A model to explain the osmotic pressure behavior of hemoglobin and serum albumin

Previously published osmotic pressure data on hemoglobin and bovine serum albumin were used to determine the osmotically unresponsive solvent volume per unit dry mass of protein. A model is presented that accounts for the osmotic pressure of globular proteins based on a surface-associated osmotically unresponsive solvent volume. The model also accounts for changes in the osmotically unresponsive solvent volume owing to changes in pH, cosolute salt concentration, protein conformation, and protein aggregation.     

Studies on isolated cell components. IV. The effect of various solutions on the isolated rat liver nucleus

1. The effects of morganic ions, electrolyte concentration, and pH on the appearance and volume of the isolated rat liver nucleus have been studied. Nuclei were isolated by differential centrifugation in a buffered salt-sucrose mixture at pH 7.1. Nuclear volumes were determined photographically. 2. In solutions of NaCl, of KCl, and in potassium phosphate buffers the nuclear volume decreased markedly with an increase in concentration from 0.001 M to 0.05 M but remained essentially constant with further increase in concentration to 1.0 M. The effects of CaCl(2) and MgCl(2) differed from those of NaCl and KCl in that a smaller volume was obtained in concentrations less than 0.15 M, and in the case of CaCl(2) an increase in volume was obtained in more concentrated solutions. The volume changes are considered to be due primarily to ionic effects on the nuclear colloids rather than to osmotic behavior. 3. Treatment of nuclei with DNAase prevented the characteristic volume changes resulting from ion effects, suggesting the importance of DNA in nuclear volume changes. 4. The optical changes in isolated nuclei in various concentrations of KCl, NaCl, CaCl(2), MgCl(2), and in potassium phosphate buffers as observed under phase contrast illumination are described. CaCl(2) gave the most marked nuclear changes from the conditions in the uninjured cell and caused shrinkage and granulation in 0.001 M concentration. The effects of CaCl(2) were also manifested in 0.88 M sucrose, in mixtures with monovalent salts, and in serum. Changes in nuclear volume and optical appearance which occurred in salt solutions and in 0.1 N HCl were readily reversible. 5. Nuclear volume remained constant between pH 8.91 and 5.12 and decreased in more acid solutions. 6. Sucrose had no appreciable osmotic effect, and in hyperosmotic solution. (0.88 M) nuclei showed swelling and rupture comparable to that in distilled water. 7. The results are considered in relation to the requirements of nuclear isolation media. 8. Rat liver nuclei isolated in a buffered salt-sucrose medium by differential centrifugation exhibited a pattern of size distribution similar to that of fixed nuclei but were of considerably larger volume. The ratio of the volumes of the peak frequencies of the two chief size groups was 1:1.9.     

The osmotic behaviour of Sephadex and its effects on chromatography

1. Sephadex in bead form shows reversible changes of inner volume when immersed in solutions of a non-penetrating solute. These changes are in accordance with the theory of Flory (1953) for the swelling of gels. This makes possible the use of single beads for measuring the osmotic pressures of polymer solutions, up to or beyond 1kg./cm.(2). 2. Measurements of the inner volume of Sephadex G-200 by equilibrium dilution, at various concentrations of dextran 500, gave values in agreement with those obtained from the dimensions of single beads. 3. Measurements by chromatography of the inner volume of Sephadex G-200 gave values that differed slightly but significantly from those obtained by the other methods. Similar small disagreements were found between the dilution and chromatographic values for bovine serum albumin measured in the presence of various concentrations of dextran 500. The major factor that affects the dependence of the chromatographic elution volume (of dextran 500) on concentration is shown to be the change of the inner volume of the Sephadex, with dynamic factors playing a minor role. 4. Contrary to the findings of Ackers (1964), the equilibrium dilution and chromatographic methods gave closely agreeing values for the distribution coefficient, K(av.), of bovine serum albumin on Sephadex G-200; this cast doubt on his explanation of the nature of chromatographic separations on Sephadex.     

NATIVE AND REGENERATED BOVINE ALBUMIN : I. PREPARATION AND PHYSICOCHEMICAL PROPERTIES

1. Whole bovine albumin, homogeneous in diffusion and sedimentation, and essentially homogeneous in electrophoresis, has been prepared by a method involving ammonium sulfate precipitation of the globulins in the cold and of the albumin at room temperature, isoelectric precipitation of the euglobulins, and reprecipitation of the albumin. 2. The product has been characterized by chemical analysis and by viscosity, diffusion, sedimentation, and electrophoresis measurements. The carbohydrate content is 0.38 per cent, the nitrogen content, 15.2 per cent. The molecular shape approximates that of a prolate ellipsoid with an axial ratio of 3.1, assuming 33 per cent hydration; the average molecular weight is 65,000. 3. Bovine albumin is readily denatured by concentrated solutions of urea or guanidine hydrochloride, gross changes in molecular shape resulting. 4. Regeneration of bovine albumin denatured in solutions of 8 M urea or guanidine hydrochloride yields a material closely resembling the native in carbohydrate content, in molecular size and shape, and in electrophoretic properties. However, the regenerated protein differs from the native in susceptibility to tryptic digestion, and, in this respect, appears to be in a denatured state. 5. In 8 M solutions of guanidine hydrochloride a limiting yield of regenerated albumin equivalent to 95 per cent of the original protein is approached. 6. Bovine crystalbumin, a crystalline carbohydrate-free fraction of the whole albumin, appears to be more susceptible to denaturation than whole bovine albumin.     

THE SPECIFIC VOLUME OF DRY PROTEIN

The specific volume of dry protein is of interest in the interpretation of electron micrographs of protein molecules. A pycnometer with helium gas as the fluid is described and measurements on dry powders of lysozyme, ovalbumin, bovine serum albumin, and fibrinogen are reported. The values obtained exceed those from determinations on solutions by an average of 1.4 per cent when the latter measurements are uncorrected for electrostriction.     

Regeneration of Nocardia orientalis protoplasts

A method for effective regeneration of the protoplasts of N. orientalis, a vancomycin-producing organism into viable cells on a rich organic medium was developed. The dependence of the regeneration on the conditions of the protoplast plating out and the level of the regeneration medium dehydration was studied. The highest positive effect was observed when the protoplasts were suspended in the agarized medium and then plated out on the regeneration medium dehydrated by 2.5 per cent. The frequency of the protoplast regeneration increased on addition of bovine serum albumin to the regeneration medium. The effect of bovine serum albumin depended on its concentration. When the albumin concentration was optimal (0.01 per cent) the regeneration amounted to 100 per cent.     

Active immunization to luteinizing hormone releasing hormone to inhibit the induction of mammary tumors in the rat

Immunization of female rats with a bovine serum albumin-luteinizing hormone releasing hormone conjugate results in suppression of dimethylbenzanthracene mammary tumor incidence. Tumor incidence was 1.3, and 1.29 tumors per rat in bovine serum albumin alone (n = 10) and unimmunized (n = 18) control groups, but no tumors were found in the bovine serum albumin-luteinizing hormone releasing hormone conjugate immunized animals (n = 10). In a second experiment immunization with bovine serum albumin-luteinizing hormone releasing hormone conjugates reduced tumor incidence to 0.3 tumors per rat (n = 10) from the 1.2 tumors per animal seen in the control animals (n = 10) immunized with bovine serum albumin alone. Bovine serum albumin-luteinizing hormone immunization caused the production of anti-LHRH antibodies, an interruption of estrous cycles, lowered serum estradiol and progesterone levels, and atrophy of the ovaries and uteri. Immunization BSA-hormone conjugates is a novel anti-tumor strategy.     

THE RHEOLOGY OF THE BLOOD. V

A study has been made of those proteins which might offer exceptions to the law that the fluidity of a protein solution is a linear function of the volume concentration; viz., egg albumin, serum albumin, pseudoglobulin, euglobulin, gelatin, and sodium caseinogenate. Solutions of egg albumin below 20 per cent by weight obey the above law but somewhat below 30 per cent the fluidities begin to be too high, presumably due to the contribution to the fluidity made by the deformation of the particles as they come into contact, as the fluidity approaches zero. The fluidity of serum albumin solutions shows a similar behavior, being exceptional above 15 per cent in weight. Pseudoglobulin and euglobulin give fluidity-concentration curves (Fig. 4) which are linear up to about 2.5 per cent each in a total range of 20 and 14 per cent respectively. From this singular point both compounds show a second range which is linear. Pseudoglobulin is the only substance whose solutions seem to show a third linear range. We have also used the data of Chick and Martin for sodium caseinogenate and found evidence for two linear régimes. It is desirable at this time to call attention to the measurements of the flow of glycogen solutions by Botazzi and d''Errico (14) which in Fluidity and See PDF for Structure plasticity, page 207, are expressed in rhes. The data show two linear fluidity curves of different slopes. In this case it was definitely known that the data for each curve were measured with different viscometers which suggested the possibility of an error in viscometry entering in to confuse the issue. We have no suspicions as to the reliability of the data studied in this paper; we only wish to caution the readers that our hypotheses based on these data must be regarded with due reserve until confirmed. We have found a formula (11) based on the supposed linear relation between logarithmic fluidities and concentration which is convenient to use within the range, but close examination reveals that it does not reproduce the data for the higher concentrations at 25° nor does it permit extrapolation to pure water It is not realistic enough because it does not contemplate any change of régime in going from viscous to non-Newtonian or plastic flow. The formula does not apply to any other of the proteins studied in this paper nor to the great majority of proteins already reported as following the linear law. These are serious objections. We have therefore offered as an alternative a simple formula (24) according to which the fluidities are additive in the viscous régime. When the emulsoid particles approach close packing, they are deformed and this deformation contributes to the flow and the fluidity volume concentration curve is again linear. In fact, there may be one or more additional changes of régime.     

Effects of alterations in endothelial cell volume on transendothelial albumin permeability

We examined the effects of alterations in endothelial cell volume on transendothelial albumin permeability. Studies were done using a confluent monolayer of bovine pulmonary artery endothelial cells grown on gelatinized microporous filters. When endothelial cells were exposed to media made hypertonic with 200 mM mannitol, the intracellular volume (measured with 14C-urea) decreased twofold and remained decreased over a 30-minute time-span, thus showing no significant regulatory volume increase (RVI) within this time period. When endothelial cells were exposed to hypotonic media, intracellular volume rapidly doubled within 2 minutes, and then decreased to baseline values within 10 minutes in spite of the sustained hypotonic environment, a process known as regulatory volume decrease (RVD). We also measured the transendothelial flux of 125I-albumin with the cells exposed to the same osmotic changes. We observed that only under hypertonic conditions was there a significant change in the 125I-albumin permeability. These results indicate that the pulmonary artery endothelial cells in culture alter their cell volume when exposed to variations in the osmotic environment, and also show RVD in response to hypotonic conditions but no RVI within 40 minutes after exposure to hypertonic conditions. The transendothelial albumin permeability did not change under hypotonic conditions but increased under hypertonic conditions. Thus, endothelial cells shrinkage may be an important mechanism of increased endothelial macromolecule permeability. These volume changes may occur in endothelial cells in situ and have a role in inducing alterations in the transendothelial permeability to proteins.     

Variability in different lots of commercial bovine serum albumin affects cell multiplication and hatching of rabbit blastocysts in culture

Rabbit morulae were cultured in vitro for 4 days in a synthetic culture medium supplemented with two different lots of commercial bovine serum albumin (BSA) and two different amino acid formulations in a factorial 2 x 2 arrangement. One lot of BSA caused complete hatching of a proportion of blastocysts and formation of more than twice as many cells per blastocyst (hatched and unhatched) as that of the second BSA lot which did not cause complete hatching of any blastocysts. The mean cell numbers of hatched blastocysts were more than twice those of non-hatched blastocysts. There was no significant effect of amino acid formulation.     

The origin and consequences of concentration dependence in gel chromatography

Concentration dependence of elution volume was determined for Blue Dextran 2000, Dextran 500, Dextran sulphate 500 and bovine serum albumin on columns of Sephadex G-100 equilibrated with sodium phosphate buffer, I 0.1, pH6.8. From the results for Dextran 500, it was shown that a linear relation exists between elution volume and the corresponding osmotic pressure calculated for the same concentration and incorporating the term containing the second virial coefficient. This relationship was used to predict the concentration dependence of elution volume for bovine serum albumin and myoglobin, proteins that partially penetrate Sephadex G-100. Possible consequences of osmotic effects are considered in relation to various types of column experiments, including differential chromatography.     

Stimulation of bone resorption by various prostaglandins in organ culture.

The ability of E, F, A and B prostaglandins to stimulate bone resorption was demonstrated in organ culture. All of the compounds tested were able to increase the release of previously incorporated 45Ca from fetal rat bone by 60 to 135 per cent at maximally effective doses, but prostaglandins of the E series were 10- to 100- fold more potent than F, A or B prostaglandins. Compounds with two double bonds in the side chain were usually more potent than those with one double bond. PGE2 stimulation of bone resorption increased linearly with the logarithm of the medium concentration over the range of 10(-9)M to 10(-5)M, then decreased at higher concentrations. PGE2 stimulated bone resorption more slowly than did parathyroid hormone but caused complete resorption after six days in the culture system. Equilibrium dialysis studies showed no significant binding of F, and 16-34% binding of E and A prostaglandins to bovine serum albumin, which was present in the medium at 1 mg/ml. These differences in albumin binding could not account for differences in potency.     

Osmotic Flow of Water across Permeable Cellulose Membranes

Direct measurements have been made of the net volume flow through cellulose membranes, due to a difference in concentration of solute across the membrane. The aqueous solutions used included solutes ranging in size from deuterated water to bovine serum albumin. For the semipermeable membrane (impermeable to the solute) the volume flow produced by the osmotic gradient is equal to the flow produced by the hydrostatic pressure RT ΔC, as given by the van't Hoff relationship. In the case in which the membrane is permeable to the solute, the net volume flow is reduced, as predicted by the theory of Staverman, based on the thermodynamics of the steady state. A means of establishing the amount of this reduction is given, depending on the size of the solute molecule and the effective pore radius of the membrane. With the help of these results, a hypothetical biological membrane moving water by osmotic and hydrostatic pressure gradients is discussed.     

Viscosity of concentrated solutions and of human erythrocyte cytoplasm determined from NMR measurement of molecular correlation times. The dependence of viscosity on cell volume

Metabolically active human erythrocytes were incubated with [alpha-13C]glycine which led to the specific enrichment of intracellular glutathione. The cells were then studied using 13C-NMR in which the longitudinal relaxation times (T1) and nuclear Overhauser enhancements of the free glycine and glutathione were measured. The T1 values of labelled glycine were also determined in various-concentration solutions of bovine serum albumin and glycerol and also of the natural abundance 13C of glycerol in glycerol solutions. From the T1 estimates the rotational correlation time (tau r) was calculated using a formula based on a model of an isotropic spherical rotor or that of a symmetrical ellipsoidal rotor; for glycine the differences in estimates of tau r obtained using the two models were not significant. From the correlation times and by use of the Stokes-Einstein equations viscosity and translational diffusion coefficients were calculated; thus comment can be made on the likelihood of diffusion control of certain enzyme-catalysed reactions in the erythrocyte. Bulk viscosities of the erythrocyte cytoplasm and the above-mentioned solutions were measured using Ostwald capillary viscometry. Large differences existed between the latter viscosity estimates and those based upon NMR-T1 measurements. We derived an equation from the theory of the viscosity of concentrated solutions which contains two phenomenological interaction parameters, a 'shape' factor and a 'volume' factor; it was fitted to data relating to the concentration dependence of viscosity measured by both methods. We showed, by using the equation and interaction-parameter estimates for a particular probe molecule in a particular solution, that it was possible to correlate NMR viscosity and bulk viscosity; in other words, given an estimate of the bulk viscosity, it was possible to calculate the NMR 'micro' viscosity or vice versa. However, the values of the interaction parameters depend upon the relative sizes of the probe and solute molecules and must be separately determined for each probe-solute-solvent system. Under various conditions of extracellular osmotic pressure, erythrocytes change volume and thus the viscosity of the intracellular milieu is altered. The volume changes resulted in changes in the T1 of [alpha-13C]glycine. Conversely, we showed that alterations in T1, when appropriately calibrated, could be used for monitoring changes in volume of metabolically active cells.     

Theoretical and experimental studies on freezing point depression and vapor pressure deficit as methods to measure osmotic pressure of aqueous polyethylene glycol and bovine serum albumin solutions

For survival in adverse environments where there is drought, high salt concentration or low temperature, some plants seem to be able to synthesize biochemical compounds, including proteins, in response to changes in water activity or osmotic pressure. Measurement of the water activity or osmotic pressure of simple aqueous solutions has been based on freezing point depression or vapor pressure deficit. Measurement of the osmotic pressure of plants under water stress has been mainly based on vapor pressure deficit. However, differences have been noted for osmotic pressure values of aqueous polyethylene glycol (PEG) solutions measured by freezing point depression and vapor pressure deficit. For this paper, the physicochemical basis of freezing point depression and vapor pressure deficit were first examined theoretically and then, the osmotic pressure of aqueous ethylene glycol and of PEG solutions were measured by both freezing point depression and vapor pressure deficit in comparison with other aqueous solutions such as NaCl, KCl, CaCl(2), glucose, sucrose, raffinose, and bovine serum albumin (BSA) solutions. The results showed that: (1) freezing point depression and vapor pressure deficit share theoretically the same physicochemical basis; (2) theoretically, they are proportional to the molal concentration of the aqueous solutions to be measured; (3) in practice, the osmotic pressure levels of aqueous NaCl, KCl, CaCl(2), glucose, sucrose, and raffinose solutions increase in proportion to their molal concentrations and there is little inconsistency between those measured by freezing point depression and vapor pressure deficit; (4) the osmotic pressure levels of aqueous ethylene glycol and PEG solutions measured by freezing point depression differed from the values measured by vapor pressure deficit; (5) the osmotic pressure of aqueous BSA solution measured by freezing point depression differed slightly from that measured by vapor pressure deficit.     

Mitochondria in the flight muscles of insects. II. Effects of the medium on the size form,and organization of isolated sarcosomes

1. The sarcosomes of Drosophila and the blowfly, Phormia, are dense, spherical, homogeneous bodies when isolated from flight muscle and promptly examined under the phase contrast, oil immersion objective. 2. Their average diameter in newly emerged flies is about 1 micro. This value increases rapidly during the 1st week of adult life and then becomes constant at approximately 2.5 micro. At each age the variation in sarcosome diameter conforms approximately to a normal distribution. 3. The degree to which isolated sarcosomes retain their initial size and organization is remarkably conditioned by the composition and the hydrogen ion concentration of the medium in which they are teased and suspended. In suboptimal media three major categories of change were encountered: (1) swelling, with or without compaction of the contents (as in distilled water and salt solutions); (2) shrinkage to rod-like, pleomorphic forms (as in blood serum); and (3) fuzzy degeneration (as in sugar solutions). 4. The membrane that surrounds each sarcosome becomes plainly visible in swollen sarcosomes. A continuation of swelling is accompanied by the escape of the sarcosomal contents, the vacated membrane persisting as a spherical, optically empty ghost. 5. Sarcosomes appear to behave like osmometers when suspended in various aqueous solutions. Solutes which penetrate the membrane show only transient effects in preventing the osmotic entry of water. 6. Under this analysis we find the membrane to be more or less freely permeable to the ions of sodium, potassium, calcium, magnesium, chloride, and phosphate, to non-electrolytes smaller than hexoses, to phosphorylated hexoses, and to several intermediates of the citric acid cycle. 7. The sarcosomal membrane appears to be less permeable to non-electrolytes larger than pentoses, provided that such molecules are not phosphorylated. 8. The membrane shows a higher permeability to ATP than to ADP. The significance of this observation is considered with respect to the ADP-ATP shuttle between sarcosomes and muscle fibrils. 9. Simple solutions of electrolytes or non-electrolytes cause more or less conspicuous changes in the microscopic appearance of sarcosomes. Prolonged preservation was achieved only in more complicated media containing protein. It is concluded that the Donnan equilibrium is the source of the principal osmotic forces regulating the movement of water through the sarcosomal membrane. 10. The optimal medium for the preservation of isolated sarcosomes was an intracellular Ringer solution containing 2.5 per cent crystalline bovine albumin in 0.16 M potassium phosphate buffer at pH 7.0.     

Exclusion in hyaluronate gels.

Osmotic pressures of solutions of hyaluronate (HA) (mol wt 117,000) and mixtures of HA and bovine serum albumin (BSA) in phosphate-buffered saline, pH 7.2 were measured with a membrane osmometer. The data were fit with a virial expansion in integral powers of total nondiffusible solute concentration. Values of number average molecular weight were calculated for HA and the mixtures from the first virial coefficients. The excluded volume of HA in the single nondiffusible solute solution was calculated from the second virial coefficient extracted from the data on the HA solution. The excluded volume of HA with respect to BSA was estimated from the "osmotic parameters" of HA and BSA by an approach developed in 1976 by Shaw. The resulting excluded volume of HA with respect to BSA was compared with those obtained from a lightly cross-linked HA gel and from solutions of HA (mol wt 1.5 x 10(6)) studied in 1964 by Laurent. The development of this cross-linked HA gel and its subsequent calibration are described.     

MOLECULAR ASSOCIATION OF HEMOCYANIN PRODUCED BY X-RAYS AS OBSERVED IN THE ULTRACENTRIFUGE

1. When normal, monodisperse hemocyanin (60.5S) from Limulus Rolyphemus was irradiated in neutral buffer with x-rays, several new, more rapidly sedimenting ultracentrifugal components (86S, 107S, 122S) were produced, with a corresponding loss in the amount of the unaffected protein. The amount of the effect was roughly proportional to the amount of irradiation. 2. The new resolvable components apparently represented an association of the primary particles into aggregates of 2, 3, and 4 primary particles respectively. 3. The proportional amount of hemocyanin affected decreased almost to the vanishing point as the concentration of the protein was raised to high levels. 4. The absolute effect, i.e. the total number of particles affected in a given volume, increased with the concentration of hemocyanin, at least for concentrations below 15 per cent. 5. The presence of 33 per cent horse serum during irradiation inhibited the effect on the hemocyanin almost completely, with hemocyanin concentrations of both 0.8 and 14 per cent. 6. The presence of 2.8 per cent egg albumin during irradiation lowered the effect by about 70 per cent in the case of dilute preparations (0.8 per cent hemocyanin), but by only about 25 per cent in the case of 14 per cent solutions. 7. A lowering of the solution''s oxygen tension during irradiation enhanced the effect, almost doubling it in some cases. 8. The probable theoretical significance of these and other observations are discussed in the text.     

Protein osmotic pressure and the state of water in frog myoplasm

We measured the osmotic pressure of diffusible myoplasmic proteins in frog (Rana temporaria) skeletal muscle fibers by using single Sephadex beads as osmometers and dialysis membranes as protein filters. The state of the myoplasmic water was probed by determining the osmotic coefficient of parvalbumin, a small, abundant diffusible protein distributed throughout the fluid myoplasm. Tiny sections of membrane (3.5- and 12-14-kDa cutoffs) were juxtaposed between the Sephadex beads and skinned semitendinosus muscle fibers under oil. After equilibration, the beads were removed and calibrated by comparing the diameter of each bead to its diameter measured in solutions containing 3-12% Dextran T500 (a long-chain polymer). The method was validated using 4% agarose cylinders loaded with bovine serum albumin (BSA) or parvalbumin. The measured osmotic pressures for 1.5 and 3.0 mM BSA were similar to those calculated by others. The mean osmotic pressure produced by the myoplasmic proteins was 9.7 mOsm (4 degrees C). The osmotic pressure attributable to parvalbumin was estimated to be 3.4 mOsm. The osmotic coefficient of the parvalbumin in fibers is approximately 3.7 mOsm mM(-1), i.e., roughly the same as obtained from parvalbumin-loaded agarose cylinders under comparable conditions, suggesting that the fluid interior of muscle resembles a simple salt solution as in a 4% agarose gel.     

A PERFUSING SOLUTION FOR THE LOBSTER (HOMARUS) HEART AND THE EFFECTS OF ITS CONSTITUENT IONS ON THE HEART

1. All inorganic perfusing solution for the heart of the lobster Homarus americanus, to allow prolonged normal beating (20 hours or more) must agree closely with the inorganic composition of the serum, which varies differentially with that of the environmental sea water. 2. All of the chief inorganic ions of the serum are necessary—Na, K, Ca, Mg, Cl, and SO₄; the critical numbers of the ions being 100, 3, 5, 2–3, 116, and 1–2 respectively. Absence of Mg and SO₄ will be tolerated for several hours. 3. The pH of the solution must agree with that of the serum within 0.2. 4. The osmotic pressure of the solution must agree with that of the serum within 15 per cent. 5. Beating of the heart will continue for several hours on improperly balanced solutions but changes in frequency, tone, or amplitude will occur. Hearts adapted to such solutions will show different responses to physical and chemical stimuli of the solution than those perfused on properly balanced solutions. 6. Arrest in systole is caused by isotonic NaCl, KCl, LiCl, and urea, and arrest in diastole by isotonic CaCl₂, MgCl₂, NaBr, NaI, MgSO₄, and glucose. 7. Lithium cannot replace sodium; neither can bromide or iodide replace chloride ions.