Isoosmotic KCl Research Articles

Use of iso-osmotic solution to understand salt stress responses in lentil (Lens culinaris Medik.)

Lentil, an important source of protein for human consumption, is considered as a salt susceptible crop species. Thus the present study was carried out to evaluate the most important lentil physiological characters that induce salt tolerance. Salt stress results in osmotic stress immediately followed by ionic toxicity. We used iso-osmotic solutions with different kinds of osmotica i.e. salt (NaCl and KCl), and polyethylene glycol (PEG) to identify the specific response of osmotic stress and ionic toxicity. All of these altered seedling height, chlorophyll, malondialdehyde (MDA), hydrogen peroxide (H2O2), proline, reduced ascorbate (AsA) and total glutathione content, and ion uptake. Moreover, these osmotica also altered the activities of antioxidant enzymes such as catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR), except monodehydroascorbate reductase (MDHAR). Among different osmotica, only NaCl exhibited leaf chlorosis, reduction in K+ and disruption in ion homeostasis, and increased MDA, H2O2 and proline content than iso-osmotic KCl and PEG, indicating susceptibility of lentil seedling to salt stress. Addition of Ca along with NaCl showed no chlorosis and improved K+ content. These results demonstrate that prevention of Na-induced K depletion in root might enhance salt tolerance in lentil.

Involvement of AQP6 in the Mercury-Sensitive Osmotic Lysis of Rat Parotid Secretory Granules

In secretory granules and vesicles, membrane transporters have been predicted to permeate water molecules, ions and/or small solutes to swell the granules and promote membrane fusion. We have previously demonstrated that aquaporin-6 (AQP6), a water channel protein, which permeates anions, is localized in rat parotid secretory granules (Matsuki-Fukushima et al., Cell Tissue Res 332:73-80, 2008). Because the localization of AQP6 in other organs is restricted to cytosolic vesicles, the native function or functions of AQP6 in vivo has not been well determined. To characterize the channel property in granule membranes, the solute permeation-induced lysis of purified secretory granules is a useful marker. To analyze the role of AQP6 in secretory granule membranes, we used Hg²⁺, which is known to activate AQP6, and investigated the characteristics of solute permeability in rat parotid secretory granule lysis induced by Hg²⁺ (Hg lysis). The kinetics of osmotic secretory granule lysis in an iso-osmotic KCl solution was monitored by the decay of optical density at 540 nm using a spectrophotometer. Osmotic secretory granule lysis was markedly facilitated in the presence of 0.5-2.0 μM Hg²⁺, concentrations that activate AQP6. The Hg lysis was completely blocked by β-mercaptoethanol which disrupts Hg²⁺-binding, or by removal of chloride ions from the reaction medium. An anion channel blocker, DIDS, which does not affect AQP6, discriminated between DIDS-insensitive and sensitive components in Hg lysis. These results suggest that Hg lysis is required for anion permeability through the protein transporter. Hg lysis depended on anion conductance with a sequence of NO(3) (-) > Br⁻ > I⁻ > Cl⁻ and was facilitated by acidic pH. The anion selectivity for NO(3) (-) and the acidic pH sensitivity were similar to the channel properties of AQP6. Taken together, it is likely that AQP6 permeates halide group anions as a Hg²⁺-sensitive anion channel in rat parotid secretory granules.

Effect of KCl Medium on Malate Oxidation in Mitochondria of Sugar Beet Taproot

Isolated mitochondria were obtained from growing and stored sugar beet (Beta vulgaris L.) taproots. These preparations were used to monitor the mitochondrial matrix volume and malate oxidation after the replacement of sucrose with KCl in the reaction medium. The transfer of mitochondria from sucrose-containing isolation medium to the isoosmotic KCl solution initiated spontaneous or energy-dependent (in the presence of respiratory substrate) swelling whose kinetic parameters (the initial rate and amplitude) were virtually independent of the plant age. At the same time, effects of KCl-induced swelling on oxidative and phosphorylating activities of mitochondria were age-dependent. In mitochondria from growing taproots, K+ ions stimulated nonphosphorylating malate oxidation, thereby decreasing the respiratory control ratio and the ADP/O coefficient. The incubation of mitochondria from stored taproots in KCl solution induced a short-term activation and subsequent progressive inhibition of malate oxidation but did not inhibit the oxidation of exogenous NADH. The inhibition of malate oxidation was not released by adding ADP or uncouplers and was enhanced in the presence of valinomycin. The swelling of mitochondria in KCl solutions did not impair the integrity of mitochondrial membranes and did not preclude stimulation of malate oxidation by exogenous NAD. It is supposed that the KCl-induced inhibition of respiration is related to a large increase in the matrix volume and a drastic decrease in the concentration of a coenzyme NAD. Previous studies with isolated mitochondria from stored taproots showed that the mitochondrial NAD level was a rate-limiting factor of malate oxidation assayed in the sucrose-containing media. A possible role of K+-transporting mechanisms in regulation of mitochondrial matrix volume and metabolic activity of plant mitochondria is discussed.

Cellular mechanisms involved in iso-osmotic high K + solutions-induced contraction of the estrogen-primed rat myometrium

The aim of the present study was to investigate the mechanisms involved in the contraction evoked by iso-osmotic high K + solutions in the estrogen-primed rat uterus. In Ca 2+-containing solution, iso-osmotic addition of KCl (30, 60 or 90 mM K +) induced a rapid, phasic contraction followed by a prolonged sustained plateau (tonic component) of smaller amplitude. The KCl (60 mM)-induced contraction was unaffected by tetrodotoxin (3 μM), ω-conotoxin MVIIC (1 μM), GF 109203X (1 μM) or calphostin C (3 μM) but was markedly reduced by tissue treatment with neomycin (1 mM), mepacrine (10 μM) or U-73122 (10 μM). Nifedipine (0.01–0.1 μM) was significantly more effective as an inhibitor of the tonic component than of the phasic component. After 60 min incubation in Ca 2+-free solution containing 3 mM EGTA, iso-osmotic KCl did not cause any increase in tension but potentiated contractions evoked by oxytocin (1 μM), sodium orthovanadate (160 μM) or okadaic acid (20 μM) in these experimental conditions. In freshly dispersed myometrial cells maintained in Ca 2+-containing solution and loaded with indo 1, iso-osmotic KCl (60 mM) caused a biphasic increase in the intracellular Ca 2+ concentration ([Ca 2+] i). In cells superfused for 60 min in Ca 2+-free solution containing EGTA (1 mM), KCl did not increase [Ca 2+] i. In Ca 2+-containing solution, KCl (60 mM) produced a 76.0 ± 16.2% increase in total [ 3H]inositol phosphates above basal levels and increased the intracellular levels of free arachidonic acid. These results suggest that, in the estrogen-primed rat uterus, iso-osmotic high K + solutions, in addition to their well known effect on Ca 2+ influx, activate other cellular processes leading to an increase in the Ca 2+ sensitivity of the contractile machinery by a mechanism independent of extracellular Ca 2+.

Ca 2+-independent contraction induced by hyperosmolar K +-rich solutions in rat uterus

The present experiments were designed to investigate the mechanisms involved in the contractile responses evoked by KCl, added either isoosmotically or hyperosmotically, in the rat uterus. Exposure of uterine strips to a Ca 2+-free, 3 mM EGTA-containing solution abolished the responses induced by isoosmotic KCl solutions. Conversely, addition of hyperosmolar KCl induced concentration-dependent tonic responses in a Ca 2+-free, 3 mM EGTA-containing solution. The maximum increase in tension was reached with 210 mM K +. The response to hyperosmotic K + was unaffected by previous depletion of intracellular Ca 2+ stores with oxytocin (1 μM), by inhibition of refilling of the intracellular Ca 2+ stores using cyclopiazonic acid (10 μM) or by increasing the concentration of EGTA in the medium to 10 mM. Sucrose and mannitol (60–420 mM) induced concentration-dependent sustained contractions which were not reproducible and were significantly smaller in size than those evoked by the maximally effective concentration of hyperosmotic K + (210 mM). The contraction induced by hyperosmotic K + in Ca 2+-free solution was not altered by the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide hydrochloride (W-7, 100 μM), the Ca 2+ calmodulin protein kinase II inhibitor 1-[ N,O-bis(1,5-isoquinoline-sulphonyl)- N-methyl- l-tyrosyl]-4-phenylpiperazine (KN-62, 10 μM) or the tyrosine kinase inhibitor genistein (10 μM). The protein kinase C inhibitor calphostin C (1–3 μM) failed to modify the K +-effect curve, which was however partially inhibited in the presence of the non-selective protein kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine dihydrochloride (H-7, 3–100 μM). The protein kinase inhibitor staurosporine (30–300 nM) depressed the contraction induced by hyperosmolar K + in a concentration-dependent manner. The contraction induced by sucrose in Ca 2+-free solution was unaffected by W-7 (100 μM) and KN-62 (10 μM) but was partially reduced by calphostin C (1 μM), H-7 (30 μM), staurosporine (100 nM) and genistein (10 μM). These results suggest that different mechanisms are involved in the responses evoked by isoosmotic and hyperosmotic KCl in the rat uterus. A component of the contraction induced by hypertonic KCl seems mainly independent of both external and internal Ca 2+ and of hyperosmolar stress. This contraction is not mediated by protein kinase C, Ca 2+ calmodulin-dependent kinases or protein tyrosine kinases but involves activation of other, at the present unknown, staurosporine-sensitive protein kinase(s).

Fate of injected glucagon taken up by rat liver in vivo. Degradation of internalized ligand in the endosomal compartment

The uptake and processing of glucagon into liver endosomes were studied in vivo by subcellular fractionation. After injection of [[125I]iodo-Tyr10]glucagon and [[125I]iodo-Tyr13]glucagon to rats, the uptake of radioactivity into the liver was maximum at 2 min (6% of the dose/g of tissue). On differential centrifugation, the radioactivity in the homogenate was recovered mainly in the nuclear (N), microsomal (P) and supernatant (S) fractions, with maxima at 5, 10 and 40 min, respectively; recovery of radioactivity in the mitochondrial-lysosomal (ML) fraction did not exceed 6% and was maximal at 20 min. On density-gradient centrifugation, the radioactivity associated first (2-10 min) with plasma membranes and then (10-40 min) with Golgi-endosomal (GE) fractions, with 2-5-fold and 20-150-fold enrichments respectively. Subfractionation of the GE fractions showed that, unlike the Golgi marker galactosyltransferase, the radioactivity was density-shifted by diaminobenzidine cytochemistry. Subfractionation of the ML fraction isolated at 40 min showed that more than half of the radioactivity was recovered at lower densities than the lysosomal marker acid phosphatase. Throughout the time of study, the [125I]iodoglucagon associated with the P, PM and GE fractions remained at least 80-90% trichloroacetic acid (TCA)-precipitable, whereas that associated with other fractions, especially the S fraction, became progressively TCA-soluble. On gel filtration and h.p.l.c., the small amount of degraded [125I]iodoglucagon associated with GE fractions was found to consist of monoiodotyrosine. Chloroquine treatment of [125I]iodoglucagon-injected rats caused a moderate but significant increase in the late recovery of radioactivity in the ML, P and GE fractions, but had little effect on the association of the ML radioactivity with acid-phosphatase-containing structures. Chloroquine treatment also led to a paradoxical decrease in the TCA-precipitability of the radioactivity associated with the P and GE fractions. Upon h.p.l.c. analysis of GE extracts of chloroquine-treated rats, at least four degradation products less hydrophobic than intact [125I]iodoglucagon were identified. Radio-sequence analysis of four of these products revealed three cleavages, affecting bonds Ser2-Gln3, Thr5-Phe6 and Phe6-Thr7. When GE fractions containing internalized [125I]iodoglucagon were incubated in iso-osmotic KCl at 30 degrees C, a rapid generation of TCA-soluble products was observed, with a maximum at pH 4. We conclude that endosomes are a major site at which internalized glucagon is degraded, endosomal acidification being required for optimum degradation.

The permeability of lysosomes to sugars. Effect of diethylstilbestrol on the osmotic activation of lysosomes induced by glucose.

We have investigated the effect on the osmotic activation of rat liver lysosomes, by glucose penetration, of different substances known to inhibit the glucose transport through the plasma membrane. Diethylstilbestrol is the most efficient, particularly when purified lysosomes are used. It has no effect on osmotic activation induced by hypo-osmotic sucrose or by iso-osmotic KCl. It is proposed that diethylstilbestrol reacts with specific sites involved in the glucose translocation through the lysosomal membrane. These sites could not be identified by binding experiments, presumably owing to the considerable unspecific binding of the compound to the membrane.

Uptake, retention, and efflux of Ca 2+ by mitochondrial preparations from skeletal muscle

Functionally intact mitochondria, substantially free of contamination, were isolated from rabbit gastrocnemius muscle after protease digestion and their Ca 2+-handling properties examined. When judged by their capacity to retain large Ca 2+ loads and the magnitude of basal and Na +-stimulated Ca 2+ effluxes, the most suitable isolation method was digestion of finely minced muscle in buffered isoosmotic KCl with low levels (0.4 mg/g) of trypsin or the bacterial protease nagarse, followed by differential centrifugation. Polytron disruption of skeletal muscle in both sucrose- and KCl-based media released mitochondria deficient in cytochrome c. Use of the divalent ion chelator EDTA rather than EGTA in the isolation medium sharply reduced Ca 2+-dependent respiratory control and tolerance of the mitochondria to Ca 2+ loads, probably by removing Mg 2+ essential to membrane integrity. ADP-dependent respiratory control was not altered in mitochondria prepared in an EDTA-containing isolation medium. Purification of mitochondria on a Percoll density gradient did not improve their Ca 2+-handling ability despite removal of minor contaminants. Mitochondria prepared by the protease method could accumulate micromole loads of Ca 2+/mg while maintaining a low basal Ca 2+ efflux. Addition of BSA to the assay medium slightly improved Ca 2+ retention but was not essential either during isolation or assay. Ca 2+-dependent state 3 respiration was maximal at pH 6.5–7.0 while respiratory control and Ca 2+ O were optimal at pH 7.0–7.5. Neither P i nor oxaloacetate induced Ca 2+ release from loaded mitochondria when monitored for 30 min after ruthenium red addition. Na +-stimulated Ca 2+ efflux had sigmoidal kinetics with a Hill coefficient of 3. Since skeletal muscle mitochondria can be isolated and assayed in simple media, functional deficiencies of mitochondria from diseased muscle are unlikely to be masked.

Effect of Osmotic Shock on Some Intracellular Solutes in Two Filamentous Fungi

SUMMARY: Hypoosmotic and hyperosmotic shock experiments confirmed the importance for osmoregulation in Penicillium chrysogenum and Chrysosporium fastidium of glycerol and the osmoticum used in the medium. Growth ceased following both types of shock treatment for a time, depending on the magnitude of the shock and the species. Regrowth following hypoosmotic shock took place some distance behind burst tips. Following hyperosmotic shock, growth was initiated by branching at the apex, but if the magnitude of the shock was greater than 10 MPa, it did not occur in either species although osmotic adjustment was observed. Shock experiments did not implicate the higher polyols in osmotic adjustment. Hypoosmotic shock to C. fastidium resulted in a rapid loss of all internal solutes, while decrease in glycerol in P. chrysogenum following this treatment was more gradual. Transfer of C. fastidium, a species which does not grow on media of high salt concentration, to isoosmotic KCl did not result in loss of turgor although growth was inhibited. Glucose was lost from the hyphae but K+ and Cl- were taken up.

Behavioral and autonomic temperature regulation in competition with food intake and water balance of pigeons.

Four adult pigeons were conditioned to adjust to a preferred ambient temperature and to receive food or water by instrumental responses. The experiments were performed in a Skinner-box under heat load conditions. Each session lasted 1 h. The birds were deprived of food and water for several days before the actual experiments with the exception of the control tests. The exposure to different heat loads (50°, 55° and 60° C) effected both an increase in the responses for cold reward and a rise in respiratory evaporation (panting). The combined effort of behavioral and autonomous responses resulted in constant body temperature (42° C) under the three heat conditions (Fig. 1). The food deprivation effected a preference of instrumental response for feeding and a negligence of behavioral temperature regulation, whereby a slight hyperthermia resulted in the first part of heat exposure (55° C, Fig. 2). Water deprivation reduced the rate of respiration and simultaneously forced the response for cold reward, through which the deep body temperature was adjusted to the control level of 42° C (Fig. 3). Intragastric infusion of 20 ml NaCl (0.8 mol) half an hour before starting heat load (55° C) produced the same result as water deprivation. Whereas infusion of isoosmotic KCl solution did not produce any deviation from the control tests (Fig. 4). The results show competition between the conditioned thermal behaviour and the motivation of feeding or drinking in animals which may permit to compare the conditioned thermal behaviour with the natural kinds of behavioral temperature regulation. The non-appearance of hyperthermia under conditions of water deprivation and NaCl loading may indicate that the animals are able to use the autonomous and the behavioral types of heat defence responses to a greater or lesser extent as circumstances demand. This question seems to be of great interest and is fully discussed here.

The influx of calcium ions into human erythrocytes during cold storage. The influences of extracellular pH, intracellular adenosine triphosphate and efflux of univalent cations.

1. When human erythrocytes are stored at 3 degrees C for several days as a suspension in iso-osmotic sucrose or KCl, containing CaCl(2), the rates of cellular ATP degradation are similar. 2. During cold storage of erythrocytes in sucrose-CaCl(2) medium, Ca(2+) influx and univalent-cation efflux occur, the pH value of the suspending medium rises and the intracellular pH falls. These pH changes correlate reasonably well with alterations in the membrane potential calculated from Cl(-) distribution. 3. The presence of Ca(2+) in the medium does not increase the rate of univalent-cation efflux from the cells. 4. When the pH of the medium is raised by addition of buffers, the rates of both Ca(2+) influx and univalent-cation efflux increase. 5. Replacement of sucrose by KCl as the main osmotic component of the medium completely suppresses Ca(2+) influx and univalent-cation efflux, although the pH of the KCl medium is higher than that of the sucrose medium. 6. When sucrose is replaced by choline chloride, Ca(2+) influx and univalent-cation efflux still occur, and the pH of the medium is similar to that found in iso-osmotic KCl. 7. When valinomycin, Pb(2+) or Cd(2+) are added to the iso-osmotic sucrose medium, the rate of efflux of univalent cations increases as also does the influx of Ca(2+). 8. From these and other observations, it was concluded that it is univalent-cation efflux rather than ATP depletion or elevated extracellular pH which is the prerequisite for Ca(2+) influx during cold storage.

The influx of calcium ions into human erythrocytes during cold storage.

1. When human erythrocytes, suspended in iso-osmotic sucrose containing CaCl(2), are stored at 3 degrees C, Ca(2+) influx into the cells occurs. Simultaneously, efflux of K(+), Na(+), Cl(-) and water takes place and cell volume diminishes. 2. The extent of Ca(2+) influx increases with duration of cold storage and with increasing concentration of Ca(2+) in the suspending medium. 3. Erythrocytes that have been thus loaded with Ca(2+) exhibit Ca(2+) efflux against a concentration gradient when subsequently incubated at 37 degrees C. 4. Ca(2+) influx likewise occurs when the sucrose of the medium is replaced by iso-osmotic solutions of other non-ionized compounds. 5. Replacement of sucrose by iso-osmotic KCl or NaCl greatly diminishes the rate of Ca(2+) influx during cold storage; however, in iso-osmotic choline chloride, Ca(2+) influx is as rapid as in sucrose. 6. Preincubation of erythrocytes in iso-osmotic sucrose at 37 degrees C causes rapid efflux of K(+) and Na(+) and renders the cell membranes highly permeable to Ca(2+) during subsequent cold storage. 7. Preincubation of erythrocytes in iso-osmotic NaCl at 37 degrees C with trypsin or neuraminidase is without effect on the permeability of the membrane towards Ca(2+). 8. The experimental results lead to the conclusion that the main prerequisite for Ca(2+) influx into erythrocytes is the partial depletion of the cells of their univalent cations.

Effect of temperature on the potential and current thresholds of axon membrane.

The effect of temperature on the potential and current thresholds of the squid giant axon membrane was measured with gross external electrodes. A central segment of the axon, 0.8 mm long and in sea water, was isolated by flowing low conductance, isoosmotic sucrose solution on each side; both ends were depolarized in isoosmotic KCl. Measured biphasic square wave currents at five cycles per second were applied between one end of the nerve and the membrane of the central segment. The membrane potential was recorded between the central sea water and the other depolarized end. The recorded potentials are developed only across the membrane impedance. Threshold current values ranged from 3.2 microa at 267deg;C to 1 microa at 7.5 degrees C. Threshold potential values ranged from 50 mv at 26 degrees C to 6 mv at 7.5 degrees C. The mean Q(10) of threshold current was 2.3 (SD = 0.2), while the Q(10) for threshold potentials was 2.0 (SD = 0.1).