Showing papers in "The Journal of General Physiology in 1967"

ATPase Activity of Myosin Correlated with Speed of Muscle Shortening

Abstract: Myosin was isolated from 14 different muscles (mammals, lower vertebrates, and invertebrates) of known maximal speed of shortening. These myosin preparations were homogeneous in the analytical ultracentrifuge or, in a few cases, showed, in addition to the main myosin peak, part of the myosin in aggregated form. Actin- and Ca++-activated ATPase activities of the myosins were generally proportional to the speed of shortening of their respective muscles; i.e. the greater the intrinsic speed, the higher the ATPase activity. This relation was found when the speed of shortening ranged from 0.1 to 24 muscle lengths/sec. The temperature coefficient of the Ca++-activated myosin ATPase was the same as that of the speed of shortening, Q10 about 2. Higher Q10 values were found for the actin-activated myosin ATPase, especially below 10°C. By using myofibrils instead of reconstituted actomyosin, Q10 values close to 2 could be obtained for the Mg++-activated myofibrillar ATPase at ionic strength of 0.014. In another series of experiments, myosin was isolated from 11 different muscles of known isometric twitch contraction time. The ATPase activity of these myosins was inversely proportional to the contraction time of the muscles. These results suggest a role for the ATPase activity of myosin in determining the speed of muscle contraction. In contrast to the ATPase activity of myosin, which varied according to the speed of contraction, the F-actin-binding ability of myosin from various muscles was rather constant.

Standing-Gradient Osmotic Flow A mechanism for coupling of water and solute transport in epithelia

Abstract: At the ultrastructural level, epithelia performing solute-linked water transport possess long, narrow channels open at one end and closed at the other, which may constitute the fluid transport route (e.g., lateral intercellular spaces, basal infoldings, intracellular canaliculi, and brush-border microvilli). Active solute transport into such folded structures would establish standing osmotic gradients, causing a progressive approach to osmotic equilibrium along the channel's length. The behavior of a simple standing-gradient flow system has therefore been analyzed mathematically because of its potential physiological significance. The osmolarity of the fluid emerging from the channel's open end depends upon five parameters: channel length, radius, and water permeability, and solute transport rate and diffusion coefficient. For ranges of values of these parameters encountered experimentally in epithelia, the emergent osmolarity is found by calculation to range from isotonic to a few times isotonic; i.e., the range encountered in epithelial absorbates and secretions. The transported fluid becomes more isotonic as channel radius or solute diffusion coefficient is decreased, or as channel length or water permeability is increased. Given appropriate parameters, a standing-gradient system can yield hypertonic fluids whose osmolarities are virtually independent of transport rate over a wide range, as in distal tubule and avian salt gland. The results suggest that water-to-solute coupling in epithelia is due to the ultrastructural geometry of the transport route.

Cell motility by labile association of molecules. The nature of mitotic spindle fibers and their role in chromosome movement.

Abstract: This article summarizes our current views on the dynamic structure of the mitotic spindle and its relation to mitotic chromosome movements. The following statements are based on measurements of birefringence of spindle fibers in living cells, normally developing or experimentally modified by various physical and chemical agents, including high and low temperatures, antimitotic drugs, heavy water, and ultraviolet microbeam irradiation. Data were also obtained concomitantly with electron microscopy employing a new fixative and through measurements of isolated spindle protein. Spindle fibers in living cells are labile dynamic structures whose constituent filaments (microtubules) undergo cyclic breakdown and reformation. The dynamic state is maintained by an equilibrium between a pool of protein molecules and their linearly aggregated polymers, which constitute the microtubules or filaments. In living cells under physiological conditions, the association of the molecules into polymers is very weak (absolute value of Δ F 25°C S 25°C ≃ 100 eu) or by the addition of heavy water. The spindle proteins tend to polymerize with orienting centers as their geometrical foci. The centrioles, kinetochores, and cell plate act as orienting centers successively during mitosis. Filaments are more concentrated adjacent to an orienting center and yield higher birefringence. Astral rays, continuous fibers, chromosomal fibers, and phragmoplast fibers are thus formed by successive reorganization of the same protein molecules. During late prophase and metaphase, polymerization takes place predominantly at the kinetochores; in metaphase and anaphase, depolymerization is prevalent near the spindle poles. When the concentration of spindle protein is high, fusiform bundles of polymer are precipitated out even in the absence of obvious orienting centers. The shift of equilibrium from free protein molecules to polymer increases the length and number of the spindle microtubules or filaments. Slow depolymerization of the polymers, which can be brought about by low concentrations of colchicine or by gradual cooling, allows the filaments to shorten and perform work. The dynamic equilibrium controlled by orienting centers and other factors provides a plasusible mechanism by which chromosomes and other organelles, as well as the cell surface, are deformed or moved by temporarily organized arrays of microtubules or filaments.

The Selective Inhibition of Delayed Potassium Currents in Nerve by Tetraethylammonium Ion

Abstract: The effect of tetraethylammonium ion (TEA) on the voltage clamp currents of nodes of Ranvier of frog myelinated nerve fibers is studied. The delayed K currents can be totally abolished by TEA without affecting the transient Na currents or the leakage current in any way. Both inward and outward currents disappear. In low TEA concentrations small K currents remain with normal time constants. The dose-response relationship suggests the formation of a complex between TEA and a receptor with a dissociation constant of 0.4 mM. Other symmetrical quaternary ammonium ions have very little effect. There is no competition between TEA and agents that affect the Na currents such as Xylocaine, tetrodotoxin, or Ca ions. The pharmacological data demonstrate that the Na, K, and leakage permeabilities are chemically independent, probably because their mechanisms occupy different sites on the nodal membrane. The data are gathered and analyzed by digital computer.

Surface density of calcium ions and calcium spikes in the barnacle muscle fiber membrane.

Abstract: The effects of various divalent cations in the external solution upon the Ca spike of the barnacle muscle fiber membrane were studied using intracellular recording and polarizing techniques. Analysis of the maximum rate of rise of the spike potential indicates that different species of divalent cations bind the same membrane sites competitively with different dissociation constants. The overshoot of the spike potential is determined by the density of Ca (Sr) ions in the membrane sites while the threshold membrane potential for spike initiation depends on the total density of divalent cations. The order of binding among different divalent and trivalent cations is the following: La+++, UO2++ > Zn++, Co++, Fe++ > Mn++ > Ni++ > Ca++ > Mg++, Sr++

The Ultrastructural Route of Fluid Transport in Rabbit Gall Bladder

Abstract: The route of fluid transport across the wall of the rabbit gall bladder has been examined by combined physiological and morphological techniques. Fluid transport was either made maximal or was inhibited by one of six physiological methods (metabolic inhibition with cyanide-iodoacetate, addition of ouabain, application of adverse osmotic gradients, low temperature, replacement of Cl by SO4, or replacement of NaCl by sucrose). Then the organ was rapidly fixed and subsequently embedded, sectioned, and examined by light and electron microscopy. The structure of the gall bladder is presented with the aid of electron micrographs, and changes in structure are described and quantitated. The most significant morphological feature seems to be long, narrow, complex channels between adjacent epithelial cells; these spaces are closed by tight junctions at the luminal surface of the epithelium but are open at the basal surface. They are dilated when maximal fluid transport occurs, but are collapsed under all the conditions which inhibit transport. Additional observations and experiments make it possible to conclude that this dilation is the result of fluid transport through the spaces. Evidently NaCl is constantly pumped from the epithelial cells into the spaces, making them hypertonic, so that water follows osmotically. It is suggested that these spaces may represent a "standing-gradient flow system," in which osmotic equilibration takes place progressively along the length of a long channel.

Alanine and Sodium Fluxes Across Mucosal Border of Rabbit Ileum

Abstract: Unidirectional influxes of L-alanine and Na from the mucosal solution into the epithelium of in vitro rabbit ileum have been determined. In the presence of 140 mM Na, alanine influx is approximately 2.2 µmoles/hr cm2, but is inhibited if the NaCl in the mucosal solution is replaced by choline Cl, Tris-Cl, mannitol, LiCl, or KCl. Although alanine influx is strongly dependent upon Na in the mucosal solution, it is uninfluenced by marked reduction of intracellular Na pools. In addition, alanine influx is unaffected by intracellular alanine concentration. Na influx is markedly inhibited by the presence of Li. Evidence is presented that Na transport across the mucosal border cannot be attributed to simple diffusion even though the net flux across this surface is in the direction of the electrochemical potential difference.

Water Permeability of Thin Lipid Membranes

Abstract: The osmotic permeability coefficient, Pf, and the tagged water permeability coefficient, Pd, were determined for thin ( Pd can be attributed to the presence of unstirred layers in the experimental determination of Pd. Thus, there is no evidence for the existence of aqueous pores in these thin phospholipid membranes. The adsorption onto the membrane of a protein that lowers its electrical resistance by a factor of 103 was found not to affect its water permeability; however, glucose and sucrose were found to interact with the membrane to modify Pf. Possible mechanisms of water transport across these films are discussed, together with the implications of data obtained on these structures for plasma membranes.

Kinetic Relations of the Na-Amino Acid Interaction at the Mucosal Border of Intestine

Abstract: The relation between unidirectional influxes of Na and amino acids across the mucosal border of rabbit ileum was studied under a variety of conditions. At constant Na concentration in the mucosal bathing solution, amino acid influx followed Michaelis-Menten kinetics permitting determination of maximal influx and the apparent Michaelis constant, Kt. Reduction in Na concentration, using choline as substitute cation, caused an increase in Kt for alanine but had no effect on maximal alanine influx. The reciprocal of Kt was a linear function of Na concentration. Similar results were obtained for valine and leucine and these amino acids competitively inhibited alanine influx both in the presence and in the absence of Na. These results lead to a model for the transport system which involves combination of Na and amino acid with a single carrier or site leading to penetration of both solutes. The model predicts that alanine should cause an increase in Na influx and the ratio of this extra Na flux to alanine flux should vary with Na concentration. The observed relation agreed closely with predicted values for Na concentrations from 5 to 140 mM. These results support the hypothesis that interactions between Na and amino acid transport depend in part on a common entry mechanism at the mucosal border of the intestine.

The effect of valinomycin on the ionic permeability of thin lipid membranes.

Abstract: Optically black membranes prepared from sheep red cell lipids have a high electrical resistance (1–3 x 108 ohm-cm2). The ionic transference numbers (Ti) for cations (Na+ or K+) are equal to each other but at least four to five times greater than for Cl-. The cyclic depsipeptide valinomycin produces a striking decrease in the membrane resistance when K+, but not when Na+ is in the solutions bathing the membrane. The ratio TNa/TK, estimated from membrane voltages in the presence of ionic concentration gradients, approaches zero. The order of membrane monovalent cation selectivity, in the presence of valinomycin, is H+ > Rb+ > K+ > Cs+ > Na+. Addition of the antibiotic to one side of a membrane which separates identical solutions of NaCl produces a substantial (up to 80 mV) membrane voltage (side opposite valinomycin negative). These data are consistent with the hypothesis that valinomycin can interact with appropriately sized cations (hydrated diameter ??? 6 A) to increase their membrane permeability, perhaps by forming hydrogen bonds between the solvation shell of the cations and carbonyl oxygens in the valinomycin molecule which are directed toward the aperture of the ring.

Junctional Membrane Uncoupling: Permeability transformations at a cell membrane junction

Abstract: The permeability of the membrane surfaces where cells are in contact (junctional membranes) in Chironomus salivary glands depends on Ca++ and Mg++ When the concentration of these ions at the junctional membranes is raised sufficiently, these normally highly permeable membranes seal off; their permeability falls one to three orders, as they approach the nonjunctional membranes in conductance This permeability transformation is achieved in three ways: (a) by iontophoresis of Ca++ into the cell; (b) by entry of Ca++ and/or Mg++ from the extracellular fluid into the cell through leaks in the cell surface membrane (eg, injury); or (c) by entry of these ions through leaks arising, probably primarily in the perijunctional insulation, due to trypsin digestion, anisotonicity, alkalinity, or chelation Ca++ and Mg++ appear to have three roles in the junctional coupling processes: (a) in the permeability of the junctional membranes; (b) in the permeability of the perijunctional insulation; and (c) a role long known— in the mechanical stability of the cell junction The two latter roles may well be closely interdependent, but the first is clearly independent of the others

The Role of Membrane Phosphoglycerate Kinase in the Control of Glycolytic Rate by Active Cation Transport in Human Red Blood Cells

Abstract: When the internal Na of human red cells is raised, both K influx and lactate production increase and become more sensitive to the inhibitory action of ouabain. This occurs with either glucose or purine nucleoside as substrate. Fresh whole hemolysates enriched with Na and Mg will convert intermediates above the triose phosphate dehydrogenase step to lactate at a rate which is slowed by ouabain. Intermediates beyond the phosphoglycerate kinase step (PGK) are metabolized at a very rapid rate which is not affected by ouabain. No metabolic effects of ouabain were found in ghost-free hemolysates. Hemoglobin-free ghosts were shown to have both triose phosphate dehydrogenase and PGK activity. The rate of this two-enzyme sequence was found to be a function of the ADP concentration, being maximal when ADP > 0.35 mM. Initial addition of ATP to the ghost system rendered the forward rate of the sequence sensitive to the inhibitory action of ouabain. When the sequence was run in reverse, no inhibitory effect of ouabain could be demonstrated. It is concluded that membrane PGK is a point at which the Na-K transport system can influence the metabolic rate and that this action is possibly exerted via a compartmentalized form of ADP which is an immediate substrate for the ghost PGK.

Effects of ATP on the Interaction of Ca ++, Mg ++, and K + with Fragmented Sarcoplasmic Reticulum Isolated from Rabbit Skeletal Muscle

Abstract: Fragmented sarcoplasmic reticulum isolated from skeletal muscle of the rabbit has a cation-binding capacity of about 350 µeq/g of protein at neutral pH The same binding sites bind Ca, Mg, K, and H ions and, consequently, the selective binding of Ca induced by ATP releases an amount of the other cations equivalent to the Ca taken up At pH values below 62, an increasing number of binding sites are associated with H+, and ATP induces exchange of Ca mostly for H+ At pH values above 62, the binding sites exist in the form of Mg and K, and Ca is bound in exchange for these cations The total bound Ca + Mg + K, expressed in microequivalents of cations bound per gram of protein, is approximately constant at various pCa values, which indicates a stoichiometric exchange of Ca for the other cations To accomplish the same degree of exchange of Ca for other cations bound, in the absence of ATP, concentrations of free Ca++ of about 1000-fold higher than those needed in the presence of ATP are required in the medium We cannot distinguish between a mechanism whereby Ca actively transported into a compartment of the microsomal vesicles containing also the binding sites is bound passively to these sites in exchange for Mg, K, and H and another in which ATP selectively increases the affinity of surface-binding sites for Ca Irrespective of the mechanism of accumulation, the Ca retained does not contribute to the activity of the cation in the membrane fraction Caffeine (10 mM) has no effect on the binding of Ca, but releases a more labile fraction of Ca, which presumably accumulates in excess of the bound Ca Procaine (5 mM) antagonizes the effect of caffeine Acetylcholine and epinephrine have no effect on the binding of Ca

Enhanced Permeability to Sugar Associated with Muscle Contraction : Studies of the role of Ca++

Abstract: When contractures were induced in isolated frog sartorius muscles with 4 mM caffeine, there was an increase in permeability of the muscle cells to 3-methylglucose. This observation suggests that the changes in permeability to sugar that are known to occur in electrically stimulated muscles may not be intimately related to the depolarization phase of the tissue response. Contractures that were elicited by exposing the muscles to a high concentration of K+ were also associated with an increased permeability to sugar. As the concentration of 45Ca in the medium was raised, more 45Ca entered the muscles during potassium contractures, and the contractures lasted longer, in agreement with the observations of other investigators. There was also a greater change in permeability to sugar when potassium contractures were elicited in the presence of higher concentrations of Ca++. The possibility that the enhanced permeability to sugar may be related to changes in the intracellular concentration of Ca++ is discussed.

Sodium Extrusion by Internally Dialyzed Squid Axons

Abstract: A method has been developed which allows a length of electrically excitable squid axon to be internally dialyzed against a continuously flowing solution of defined composition. Tests showed that diffusional exchange of small molecules in the axoplasm surrounding the dialysis tube occurred with a half-time of 2–5 min, and that protein does not cross the wall of the dialysis tube. The composition of the dialysis medium was (mM): K isethionate 151, K aspartate 151, taurine 275, MgCI2 4–10, NaCl 80, KCN 2, EDTA 0.1, ATP 5–10, and phosphoarginine 0–10. The following measurements were made: resting Na influx 57 pmole/cm2sec (n = 8); resting potassium efflux 59 pmole/ cm2sec (n = 4); stimulated Na efflux 3.1 pmole/cm2imp (n = 9); stimulated K efflux 2.9 pmole/cm2imp (n = 3); resting Na efflux 48 pmole/cm2sec (n = 18); Q10 Na efflux 2.2 (n = 5). Removal of ATP and phosphoarginine from the dialysis medium (n = 4) or external application of strophanthidin (n = 1) reversibly reduced Na efflux to 10–13 pmole/cm2sec. A general conclusion from the study is that dialyzed squid axons have relatively normal passive permeability properties and that a substantial fraction of the Na efflux is under metabolic control although the Na extrusion mechanism may not be working perfectly.

Decreased K+ Conductance Produced by Ba++ in Frog Sartorius Fibers

Abstract: The action of Ba(++) on membrane potential (E(m)) and resistance (R(m)) of frog (R. pipiens) sartorius fibers was studied. In normal Cl(-) Ringer's, Ba(++) (<9 mM) did not depolarize or induce contractions, but increased R(m) slightly above the control value of 3.8 +/- 0.6 K ohm-cm(2). In Cl(-)-free Ringer's (methane sulfonate) R(m) was 28.8 +/- 2.8 K ohm-cm(2), and low concentrations of Ba(++) (0.05-5.0 mM) depolarized and induced spontaneous contractions (fibrillation), even in tetrodotoxin. To stop disturbance of the microelectrodes, contractions were prevented by using two Cl(-)-free solutions: (a) twice hypertonic with sucrose (230 mM), or (b) high K(+) (83 mM) partially replacing Na(+). In the hypertonic solution, the fiber diameters decreased, E(m) increased slightly, and R(m) decreased to 9.0 +/- 0.6 K ohm-cm(2) (perhaps due to swelling of sarcotubules). Ba(++) (0.5 mM) rapidly increased R(m) to 31.3 +/- 3.8, decreased E(m) (e.g., to -30 mv), and induced spontaneous "action potentials;" Sr(++) had no effect. In the high K(+) solution, the fibers were nearly completely depolarized, and R(m) was decreased markedly to 1.5 +/- 0.2 K ohm-cm(2); Ba(++) increased R(m) to 6.7 +/- 0.5 K ohm-cm(2). The Ba(++) actions usually began within 0.5 min and reached a maximum within 5 min. Addition of SO(4) (=), to precipitate the Ba(++), rapidly reversed the increase in R(m). Ba(++) must act by decreasing K(+) conductance (g(K)). In Cl(-) Ringer's, the high g(Cl)/g(K) ratio masked the effect of Ba(++) on g(K). Thus, small concentrations of Ba(++) specifically and rapidly decrease g(K).

The effect of the unstirred layer on human red cell water permeability.

Abstract: A study has been made of water entry into human red blood cells under an osmotic pressure gradient. The measurements were made using a rapid reaction stop flow apparatus, whose construction, calibration, and performance are described in detail. Red cell volume changes were determined from 90° scattered light. The permeability coefficient for water entry under a relative isosmolar concentration of 1 to 1.5 was found to be 0.22 ± 0.01 cm4/sec osmol, which agrees well with our previously published value. The experiments were also designed to measure the thickness of the unstirred layer around the6 red cells. This was found to be 5.5 ± 0.8 µ under the present experimental conditions. It is concluded that our previously measured permeability coefficient for water entrance under a diffusion gradient does not require correction on account of the unstirred layer.

Depolarization of the internal membrane system in the activation of frog skeletal muscle.

Abstract: "Skinned" muscle fibers, single fibers from the frog semitendinosus muscle in which the sarcolemma had been removed, could be reversibly activated by electrical stimulation. Electrical responsiveness was abolished when the skinned fiber was prepared from a muscle exposed to a cardiac glycoside, and the development of responsiveness was delayed when the muscle was bathed in high potassium solution. The findings were taken as evidence that active sodium-potassium exchange across the internal membranes restored electrical excitability, after the sarcolemma had been removed, by establishing a potential gradient across the internal membranes. In general, the contractions were graded with the strength of the applied current. On occasion, however, "all-or-none" type responses were seen, raising the possibility that the internal membranes were capable of an electrically regenerative response. Activation could also be produced by an elevation of the intracellular chloride ion concentration or a decrease in the intracellular potassium, ion concentration, suggesting that depolarization of some element of the internal membrane system, that is, a decrease in the potential of the lumen of the internal membrane system relative to the potential of the myofibrillar space, was responsible for activation in these experiments. The distribution of both the electrically induced contractions and those produced by changes in the intracellular ion concentrations indicated that the responsive element of the internal membrane system was electrically continuous over many sarcomeres.

Basis of tetrodotoxin's selectivity in blockage of squid axons.

Abstract: The blockage of nerve activity by tetrodotoxin is unusually potent and specific. Our experiments were designed to distinguish whether its specificity of action was based on the identification of ions, the direction of cation flow, or differences in the early transient and late steady conductance pathways. Alkali cations were substituted for sodium in the sea water, bathing an "artificial node" in a voltage-clamped squid axon. When tetrodotoxin was added to the artificial sea waters at a concentration of 100 to 150 mM, it was found to always block the flow of cations through the early transient channel, both inward and outward, but it never blocked the flow of ions using the late steady pathway. We conclude that the selectivity of tetrodotoxin is based on some difference in these two channels.

NMR Evidence for Complexing of Na+ in Muscle, Kidney, and Brain, and by Actomyosin. The Relation of Cellular Complexing of Na+ to Water Structure and to Transport Kinetics

Abstract: The nuclear magnetic resonance (NMR) spectrum of Na+ is suitable for qualitative and quantitative analysis of Na+ in tissues. The width of the NMR spectrum is dependent upon the environment surrounding the individual Na+ ion. NMR spectra of fresh muscle compared with spectra of the same samples after ashing show that approximately 70% of total muscle Na+ gives no detectable NMR spectrum. This is probably due to complexation of Na+ with macromolecules, which causes the NMR spectrum to be broadened beyond detection. A similar effect has been observed when Na+ interacts with ion exchange resin. NMR also indicates that about 60% of Na+ of kidney and brain is complexed. Destruction of cell structure of muscle by homogenization little alters the per cent complexing of Na+. NMR studies show that Na+ is complexed by actomyosin, which may be the molecular site of complexation of some Na+ in muscle. The same studies indicate that the solubility of Na+ in the interstitial water of actomyosin gel is markedly reduced compared with its solubility in liquid water, which suggests that the water in the gel is organized into an icelike state by the nearby actomyosin molecules. If a major fraction of intracellular Na+ exists in a complexed state, then major revisions in most theoretical treatments of equilibria, diffusion, and transport of cellular Na+ become appropriate.

Active state in heart muscle. Its delayed onset and modification by inotropic agents.

Abstract: The course of active state in heart muscle has been analyzed using a modified quick release method. The onset of maximum active state was found to be delayed, requiring 110–500 msec from time of stimulation, while the time to peak isometric tension required 250–650 msec. Further, the time from stimulation to peak tension was linearly related to the time required to establish maximum intensity of active state as well as to the duration of maximum active state. The duration of maximum active state was prolonged (90–220 msec), occupying most of the latter half of the rising phase of the isometric contraction. Norepinephrine (10-5 M) shortened the latency from electrical stimulus to mechanical response, accelerated the onset of maximum active state, increased its intensity, decreased its duration, and accelerated its rate of decline. These changes were accompanied by an increase in the rate of tension development and the tension developed while the time from stimulation to peak isometric tension was abbreviated. Similar findings were shown for strophanthidin (1 µg/ml) although lesser decrements in the duration of maximum active state and time to peak tension were found than with norepinephrine for similar increments in the maximum intensity of active state.

Correlation of Transmitter Release with Membrane Properties of the Presynaptic Fiber of the Squid Giant Synapse

Abstract: Depolarization of the presynaptic terminal by current produced a postsynaptic potential (PSP) which increased with increasing presynaptic polarization and then reached a plateau. Iontophoretic injection of tetraethylammonium ions (TEA) into the presynaptic axon near the terminal produced a prolonged presynaptic spike. The resulting PSP is increased in size and its time course closely followed that of the presynaptic spike. The presynaptic fiber no longer exhibited rectification and strong depolarizations revealed that the PSP reached a maximum with about 110 mv depolarization. Further depolarization produced a decrease in PSP amplitude and finally transmission was blocked. However, a PSP then always appeared on withdrawal of the depolarizing current. Under the conditions of these experiments, the PSP could be considered a direct measure of transmitter release. Bathing the TEA-injected synapse with concentrations of tetrodotoxin (TTX) sufficient to block spike activity in both pre- and postsynaptic axons did not greatly modify postsynaptic electrogenesis. However, doubling TTX concentration reversibly blocked PSP. Thus the permeability changes to Na and K accompanying the spike do not appear necessary for transmitter release. Some other processes related to the level of presynaptic polarization must be involved to explain the data. The inhibition of transmitter release by strong depolarizations appears to be related to Ca action. A membrane Ca current may also be necessary for normal transmitter release.

Temperature Effects on Pacemaker Generation, Membrane Potential, and Critical Firing Threshold in Aplysia Neurons

Abstract: Temperature increases cause a regular and reproducible increase in the frequency of generation of pacemaker potentials in most Aplysia neurons specialized for this type of activity which can only be explained as a direct stimulating effect of temperature upon the ionic mechanisms responsible for pacemaker potentials. At the same time all cells in the visceral ganglion undergo a membrane potential hyperpolarization of approximately 1–2 mv/°C warmed. In spite of the marked variation in resting membrane potential the critical firing threshold remains at a constant membrane potential level at all temperatures in the absence of accommodative changes. The temperature-frequency curves of all types of cells are interpreted as a result of the interaction between the effects of temperature on the pacemaker-generating mechanism and resting membrane potential. Previous observations on the effects of temperature on excitability of mammalian neurons suggest that other types of neurons may undergo similar marked shifts in resting membrane potential with temperature variation.

The Effect of Valinomycin on Potassium and Sodium Permeability of HK and LK Sheep Red Cells

Abstract: A cyclic depsipeptide antibiotic, valinomycin, was found to produce increased selective permeability of the plasma membranes of HK and LK sheep red blood cells to potassium but not to sodium ions. The compound had relatively little effect on the active extrusion of sodium from HK sheep red blood cells or on the Na + K-stimulated ATPase activity of membranes derived from these cells. It is proposed that the selective cation permeability produced by this compound depends primarily on steric factors, particularly the relationship between the diameter of the ring and the effective diameter of the ion. The significance of these results for the problem of the mechanism of ionic selectivity in natural membranes is discussed.

Quinine and caffeine effects on 45Ca movements in frog sartorius muscle.

Abstract: 1 mM caffeine, which produces only twitch potentiation and not contracture in frog sartorius muscle, increases both the uptake and release of (45)Ca in this muscle by about 50 %, thus acting like higher, contracture-producing concentrations but less intensely. Quinine increases the rate of release of (45)Ca from frog sartorius but not from the Achilles tendon. The thresholds for the quinine effect on (45)Ca release and contracture tension are about 0.1 and 0.5 mM, respectively, at pH 7.1. Quinine (2 mM) also doubles the uptake of (45)Ca by normally polarized muscle. However, there are variable effects of quinine upon (45)Ca uptake in potassium-depolarized muscle. Quinine (2 mM), increases the Ca, Na, and water content of muscle while decreasing the K content. Both caffeine (1 mM) and quinine (2 mM) act to release (45)Ca from muscles that have been washed in Ringer's solution from which Ca was omitted and to which EDTA (5 mM) was added. These results, correlated with those of others, indicate that a basic effect of caffeine and quinine on muscle is to directly release activator Ca(2+) from the sarcoplasmic reticulum in proportion to the drug concentration. The drugs may also enhance the depolarization-induced Ca release caused by extra K(+) or an action potential. In respect to the myoplasmic Ca(2+) released by direct action of the drugs, a relatively high concentration is required to activate even only threshold contracture, but a much lower concentration, added to that released during excitation-contraction coupling, is associated with the condition causing considerable twitch potentiation.

Comparison of tetrodotoxin and procaine in internally perfused squid giant axons.

Abstract: Squid giant axons were internally perfused with tetrodotoxin and procaine, and excitability and electrical properties were studied by means of current-clamp and sucrose-gap voltage-clamp methods. Internally perfused tetrodotoxin was virtually without effect on the resting potential, the action potential, the early transient membrane ionic current, and the late steady-state membrane ionic current even at very high concentrations (1,000–10,000 nM) for a long period of time (up to 36 min). Externally applied tetrodotoxin at a concentration of 100 nM blocked the action potential and the early transient current in 2–3 min. Internally perfused procaine at concentrations of 1–10 mM reversibly depressed or blocked the action potential with an accompanying hyperpolarization of 2–4 mv, and inhibited both the early transient and late steady-state currents to the same extent. The time to peak early transient current was increased. The present results and the insolubility of tetrodotoxin in lipids have led to the conclusion that the gate controlling the flow of sodium ions through channels is located on the outer surface of the nerve membrane.

The Regulation of Catch in Molluscan Muscle

Abstract: Molluscan catch muscles are smooth muscles. As with mammalian smooth muscles, there is no transverse ordering of filaments or dense bodies. In contrast to mammalian smooth muscles, two size ranges of filaments are present. The thick filaments are long as well as large in diameter and contain paramyosin. The thin filaments contain actin and appear to run into and join the dense bodies. Vesicles are present which may be part of a sarcoplasmic reticulum. Neural activation of contraction in Mytilus muscle is similar to that observed in mammalian smooth muscles, and in some respects to frog striated muscle. The relaxing nerves, which reduce catch, are unique to catch muscles. 5-Hydroxytryptamine, which appears to mediate relaxation, specifically blocks catch tension but increases the ability of the muscle to fire spikes. It is speculated that Mytilus muscle actomyosin is activated by a Ca++-releasing mechanism, and that 5-hydroxytryptamine may reduce catch and increase excitability by influencing the rate of removal of intracellular free Ca++.

Oxidative and Glycolytic Recovery Metabolism in Muscle Fluorometric observations on their relative contributions

Abstract: The average degree of reduction of mitochondrial NAD has been measured in the intact toad sartorius by a fluorometric technique. It has been shown that cytoplasmic NADH does not interfere materially with these measurements. The percentage reduction of this respiratory coenzyme has been determined in a number of physiological steady states which are well correlated with fluorometrically determined levels of NADH in suspensions of mitochondria from the hind leg musculature of the toad. In addition, these findings are closely comparable to similar, spectrophotometric measurements on mitochondria from other sources. In the presence of an adequate O2 level a single twitch produces a decrease in fluorescence from the resting steady state which is followed by a slow return to the base line condition. This cycle indicates the intensity and the time course of the oxidative recovery metabolism. The area under this curve is directly related to the number of twitches up to three or four. Greater activity produces a curtailment of oxidative recovery due to glycolysis. In the presence of iodoacetate the linear relation holds for five to seven twitches. At still higher levels of activity a curtailment of the change in NAD level sets in, probably due to the removal of AMP by catabolic reactions.

In vivo effect of uncoupling agents on the incorporation of calcium and strontium into mitochondria and other subcellular fractions of rat liver.

Abstract: After injection of 45Ca++ or 89Sr++ into rats, the largest part of the radioactivity in the liver cell is associated with the subcellular structures, only negligible amounts of it being found in the soluble hyaloplasm. 50 % or more of the 45Ca++ and 89Sr++ in the liver cell is recovered in the mitochondrial fraction. The specific activity of Ca++ after injection of 45Ca++ is far greater in mitochondria than in microsomes. Pretreatment of the rats with uncouplers of oxidative phosphorylation markedly decreases the amount of radioactivity associated with the mitochondrial fraction. The amount of radioactivity recovered in the microsomes and in the final supernatant on the contrary increases. These effects are present only when mitochondrial oxidative phosphorylation is completely uncoupled. The Ca++ content of mitochondria from the livers of rats pretreated with uncouplers is sharply decreased with respect to the controls. It is concluded that in the liver cells of the intact animal energy-linked movements of Ca++ and Sr++ take place in mitochondria.

Discrimination between Alkali Metal Cations by Yeast II. Cation interactions in transport

Abstract: K+ is a competitive inhibitor of the uptake of the other alkali metal cations by yeast. Rb+ is a competitive inhibitor of K+ uptake, but Li+, Na+, and Cs+ act like H+. At relatively low concentrations they behave as apparent noncompetitive inhibitors of K+ transport, but the inhibition is incomplete. At higher concentrations they inhibit the remaining K+ transport competitively. Ca++ and Mg++ in relatively low concentrations partially inhibit K+ transport in an apparently noncompetitive manner although their affinity for the transport site is very low. In each case, in concentrations that produce "noncompetitive" inhibition, very little of the inhibiting cation is transported into the cell. Competitive inhibition is accompanied by appreciable uptake of the inhibiting cation. The apparently noncompetitive effect of other cations is reversed by K+ concentrations much higher than those necessary to essentially "saturate" the transport system. A model is proposed which can account for the inhibition kinetics. This model is based on two cation-binding sites for which cations compete, a carrier or transporting site, and a second nontransporting (modifier) site with a different array of affinities for cations. The association of certain cations with the modifier site leads to a reduction in the turnover of the carrier, the degree of reduction depending on the cation bound to the modifier site and on the cation being transported.