicating epithelial barrier function was determined by transmural alternating current impedance analysis. It was reduced by 56% in acute sprue (9±1 Ohm cm2)
as compared to controls (20±2 Ohm cm2). In gluten-free nourished sprue patients Re
was only partly recovered (15±1 Ohm cm2). Subepithelial resistance (Rsub) was also altered from 28±1 Ohm cm2 in control to 17±1 Ohm cm2 in acute sprue due
to the change in mucosal architecture, but was unchanged in gluten-free nourished sprue patients (29±4 Ohm cm2).
In acute sprue, unidirectional Na+ and Cl- fluxes were increased in both directions as a consequence of the decreased resistance. However, short circuit current (ISC)
as well as Na+ and Cl-
net fluxes were not significantly different from control.
Subsequently, the electrogenic Cl- secretory system was investigated. After maximal stimulation with theophylline and PGE1, a Cl--dependent increase in ISC
was obtained in the sprue mucosa and control jejunum. It showed saturation characteristics and was blockable by serosal bumetanide. When compared to control, neither Km nor Vmax
of this electrogenic Cl- secretion was significantly altered in celiac sprue.
In conclusion, a miniaturized Ussing-device was used for transport measurements on intestinal biopsies. In acute celiac disease, the epithelial barrier of the jejunum was seriously disturbed. The
active electrogenic Cl-
secretory transport system was present in the sprue mucosa, but was not activated in the Ussing-chamber in vitro when compared to control jejunum.
Am. J. Physiol. 268: G725-G731 (1995)
SUMMARY: Distension of rat rectal colon causes electrogenic Cl- secretion via the plexus submucosus Meissner. This study aimed to identify the neurotransmitter(s) of this reflex pathway. Distension was applied to partially stripped rat rectal colon in Ussing-chambers. Baseline short-circuit current (ISC) increased and then slowly declined again within 30 min. The increase in ISC 10 min after distension ({delta} ISC10) was 1.8±0.3 µmol h-1cm-2. Atropine (1 µM) did not alter {delta} ISC10. Thus, cholinergic neurons with muscarinic synapses were not involved. Then tissues were desensitized to VIP or substance P. This required continuous infusion of VIP or of substance P into the chamber, since otherwise desensitization was only temporary due to rapid degradation of VIP or substance P. During substance P-desensitization distension still induced a secretory response ({delta} ISC10 n.s. versus control), while during VIP-desensitization distension had no effect anymore. Furthermore, a polyclonal anti-VIP antiserum blocked 81% and the VIP-antagonist (p-Cl-D-Phe6,Leu17)-VIP 89% of the distension-induced {delta} ISC10 supporting the results of the desensitization-experiments. To localize the site of VIP action, TTX was used. The TTX-effect on ISC during VIP-stimulation was not different from its effect on baseline ISC. This is in accord with the concept that the VIP-receptors are mainly located on the enterocytes. We conclude that VIP, but not substance P or acetylcholine (via muscarinic receptors), acts as a neurotransmitter in the distension-induced reflex pathway causing Cl- secretion in rat rectal colon.