Liquid Ion-exchanger Microelectrodes Research Articles

B-6. 生体膜細胞への液体イオン交換膜微小電極の応用

B-6. 生体膜細胞への液体イオン交換膜微小電極の応用

Thermodynamics of Symport and Antiport Catalyzed by Cloned or Native Transporters

Thermodynamic measurements are required to confirm whether cloned transport-associated proteins in a membrane truly constitute a functional transport system. Symport or antiport, catalyzed by native systems or by cloned proteins in membranes, can lead to steady-state intracellular accumulation of solute when the electrochemical potentials of activator ion and solute are energetically coupled. Secondary active transport can occur if an appropriate physical coupling mechanism exists in the membrane. Driving forces for secondary active transport are ultimately established by primary active transport or respiration. Steep steady-state gradients of solute can be maintained when the ion:solute coupling ratio is greater than one and/or when coupling includes an electrical component. Although the steady-state accumulation of substrate is independent of the exact physical mechanism of transport, non-equilibrium and equilibrium transport kinetics aid in interpreting the rate, direction (symport versus antiport) and control of ion-coupled flux across a membrane. In some cases, the activator ion's chemical gradient alone is energetically adequate to maintain steady-state intracellular accumulation of solute, as demonstrated in invertebrate epithelial cells. To interpret accumulation ratios accurately, it is necessary to measure the intracellular activity coefficients for ions. For example, liquid ion-exchange microelectrode measurements demonstrate that over 30% of intracellular Na+ can be sequestered in epithelial cells.

Evidence for an acid pH in rat renal inner medulla: paired measurements with liquid ion-exchange microelectrodes on collecting ducts and vasa recta.

We reevaluated the pH in the renal medulla in rats. pH of the vasa recta blood was about 1 pH unit acidic in comparison to the pH of renal artery blood. During furosemide-induced diuresis pH of vasa recta blood increased whereas pH of collecting duct urine further decreased. The acidic pH in the rat renal inner medulla during antidiuresis raises important questions about the source of H+ in inner medulla.

Ion-selective microelectrodes for intracellular measurements

Ion-selective electrodes have been used for many years to analyze ion activities in electrolyte solutions. In recent years, these electrodes have been miniaturized and applied for measurements in the cell interior. This article describes basic principles for both the fabrication and the intracellular application of ion-selective microelectrodes, particularly focusing on double barreled liquid ion-exchanger microelectrodes. The use of ion-selective microelectrodes allows continuous and real-time measurements of intracellular ion activities of a single cell in both multicellular and isolated cell preparations under various conditions, such as changes in ion composition of the extracellular bathing fluid and exposure to certain drugs. With double-barreled ion-selective microelectrodes, the transmembrane potential and intracellular ion activities can be measured simultaneously in the same cell. Although there are still some problems or limitations to the intracellular application of ion-selective electrodes, this technique is useful for determining the actual movement of intracellular ions, and thereby to elucidate cellular mechanisms of membrane transport and other physiological functions.

Electrochemical potentials of potassium in skeletal muscle under different metabolic states.

Intracellular potassium and membrane potential were measured simultaneously by means of double-barrelled liquid ion-exchange microelectrodes in single fibers of rat thigh muscle in vivo in rats maintained in seven different metabolic states. The K+ equilibrium potential (EK) was more negative than the simultaneously measured membrane potential (Em) in the normal state by 18.4 mV. K+ loading, acute and chronic, resulted in depolarization of Em due to increased serum K+ (hyperkalemia) with no increase in intracellular K+. K+ depletion resulted in hyperpolarization of Em as plasma K+ decreased proportionately more than intracellular K+. Low Na+ diet had no effect. Intracellular K+ was decreased in acute acidosis but not in the chronic state. Thus K+ depletion and acute acidosis are associated with intracellular K+ decrease. The fact that hyperpolarization exists in the former and not the latter is a reflection that hypokalemia accompanies the former condition. The hyperpolarizing states of K+ depletion and chronic acidosis are accompanied by decreased excitability and muscle weakness.

Liquid Ion Exchanger Microelectrodes Used to Study Soil Solute Concentrations near Plant Roots

AbstractA technique for using Na+ liquid ion exchanger (Na+‐LIX) microelectrodes to accurately measure, repetitively, and in a nondestructive fashion, Na+ concentrations in the soil solution at single points is described. The method has been used to examine solute (NaCl) accumulation in proximity to single roots of lupine (Lupinus angustifolius L.) and radish (Raphanus sativus L.) plants subjected to two levels of transpirational demand and five soil solute concentrations. The results obtained indicate that, at soil water contents corresponding to matric water potentials >−140 kPa, back diffusion of solute significantly counteracts the tendency for solute accumulation at root surfaces during transpiration.

Measurement of Na+ and K+ in nanoliter droplets by ion-specific microelectrodes

The measurement of electrolyte concentration in nanoliter quantities of fluid is a fundamental step in the utilization of micropuncture and isolated tubule microperfusion techniques for studying ion transport. A number of methodologies have been developed for these analyses, including helium glow photometry [1], electron probe microanalysis [2], and atomic absorption spectrophotometry [3]. Because of the significant cost of the equipment for performing atomic absorption spectrophotometry or electron probe microanalysis, and the lack of commercial availability of helium glow photometers, we decided to utilize ion-specific electrodes for the measurement of sodium and potassium in samples from isolated tubule microperfusion experiments. The application of ion-specific micro-electrodes to the measurement of intracellular ion activity and macroanalysis is well established [4], but the technique has not been widely applied to the analysis of electrolyte concentration in nanoliter droplets, although Garvin has recently developed a flow-through ion-selective electrode apparatus for measurement of potassium concentrations [5]. Since aqueous droplets must be analyzed under oil to prevent evaporation, one problem we had to overcome was the deterioration in the sensitivity of liquid ion exchanger microelectrodes on contact with oil. Other investigators have utilized ion-selective microelectrodes to study intracellular ion activities or ion activities in tubular fluid in situ, circumstances in which the tissue is bathed in an aqueous electrolyte solution rather than mineral oil [6–8]. In the case where measurements have been made in aqueous droplets under oil, an ion-selective glass rather than a liquid ion exchanger microelectrode was used [9]. With liquid ion exchangers, we found that the sensitivity of the electrode decreased markedly on contact with mineral oil, probably due to the lipophilicity of the ionophore. Although it is possible to utilize very small tips and holders with sidearms to express contaminated ionophore (S. Agulian, personal communication), we found this technique cumbersome and have adapted the methods described by Vogel et al [10], which utilizes a desilanization step to allow a droplet of aqueous solution to protect the ionophore from oil contamination. We have applied electrodes constructed using this technique to the measurement of electrolyte concentrations in aqueous nanoliter droplets under oil. The equipment for this analysis is relatively inexpensive, available in many microperfusion laboratories, and can be applied to a variety of ions, and therefore the technique may be valuable to a wide spectrum of investigators.

The changes in CiNa of sheep cardiac purkinje fibres in hyperosmotic solutions

1. 1. The changes in intracellular sodium ion concentration ( C i Na ) of sheep cardiac Purkinje fibres in hyperosmotic solutions were studied using Na-sensitive liquid ion-exchanger microelectrodes. 2. 2. C i Na was increased in hyperosmotic solutions containing different concentrations of sucrose from 0 to 300 mM. 3. 3. The changes in resting membrane potential (RMP) in hyperosmotic solutions had no regularity. In most of the experiments there was hyperpolarization of the membrane but in a few cases a depolarization or no change of RMP were also observed. 4. 4. The N-shape of I- V relations of the fibres became more pronounced in hyperosomotic solutions.

Demonstration and modification of intervillous pH profiles in rat small intestine in vitro

The intervillous pH profiles along the crypt villus axis in different regions of the rat small intestine were measured in vitro by using pH-sensitive liquid ion-exchanger microelectrodes. A characteristic pH profile was observed in the duodenum and jejunum. A region of low pH was detected in the upper parts of the villi (pH 6.65 +/- 0.06 to 6.85 +/- 0.07), whereas pH at the villus base was always higher. In the ileum no gradient was observed (pH 7.26 +/- 0.05 to 7.31 +/- 0.05). Preincubation of the tissue in situ with 10 mM theophylline for 1 h caused an increase in the villus base pH in the jejunum (pH 7.24 +/- 0.04) and ileum (7.44 +/- 0.04) followed by a subsequent increase of the pH in the upper part of the villi. These results indicate that the low pH in the upper intervillous space may be related to H+ secretion occurring from the mature enterocytes, whereas the crypt cells may secrete a rather neutral or slightly alkaline fluid. Alkaline secretion from the crypts may be increased by theophylline, which changes the levels of cyclic nucleotides in the mucosa.

PH in eggs of the ascidian Ciona intestinalis at fertilization and activation

AbstractUsing liquid ion exchanger microelectrodes, we have measured the intracellular pH of unfertilized ascidian eggs. The pH in these eggs ranges from 7.1 to 7.6 (similar to values for fertilized sea urchin eggs) and does not change during the first 7 minutes of activation, whether induced by spermatozoa or by the Ca2+ ionophore A23187. Factors regulating metabolic derepression in regulative and mosaic eggs are discussed.

Double-barreled K+-selective microelectrodes based on dibenzo-18-crown-6.

Liquid ion-exchanger microelectrodes based on Corning code 477317 K+ exchanger are known to be much more sensitive to quaternary ammonium ions than to K+. In the presence of such cations, the capability of measuring K+ activities with Corning microelectrodes may be seriously impaired. We have developed a neutral carrier K+-selective microelectrode based on the crown ether dibenzo-18-crown-6. The crown ether cocktail contained (wt/wt) 2.3% dibenzo-18-crown-6, 0.8% Na-tetraphenylborate, 30.1% 2-nitrophenylocylether, and 66.8% O-nitrotoluene. Double-barreled crown ether and Corning microelectrodes were calibrated in KCl solutions with or without choline, acetylcholine, tetramethylammonium, imidazole, Na+, tris(hydroxymethyl)aminomethane (Tris), and N-methyl-D-glucamine. Both kinds of microelectrodes showed similar K+ over Na+, Tris, and N-methyl-D-glucamine selectivities. However, crown ether microelectrodes had immensely greater selectivities of K+ over quaternary ammonium ions and imidazole than Corning microelectrodes. Selectivity factors, defined as log K(ij)K, of crown ether microelectrodes with respect to K+ for tetramethylammonium, choline, acetylcholine, and imidazole were -1.92 +/- 0.13, -2.97 +/- 0.03, -1.75 +/- 0.15, and -1.30 +/- 0.20, respectively. Intracellular K+ activities measured in the same Necturus gallbladders with both kinds of microelectrodes did not differ significantly.

Dependence of cell pH and buffer capacity on the extracellular acid-base change in the skeletal muscle of bullfrog.

Intracellular pH was measured with single- or double-barreled liquid ion-exchanger microelectrodes in the bullfrog sartorius muscle perfused in vitro. A neutral carrier ligand was used for pH sensor of microelectrodes. Average slopes of the single-barreled microelectrodes were -56.4 +/- 1.34 mV/pH (n = 30) and the double-barreled -52.6 +/- 1.34 mV/pH (n = 65). While changing acid-base parameters of bathing media (pHe from 6.7 to 8.4, PCO2 from 3.7 to 37 mmHg, and HCO3- concentrations from 5 to 75 mM), paired muscle cell pH (pHi) and membrane potential (EM) values were determined at 23 degrees C. In control conditions (pHe = 7.6, HCO3- = 15 mM, PCO2 = 11 mmHg), pHi and EM (n = 20) averaged 6.99 +/- 0.04 (S.E.) and -69.2 +/- 2.2 mV, respectively. A negative correlation was observed between pHi and EM (correlation coefficient r = -0.564, p less than 0.002). The change in EM per unit pH change was approximately -30mV, indicating that the H+ distribution across the cell membrane only incompletely obeys the Donnan rule. The pHi varied more or less with pHe. Namely, changes in pHe and PCO2 at constant HCO3- produced relatively large changes in pHi, but elevation of pHe and HCO3- at constant PCO2 produced relatively minor rise in pHi. The stability of pHi or the size of buffer capacity were proportional to external HCO3- concentrations. These data suggested that a transmembrane distribution of buffer pairs depends largely on non-ionic diffusion of CO2-HCO3- buffer system and partly on ion fluxes of HCO3- or H+.

PH in the Jelly Layer of Starfish Oocytes: (starfish oocytes/jelly layer/pH).

Using liquid ion exchanger micro-electrodes we have studied the pH in the vicinity of starfish oocytes and eggs and also in their jelly layers. Although highly variable, the pH in these zones was consistently lower (6.3-7.6) than the pH of sea water (7.8-8.2). Aged oocytes/eggs presented more acid environments than fresh oocytes. The pH indicator Phenol Red shows that the ovarian fluid is more acid than seawater, ranging from 6.0 to 7.5. Our data suggests that starfish oocytes, from their time in the ovary until several hr after their release, are surrounded by an acid environment. This micro-environment may play a role in metabolic repression in the ovary and in the regulation of sperm-egg interaction following spawning.

Thirty years of ion-selective microelectrodes: disappointments and successes.

The need to know the intracellular activity of an ion and how it changes under controlled conditions is as important today as it was 30 years ago. In 1956, one could fabricate only a H+-selective microelectrode and with a tip size not much smaller than 100 micron. Today, one can fabricate microelectrodes selective to H+, Na+, K+, Cl-, HCO3-, Ca2+, or Mg2+ (plus others) and with active tips less than 1 micron. The reduction of active tip size can be attributed mainly to the introduction of liquid ion exchanger (LIX) and neutral carrier ligands. Unfortunately, the LIX microelectrodes, as currently fabricated, do not yet function optimally as reliable and stable electrochemical measuring devices. A durable bond between the active membrane and its insulated container continues to remain the major design problem even after 30 years of development.

Electrophysiological evidence for ammonium as a substitute for potassium in activating the sodium pump in a crayfish sensory neuron.

Electrophysiological studies were performed on slowly adapting cells of the crayfish (Astacus astacus) stretch receptor to examine some aspects of the operation of the sodium pump. Intracellular sodium activity (aiNa) and pH (pHi) were measured with liquid ion exchanger microelectrodes and the effects of NH3/NH+4 were observed. In cells in which the sodium pump was inhibited by K+-free solution, NH+4 induced a decrease of aiNa that can be explained only in Na+ extrusion is assumed. pHi measurements provide indirect evidence that NH+4 was taken up at the same time as Na+ was extruded. Ouabain blocks the operation of the sodium pump in the presence of K+ and NH+4. This result suggests that the ammonium-mediated decrease in aiNa in K+-free solution was caused by activation of the sodium pump. The results obtained by electrophysiological methods in a living cell are qualitatively in good agreement when compared with biochemical investigations on assays of crustacean Na+-K+ ATPase.

Potassium microclimate at the mucosal surface of the proximal and the distal colon of guinea pig.

K+ concentrations were measured with K+ sensitive liquid ion exchanger microelectrodes in situ and in vitro in the mucus layer at the luminal cell surface of the proximal and the distal colon in guinea pig. In a first series of experiments K+ concentrations were increased in the luminal solution from 0 to 70 mmol X l-1; the serosal K+ concentrations were kept in vitro at 5.4 mmol X l-1. In the proximal colon mean K+ concentration in the microclimate was in vitro 7.9 +/- 3.5 mmol X l-1, and independent from mucosal concentrations. In the distal colon in vitro, and in situ in the proximal as well as in the distal colon, K+ concentrations in the microclimate were increased slightly when K+ concentrations were elevated in the luminal solution up to 70 mmol X l-1. In a second series of in vitro studies K+ concentrations were also altered in the serosal fluid. In the proximal and in the distal colon K+ concentrations increased linearly with elevated K+ concentrations in the serosal solutions. A temporarily interrupted mucosal blood flow resulted in a significant increase in the K+ concentration in the microclimate. A paracellular shunt pathway and a high preepithelial diffusion barrier for K+ would explain the observed K+ concentrations in the microclimate at the luminal cell surface.

Intracellular free magnesium in frog skeletal muscle fibres measured with ion‐selective micro‐electrodes.

Intracellular free Mg2+ concentration [( Mg2+]i) was measured in frog skeletal muscle fibres, using Mg2+-selective micro-electrodes based on the neutral ligand ETH-1117. In calibration solutions the electrodes showed significant interference from K+, and to a lesser extent from Na+, at concentrations found intracellularly. Therefore, in order to calibrate the electrodes properly, it was necessary first to obtain an accurate value for intracellular free Na+ and K+ concentrations ([Na+]i and [K+]i), using the appropriate liquid ion exchanger micro-electrodes. In fibres from muscles maintained in Ringer solution, the mean value for [Na+]i was 6.2 +/- 0.4 mM (S.E. of mean; n = 20 fibres in five muscles), while [K+]i was 104 +/- 1.7 mM (range 83-122 mM; n = 25 fibres in eight muscles). Due to the substantial variability found for [K+]i, not only between fibres from different muscles, but also between fibres belonging to the same muscle, it was necessary to measure [Mg2+]i and [K+]i simultaneously in the same fibre to determine as accurately as possible the degree of K+ interference on Mg2+-selective micro-electrode response. In nineteen fibres from six muscles maintained in Ringer solution, the mean [K+]i was 91.7 +/- 2.7 mM (range 71-110 mM), while the mean [Mg2+]i was 0.80 +/- 0.07 mM (range 0.2-1.2 mM). The mean resting potential was -79.3 +/- 0.4 mV (S.E. of mean). In fifteen fibres from four muscles equilibrated in Ringer solution containing 0.5 mM-Mg2+, the mean [K+]i was 115.5 +/- 0.1 mM (range 97-129 mM) and the mean [Mg2+]i measured simultaneously in the same fibres was 1.69 +/- 0.21 mM (range 0.2-2.7 mM). The mean resting potential was -83 +/- 0.7 mV. The mean [K+]i and [Mg2+]i found in these fibres was significantly higher (P less than 0.0001) than those measured in fibres from muscles maintained in standard Ringer solution (i.e. without external Mg2+). Possible explanations for this finding are discussed. Whether in the presence (0.5 mM) or in the absence of external Mg2+, our values for [Mg2+]i are distinctly lower than those previously reported by others, using the same type of Mg2+-selective micro-electrodes but calibrated simply from assumptions about the actual level of K+ and Na+ interference on Mg2+-selective micro-electrode response.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrophysiological properties of gap junctions between dissociated pairs of rat hepatocytes.

Physiological properties of isolated pairs of rat hepatocytes were examined within 5 h after dissociation. These cells become round when separated, but cell pairs still display membrane specializations. Most notably, canaliculi are often present at appositional membranes which are flanked by abundant gap and tight junctions. These cell pairs are strongly dye-coupled; Lucifer Yellow CH injected into one cell rapidly diffuses to the other. Pairs of hepatocytes are closely coupled electrically. Conductance of the junctional membrane is not voltage sensitive: voltage clamp studies demonstrate that gj is constant in response to long (5 s) transjunctional voltage steps of either polarity (to greater than +/- 40 mV from rest). Junctional conductance (gj) between hepatocyte pairs is reduced by exposure to octanol (0.1 mM) and by intracellular acidification. Normal intracellular pH (pHi), measured with a liquid ion exchange microelectrode, was generally 7.1-7.4, and superfusion with saline equilibrated with 100% CO2 reduced pHi to 6.0-6.5. In the pHi range 7.5-6.6, gj was constant. Below pH 6.6, gj steeply decreased and at 6.1 coupling was undetectable. pHi recovered when cells were rinsed with normal saline; in most cases gj recovered in parallel so that gj values were similar for pHs obtained during acidification or recovery. The low apparent pK and very steep pHi-gj relation of the liver gap junction contrast with higher pKs and more gradually rising curves in other tissues. If H+ ions act directly on the junctional molecules, the channels that are presumably homologous in different tissues must differ with respect to reactive sites or their environment.

The ionic mechanism of intracellular pH regulation in crayfish muscle fibres.

The ionic mechanism of intracellular pH (pHi) regulation was investigated in isolated muscle fibres of the carpopodite adductor in the crayfish Astacus fluviatilis by electrophysiological means with pH, Na+ and Cl- -sensitive liquid ion exchanger micro-electrodes. In eighty-six cells a mean pHi of 7.14 +/- 0.12 (S.D.) at a membrane potential of--79.7 +/- 3.4 mV was found under control conditions which is about one pH unit more alkaline than predicted from passive distribution and indicates the presence of an acid-extrusion mechanism. In order to study pHi recovery the cells were acid loaded by exposure either to NH4 Cl or CO2. The effects of HCO3- and DIDS (an inhibitor of the anion exchange) on pHi recovery as well as the HCO3- -dependent decrease of intracellular Cl- during pHi recovery indicate that in pHi regulation a mechanism of acid extrusion is involved which exchanges extracellular HCO3- for intracellular Cl-. In CO2/HCO3- -free solution or in salines with DIDS, pHi recovery was retarded to the same degree, but the effects were not additive. Because of this the remaining pHi recovery must originate from an HCO3- -independent acid-extrusion mechanism. In Na+ -free solution any pHi recovery was blocked; if pHi recovery occurred it was accompanied by an increase of intracellular Na+ activity (aiNa). From these results it was concluded that all acid extrusion mechanisms which contributed to pHi recovery are coupled to an influx of Na+. A Na+/H+/HCO3-/Cl-, and a separate Na+/H+, exchange are proposed as a model of pHi regulation in the crayfish muscle fibre. Similar kinds of acid extrusion mechanisms are found in the neurone of the crayfish (Moody, 1981), with the difference that in the muscle fibre pHi regulation is achieved mainly by the former process. The rate of pHi recovery is considerably lower in the muscle fibre than in the neurone or in the sensory cell (Moser, 1985) of crayfish.

Extra- and intracellular pH during near-complete forebrain ischemia in the rat.

The objective of the present study was to estimate extracellular pH (pHe) and intracellular pH (pHi) during near-complete forebrain ischemia in the rat, and to evaluate the relative importance of lactic acidosis and rise in tissue Pco2 (Ptco2) in causing pHe and pHi to fall. The animals, which were ventilated, normoxic, normocapnic, and normothermic, were subjected to 15 min of ischemia, either without or with 30-60 min of recirculation. Ptco2 was measured with a tissue electrode, pHe with a double-barrel liquid ion-exchanger microelectrode, changes in extracellular fluid (ECF) volume by impedance measurements, tissue CO2 content by a microdiffusion technique, and labile tissue metabolites by enzymatic fluorometric methods. Ischemia caused Ptco2 to rise to between 95 and 190 mm Hg (mean 149 mm Hg), and pHe to fall by 0.45-1.05 units (mean 0.70 units). During recovery, Ptco2 normalized within 5 min and pHe after 15-30 min. During ischemia, high-energy phosphates were depleted and tissue lactate content increased to 15 mumol X g-1. The total CO2 content (Tco2) was minimally or moderately reduced (normal, 11.9 mumol X g-1; range of ischemic values, 7.9-12.1 mumol X g-1), this range probably reflecting variable amounts of remaining blood flow. Impedance measurements demonstrated that ECF volume during ischemia was reduced to 55% of control, with gradual normalization during the first 15-30 min of recirculation. From values for Ptco2, Tco2, [HCO3-]e, and ECF volume, [HCO3-]i and pHi could be calculated. These values pertain to an idealized homogeneous intracellular compartment, and the methods used cannot detect whether different intracellular compartments diverge in their acid-base responses.(ABSTRACT TRUNCATED AT 250 WORDS)