Membrane lipid layers vs. polarized water dominated by fixed ions: a comparative study of the effects of three macrocyclic ionophores on the K+ permeability of frog skeletal muscle, frog ovarian eggs, and human erythrocytes

The effects of 10(-7) M valinomycin, nonactin, and monactin on human erythrocytes, frog sartorius muscle, and frog ovarian oocytes in the presence of varying external K+ concentration were studied. The results showed essentially a consistent but relatively modest increase of the K+ permeability constant in cm/sec with all three antibiotics on human erythrocytes. No change in response to any one of the antibiotics was observed in frog muscles or in frog ovarian eggs. These results and reports of similar failure to demonstrate ionophore-mediated increase of K+ permeability in squid axon and inner membrane of the liver mitochondria led to the conclusion that lipid membrane barrier to ionic traffic may be significant in the human erythrocytes but even here one must regard the evidence as tentative. In contrast, for the majority of other cell types studied, the data indicate the primary, if not exclusive route of ion traffic, is via the nonlipid component of the cell membrane. The evidence that these nonlipid paths are the fixed charge-polarized water layer complex and that they cover much of the cell surface of many types of living cells was discussed.     

Cooperative interaction among surface beta- and gamma-carboxyl groups mediating the permeation of ions into frog muscle cells

A new equation for the ion permeation into living cells is described. This equation, differs from earlier ones, in that cooperative interaction among the fixed surface beta- and gamma-carboxyl groups mediating ion entry via the adsorption-desorption route is taken into account. Results of a single set of experiments describing labeled Cs+ into frog sartorius muscles at 0 degree C affirms the existence of the predicted cooperative interaction which endows the cell membrane and other organelles with a mechanism for coherence and control.     

Studies on Ion Accumulation in Muscle Cells

A comparison is made between the quantitative predictions of equilibrium ionic distribution in living cells according to the membrane theory (Donnan equilibrium) and according to the association-induction hypothesis. This comparison shows that both theories predict competitive effects of one permeant ion on the equilibrium concentration of another permeant ion; but within the limit of experimental accuracy only the association-induction model predicts quantitatively significant specific competition of one specified ion with the accumulation of another specified ion. The equilibrium distributions of K⁺, Rb⁺, and Cs⁺ ions in frog sartorius muscle were studied and quantitatively significant specific competition was demonstrated; these results favor the association-induction hypothesis (adsorption on cell proteins and protein complexes and partial exclusion from cell water). Based on this model we estimated that at 257deg;C, the apparent association constants for K⁺, Rb⁺, and Cs⁺ ion are 665, 756, and 488 (mole/liter)^-1. We found that the total concentration of adsorption sites (no less than 240 mmole/kg of fresh cells) agrees with the analytically determined concentrations of β- and γ-carboxyl groups of muscle cell proteins (260 to 288 mmole/kg).     

An electronic mechanism in the actions of drugs and other cardinal adsorbents. I. Effects of ouabain on the relative affinities of the cell surface beta- and gamma-carboxyl groups for K+, Na+, glycine and other ions

The effects of ouabain on the effectiveness of glycine, Li+, Na+, K+, Rb+, and Cs+ in the external medium in reducing the rate of entry of labeled Cs+ into frog sartorius muscles were studied. The results showed that in the absence of ouabain the effectiveness of glycine and alkali-metal ions in inhibiting labeled Cs+ entry follows the rank order: K+ greater than Cs+, Rb+ greater than Na+, Li+ greater than glycine. Exposure to ouabain in essence reverses this order which then becomes: glycine greater than Li+, Na+ greater than K+, Rb+, greater than Cs+. These results confirm the prediction of the basic electronic interpretation of drug action according to the association-induction hypothesis. In addition, it shows that the action of ouabain on the surface beta- and gamma-carboxyl groups of frog muscle mediating Cs+ entry is quite similar to its action on the cytoplasmic beta- and gamma-carboxyl groups that are the seats of K+ accumulation in the bulk phase cytoplasm as well as to its action on the cell surface beta- and gamma-carboxyl groups responsible for the generation of the resting potential. In all these cases, ouabain acts as an electron-donating cardinal adsorbent (EDC). Finally the marked increase of the binding strength of glycine on the surface beta- and gamma-carboxyl groups was used to explain the primary pharmacodynamic effect of cardiac glycosides in combating heart failure.     

Optimization criteria of the mechanism governing the stability of the membrane potential

For small changes in ion concentration within the physiological range the membrane potential transients can be explained in terms of two linear models both for passive and active transport. Using frog sartorius muscle as a suitable model system the ion pump is considered to work within the steepest range of the flux-concentration characteristic. Further for the small perturbations the equations describing passive ion transport can be safely linearized. The conclusion appears inescapable that for the muscle membrane the intracellular ion concentration adjusts itself in some optimal manner to the level of the extracellular ions. The active ion transport represents a control parameter for the membrane potential. The model structure corresponds to a dynamic system, the control processes of which are optimized with respect to a quadratic integral-criterion function. Here, both the performance index of the control sequence in the membrane processes and the energy consumed by the ion fluxes have been considered for small perturbations of Na⁺, K⁺, and Cl^? in the neighbourhood of the physiological working point. As it is, the control system governing the active and passive ion transport processes is essentially optimized with respect to a minimal energy usage. The amount of energy consumed during the transients predicted by the model has been calculated.     

An electronic mechanism in the action of drugs, ATP, transmitters and other cardinal adsorbents. II. Effect of ouabain on the relative affinities for Li+, Na+, K+, and Rb+ of surface anionic sites that mediate the entry of Cs+ into frog ovarian eggs

Ouabain enhanced the inhibitory effects of Li+, Na+, and K+ on the rate of Cs+ permeation into frog ovarian eggs while it reduced the inhibiting effect of Rb+. The data agree with earlier demonstrated effects of ouabain on the rank order of selective accumulation of the five alkali-metals in frog muscles and on the relative effectiveness of glycine, Li+, Na+, K+, Rb+, and Cs+ in inhibiting the rate of entry of Cs+ into frog sartorius muscle. In all three cases, the ouabain behaved as an electron-donating cardinal adsorbent (EDC) causing a rise of the electron density (c-value) of the beta- and gamma-carboxyl groups in the cell cytoplasm (for selective accumulation) and on the cell surface (for selective ion permeation). Explanations based on the association-induction hypothesis were offered why an EDC like ouabain does not initiate cell activation (like veratridine does) and why Ca++ and tetradotoxin delays or inhibits physiological and artificial cell activation.     

Lytic agents, cell permeability, and monolayer penetrability

Cell lysis induced by lytic agents is the terminal phase of a series of events leading to membrane disorganization and breadkdown with the release of cellular macromolecules. Permeability changes following exposure to lytic systems may range from selective effects on ion fluxes to gross membrane damage and cell leakage. Lysis can be conceived as an interfacial phenomenon, and the action of surface-active agents on erythrocytes has provided a model in which to investigate relationships between hemolysis and chemical structure, ionic charge, surface tension lowering, and ability to penetrate monolayers of membrane lipid components. Evidence suggests that lysis follows the attainment of surface pressures exceeding a "critical collapse" level and could involve membrane cholesterol or phospholipid. Similarities of chemical composition of membranes from various cell types could account for lytic responses observed on interaction with surface-active agents. Cell membranes usually contain about 20–30 % lipid and 50–75 % protein. One or two major phospholipids are present in all cell membranes, but sterols are not detectable in bacterial membranes other than those of the Mycoplasma group. The rigid cell wall in bacteria has an important bearing on their response to treatment with lytic agents. Removal of the wall renders the protoplast membrane sensitive to rapid lysis with surfactants. Isolated membranes of erythrocytes and bacteria are rapidly dissociated by surface-active agents. Products of dissociation of bacterial membranes have uniform behavior in the ultracentrifuge (sedimentation coefficients 2–3S). Dissociation of membrane proteins from lipids and the isolation and characterization of these proteins will provide a basis for investigating the specificity of interaction of lytic agents with biomembranes.     

Muscle fiber types in tetrapods. A comparative histochemical and morphometric study

A comparative histochemical and morphometric study in two groups of homologous muscles from different tetrapods (rat, pigeon, lizard and frog) was performed. On the basis of their fiber diameters and oxidative enzyme activities, an initial correlation between fiber types of all animals is observed, although in the lizard and frog muscles, another fiber type does exists that could not be demonstrated in higher vertebrates. When more than one histochemical techniques are used for the identification of each tetrapod fiber types, the lack of correlation between them becomes obvious. Thus, different animals groups, each showing a characteristic muscle metabolic pattern, could be distinguished.     

Direct evidence for cholesterol crystalline domains in biological membranes: role in human pathobiology

This review will discuss the use of small-angle X-ray diffraction approaches to study the organization of lipids in plasma membranes derived from two distinct mammalian cell types: arterial smooth muscle cells and ocular lens fiber cells. These studies indicate that cholesterol at an elevated concentration can self-associate and form immiscible domains in the plasma membrane, a phenomenon that contributes to both physiologic and pathologic cellular processes, depending on tissue source. In plasma membrane samples isolated from atherosclerotic smooth muscle cells, the formation of sterol-rich domains is associated with loss of normal cell function, including ion transport activity and control of cell replication. Analysis of meridional diffraction patterns from intact and reconstituted plasma membrane samples indicates the presence of an immiscible cholesterol domain with a unit cell periodicity of 34 Å, consistent with a cholesterol monohydrate tail-to-tail bilayer, under disease conditions. These cholesterol domains were observed in smooth muscle cells enriched with cholesterol in vitro as well as from cells obtained ex vivo from an animal model of atherosclerosis. By contrast, well-defined cholesterol domains appear to be essential to the normal physiology of fiber cell plasma membranes of the human ocular lens. The organization of cholesterol into separate domains underlies the role of lens fiber cell plasma membranes in maintaining lens transparency. These domains may also interfere with cataractogenic aggregation of soluble lens proteins at the membrane surface. Taken together, these analyses provide examples of both physiologic and pathologic roles that sterol-rich domains may have in mammalian plasma membranes. These findings support a model of the membrane in which cholesterol aggregates into structurally distinct regions that regulate the function of the cell membrane.     

pH Effect of the Sphingomyelin Membrane Interfacial Tension

The effect of pH on the interfacial tension of a sphingomyelin membrane in aqueous solution has been studied. Three models describing H⁺ and OH⁻ ion adsorption on the bilayer lipid surface are presented. In models I and II, the membrane surface is continuous, with uniformly distributed functional groups as centers of H⁺ and OH⁻ ion adsorption. In model III, the membrane surface is composed of lipid molecules, with and without adsorbed H⁺ and OH⁻ ions. The contribution of each individual lipid molecule to the overall interfacial tension of the bilayer was assumed to be additive in models I and II. In model III, the Gibbs isotherm was used to describe adsorption of H⁺ and OH⁻ ions at the bilayer surface. Theoretical equations are derived to describe the interfacial tension as a function of pH for all three models. Maximum interfacial tension was observed experimentally at the isoelectric point.     

Amino acid accumulation in frog muscle: I. Steady-state glycine accumulation at o °C

Glycine is not significantly metabolized by frog muscle maintained at o °C in vitro. Nevertheless, in this preparation steady-state levels of [¹⁴C]glycine as high as 20 times the external concentration are attained after 3–6 days at o °C. The concentration gradient at the steady state depends on the external concentration, being highest at low external concentrations (approx. 0.1 mM) and reversed at external concentrations above 10 mM.A plot of the steady-state cellular levels of glycine vs the external concentration reveal linear and saturable components. The linear fraction has an average distribution ratio of 0.54 indicating that glycine is partially excluded from the muscle water at this temperature.Efflux of labeled glycine at o °C from previously loaded frog muscle follows first-order kinetics. The rate constant increases with increasing concentrations of glycine in the external medium (efflux facilitation).The steady-state results are shown to be consistent with an adsorption model for amino acid accumulation as well as a model in which amino acid enters the cell via a carrier and exits via a bidirectional leakage pathway. A model in which efflux proceeds through the carrier does not fit the data. This indicates that an alternative to exchange diffusion is needed to explain the observed efflux facilitation.     

Diffusion-limited forward rate constants in two dimensions. Application to the trapping of cell surface receptors by coated pits.

A variety of receptors are known to aggregate in specialized cell surface structures called coated pits, prior to being internalized when the coated pits close off. At 37 degrees C on human fibroblasts, as well as on other cell types, a recycling process maintains a constant number of coated pits on the cell surface. In this paper, we explore implications for receptor aggregation and internalization of the two types of recycling models that have been proposed for the maintenance of the coated pit concentration. In one model, coated pits alternate between accessible and inaccessible states at fixed locations on the cell surface, while in the other model, coated pits recycle to random locations on the cell surface. We consider receptors that are randomly inserted in the membrane, move by pure diffusion with diffusion coefficient D, and are instantly and irreversibly trapped when they reach a coated pit boundary (the diffusion limit). For such receptors, we calculate for each of the two models: the mean time tau to reach a coated pit, the forward rate constant k+ for the interaction of a receptor with a coated pit, and the fraction phi of receptors aggregated in coated pits. We show that for the parameters that characterize coated pits on human fibroblasts, the way in which coated pits return to the surface has a negligible effect on the values of tau, k+, and phi for mobile receptors, D greater than or equal to 1.0 X 10(-11) cm2/s, but has a substantial effect for "immobile" receptors, D much less than 1 X 10(-11) cm2/s. We present numerical examples to show that it may be possible to distinguish between these models if one can monitor slowly diffusing receptors (D less than 1 X 10(-11) cm2/s) on cells whose coated pits have relatively short lifetimes (less than or equal to 1 min). Finally, we show that for the low-density lipoprotein (LDL) receptor on human fibroblasts (D = 4.5 X 10(-11) cm2/s), the predicted and observed values of K+ and phi are in close agreement. Therefore, even for slowly diffusing LDL receptor, unaided diffusion as the transport mechanism of receptors to coated pits is consistent with measured rates of LDL internalization.     

Direct evidence for cholesterol crystalline domains in biological membranes: role in human pathobiology

This review will discuss the use of small-angle X-ray diffraction approaches to study the organization of lipids in plasma membranes derived from two distinct mammalian cell types: arterial smooth muscle cells and ocular lens fiber cells. These studies indicate that cholesterol at an elevated concentration can self-associate and form immiscible domains in the plasma membrane, a phenomenon that contributes to both physiologic and pathologic cellular processes, depending on tissue source. In plasma membrane samples isolated from atherosclerotic smooth muscle cells, the formation of sterol-rich domains is associated with loss of normal cell function, including ion transport activity and control of cell replication. Analysis of meridional diffraction patterns from intact and reconstituted plasma membrane samples indicates the presence of an immiscible cholesterol domain with a unit cell periodicity of 34 A, consistent with a cholesterol monohydrate tail-to-tail bilayer, under disease conditions. These cholesterol domains were observed in smooth muscle cells enriched with cholesterol in vitro as well as from cells obtained ex vivo from an animal model of atherosclerosis. By contrast, well-defined cholesterol domains appear to be essential to the normal physiology of fiber cell plasma membranes of the human ocular lens. The organization of cholesterol into separate domains underlies the role of lens fiber cell plasma membranes in maintaining lens transparency. These domains may also interfere with cataractogenic aggregation of soluble lens proteins at the membrane surface. Taken together, these analyses provide examples of both physiologic and pathologic roles that sterol-rich domains may have in mammalian plasma membranes. These findings support a model of the membrane in which cholesterol aggregates into structurally distinct regions that regulate the function of the cell membrane.     

Amino acid accumulation in frog muscle. II. Are cycloleucine fluxes consistent with an adsorption model for concentrative uptake of amino acid?

Cycloleucine accumulation by frog muscle was studied at o °C and 25 °C. At external concentrations less than 5 mM the distribution ratio of cycloleucine is higher at 0 °C than at 25 °C. At concentrations greater than 5 mM the converse is true due to apparent exclusion of cycloleucine from a larger portion of the cell water at 0 °C.The steady state data are consistent with an absortion model for amino acid accumulation. Flux studies provide a means to rule out this model if all the possible rate-limiting steps in the movement of amino acid into and out of the cell are considered. These steps include intra-cytoplasmic diffusion, desorption from cytoplasmic or membrane sites and passage through the cell membrane. The assumption is made that the rate-limiting step for influx and efflux is the same, allowing the use of either influx or efflux data to examine the model.Diffusion-limited flux is ruled out on the basis of“influx profile analysis” of the time course of cycloleucine entry at both 0 °C and 25 °C.At least 95% of all intracellular cycloleucine leaves frog muscle cells with a single exponential time course at both 0 °C. The rate constant of efflux does not vary with cellular concentration.These findings are shown to be incompatible with desorption-limited efflux. They are compatible with membrane-limited efflex only if (i) adsorption sites are located on membranes with direct access to the extracellular space and (ii) the rate constant for desorption is equal to the rate constant of membrane-limited efflux of free amino acid. It is considered unlikely that such a coincidence would occur at both 0 °C and 25 °C. Therefore, an absorption model for cycloleucine accumulation in frog muscle appears to be untenable.     

Temperature dependence of Na currents in rabbit and frog muscle membranes

The effect of temperature (0-22 degrees C) on the kinetics of Na channel conductance was determined in voltage-clamped rabbit and frog skeletal muscle fibers using the triple-Vaseline-gap technique. The Hodgkin-Huxley model was used to extract kinetic parameters; the time course of the conductance change during step depolarization followed m3h kinetics. Arrhenius plots of activation time constants (tau m), determined at both moderate (-10 to -20 mV) and high (+100 mV) depolarizations, were linear in both types of muscle. In rabbit muscle, Arrhenius plots of the inactivation time constant (tau h) were markedly nonlinear at +100 mV, but much less so at -20 mV. The reverse situation was found in frog muscle. The contrast between the highly nonlinear Arrhenius plot of tau h at +100 mV in rabbit muscle, compared with that of frog muscle, was interpreted as revealing an intrinsic nonlinearity in the temperature dependence of mammalian muscle Na inactivation. These results are consistent with the notion that mammalian cell membranes undergo thermotropic membrane phase transitions that alter lipid-channel interactions in the 0-22 degrees C range. Furthermore, the observation that Na channel activation appears to be resistant to this effect suggests that the gating mechanisms that govern activation and inactivation reside in physically distinct regions of the channel.     

Stochastic Ion Channel Gating in Dendritic Neurons: Morphology Dependence and Probabilistic Synaptic Activation of Dendritic Spikes

Neuronal activity is mediated through changes in the probability of stochastic transitions between open and closed states of ion channels. While differences in morphology define neuronal cell types and may underlie neurological disorders, very little is known about influences of stochastic ion channel gating in neurons with complex morphology. We introduce and validate new computational tools that enable efficient generation and simulation of models containing stochastic ion channels distributed across dendritic and axonal membranes. Comparison of five morphologically distinct neuronal cell types reveals that when all simulated neurons contain identical densities of stochastic ion channels, the amplitude of stochastic membrane potential fluctuations differs between cell types and depends on sub-cellular location. For typical neurons, the amplitude of membrane potential fluctuations depends on channel kinetics as well as open probability. Using a detailed model of a hippocampal CA1 pyramidal neuron, we show that when intrinsic ion channels gate stochastically, the probability of initiation of dendritic or somatic spikes by dendritic synaptic input varies continuously between zero and one, whereas when ion channels gate deterministically, the probability is either zero or one. At physiological firing rates, stochastic gating of dendritic ion channels almost completely accounts for probabilistic somatic and dendritic spikes generated by the fully stochastic model. These results suggest that the consequences of stochastic ion channel gating differ globally between neuronal cell-types and locally between neuronal compartments. Whereas dendritic neurons are often assumed to behave deterministically, our simulations suggest that a direct consequence of stochastic gating of intrinsic ion channels is that spike output may instead be a probabilistic function of patterns of synaptic input to dendrites.     

A Model for the Long Time-Constant Transient Voltage Response to Current in Epithelial Tissues

Upon passing a step current through the frog gastric mucosa, a transient response in voltage is observed, which can formally be represented by several types of model systems, although some models require elements which are hard to visualize in terms of the known morphology of the mucosa. A physically reasonable model can be constructed by considering the changes in intracellular ionic composition which arise due to current flow, and the consequent changes in diffusion potentials across the two cell membranes. A simple model has been developed which fits the observed long time-constant portion of the transient at low current densities, and predicts departures from exponential behavior at larger currents. Since reasonable values for membrane resistance and cell volume give a fit, it is proposed that this model may account for the long time-constant portion of the transient response. There is no reason to expect that similar considerations do not hold for epithelial tissues in general.     

Proton conductance of cell membranes

Several workers have suggested that cell membranes have a high proton conductance. Our interest in this concept arose from the possibility that the nutrient (submucosal-facing) membrane of the gastric mucosa may have a high proton or hydroxyl ion conductance which would play a role in the regulation of the acid-base balance of the cell. We found that wide changes in the H⁺ concentration of the fluid bathing the nutrient side of the in vitro frog gastric mucosa did not result in significant changes in p.d. However, a maintained change of the H⁺ concentration of the bathing fluid would be expected to produce only a temporary change in p.d. Since a diffusion barrier is present on the nutrient side the temporary change in p.d. might be masked. An analysis of this possibility was made on the basis of a conceptual model and as a result of the analysis it is concluded that the proton (and/or OH^?) conductance of the nutrient membrane of the frog gastric mucosa is not a significant fraction of its total conductance. The present status of the proton conductance hypothesis with respect to striated muscle and to the secretory membrane of the gastric mucosa is reviewed.     

Physicochemical studies of taste reception. III. Interpretation of the water response in taste reception

The model membrane composed of a Millipore filter paper and the total lipids from bovine tongue epithelium or phosphatidylcholine from egg yolk simulated well the water response of a living taste cell. The water response observed with the model membrane adapted to various salt solutions was interpreted in terms of changes in electric potential at the membrane-solution interface, i.e. the water response was attributed to the e.m.f. change produced by diffusion of the electrolytes dissolved in (or adsorbed on) the membrane surface into the bulk solution.The water response of the frog tongue was also investigated by measuring the neural response of the glossopharyngeal nerve. The results obtained were consistent with the mechanism proposed in the present paper. The response of the frog to Ca²⁺ was examined under the condition where the water response was suppressed, and it was concluded that the water response of the frog is different from the response to Ca²⁺.