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W Van Driessche

Publications and source records attributed to W Van Driessche.

At least 91 records · Page 5Linked to original sources

Oxytocin stimulates the apical K+ conductance in frog skin.

We measured the effects of oxytocin (0.1 U/ml) on the current (Isc) recorded through skins of Rana temporaria incubated with an isotonic K+ solution on the apical side while the transepithelial potential was clamped to zero. Under these conditions, Isc is carried by inward K+ movements. Oxytocin markedly stimulated this inward K+ current. When the spontaneous fluctuations were analyzed we found that oxytocin increased the plateau (S(1)o) of the spontaneous Lorentzian component without modifying the corner frequency (f(1)c). Addition of Ba2+ to the mucosal solution blocked Isc both in the presence and absence of oxytocin. Moreover, with mucosal Ba2+ a characteristic blocker-induced Lorentzian component appeared in the power spectrum. Analysis of this blocker-induced noise showed that oxytocin increased the number of active K+ channels in the apical membrane, while the changes in single channel current were in agreement with the expected alterations of the electrochemical driving force.

Animals↗

Current-noise analysis of the basolateral route for K+ ions across a K+-secreting insect midgut epithelium (Manduca sexta).

The isolated midgut of a lepidopteran larva (Manduca sexta), 5th instar was investigated with voltage-clamp and fluctuation analysis techniques. With high K+ insect saline on both sides the outward-directed short-circuit current (Isc) was carried by K+ (IK) from serosal to mucosal compartment. IK could be blocked, in a dose-dependent manner by serosal Ba2+ ions. There was no current with serosal Na+. Noise analysis of IK revealed a Lorentzian component in the power spectrum when Ba2+ was present in the serosal solution. The Ba2+/receptor kinetics show pseudo-first order characteristics only at low [Ba2+]s. For [Ba2+]s greater than KBa, the apparent Ba2+ association rate decreases with a hyperbolic course as a function of serosal [Ba2+] which could indicate some "substrate-inhibition"-like interaction of Ba2+ at its receptor site. It is concluded that the serosal membranes of the K+-secreting intestinal cells contain the common type of Ba2+-blockable K+ channel which provides the serosal pathway for K+ during secretion which is ultimately driven by the mucosally-located electrogenic K+-ATPase.

Animals↗

Effect of sodium on amiloride- and triamterene-induced current fluctuations in isolated frog skin.

The apparent association constants of two agents, amiloride and triamterene, that block the Na-selective channel of apical membrane of frog skin are shown to decrease as the Na concentration is increased in the apical bathing solution in isolated skin of the frog, Rana temporaria, Rana esculenta, and Rana pipiens. These results were obtained in "normally polarized" skins. These effects were independent of the anion used (chloride or methylsulfate) or the cation used as the Na substitute (Tris, DDA, or K ion). When NaCl was replaced with mannitol, the Na effect on the amiloride association rate constant persisted, which shows that ionic strength was not critically involved. The amiloride corner frequency was unaffected when the clamp potential was altered from +100 to -60 mV. The Na dependence was greatly attenuated or absent when the serosal surface was bathed in 120 mM K Ringer's, an effect that appears to be attributable to some pharmacological effect of high serosal K. A previously described three-state model is used to analyze the inhibitory effect of Na on the blocker association rate constant.

Amiloride↗

Lidocaine blockage of basolateral potassium channels in the amphibian urinary bladder.

1. Basolateral membranes of the frog urinary bladder were investigated after increasing the cationic conductance of the apical membrane by the incorporation of nystatin. 2. K+ currents were recorded in the presence of a mucosa to serosa oriented K+ gradient (SO4(2-) Ringer solution). Nystatin caused a rapid rise of the short-circuit current (Isc) followed by a slow increase over a period of 1-2 h. 3. Impedance analysis showed that the apical membrane resistance was drastically reduced by nystatin. The slow increase in Isc was accompanied by a progressive increase in basolateral conductance. 4. The transepithelial current and conductance recorded in the presence of nystatin could be depressed with lidocaine added to the mucosal and serosal solution. The effects of lidocaine were completely reversible. 5. Noise analysis showed that lidocaine induced additional fluctuations in Isc. The spectrum of these fluctuations was of the Lorentzian type. This noise component is caused by the random interruption of the current through the basolateral K+ channels. The Lorentzian parameters were used to calculate the microscopic parameters of the basolateral K+ channels.

Action Potentials↗

Voltage-dependent Ba2+ block of K+ channels in apical membrane of frog skin.

The characteristics of the blockage of apical K+ channels in the frog skin (Rana temporaria) by Ba2+ were investigated with current-voltage measurements and current-fluctuation analysis. Inward K+ currents were recorded with a transepithelial K+ concentration gradient oriented from mucosa to serosa. When Ba2+ (5-10,000 microM) was added to the mucosal solution, the inward K+ current was progressively depressed. This blockage was strongly voltage dependent. The apparent macroscopic Michaelis-Menten constant decreased exponentially with increasing transepithelial voltage (Vt) (mucosa positive). According to Eyring rate theory, the binding site for the Ba2+ ion in the K+ channel was found to be located at a relative electrical distance of 0.72 of the apical membrane potential field, measured from the cytosolic side. From current-voltage (I-V) measurements and Michaelis-Menten kinetics, the Ba2+ dissociation constant was calculated: KBa = 57 microM (Vt = 0 mV). The voltage dependence of the on- and off-rate of the Ba2+-receptor interaction was obtained from the analysis of current fluctuations induced by Ba2+. An exponential relationship between the rates and Vt was obtained, as was found for KBa from the I-V measurements. KBa (at Vt = 0 mV) calculated from the noise experiments was 66 microM.

Animals↗

Quinidine blockage of K+ channels in the basolateral membrane of larval bullfrog skin.

The skin of frog larvae (Rana catesbeiana) was used to study the characteristics of basolateral K+ channels with fluctuation analysis. K2SO4 and Na2SO4 Ringer's were used as mucosal and serosal solution, respectively. After addition of Nystatin (138 U/ml) the transepithelial conductance and short-circuit current (Isc) increased considerably. Most of Isc was carried by K+, moving from the mucosal to the serosal side. This current could be depressed by quinidine, added to both compartments or to mucosal side only. Fluctuation analysis showed that quinidine induced a Lorentzian component in the power density spectrum. Assuming pseudo-first order kinetics for the channel occlusion by quinidine the current through the open K+ channel and channel density were calculated: iK = 0.22 pA, M = 7.7 channels/microns2.

Animals↗

Ca2+-sensitive, spontaneously fluctuating, cation channels in the apical membrane of the adult frog skin epithelium.

The fluctuations in transepithelial current through the abdominal skin of bullfrogs (Rana catesbeiana) were analysed while the transepithelial voltage was clamped to zero. A Lorentzian component in the power spectrum was recorded when the skin was bathed with Ca2+ free NaCl Ringer's on both sides. After replacement of all mucosal Na+ by choline the Lorentzian component disappeared. The application of mucosa positive potentials enhanced the plateau of the relaxation noise component while it was depressed by mucosa negative potentials. These observations showed that the current associated with the relaxation noise, was carried by Na+ moving in the inward direction. Divalent cations added to the mucosal solution in micromolar concentrations depressed the relaxation noise immediately, which is indicative for an apical localization of the fluctuating channels. The relaxation noise depended strongly on the pH of the mucosal medium: alkalinization enhanced the relaxation noise while acidification depressed the fluctuations. Micromolar concentrations of the diuretic amiloride, which is known to block the Na+ entry into the cellular compartment, enhanced the Na+-dependent relaxation noise while at higher concentrations an inhibitory effect was observed. From these observations it was concluded that the relaxation noise is caused by inward Na+ movement through fluctuating channels which are localized in the apical membrane. These channels seem to constitute a pathway in parallel with the amiloride-blockable channels. Ionic substitution of Na+ by other monovalent cations showed that these channels are also permeable for K+, Rb+, NH4+, Cs+ and Tl+, but not for Li+. Divalent cations in micromolar concentrations completely occlude these fluctuating channels. Therefore, this pathway will be blocked for monovalent cations when normal Ca2+ containing Ringer's are used as mucosal bathing medium.

Amiloride↗

Autoregulation of apical membrane Na+ permeability of tight epithelia. Noise analysis with amiloride and CGS 4270.

Noise analysis of the Na+ channels of the apical membranes of frog skin bathed symmetrically in a Cl-HCO3 Ringer solution was done with amiloride and CGS 4270. Tissues were studied in their control states and after inhibition of transepithelial Na+ transport (Isc) by addition of quinine or quinidine to the apical solution. A critical examination of the amiloride-induced noise indicated that the single channel Na+ currents (iNa) were decreased by quinine and quinidine, probably because of depolarization of apical membrane voltage. Despite considerable statistical uncertainty in the methods of estimation of the Na+ channel density with amiloride-induced noise (NA, see text), the striking observation was a large increase of NA with amiloride inhibition of the rate of Na+ entry into the cells. NA was increased to 406% of control, whereas Isc was inhibited to 8.6% of control by 6 microM amiloride. Studies were done also with the Na+ channel blocker CGS 4270. Noise analysis with this compound was advantageous, permitting iCGSNa and NCGS to be measured in individual tissues with a relatively small inhibition of Isc. As with amiloride, inhibition of Isc with CGS 4270 caused large increases of the Na+ channel density (approximately 200% at approximately 35% inhibition of the Isc). Quinine and quinidine caused an approximately 50% increase of Na+ channel density while inhibiting iNa by approximately 60-70%. As inhibition of Na+ entry leads to an increase of Na+ channel density, a mechanism of autoregulation appears to be a major factor in adjusting the apical membrane Na+ permeability of the cells.

Amiloride↗

Electrophysiology and noise analysis of K+-depolarized epithelia of frog skin.

Epithelia of frog skin bathed either symmetrically with a sulfate-Ringer solution or bathed asymmetrically and depolarized with a 112 mM K+ basolateral solution (Kb+) were studied with intracellular microelectrode techniques. Kb+ depolarization caused an initial decrease of the short-circuit current (Isc) with a subsequent return of the Isc toward control values in 60-90 min. Whereas basolateral membrane resistance (Rb) and voltage were decreased markedly by high [Kb+], apical membrane electrical resistance (Ra) was decreased also. After 60 min, intracellular voltage averaged -27.3 mV, transcellular fractional resistance (fRa) was 86.8%, and Ra and Rb were decreased to 36.1 and 13.0%, of their control values, respectively. Amiloride-induced noise analysis of the apical membrane Na+ channels revealed that Na+ channel density was increased approximately 72% while single-channel Na+ current was decreased to 39.9% of control, roughly proportional to the decrease of apical membrane voltage (34.0% of control). In control and Kb+-depolarized epithelia, the Na+ channel density exhibited a phenomenon of autoregulation. Inhibition of Na+ entry (by amiloride) caused large increases of Na+ channel density toward saturating values of approximately 520 X 10(6) channels/cm2 in Kb+-depolarized tissues.

Amiloride↗

Ionic channels in epithelial cell membranes.

This review focused on results obtained with methods that allow studies of ionic channels in situ, namely, patch clamping and current-noise analysis. We reported findings for ionic channels in apical and basolateral plasma membranes of various tight and leaky epithelia from a wide range of animal species and tissues. As for ionic channel "species," we restricted ourselves to the discussion of cation-specific (Na+ or K+), hybrid (Na+ and K+), and Cl- channels. For the K+-specific channels it can be said that their properties in conduction (multisite, single file), selectivity (only "K+-like" cations), and blocking behavior (Ba2+, Cs+, TEA) much resemble those observed for K+ channels in excitable membranes. This seems to include also the Ca2+-activated "maxi" K+ channel. Thus, K+ channels in excitable membranes and K+ channels in epithelia appear to be very closely related in their basic structural principles. This is, however, not at all unexpected, because K+ channels provide the dominant permeability characteristics of nearly all plasma membranes from symmetrical and epithelial cells. An exception is, of course, apical membranes of tight epithelia whose duty is Na+ absorption against large electrochemical gradients in a usually anisosmotic environment. Here, Na+ channels dominate, although a minor fraction of membrane permeability comes from K+ channels, as in frog skin, colon, or distal nephron. Epithelial Na+ channels are different from excitable Na+ channels in that they 1) are far more selective and 2) seem to be chemically rather than electrically gated. Furthermore, their specific blockers belong to very different chemical families, although a guanidinium/amidinium moiety is a common feature (TTX vs. amiloride). [For a more detailed summary of Na+ channel properties see sect. IV H.] Most interesting is the occurrence of relatively nonselective cationic (hybrid) channels in apical membranes of tight epithelia, like larval or adult frog skin. Here, not only the weak selectivity is astonishing but also the fact that these channels react with so-called K+-channel-specific (Ba2+, TEA) as well as with Na+-channel-specific (amiloride, BIG) compounds. Moreover, this cross-reactivity does not seem to be inhibitory but, on the contrary, stimulating. Clearly these channels may become a fascinating object with which to assess whether Na+ and K+ channels are not only structurally but also genetically related and whether they can somehow be converted into each other.(ABSTRACT TRUNCATED AT 400 WORDS)

Aldosterone↗

The sensitivity of apical Na+ permeability in frog skin to hypertonic stress.

Na+ transport across abdominal skins of the frog species Rana esculenta and Rana pipiens was analyzed by recording short-circuit current (Isc), transepithelial conductance (Gt), and the current noise generated by the random blockage of apical Na+ channels by the diuretic, amiloride. Specific Na+ current (INa) and conductance (GNa), as reflected by the amiloride-sensitive part of Isc and Gt, respectively, were markedly depressed after addition of some osmotically active substances, like sugars or alcohols to the mucosal Na+-Ringer solution. These hypertonicity-induced reactions were fast and fully reversible, even at mucosal osmolarities of 1 Osmol. With mucosal solutions of moderate hyperosmolarity a recovery of INa and GNa was observed in presence of the osmotic gradient. This "regulatory" current showed to be carried by Na+ through the Na+-specific apical channels. Contrary to the fast current drop during the initial phase of hyperosmotic shocks, the "osmoregulation" was considerably slower. The recovery of INa was only complete at smaller osmotic gradients but became more and more suppressed at higher osmolarities. Steady-state analysis of the kinetics of the Na+-specific current revealed that the current depression by osmotic shocks obeys Michaelis-Menten kinetics. This current depression at high osmolarities, as well as during the initial phase before "osmoregulation" with small osmotic gradients, can be described in terms of a non-competitive inhibition. This was also suggested by Na+-concentration jump experiments indicating a reduction of the maximal, apical Na+ permeability as mechanism of the hypertonicity-induced drop in INa. The INa kinetics after complete "osmoregulation" were, however, indistinguishable from the isotonic control condition.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗

Physiological role of apical potassium ion channels in frog skin.

The K+ permeability of the apical membrane of frog skin (Rana temporaria) was analysed by recording the short-circuit current and its fluctuations in the presence of a mucosa-to-serosa-oriented K+ concentration gradient. Loading of the animals with KCl resulted in an augmentation of the Ba2+-blockade component of the short-circuit current and the plateau value of the K+-dependent relaxation noise. Poisoning of active transport and exposing both sides of the epithelium to KCl Ringer solution caused an increase of the K+ current and its fluctuations recorded after restoring the inward-oriented K+ gradient. Serosal quinidine (5 X 10(-4) M), which is thought to increase intracellular Ca2+ activity, depressed the K+ current and the relaxation noise. This effect was completely reversible. Removal of Na+ from the serosal solution, which is known to result in an elevation of intracellular Ca2+ by abolishing the driving force for the Na+/Ca2+ exchanger, also reduced the K+ current and the Lorentzian plateau. Both parameters returned to their control values after restoring the Na+ gradient across the basolateral membranes. It is concluded from these experiments that the apical K+ permeability is controlled by factors which depend on the intracellular K+ and Ca2+ concentration and that the apical K+ channels may constitute a pathway for K+ secretion.

Animals↗

Noise analysis of inward and outward Na+ currents across the apical border of ouabain-treated frog skin.

The passive Na+ transport across the apical membrane of frog skin (Rana catesbeiana) was studied under the following circumstances: (1) control conditions (sulfate Ringer's, K+ depolarised serosal membranes); (2) after blocking the active transport step with ouabain; (3) with an outward oriented Na+ current. The amiloride-induced Na+ current fluctuations were analysed to calculate the density of amiloride blockable channels and the current through one single channel. Despite the large reduction of the macroscopic current by ouabain, the single channel current remained unchanged, while the number of amiloride blockable Na+ channels was reduced by a factor of eight. It is concluded from these observations that the earlier described reduction of the permeability of the apical membrane is caused by a decrease of the number of electrically conductive Na+ channels. The outward oriented single channel currents were less than 50% of the currents in the opposite direction. After ouabain, the number of Na+ channels was independent from the current direction.

Amiloride↗

Hormonal control of apical membrane Na transport in epithelia. Studies with fluctuation analysis.

To study the mechanisms by which antidiuretic hormone and prostaglandins regulate Na transport at the apical membranes of the cells of anuran tissues, studies were done with fluctuation analysis. Epithelia of frog skin (Rana pipiens) were treated with vasopressin alone, or treated with vasopressin after inhibition of Na transport by indomethacin. The tissues were bathed symmetrically with a Cl-HCO3 Ringer solution and short-circuited continuously. In this experimental circumstance, the amiloride-induced current noise power density spectra were of the Lorentzian type with little or no l/f noise, provided that "scraped" skins were used for study. Despite large changes of Na transport, especially in epithelia treated with indomethacin and vasopressin, the single-channel Na current remained essentially unchanged, whereas the density of amiloride-inhibitable, electrically conductive Na channels was increased by vasopressin and decreased by indomethacin.

Amiloride↗

Cl- - and K+-related fluctuations of ionic current through oxyntic cells in frog gastric mucosa.

Transport and conductance pathways for Cl- and K+ were studied in frog gastric mucosa using noise analysis techniques. The current-noise power spectra exhibited both Cl- - and K+-dependent characteristics. In Cl- -containing solutions, reductions in Cl- transport were associated with reductions of the overall noise power. Such changes appeared to reflect the movement of Cl- through the apical (mucosal) membranes of oxyntic cells. In Cl- -free solutions a K+-dependent Lorentzian component was detected in the power spectrum when applying a mucosally or serosally directed transepithelial K+ concentration gradient. This component was enhanced by 1) stimulating the oxyntic cells with histamine and 2) appropriate voltage clamping. It was reduced by mucosal Ba2+ in resting tissues and enhanced by mucosal Ba2+ in stimulated tissues. The K+ noise measured in gastric mucosae in Cl- -free solutions appeared also to be generated at the apical membranes of oxyntic cells. This is in analogy to previous findings in other gastrointestinal epithelia with fluctuating apical K+ channels. In the gastric mucosa these channels may play a key role in the mechanism of electrogenic H+ secretion.

Animals↗

A fluctuation analysis study of the development of amiloride-sensitive Na+ transport in the skin of larval bullfrogs (Rana catesbeiana).

In the presence of the Na+ -channel blocker amiloride, the short-circuit current across the skins of bullfrog tadpoles in metamorphic stages XIX-XXIV was subjected to fluctuation analysis. The resulting power spectra contained a Lorentzian component of which the plateau value (S0) decreased while the corner frequency (fc) increased as the mucosal amiloride concentration was increased from 0.5 to 24 microM. From the linear relationship between the fc values and the amiloride concentrations it was possible to determine the binding (k'01) and unbinding (k10) constants for amiloride to its receptor on the Na+ channel. With these parameters as well as short-circuit current and S0 values, the current through the individual Na+ channels (i) was calculated (average 0.58 pA). It did not increase significantly during late metamorphosis. The density of Na+ channels (M) in the apical membrane, on the other hand, increased significantly. It would appear that the increase in short-circuit current which occurs at this time is due primarily to an increase in amiloride-blockable Na+ channels. Unexpectedly, a Lorentzian component could be fitted to power spectra in amiloride-treated skins (stages XIX-XXI) which showed no amiloride-sensitive short-circuit current. Moreover, the typical increase in fc with the amiloride concentration did not occur in these animals.

Amiloride↗

Poorly selective cation channels in the skin of the larval frog (stage less than or equal to XIX).

The abdominal skin of bullfrog larvae (Rana catesbeiana) was placed in an Ussing-type chamber, and its transepithelial electrical parameters were recorded with mucosal solutions of different ionic composition. With "K+-like" cations (K+, NH+4, RB+, Cs+) the power spectra of the fluctuations in short-circuit current displayed a Lorentzian component (fc = 30 - 40 Hz). The relaxation noise could be suppressed by addition of the K+ -channel blockers Ba2+ and TEA to the mucosal solution. Also, in presence of the ionophore antibiotic nystatin the Lorentizian noise was abolished. The Na+ -channel probes amiloride and benzimidazolyl-2-guanidine (BIG) both enhanced the relaxation noise obtained with the K+-like cations but, with Na+ and Li+, also caused the rise of a relaxation component above the background noise. In presence of amiloride or BIG, the addition of Ba2+, TEA and nystatin still abolished the Lorentizian noise. It can be concluded that the relaxation-noise source is located in the apical cell membranes of the tadpole skin. These spontaneously fluctuating cation channels do not seem to strictly discriminate between K+-like ions (K+, NH+4, Rb+, Cs+) and Na+-like ions (Na+, Li+). On the other hand, well-known specific probes for K+ channels (Ba2+, TEA) and for Na+ channels (amiloride, BIG) interact with this apical cation channel. It is possible that the poorly selective channel plays a role in the ontogenesis of the specific Na+ transport in the maturing frog skin.

Amiloride↗

Na+ channels and amiloride-induced noise in the mammalian colon epithelium.

(1) The effects of the Na+-channel blocker, amiloride, on the short-circuit current carried by Na+ was studied with fluctuation analysis, in rabbit descending colon epithelium. (2) In the presence of mucosal amiloride, the power spectrum of the Na+-current noise showed a Lorentzian component. When the Na+ current was reduced by increasing the blocker concentrations, the Lorentzian plateau decreased and corner frequency increased. Macroscopic short-circuit current and current-noise data are evidence for a two-state mechanism of the blocker interaction with the Na+ channel. (3) On- and off-rate constants for the blocker-receptor reaction, single-channel currents and Na+-channel density were calculated at room temperature and at 37 degrees C. Also, the activation energy for the amiloride-receptor reaction was estimated. The microscopic parameters obtained for the Na+ channel in the colon were similar to those found for Na+ channels in other tight epithelia.

Amiloride↗