Wakoh Tsuchiya

Electrical properties and active solute transport in rat small intestine

SummaryAddition ofd-glucose to the mucosal fluid resulted in a significant depolarization of the mucosal membrane potential (Vm) in rat duodenum, jejunum, and ileum accompanied by an increase in the transepithelial potential difference (PDt). On the other hand,l-glucose did not inducePDt andVm changes. Glycine applied from the mucosal side also inducedVm-depolarization andPDt-increment in the ileum. Phlorizin added to the mucosal fluid or ouabain added to the serosal fluid inhibited the sugar-dependent changes inPDt andVm.According to the analysis with an equivalent circuit model for the epithelium, it was concluded that an actively transported solute induced not only a depolarization of the mucosal (brush border) membrane but also a hyperpolarization of the serosal (baso-lateral) membrane of an epithelial cell, so that the origin of solute-inducedPDt changes should be attributed to changes in emfs at both membranes. The hyperpolarization of the serosal membrane in the presence of an actively transported solute was attributed to a mechanism of serosal electrogenic sodium pump stimulated by the increase in the extrusion rate of Na+ co-transported into the cell with sugar or amino acid.

Oscillations of membrane potential in L cells

SummaryEffects of divalent cations on oscillations of membrane potentials (i.e., spontaneous repetitive hyperpolarizing responses) and on hyperpolarizing responses induced by electrical stimuli as well as on resting potentials were studied in large nondividing L cells. Deprivation of Ca2+ from the external medium inhibited these hyperpolarizing responses accompanying slight depolarization of the resting potential. Sr2+ or Mn2+ applied to the external medium in place of Ca2+ was able to substitute for Ca2+ in the generation of hyperpolarizing responses, while Mg2+, Ba2+ or La3+ suppressed hyperpolarizing responses. The addition of A23187 to the bathing medium or intracellular injection of Ca2+, Sr2+, Mn2+ or La3+ induced membrane hyperpolarization. When the external Ca2+, Sr2+ or Mn2+ concentration was increased, the resting potential also hyperpolarized, in a saturating manner. The amplitude of maximum hyperpolarization produced by high external Ca2+ was of the same order of magnitude as those of hyperpolarizing responses and was dependent on the external K+ concentration. In the light of these experimental observations, it was deduced that the K+ conductance increase associated with the hyperpolarizing excitation is the result of an increase in the intracellular concentration of free Ca2+ mainly derived from the external solution.

Oscillations of membrane potential in L cells. I. Basic characteristics.

SummaryThe membrane potentials and resistances of L cells were measured using a standard electrophysiological technique. The values obtained in physiological media were around −15 mV and 37 MΩ, respectively. Almost all the large nondividing L cells (giant L cells) showed spontaneous oscillations of the membrane potential between around −15 and −40 mV. Application of an appropriate electrical or mechanical stimulus was also capable of eliciting responses but such were usually induced only once. The total membrane conductance increased significantly and in parallel with such a hyperpolarizing response. Cooling of the cells and application of metabolic inhibitors to the cells completely blocked the spontaneous oscillation despite the fact that the electrically induced hyperpolarizing response remained. Intracellular K+, Na+ and Cl− concentrations were measured by means of a flame photometer and a chloridometer, and the equilibrium potential for each ion was estimated.

Calcium channel and calcium pump involved in oscillatory hyperpolarizing responses of L-strain mouse fibroblasts

1. In fibroblastic L cells, spontaneously repeated hyperpolarizing responses (oscillation of membrane potential) and hyperpolarizing responses evoked by electrical stimuli were suppressed by the external application of a K+ channel blocker, nonyltriethylammonium (C9). This hydrophobic TEA‐analogue also inhibited the hyperpolarization induced by intracellular Ca2+ injection.

Phagocytic activity and hyperpolarizing responses in L-strain mouse fibroblasts.

1. Fibroblastic L cells not only respond with a slow hyperpolarizing potential change to a mechanical or electrical stimulus but also show spontaneous, repetitive hyperpolarizations (i.e. membrane potential oscillation). 2. Almost all the cells can actively take up latex beads whose surfaces were treated by U.V. irradiation. 3. Non‐phagocytic L cells hardly showed hyperpolarizing responses, while hyperpolarizing responses were obtained in all the phagocytic L cells. The exposure of the cell surface to beads, however, did not trigger the generation of hyperpolarizing responses. 4. Metabolic inhibitors, low temperature and cytochalasin B inhibited both the uptake of beads and the hyperpolarizing responses. 5. Increasing the external concentration of Ca2+ induced a remarkable stimulation of the phagocytosis of beads. Mg2+ and Ba2+, which inhibited hyperpolarizing responses due to competition for Ca2+ sites on the outer surface of the membrane, significantly suppressed the uptake of beads. 6. Verapamil, a Ca2+ channel blocker, inhibited not only hyperpolarizing membrane responses but also ingestion of beads. 7. It is concluded that the Ca2+ inflow on the hyperpolarizing membrane responses is closely associated with the phagocytic activity in L cells, probably through activation of the microfilament assembly.

Oscillations of membrane potential in L cells. II. Effect of monovalent ion concentrations and conductance changes associated with oscillations.

SummaryOscillation and activated hyperpolarizing responses induced by electrical stimuli (H.A. responses) were studied in large nondividing L cells (giant L cells) under a variety of ionic conditions. When Cl− in the bathing fluid was partially replaced with SO42− at fixed external Na+ and K+ concentrations, the membrane potential depolarized transiently, but recovered to the original potential level after about 10 min. Under such a steady state in a low-Cl− medium, the amplitudes of oscillations and H.A. responses remained almost identical with those in the control medium. On exposure to a low-Na+ medium, both membrane potentials in the resting and hyperpolarized states were slightly hyperpolarized, but the pattern and the amplitude of oscillations and H.A. responses remained much the same. Changes in external K+ concentrations remarkably affected the amplitudes of oscillations and H.A. responses: the amplitudes decreased with increases in external K+ concentration. Calculation of the changes in K+, Na+ and Cl− conductances during oscillations and H.A. responses under these various ionic conditions showed that the change in K+ conductance is the only factor responsible for the oscillation and the H.A. response. The reversal potential for the potential oscillation is about −94 mV under normal conditions, this value being quite close to that of the equilibrium potential of K+. The reversal potentials in various external K+ concentrations satisfied the Nernst equation for a K+ electrode. Valinomycin induced remarkable hyperpolarization of the resting potential, resulting in an inhibition of oscillations. The level of valinomycin-induced hyperpolarization of the resting potential required to inhibit H.A. responses was the same as that of the peak potentials of the oscillation and H.A. response. In the light of these observations, it is concluded that the spontaneous potential oscillation and the H.A. response are caused solely by increase in the K+ conductance of the cell membrane.

Effects of cytochalasin B and local anesthetics on electrical and morphological properties in L cells

Abstract Spontaneous oscillations of membrane potential observed in L cells were inhibited rapidly and reversibly in the presence of cytochalasin B (CB). Sustained hyperpolarization induced by high external Ca 2+ was also depressed by the drug. However, Ca 2+ injection into the cytoplasm elicited a sustained hyperpolarization, even in the presence of CB. These observations strongly suggest that CB inhibits calcium transport system in cell membrane. Morphological alterations associated with the CB treatment were decreased adhesiveness and rounding of the cells, with concomitant changes in surface architecture. Similar changes in electrophysiological and morphological properties were observed in cells treated with local anesthetics. Since such morphological changes induced by CB and local anesthetics were always preceded by electrical changes, it was suggested that the morphological changes are secondary phenomena resulting from inhibition of the Ca 2+ transport.

Membrane potential changes associated with differentiation of enterocytes in the rat intestinal villi in culture

Abstract Fragmented villi of the small intestine isolated from newborn rats were maintained in culture for periods of up to 4 weeks. In culture, floating globular villi and adherent villi were distinguished. Intracellular recordings were made from both types of villi. The membrane potential was highest (about −70 mV) in the cells at the tip of the villus and was lowest (about −18 mV) in the cells near the crypt region, showing a continuous gradation according to the cell location. The membrane potential in cultured mature enterocytes was due to the contribution of the electrogenic Na + pump as well as to the Ca 2+ -activated K + conductance. Both the components were temperature and metabolic energy dependent. Based on these data, it is suggested that the Ca 2+ transport mechanism and the electrogenic Na + pump are involved in the process of cell differentiation or maturation of the intestinal epithelia.

Membrane potential changes associated with pinocytosis of serum lipoproteins in L cells

Abstract L cells exhibit spontaneous oscillations of membrane potential in accord with fluctuations of the cytoplasmic Ca 2+ concentration. Upon addition of low-density lipoprotein (LDL), L cells show a prolonged hyperpolarization which is followed by an increase in the frequency of membrane potential oscillations. These membrane potential changes induced by LDL were inhibited by Ca 2+ -channel blockers. LDL-induced membrane potential changes were accompanied by a vigorous pinocytosis which was coupled with the formation of ring-like ridge structures on the cell surface. These electrical and morphological changes were also induced by high-density lipoprotein (HDL) but not by very-low-density lipoprotein (VLDL). These results suggest that the application of LDL or HDL to the membrane surface elicits a rapid influx of Ca 2+ into the cytosol, resulting in membrane hyperpolarization. A rise in cytoplasmic Ca 2+ may be implicated in the primary factor for the pinocytic process.

Dependence of membrane potential on Ca2+ transport in cultured cytotrophoblasts of human immature placentas

The electrical membrane properties of cultured human cytotrophoblast were examined by means of a standard electrophysiological technique. The mean values of the membrane potential (Rm) and the membrane resistance in a physiological medium were around -49 mV and 12 M omega , respectively. The membrane potential was dependent, to a large extent, on the external Ca2+ concentration ([Ca2+]o). Deprivation of external Ca2+ reduced membrane potential to about -20 mV, and an increase in [Ca2+]o caused a hyperpolarization in a saturable manner. The Ca2+-dependency of membrane potential was affected remarkably by [K+]o, but not by [Na+]o or [Cl-]o. The intracellular Ca2+ injection hyperpolarized the membrane in a Ca2+-free medium. A Ca2+ channel blocker, verapamil, completely abolished the Ca2+-dependent Em. The Ca2+-dependent Em was also suppressed by cooling or by the application of metabolic inhibitors. It is suggested that the Ca2+-dependent Em in cultured human cytotrophoblast is caused by a Ca2+ influx which, in turn, increases the K+ conductance of the cell membrane, presumable due to stimulation of Ca2+-activated K+ channel.