Willy Van Driessche

Ba2+-induced conductance fluctuations of spontaneously fluctuating K+ channels in the apical membrane of frog skin (Rana temporaria)

SummaryWe studied the influence of mucosal Ba2+ ions on the recently described (Zeiske & Van Driessche, 1979a, J. Membrane Biol.47:77) transepithelial, mucosa towards serosa directed K+ transport in the skin ofRana temporaria. The transport parametersG (conductance), PD (potential difference),Isc (short-circuit current, “K+ current”), as well as the noise ofIsc were recorded. Addition of millimolar concentrations of Ba2+ to the mucosal K+-containing solution resulted in a sudden but quickly reversible drop inIsc.G andIsc decreased continuously with increasing Ba2+ concentration, (Ba2+)o. The apparent Michaelis constant of the inhibition by Ba2+ lies within the range 40–80 μm. The apical membrane seems to remain permselective for K+ up to 500 μm (Ba2+)o. Higher (Ba2+)o, however, appears to induce a shunt (PD falls,G increases). This finding made an accurate determination of the nature of the inhibition difficult but our results tend to suggest a K+-channel block by K+−Ba2+ competition. In the presence of Ba2+, the power spectrum of the K+ current shows a second Lorentzian component in the low-frequency range, in addition to the high-frequency Lorentzian caused by spontaneous K+-channel fluctuations (Van Driessche & Zeiske, 1980). Both Lorentzian components are only present with mucosal K+ and can be depressed by addition of Cs+ ions, thus indicating that Ba2+ ions induce K+-channel fluctuations. The dependence of the parameters of the induced Lorentzian on (Ba2+)o, shows a rise in the plateau values to a maximum around 60 μm (Ba2+)o, followed by a sharp and progressive decrease to very low values. The corner frequency which reflects the rate of the Ba2+-induced fluctuations, however, increases quasi-linearly up to 1mm (Ba2+)o with a tendency to saturate at higher (Ba2+)o. Based on a three-state model for the K+ channel (having one open state, one closed by the spontaneous fluctuation and one blocked by Ba2+) computer calculations compared favorably with our results. The effect of Ba2+ could be explained by assuming reversible binding at the outer side of the apical K+ channel, thereby blocking the open channel in competition with K+. The association-dissociation of Ba2+ at its receptor site is thought to cause a chopping of the K+ current, resulting in modulated current fluctuations.

The role of epithelial P2Y2 and P2Y4 receptors in the regulation of intestinal chloride secretion

UTP‐induced chloride secretion by the intestinal mucosa mounted in Ussing chambers was assessed by measurement of the short‐circuit current (Isc) in the presence of phloridzin in the case of jejunum or amiloride in the case of colon to eliminate any contribution of electrogenic Na+ movement to the net ionic transport. Since we have previously demonstrated the absence of chloride‐secretory response to apical UTP in the jejunum from P2Y4‐null mice, in the present study we studied the response to basolateral UTP in the jejunum and to either apical or basolateral UTP in the colon, in both P2Y2‐ and P2Y4‐deficient mice. In the jejunum, the chloride‐secretory response to basolateral UTP was partially reduced in both P2Y2‐ (40%) and P2Y4‐ (60%) null mice. In the colon, both apical or basolateral UTP increased the Isc. That response was abolished in a chloride‐free medium. The colonic chloride‐secretory response to either basolateral or apical UTP was abolished in P2Y4‐deficient mice, but not significantly affected in P2Y2‐deficient mice. The chloride‐secretory response to forskolin was potentiated by prior basolateral addition of UTP and this potentiation was abolished in P2Y4‐null mice. The jejunum of mice homozygous for the ΔF508 mutation of cystic fibrosis transmembrane conductance regulator was responsive to UTP, but the magnitude of that response was smaller than in the wild‐type littermates. In conclusion, the P2Y4 receptor fully mediates the chloride‐secretory response to UTP in both small and large intestines, except at the basolateral side of the jejunum, where both P2Y2 and P2Y4 receptors are involved.

A high-throughput silicon microphysiometer

Abstract We report on a micromachined silicon chip that is capable of providing a high-throughput functional assay based on calorimetry. A calorimeter has been fabricated by IC technology process steps in combination with micromachined techniques. A rubber membrane supports two identical chambers, situated at the cold and hot junction sites of a thermopile. The thermopile consists of 666 aluminum/p + -polysilicon thermocouples. The chambers can house up to 10 6 eukaryotic cells cultured to confluence, in volumes of 10–600 μl. Power and temperature sensitivity of the sensor are 23 V/W and 130 mV/K, respectively. The response time of the sensor is 70 s, when filled with 50 μl of water. Biological experiments were done with cultured kidney cells of Xenopus laevis (A6). The thermal equilibration time of the device is 45 min. Basal metabolism is measured to be 330 pW/cell. Stimulation of transport mechanisms by reducing bath osmolality by 50% increased metabolism by 40 pW/cell. Stimulation of transport mechanisms by adding the oxytocin hormone increased metabolism by 106 pW/cell.

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 Ringers, 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 calculated the density of amiloride blockable channels and the current through one single channel. Despite the large reduction of the macroscopic current by oubain, 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.

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 Ringers 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 monovalents cations when normal Ca2+ containing Ringers are used as mucosal bathing medium.

Hypotonic treatment evokes biphasic ATP release across the basolateral membrane of cultured renal epithelia (A6)

In renal A6 epithelia, an acute hypotonic shock evokes a transient increase in the intracellular Ca2+ concentration ([Ca2+]i) through a mechanism that is sensitive to the P2 receptor antagonist suramin, applied to the basolateral border only. This finding has been further characterized by examining ATP release across the basolateral membrane with luciferin‐luciferase (LL) luminescence. Polarized epithelial monolayers, cultured on permeable supports were mounted in an Ussing‐type chamber. We developed a LL pulse protocol to determine the rate of ATP release (RATP) in the basolateral compartment. Therefore, the perfusion at the basolateral border was repetitively interrupted during brief periods (90 s) to measure RATP as the slope of the initial rise in ATP content detected by LL luminescence. Under isosmotic conditions, 1 μl of A6 cells released ATP at a rate of 66 ± 8 fmol min−1. A sudden reduction of the basolateral osmolality from 260 to 140 mosmol (kg H2O)−1 elevated RATP rapidly to a peak value of 1.89 ± 0.11 pmol min−1 (RATPpeak) followed by a plateau phase reaching 0.51 ± 0.07 pmol min−1 (RATPplat). Both RATPpeak and RATPplat values increased with the degree of dilution. The magnitude of RATPplat remained constant as long as the hyposmolality was maintained. Similarly, a steady ATP release of 0.78 ± 0.08 pmol min−1 was recorded after gradual dilution of the basolateral osmolality to 140 mosmol (kg H2O)−1. This RATP value, induced in the absence of cell swelling, is comparable to RATPplat. Therefore, the steady ATP release is unrelated to membrane stretching, but possibly caused by the reduction of intracellular ionic strength during cell volume regulation. Independent determinations of dose‐response curves for peak [Ca2+]i increase in response to exogenous ATP and basolateral hyposmolality demonstrated that the exogenous ATP concentration, required to mimic the osmotic reduction, was linearly correlated with RATPpeak. The link between the ATP release and the fast [Ca2+]i transient was also demonstrated by the depression of both phenomena by Cl− removal from the basolateral perfusate. The data are consistent with the notion that during hypotonicity, basolateral ATP release activates purinergic receptors, which underlies the suramin‐sensitive rise of [Ca2+]i during the hyposmotic shock.

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.

Saturable K+ pathway across the outer border of frog skin (rana temporaria): kinetics and inhibition by Cs+ and other cations.

SummaryThe reaction of abdominal skins of the frog speciesRana temporaria on mucosal K+-containing solutions was studied in an Ussing-type chamber by recording transepithelial potential difference (PD), short-circuit current (SCC) and conductance (G). With Na-Ringers as serosal medium, a linear correlation between PD and the logarithm of the mucosal K+-concentration ([K]o) was obtained. The K+-dependent SCC saturated with increasing [K]o, and could quickly and reversibly be depressed by addition of Rb+, Cs+, and H+, Li+, Na+, and NH4+ did not influence K+ current. A large scatter was obtained for kinetic parameters like the slope of the PD-log [K]o-line (18–36.5 mV/decade), the apparent Michaelis constant (13–200mm), and the maximal current of the saturable SCC (6–50 μA·cm−2), as well as for the degree of inhibition by Cs+ ions. This seemed to be caused by a time-dependent change during long time exposure to high [K]o (more than 30 sec), thereby inducing a selectivity loss of K+-transporting structures, together with an increase in SCC andG and a decrease in PD. Short time exposure to K+-containing solutions showed a competitive inhibition of K+ current by Cs+ ions, and a Michaelis constant of 6.6mm for the inhibitory action of Cs+. Proton titration resulted in a decrease of K+ current at pH<3. An acidic membrane component (apparent dissociation constant 2.5×10−3m) is virtually controlling K+ transfer. Reducing the transepithelial K+-concentration gradient by raising the serosal potassium concentration was accompanied by the disappearance of SCC and PD.

Noise analysis of the K+ current through the apical membrane of Necturus gallbladder

SummaryCurrent noise power spectra of the voltage-clamped(V=0) Necturus gallbladder, exposed to NaCl-Ringers on both sides contained a relaxation noise component, which overlapped with a 1/fα noise component, with α being about 2. Substitution of all Na+ by K+ on either the serosal or mucosal side increased the relaxation as well as the 1/fα noise component considerably. InNecturus gallbladder both noise components are reduced by addition of 10mm, 2,4,6-triaminopyrimidine (TAP) or 5mm Ba2+ to the mucosal side, as well as by acidification of the mucosal solution to pH5 and lower. Fivemm of tetraethylammonium (TEA+) added to the mucosal solution, abolished K+ relaxation noise and decreased the 1/fα noise component. Applying a Cs+ concentration gradient across the epithelium did not yield relaxation noise. However, if Rb+ was substituted for all Na+ on one side, a Lorentzian noise component appeared in the spectrum. Its plateau was smaller than with KCl-Ringers on the respective side. These data confirm the existence of fluctuating K+ channels in the apical membrane of theNecturus gallbladder. Furthermore it can be concluded that these channels have the permeability sequence K+>Rb+>Cs+. The inhibition of the fluctuation by mucosal acidification indicates the existence of acidic sites in the channel. The singlechannel conductance was estimated to be between 6.5 and 40 pS.

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

Abstract1.The isolated midgut of a lepidopteran larva (Manduca sexta), 5th instar was investigated with voltageclamp and fluctuation analysis techniques.2.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+.3.Noise analysis ofIK 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>KBa, the apparrent 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.4.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.