Ischemia-reperfusion injury in rats affects hydraulic conductivity in two phases that are temporally and mechanistically separate

Ischemia-reperfusion (IR) injury is a major insult to postcapillary venules. We hypothesized that IR increases postcapillary venular hydraulic conductivity and that IR-mediated changes in hydraulic conductivity result from temporally and mechanistically separate processes. A microcannulation technique was used to determine hydraulic conductivity (Lp) in rat mesenteric postcapillary venules serially throughout ischemia (45 min) and reperfusion (5 h) induced by superior mesenteric artery occlusion and release. Mesenteric IR resulted in a biphasic increase in Lp. White blood cell (WBC) adhesion slowly increased with maximal adhesion corresponding to the second peak (P < 0.005). After IR, tissue was harvested for RT-PCR analysis of ICAM-1, E-selectin, and P-selectin mRNA. Intercellular adhesion molecule-1 (ICAM-1) mRNA in the gut showed the most significant upregulation. Quantitative real-time PCR revealed that ICAM-1 mRNA was upregulated 60-fold in the gut. An ICAM-1 antibody was therefore used to determine the effect of WBC adhesion on Lp during IR. ICAM-1 inhibition attenuated Lp during the first peak and completely blocked the second peak (P < 0.005). When rats were fed a tungsten diet to inhibit xanthine oxidase and then underwent IR, Lp was dramatically attenuated during the first peak and mildly decreased the second peak (P < 0.005). Inhibition of xanthine oxidase by oxypurinol decreased Lp during IR by over 60% (P < 0.002). Tempol, a superoxide dismutase mimetic, decreased Lp during IR by over 30% (P < 0.01). We conclude that IR induces a biphasic increase in postcapillary hydraulic conductivity. Reactive oxygen species impact both the first transient peak and the sustained second peak. However, the second peak is also dependent on WBC-endothelial cell adhesion. These serial measurements of postcapillary hydraulic conductivity may lead the way for optimal timing of pharmaceutical therapies in IR injury.     

氮磷亏缺对玉米根系水流导度的影响

在人工气候室水培条件下,从单根和整株根系两个层次研究了N、P营养与玉米(Zea mays L.)根系水流导度(root hydraulic conductivity,Lpr)间的关系。结果表明：表型抗旱的杂交种F1代户单4号和母本天四的单根水导和整株根系水导均高于不抗旱的父本478,其中天四的单根水导最高,而户单4号的整株根系水导最高。N、P亏缺均使玉米单根水导和整株根系水导降低,但与N亏块相比,P亏缺的植株具有较高的整株根系水导和较低的单根水导。整株根系的水导更能反映植物根系的输水性能。     

Comparison between gradient-dependent hydraulic conductivities of roots using the root pressure probe: the role of pressure propagations and implications for the relative roles of parallel radial pathways

Hydrostatic pressure relaxations with the root pressure probe are commonly used for measuring the hydraulic conductivity (Lp(r)) of roots. We compared the Lp(r) of roots from species with different root hydraulic properties (Lupinus angustifolius L. 'Merrit', Lupinus luteus L. 'Wodjil', Triticum aestivum L. 'Kulin' and Zea mays L. 'Pacific DK 477') using pressure relaxations, a pressure clamp and osmotic gradients to induce water flow across the root. Only the pressure clamp measures water flow under steady-state conditions. Lp(r) determined by pressure relaxations was two- to threefold greater than Lp(r) from pressure clamps and was independent of the direction of water flow. Lp(r) (pressure clamp) was two- to fourfold higher than for Lp(r) (osmotic) for all species except Triticum aestivum where Lp(r) (pressure clamp) and Lp(r) (osmotic) were not significantly different. A novel technique was developed to measure the propagation of pressure through roots to investigate the cause of the differences in Lp(r). Root segments were connected between two pressure probes so that when root pressure (P(r)) was manipulated by one probe, the other probe recorded changes in P(r). Pressure relaxations did not induce the expected kinetics in pressure in the probe at the other end of the root when axial hydraulic conductance, and probe and root capacitances were accounted for. An electric circuit model of the root was constructed that included an additional capacitance in the root loaded by a series of resistances. This accounted for the double exponential kinetics for intact roots in pressure relaxation experiments as well as the reduced response observed with the double probe experiments. Although there were potential errors with all the techniques, we considered that the measurement of Lp(r) using the pressure clamp was the most unambiguous for small pressure changes, and provided that sufficient time was allowed for pressure propagation through the root. The differences in Lp(r) from different methods of measurement have implications for the models describing water transport through roots and the potential role of aquaporins.     

Gating of aquaporins by low temperature in roots of chilling-sensitive cucumber and chilling-tolerant figleaf gourd

Effects of low temperature (8 degrees C) on the hydraulic conductivity of young roots of a chilling-sensitive (cucumber, Cucumis sativus L.) and a chilling-resistant (figleaf gourd, Cucurbita ficifolia Bouche) crop have been measured at the levels of whole root systems (root hydraulic conductivity, Lp(r)) and of individual cortical cells (cell hydraulic conductivity, Lp). Exposure of roots to low temperature (LRT) for up to 6 d caused a stronger suberization of the endodermis in cucumber compared with figleaf gourd, but no development of exodermal Casparian bands in either species. Changes in anatomy after 6 d of LRT treatment corresponded with a reduction in hydrostatic root Lp(r) of cucumber roots by a factor of 24, and by a factor of 2 in figleaf gourd. In figleaf gourd, there was a reduction only in hydrostatic Lp(r) but not in osmotic Lp(r) suggesting that the activity of water channels was not much affected by LRT treatment in this species. Changes in cell Lp in response to chilling and recovery were similar to the root levels, although they were more intense at the root level. Activation energies (E(a)) and Q10 of water flow as measured at the cell level were high in cucumber (E(a)=109+/-13 kJ mol(-1); Q(10)=4.8+/-0.7; n=6-10 cells), but small in figleaf gourd (E(a)=11+/-2 kJ mol(-1); Q10=1.2+/-0.1; n=6-10 cells). Roots of figleaf gourd recovered better from LRT treatment than those of cucumber. In figleaf gourd, recovery (at both the root and cell level) often resulted in Lp and Lp(r) values which were even bigger than the original, i.e. there was an overshoot in hydraulic conductivity. These effects were larger for osmotic (representing the cell-to-cell passage of water) than for hydrostatic Lp(r). After a short-term (1 d) exposure to 8 degrees C followed by 1 d at 20 degrees C, hydrostatic Lp(r) of cucumber nearly recovered and that of figleaf gourd still remained higher due to the overshoot. By contrast, osmotic Lp(r) and cell Lp in both species remained high by a factor of 3 compared with the control, possibly due to an increased activity of water channels. After preconditioning of roots at LRT, increased hydraulic conductivity was completely inhibited by HgCl2 at both the root and cell levels. Different from figleaf gourd, recovery from chilling was not complete in cucumber after longer exposure to LRT. It is concluded that at LRT, both changes in the activity of aquaporins (AQPs) and alterations of root anatomy determine the water uptake in both species. The high temperature dependence of cell Lp in cucumber suggests conformational changes of AQPs during LRT treatment which result in channel closure and in a strong gating of AQP activity by low temperature. This mechanism is thought to be different from that in figleaf gourd where AQPs reacted in the conventional way, i.e. low temperature affected the mobility of water molecules in AQPs rather than their open/closed state, and Q(10) was low.     

An experimental study of some indigenous drugs with special reference to hydraulic permeability

The effect of commonly used indigenous drugs for hepatic disorders i.e. Tinospora cordifolia, (Guduchi/Amrita), Andrographis paniculata (Kalmegha), Picrorhiza kurroa (Kutki), Phyllantnus niruri (Bhoomyamalaki) and Berberis aristata (Daruharidra) was tested on the hydraulic permeability of water in the presence of bile salt through a transport cell model. The data on hydraulic permeability were calculated as t (time). JV = Lp x AP, where Lp = hydraulic conductivity and AP is the pressure difference. It was observed that the value of controlled hydraulic permeability (0.49 x 10(-8) M3 S(-1) N(-1)) decreased in the presence of indigenous drugs and bile salt. The results suggest that these drugs might have the cell membrane stabilizing property which may lead to prevention of the toxic effect of bile salts in various hepatic disorders.     

Regulation of hydraulic conductivity in response to sustained changes in pressure

The present study addresses the effect of a sustained change in pressure on microvascular permeability assessed by hydraulic conductivity (Lp) measurements from microvessels of the rat mesentery. With a microperfusion technique, transvascular filtration (normalized to surface area; Jv/S) and Lp were measured in small arterioles (baseline Lp= 0.26 x 10(-7) cm.s(-1).cmH2O(-1)) and venules (baseline Lp= 2.88 x 10(-7) cm.s(-1).cmH2O(-1)). The main finding of this study is that step increases in microvascular pressure led to time-dependent alterations of L(p). Immediately after a twofold step increase in pressure, Jv/S increased in proportion to the pressure change. This observation is consistent with Starling's law that predicts filtration proportional to the overall pressure gradient when Lp is constant. However, when Jv/S measurements continued for 60-90 min past the step in pressure, there was an initial decrease in Jv/S for 30 min ("sealing effect") followed by a substantial increase in Jv/S out to 90 min. The sustained increase in Jv/S suggests an increase in Lp of 36 +/- 7% for small arterioles and 42 +/- 5% for small venules (P < 0.05 for both). In addition, the increase in Lp in response to an increase in pressure was attenuated significantly by nitric oxide synthase inhibition. These results indicate that a pressure-induced mechanical stimulus (possibly Jv) activates a NO-dependent biochemical response that leads to an increase in hydraulic conductivity.     

Control of water uptake by rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.): role of the outer part of the root

A new pressure-perfusion technique was used to measure hydraulic and osmotic properties of the outer part of roots (OPR) of 30-day-old rice plants (lowland cultivar: IR64, and upland cultivar: Azucena). The OPR comprised rhizodermis, exodermis, sclerenchyma and one cortical cell layer. The technique involved perfusion of aerenchyma of segments from two different root zones (20-50 mm and 50-100 mm from the tip) at precise rates using aerated nutrient solution. The hydraulic conductivity of the OPR (Lp(OPR)=1.2x10(-6) m s(-1) MPa(-1)) was larger by a factor of 30 than the overall hydraulic conductivity (Lp(r)=4x10(-8) m s(-1) MPa(-1)) as measured by pressure chamber and root pressure probe. Low reflection coefficients were obtained for mannitol and NaCl for the OPR (sigma(sOPR)=0.14 and 0.09, respectively). The diffusional water permeability ( P(dOPR)) estimated from isobaric flow of heavy water was smaller by three orders of magnitude than the hydraulic conductivity (Lp(OPR)/ P(fOPR)). Although detailed root anatomy showed well-defined Casparian bands and suberin lamellae in the exodermis, the findings strongly indicate a predominantly apoplastic water flow in the OPR. The Lp(OPR) of heat-killed root segments increased by a factor of only 2, which is in line with the conclusion of a dominating apoplastic water flow. The hydraulic resistance of the OPR was not limiting the passage of water across the root cylinder. Estimations of the hydraulic properties of aerenchyma suggested that the endodermis was rate-limiting the water flow, although the aerenchyma may contribute to the overall resistance. The resistance of the aerenchyma was relatively low, because mono-layered cortical septa crossing the aerenchyma ('spokes') short-circuited the air space between the stele and the OPR. Spokes form hydraulic bridges that act like wicks. Low diffusional water permeabilities of the OPR suggest that radial oxygen losses from aerenchyma to medium are also low. It is concluded that in rice roots, water uptake and oxygen retention are optimized in such a way that hydraulic water flow can be kept high in the presence of a low efflux of oxygen which is diffusional in nature.     

A Negative Hydraulic Message from Oxygen-Deficient Roots of Tomato Plants? (Influence of Soil Flooding on Leaf Water Potential,Leaf Expansion,and Synchrony between Stomatal Conductance and Root Hydraulic Conductivity)

Four to 10 h of soil flooding delayed and suppressed the normal daily increase in root hydraulic conductance (Lp) in tomato (Lycopersicon esculentum Mill. cv Ailsa Craig) plants. The resulting short-term loss of synchrony between Lp and stomatal conductance decreased leaf water potential ([psi]L) relative to well-drained plants within 2 h. A decrease in [psi]L persisted for 8 h and was mirrored by decreased leaf thickness measured using linear displacement transducers. After 10 h of flooding, further closing of stomata and re-convergence of Lp in flooded and well-drained roots returned [psi]L to control values. In the second photoperiod, Lp in flooded plants exceeded that in well-drained plants in association with much increased Lp and decreased stomatal conductance. Pneumatic balancing pressure applied to roots of intact flooded plants to prevent temporary loss of [psi]L in the 1st d did not modify the patterns of stomatal closure or leaf expansion. Thus, the magnitude of the early negative hydraulic message was neither sufficient nor necessary to promote stomatal closure and inhibit leaf growth in flooded tomato plants. Chemical messages are presumed to be responsible for these early responses to soil flooding.     

Hydraulic conductivity of rice roots

A pressure chamber and a root pressure probe technique have been used to measure hydraulic conductivities of rice roots (root Lp(r) per m(2) of root surface area). Young plants of two rice (Oryza sativa L.) varieties (an upland variety, cv. Azucena and a lowland variety, cv. IR64) were grown for 31-40 d in 12 h days with 500 micromol m(-2) s(-1) PAR and day/night temperatures of 27 degrees C and 22 degrees C. Root Lp(r) was measured under conditions of steady-state and transient water flow. Different growth conditions (hydroponic and aeroponic culture) did not cause visible differences in root anatomy in either variety. Values of root Lp(r) obtained from hydraulic (hydrostatic) and osmotic water flow were of the order of 10(-8) m s(-1) MPa(-1) and were similar when using the different techniques. In comparison with other herbaceous species, rice roots tended to have a higher hydraulic resistance of the roots per unit root surface area. The data suggest that the low overall hydraulic conductivity of rice roots is caused by the existence of apoplastic barriers in the outer root parts (exodermis and sclerenchymatous (fibre) tissue) and by a strongly developed endodermis rather than by the existence of aerenchyma. According to the composite transport model of the root, the ability to adapt to higher transpirational demands from the shoot should be limited for rice because there were minimal changes in root Lp(r) depending on whether hydrostatic or osmotic forces were acting. It is concluded that this may be one of the reasons why rice suffers from water shortage in the shoot even in flooded fields.     

Role of albumin arginyl sites in albumin-induced reduction of endothelial hydraulic conductivity

We determined the effect of albumin on endothelial hydraulic conductivity (Lp) and the contributions of the positively charged arginyl and lysinyl residues of albumin in mediating the effect. Studies were made using monolayers of cultured sheep pulmonary artery endothelial cells grown to confluence on polycarbonate filters. Water flux was measured as transendothelial hydrostatic pressure was varied from 5 to 20 cm H2O. Lp was calculated from the slope of the relationship of water flux versus pressure. The Lp of endothelial monolayers perfused with albumin-free Hanks Balanced Salt Solution (HBSS) was compared to perfusion with HBSS containing either native albumin, or albumin in which the arginyl residues were modified by a condensation reaction with 1,2-cyclohexanedione (CHD-albumin), or albumin in which the lysinyl residues were modified by a substitution reaction with succinic anhydride (SC-albumin). Baseline Lp at 2.5 mg/ml native albumin was 1.6 +/- 0.1 X 10(-6) cm/s/cm H2O compared to the filter Lp after removing cells of 3.0 +/- 0.3 X 10(-4) cm/s/cm H2O. Endothelial Lp increased by 60% when albumin concentration was decreased from 2.5 mg/ml to 0.5 mg/ml (P less than 0.05), but did not change with an increase in concentration to 10 mg/ml. Albumin-free buffer and CHD-albumin increased endothelial Lp by 2.2 +/- 0.3-fold and 1.9 +/- 0.3-fold, respectively (P less than 0.05). All endothelial Lp values were restored to baseline when the native albumin concentration was returned to 2.5 mg/ml. Excess l-arginine (2 X 10(-3) M) inhibited the effect of native albumin and increased endothelial Lp 1.5 +/- 0.02-fold (P less than 0.05), but excess l-lysine (4 X 10(-3) in the presence of native albumin had no effect on Lp. None of the perfusates altered the filter Lp value. Neutral dextran (70 kD), in contrast to native albumin, had no effect on endothelial Lp. These results indicate that albumin reduces the hydraulic conductivity of endothelial monolayers in a concentration-dependent fashion and that the arginyl residues of albumin are required for the response. The effect of albumin may be mediated by a charge interaction of albumin with the endothelium.     

Light-induced transpiration alters cell water relations in figleaf gourd (Cucurbita ficifolia) seedlings exposed to low root temperatures

Water relation parameters including elastic modulus (epsilon), half-times of water exchange (T(w)(1/2)), hydraulic conductivity and turgor pressure (P) were measured in individual root cortical and cotyledon midrib cells in intact figleaf gourd (Cucurbita ficifolia) seedlings, using a cell pressure probe. Transpiration rates (E) of cotyledons were also measured using a steady-state porometer. The seedlings were exposed to low ambient (approximately 10 micromol m(-2) s(-1)) or high supplemental irradiance (approximately 300 micromol m(-2) s(-1) PPF density) at low (8 degrees C) or warm (22 degrees C) root temperatures. When exposed to low irradiance, all the water relation parameters of cortical cells remained similar at both root temperatures. The exposure of cotyledons to supplemental light at warm root temperatures, however, resulted in a two- to three-fold increase in T(w)(1/2) values accompanied with the reduced hydraulic conductivity in both root cortical (Lp) and cotyledon midrib cells (Lp(c)). Low root temperature (LRT) further reduced Lp(c) and E, whether it was measured under low or high irradiance levels. The reductions of Lp as the result of respective light and LRT treatments were prevented by the application of 1 microM ABA. Midrib cells required higher concentrations of ABA (2 microM) in order to prevent the reduction in Lp(c). When the exposure of cotyledons to light was accompanied by LRT, however, ABA proved ineffective in reversing the inhibition of Lp. LRT combined with high irradiance triggered a drastic 10-fold reduction in water permeability of cortical and midrib cells and increased epsilon and T(w)(1/2) values. Measurement of E indicated that the increased water demand by the transpiring plants was fulfilled by an increase in the apoplastic pathway as principal water flow route. The importance of water transport regulation by transpiration affecting the hydraulic conductivity of the roots is discussed.     

Apoplastic barriers,aquaporin gene expression and root and cell hydraulic conductivity in phosphate-limited sheepgrass plants

Mineral nutrient supply can affect the hydraulic property of roots. The aim of the present work on sheepgrass (Leymus chinensis L.) plants was to test whether any changes in root hydraulic conductivity (Lp; exudation analyses) in response to a growth-limiting supply of phosphate (P) are accompanied by changes in (1) cell Lp via measuring the cell pressure, (2) the aquaporin (AQP) gene expression by performing qPCR and (3) the formation of apoplastic barriers, by analyzing suberin lamella and Casparian bands via cross-sectional analyses in roots. Plants were grown hydroponically on complete nutrient solution, containing 250 µM P, until they were 31–36 days old, and then kept for 2–3 weeks on either complete solution, or transferred on solution containing 2.5 µM (low-P) or no added P (no-P). Phosphate treatments caused significant decreases in root and cell-Lp and AQP gene expression, while the formation of apoplastic barriers increased, particularly in lateral roots. Experiments using the AQP inhibitor mercury (Hg) suggested that a significant portion of radial root water uptake in sheepgrass occurs along a path involving AQPs, and that the Lp of this path is reduced under low- and no-P. It is concluded that a growth-limiting supply of phosphate causes parallel changes in (1) cell Lp and aquaporin gene expression (decrease) and (2) apoplastic barrier formation (increase), and that the two may combine to reduce root Lp. The reduction in root Lp, in turn, facilitates an increased root-to-shoot surface area ratio, which allocates resources to the root, sourcing the limiting nutrient.     

Water uptake by roots: effects of water deficit

The variable hydraulic conductivity of roots (Lp(r)) is explained in terms of a composite transport model. It is shown how the complex, composite anatomical structure of roots results in a composite transport of both water and solutes. In the model, the parallel apoplastic and cell-to-cell (symplastic and transcellular) pathways play an important role as well as the different tissues and structures arranged in series within the root cylinder (epidermis, exodermis, cortex, endodermis, stelar parenchyma). The roles of Casparian bands and suberin lamellae in the root's endo- and exodermis are discussed. Depending on the developmental state of these apoplastic barriers, the overall hydraulic resistance of roots is either more evenly distributed across the root cylinder (young unstressed roots) or is concentrated in certain layers (exo- and endodermis in older stressed roots). The reason for the variability of root Lp(r), is that hydraulic forces cause a dominating apoplastic flow of water around protoplasts, even in the endodermis and exodermis. In the absence of transpiration, water flow is osmotic in nature which causes a high resistance as water passes across many membranes on its passage across the root cylinder. The model allows for a high capability of roots to take up water in the presence of high rates of transpiration (high demands for water from the shoot). By contrast, the hydraulic conductance is low, when transpiration is switched off. Overall, this results in a non-linear relationship between water flow and forces (gradients of hydrostatic and osmotic pressure) which is otherwise hard to explain. The model allows for special root characteristics such as a high hydraulic conductivity (water permeability) in the presence of a low permeability of nutrient ions once taken up into the stele by active processes. Low root reflection coefficients are in line with the idea of some apoplastic bypasses for water within the root cylinder. According to the composite transport model, the switch from the hydraulic to the osmotic mode is purely physical. In the presence of heavily suberized roots, the apoplastic component of water flow may be too small. Under these conditions, a regulation of radial water flow by water channels dominates. Since water channels are under metabolic control, this component represents an 'active' element of regulation. Composite transport allows for an optimization of the water balance of the shoot in addition to the well-known phenomena involved in the regulation of water flow (gas exchange) across stomata. The model is employed to explain the responses of plants to water deficit and other stresses. During water deficit, the cohesion-tension mechanism of the ascent of sap in the xylem plays an important role. Results are summarized which prove the validity of the coehesion/tension theory. Effects of the stress hormone abscisic acid (ABA) are presented. They show that there is an apoplastic component of the flow of ABA in the root which contributes to the ABA signal in the xylem. On the other hand, (+)-cis-trans-ABA specifically affects both the cell level (water channel activity) and water flow driven by gradients in osmotic pressure at the root level which is in agreement with the composite transport model. Hydraulic water flow in the presence of gradients in hydrostatic pressure remains unchanged. The results agree with the composite transport model and resemble earlier findings of high salinity obtained for the cell (Lp) and root (Lp(r)) level. They are in line with known effects of nutrient deprivation on root Lp(r )and the diurnal rhythm of root Lp(r )recently found in roots of LOTUS.     

Chemical composition of apoplastic transport barriers in relation to radial hydraulic conductivity of corn roots (Zea mays L.)

The hydraulic conductivity of roots (Lp_r) of 6- to 8-d-old maize seedlings has been related to the chemical composition of apoplastic transport barriers in the endodermis and hypodermis (exodermis), and to the hydraulic conductivity of root cortical cells. Roots were cultivated in two different ways. When grown in aeroponic culture, they developed an exodermis (Casparian band in the hypodermal layer), which was missing in roots from hydroponics. The development of Casparian bands and suberin lamellae was observed by staining with berberin-aniline-blue and Sudan-III. The compositions of suberin and lignin were analyzed quantitatively and qualitatively after depolymerization (BF₃/methanol-transesterification, thioacidolysis) using gas chromatography/mass spectrometry. Root Lp_r was measured using the root pressure probe, and the hydraulic conductivity of cortical cells (Lp) using the cell pressure probe. Roots from the two cultivation methods differed significantly in (i) the Lp_r evaluated from hydrostatic relaxations (factor of 1.5), and (ii) the amounts of lignin and aliphatic suberin in the hypodermal layer of the apical root zone. Aliphatic suberin is thought to be the major reason for the hydrophobic properties of apoplastic barriers and for their relatively low permeability to water. No differences were found in the amounts of suberin in the hypodermal layers of basal root zones and in the endodermal layer. In order to verify that changes in root Lp_r were not caused by changes in hydraulic conductivity at the membrane level, cell Lp was measured as well. No differences were found in the Lp values of cells from roots cultivated by the two different methods. It was concluded that changes in the hydraulic conductivity of the apoplastic rather than of the cell-to-cell path were causing the observed changes in root Lp_r. Received: 17 March 1999 / Accepted: 22 June 1999     

Light and turgor affect the water permeability (aquaporins) of parenchyma cells in the midrib of leaves of Zea mays

In response to light, water relation parameters (turgor, half-time of water exchange, T(1/2), and hydraulic conductivity, Lp; T(1/2) proportional 1/Lp) of individual cells of parenchyma sitting in the midrib of leaves of intact corn (Zea mays L.) plants were investigated using a cell pressure probe. Parenchyma cells were used as model cells for the leaf mesophyll, because they are close to photosynthetically active cells at the abaxial surface, and there are stomata at both the adaxial and abaxial sides. Turgor ranged from 0.2 to 1.0 MPa under laboratory light condition (40 micromol m(-2) s(-1) at the tissue level), and individual cells could be measured for up to 6 h avoiding the variability between cells. In accordance with earlier findings, there was a big variability in T(1/2)s measured ranging from 0.5 s to 100 s, but the action of light on T(1/2)s could nevertheless be worked out for cells having T(1/2)s greater than 2 s. Increasing light intensity ranging from 100 micromol m(-2) s(-1) to 650 micromol m(-2) s(-1) decreased T(1/2) by a factor up to five within 10 min and increased Lp (and aquaporin activity) by the same factor. In the presence of light, turgor decreased due to an increase in transpiration, and this tended to compensate or even overcompensated for the effect of light on T(1/2). For example, during prolonged illumination, cell turgor dropped from 0.2 to 1.0 MPa to -0.03 to 0.4 MPa, and this drop caused an increase of T(1/2) and a reduction of cell Lp, i.e. there was an effect of turgor on cell Lp besides that of light. To separate the two effects, cell turgor (water potential) was kept constant while changing light intensity by applying gas pressure to the roots using a pressure chamber. At a light intensity of 160 micromol m(-2) s(-1), there was a reduction of T(1/2) by a factor of 2.5 after 10-30 min, when turgor was constant within +/-0.05 MPa. Overall, the effects of light on T(1/2) (Lp) were overriding those of turgor only when decreases in turgor were less than about 0.2 MPa. Otherwise, turgor became the dominant factor. The results indicate that the hydraulic conductivity increased with increasing light intensity tending to improve the water status of the shoot. However, when transpiration induced by light tends to cause a low turgidity of the tissue, cell Lp was reduced. It is concluded that, when measuring the overall hydraulic conductivity of leaves, both the effects of light and turgor should be considered. Although the mechanism(s) of how light and turgor influence the cell Lp is still missing, it most likely involves the gating of aquaporins by both parameters.     

Osmotic water permeability of human red cells

The osmotic water permeability of human red cells has been reexamined with a stopped-flow device and a new perturbation technique. Small osmotic gradients are used to minimize the systematic error caused by nonlinearities in the relationship between cell volume and light scattering. Corrections are then made for residual systematic error. Our results show that the hydraulic conductivity, Lp, is essentially independent of the direction of water flow and of osmolality in the range 184-365 mosM. the mean value of Lp obtained obtained was 1.8 +/- 0.1 (SEM) X 10-11 cm3 dyne -1 s-1.     

Determination of bull sperm membrane permeability to water and cryoprotectants using a concentration-dependent self-quenching fluorophore

The objective of this study was to determine the membrane permeability characteristics of bovine spermatozoa. These included the hydraulic conductivity (Lp), the permeability coefficients (Ps) of four common cryoprotective agents (CPAs) and the associated reflection coefficients (sigma). Stopped-flow fluorometry was applied in order to capture rapid cell volume changes under anisosmotic conditions in the absence or presence of permeant solutes (CPAs). This technique utilizes a concentration-dependent self-quenching entrapped fluorophore. The resulting cell volume changes were used in three-parameter fitting calculations to compute Lp in the absence of permeant solutes and Ps and Lp in the presence of permeating solutes (CPAs) at 22 degrees C. The hydraulic conductivity in the absence of permeating solutes was estimated to be 0.68+/-0.05 microm/min/atm (mean+/-SEM). Hydraulic conductivity (Lp) in the presence of CPAs was 0.91+/-0.27 (mean+/-SEM), 0.29+/-0.04, 0.42+/-0.05, and 0.39+/-0.03 microm/min/atm in the presence of dimethylsulfoxide (Me(2)SO), glycerol, propylene glycol (PG), and ethylene glycol (EG), respectively. The values for Ps were estimated to be 1.72+/-0.36, 1.75+/-0.03, 2.47+/-0.24, and 1.49+/-0.33 x 10(-3)cm/min for Me(2)SO, glycerol, PG, and EG, respectively. The data were used to simulate volume excursions during addition and removal of CPA, to predict the different effects of the four CPAs.     

水曲柳幼苗根系在不同浓度NH4NO3溶液中水流导度的变化

水分吸收过程是根系重要的生理过程。水孔蛋白在根系水分径向运输中起着重要的作用,根系水流导度(L_p)的测定是研究水孔蛋白的重要途径。该研究采用压力流的方法,对相同生长条件下的水曲柳(Fraxinus mandshurica)幼苗根系进行研究,测定了根系在去离子水和不同浓度NH₄NO₃溶液中的L_p。结果表明:未经处理的水曲柳幼苗根系,L_p随NH₄NO₃浓度的增加而上升,而且NH₄NO₃溶液中的L_p比去离子水中的L_p平均高77%;经HgCl₂处理后,水曲柳幼苗根系的L_p仍然随NH₄NO₃浓度的增加而增大,但是根系L_p在去离子水下降了22%,而在NH₄NO₃溶液中下降了68%,与以前的研究相比发现,经HgCl₂处理后,以营养液为吸水基质的根系L_p的降低值普遍高于以去离子水为基质的试验。因此,基质中养分离子的存在对根系中水孔蛋白活性产生了重要的影响,进而影响根系水分的吸收过程。     

Permeability of cultured megakaryocytopoietic cells of the rat to dimethyl sulfoxide

The permeability of the membrane of the rat megakaryocytopoietic cell to dimethyl sulfoxide was measured to assess its availability to the intracellular compartment. The method used was osmotic, and measured the initial loss of cell water followed by a reswelling to isotonic volume when cells were placed in culture media containing 0.6 M DMSO. Values for the hydraulic coefficient, Lp, the permeability of the membrane to DMSO, wRT , and the reflection coefficient were calculated from the equations of Kedem and Katchalsky . The average value at 25 degrees C for Lp was 0.46 micron min-1 atm1 ; wRT was 9.3 micron min-1, and the reflection coefficient was 0.65. At these cell volumes, 50% equilibration occurred in 5 sec. Cells equilibrated in 0.6 M DMSO increased their volume of osmotically inactive water. Coupled with this phenomenon of stabilization of water was a reduction in the hydraulic coefficient by 50%. These findings are discussed in the context of current hypotheses about cellular viability during freezing and thawing in the presence and absence of cryoprotectants.     

Osmotic response of oocytes using a microscope diffusion chamber: a preliminary study comparing murine and human ova

The hydraulic conductivity (Lp) of the plasma membrane determines how cells respond to the stresses of dehydration encountered during cryopreservation. We have used a microscope diffusion chamber which allows for direct real-time observation of the dynamic osmotic response of individual cells in microvolume suspension to compare the Lp of murine and human unfertilized ova. In this system, the response of an individual cell to the induced osmotic imbalance is documented via a series of photomicrographs or videotape; from these data the Lp can be computed. Donated human preovulatory oocytes were compared with macroscopically normal human ova, 43 hr after insemination, which had failed to fertilize (Ff) and with murine ova collected 13 hr post human chorionic gonadotropin injection. The permeability coefficients were 0.65 +/- 0.43, 0.84 +/- 0.39, and 0.36 +/- 0.07 micron3/micron2/atm/min. The results suggest that it may be possible to use Ff ova for experiments to design suitable cryopreservation procedures.