Osmotic Volume Changes Research Articles

The fern cavitation catapult: mechanism and design principles.

Leptosporangiate ferns have evolved an ingenious cavitation catapult to disperse their spores. The mechanism relies almost entirely on the annulus, a row of 12-25 cells, which successively: (i) stores energy by evaporation of the cells' content, (ii) triggers the catapult by internal cavitation, and (iii) controls the time scales of energy release to ensure efficient spore ejection. The confluence of these three biomechanical functions within the confines of a single structure suggests a level of sophistication that goes beyond most man-made devices where specific structures or parts rarely serve more than one function. Here, we study in detail the three phases of spore ejection in the sporangia of the fern Polypodium aureum. For each of these phases, we have written the governing equations and measured the key parameters. For the opening of the sporangium, we show that the structural design of the annulus is particularly well suited to inducing bending deformations in response to osmotic volume changes. Moreover, the measured parameters for the osmoelastic design lead to a near-optimal speed of spore ejection (approx. 10 m s(-1)). Our analysis of the trigger mechanism by cavitation points to a critical cavitation pressure of approximately -100 ± 14 bar, a value that matches the most negative pressures recorded in the xylem of plants. Finally, using high-speed imaging, we elucidated the physics leading to the sharp separation of time scales (30 versus 5000 µs) in the closing dynamics. Our results highlight the importance of the precise tuning of the parameters without which the function of the leptosporangium as a catapult would be severely compromised.

Osmotic tolerance limits of red blood cells from umbilical cord blood

Effective methods for long-term preservation of cord red blood cells (RBCs) are needed to ensure a readily available supply of RBCs to treat fetal and neonatal anemia. Cryopreservation is a potential long-term storage strategy for maintaining the quality of cord RBCs for the use in intrauterine and neonatal transfusion. However, during cryopreservation, cells are subjected to damaging osmotic stresses during cryoprotectant addition and removal and freezing and thawing that require knowledge of osmotic tolerance limits in order to optimize the preservation process. The objective of this study was to characterize the osmotic tolerance limits of cord RBCs in conditions relevant to cryopreservation, and compare the results to the osmotic tolerance limits of adult RBCs. Osmotic tolerance limits were determined by exposing RBCs to solutions of different concentrations to induce a range of osmotic volume changes. Three treatment groups of adult and cord RBCs were tested: (1) isotonic saline, (2) 40% w/v glycerol, and (3) frozen–thawed RBCs in 40% w/v glycerol. We show that cord RBCs are more sensitive to shrinkage and swelling than adult RBCs, indicating that osmotic tolerance limits should be considered when adding and removing cryoprotectants. In addition, freezing and thawing resulted in both cord and adult RBCs becoming more sensitive to post-thaw swelling requiring that glycerol removal procedures for both cell types ensure that cell volume excursions are maintained below 1.7 times the isotonic osmotically active volume to attain good post-wash cell recovery. Our results will help inform the development of optimized cryopreservation protocol for cord RBCs.

127 Measuring of Sf21 cell membrane biophysical parameters using a new nondilute solute transport equation

In cryobiology, Kedem-Katchalsky model (KK) and two-parameter model (2P) has been used to predict cell volume changes. However, the KK model might lead to conceptual errors when used for cell membranes and the 2P model is based on ideal and dilute solution. Recently, Heidi Y. Elmoazzen et al. (Heidi Y. Elmoazzen, Janet A. W. Elliott, and Locksley E. McGann, “Osmotic Transport across Cell Membranes in Nondilute Solutions: A New Nondilute Solute Transport Equation,” Biophysical Journal, vol. 96, pp. 2559–571, 2009) had proposed a new nondilute solute transport equation which could fit or predict cell osmotic volume changes more appropriate. The purpose of the article is to verify the validity of the new equation. We used SF21 cell volume changes in different concentrations of Glycerol and different temperatures to fit the new model to water permeability coefficient ( L ) and solute permeability coefficient ( P ) of cell membrane. The experiment was carried out by using microperfusion system designed by our lab. What’s more, the extracellular concentration of CPA was simulated by using a universal self-adaptive time-varying function, which is derived by Gang Zhao (Gang Zhao, “A Universal Self-Adaptive Time-Varying Function for Extracellular Concentration during Osmotic Shift for Curve-Fitting Permeability Coefficients of Cell Membrane,” Cryoletters, vol. 31, pp. 279–90, 2010). Mean (means ± SD) L values in the presence of 2.0 M Glycerol at 25, 20, 15, and 10 °C were determined to be 0.68 ± 0.29, 0.78 ± 0.33, 0.77 ± 0.20, and 0.67 ± 0.16 10-28mol2/ (minatm μ m5). The P values were 4.6 ± 0.1.69, 1.11 ± 0.99, 3.84 ± 0.65, and 2.96 ± 2.81 10–32 ml 2 / (minatm μ m 5 ). L values in the presence of 1.5 M Glycerol at 25, 20, 15, and 10 °C were determined to be 0.97 ± 0.61, 1.27 ± 0.64, 0.818 ± 0.29, and 0.533 ± 0.17 10–28mol 2 / (minatm μ m 5 ). The P values were 4.18 ± 2.51, 3.00 ± 1.62, 1.14 ± 0.58, and 2.3 ± 1.53 10–32 mol 2 / (minatm μ m 5 ). L values in the presence of 1.0 M Glycerol at 25, 20, 15, and 10 °C were determined to be 1.78 ± 0.65, 1.24 ± 0.66, 1.02 ± 0.26, and 1.15 ± 0.39 10–28 mol 2 / (minatm μ m 5 ). The P values were 2.67 ± 0.94, 2.70 ± 1.53, 1.45 ± 0.96, and 0.79 ± 0.40 10–32 mol 2 / (minatm μ m 5 ). From fitting lines of the experiment data, we could find out the new nondilute equation is very similar to 2 P model, which demonstrates, in our opinion, the new nondilute equation can be used to fit and predict cell volume changes. Therefore, more experiments should be done to prove which one might lead to a better result. Source of funding: This work was supported by the National Natural Science Foundation of China (Nos. 51076149, 51276179) Conflict of interest: None declared. zhaog@ustc.edu.cn

Effects of all-trans-retinoic acid on the permeability transition and bioenergetic functions of rat liver mitochondria in combination with endoxifen

AimsThe clinical utilization of the combinations of all-trans-retinoic acid (RA) with antiestrogens, which present synergism of action in breast cancer, has been limited by RA adverse effects, including hepatotoxicity, which may be related with mitochondrial damage. This work evaluated the effects of RA alone and in combination with the antiestrogen endoxifen (EDX) on liver mitochondria. Main methodsMitochondrial permeability transition (MPT) was assessed by using Calcium Green-5N fluorescence and a tetraphenylphosphonium selective electrode. Oxidative stress was evaluated by oxygen consumption and thiobarbituric acid method. Mitochondrial bioenergetic was monitored by measuring oxygen consumption and mitochondrial membrane potential (ΔΨ). Osmotic volume changes of mitochondria were followed at 540nm. Key findingsEDX prevents the MPT induced by RA, allowing mitochondria pre-incubated with RA to accumulate Ca2+ and inhibiting the depolarization of ΔΨ. RA above 10nmol/mg protein depresses the phosphorylation capacity of mitochondria, as shown by the increase in the time required for ADP phosphorylation as well as by the decrease in state 3 respiration. At 20nmol/mg protein, RA decreases the ΔΨ and increases the state 4 respiration, suggesting that high concentrations of RA permeabilize the membrane to protons, possibly due to a proton leak through the Fo fraction of complex V. Moreover, the effects of RA on mitochondrial bioenergetics are not changed by EDX. SignificanceRA-induced hepatotoxicity may be related with induction of MPT and alterations in bioenergetic parameters; the combination with EDX, which reduces mitochondrial dysfunction and synergistically potentiates the anticancer activity, may provide a safer therapeutic strategy.

35 Protective action of synthetic diarylamines on mitochondrial permeability transition pore

Mitochondria can undergo a generalised increase of the permeability of the inner membrane, called mitochondrial permeability transition (MPT), due to the formation of non-specific pores (MPTP), which is an early...

Inhibition of Protein Kinase Akt1 by Apoptosis Signal-regulating Kinase-1 (ASK1) Is Involved in Apoptotic Inhibition of Regulatory Volume Increase

Most animal cell types regulate their cell volume after an osmotic volume change. The regulatory volume increase (RVI) occurs through uptake of NaCl and osmotically obliged water after osmotic shrinkage. However, apoptotic cells undergo persistent cell shrinkage without showing signs of RVI. Persistence of the apoptotic volume decrease is a prerequisite to apoptosis induction. We previously demonstrated that volume regulation is inhibited in human epithelial HeLa cells stimulated with the apoptosis inducer. Here, we studied signaling mechanisms underlying the apoptotic inhibition of RVI in HeLa cells. Hypertonic stimulation was found to induce phosphorylation of a Ser/Thr protein kinase Akt (protein kinase B). Shrinkage-induced Akt activation was essential for RVI induction because RVI was suppressed by an Akt inhibitor, expression of a dominant negative form of Akt, or small interfering RNA-mediated knockdown of Akt1 (but not Akt2). Staurosporine, tumor necrosis factor-alpha, or a Fas ligand inhibited both RVI and hypertonicity-induced Akt activation in a manner sensitive to a scavenger for reactive oxygen species (ROS). Any of apoptosis inducers also induced phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) in a ROS-dependent manner. Suppression of (ASK1) expression blocked the effects of apoptosis, in hypertonic conditions, on both RVI induction and Akt activation. Thus, it is concluded that in human epithelial cells, shrinkage-induced activation of Akt1 is involved in the RVI process and that apoptotic inhibition of RVI is caused by inhibition of Akt activation, which results from ROS-mediated activation of ASK1.

Über Beziehungen zwischen der Spermienmotilität und dem Gehalt an ungesättigten Fettsäuren sowie dem osmotischen Verhalten der menschlichen Spermienzelle

The relations between sperm motility and the content of lipids and fatty acids in sperm cells were investigated. In addition the osmotic volume changes of sperm cells were studied, by determination of progressive sperm motility, measurement of relative concentration of lipid-peroxy radicals in seminal plasma using chemoluminescence technique, gas-chromatographic determination of fatty acid pattern in sperm cells and in seminal plasma and by electronic measurement of the sperm cell volume. The impulse installment of chemoluminescence increased during the time of storage of the ejaculate. In contrast, this phenomenon was not observed in pure seminal plasma prepared by centrifugation immediately after liquefaction. The pattern of phospholipids and free fatty acids changed during storage of the ejaculate both in sperm cells and in seminal plasma. Sperm cells showed a significant increase of volume under hypotonic stress. The increase of volume is more distinct in spermatozoa with high progressive motility as in spermatozoa with low progressive motility. The determination of initial and late progressive motility enables the differentiation of two groups of sperm populations: one group with a medium initial motility and a normal decrease of motility during the following time and another group with excellent initial motility and an extreme decrease of motility following. The second group is characterized by an especially distinct increase of volume under hypotonic conditions.

Computer simulations of neuron-glia interactions mediated by ion flux

Extracellular potassium concentration, [K(+)](o), and intracellular calcium, [Ca(2+)](i), rise during neuron excitation, seizures and spreading depression. Astrocytes probably restrain the rise of K(+) in a way that is only partly understood. To examine the effect of glial K(+) uptake, we used a model neuron equipped with Na(+), K(+), Ca(2+) and Cl(-) conductances, ion pumps and ion exchangers, surrounded by interstitial space and glia. The glial membrane was either "passive", incorporating only leak channels and an ion exchange pump, or it had rectifying K(+) channels. We computed ion fluxes, concentration changes and osmotic volume changes. Increase of [K(+)](o) stimulated the glial uptake by the glial 3Na/2K ion pump. The [K(+)](o) flux through glial leak and rectifier channels was outward as long as the driving potential was outwardly directed, but it turned inward when rising [K(+)](o)/[K(+)](i) ratio reversed the driving potential. Adjustments of glial membrane parameters influenced the neuronal firing patterns, the length of paroxysmal afterdischarge and the ignition point of spreading depression. We conclude that voltage gated K(+) currents can boost the effectiveness of the glial "potassium buffer" and that this buffer function is important even at moderate or low levels of excitation, but especially so in pathological states.

On the mechanism of cell lysis by deformation

In this study, we identify the extent of deformation that causes cell lysis using a simple technique where a drop of cell suspension is compressed by two flat plates. The viability of human prostatic adenocarcinoma PC-3 cells in solutions of various concentrations of NaCl is determined as a function of the gap size between the plates. The viability declines with decreasing gap size in the following order: 700 mM>150 mM>75 mM NaCl. This is considered to be due to the difference in cell size, which is caused by the osmotic volume change before deformation; cell diameter becomes smaller in a solution of higher NaCl concentration, which appears to increase the survival ratio in a given gap size. The deformation-induced decrease in cell viability is correlated with the cell surface strain, which is dependent on the increase in surface area, irrespective of NaCl concentration. In addition, the treatment of cells with cytochalasin D results in the disappearance of cortical actin filaments and a marked drop in the viability, indicating that cell lysis is closely related to the deformation of the cytoskeleton.

Functional Characterization of Contractile Vacuole Isolated from Amoeba proteus

Contractile vacuoles (CVs) released from cells of Amoeba proteus were used to analyze its function in vitro. When CV was transferred to a hypertonic medium, its volume decreased within 10 sec. When it was subsequently returned to its original medium, it quickly started swelling. However, it ruptured before recovering its initial volume. These results suggested that the CV membrane is semi-permeable and that the fluid is collected by the osmotic gradient in vivo. The water permeability of membrane of isolated CV was calculated from the rate of osmotic volume change to be 0.94 microm/sec . OsM. This high value suggested that CV membrane is equipped with water channel. CV contracted (or burst) quickly upon addition of 1 mM ATP. Contraction was induced by ATP, but not by other nucleotides, GTP, ITP, ADP, or the analogues of ATP, AMP-PNP and ATPgammaS. It was suggested that the contraction of isolated CV was caused by increase in the tension of its membrane by ATP.

Influence of salinity on the bioaccumulation and photoinduced toxicity of fluoranthene to an estuarine shrimp and oligochaete.

The effect of salinity on the photoinduced toxicity of waterborne fluoranthene to larvae of the grass shrimp (Palaemonetes pugio) and tubificid oligochaete worms (Monopylephorus rubroniveus) was studied in a laboratory system under simulated sunlight. In the grass shrimp toxicity tests, five concentrations of fluoranthene (0, 3.6, 7.3, 13.8, and 29.0 microg/L) and four salinities (6.9, 14.5, 21.2, and 28.6 per thousand) were achieved. In the oligochaete toxicity tests, five concentrations of fluoranthene (0, 0.8, 1.4, 3.3, and 7.7 microg/L) and four salinities (7.1, 13.3, 20.5, and 27.6 per thousand) were achieved. Salinity had no effect on either the photoinduced toxicity or the bioaccumulation of fluoranthene in the grass shrimp. However, the highest level of salinity decreased the median lethal time for the oligochaete. Bioaccumulation of fluoranthene was inversely related to salinity for the oligochaete. Additional experiments demonstrated an inverse relationship between salinity and short-term osmotic weight change in the oligochaete. Weight of the grass shrimp larvae was not affected by salinity. These findings show that salinity can influence the toxicity and bioaccumulation of fluoranthene in some estuarine organisms. The influence of salinity on these populations may be related to physiological responses associated with internal osmotic volume changes. Thus, salinity needs to be taken into account when assessing the risk of photoactivated polycyclic aromatic hydrocarbon (PAH) to at least some estuarine species.

138 細胞の圧迫変形障害に及ぼす浸透的体積変化の影響

138 細胞の圧迫変形障害に及ぼす浸透的体積変化の影響

Canine RBC osmotic tolerance and membrane permeability

The objective of this study was to determine the cryobiological characteristics of canine red blood cells (RBC). These included the hydraulic conductivity ( L p), the permeability coefficients ( P s) of common cryoprotectant agents (CPAs), the associated reflection coefficient ( σ), the activation energies ( E a) of L p and P s and the osmotic tolerance limits. By using a stopped-flow apparatus, the changes of fluorescence intensity emitted by intracellularly entrapped 5-carboxyfluorescein diacetate (CFDA) were recorded when cells were experiencing osmotic volume changes. After the determination of the relationship between fluorescence intensity and cell volume, cell volume changes were calculated. These volume changes were used in three-parameter fitting calculations to determine the values of L p, P s, and σ for common CPAs. These volume measurements and data analyses were repeated at three different temperatures (22, 14, 7 °C). Using the Arrhenius equation, the activation energies of L p and P s in the presence of CPAs were determined. The osmotic tolerance limits for canine RBC were determined by measuring the percentage of free hemoglobin in NaCl solutions with various osmolalities compared to that released by RBC incubated in double distilled water. The upper and lower osmotic tolerance limits were found to be 150 mOsm (1.67 V iso) and 1200 mOsm (0.45 V iso), respectively. These parameters were then used to calculate the amount of non-permeating solute needed to keep cell volume excursions within the osmotic tolerance limits during CPA addition and removal.

Osmotic Volume Change and Cellular Injury by Hypertonic Sodium Choloride Solution.

It has been believed that exposure to hypertonic solution during freezing is the major cause of the post-thaw cellular injury after freezing at low cooling rates. To develop a fundamental understanding of cell damage due to the hypertonic solution, we have developed a new perfusion technique that isolates the effect of exposure to hypertonic solutions from other effects associated with ice crystals. Using human prostatic adenocarcinoma cells, we characterized the post-hypertonic survival as a function of hypertonic solute concentration, the time of exposure, and the rates of increase and decrease in concentration. Also measured were the membrane permeability and the osmotically inactive volume of cells that are required for the description of osmotic change in cell volume.

Single water channels of aquaporin-1 do not obey the Kedem-Katchalsky equations.

The Kedem-Katchalsky (KK) equations are often used to obtain information about the osmotic properties and conductance of channels to water. Using human red cell membranes, in which the osmotic flow is dominated by Aquaporin-1, we show here that compared to NaCl the reflexion coefficient of the channel for methylurea, when corrected for solute volume exchange and for the water permeability of the lipid membrane, is 0.54. The channels are impermeable to these two solutes which would seem to rule out flow interaction and require a reflexion coefficient close to 1.0 for both. Thus, two solutes can give very different osmotic flow rates through a semi-permeable pore, a result at variance with both classical theory and the KK formulation. The use of KK equations to analyze osmotic volume changes, which results in a single hybrid reflexion coefficient for each solute, may explain the discrepancy in the literature between such results and those where the equations have not been employed. Osmotic reflexion coefficients substantially different from 1.0 cannot be ascribed to the participation of other 'hidden' parallel aqueous channels consistently with known properties of the membrane. Furthermore, we show that this difference cannot be due to second-order effects, such as a solute-specific interaction with water in only part of the channel, because the osmosis is linear with driving force down to zero solute concentration, a finding which also rules out the involvement of unstirred-layer effects. Reflexion coefficients smaller than 1.0 do not necessitate water-solute flow interaction in permeable aqueous channels; rather, the osmotic behaviour of impermeable molecular-sized pores can be explained by differences in the fundamental nature of water flow in regions either accessible or inaccessible to solute, created by a varying cross-section of the channel.

Effect of Osmolality and Oxygen Tension on the Survival of Mouse Sperm Frozen to Various Temperatures in Various Concentrations of Glycerol and Raffinose

Cryopreserved mouse sperm are beginning to be used to meet the demand of a reliable cost-effective method for maintaining the rapidly expanding numbers of lines of mutant mice. However, successful and reproducible cryopreservation has proven to be a difficult problem. Furthermore, the underlying factors responsible for success or failure are mostly obscure. Several contributors to these difficulties have been identified. Our laboratory has found that mouse sperm are extremely susceptible to the mechanical stresses associated with pipetting, mixing, and centrifugation, and others have found that they are severely limited in their tolerance to osmotic volume changes. We have hypothesized two other contributors to the difficulties. One is that the concentrations of glycerol used in published protocols are substantially lower than those found to be optimal for most mammalian cells. The other hypothesis relates to the fact that mouse sperm membranes are especially susceptible to damage from oxygen-derived free radicals. That damage may reduce their ability to survive freezing. If so, survival ought to increase if the concentration of oxygen is kept low throughout the procedure. To achieve low levels, we have incorporated an Escherichia coli membrane fraction, Oxyrase, into all media. A previous report showed a protective effect. That is confirmed here under a broader range of conditions. The conditions studied have been the individual and interactive effects of the concentrations of glycerol, raffinose, and phosphate-buffered saline (PBS) on motility after freezing at 21°C/min to −70°C. Cryoprotection increased with increasing raffinose concentration, provided that the concentration of PBS was appropriately reduced to hold the total osmolality of nonpermeating solutes to within tolerated limits. Surprisingly, the best results were achieved in the total absence of glycerol. The highest motilities to date (68 ± 8%) after freezing to −70°C have been achieved using media containing Oxyrase, 0 M glycerol, and 18% raffinose in 14× strength modified PBS. We also determined the motility loss after freezing to intermediate temperatures, i.e., −10 and −30°C. The major motility loss occurred by −10°C, especially in the absence of Oxyrase. These results suggest that a major problem in the freezing of mouse sperm is the physical stress resulting from extracellular ice crystal formation. Oxyrase appears to lessen that damage substantially.

Light-Enhanced Uptake of Metabolites in Mesophyll Protoplasts: Evidence for a Novel Pyruvate Transport System

) and sorghum (C4) leaves for the measurements of osmotic volume change and metabolite uptake. We first investigated whether the silicone oil layer filtering centrifugation method could be applied to the protoplasts. The density of the silicone oil was optimized (ρ =1.026) and 0.5M betaine was chosen as an osmoticum in the protoplast suspending medium. By using [14C] sorbitol and [14C] inulin as the marker of the medium carried over into the pellet, protoplast osmotic or internal volume was estimated to be 200–300 μl (mg Chl)−1, with the medium space in the pellet of 8–15 μl (mg Chl)−1. Lowering of the osmotic pressure of the medium induced protoplast swelling as expected. Light also induced swelling. Using this system, we could detect light-enhanced uptake of ascorbate, glutamate and pyruvate in both barley and sorghum protoplasts. Pyruvate uptake was far higher in barley than in sorghum and inhibited by various inhibitors, showed saturation kinetics and, therefore, seemed to be mediated by a translocator protein.

Sodium Chloride-Induced Volume Changes of Freshwater Cyanobacterium Synechococcus sp. PCC 7942 Cells Can Be Probed by Chlorophyll a Fluorescence

Freshwater species of the cyanobacterial genus Synechococcus import NaCl passively, and export Na+ actively, by means of primary and secondary extrusion mechanisms. As a result of the ion and water fluxes, cell volumes are enlarged. We show in this paper that the NaCl-induced volume enlargement of Synechococcus sp. PCC 7942 cells is attended by a rapid (k = 0.39 s−1) increase in chlorophyll (Chl) a fluorescence. The cell turgor threshold (measured by osmotic titration of Chl a fluorescence) was lower in the absence of NaCl (0.195 Osm kg−1) than in the presence of 0.4 M NaCl (0.248 Osm kg−1) indicating NaCl uptake by the cells. Turgor thresholds of cells suspended in NaCl-containing medium were enlarged further by protonophoric uncouplers, P-type ATPase inhibitors, and light starvation, conditions that are known to interfere with the active extrusion of Na+ ions. Cell swelling exerts probably a regulation on the distribution of phycobilisome (PBS) excitation between photosystem II (fluorescent Chl a) and photosystem I (nonfluorescent Chl a), since it affects PBS-sensitized Chl a fluorescence, but not directly excited Chl a fluorescence. The dependence of the Chl a fluorescence of cyanobacteria on cell volumes allows probing of bioenergetic phenomena that are related to dynamic osmotic volume changes, transmembrane solute and water fluxes, plasma membrane permeabilities, and internal osmotic conditions of cyanobacterial cells. Thus, cyanobacteria may serve as quite convenient models of aquatic microorganisms in experimental studies directed toward the elucidation of perception mechanisms and defense mechanisms of water and solute stresses.

On the relationship between matrix free Mg 2+ concentration and total Mg 2+ in heart mitochondria

The matrix free magnesium ion concentration, [Mg 2+] m, estimated using the fluorescent probe furaptra, averaged 0.67 mM in 15 preparations of beef heart mitochondria containing an average of 21 nmol total Mg 2+ per mg protein. [Mg 2+] m was compared with total Mg 2+ during respiration-dependent uptake and efflux of Mg 2+ and during osmotic swelling. In the absence of external P i these mitochondria contain about 32 nmol/mg non-diffusible Mg-binding sites with an apparent K d of 0.34 mM. [Mg 2+] m depends on both the size of the total Mg 2+ pool and the ability of matrix anions to provide Mg-ligands. P i interacts strongly with Mg 2+ to decrease [Mg 2+] m and, in the absence of external Mg 2+, promotes respiration-dependent Mg 2+ efflux and a decrease in [Mg 2+] m to very low levels. The uptake of P i by respiring mitochondria converts ΔpH to membrane potential (Δ Ψ) and provides additional Mg-binding sites. This permits large accumulations of Mg 2+ and P i with little change in [Mg 2+] m. Nigericin also converts ΔpH to Δ Ψ in respiring mitochondria and induces a large and rapid increase in both total Mg 2+ and [Mg 2+] m. Mersalyl increases the permeability of the mitochondrial membrane to cations and this also induces a marked increase in both total Mg 2+ and [Mg 2+] m. These results suggest that mitochondria take up Mg 2+ by electrophoretic flux through membrane leak pathways, rather than via a specific Mg 2+ transporter. Mitochondria swollen by respiration dependent uptake of potassium phosphate show decreased [Mg 2+] m, whereas those swollen to the same extent in potassium acetate do not. This suggests that [Mg 2+] m is well-buffered during osmotic volume changes unless there is also a change in ligand availability.

Regulatory volume decrease of cardiac myocytes induced by beta-adrenergic activation of the Cl- channel in guinea pig.

A new method was developed to automatically measure the thickness of a single ventricular myocyte of guinea-pig heart. A fine marker was attached on the cell's upper surface and changes in its vertical position were measured by focusing it under the microscope. When the osmolarity of the bath solution was varied, the cell thickness reached a new steady level without any obvious regulatory volume change within the period of observation up to 15 min. The cell thickness was 7.8 +/- 0.2 microns (n = 94) in the control Tyrode solution and was varied to 130.4 +/- 3.1% (n = 10), 119.1 +/- 1.1% (n = 50), 87.2 +/- 1.9% (n = 9), and 75.6 +/- 3.2% (n = 5) of control at 50, 70, 130, and 200% osmolarity, respectively. The application of a Cl- channel blocker, 500 microM anthracene-9-carboxylic acid (9AC) did not modify these osmotic volume changes. We discovered that the application of isoprenaline induced a regulatory volume decrease (RVD) in cells inflated by hypotonic solutions. This isoprenaline-induced RVD was inhibited by antagonizing beta-adrenergic stimulation with acetylcholine. The isoprenaline-induced RVD was mimicked by the external application of 8-bromoadenosine 3':5'-cyclic monophosphate. The RVD was inhibited by blocking the cAMP-dependent Cl- channel (ICl, rAMP) with 9AC but was insensitive to 4,4'-diisothiocyanostilbene-2,2'-dissulphonate (DIDS). Taken together these data suggest an involvement of ICl, cAMP activation in the RVD. Whole cell voltage clamp experiments revealed activation of ICl, cAMP by isoprenaline under the comparable conditions. The cardiac cell volume may be regulated by the autonomic nervous activity.