Wolfgang Clauss

A low-salt diet facilitates Cl secretion in hen lower intestine

SummaryThe regulation of sodium and chloride transport in hen coprodeum by mineralocorticoids was investigated with isolated epithelia under short-circuit conditions. Unidirectional fluxes of Na and Cl were measured by isotopes and modulated by amiloride, theophylline and bumetanide. Hens were maintained either on low-NaCl diet (LS) or on high-NaCl diet (HS). Plasma aldosterone (PA) levels of these groups were measured with radioimmunoassay. A group of HS hens received injections of aldosterone on a 6-hr schedule before experiments. Another group of LS hens was resalinated, and experiments carried out on a 24-hr interval.Salt deprivation stimulated PA levels ninefold, compared to HS hens. Na absorption was stimulated according to previous reports. Electrogenic Cl secretion was elicited by theophylline and partially inhibited by bumetanide. Modulation of PA levels by diet, resalination or aldosterone injection changed the magnitude of electrogenic Cl secretion in parallel between 0.5 μeq/cmau2 · hr (HS) and 4 μeq/cm2 · hr (LS), with pronounced alteration in tissue resistance.The results demonstrate a new action of aldosterone which besides stimulating Na absorption also directly or indirectly elicits Cl secretion. Evidence is presented for a hormonal adaptation of chloride transport in this epithelium. There was a morphological change of the apical plasma membrane and further experiments will have to clarify the exact cellular nature of this process.

Noise analysis of cAMP-stimulated Na current in frog colon.

The effects of oxytocin and cAMP on the electrogenic Na+-transport in the short-circuited epithelium of the frog colon (Rana esculenta, Rana temporaria) were investigated. Oxytocin (100 mU · ml−1) elevated the shortcircuit current (Isc) transiently by 70% whereas cAMP (1 mmol · l−1) elicited a comparable sustained response. The mechanism of the natriferic action of cAMP was studied by analysing current fluctuations through apical Na+-channels induced by amiloride or CDPC (6-chloro-3,5-diaminopyrazine-2-carboxamid). The noise data were used to calculate Na+-channel density (M) and single apical Na+-current (iNa).iNa-Values obtained with amiloride and CDPC were 1.0±0.1 pA (n=5) and 1.1±0.2 pA (n=6) respectively and unaffected by cAMP. On the other hand, cAMP caused a significant increase in M from 0.23±0.08 μm−2 (n=5) to 0.49±0.17 μm−2 (n=5) in the amiloride experiments. In our studies with CDPC we obtained smaller values for M in control (0.12±0.04 μm−2;n=6) as well as during cAMP treatment (0.19±0.06 μm−2;n=6). However, the cAMP-induced increase in M was also significant. We conclude that cAMP stimulates Na+-transport across the frog colon by activating “silent” apical Na+-channels. Thus, the mechanism of regulation of colonic Na-transport in frogs differs considerably from that in other vertebrates as mammals and birds.

The outwardly rectifying Cl− channel is not involved in cAMP-mediated Cl− secretion in HT-29 cells: evidence for a very-low-conductance Cl− channel

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, NPPBs inhibitory effects were also very small. Noise analysis of this gradient-driven Cl− current showed a very-low-frequency Lorentzian noise component (fc=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.

Regulation of Na+ channels in frog lung epithelium: a target tissue for aldosterone action

Sodium transport across isolated lung tissue of the frog Xenopus laevis was measured in Ussing chambers under voltage-clamp conditions. Perfusing the lungs with NaCl-Ringers solutions on both sides, a basal distinct amiloride-blockable Na+ current was present. Incubating the lungs with 1 μmol/l aldosterone from the pleural side raised the short circuit current after a 1-h latent period. Maximal values were reached after 4–5 h of aldosterone treatment, at which time the transepithelial Na+ current was more than doubled compared to the control. The stimulatory effect was totally inhibited when the aldosterone treatment was preceded by incubation of the lung tissues with spironolactone in 2000-fold excess. In the presence of amiloride (0.5–8 μmol/l) in the alveolar compartment, a Lorentzian noise component appeared in the power spectrum of the fluctuations in the short circuit current. This enabled the calculation of single Na+ channel current and Na+ channel density under both experimental conditions. Aldosterone stimulation did not change single Na+ channel current. On the other hand, the number of conducting Na+ channels increased in parallel with the transepithelial Na+ transport. This suggests that the alveolar epithelium may be a physiological target tissue for aldosterone. Since fluid absorption in the lung is secondary to active Na+ transport, aldosterone may be a potent regulator for maintaining the relatively fluid-free state of the lumen of the lung in some cases of fluid accumulation.

MODULATION OF Na AND Cl TRANSPORT BY MINERALOCORTICOIDS

1. The epithelia of the hen lower intestine show a Na-channel, Na-cotransport, chloride cells, and chloride absorption and secretion. 2. The short circuit current is affected by low salt levels, amiloride, glucose, lysine, leucine, galactose, ouabain, bumetanide, aldosterone, dexamethasone and spironolactone. 3. The properties of the different sodium and chloride channels are described.

Electrophysiological analysis of sodium-transport in the colon of the frog (Rana esculenta)

Sodium transport and apical bioelectrical membrane properties were investigated in frog colonic epithelium in the absence and presence of the antidiuretic hormone arginine-vasotocin (AVT). Apical Na-permeability and intracellular Na-activity were evaluated by analysis of current-voltage relationships in the serosally K-depolarized tissue. Tissue- and apical membrane capacitance were measured by voltages step analysis. The frog colon was found to be a tight epithelium with a transepithelial resistance of 2.63±0.25 kω·μF (n=17). 85–90% of short circuit current (11.2±1.1 μA·μF·l−1;n=17) was related to electrogenic Na-transport from mucosa to serosa. Graded doses of amiloride (<50 μmol·l−1) induced Michaelis-Menten-type inhibition kinetics. Serosal addition of 10−6 mol·l−1 AVT induced a significant increase in sodium current (25%), apical sodium permeability (19%) and tissue capacitance (4.3%) whereas intracellular Na-activity remained unchanged. There was a good correlation between increased Na-current and apical Na-permeability. No correlation was found between Na-current and membrane capacitance. Our results demonstrate that in contrast to other species the amphibian colon shows a natriferic reaction to AVT. We suggest that the regulation of Na-transport in frog colon is similar to that in the toad urinary bladder. It is caused by an activation of preexisting apical Na-channels and not by fusion of subapical cytoplasmic vesicles with the apical membrane.

Aldosterone regulates paracellular pathway resistance in rabbit distal colon

SummaryRegulation of the paracellular pathway in rabbit distal colon by the hormone aldosterone was investigated in vitro in Ussing chambers by means of transepithelial and microelectrode techniques. To evaluate the cellular and paracellular resistances an equivalent circuit analysis was used. For the analysis the apical membrane resistance was altered using the antibiotic nystatin. Under control conditions two groups of epithelia were found, each clearly dependent on the light: dark regime. Low-transporting epithelia (LT) were observed in the morning and high-transporting epithelia (HT) in the afternoon. Na+ transport was about 3-fold higher in HT than in LT epithelia. Incubating epithelia of both groups with 0.1 μmol·1-1 aldosterone on the serosal side nearly doubled in LT epithelia the short circuit current and transepithelial voltage but the transepithelial resistance was not influenced. Maximal values were reached after 4–5 h of aldosterone treatment. In HT epithelia due to the effect of aldosterone all three transepithelial parameters remained constant over time. Evaluation of the paracellular resistance revealed a significant increase after aldosterone stimulation in both epithelial groups. This increase suggests that tight junctions might have been regulated by aldosterone. The hormonal effect on electrolyte transport was also dependent on the physiological state of the rabbit colon. Since net Na+ absorption in distal colon is, in addition to transcellular absorption capacity, also dependent on the permeability of the paracellular pathway, the regulation of tight junctions by aldosterone may be a potent mechanism for improving Na+ absorption during hormone-stimulated ion transport.

Different modes of electrogenic Na+ absorption in the coprodeum of the chicken embryo role of extracellular Ca2+

SummaryTransepithelial electrogenic Na+ transport (INa) was investigated in the coprodeum of 20-days-old chicken embryos in Ussing chambers. Short circuit current (Isc) and transepithelial resistance (Rt) were 14.7±4.8 μA · cm-2 (n=12) and 0.53±0.09 kΩ · cm-2 (n=12), respectively. INa was calculated from changes in Isc by substitution of mucosal Na+ by (N-methyl-d-glucamine) (NMDG). Isc inversed during Na+ removal, and INa was found to be 27.8±4.7 μA · cm-2 (n=12). Amiloride (100 μmol · l-1) inhibited only about 60% of INa. Analysis of Isc fluctuations revealed a Lorentzian component in the power density spectrum with a corner frequency of about 57 Hz. This component was not correlated to INa, and its origin is still unclear. Removal of mucosal Ca2+ increased INa about 2.5-fold due to an increase of the amiloride-insensitive component of INa in additionally investigated adult tissues. The results clearly show that this is due to a non-selective cation channel with an “apparent” order of selectivity Cs+>Na+=K+>Rb+>Li+. The Ca2+ concentration required to block 50% of the Isc was about 18 μmol · l-1. The IscCacould also be supressed by other divalent cations such as Mg2+ and Ba2+. Additionally, an INa-linked Lorentzian component occurred which dominated the control spectrum with a significantly higher corner frequency (about 88 Hz). The results indicate that Na+ absorption in the coprodeum of the chicken embryo is more complex than in adult hens. However, the Ca2+ sensitivity of INa is similar to comparable effects described for other epithelia. This possibly reflects the existence of two types of amiloride-insensitive apical cation channels as pathways for Na+ absorption, which may be involved to differing degrees in ontogenetic developments of nonselective channels to Na+-specific ion channels.

Ca-sensitive sodium absorption in the colon of Xenopus laevis

SummaryTransepithelial electrogenic Na transport (INa) was investigated in the colon of the frog Xenopus laevis with electrophysiological methods in vitro. The short circuit current (Isc) of the voltage-clamped tissue was 24.2±1.8 μA·cm-2 (n=10). About 60% of this current was generated by electrogenic Na transport. Removal of Ca2+ from the mucosal Ringer solution stimulated INa by about 120%. INa was not blockable by amiloride (0.1 mmol·l-1), a specific Na-channel blocker in epithelia, but a fully and reversible inhibition was achieved by mucosal application of 1 mmol·l-1 lanthanum (La3-). No Na-self-inhibition was found, because INa increased linearly with the mucosal Na concentration. A stimulation of INa by antidiuretic hormones was not possible. The analysis of fluctuations in the short circuit current (noise analysis) indicated that Na ions pass the apical cell membrane via a Ca-sensitive ion channel. The results clearly demonstrate that in the colon of Xenopus laevis Na ions are absorbed through Ca-sensitive apical ion channels. They differ considerably in their properties and regulation from the amiloride-sensitive Na channel which is “typically” found in the colon of vertebrates.

Segmental differences in K-transport across rabbit proximal and distal colon in vivo and in vitro

Net K-transport was investigated in three different segments of rabbit proximal and distal colon. In vivo studies used a dialysis method, and in vitro studies an everted sac preparation. Results from both methods are in good agreement and show net K-absorption in the two segments of the proximal colon, and net K-secretion in the distal colon. K-absorption in the proximal colon seems to be an active mechanism, because K is transported against an electrical potential gradient. The present study is a comparative approach to demonstrate the heterogeneity of an organ function, and represents the first direct comparison of K-transport in various segments of rabbit colon under in vitro and in vivo conditions.