Urea tolerance and osmoregulation in Bufo viridis and Rana ridibunda

1. The tolerance and adaptation to urea solutions by terrestrial green toads (Bufo viridis) and semi-aquatic frogs (Rana Ridibunda) were studied. 2. the green toad showed tolerance to urea solution of 800 mM and the frogs showed tolerance only to about 400 mM urea solution. 3. The plasma concentrations of both species was hyperosmotic to the external medium in all the different urea solutions. 4. Blood osmolality, urea, Na+ and Cl- concentrations of B. viridis were always higher than in R. ridibunda. 5. The urea concentration in muscle of R. ridibunda was higher than the urea concentration in muscle of B. viridis. 6. The muscle tissue weight loss of B. viridis was significantly lower than R. ridibunda.     

Coupled Solute Fluxes in Toad Skin

Net inward flux of mannitol across toad skin induced by making the outside solution hypertonic with urea has been investigated. No significant relation between net mannitol flux and net Na flux could be detected when both fluxes were measured simultaneously. In addition, the net mannitol flux caused by hypertonic solution was not altered by inhibition of Na transport with ouabain or by replacement of all Na in the bathing solutions by choline. The rate of net mannitol flux was dependent on the magnitude of the urea concentration difference across the skin and the direction of net flux could be reversed by reversing the direction of the urea concentration difference. These observations suggest that the mannitol transfer is the result of a coupling between the flows of urea and mannitol.     

Effects of osmolarity on taste receptor cell size and function

Osmotic effects onsalt taste were studied by recording from the rat chorda tympani (CT)nerve and by measuring changes in cell volume of isolated rat fungiformtaste receptor cells (TRCs). Mannitol, cellobiose, urea, or DMSO didnot induce CT responses. However, the steady-state CT responses to 150 mM NaCl were significantly increased when the stimulus solutions alsocontained 300 mM mannitol or cellobiose, but not 600 mM urea or DMSO.The enhanced CT responses to NaCl were reversed when the saccharideswere removed and were completely blocked by addition of 100 µMamiloride to the stimulus solution. Exposure of TRCs to hyperosmoticsolutions of mannitol or cellobiose induced a rapid and sustaineddecrease in cell volume that was completely reversible, whereasexposure to hypertonic urea or DMSO did not induce sustained reductionsin cell volume. These data suggest that the osmolyte-induced increasein the CT response to NaCl involves a sustained decrease in TRC volumeand the activation of amiloride-sensitive apicalNa⁺ channels.

     

Effect of hyperosmotic solutions on salt excretion and thirst in rats

We investigated urinary changes and thirst induced by infusion of hyperosmotic solutions in freely moving rats. Intracarotid infusions of 0.3 M NaCl (4 ml/20 min, split between both internal carotid arteries) caused a larger increase in excretion of Na(+) and K(+) than intravenous infusions, indicating that cephalic sensors were involved in the response to intracarotid infusions. Intravenous and intracarotid infusions of hyperosmotic glycerol or urea (300 mM in 150 mM NaCl) had little or no effect, suggesting the sensors were outside the blood-brain barrier (BBB). Intracarotid infusion of hypertonic mannitol (300 mM in 150 mM NaCl) was more effective than intravenous infusion, suggesting that cell volume rather than Na(+) concentration of the blood was critical. Similarly, intracarotid infusion (2 ml/20 min, split between both sides), but not intravenous infusion of hypertonic NaCl or mannitol caused thirst. Hyperosmotic glycerol, infused intravenously or into the carotid arteries, did not cause thirst. We conclude that both thirst and electrolyte excretion depend on a cell volume sensor that is located in the head, but outside the BBB.     

Changes in red blood cell volume on fixation in glutaraldehyde solutions

Summary Light scattering (nephelometry) was used to determine directly the change in volume of red blood cells immersed in a variety of buffer and fixative solutions. Cells immersed in saline or phosphate buffer solutions showed a change in volume that reflected the osmolarity of the solution, shrinkage taking place in hypertonic solutions and swelling and haemolysis occurring in strongly hypotonic solutions. On the other hand, while there was considerable shrinkage in hypertonic glutaraldehyde solutions, swelling was more restricted and haemolysis was prevented in the weaker glutaraldehyde solutions. Thus, while glutaraldehyde exerts a definite osmotic effect on cells in fixative solutions, the magnitude of this effect seems to be limited by its direct action as a fixative.     

Hypertonic preconditioning inhibits macrophage responsiveness to endotoxin.

Hypertonic saline has been shown to modulate cell shape and the response of components of the innate immune response. However, the effect of hypertonic saline on the macrophage remains unknown. We hypothesized that hypertonic preconditioning would impair subsequent inflammatory mediator signaling through a reduction in stress fiber polymerization and mitogen-activated protein kinase activity after LPS stimulation. Rabbit alveolar macrophages were stimulated with 100 ng/ml of LPS. Selected cells were preconditioned with 40-100 mM of NaCl, mannitol, or urea for 4 h and returned to isotonic medium before LPS stimulation. Cellular protein was harvested and subjected to Western blot analysis for the dually phosphorylated active forms of p38 and extracellular signal-related kinase (ERK) 1/2. TNF production was determined by an L929 bioassay, and stress fiber polymerization was evaluated by confocal microscopy. Preconditioning of macrophages with NaCl or mannitol resulted in dose-dependent reduction in ERK 1/2 phosphorylation with no effect on p38 phosphorylation. Urea preconditioning had no effect on either mitogen-activated protein kinase. A dose-dependent attenuation of TNF production was seen with NaCl and mannitol preconditioning (p < 0.05), but not with urea. NaCl and mannitol preconditioning resulted in failure of LPS-induced stress fiber polymerization, whereas urea did not. Extracellular hypertonic conditions (i.e., NaCl and mannitol) have an immunomodulatory effect on macrophages, demonstrated through failure of optimal stress fiber polymerization, ERK 1/2 activity, and TNF production. Intracellular hypertonic conditions (i.e., urea) had no significant effect. Hypertonic saline or mannitol resuscitation, therefore, may help protect against multiple-organ dysfunction syndrome as a result of this reduced proinflammatory responsiveness.     

Drinking induced by cellular dehydration in the quail, Coturnix coturnix japonica

1. Drinking was induced in water-replete quail 5-10 min after intravenous injection of hypertonic NaCl (0.69 osmol/l) or sucrose (1.06 osmol/l), but hypertonic urea (2.78 osmol/l) failed to induce drinking. 2. The birds drank approximately the amount required to dilute the injected solutes to isotonicity for each given dose of NaCl or sucrose. 3. The plasma angiotensin II level decreased after injection of 7% NaCl (2.5 osmol/l), but it increased after injection of an equi-osmolar solution of sucrose (65%). 4. Plasma osmolality and Na+ concentration returned quickly to control levels, and then decreased further, after injection of 7% NaCl or 65% sucrose. 5. Blood volume and blood pressure increased immediately after injection of 7% NaCl or 65% sucrose. 6. These results show that drinking is induced after injection of hypertonic solutions exclusively by cellular dehydration, and other regulatory mechanisms for thirst, such as extracellular dehydration and the renin-angiotensin system, are rather inhibitory after injection of hypertonic NaCl.     

Isoosmotic transport of fluid across the hamster small intestine in the presence of phlorizin-induced inhibition of sugar transport.

Experiments were performed to investigate whether the fluid transported across the small intestine is isoosmotic with the mucosal solution when the active transport of glucose is partially inhibited. Everted hamster mid small intestine was incubated in one of the following four mucosal solutions: (1) Isotonic control, Krebs-Ringer bicarbonate solution containing 10 mM glucose (KRBSG), (2) Isotonic with phlorizin, KRBSG + 5X10-5 M phlorizin, (3) Hypertonic control, KRBSG + 50 mM mannitol, (4) Hypertonic with phlorizin, KRBSG + 50 MM mannitol + 5x10-5 M phlorizin. The serosal surface of the intestine was not bathed. Results indicate that the transported fluid was always isoosmotic with any of the mucosal solutions used. When the mucosal solution was made hypertonic with mannitol, the concentration of glucose and electrolytes in the absorbate increased, and as a result, the absorbate became hypertonic and isoosmotic with the mucosal solution. The presence of phlorizin either in the isotonic or in the hypertonic mucosal solution decreased the glucose concentration of the absorbate, but the transported fluid became isoosmotic with the mucosal solution due to a higher concentration of Na, K, and their associated anions. Phlorizin caused a decrease in the transmural potential difference. In spite of this, the presence of this glucoside in the mucosal solution increased the transport of sodium in relation to glucose transport. It is suggested that, at the concentrations used, phlorizin inhibits sodium movement through the electrogenic pathway, but increases the transport of this ion through the non-electrogenic route. This increase in neutral sodium transport seems to compensate for the low concentration of glucose in the absorbate, so that the absorbate becomes isoosmotic with the mucosal solution whether the latter is isotonic or hypertonic. It is suggested further that isoosmotic transport of fluid is an inherent property of the small intestine and that there may be an osmoregulatory mechanism in the gut which controls this process.     

Efflux of Red Cell Water into Buffered Hypertonic Solutions

Buffered NaCl solutions hypertonic to rabbit serum were prepared and freezing point depressions of each determined after dilution with measured amounts of water. Freezing point depression of these dilutions was a linear function of the amount of water added. One ml. of rabbit red cells was added to each 4 ml. of the hypertonic solutions and after incubation at 38°C. for 30 minutes the mixture was centrifuged and a freezing point depression determined on the supernatant fluid. The amount of water added to the hypertonic solutions by the red cells was calcuated from this freezing point depression. For each decrease in the freezing point of -0.093°C. of the surrounding solution red cells gave up approximately 5 ml. of water per 100 ml. of red cells in the range of -0.560 to -0.930°C. Beyond -0.930°C. the amount of water given up by 100 ml. of red cells fits best a parabolic equation. The maximum of this equation occurred at a freezing point of the hypertonic solution of -2.001°C. at which time the maximum amount of water leaving the red cells would be 39.9 ml. per 100 ml. of red cells. The data suggest that only about 43 per cent of the red cell water is available for exchange into solutions of increasing tonicity.     

The effect of hypertonic urea solution on Na+ and K+ transport through the smooth muscle membrane.

The aim of this work was to examine the effect of a hypertonic solution (Krebs solution + 290 mM urea) on K+ and Na+ transport. The experiments were carried out on the guinea-pig taenia coli preparations using the method of Na-24 and K-24 loading and washout. The efflux curves were analysed by means of the digital computer technique. The following parameters were determined: efflux rate constant k2, influx rate constant k1, intracellular ion concentration C1 ion flux M and permeability P. Any significant difference between PNa/PK ratio in hypertonic urea and isotonic Krebs solutions was found.     

Quantitative examination of a perfusion microscope for the study of osmotic response of cells

The perfusion microscope was developed for the study of the osmotic response of cells. In this microscope, the cells are immobilized in a transparent chamber mounted on the stage and exposed to a variety of milieus by perfusing the chamber with solutions of different concentrations. The concentration of the supplied solution is controlled using two variable-speed syringe pumps, which supply an isotonic solution and a hypertonic solution. Before using this system to characterize the osmotic response of cells, the change in the concentration of NaCl solution flowing through the chamber is examined quantitatively using a laser interferometer and an image processing technique. The NaCl concentration is increased from an isotonic condition to a hypertonic condition abruptly or gradually at a given constant rate, and decreased from a hypertonic condition to an isotonic condition. It is confirmed that the concentration is nearly uniform in the cross direction at the middle of the chamber, and the change in the NaCl concentration is reproducible. The average rate of increase or decrease in the measured concentration agrees fairly well with the given rate when the concentration is changed gradually at a constant rate. The rate of the abrupt change is also determined to be the highest limit achieved by the present method. As the first application of using the perfusion microscope for biological studies, the volume change of cells after exposure to a hypertonic solution is measured. Then, the hydraulic conductivity of the cell membrane is determinedfrom the comparison of the volume change between the experiment and the theoretical estimation for the measured change in the NaCl concentration of the perfused solution.     

Study in the anuran amphibian Xenopus laevis, some blood characteristics and nitrogen excretion based on changes in the water osmolarity]

The pH, the osmolality and the urea and ammonia concentrations in blood, as well as the net urea and ammonia excretions, were studied in the amphibian Xenopus laevis exposed for several weeks to increased osmotic pressure (OP) of the ambient water, as a result of the addition of either NaCl or mannitol to the water. The pH and the ammonia concentration of the blood were independent of the variations of the ambient osmolarity. On the contrary, the blood osmolality and its urea concentration increased markedly when the ambient OP was augmented. The increase of ambient OP by NaCl addition to the medium augmented the urea net excretion and slightly decreased the ammonia excretion. When the increase of ambient OP resulted from the addition of mannitol in the water, excretions of urea and ammonia became negligible.     

Mechanisms of hyperosmotic acclimation in Xenopus laevis (salt,urea or mannitol)

The acclimation of the clawed toad Xenopus laevis to hyperosmotic solutions of NaCl (balanced solution of sea salt), urea or mannitol was studied. The animals could not be acclimated to salt solutions more concentrated centrated than 400 mosm·l^-1. Urea was tolerated till 500 mmol·l^-1. Plasma osmolality was always hyperosmotic to the environmental solution, but with diminished osmotic gradient at the highest tolerated solutions. Plasma urea concentration approached 90 mmol·l^-1, similar in the three solutions of acclimation. Urine volume was very small under all conditions. Serum aldosterone and corticosterone did not differ significantly, although there was a slight tendency towards lower aldosterone in the NaCl solution. In vivo water uptake in tap water acclimated animals was very small, and was higher in the other groups. Only the salt- and urea-acclimated, but not the tap water and mannitol-acclimated groups responded with a clear increase following injection of oxytocin or theophylline. In vitro urea fluxes were similar and invariable in both directions under all conditions. No significant effect of theophylline was observed. Sodium transport measured by the short-circuit technique in vitro was lower in salt- and mannitol-acclimation conditions, and was stimulated significantly under all conditions in response to serosal oxytocin or theopylline. It is concluded that Xenopus laevis can osmoregulate at a limited range of external solutions. It is limited in the increase of its plasma urea concentration; the transport properties of the skin do not change very much upon acclimation, except for the hydroosmotic response to oxytocin.Abbreviations I _sc short circuit current - PD potential difference - SW balanced sea water - TW tap water     

Cerebral Na concentration, Na appetite and thirst of sheep: influence of somatostatin and losartan

Na and water intakes of Na-depleted sheep are influenced by changes in cerebral Na concentration. The effect of intracerebroventricular infusion of somatostatin or losartan, the ANG II type 1 receptor antagonist, on the Na appetite and thirst of Na-depleted sheep during infusions that decrease (intracerebroventricular hypertonic mannitol) or increase (intracerebroventricular or systemic hypertonic NaCl) cerebral Na concentration was investigated. Na intake was increased but water intake was unchanged during intracerebroventricular infusion of hypertonic mannitol. The increased Na appetite caused by intracerebroventricular infusion of hypertonic mannitol was decreased by concurrent intracerebroventricular infusion of either somatostatin or losartan, with somatostatin being most effective. Water intake was increased during intracerebroventricular infusion of hypertonic mannitol and somatostatin. Na intake was decreased and water intake was increased during systemic or intracerebroventricular infusion of hypertonic NaCl. Intracerebroventricular infusion of losartan blocked both (Na and water intake), whereas somatostatin did not influence either of these changes in intake. The results further consolidate a role for somatostatin and ANG II in the central mechanisms controlling Na appetite and thirst of sheep.     

Gallbladder epithelial cell hydraulic water permeability and volume regulation

The hydraulic water permeability (Lp) of the cell membranes of Necturus gallbladder epithelial cells was estimated from the rate of change of cell volume after a change in the osmolality of the bathing solution. Cell volume was calculated from computer reconstruction of light microscopic images of epithelial cells obtained by the "optical slice" technique. The tissue was mounted in a miniature Ussing chamber designed to achieve optimal optical properties, rapid bath exchange, and negligible unstirred layer thickness. The control solution contained only 80% of the normal NaCl concentration, the remainder of the osmolality was made up by mannitol, a condition that did not significantly decrease the fluid absorption rate in gallbladder sac preparations. The osmotic gradient ranged from 11.5 to 41 mosmol and was achieved by the addition or removal of mannitol from the perfusion solutions. The Lp of the apical membrane of the cell was 1.0 X 10(-3) cm/s . osmol (Posm = 0.055 cm/s) and that of the basolateral membrane was 2.2 X 10(-3) cm/s . osmol (Posm = 0.12 cm/s). These values were sufficiently high so that normal fluid absorption by Necturus gallbladder could be accomplished by a 2.4-mosmol solute gradient across the apical membrane and a 1.1-mosmol gradient across the basolateral membrane. After the initial cell shrinkage or swelling resulting from the anisotonic mucosal or serosal medium, cell volume returned rapidly toward the control value despite the fact that one bathing solution remained anisotonic. This volume regulatory response was not influenced by serosal ouabain or reduction of bath NaCl concentration to 10 mM. Complete removal of mucosal perfusate NaCl abolished volume regulation after cell shrinkage. Estimates were also made of the reflection coefficient for NaCl and urea at the apical cell membrane and of the velocity of water flow across the cytoplasm.     

高张NaCl溶液对失血大鼠血压代偿的作用

使麻醉大鼠在5min 内失血,致使动脉血压下降至25mmHg,然后静脉注射1/10失血量的高张 NaCl 溶液(Hypertonic solution,HS,7.5%NaCl)或生理盐水(NS)。静脉注射少量 HS 能明显促进失血后血压回升,这个作用能为6-羟多巴胺或巯甲丙脯酸显著减弱,若将这两个药物同时应用,则 HS 的升压作用完全被解除。HS 使血浆 Na~+浓度明显升高,而 NS使之下降。侧脑室微量注射 HS 后也明显促进动脉血压的恢复。这些实验结果表明失血后静脉注射少量 HS 可升高血浆 Na~+浓度,高 Na~+作用于中枢神经系统,激活交感神经系统和肾素-血管紧张素系统,从而促使血压迅速恢复。     

Hemolysis of human red blood cells by freezing and thawing in solutions containing sucrose: relationship with posthypertonic hemolysis and solute movements

Human red blood cells were frozen at temperatures down to ?9 °C in solutions containing sucrose, and the hemolysis on thawing was measured. This was compared with the hemolysis caused by exposing the cells to high concentrations of sucrose and then resuspending them in more dilute solutions at 4 °C. The effects of the hypertonic solutions of sucrose on potassium, sodium, and sucrose movements were also investigated. It was found that sucrose does not prevent damage to the cells by very hypertonic solutions (whether during freezing and thawing or at 4 °C) but it does reduce hemolysis of cells previously exposed to these solutions if present in the resuspension (or thawing) solution. Evidence is presented that the damaging effects of the hypertonic solutions of sucrose occurring during freezing are associated with changes in cell membrane permeability but that posthypertonic hemolysis is not primarily associated with a “loading” of the cells with extracellular solutes in the hypertonic phase. It is concluded that sucrose may reduce hemolysis of red blood cells by slow freezing and thawing by reducing colloid osmotic swelling of cells with abnormally permeable membranes.     

Deglycerolization of red blood cells: A new dilution-filtration system

In this work, we present a new version of the dilution-filtration system for rapidly deglycerolizing a large volume of cryopreserved blood. In our earlier system, one of the major problems was the damage induced to the red blood cells (RBCs) due to high osmolality change at the dilution point. Therefore, we devised a new system to solve this problem. First, we theoretically simulated the osmolality variation in the new system and the variation of the maximum and minimum volumes of the RBCs at the dilution point to examine the effects of operating parameters/conditions. Next, we experimentally validated the effects of these operating parameters by deglycerolizing porcine blood. The results show that when the initial NaCl concentration in the hypertonic solution is 18%, the volume of the hypertonic solution is 200 mL, and the flow rate of the filtrate is 50 mL/min, the system can effectively remove glycerin from 200 mL of porcine blood in 30 min, with ∼87% RBC survival rate and ∼73% RBC recovery rate. Our results indicated that in the new system the concentration and the volume of the hypertonic solution used to dilute the blood are the important parameters that need to be adjusted to reduce osmotic damage to the RBCs. In addition, a fast filtrate flow rate is highly recommended. This work can significantly contribute to the development of a more efficient and effective system for deglycerolizing large volumes of cryopreserved blood in clinic.     

Some combined effects of hypertonic solutions and changes in temperature on posthypertonic hemolysis of human red blood cells

The combined effects of hypertonic solutions and temperature changes on the posthypertonic hemolysis of human red blood cells have been investigated. Cells were exposed to hypertonic solutions of sodium chloride and also to hypertonic solutions of the extracellular cryoprotective additive sucrose, such as would occur during the freezing of cells in an isotonic salt solution to which 15% $\text{w}\text{v}$ sucrose had been added. In both cases the extent of posthypertonic hemolysis was increased by temperature reduction per se when the osmolality of the extracellular solution exceeded about 1400 mOsm/kg water. The posthypertonic hemolysis of cells exposed to a hypertonic solution at 0 °C was reduced with the temperature of the resuspension solution up to 35 °C.     

Hemolysis of human red blood cells by freezing and thawing in solutions containing polyvinylpyrrolidone: relationship with postthypertonic hemolysis and solute movements

An investigation was made into the effects of the presence of polyvinylpyrrolidone (PVP) on changes in human red blood cells suspended in hypertonic solutions, on posthypertonic hemolysis, and on freezing at temperatures down to ?12 °C.PVP is very effective at reducing hemolysis when the red blood cells are frozen at temperatures down to ?12 °C. However, the membranes of the cells recovered on thawing have become very permeable to sodium and potassium ions and there is a much increased hemolysis if the cells are resuspended in an isotonic solution of sodium chloride.The presence of PVP does not affect the dehydration of the cells or the development of a change in membrane permeability when the cells are shrunken in hypertonic solutions at 0 °C. Neither does its presence in the hypertonic solution reduce the extent of posthypertonic hemolysis at 4 °C (as measured by the hemolysis on resuspension in an isotonic solution of sodium chloride), but it is more effective than sucrose at reducing hemolysis when present in the resuspension solution. It is concluded that the PVP is able to prevent swelling and hemolysis of cells which are very permeable to cations by opposing the colloid osmotic pressure due to the hemoglobin. However, this does not explain how PVP is able to protect cells against freezing damage at high cooling rates, and a mechanism by which it might do this is discussed.