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Effects 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 Ca from the external medium inhibited these hyperpolarizing responses accompanying slight depolarization of the resting potential. Sr or Mn applied to the external medium in place of Ca was able to substitute for Ca in the generation of hyperpolarizing responses, while Mg, Ba or La suppressed hyperpolarizing responses. The addition of A23187 to the bathing medium or intracellular injection of Ca, Sr, Mn or La induced membrane hyperpolarization. When the external Ca, Sr or Mn concentration was increased, the resting potential also hyperpolarized, in a saturating manner. The amplitude of maximum hyperpolarization produced by high external Ca 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 Ca mainly derived from the external solution. [ABSTRACT FROM AUTHOR]

Dielectric dispersion measurements over a frequency range 0.01-100 MHz were made with the suspensions of a cultured cell line, mouse lymphoma L5178Y, and an attempt to explain the observed dielectric behavior by taking explicitly into consideration the possible involvement of cell nucleus has been presented. The use of a conventional 'single-shell' model in which the cell is represented by a homogeneous sphere coated with a thin limiting shell phase did not duplicate the observed dispersion curves, whereas a 'double-shell' model in which one additional concentric shell is incorporated into the 'single-shell' model gave a much better fit between the observed and the predicted dispersion curves. Based on the latter model, we analyzed the raw data of dielectric measurements to yield a set of plausible electrical parameters for the lymphoma cell: C≅1.0μF/cm, C≅0.4μF/cm, ε≅300, κ/κ≅0.9, and κ/κ≅0.7. Here, C and C are the specific capacities of plasma and nuclear membranes; ε and κ are the dielectric constant and conductivity with subscript a, c and k referring respectively to the extracellular, the cytoplasmic and the karyoplasmic phases. [ABSTRACT FROM AUTHOR]

The 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. [ABSTRACT FROM AUTHOR]

Oscillation 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 SO 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. [ABSTRACT FROM AUTHOR]

Addition of d-glucose to the mucosal fluid resulted in a significant depolarization of the mucosal membrane potential ( V ) in rat duodenum, jejunum, and ileum accompanied by an increase in the transepithelial potential difference ( PD ). On the other hand, l-glucose did not induce PD and V changes. Glycine applied from the mucosal side also induced V -depolarization and PD -increment in the ileum. Phlorizin added to the mucosal fluid or ouabain added to the serosal fluid inhibited the sugar-dependent changes in PD and V . 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-induced PD changes should be attributed to changes in emf's 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. [ABSTRACT FROM AUTHOR]

The transepithelial resistance, the cell membrane resistance and the ratio of resistances of the serosal (baso-lateral) to the mucosal (brush border) cell membrane were measured in rat duodenum, jejunum and ileum by means of microelectrode techniques. These measured values were not affected in the presence of actively transported solutes in the mucosal bathing fluid. Contribution of an electrical conductance through the extracellular shunt pathway to the total transepithelial conductance was quantitatively estimated using an electrically equivalent circuit analysis. These values estimated in respective tissues of small intestine were approx. 95% of the total transepithelial conductance, remaining unaffected by an active solute transport. From these data, the changes in emf's of the mucosal and serosal membrane induced by d-glucose or glycine were separately evaluated. [ABSTRACT FROM AUTHOR]

Using nitroxide fatty acid spin labels, the effects of some cations such as La, Cd and Hg on synaptosomal membranes were studied by observing changes in their ESR spectra. The labels were incorporated almost instantaneously into synaptosomes isolated from rat brain cortex. ESR spectra of the spin-labeled synaptosomes were significantly broadened immediately upon adding La, Ce, Cd or Hg but hardly affected by Ca, Sr and Ba. The magnitude of the change in the separation of the outer two peaks in ESR spectra (2T) depends on the number ( n) of methylene units between the polar head group and the spin-label (nitroxide) group; that is, it increases with decreasing n. Among these ions, the effect of La was the greatest and appeared to be in parallel with the amount of La bound with the synaptosomes. On the other hand, K, Rb or Li causes hardly any significant changes. [ABSTRACT FROM AUTHOR]

Ruthenium red combines with isolated synaptosomes, resulting in strong inhibition of their Ca-binding. In isotonic saline media, however, the dye-induced inhibition of Ca-binding is significantly greater than that expected for the amount of bound dye and Hill's exponent of the Ca-binding decreases to 1 with an increase in the amount of the dye bound. On the other hand in isotonic mannitol-sucrose solution, inhibition of synaptosomal Ca-binding brought about by the dye is proportional to the amount of dye bound. Based on these results, the effects of the dye on the co-operative nature of synaptosomal Ca-binding is discussed. [ABSTRACT FROM AUTHOR]

Free ion concentration of some divalent heavy metal ions such as Mn, Co, Ni, Cd and Zn in the synaptosomal suspension was measured to determine binding with synaptosomes isolated from rat brain cortex. A dual wavelength spectrophotometer was utilized to monitor the absorbance changes of murexide raised by stepwise addition of these ions (as chloride salts). Such titration experiments of the synaptosomal suspension revealed that a part of the added divalent cation such as Mn, Co or Ni was almost instantaneously bound to synaptosomes in isotonic NaCl media. Our previous study (Kamino, Uyesaka & Inouye, J. Membrane Biol. 17∶13, 1974) demonstrated that raised external K resulted in a specific noncompetitive inhibition of synaptosomal Ca-binding. Just like the Ca-binding, Mn-, Co- or Ni-binding was almost completely depressed by high external K or ruthenium red when the free concentration of the cations was 10 μ m or less, while at higher concentrations the binding was not affected. The present results indicate that tested divalent cations bind with both 'Ca-binding sites' and 'non-Ca-binding sites' of synaptosomal membrane, the nature of the binding sites of both being quite different: the former is sensitive to high external K and to ruthenium red but the latter is not. [ABSTRACT FROM AUTHOR]

As in our previous report (Kamino, Uyesaka & Inouye, J. Membrane Biol. 17:13, 1974), the absorbance changes of murexide caused by Ca and followed up by a dual wavelength spectrophotometer were applied to measure synaptosomal Ca-binding in the presence of cations such as Rb, Mn or La. All the cations tested showed a significant inhibition of synaptosomal Ca-binding except Li. The inhibitory effects could be divided into the following three categories: (1) noncompetitive, co-operative K-type, which includes alkali metal ions. The potency of inhibition is K>Rb>Cs>Li, Na=0; (2) competitive Mn-type which includes many divalent cations. The inhibitory potency was found to be in the following order: Mn≫Sr>Cd, Ba>Mg; (3) nonspecific, noncompetitive La-type: among the cations tested, La and Ce were found to markedly reduce the Ca-binding capacity of synaptosomal particles, resulting in a noncompetitive inhibition, at least in the range of Ca concentration used. [ABSTRACT FROM AUTHOR]

Ca concentration in the synaptosomal suspension was measured to determine the liberation from and binding to isolated brain cortex synaptosomes by utilizing a dual wavelength spectrophotometer to monitor the absorbance changes of murexide raised by Ca. When synaptosomes were suspended in isotonic solutions of a NaCl−KCl mixture containing more than 30 to 40 mM of KCl, a marked liberation of calcium ion was observed in proportion to the rise in KCl concentration, whereas there was rarely any significant release of Ca observed with an external KCl concentration of less than 20 mM. Titration of the synaptosomal suspension with Ca revealed that, in the absence of external K, a part of added Ca was almost instantaneously bound to the synaptosomal particles so far as these particles had not been saturated with Ca. Such a Ca-binding was markedly depressed by a higher external K concentration. Ouabain and cyanide did not have that effect on such a K-induced inhibition of Ca-binding. The present results indicate that the inhibition of Ca-binding by high external K concentration probably results from a change in synaptosomal membrane which is of a cooperative nature. [ABSTRACT FROM AUTHOR]

Dielectric measurements were performed on the suspensions of synaptosomes isolated from rat brain cortex. The synaptosomes in buffered salt media showed typical dielectric dispersions caused by the presence of a thin limiting membrane of sufficiently low conductivity. An analysis of the dielectric data revealed that the electric conductivity of the synaptosome interior was about 37 % of the external medium conductivity under isotonic conditions and that the dielectric constant for the interior phase was about 35. The membrane capacitance (0.7 ΜF cm) remained constant irrespective of nature and concentration of the univalent salts examined. Significant reduction in both the conductivity and the dielectric constant of the internal phase can be explained theoretically provided that some intra-synaptosomal structure (synaptic vesicles and/or small mitochondria) of non-conducting nature occupies about 50 % of the particulate volume, the remainder being in equilibrium with the external salt medium. [ABSTRACT FROM AUTHOR]