Intracellular gradients of electrical potential in the epithelial cells of theNecturus gallbladder

Summary When single-barrelled electrodes (5–60 M) were advanced under manual control from the mucosal side of the epithelium the mucosal membrane was on average indented by about 40 m before the microelectrode penetrated the cell. Since this dimpling was comparable with the total depth of the cell, which recovered its original shape within 0.5 sec, the steady intracellular potential was recorded only about 14 m from the basal (serosal) membrane. Fast recording of the associated change in potential revealed an abrupt drop to –26 mV at a mean rate of 84 V/sec, followed by a further slow drop to a steady value of about –50 mV at a mean rate of 0.28 V/sec. The initial level of –26 mV may be regarded as the potential difference across the mucosal membrane. This conclusion was confirmed by mounting the microelectrode on a piezoelectric probe, which delivered 3 m jabs in less than 0.5 msec. With this device in operation to prevent dimpling, the mean potential difference across the mucosal membrane was recorded as –29 mV. In all cases the potential across the basal membrane was recorded as –52 mV. Manual advance of the microelectrode tip within the cytoplasm yielded an intracellular potential gradient of 0.6 mV/m. The same potential profile and membrane potentials were demonstrated on penetrating the epithelium from the serosal side, and measurements with multibarrelled electrodes whose tips were staggered in depth gave roughly the same internal potential gradient. The resistivity of the cytoplasm was determined by a triple-barrelled microelectrode, and varied from 10 times that ofNecturus saline at the mucosal end of the cell to 4 times in the middle and 6 times at the serosal end.     

Measuring extracellular ion gradients from single channels with ion-selective microelectrodes

Under many different conditions activated plasma membrane ion channels give rise to changes in the extracellular concentration of the permeant ion(s). The magnitude and duration of these changes are dependent on the electrochemical driving force(s) on the permeant ion(s) as well as conductance, open time, and channel density. We have modeled the change in the extracellular [K+] due to efflux through Ca2+-activated K+ channels, mSlo, to determine the range of parameters that would give rise to measurable signals in the surrounding media. Subsequently we have used extracellular, K+-selective microelectrodes to monitor localized changes in [K+]ext due to efflux through mSlo channels expressed in Xenopus oocytes. The rapid changes in [K+] show a close fit with the predicted model when the time response of the ion-selective microelectrode is taken into account, providing proof of the concept. Measurement of the change in extracellular ion concentration with ion-selective microelectrodes provides a noninvasive means for functional mapping of channel location and density, as well as characterizing the properties of ion channels in the plasma membrane.     

Intracellular measurement of ammonium in Chara corallina using ion-selective microelectrodes

The study of ammonium (NH₄ ⁺) transport across plant cell membranes requires accurate measurement of NH₄ ⁺ gradients across subcellular gradients. We have developed an ammonium-selective microelectrode based on the ionophore nonactin. This electrode can detect NH₄ ⁺ activities (a_NH4) in vivo in the millimolar range in the presence of cytosolic levels of potassium, the main interfering ion. The electrode was used to measure intracellular a_NH4 in internodal cells of the giant alga Chara corallina. Results from cells incubated in media supplemented with 1 mM NH₄ ⁺ produced two populations, with means of 7.3 and 30.8 mM, respectively. HPLC analysis of vacuolar sap suggests the higher population represents vacuolar impalements, and the lower population can thus be assumed to be cytosolic. These results suggest a four-fold accumulation of NH₄ ⁺ in the vacuolar compartment of Chara. This revised version was published online in June 2006 with corrections to the Cover Date.     

Regulation of paracellular ion conductances by NaCl gradients in renal epithelial cells

In the present study, we clarified how the NaCl gradient across the epithelial cells regulates the paracellular ion conductance. Under isotonic conditions, the absorption-directed NaCl gradient elevated the paracellular conductances of Na(+) (G(Na)) and Cl(-) (G(Cl)), while the secretion-directed NaCl gradient diminished the G(Na) and G(Cl). We further investigated the paracellular ionic conductances of NMDG (G(NMDG)) and gluconate (G(gluconate)) by replacing Na(+) with NMDG or Cl(-) with gluconate. The G(NMDG) was lower than the G(Na) and the replacement of Na(+) with NMDG decreased the G(Cl). The G(gluconate) was lower than the G(Cl) and the replacement of Cl(-) with gluconate also decreased the G(Na). These observations suggest the interaction of cations and anions on paracellular ionic conductances; i.e., cations affect paracellular anion conductances and anions affect paracellular cation conductances.     

Intracellular pH gradients in migrating cells

Cell polarization along the axis of movement is required for migration. The localization of proteins and regulators of the migratory machinery to either the cell front or its rear results in a spatial asymmetry enabling cells to simultaneously coordinate cell protrusion and retraction. Protons might function as such unevenly distributed regulators as they modulate the interaction of focal adhesion proteins and components of the cytoskeleton in vitro. However, an intracellular pH (pH(i)) gradient reflecting a spatial asymmetry of protons has not been shown so far. One major regulator of pH(i), the Na(+)/H(+) exchanger NHE1, is essential for cell migration and accumulates at the cell front. Here, we test the hypothesis that the uneven distribution of NHE1 activity creates a pH(i) gradient in migrating cells. Using the pH-sensitive fluorescent dye BCECF, pH(i) was measured in five cell lines (MV3, B16V, NIH3T3, MDCK-F1, EA.hy926) along the axis of movement. Differences in pH(i) between the front and the rear end (ΔpH(i) front-rear) were present in all cell lines, and inhibition of NHE1 either with HOE642 or by absence of extracellular Na(+) caused the pH(i) gradient to flatten or disappear. In conclusion, pH(i) gradients established by NHE1 activity exist along the axis of movement.     

Intracellular stores maintain stable cytosolic Ca gradients in epithelial cells by active Ca redistribution

A stable localized region of high calcium concentration near the plasma membrane has been postulated to exist as an outcome of prolonged calcium influx and to play a crucial role in regulation of cellular life. However, the mechanism supporting this phenomenon is a perplexing problem. We show here that a sustained localized region of high cytosolic Ca(2+) concentration is formed near the plasma membrane. Calcium influx, calcium uptake by intracellular stores and calcium release from the stores are essential for this phenomenon. Our results strongly suggest that the mechanism of formation of stable calcium gradient near the plasma membrane involves a process of active redistribution-uptake of entering calcium into intracellular stores and its release from the stores toward the plasma membrane.     

Intracellular ion activities in Necturus proximal tubule

Ion-sensitive microelectrodes were used to measure the intracellular activities of Na, K, and Cl in proximal tubules of the perfused Necturus kidney. Cell Cl was 2-3 times higher than the value predicted for passive distribution during perfusion with normal Ringer; intracellular Na was far below the level for passive distribution. Cell Na and Cl fell to very low values when the lumen was NaCl-free. Cl entry into the tubule cell from the lumen required luminal Na. Na entered the cell across the luminal membrane both by diffusion and by coupled movement with Cl.     

Streaming potentials in gramicidin channels measured with ion-selective microelectrodes.

Streaming potentials have been measured for gramicidin channels with a new method employing ion-selective microelectrodes. It is shown that ideally ion-selective electrodes placed at the membrane surface record the true streaming potential. Using this method for ion concentrations below 100 mM, approximately seven water molecules are transported whenever a sodium, potassium, or cesium ion, passes through the channel. This new method confirms earlier measurements (Rosenberg, P.A., and A. Finkelstein. 1978. Interaction of ions and water in gramicidin A channels. J. Gen. Physiol. 72:327-340) in which the streaming potentials were calculated as the difference between electrical potentials measured in the presence of gramicidin and in the presence of the ion carriers valinomycin and nonactin.     

The effect of electrical gradients on current fluctuations and impedance recorded fromNecturus gallbladder

Summary Transepithelial current fluctuations were recorded inNecturus gallbladder, clamped at negative as well as positive potentials up to 64 mV. With NaCl-Ringer's (+10mm TAP) on both sides a mucosa-negative potential enhanced the relaxation noise component, present at zero potential, and produced peaking in the power spectrum at potentials above –36mV. Concomitantly at these potentials an inductive as well as a capacitive low-frequency feature appeared in the impedance locus. Clamping at positive potentials of 18 mV suppressed the relaxation noise component. At potentials above 51mV the spectral values increased predominantly at low frequencies. In this case the power spectrum showed only a 1/f noise component. The experiments confirm the previous finding that a K⁺ efflux through fluctuating apical K⁺ channels exists under normal conditions. With serosal KCl-Ringer's the initial Lorentzian component was enhanced at negative but suppressed at positive potentials. The increase at negative potentials was less pronounced than in experiments with NaCl-Ringer's on both sides, indicating saturation of the fluctuating K⁺ current component. With mucosal KCl-Ringer's a negative potential depressed the initial relaxation noise component, whereas it was enhanced at +18 mV clamp potential. In the latter case an additional Lorentzian component became apparent at higher frequencies. At potentials of 36 mV and above the low-frequency Lorentzian disappeared whereas the corner frequency of the high-frequency component increased. The latter experiments demonstrate that the relaxation noise component inNecturus gallbladder consists of two superimposed Lorentzians. As the relaxation times of these two components behave differently under an electrical field, there may exist two different types of K⁺ channels. It is demonstrated that peaking in the plateau of power spectra can be explained by frequency-dependent attenuation effects, caused by a polarization impedance.     

Schistosoma mansoni: measurement of Na+ ion activity in the tegument and the extracellular spaces using ion-selective microelectrodes

Ion-selective microelectrodes were used to measure sodium ion activity (aNa) in the tegument and interstitial spaces in adult male Schistosoma mansoni. In RPMI 1640, aNa averaged 31 +/- 13 mM in the tegument, a value significantly less than that in the bathing medium. In the interstitial spaces, it averaged 72 +/- 17 mM, a value nearly the same as that in the bathing medium. In hypo- or hyperosmotic media, aNa in the interstitial spaces varied by a value commensurate with change in aNa in the medium, but aNa in the tegument was changed by only a small amount. Monensin (10 microM), low temperature (20 C), and ouabain (0.3 to 10 microM) all caused significant increases in aNa in the tegument. Hypo- and hyperosmotic media produced initial weight changes followed by gradual recovery back toward original weights. It is concluded that the schistosome is a volume regulating osmoconformer with osmolality of the extracellular fluid approximating that of the bathing medium, but that within the tegument of the parasite, Na+ concentration is controlled by active transport processes.     

Intracellular ionic activities and transmembrane electrochemical potential differences in gallbladder epithelium

Summary Intracellular ion activities inNecturus gallbladder epithelium were measured with liquid ion-exchanger microelectrodes. Mean values for K, Cl and Na activities were 87, 35 and 22mm, respectively. The intracellular activities of both K and Cl are above their respective equilibrium values, whereas the Na activity is far below. This indicates that K and Cl are transported uphill toward the cell interior, whereas Na is extruded against its electrochemical gradient. The epithelium transports NaCl from mucosa to serosa. From the data presented and the known Na and Cl conductances of the cell membranes, we conclude that neutral transport driven by the Na electrochemical potential difference can account for NaCl entry at the apical membrane. At the basolateral membrane, Na is actively transported. Because of the low Cl conductance of the membrane, only a small fraction of Cl transport can be explained by diffusion. These data suggest that Cl transport across the basolateral membrane is a coupled process which involves a neutral NaCl pump, downhill KCl transport, or a Cl-anion exchange system.     

Steady states and the effects of ouabain in theNecturus gallbladder epithelium: A model analysis

Summary A simple numerical model for theNecturus gallbladder epithelium is presented. K⁺, Na⁺ and Cl^– cross the mucosal and serosal membranes as well as the junctions by means of electrodiffusion; furthermore the mucosal membrane contains a neutral entry mechanism for NaCl and the serosal membrane contains an active pump for K⁺ and Na⁺. The values which have been used for the model are taken from the literature. The model can only attain steady states if the resistance of the serosal membrane is lower than 1000 cm². Values reported in the literature for the resistance of this membrane vary from about 3000 to about 100 cm². We shall argue, however, that the higher estimates are in error because they are derived from a model of the tissue in which each membrane and the junction are modeled by a resistor; this procedure is invalid because the resistance of the lateral intercellular space relative to the resistance of the tight junctions is neglected and consequently the resistance of the serosal membrane is overestimated by a factor of about four. Apart from predicting a realistic steady state at normal external concentrations the model can predict quantitatively several experimental results obtained from the living epithelium. We have focused on the experiments which test the permeabilities of the serosal membrane and the properties of the pump:i) Replacement of serosal Cl^– by an impermeant ion.ii) Replacement of serosal K⁺ by Na⁺.iii) Inhibiting the (Na⁺, K⁺)-pump. The best correspondence between model and experiments is obtained when the pump is assumed to be electrogenic (or rheogenic) with a ratio of coupling between Na⁺ and K⁺ of 32. In this case both model and direct experiments (also presented in this paper) show an initial abrupt depolarization of 6 to 7 mV. The model also shows that it cannot be concluded fromi andii that the Cl^– permeability of the serosal membrane is low. The model explains, even with high passive Cl^– permeabilities, why the intracellular Cl^– concentration is relatively unaffected by paracellular currents, a fact which in other epithelia has been taken as an implication of a low Cl^– permeability of the serosal membranes.     

Ion fluxes in giant excised cardiac membrane patches detected and quantified with ion-selective microelectrodes

We have used ion-selective electrodes (ISEs) to quantify ion fluxes across giant membrane patches by measuring and simulating ion gradients on both membrane sides. Experimental conditions are selected with low concentrations of the ions detected on the membrane side being monitored. For detection from the cytoplasmic (bath) side, the patch pipette is oscillated laterally in front of an ISE. For detection on the extracellular (pipette) side, ISEs are fabricated from flexible quartz capillary tubing (tip diameters, 2-3 microns), and an ISE is positioned carefully within the patch pipette with the tip at a controlled distance from the mouth of the patch pipette. Transport activity is then manipulated by solution changes on the cytoplasmic side. Ion fluxes can be quantified by simulating the ion gradients with appropriate diffusion models. For extracellular (intrapatch pipette) recordings, ion diffusion coefficients can be determined from the time courses of concentration changes. The sensitivity and utility of the methods are demonstrated with cardiac membrane patches by measuring (a) potassium fluxes via ion channels, valinomycin, and Na/K pumps; (b) calcium fluxes mediated by Na/Ca exchangers; (c) sodium fluxes mediated by gramicidin and Na/K pumps; and (d) proton fluxes mediated by an unknown electrogenic mechanism. The potassium flux-to-current ratio for the Na/K pump is approximately twice that determined for potassium channels and valinomycin, as expected for a 3Na/2K pump stoichiometery (i.e., 2K/charge moved). For valinomycin-mediated potassium currents and gramicidin-mediated sodium currents, the ion fluxes calculated from diffusion models are typically 10-15% smaller than expected from the membrane currents. As presently implemented, the ISE methods allow reliable detection of calcium and proton fluxes equivalent to monovalent cation currents <1 pA in magnitude, and they allow detection of sodium and potassium fluxes equivalent to <5 pA currents. The capability to monitor ion fluxes, independent of membrane currents, should facilitate studies of both electrogenic and electroneutral ion-coupled transporters in giant patches.     

Measurement of intracellular calcium ion activity with neutral exchanger ion sensitive microelectrodes

Micropipettes filled with the neutral liquid ion exchanger ETH 1001 can be used to make microelectrodes that are sensitive to cytoplasmic levels of Ca2+. They are high resistance electrodes, so that care is required in order to record the low current signal. The electrodes often yield 10-15 mV change between intracellular Ca2+ activities of 10(-6) and 10(-7) M, according to a log relation. The microelectrodes are non-destructive, even in rather small cells, and can be used to monitor Ca2+ changes during experimental interventions.     

Intracellular activities of Salmonella enterica in murine dendritic cells

Dendritic cells (DC) efficiently phagocytose invading bacteria, but fail to kill intracellular pathogens such as Salmonella enterica serovar Typhimurium (S. Typhimurium). We analysed the intracellular fate of Salmonella in murine bone marrow-derived DC (BM-DC). The intracellular proliferation and subcellular localization were investigated for wild-type S. Typhimurium and mutants deficient in Salmonella pathogenicity island 2 (SPI2), a complex virulence factor that is essential for systemic infections in the murine model and intracellular survival and replication in macrophages. Using a segregative plasmid to monitor intracellular cell division, we observed that, in BM-DC, S. Typhimurium represents a static, non-dividing population. In BM-DC, S. Typhimurium resides in a membrane-bound compartment that has acquired late endosomal markers. However, these bacteria respond to intracellular stimuli, because induction of SPI2 genes was observed. S. Typhimurium within DC are also able to translocate a virulence protein into their host cells. SPI2 function was not required for intracellular survival in DC, but we observed that the maturation of the Salmonella-containing vesicle is different in DC infected with wild-type bacteria and a strain deficient in SPI2. Our observations indicate that S. Typhimurium in DC are able to modify normal processes of their host cells.