Intracellular Ionic Strength Research Articles

Preparation and properties of highly-purified Vibrio costicola polynucleotide phosphorylase

Vibrio costicola polynucleotide phosphorylase (polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8) has been purified to electrophoretic homogeneity. It has an approximate molecular weight of 220000 and consists of identical subunits with an approximate molecular weight of 72000. The enzyme appears to be a fairly typical polynucleotide phosphorylase with respect to its pH optima, substrate specificity and requirement for a divalent cation cofactor. However, the effect of salt concentration on its physiologically important phosphorolysis activity suggests that it is a moderately halophilic enzyme, able to function at the intracellular ionic strength of the bacterium. In addition, its ADP polymerization activity is remarkably stimulated by polylysine.

Intrinsic shortening speed of temperature-jump-activated intact muscle fibers. Effects of varying osmotic pressure with sucrose and KCl

Effects of intracellular ionic strength on the isotonic contraction properties of both intact fibers and skinned fibers give insights into the cross-bridge mechanism, but presently there is fundamental disagreement in the results on the two fiber preparations. This paper, which studies the effects on contraction of varying the osmotic pressure of the bathing medium with impermeant and permeant solutes, explains the above controversy and establishes the physiological significance of the previous results on skinned fibers. Fast-twitch fibers, isolated singly from tibialis and semitendinosus muscles of frogs, were activated by a temperature-jump technique in hyperosmotic solutions with either 100 or 150 mM sucrose (impermeant), or 50 or 75 mM KCl (permeant). Intracellular ionic strength was expected to rise in these solutions from the standard value of approximately 190 to 265 mM. Cell volume and the speed of unloaded shortening both decreased with sucrose and were constant with KCl. On the other hand, isometric force decreased equally with equiosmolar addition of either solute; this is additional evidence that contractile force decreases with ionic strength and is independent of fiber volume. Therefore, for the main cross-bridges, force per bridge is constant with changes in the lateral separation between the myofilaments. The next finding, that at a fixed cell volume the contraction speed is constant with KCl, provides clear evidence in intact fibers that the intrinsic speed of shortening is insensitive to increased ionic strength. The data with KCl are in agreement with the results on skinned fibers. The results suggest that in the cross-bridge kinetics in vivo the rate-limiting step is different for force than that for shortening. On the other hand, the decrease in speed with sucrose is associated with the shrinkage in cell volume, and is explained by the possibility of an increased internal load. A major fraction of the internal load may arise from unusual interactions between the sliding filaments; these interactions are enhanced in the fibers compressed with sucrose, but this does not affect the intrinsic kinetics of the main cross-bridges.

Effects of hypertonic solutions on contraction of frog tonic muscle fibers.

The influence of solution hypertonicity on contraction was studied in small bundles of tonic muscle fibers from the iliofibularis muscle of the frog Rana pipiens. Muscles were activated with high-K+ solutions that had osmolalities which were increased with tris(hydroxymethyl)aminomethanepropionate. Peak potassium contracture force decreased monotonically with tonicity and was zero in solutions with 2.5 or 3 times the osmolality of control Ringer. Contracture force at all tonicities studied (less than or equal to 3 X Ringer) was increased by increasing Ca2+ in the media 10-fold (to 20 mM) and/or by adding caffeine (10-20 mM). Nevertheless, this potentiated force also was diminished as tonicity increased. Force of single, mechanically skinned tonic fibers taken from these bundles was activated by Ca2+ over the same concentration range as that of twitch fibers. Moreover, maximal Ca2+-activated force, normalized per cross-sectional area, was similar in skinned tonic and twitch fibers. As was shown previously in twitch fibers, maximal Ca2+-activated force was decreased when ionic strength was increased. These data suggest that, as with twitch fibers, increased tonicity depresses contraction of tonic fibers by increasing the intracellular ionic strength, which in turn inhibits the ability of the contractile apparatus to generate force. Unlike twitch fibers, however, disruption of the excitation-contraction coupling process probably plays a more significant role in the action of hypertonicity on tonic fibers.

Hypertonicity and force development in frog skeletal muscle fibres.

Mechanical experiments were performed on isolated fibre bundles of frog semitendinosus muscle, using solutions made hypertonic by the addition of sucrose or NaC1. Muscle stiffness was measured with 1-ms step changes of length, from the plateau of an isometric tetanus. Within 20 min in hypertonic Ringer, isometric tetanus tension fell by 50% or more. The rate of tension redevelopment following stepwise shortening was reduced in hypertonic solution. Point voltage clamp studies showed that even when isometric tension had fallen to this extent, the stimulus strength--duration relation for mechanical activation was identical to that for controls. Point voltage clamp was used to intermittently repolarize fibres depolarized in solutions of high ionic strength, reactivating the excitation contraction coupling mechanism. Subsequent depolarization of the fibres allowed a visual test to be made of the fibres' ability to contract. The period during which contraction could be seen grew shorter as the ionic strength of the bathing medium was raised. The results of these different types of mechanical experiments suggested that the increased intracellular ionic strength caused by osmotic shrinkage in hypertonic Ringer's solution slows the rate at which individual cross bridges can form and develop tension, as well as reducing the maximum amount of tension they can generate. Muscle stiffness did not remain at control level in hypertonic solution; nor did it fall as much as did developed tension. The most obvious, although not the only, interpretation of this observation was that cross bridges are less stretched when isometric force is reduced and the length-tension relation of the elastic element is nonlinear.

The influence of varied tonicity of the extracellular medium on the depressant effect of active shortening in vertebrate striated muscle.

The depressant effect of active shortening was studied during isometric twitch contraction in intact single muscle fibres of the frog at varied tonicity of the extracellular medium. The shortening effect was calculated as the difference in peak redeveloped force after a small (control) and a larger (test) release step and was expressed in per cent of the isometric tetanic force. The solutions were made hypertonic by addition of sucrose (relative tonicity 1.22T and 1.44T) and hypotonic by reduction of NaCl (relative tonicity 0.81T and 0.62T). The shortening induced depression decreased from 13.0 +/- 1.2% in normal Ringer solution to 7.8 +/- 1.3% after immersion of the fibre in 1.22T solution (mean +/- SE, n = 7). This reduction of the depressant effect by shortening was less than half the size of that obtained in 1.44T solution. An increased force depression by shortening, from 12.7 +/- 1.2% to 17.1 +/- 1.5% (mean +/- SE, n = 7), was obtained when normal Ringer was replaced by 0.81T solution. This enhancement was further augmented in 0.62T solution. Experimental evidence is presented supporting the view that the influence of tonicity on the depressant effect of shortening is not due to tonicity induced changes in fibre width. The effect of varied tonicity on the shortening induced depression appears to be essentially related to alterations in intracellular ionic strength.

A generalized method for the minimization of cellular osmotic stresses and strains during the introduction and removal of permeable cryoprotectants.

The successful freeze preservation of mammalian cells and tissues usually requires the presence of high concentrations of cryoprotective agents (CPAs) such as glycerol, ethylene glycol, or dimethylsulfoxide. Unfortunately, the addition of these permeable agents to cells and tissues prior to freezing and their removal after thawing has been documented to be as damaging as the freeze-thaw process itself. This damaging process has been hypothesized to result from the drastic alterations in cell size caused by the osmotic stresses usually imposed upon cells during the introduction and removal of the cryoprotectants. Consequently, on the basis of a nonequilibrium thermodynamic model for the transport of water and a permeable CPA across cell membranes, a method has been developed to minimize these potentially lethal transient changes in cell size. This method involves the simultaneous variation of both the extracellular CPA and electrolyte or osmotic extender osmolalities in a balance, prescribed manner so that both the cellular water content and the total intracellular ionic strength remain constant as the intracellular CPA osmolarity is either raised or lowered. The theoretical analysis indicates that many of the resulting protocols are practical from the clinical point of view.

The control by Ca2+ of the polyphosphoinositide phosphodiesterase and the Ca2+-pump ATPase in human erythrocytes.

1. Both the Ca(2+)-pump ATPase and the polyphosphoinositide phosphodiesterase of the erythrocyte membrane can, when assayed under appropriate conditions, be activated by Ca(2+) in the micromolar range. We have therefore compared the mechanisms and affinities for Ca(2+) activation of the two enzymes in human erythrocyte membranes, to see whether the polyphosphoinositide phosphodiesterase would be active in normal healthy erythrocytes. 2. At physiological ionic strength and in the presence of calmodulin, the Ca(2+)-pump ATPase was activated by Ca(2+) in a highly co-operative manner, with half-maximal activation occurring at about 0.3mum-Ca(2+). At an optimal Ca(2+) concentration, calmodulin stimulated the Ca(2+)-sensitive ATPase activity about 10-fold. 3. Ca(2+) activated the polyphosphoinositide phosphodiesterase in a non-co-operative manner. The Ca(2+) requirements for breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were identical, which supports our previous conclusion that Ca(2+) activates a single polyphosphoinositide phosphodiesterase that degrades both lipids with equal facility. Added calmodulin did not affect the activity of the polyphosphoinositide phosphodiesterase. 4. At low ionic strength in the absence of Mg(2+), half-maximal activation of the phosphodiesterase was at about 3mum-Ca(2+). The presence of 1mm-Mg(2+) shifted the Ca(2+) activation curve to the right, as did elevation of the ionic strength. When the Ca(2+)-pump ATPase and the polyphosphoinositide phosphodiesterase were assayed in the same incubations and under conditions of intracellular ionic strength and Mg(2+) concentration, the ATPase was fully activated at 3mum-Ca(2+), whereas no polyphosphoinositide phosphodiesterase activity was detected below 100mum-Ca(2+). 5. The Ca(2+)-pump ATPase of the erythrocyte membrane normally maintains the Ca(2+) concentration of healthy erythrocytes below approx. 0.1mum. It therefore seems unlikely that the polyphosphoinositide phosphodiesterase of the erythrocyte membrane ever expresses its activity in a healthy erythrocyte.

Intracellular pH measurements by 31P nuclear magnetic resonance. Influence of factors other than pH on 31P chemical shifts.

Titration curves plotting chemical shift vs. pH for inorganic phosphate and glucose 6-phosphate in solutions of various composition are presented. Physiological concentrations of K+ (0.1 M) and Mg2+ (5 mM) are shown to significantly shift the titration curve. The Mg2+ effect can be partly or completely reversed by addition of sufficient quantities of adenosine triphosphate or organic acids. The acidic protein bovine serum albumin and soluble maize root tip protein have no noticeable effect on the titration curves, whereas the basic protein protamine exerts a profound effect. The results clearly indicate that knowledge of intracellular ionic strength and free Mg2+ concentrations in the sample are required if the determination of intracellular pH by 31P NMR is to be considered accurate within +/- 0.05-01 pH unit.

A membrane hypothesis of aging

Experimental data show that cell membranes become more rigid during aging. If this involves a decrease of resting potassium permeability, the intracellular potassium concentration will increase. Such an increase is beneficial for the maintenance of cell excitability, however, it represents a drawback for the nuclear functions, since the intracellular ionic strength may reach values even above 400 mEq kg −1 cell water, where the chromatin becomes more condensed and the activity of DNA-dependent RNA-polymerase as well as other enzymes probably decreases. This “membrane hypothesis” of aging may explain the decreased protein synthetic activity of old cells, especially of postmitotic ones. X-ray microanalysis has revealed that potassium concentrations significantly increase in the nucleus and cytoplasm of brain and liver cells of old rats. The chromatin of old nerve cells is more condensed than that of the young ones, the rates of nucleolar and nucleoplasmic RNA-synthesis are significantly lower in the old brain cells, and also the number of perichromatin granules decreases with age. A decrease of intracellular potassium content, in brain cells of old rats can be brought about by phytohemagglutinin-P and centrophenoxine. This is accompanied by a sort of rejuvenation: the chromatin becomes decondensed, the rates of nucleolar and nucleoplasmic RNA-synthesis increase together with the number of perichromatin granules, the medium life-span and the learning capacity of the animals increase. These experimental results support the “membrane hypothesis” of aging.

The force‐velocity relationship in vertebrate muscle fibres at varied tonicity of the extracellular medium

1. The relationship between active force and velocity of shortening was studied during tetanic contraction of isolated semitendinosus muscle fibres of the frog (0.5-2.0 degrees C). Measurements were carried out with the fibre immersed in isotonic (1.00R) Ringer solution and in solutions that were made hypotonic by reduction of NaCl (osmolality 0.62 and 0.81 of normal Ringer) and hypertonic by addition of sucrose (osmolality 1.22 and 1.44 of normal Ringer).2. The force-velocity relation was hyperbolic at loads lower than 80% of measured isometric force (P(0)) but exhibited a reversed curvature between 0.8P(0) and P(0). The maximum velocity of shortening was determined in two different ways: (i) by extrapolation to zero load from force-velocity data truncated at 0.8P(0) (computer fitting of hyperbola, leastsquares method) and (ii) by recording the time required to take up the slack of the fibre after a quick release during tetanus.3. Isometric force and maximum speed of shortening both changed inversely with the tonicity of the extracellular medium. Immersion of the fibre in 0.81R hypotonic solution caused active tension and shortening velocity to increase by 10 +/- 1% (mean +/- S.E. of mean, n = 14) and 12 +/- 1%, respectively. Conversely, force and shortening velocity decreased by 12 +/- 1% (n = 13) and 22 +/- 2% when normal Ringer was replaced by 1.22R hypertonic solution. These changes doubled when the tonicity was altered from normal Ringer to 0.62R and 1.44R, respectively.4. Changes in fibre cross-sectional area equivalent to those obtained in the 0.81R and 1.22R solutions (+11% and -13%, respectively) were produced by varying the sarcomere length within the range 2.0-2.5 mum in the normal Ringer solution. Maximum velocity of shortening remained very nearly constant under these conditions, indicating that the shortening velocity, like the isometric force, is not critically dependent on changes in myofilament lattice width over the range considered.5. The results support the view that both shortening velocity and active force are modulated by changes of the intracellular ionic strength above and below the level that normally exists in the intact muscle fibre.

Effects of salinity and osmolarity of the medium on amino acid metabolism in Cyclotella cryptica

The amino acid metabolism of Cyclotella cryptica is strongly influenced by changes in salinity of the medium. A sudden increase in salinity (water stress) reduces the total uptake of amino acids by the cells and also their assimilation into proteins. Under water-stress conditions, proline is readily synthesized from glutamate, arginine, and ornithine. and proline synthesized under these conditions turns over very slowly and accumulates.Increases in osmolarity of the medium, whether caused by salts, mannitol, or sucrose (but not by glycerol), is followed immediately by rapid synthesis of proline. Proteolysis is not observed under water-stress conditions. The high level of proline accumulated under these conditions is rapidly reduced when the cells are transferred back to low osmolarity media. The loss of 14C from proline is accompanied by rapid incorporation into proteins and also conversion of proline into other cell constituents.The proline level is relatively low in high-salinity-adapted cells, and proline turns over rapidly in such cells. This indicated that the accumulation of proline occurs mainly in response to abrupt changes of intracellular ionic strength during adaptation to increased salinities. Additions of salts and organic substances to cell suspensions in low-salinity media cause plasmolysis followed by rapid deplasmolysis. However, when cells are transferred to high-osmolarity solutions of single salts or organic substances, deplasmolysis is only observed in media containing KCl or glycerol. Increases of external osmolarity cause increases in concentrations of intracellular potassium and free amino acids.

Activities of potassium and sodium ions in rabbit heart muscle.

Activities (a) of intracellular K and Na in rabbit ventricular papillary muslces were determined with cation-selectivve glass microelectrodes and concentrations (C) were estimated with flame photometry. The CK and aK of the muscles were 134.9 +/- 3.1 mM (mean value +/- SE) and 82.6 mM, respectively, at 25 degrees C. The corresponding CNa and aNa were 32.7 +/- 2.7 and 5.7, respectively. The apparent intracellular activity coefficients for K (gammaK) and Na (gammaNa) were 0.612 and 0.175, respectively. Similar results were obtained at 35 +/- 1 degree C. gammaK was substantially lower than the activity coefficient (0.745) of extracellular fluid (Tyrode's solution), which might be expected on the basis of a different intracellular ionic strength. gammaNa was much lower than that of extracellular fluid, and suggest that much of the Na was compartmentalized or sequestered. For external K concentrations greater than 5 mM, the resting membrane potentials agreed well with the potential differences calculated from the K activity gradients across the cell membrane as a potassium electrode. These results emphasize that potassium equilibrium potentials in heart muscle should be calculated by activities rather than concentrations.

Effect of Osmotic Shrinking and Swelling of Red Cells on Whole Blood Oxygen Affinity

The effect of osmotically induced alterations in mean corpuscular hemoglobin concentration (MCHC) on oxygen affinity of hemoglobin was studied in blood cells containing various concentrations of 2,3-diphosphoglycerate (DPG). At normal DPG concentration (0.88 mol/mol Hb) there was a positive correlation between MCHC and whole blood oxygen affinity (P50); the magnitude of the effect, expressed as δP50/δMCHC, was 0.17, a value considerably smaller than that reported by previous investigators. At low DPG concentration (< 0.2 mol/mol Hb) the value of δP50/δAMCHC was 0.40; at DPG levels above normal (> 1.3 mol/mol Hb) no relationship between MCHC and P50 was found. The results are discussed in relation to the various changes in the intracellular milieu on osmotic manipulation, and it is concluded that the alterations of P50 are due mainly to changes in intracellular ionic strength.

Effect of osmotic shrinking and swelling of red cells on whole blood oxygen affinity.

The effect of osmotically induced alterations in mean corpuscular hemoglobin concentration (MCHC) on oxygen affinity of hemoglobin was studied in blood cells containing various concentrations of 2,3-diphosphoglycerate (DPG). At normal DPG concentration (0.88 mol/mol Hb) there was a positive correlation between MCHC and whole blood oxygen affinity (P50); the magnitude of the effect, expressed as δP50/δMCHC, was 0.17, a value considerably smaller than that reported by previous investigators. At low DPG concentration ( 1.3 mol/mol Hb) no relationship between MCHC and P50 was found. The results are discussed in relation to the various changes in the intracellular milieu on osmotic manipulation, and it is concluded that the alterations of P50 are due mainly to changes in intracellular ionic strength.

Equilibrium dialysis of ions in nystatin-treated red cells.

THE human red cell membrane is as impermeable to K and Na ions (P=10−11 cm s−1) as an unmodified lipid bilayer1 and has a low permeability (P=10−8 cm s−1) to Cl moving as a free ion. A permeability coefficient of 10−11 cm s−1 is as low as that estimated for unmodified lipid bilayer membranes, which are essentially impermeable to alkali metal cations and anions. Because of this low permeability, studies of active and passive ion transport that require control of both ion concentration and ionic strength have been virtually restricted to work on red cell ghosts prepared by haemolysis and on perfused giant squid axons3. Here we give a principle for altering rapidly and drastically the intracellular ionic strength by varying the concentrations of monovalent cations and anions.

Polyamine metabolism in potassium-deficient bacteria.

The metabolism of polyamines was studied in K(+)-dependent strains of Escherichia coli. When these stringent organisms were in a medium containing Na(+) instead of K(+), protein synthesis was arrested, but synthesis of ribonucleic acid continued as it would in a relaxed organism. The Na(+) medium inhibited synthesis of spermidine and S-adenosylmethionine. However, the synthesis of putrescine was accelerated at least five- to eightfold. Exogenous ornithine doubled even this rate of putrescine synthesis but did not increase the low level of putrescine synthesis in the K(+) medium. In K(+) or Na(+) media, with or without 0.3 mm arginine, putrescine was derived almost entirely from ornithine via ornithine decarboxylase. Addition of spermidine (5 mm) to a Na(+) culture markedly inhibited putrescine synthesis. The ornithine decarboxylase of an extract of a K(-)-dependent strain prepared at low ionic strength was separated from ribosomes, deoxyribonucleic acid, and associated polyamines by centrifugation, and from many ions by ultrafiltration and fractionation on Sephadex G-100. Addition of Na(+) and K(+) salts to 200 mm was markedly inhibitory. The combined reductions both in synthesis of the inhibitor spermidine and in intracellular ionic strength may explain the in vivo activation of this enzyme.

Halobacterium cutirubrum RNA polymerase: Subunit composition and salt-dependent template specificity

We have described [ 1,2] the partial purification and properties of DNA-dependent RNA polymerase from the extreme halophile Halobacterium cutirubrum. Since extremely halophilic bacteria-have an intracellular ionic strength of about 5.9 and enzymes isolated from them usually require high salt concentrations for stability and activity [3] , it was surprising that the RNA polymerase was inhibited by salt when calf thymus DNA was used as template. The present paper describes (1) the isolation and purification of two proteins that together form the active enzyme, and (2) the effect of salt concentration on the template specificity of the polymerase.

Muscle contraction: the effect of ionic strength.

CONTRACTILE responses are depressed and may be abolished in muscles that are shrunken by exposure to hyperosmotic media; they are enhanced when the muscles are swollen in hyposmotic media1–4. The effects of hyperosmotic solutions have been ascribed5 to “dissociation of electromechanical coupling”, but earlier work4,6 had shown that the processes of excitation–contraction coupling were unaffected. Howarth4 therefore proposed two alternative mechanisms. He suggested that the movement of water into or out of the cell in the different osmotic conditions (i) may modify the viscosity of the myoplasm, or (ii) may affect the contractile machinery by the associated changes in the intracellular ionic strength. In our experiments, intracellular applications of calcium were used to activate contraction; thus they bypassed the factor of e–c coupling. Furthermore, they were designed so as to discriminate between effects on the volume of the fibres and on the ionic strength of the intracellular phase which are both a result of changes in water content. The results demonstrate that the predominant and, perhaps, the only factor affecting the tension is the change in ionic strength. Only data on ionophoretic applications of calcium are reported here, but similar findings have also been obtained with other agents that activate contractile responses7.