Studies of excitable membrane formed on the surface of protoplasmic drops isolated from Nitella III. Impedance of the surface membrane

A protoplasmic drop isolated from an internodal cell of Nitella in an initial solution composed of 70 mM KNO₃, 50 mM NaNO₃ and 5 mM CaCl₂ became electrically excitable when the drop was placed in the final solution containing 0.5 mM KNO₃, 0.5 mM NaCl, 1 mM Ca(NO₃)₂ and 2 mM Mg(NO₃)₂. The electrical impedance of the surface membrane of the drop was measured both in the initial and final solutions at frequencies between 60 Hz and 100 kHz.The impedance and admittance loci of the surface membrane fell on circular arcs. The d.c. resistance R_m°, and the d.c. capacitance C_m° were determined by extrapolating the circular arcs to the low frequency limit. R_m° thus determined was in the range of 50–200 Ω·cm² in the initial solution, and increased to a steady value of 0.4–4.0 kΩ·cm² when the external solution was replaced by the final solution. After the protoplasmic drop was isolated from the internodal cell of Nitella, C_m° decreased monotonically from about 1.5 μF/cm² within 20 min and approached $\text{1.25±0.1 μ}\text{F/cm}{}^2$ both in the initial and final solutions. No appreciable difference was observed for C_m° in these two solutions.The impedance data were discussed in relation to the process of formation of the membrane at the surface of the protoplasmic drop. After the excitable stage was reached, the drop membrane impedance was found to decrease by a factor of 10 during excitation.     

Hormone-induced changes in pyruvate kinase activity in isolated hepatocytes II. Relation to the hormonal regulation of gluconeogenesis gluconeogenesis

1. 1. Incubation of isolated hepatocytes with glucagon (10⁻⁶ M) or dibutyryl cyclic AMP (0.1 mM) causes a decrease in pyruvate kinase activity of 50%, measured at suboptimal substrate (phosphoenolpyruvate) concentrations and 1 mM Mg_free²⁺. The magnitude of the decrease in activity is not influenced by the applied extracellular concentrations of lactate (1 and 5 mM), glucose (5 and 30 mM) or fructose (10 and 25 mM). With all three substrates comparable inhibition percentages are induced by glucagon or dibutyryl cyclic AMP.

2. 2. The extent of inhibition of pyruvate kinase induced by incubation of hepatocytes with glucagon or dibutytyl cyclic AMP is not influenced by the extracellular Ca²⁺ concentration nor by the presence of 2 mM EGTA. The reactivation of pyruvate kinase seems to be inhibited by a high concentration of extracellular Ca²⁺ (2.6 mM) as compared to a low concentration of extracellular Ca²⁺ (0.26 mM).

3. 3. Incubation of hepatocytes in a Na⁺-free, high K⁺-concentration medium does not influence the magnitude of the pyruvate kinase inhibition induced by dibutyryl cyclic AMP. However, the reactivation reaction is stimulated under these incubation conditions.

4. 4. Incubation of hepatocytes with dibutyryl cyclic GMP (0.1 mM) leads to a 25% decrease in pyruvate kinase activity. The magnitude of the inhibition by dibutyryl cyclic (GMP) is not influenced by the presence of pyruvate (1 mM) or glucose (5 mM and 30 mM).

5. 5. The relative insensitivity of the pyruvate kinase inhibition induced by glucagon, dibutyryl cyclic AMP and dibutyryl cyclic GMP to the extracellular environment leads to the conclusion that the hormonal regulation of pyruvate kinase is not the only site of hormonal regulation of glycolysis and gluconeogenesis. It is concluded that hormonal regulation of pyruvate kinase activity is exerted by changes in the degree of (de)phosphorylation of the enzyme reflecting acute hormonal control as well as by changes in the concentration of the allosteric activator fructose 1,6-diphosphate. The latter depends at least in part on the hormonal control of the phosphofructokinase-fructose-1,6-phosphatase cycle.

The role of water in protoplasmic permeability and in antagonism

The behavior of the cell depends to a large extent on the permeability of the outer non-aqueous surface layer of the protoplasm. This layer is immiscible with water but may be quite permeable to it. It seems possible that a reversible increase or decrease in permeability may be due to a corresponding increase or decrease in the water content of the non-aqueous surface layer. Irreversible increase in permeability need not be due primarily to increase in the water content of the surface layer but may be caused chiefly by changes in the protoplasm on which the surface layer rests. It may include desiccation, precipitation, and other alterations. An artificial cell is described in which the outer protoplasmic surface layer is represented by a layer of guaiacol on one side of which is a solution of KOH + KCl representing the external medium and on the other side is a solution of CO₂ representing the protoplasm. The K⁺ unites with guaiacol and diffuses across to the artificial protoplasm where its concentration becomes higher than in the external solution. The guaiacol molecule thus acts as a carrier molecule which transports K⁺ from the external medium across the protoplasmic surface. The outer part of the protoplasm may contain relatively few potassium ions so that the outwardly directed potential at the outer protoplasmic surface may be small but the inner part of the protoplasm may contain more potassium ions. This may happen when potassium enters in combination with carrier molecules which do not completely dissociate until they reach the vacuole. Injury and recovery from injury may be studied by measuring the movements of water into and out of the cell. Metabolism by producing CO₂ and other acids may lower the pH and cause local shrinkage of the protoplasm which may lead to protoplasmic motion. Antagonism between Na⁺ and Ca⁺⁺ appears to be due to the fact that in solutions of NaCl the surface layer takes up an excessive amount of water and this may be prevented by the addition of suitable amounts of CaCl₂. In Nitella the outer non-aqueous surface layer may be rendered irreversibly permeable by sharply bending the cell without permanent damage to the inner non-aqueous surface layer surrounding the vacuole. The formation of contractile vacuoles may be imitated in non-living systems. An extract of the sperm of the marine worm Nereis which contains a highly surface-active substance can cause the egg to divide. It seems possible that this substance may affect the surface layer of the egg and cause it to take up water. A surface-active substance has been found in all the seminal fluids examined including those of trout, rooster, bull, and man. Duponol which is highly surface-active causes the protoplasm of Spirogyra to take up water and finally dissolve but it can be restored to the gel state by treatment with Lugol solution (KI + I). The transition from gel to sol and back again can be repeated many times in succession. The behavior of water in the surface layer of the protoplasm presents important problems which deserve careful examination.     

Renaissance of cytochemical localization of membrane ATPases in the myocardium

ATPases of cardiac cells are known to be among the most important enzymes to maintain the fluxes of vital cations by hydrolysis of the terminal high-energy phosphate of ATP. Biochemically the activities of Ca²⁺-pump ATPase, Ca²⁺/Mg²⁺-ecto ATPase, Na⁺,K⁺-ATPase and Mg²⁺-ATPase are determined in homogenates and isolated membranes as well as in myofibrillar and mitochondrial fractions of various purities. Such techniques permit estimation of enzyme activitiesin vitro under optimal conditions without precise enzyme topography. On the other hand, cytochemical methods demonstrate enzyme activityin situ, but not under optimal conditions. Until recently several cytochemical methods have been employed for each enzyme in order to protect its specific activity and precise localization but the results are difficult to interpret. To obtain more consistent data from biochemical and cytochemical point of view, we modified cytochemical methods in which unified conditions for each ATPase were used. The fixative solution (1% paraformaldehyde –0.2% glutaraldehyde in 0.1 M Tris Base buffer, pH 7.4), the same cationic concentrations of basic components in the incubation medium (0.1 M Tris Base, 2mM Pb(NO₂)₃, 5 mM MgSO₄, 5 mM ATP) and selective stimulators or inhibitors were employed. The results reveal improved localization of Ca²⁺-pump ATPase, Na⁺–K⁺ ATPase and Ca²⁺/Mg²⁺-ecto ATPase in the cardiac membrane.     

Inhibition of red cell Ca-ATPase by vanadate

1. 1. The Mg²⁺- plus Ca²⁺-dependent ATPase (Ca²⁺-ATPase) in human red cell membranes is susceptible to inhibition by low concentrations of vanadate.

2. 2. Several natural activators of Ca²⁺-ATPase (Mg²⁺, K⁺, Na⁺ and calmodulin) modify inhibition by increasing the apparent affinity of the enzyme for vanadate.

3. 3. Among the ligands tested, K⁺, in combination with Mg²⁺, had the most pronounced effect on inhibition by vanadate.

4. 4. Under conditions optimal for inhibition of Ca²⁺-ATPase, the K for vanadate was 1.5 μM and inhibition was nearly complete at saturating vanadate concentrations.

5. 5. There are similarities between the kinetics of inhibition of red cell Ca²⁺-ATPase and (Na⁺ + K⁺)-ATPase prepared from a variety of sources; however, (Na⁺ + K⁺)-ATPase is approx. 3 times more sensitive to inhibition by vanadate.

Vanadate inhibition of the Ca-ATPase from human red cell membranes

1. (1) VO₃⁻ combines with high affinity to the Ca²⁺-ATPase and fully inhibits Ca²⁺-ATPase and Ca²⁺-phosphatase activities. Inhibition is associated with a parallel decrease in the steady-state level of the Ca²⁺-dependent phosphoenzyme.

2. (2) VO₃⁻ blocks hydrolysis of ATP at the catalytic site. The sites for VO₃⁻ also exhibit negative interactions in affinity with the regulatory sites for ATP of the Ca²⁺-ATPase.

3. (3) The sites for VO₃⁻ show positive interactions in affinity with sites for Mg²⁺ and K⁺. This accounts for the dependence on Mg²⁺ and K⁺ of the inhibition by VO₃⁻. Although, with less effectiveness, Na⁺ substitutes for K⁺ whereas Li⁺ does not. The apparent affinities for Mg²⁺ and K⁺ for inhibition by VO₃⁻ seem to be less than those for activation of the Ca²⁺-ATPase.

4. (4) Inhibition by VO₃⁻ is independent of Ca²⁺ at concentrations up to 50 μM. Higher concentrations of Ca²⁺ lead to a progressive release of the inhibitory effect of VO₃⁻.

Direct measurement of intracellular free magnesium in frog skeletal muscle using magnesium-selective microelectrodes

Mg²⁺-selective microelectrodes have been used to measure the intracellular free Mg²⁺ concentration in frog skeletal muscle fibers. Glass capillaries with a tip diameter of less than 0.4 μm were backfilled with the Mg²⁺ sensor, ETH 1117. In the absence of interfering ions, they gave Nernstian responses between 1 and 10 mM free Mg²⁺. In the presence of an ionic environment resembling the myoplasm, the microelectrode response was sub Nernstian (18–24 mV) but still useful. The electrodes were calibrated before and after muscle-fiber impalements. In quiescent fibers from sartorius muscle (Rana pipiens), with resting membrane potentials not less than ?82 mV, the intracellular free Mg²⁺ concentration was 3.8±0.41 (S.E.) mM (n=58) at 22°C. No significant change in the intracellular free Mg²⁺ was observed following extensive (approx. 6 h) incubation in Mg²⁺-free media. Increasing the external concentration of magnesium from 4 to 20 mM (approx. 15 min) produced a slow and small enhancement (1.8 mM) of [Mg²⁺]_i, which was fully reverted when the divalent cation was removed from the bathing solution. No change in ionic magnesium resting concentration was observed when the muscle fibers were treated either with caffeine 3 mM or with Na⁺-free solutions. In depolarized muscle fibers (?23±2.7 mV) treated with 100 mM K⁺, the myoplasmic [Mg²⁺] was 3.7±0.45 (S.E.) mM, n=6, immediately after the spontaneous relaxation of the contracture. Similar determinations in muscle fibers during stimulation at low frequency (5 Hz), and after fatigue development, showed no changes in the concentration of free cytosolic Mg²⁺. These results point out that [Mg²⁺]_i is not modified under these three different experimental conditions.     

A theory of self-organization of an excitable membrane formed on the protoplasmic droplet isolated from Nitella

The process of self-organization of an excitable membrane of protoplasmic droplet of Nitella is studied theoretically by taking the interaction of the local electric current caused by the spatially non-uniform distribution of active domains into account. The theoretical model employed is that the surface membrane forms a mosaic structure composed of lipids and protein molecules, and that each element of the mosaic structure (domain) on the membrane has two distinct conformations corresponding to excited and resting states. The molecules constituting the membrane are derived from the inside of the protoplasm by diffusion. The excitability of the surface membrane appears suddenly after a morphogenetic structure of the membrane is formed with time on the surface of droplet. Time courses of the variation in membrane potential and in membrane resistance are calculated, and the results are compared with experimental data obtained with the protoplasmic droplet of Nitella.     

Resting potential of the mouse neuroblastoma cells: II. Significant contribution of the surface potential to the resting potential of the cells under physiological conditions

In the previous paper, we showed that the K⁺ channels of the mouse neuroblastoma cell (clone N-18) are closed at low concentration of external K⁺ ([K⁺]₀) including the physiological concentration for the cells. In the present study, the origin of the resting membrane potential of N-18 cells has been examined. (1) The resting membrane potential of N-18 cells was depolarized by increasing concentration of the polyvalent cations (La³⁺, Fe³⁺, Co²⁺, Ca²⁺, Sr²⁺, Mg²⁺) and by decreasing the pH of the medium. The input membrane resistance was slightly increased during the depolarization. The depolarization was not explained in terms of the diffusion of the cations across the membrane, since the trivalent cations of greater ionic size were effective at much lower concentrations than the divalent cations. The results obtained from the measurements of ⁸⁶Rb efflux suggested that the depolarization cannot be explained in terms of blocking of the K⁺ channels by the cations. (2) An increase in Ca²⁺ concentration from 0.3 to 1.8 mM induced depolarization of about 10 mV at low [K⁺]₀ where the K⁺ channels are closed, but did not induce any depolarization at high [K⁺]₀ where the channels are open. (3) In order to estimate the changes in the zeta-potential, the electrophoretic mobility of N-18 cells was measured under various conditions. There was a close correlation between the changes in the zeta-potential and those in the membrane potential in response to the polyvalent cations and proton. On the other hand, an increase in K⁺-concentration in the medium, which induced a large depolarization in the cells, did not affect the zeta-potential. (4) The results obtained were explained by an electrical circuit model for the membranes of N-18 cells. In this model, an electrical circuit for the membrane part carrying no selective ionic channels, in which changes in the surface potential directly affect the transmembrane potential, is connected in parallel to the usual circuit model representing selective ionic channel systems. It was concluded that the surface potential contributes significantly to the resting membrane potential of N-18 cells at low [K⁺]₀ where the K⁺ channels are closed.     

Effects of anesthetic alcohols on membrane transport processes in human erythrocytes

1. 1. Anesthetic alcohols (pentanol, hexanol and heptanol) were found to increase the fluidity of red cell membrane lipids as monitored by the fluorescence depolarization of diphenylhexatriene. The relative potency of the alcohols was found to be parallel to their relative membrane/water partition coefficients.

2. 2. Hexanol had biphasic effect on erythritol uptake by simple diffusion by red cells. At concentrations less than 9 mM, hexanol had no significant effect. At concentrations greater than 9 mM, there was an approximately linear increase in erythritol permeability with increasing alcohol concentration.

3. 3. The facilitated transport of uridine was markedly inhibited by hexanol. Hexanol at 6 mM produced a 65% inhibition of uridine (4 mM) uptake. Hexanol decreased both the apparent K_m and V values for the equilibrium exchange of uridine.

4. 4. The facilitated transport of galactose was only slightly inhibited by hexanol.

5. 5. Hexanol was without effect on the passive and active fluxes of Na⁺ and K⁺ in red cells with altered cation contents. Cells that were slightly depleted of K⁺ and cells that were highly K⁺-depleted were both insensitive to hexanol.

Intrasynaptosomal Free Mg2+ Concentration Measured with the Fluorescent Indicator Mag-Fura-2: Modulation by Na+ Gradient and by Extrasynaptosomal ATP

Abstract: Synaptosomes can be loaded with mag-fura-2 without significant perturbation of their ATP content by incubation for 10 min at 37°C with 10 µM mag-fura-2 acetoxymethyl ester in Hanks'-HEPES buffer (pH 7.45). The intrasynaptosomal free Mg²⁺ concentration ([Mg²⁺]_i) was found to be dependent on external Mg²⁺ concentration, increasing from 0.8 to 1.25 mM when the concentration of Mg²⁺ in the incubation medium increased from 1 to 8 mM. Dissipation of the Na⁺ gradient across the plasma membrane of synaptosomes by treatment with the Na⁺ ionophore monensin (0.2 mM) or with veratridine (0.2 mM) and ouabain (0.6 mM) produced a moderate increase of [Mg²⁺]_i, from 1.0 to 1.2–1.3 mM in an incubation medium containing 5 mM Mg²⁺. Plasma membrane depolarization by incubation of synaptosomes in a medium containing 68 mM KCl and 68 mM NaCl had no effect on [Mg²⁺]_i. Reversal of the Na⁺ gradient by incubation of synaptosomes in a medium in which external Na⁺ was replaced by choline increased [Mg²⁺]_i up to 1.6 and 2.2 mM for extrasynaptosomal Mg²⁺ concentrations of 1 and 8 mM, respectively. We conclude that a Na⁺/Mg²⁺ exchange operates in the plasma membrane of synaptosomes. In the presence of Mg²⁺ in the incubation medium, extrasynaptosomal ATP, but not ADP or adenosine, increased [Mg²⁺]_i from 1.1 ± 0.1 up to 1.6 ± 0.1 mM. The nonhydrolyzable ATP analogue adenosine 5′-(βγ-imido)triphosphate antagonized the effect of ATP, but had no effect by itself on [Mg²⁺]_i. It is concluded that Mg²⁺ transport across the plasma membrane of synaptosomes is modulated by the activity of an ecto-ATPase or an ecto-protein kinase.     

Ciliary reversal inParamecium: Temperature dependence of K+-induced excitability decrease and of recovery

Summary The duration of K⁺-induced reversed swimming ofParamecium caudatum was used as a measure of excitability. The time course of excitability decrease following an exposure to high [K⁺]_o and of the subsequent recovery in a solution devoid of K⁺ was measured at various temperatures.When the temperature was increased during the exposure to K⁺ the loss of excitability increased with a Q₁₀ of ca. 1.3 (Fig. 2). The recovery of excitability was enhanced with a Q₁₀ of 2.0–3.0 (Fig. 4). The renormalization of ciliary beat in K⁺-rich medium showed Q₁₀-values between 2.1 and 2.5 (Fig. 5).The observed alterations in excitability are interpreted in terms of passive and active Ca²⁺ fluxes. Ca channels in the excitable membrane are assumed to inactivate as a result of increased [Ca²⁺]_i. Recovery of excitability is thought to be mediated by active Ca²⁺ extrusion.I would like to thank Mrs. A. Müller-Holtkamp, who carried out the experiments, for her invaluable help. This work is part of the research program SFB 160 of the Deutsche Forschungsgemeinschaft.     

Cytoplasmic streaming and ionic regulation inNitella flexilis having artificial cell saps of low ionic strength

The cell sap of the internode ofNitella flexilis was replaced with the isotonic artificial pond water of high Ca²⁺-concentration (0.1 mM KCl, 0.1 mM NaCl, 10 mM CaCl₂ and 275 mM mannitol) and changes in osmotic value and concentrations of K⁺, Na⁺ and Cl⁻ of the cells were followed. When the operated cells were incubated in the artificial pond water containing 0.1 mM each of KCl, NaCl, CaCl₂, they survived for only a short period of time (<10 hr). The cells did not absorb ions from the artificial pond water and showed a conspicuous decrease in the rate of cytoplasmic streaming. In such cell the concentration of K⁺ in the protoplasm decreased significantly. In order to reverse normal concentration gradients of K⁺ and Na⁺ across the protoplasmic layer, the cells of low vacuolar ionic concentrations were incubated in the artificial cell sap (90 mM KCl, 40 mM NaCl, 15 mM CaCl₂, 10 mM MgCl₂). It was found that the cells rapidly absorbed much K⁺, Na⁺ and Cl⁻ and survived for a longer period (1–2 days). During this period the rate of cytoplasmic streaming was nearly normal. Furthermore, the cell lost much mannitol, indicating an enormous increase in permeability to it. Since both absorption of ions and leakage of mannitol at 1 C occurred at nearly the same rates as at 22 C, the processes are assumed to be passive.