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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 87 (1985), S. 141-150 
    ISSN: 1432-1424
    Keywords: protamine ; Necturus gallbladder ; tight junction ; impedance analysis ; transepithelial resistance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Protamine, a naturally occurring arginine-rich polycationic protein (pI 9.7 to 12), was tested inNecturus gallbladder using a transepithelial AC-impedance technique. Protamine sulfate or hydrochloride (100 μg/ml=20 μm), dissolved in the mucosal bath, increased transepithelial resistance by 89% without affecting the resistance of subepithelial layers. At the same time, transepithelial voltage (ψ ms ) turned from slightly mucosapositive values to mucosa-negative values of approximately +1 to −5 mV. The effect of protamine on transepithelial resistance was minimal at concentrations below 5 μg/ml but a maximum response was achieved between 10 and 20 μg/ml. Resistance started to increase within 1 min and was maximal after 10 min. These effects were not inhibited by serosal ouabain (5×10−4 m) but could be readily reversed by mucosal heparin. The sequence of protamine effect and heparin reversal could be repeated several times in the same gallbladder. Mucosal heparin, a strong negatively charged mucopolysaccharide, or serosal protamine were without effect. Mucosal protamine reversibly decreased the partial ionic conductance of K and Na by a factor of 3, but did not affect Cl conductance. Net water transport from mucosa to serosa was reversibly increased by 60% by protamine. We conclude that protamine reversibly decreases the conductance of the cation-selective pathway through the tight junction. Although this effect is similar to that reported for 2,4,6-triamino-pyrimidinium (TAP), the mechanism of action may differ. We propose that protamine binds to the apical cell membrane and induces a series of intracellular events which leads to a conformational alteration of the tight junction structure resulting in decreased cationic permeability.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-1424
    Keywords: protamine ; tight junction ; transepithelial resistance ; tight junction morphology ; tight junction strands ; Necturus gallbladder
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Protamine is a naturally occurring basic protein (pI; 9.7 to 12.0). We have recently reported that protamine dissolved in the mucosal bath (2 to 20 μm), induces about a twofold increase in transepithelial resistance inNecturus gallbladder within 10 min. Conductance decreased concomitantly with cation selectivity. In this leaky epithelium, where 〉90% of an applied current passes between cells, an increment in resistance of this magnitude suggests a paracellular actiona priori. To confirm this, ionic conductance across the apical cell membrane was studied with microelectrodes. Protamine increased transepithelial resistance without changing apical cell membrane voltage or fractional membrane resistance. Variation in extracellular K concentration (6 to 50mm) caused changes in apical membrane voltage not different from control. To determine if protamine-induced resistance changes were associated with structural alteration of tight junctions, gallbladders were fixedin situ at peak response and analyzed by freeze-fracture electron microscopy. According to a morphometrical analysis, the tight junctional intramembranous domain expands vertically due to incorporation of new strands (fibrils) into the main compact fibrillar meshwork. Since morphologic changes are complete within 10 min, strands are probably recycled into and out of the tight junctional membrane domain possibly by the cytoskeleton either from cytoplasmic vesicles or from intramembranous precursors. Regulation of tight junctional permeability by protamine and other perturbations may constitute a common mechanism by which leaky epithelia regulate transport, and protamine, in concentrations employed in this study, seems reasonably specific for the tight junction.
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  • 3
    ISSN: 1432-2013
    Keywords: Large intestine ; Aldosterone ; Anaesthesia ; Amiloride ; Ion transport ; Osmolyte transport ; Water transport ; Luminal steady state concentrations ; Transepithelial voltage ; Segmental heterogeneity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Thiobutabarbital anaesthetized and abdominally operated control rats develop high endogenous plasma levels of both aldosterone and corticosterone during the course of a 12 h experiment. This effect was used as a model for examining ‘acute’ steroid action (i) on net ion and water fluxes and (ii) on zero flux luminal limiting concentrations in rat upper colon (proximal 50% of large intestine) and rectum (distal 40%). Experiments of both kinds consisted of 8 independent 90 min measuring periods. (i) In rectum net fluxes of Na, K, osmolytes (sum of all solutes) and water started at low levels around zero, began to rise about 2 h after plasma levels of aldosterone had increased, and reached plateau values around the 6th hour of anaesthesia. In upper colon, fluxes of Na, K, Cl, and osmolytes were high from the beginning and did not vary significantly with time. (ii) At zero flux conditions limiting concentrations of Na in the hormonally unstimulated phase of the experiment were 20±3 mM in upper colon and 22±3 mM in rectum. After maximal endogenous aldosterone liberation zero flux concentrations were 5.2 mM in upper colon and 2.2 mM in rectum, corresponding to luminal fluid to plasma ratios (LF/P) of 0.040 and 0.016, respectively. Amiloride reduced the maximal Na gradient in rectum to aLF/P of 0.3 but was not effective in upper colon and did not prevent the stimulating effect of aldosterone in this segment. Under all experimental conditions zero flow concentrations of K were higher than consistent with a solely passive distribution, indicating simultaneous passive and active secretion in both segments. In contrast to the findings of others, the luminal fluid remained isoosmolar with plasma in all zero flux experiments. Conclusions: Under acute elevation of steroid levels not higher than reached in a living animal the rectum varies net fluxes of Na, K, osmolytes, and water between zero and high rates. At this range of steroid levels, the upper colon practically does not respond with variations in net fluxes. Under zero flux conditions aldosterone acutely increases maximal Na gradients in rectum and, to a lesser degree, also in upper colon. Thus, acutely elevated aldosterone levels affect Na transport in upper colon and rectum by different mechanisms and intensities: in rectum, which is the most sensitive site of transport regulation, via an amiloridesensitive Na-pathway, and in upper colon, via an amiloride-insensitive pathway.
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  • 4
    ISSN: 1432-2013
    Keywords: Colon ; Rectum ; Epithelial resistance ; AC impedance ; Stripping ; Subepithelial resistance ; Aldosterone
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Epithelial and subepithelial electrical resistances of rat large intestine were measured by means of a 4-electrode AC impedance technique in three segments, colon ascendens, colon descendens and rectum. (i) Epithelial resistance of colon ascendens and colon descendens was about 35 Ω · cm2 and not different between these two segments. It was, however, about 3 times higher in rectum (99 Ω · cm2). This finding is in accord with our previous observation of about 3-fold higher net fluxes of ions and water in colon ascendens and colon descendens than in rectum. It confirms the concept of a main functional difference between the terminal part of the large intestine (rectum) and the more proximal segments (colon). (ii) The acutely (within hours) varied level of aldosterone by keeping the rats for 7 h in anaesthesia caused in the rectum a more than 10-fold increase in short circuit current (I SC) and transepithelial voltage but no significant decrease in resistance. Similarly, the decline inI SC, as regularly observed in the early phase of in vitro measurements on partially stripped large intestine, was paralleled by voltage changes but not by changes in resistance. We conclude that the wide range of resistance values published so far was caused to a great extent by including various portions of colon or rectum. (iii) By comparing intact (not stripped) and partially stripped preparations (muscularis propria removed) of the rectum it was shown that partial stripping did not alter the epithelial resistance but reduced the subepithelial resistance in this segment from 26 to 8 Ω · cm2, or by 68%. Subepithelial resistances of stripped rectum, colon ascendens and colon descendens were 8, 12 and 13 Ω · cm2, respectively. Based on these figures,I SC of conventional voltage clamp measurements is underestimated due to subepithelial tissue layers in intact rectum by 23% and in the partially stripped preparations of rectum, colon ascendens and colon descendens by 9%, 34%, and 37%, respectively.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1432-2013
    Keywords: Epithelial monolayers ; HT-29/B6 cells ; Single-channel patch clamp ; Ussing chamber ; Noise analysis ; Fura-2 imaging ; NPPB ; DNDS
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The patch-clamp technique and transepithelial current measurements in conjunction with analysis of transepithelial current noise were employed in order to clarify the role of the outwardly rectifying, depolarization-induced Cl− channel (ORDIC) during cAMP-mediated Cl− secretion in HT-29/B6 cells. Confluent monolayers growing on permeable supports were used in order to ensure the apical location of measured Cl− channels. The ORDIC needed to be activated by excision and/or depolarization, and was found in both cAMP-stimulated and non-stimulated cells. Both 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and 4,4′-dinitro-2,2′-stilbenedisulphonate (DNDS) induced fast flickery-type blocks of the ORDIC at low, micromolar blocker concentrations and were used as a probe for ORDIC. However, these substances were ineffective in blocking transepithelial forskolin-induced Cl− secretion of monolayers in Ussing chambers. No inhibitory effect at all was detected for DNDS up to 1 mmol/l. NPPB blocked the ORDIC at low concentrations (IC50=0.5±0.3 μmol/l) by reducing its open probability, but NPPB did not block forskolin-induced Cl− secretion unless high concentrations were used (IC50=240±10 μmol/l). In order to exclude effects of NPPB other than on the apical Cl− channel, trans-epithelial measurements were performed in basolaterally amphotericin-permeabilized, forskolin-stimulated preparations, and a serosal-to-mucosal Cl− gradient was applied as a driving force. Under these conditions, NPPB's inhibitory effects were also very small. Noise analysis of this gradient-driven Cl− current showed a very-low-frequency Lorentzian noise component (f c=1.4±0.2 Hz), which was not compatible with Lorentzians predicted from single-channel gating of ORDIC. As revealed from fura-2 fluorescence measurements, forskolin-stimulated Cl− secretion occurred in the absence of changes in intracellular Ca2+. Thus, we conclude that there is an apical Cl− channel in HT-29/B6 that is activated through the cAMP-mediated pathway and is insensitive to NPPB and DNDS, and the kinetics of which are incompatible with ORDIC kinetics. Therefore, despite its prevalence in isolated patches and even in cell-attached recordings, the ORDIC appears not to be involved in cAMP-mediated Cl− secretion by HT-29/B6 cells. From noise analysis, a very-small-conductance (probably below 1 pS), slow-gating Cl− channel was calculated as the conductive site in the apical membrane during forskolin stimulation.
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  • 6
    ISSN: 1432-2013
    Keywords: Mammalian small intestine ; in vitro technique ; AC impedance ; epithelial resistance ; subepithelial resistance ; leaky epithelia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Epithelial and subepithelial resistance of rat jejunum was measured in vitro by two independent methods. (i) Transepithelial AC impedance data were interpreted in terms of a simple parallel RPCP element (representing the epithelial cell layer) in series with an ohmic resistor RS (representing the subepithelial layers). (ii) In separate experiments, the tip of a microelectrode was positioned between epithelium and subepithelial layers and the respective resistances were obtained from DC-pulse voltage divider ratios between both structures. The total tissue resistance as measured in conventional Ussing-chamber experiments (49±4 Ohm·cm2, mean of both methods) was formed to 81±6% (40±3 Ohm·cm2) by subepithelial layers and to only 19±3% (9±1 Ohm·cm2) by the epithelial cell line. We conclude that rat jejunum is more conductive than assumed so far. In in vitro flux studies on intact jejunal sheets a pronounced back-diffusion of absorbed substances will lead to an underestimation of the true net transport capacity of this structure. This error averages about fivefold and will be found likewise in conventional short-circuit measurements.
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  • 7
    ISSN: 1432-2013
    Keywords: Amiloride ; Bumetanide ; Cl-secretion ; Electrical field stimulation ; Large intestine ; Na-absorption ; Rat ; Stripping ; Submucosal plexus ; Tetrodotoxin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The initial phase of in vitro experiments in Ussing-type chambers on large intestine is characterized by short-circuit currents (ISC) declining from high starting values to a lower plateau within 0.5 h. The origin of this “initial ISC-transient” was investigated by ISC measurements on partially stripped segments of rat rectal colon. Transport was pre-stimulated in vivo by keeping animals in barbiturateanesthesia for 5 h prior to tissue preparation. This procedure caused by endogenous aldosterone-liberation amiloride-sensitive Na-absorption to become the predominant electrogenic transport. The initial ISC-transient was abolished by tetrodotoxin (TTX, 1 μM), indicating a neuronal mediation of this phenomenon. In order to identify the transport which was subject to neuronal control, the amiloride-sensitive Na-absorption was measured during electrical field stimulation (bipolar rectangular pulses: 5 Hz, 1 ms, ±6 mA). There was no difference to unstimulated controls. In contrast, the initial ISC-transient was dependent on Cl in the bath following Michaelis-Menten-kinetics (K M=20 mM) and could be prevented by 10 μM serosal bumetanide. Then, initial filling of the Ussing-chamber was imitated during the course of the experiment by removal and immediate readdition of the bathing fluid. This procedure caused ISC-changes of similar appearance as the initial ISC-transient. To verify that indeed mechanical stretch is the sensory stimulus triggering the initial ISC-transient, the effect of small pressure oscillations was studied. This also produced an ISC-transient which was TTX-sensitive and was abolished after removal of the submucosal plexus Meissner by total stripping. It is concluded that amiloride-sensitive Na-absorption does not contribute to the initial transient and is not affected by the enteric nervous system. Initial ISC-transients asobserved during the first half hour of Ussing experiments are due to electrogenic Cl-secretion which is stimulated by mechanical stretch during tissue preparation and filling of the chamber via a submucosal neuronal reflex pathway. The possible biological meaning of this stretch-induced secretory process could be facilitation of transit during imminent stasis of the gut contents.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1432-2013
    Keywords: Adrenalectomy ; Aldosterone ; K absorption ; K secretion ; Na transport ; Osmolyte transport ; Proximal colon ; Rectal colon
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The short-term action of aldosterone in physiological concentration on net fluxes of Na, K, Cl, HCO3, osmolytes, and water was examined in the proximal colon and rectal colon of adrenalectomized (ADX) rats in vivo. The measuring time was 12 h, divided in eight periods of 90 min. (a) Aldosterone alone (6 nmol h−1kg−1) did not stimulate transport in ADX rats. In these experiments plasma [K] increased to fatal values. A basal glucocorticoid substitution of 24 nmol h−1kg−1 corticosterone caused plasma K to stay constant throughout the experiment, so that epithelial transport was not handicapped by non-specific effects of ADX, but this also did not restore the decreased transport of ADX rats to control values. Under these conditions (absence of aldosterone) in the rectal colon Na and H2O transport was zero, whereas in the proximal colon flux rates were depressed by between 30% and 50%. In contrast, basal glucocorticoid substitution of 18 nmol h−1kg−1 corticoster-one plus infusion of 6 nmol h−1kg−1 aldosterone caused transport stimulation to values not significantly different from those of non-ADX controls. We conclude that after ADX, aldosterone at physiological concentrations increases transport if, as a prerequisite, a basal glucocorticoid substitution is provided. Transport of Na, K, and H2O is under the total control of aldosterone in the rectal colon but is only moderately altered in the proximal colon. (b) In the proximal colon aldosterone is effective on electroneutral Na transport consistent with the Na/H, Cl/HCO3 double exchanger, while in the rectal colon aldosterone controls the amiloride-sensitive Na channel, which is the sole apical Na transporter in that segment. In this respect the rectal colon is functionally distinct from the distal colon of other studies in the rat, where under unstimulated conditions the double exchanger is present. Regarding Na transport mechanisms, the rat large intestine thus consists of three distinct segments, the proximal, distal, and rectal colon. (c) In the rectal, but not the proximal colon, active net K absorption against the electrochemical gradient took place in the absence of aldosterone, but was suppressed in its presence. (d) The flux ratio of osmolytes over H2O was constantly hypertonic as compared to the plasma (387±1 mosmol l−1), independent of absolute flux size, the colonic segment examined, or the steroid concentration used. This effect may be due to a hypertonic unstirred layer within the lamina propria.
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  • 9
    ISSN: 1432-2013
    Keywords: Aldosterone plasma concentration ; Corticosterone plasma concentration ; Anaesthesia ; Barbiturates ; Abdominal surgery ; Large intestine ; Transepithelial potential difference
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The transepithelial voltage (Ψ ms) of rat rectum in vivo increases for several hours in experiments under general anaesthesia. So far this was attributed by indirect evidence to increasing aldosterone plasma levels during the course of the experiment. We performed direct measurements of aldosterone and corticosterone plasma concentrations during intestinal perfusion experiments on barbiturate anaesthetized rats. Experiments were terminated for blood sampling at 10, 75, 300, 400, 800, or 1,800 min, respectively. (i) After 75 min of anaesthesia, surgical preparation was finished and plasma levels of aldosterone and of corticosterone were found increased by the factors 5 and 3, respectively, as compared to conscious controls. (ii) During the following 12 h, aldosterone further increased to levels 10 times as high as those of controls. In contrast, during the same period corticosterone slowly decreased but still remained elevated as compared to controls. (iii) The increase of both hormones was attenuated when abdominal surgery was omitted. (iv) The use of pentobarbital (Nembutal) instead of thiobarbital (Inactin) did not influence the adrenal response. (v) In adrenalectomized rats a continous substitution with 65 ng·h−1·kg−1 BWT aldosterone resulted in plasma levels as high as in conscious intact animals. (vi) RectalΨ ms started to move to higher lumen-negative values with a time delay of 1–1 1/2 h as compared with the increase of hormone levels.Ψ ms then stayed elevated until to the end of the experiments. We conclude that in vivo experiments of several hours duration in thio- or pentobarbital anaesthetized rats take place under conditions of aldosterone and corticosterone plasma levels which are high as compared to those of conscious unstressed animals. The different time course of aldosterone and corticosterone plasma levels after the end of surgery would be in accord with a stimulatory effect of anaesthesia on both renin and ACTH, plus an additional stimulatory effect of abdominal surgery on ACTH alone.
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  • 10
    ISSN: 1432-2013
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Zusammenfassung 1. Der Knäuelschweiß ist isoton (Mittelwert 318 mosmol/l). Er enthält im Durchschnitt 147 mÄq/l Na und 123 mÄq/l Cl. 2. Der Schweiß am Ende des Ausführungsganges ist hypoton (154 mosmol/l) und enthält im Mittel 22 mÄq/l Na und 28 mÄq/l Cl. 3. Zwischen dem Lumen des engen Gangabschnittes und dem Interstitium wurde eine elektrische Potentialdifferenz in der Größenordnung von 40 mV gemessen (Lumen negativ). 4. Die Potentialdifferenz fiel nach lokal-subcutaner Injektion von G-Strophantin in die Haut ab. Die Na-Konzentration des Endschweißes stieg dabei von 22 mÄq/l auf 90–107 mÄq/l. Aus den Befunden wird der Schluß gezogen, daß aus dem isotonen Primärsekret durch einen strophantinempfindlichen Natriumtransportmechanismus im dünnen Gangabschnitt NaCl rückresorbiert wird und dadurch der hypotone Endschweiß resultiert.
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