Hypertonic Salt Solution Research Articles

Introductory class exercises in experimental embryology usingXenopus laevis

Abstract This paper describes classical experiments of experimental embryology which may be repeated at class level using the early embryos of Xenopus laevis. The experiments demonstrate the effects of lithium on the early development of the nervous system, and the use of hypertonic salt solutions to produce exogastrulation. Both experiments involve the removal of the jelly coats around the embryo, and for this purpose a rapid chemical method using a cysteine hydrochloride/papain solution is described. Both experiments are theoretically important: the effects of lithium are used as evidence for the existence of an anterior-posterior gradient in the early embryo, while exogastrulation provides a convenient system for interfering with the primary inductive effect of the chordo-mesoderm, necessary for ectodermal neuralization. The procedure for obtaining fertile eggs from mature Xenopus specimens by injection of mammalian chorionic gonadotrophin is described.

Circus movement in dissociated embryonic cells of a teleost,Oryzias latipes

The dissociated early embryonic cells of the fresh water fish, Oryzias latipes, protrude hyaline lobopodia, which tend to rotate around the cell circumference in a propagating wave. Cells from late blastula or gastrula continuously show this "circus movement", while most cells up to early blastula are rounded. The linear velocity of the lobopodium was estimated by means of time-lapse cinemicrography. The velocity increases slightly as cell diameter increases. The effects of pH, temperature and osmotic pressure of the immersion media on the movement were also quantitatively investigated. Cells become rounded and do not form lobopodial blebs when immersed in media below pH 5. The velocity is reduced by decreasing temperature, but the movement continues even at 5 degrees C. Cells placed in hypertonic salt solutions become crenated and do not continuously demonstrate the circus movement.

Eutectics: a neglected area in clinical chemistry

When serum is cooled in the deep freeze, ice crystals of pure water form progressively. The progressive removal of water leads to increasing concentrations of salts and proteins in the residual unfrozen solution. This continues until the eutectic point for serum is reached (−23° C) at which point the remaining water, salts and proteins solidify together as an eutectic mixture. Thus at −20° C small lakes of a liquid concentrate of proteins in hypertonic salt solution exist inside a solid matrix of water ice. Under such circumstances denaturation of proteins may occur especially as the pH may fall to as low as 3. Accordingly −20° C may be about the worst temperature at which to store serum, at least for some substances. It may therefore be advisable to use −70° C refrigerators for storage of sera for susceptible substances such as clotting factor standards and controls.

Characterization of cell-associated dextransucrase activity from glucose-grown cells of Streptococcus mutans.

Two distinct forms of cell-associated dextransucrase activity have been extracted from glucose-grown cells of Streptococcus mutans GS-5. One form can be washed from cells by treatment with hypertonic salt solutions. This activity is located on the exterior of the cell since it is susceptible to inactivation by proteolytic enzymes. Gel filtration of the extracted enzyme indicates that the enzyme exists as a highly aggregated complex which dissociates into a smaller soluble species in the presence of hypertonic salt solutions. This form of the enzyme also plays a role in sucrose-dependent adherence of whole cells to smooth surfaces. Hypertonic salt solutions also elute most of the cell-associated dextransucrase activity from strain FA-1 but not from strain HS-6. The second cell-associated activity of strain GS-5 is released after disruption of washed cells, is not susceptible to proteolytic enzyme treatment of whole cells, and represents the intracellular form of the enzyme. A comparison of the gel filtration properties. pH and temperature optima, kinetic constants, and insoluble dextran-synthesizing activities of the two cell-associated forms of the enzyme with the extracellular enzyme did not reveal any major differences.

Experimental modification of the “tertiary structure” of chromosomes in human lymphocytes: The effects of polyanionic substances and hypertonic salt solutions

The chromosomes in G 0 lymphocytes in freshly drawn human blood react to radiation as though they were single-stranded: chromosome, rather than chromatid, aberrations are induced. Treatment of the cells with glycerol or urea has previously been found to change this response. The present experiments show that treatment with the polyanionic substances spermine or spermidine can cause loosening of chromosomal subunits in G 1, but not G 0, lymphocytes. Cleland's reagent, which breaks disulfide bonds, and EDTA, which chelates divalent metal ions, do not affect the structure. The response of chromosomes in very late G 1 cells can also be modified. At this point in the cell cycle the subunits separate spontaneously to react to radiation by forming chromatid aberrations. Treatment with hypertonic salt solutions to induce plasmolysis tightens the tertiary structure so that now fewer chromatid aberrations can be induced.

Salt extraction of soluble HL-A antigens.

Extraction of cultured human lymphoid cells with hypertonic salt solutions (3 molar potassium chloride) resulted in high recoveries of membrane-associated histocompatibility (HL-A) antigens in soluble form with potent activity and marked immunologic specificity. The active principle was purified by preparative acrylamide-gel electrophoresis. Application of the hypertonic salt extraction method is now yielding sufficient HL-A antigen to begin the elucidation of the molecular basis of transplantation individuality.

Infection of mouse fibroblasts by cardioviruses: Premature uncoating and its prevention by elevated pH and magnesium chloride

The plating efficiencies of ME-, EMC-, MM-, Columbia SK-, and mengoviruses, collectively called “cardioviruses,” were enhanced 2- to 5-fold by conducting the virus attachment step at 22° rather than at 37°. This behavior was traced to a temperature-dependent loss of infectivity (abortive infection) following attachment of the virion to the cell. Abortive infection was virus specific; it was detectable with cardioviruses but not with poliovirus type 1 or coxsackievirus B1 and took place on both HeLa and L cells. Studies with purified, isotopically labeled ME-virus showed that loss of infectivity was accompanied by release of the RNA genome from cell-attached virions into the extracellular fluid. The coat protein initially remained attached to the cell after RNA release but subsequently dissociated from the cell in the form of 14 S subunits. Premature uncoating was inhibited by alkaline pH and by hypertonic salt solutions.

Vasopressin release from the rat supraoptico-neurohypophysial system in vitro in response to hypertonicity and acetylcholine.

ABSTRACT Vasopressin release from isolated supraoptico-neurohypophysial systems of rats was studied by means of an antidiuretic bioassay procedure using iv hydration. Preparations which included adjacent areas released less hormone during the post-isolation recovery period. Following the recovery period less than 20 pg/min of arginine-vasopressin was released from a single preparation; but on challenge with acetylcholine (1 mg/l) or with strongly hypertonic salt solutions (950 mosm/kg) the hormone release was increased to over 100 pg/min/preparation. Cross-tachyphylaxis between these two forms of stimulation occurred after less than 1% of the total vasopressin content of the supraoptico-neurohypophysial system had been released. A concentration of potassium of 56 mmol/l caused release of much larger amounts of vasopressin than stimulation with acetylcholine or hypertonicity.

Daily patterns of feeding and excretion in rats confined to metabolism cages: strain and age differences.

Data are presented on the daily feeding and excretion patterns of Wistar, Sprague-Dawley and Wistar/Sprague-Dawley hybrid rats confined to metabolism cages. All strains were characterized by the formation of a grossly hypertonic urine which contained high concentrations of both sodium and potassium. Daily concentration rhythms were found in urinary sodium, potassium, calcium and magnesium, but the magnitude of these rhythms depended on strain and age. Rats of all ages were characterized by a high turnover of water and salts. A large proportion of the water was lost by routes other than urine and faeces, and the animals appeared to absorb a slightly hypertonic salt solution from the intestine (the result of eating and drinking) in order to counteract this water loss.

Water, sodium chloride, and food intake induced by injections of cholinergic and adrenergic drugs into the third ventricle of the rat brain.

SummaryThe effects of microinjection of adrenergic and cholinergic drugs and hypertonic saline into the third ventricle on the intake of water, hypertonic salt (NaCl) solution, and food were studied. Carbachol induced a dramatic, rapid, 15-fold increase in water intake, whereas none of the other drugs were active. Both carbachol and isoproterenol evoked large increases in salt intake. Again, all other drugs failed to produce significant effects. Food intake was increased by the following adrenergic compounds: epinephrine, norepinephrine, metaraminol, isoproterenol, and dopamine. Carbachol was also effective in augmenting food intake but the effect was delayed. Hypertonic saline produced a delayed increase in both water and food intake but did not alter salt intake. The results are interpreted to mean that a cholinergic synapse lies in the pathways which mediate water intake, whereas both cholinergic and adrenergic synapses may be involved in the mediation of salt and food intake.

Interactions of red cells with phagocytes of the wax-moth ( Galleria mellonella, L.) and mouse

Phagocytic recognition was examined in the wax-moth Galleria and in the mouse, by means of a range of modified erythrocytes. Particles were injected into the body cavity of the larvae or incubated in vitro with the insect phagocytes or with mouse macrophages. Whereas untreated Alsever's stored sheep erythrocytes (SE) were taken up at a low rate, SE treated with aldehyde, Fe 2+, tannic acid, colloidal silica, saponin, carbodiimide, or acid sucrose solutions, were taken up by or attached themselves to moth and mouse phagocytes. No significant uptake above that of control SE was found of SE exposed to hypertonic or hypotonic salt solutions, or of SE treated with Na dodecylsulfate, trypsin or chymotrypsin. SE made effete by incubation in saline at 37°C, or SE treated with salt-free sugar solutions or with homologous antiserum, were taken up in vivo but not in vitro by the insect phagocytes. Fetal bovine serum or Ca 2+ added to the saline lowered the subsequent uptake of the SE. Salt added to the sugar solutions likewise reduced the in vivo uptake of SE by the hemocytes. The uptake of glutaraldehyde-horse red cells by insect phagocytes in vitro was increased when the particles were treated with chymotrypsin and decreased when particles were treated with ovalbumin, in fashion similar to that previously reported for mouse macrophages and L2 cells. Thus, (1) hemocytes ingested in vivo a broader range of particles than in vitro; it is not known whether hemolymph recognition factors can explain the discrepancy; (2) rather extensive modification or damage of the SE was required for their interaction in vitro with insect or with mouse phagocytes; (3) despite some irregularities, similar patterns emerged of particle recognition by insect phagocytes and by mouse macrophages when these cells were incubated with particles in the absence of added hemolymph or serum. It is suggested that common features of the cell membranes of the two phylogenetically distant phagocytes may underlie this form of recognition.

Apparent nonsolvent water and osmotic behavior of yeast cells

1. 1. l-Xylose and dimethylsulfoxide permeate the yeast cell, reaching an intracellular eqiulibrium concentration significantly lower than the medium concentration. At equilibrium the intracellular dimethylsulfoxide concentration is 70–85%, the l-xylose concentration about 40% of that in the medium. In anaerobically grown yeast, lacking intracellular membranous structures, the same equilibrium concentrations are reached as in normal yeast. 2. 2. Both l-xylose and dimethylsulfoxide are present inside the cells in an osmotically active form. 3. 3. The osmotic response of yeast cells in hypertonic NaCl solutions shows a deviation from ideal osmotic behavior. The deviation could be explained by increase of the number of intracellular particles in hypertonic salt solutions and by elasticity forces in the cell wall, causing some reduction of the cellular volume with a connected increase in the intracellular tonicity. 4. 4. With equilibrium dialysis on disintegrated cells, it could be shown that there exists no bound water or exclusion water with respect to l-xylose and dimethylsulfoxide in the yeast cell. 5. 5. The experimental results make the existence of osmotically inactive compartments in the yeast cell, as suggested in recent literature, highly improbable.

Pathogenesis of hemolytic anemia in homozygous hemoglobin C disease.

Hemoglobin C is less soluble than hemoglobin A in red cells, in hemolysates, and in dilute phosphate buffer. Its relative insolubility may be explained by electrostatic interactions between positively charged beta6-lysyl groups and negatively charged groups on adjacent molecules. Red cells from patients with homozygous hemoglobin C (CC) disease exhibit aberrant physical properties which suggest that the cells are more rigid than normal erythrocytes. They pass through membrane filters less readily than normal red cells do, and their viscosity is higher than that of normal cells. Differences from normal cells are exaggerated if mean corpuscular hemoglobin concentration (MCHC) is increased, by suspension in hypertonic salt solution. Increased rigidity of CC cells, by accelerating their fragmentation, may be responsible for formation of microspherocytes. These small dense cells are exceptionally rigid, and probably are even more susceptible to fragmentation and sequestration. Rigidity of CC cells can be attributed to a "precrystalline" state of intracellular hemoglobin, in which crystallization does not occur, although the MCHC exceeds the solubility of hemoglobin in hemolysates.

Increased Tolerance to Intravenous Hypertonic Saline in Patients with Essential Hypertension

THE application of hypertonic solutions speeds the appearance of clinical signs of cellular dehydration in proportion to the impermeability of the cellular membrane. Intravenous infusion of hypertonic saline, which remains effectively in the extracellular fluid space, stimulates cellular dehydration more intensely than an equivalent amount of urea, which readily penetrates cell membranes1,2. Rapid intravenous application of a hypertonic solution of sodium chloride brings about a hypertonic expansion of the physiologically active part of the extracellular fluid space3,4. There is evidence that intravenous loading with strongly hypertonic salt solutions is partially dependent on the mean arterial blood pressure if the renal function is not or is only slightly affected.

Fresh-water fish chemoreceptors responsive to dilute solutions of electrolytes.

The existence of the palatal chemoreceptors responding specifically to dilute solutions of salts with monovalent cations was demonstrated in carp. The distilled water effect (a response produced by the application of distilled water after chemoreceptors had been rinsed out with hypertonic salt solutions) was assigned to the activity of the same receptor. Intensity of the response to dilute solutions of salts depended on the valency of the anion: the larger the valency, the greater the response. Positively charged sites of the receptor responsive to dilute salt solutions were suggested by previous treatments with acid and alkali, and dye salts. Increase in the ionic strength of the stimulating solution by the addition of supporting electrolytes caused a depression of response. In particular, strong depression of response was caused by the addition of a supporting electrolyte with a divalent cation. Effects of polarizing current on the chemoreceptor activity were investigated. Based upon the findings in this paper, a hypothesis is presented, which explains mechanisms underlying chemoreceptor responses to dilute solutions of electrolytes in terms of an interfacial electro-kinetic process.

The role of cytoplasmic membranes in the regulation of water concentration within frog oocytes

Large ovarian oocytes from frog ovaries were treated for 1 sec in water of 80°C. Such “nudoplasts” were shown to lose ultraviolet-absorbing and trichloroacetic acid-precipitable substances into a physiological saline medium. They were also shown to be permeable to labeled gamma globulin protein and to be unable to retain their normal appearance. Despite the fact that they had become highly permeable, nudoplasts could still change volume in hyper- and hypotonic salt solutions for a period of one to several hours. They could swell markedly in hypotonic media but sometimes could not shrink in hypertonic salt solutions. These data were interpreted as evidence that the oocytes consist of two osmotically active “compartments”. One, the protoplasmic mass, owing its osmotic properties to the plasma membrane and the other consisting of interior vesicles owing their osmotic properties to their investing membranes. In nudoplasts the surface damage renders the protoplasmic compartment osmotically non-functional but the vesicular compartment is though to retain its osmotic properties. Cytological evidence of swollen cortical vesicles in normal cells was presented to support the argument. Examination of the magnitude of swelling led to the conclusion that the interior vesicles could not increase in volume to the observed extent simply with semi-permeable membranes and a fixed solute content. It was suggested that ion-pumping takes place into the vesicles of the cytoplasm, especially those of the cell cortex.

Pharmacologically active constituents of the cockroach corpus cardiacum: Resolution and some characteristics

By means of paper chromatography, chymotrypsin digestion, and bioassay, six to nine active substances were detected in extracts of the corpus cardiacum of Periplaneta americana (Linnaeus). These substances affect the cockroach foregut, hindgut, heart, nerve cord, and hemolymph trehalose levels with some degree of specificity. Three components, designated as Factors P 1, P 2 and P 3, are responsible for most, if not all, of the heart and hindgut activity of crude extracts of the corpus cardiacum. These three substances appear to be peptides. They are heat-stable, chymotrypsin-sensitive and dialysable; their rates of dialysis differ. Factor P 1, according to its rate of dialysis, is the largest of the three peptides and is equally active on the hindgut and heart. Two smaller peptides, Factors P 2 and P 3, are specific activators of the hindgut and heart, respectively. Isolated “intact” corpora cardiaca retained Factors P 1 and P 2, quantitatively, during a 60-minute incubation in isotonic and hypertonic salt solutions, but not in hypotonic solutions. Considerably less of these peptides were retained within the “intact” gland during incubation in glucose or sucrose solutions. Thus, retention of the peptides under these conditions was salt-dependent; this salt requirement appeared to be non-specific. Factors P 1, P 2 and P 3 were associated with a particulate fraction in homogenates of corpora cardiaca. Hypotonic solutions disrupted this association. Unlike the retention of these factors by the “intact” gland, this association does not appear to be salt-dependent. Finally, transplanted corpora cardiaca were gradually depleted of their hindgut-active peptides.

CORTICOSTEROIDOGENESIS IN THE FRESH WATER- AND SALINE-MAINTAINED DUCK (ANAS PLATYRHYNCHOS).

SUMMARY No differences were observed in the patterns of adrenal steroidogenesis in vitro by ducks maintained on fresh water or on a hypertonic salt solution. Determinations in vivo of the biological half-life of corticosterone showed no significant difference between ducks maintained on or loaded with saline and fresh water controls. An increase in the apparent volume of distribution of corticosterone was, however, observed in birds loaded with the salt solution after maintenance on fresh water or salt solution. Estimations were made of the secretory rate of corticosterone in vivo. In the ducks maintained on the salt solution an increased adrenal weight was responsible for the enhanced secretory rate but ducks maintained on fresh water and loaded with saline secreted corticosterone at a higher rate per unit weight of adrenal. The implications of the increased rate of corticosterone secretion are discussed in relation to the known dependence of nasal gland function in marine birds on a functional adrenal gland.

The dissociability of different poliovirus-antibody complexes as tested by hypertonic salt solutions

The dissociability of complexes formed by poliovirus and early 19Sγ1and late 7Sγ2-globulin antibody respectively was studied by the use of hypertonic sodium chloride solutions (0.6–5.0 M). The effect of high ionic strength on the neutralization kinetics was also investigated. Complexes of poliovirus and early rabbit 19Sγ1-globulin antibody were dissociated by hypertonic salt solutions; the dissociation being proportional to the hypertonicity. Complexes of virus and late 7Sγ2-globulin antibody did not dissociate in the same hypertonic milieu. In a hypertonic environment the neutralization reaction rate was reduced and equilibrium was reached at high levels of surviving virus. Following a change from iso- to hypertonic milieu the reaction between virus and 19Sγ1-globulin antibody rapidly reached a new level of equilibrium equal to that established when the reaction proceeded in this hypertonicity from its start. The same change in tonicity evoked no shift in the state of equilibrium established by virus and late 7Sγ2-globulin antibody.

Hyponatremia and its treatment in the hospitalized patient

Analysis of 136 cases of hyponatremia observed in this hospital shows that 50 per cent occurred in patients over 60 years old. Fluid evaluation showed that 40 per cent had excess fluids, 48 per cent normal hydration, and 12 per cent were dehydrated. Approximately 58 per cent were asymptomatic. In about half of the symptomatic group, a loss of salt had occurred and in half an excess of water. In approximately 10 per cent of the cases, the low serum sodium value proved to be a laboratory error. Effective therapy included hypertonic salt solution, 0.9% salt solution, oral sodium chloride and dialysis. Emphasis was placed on the need for complete evaluation of the patient with suspected hyponatremia and restriction of therapy to those patients with definite symptoms and with demonstrated loss of salt or water excess.