Free radical and freezing injury to cell membranes of winter wheat

The symptoms of injury in microsomal membranes isolated from crowns of seedlings of Triticum aestivum , L. cultivar Fredrick after a lethal freeze-thaw stress included an increased lipid phase transition temperature, loss of lipid phosphate (lipid-P), and increased free fatty acid levels. However, minimal changes in fatty acid saturation were observed, suggesting minimal amounts of lipid peroxidation. All of these injury symptoms, including the lack of lipid peroxidation, were simulated in vitro by treatment of isolated membranes with oxygen free radicals, generated from either xanthine oxidase (EC 1.1.3.22) or paraquat (l,r-dimethyl-4,4'-bipyridinium dichloride). Further evidence indicating a relationship between free radicals and freezing injury comes from the observation that both protoplasts and microsomal membranes isolated from wheat seedlings, that had been acclimated to induce freezing tolerance, also had increased tolerance of oxygen free radicals, and contained higher lipid-soluble antioxidant levels, than those from non-acclimated seedlings. Lipid-soluble antioxidants accumulated in the crown tissue of the wheat seedling during the acclimation period. Freezing stress accelerated the formation of oxygen free radicals. Membranes isolated from crowns after a freeze–thaw stress tended to produce higher levels of superoxide as shown by the reduction of Tiron (1,2-dihydroxy-l,3-benzenedisulfonic acid). In protoplasts, increased superoxide production coincided with lethal freezing injury. These results are discussed in terms of the possible involvement of oxygen free radicals in mediating aspects of freezing injury to cell membranes.     

Physiological and metabolic responses of winter wheat to prolonged freezing stress

Survival and cold hardiness declined gradually when cold-hardened Fredrick winter wheat (Triticum aestivum L.) was maintained at −6°C for several weeks. Moisture content of crown and root tissue did not change significantly during this period. Uptake of O₂ and accumulation of ⁸⁶Rb by root tissue declined abruptly upon exposure to −6°C, whereas a concomitant negative effect of freezing on these metabolic processes was not observed in crown tissue. Electron spin resonance spectroscopic analysis of microsomal membrane preparations from crown tissue revealed no evidence of gross changes in the physical properties of the bulk lipids even when seedlings were killed. The results provide biochemical evidence that seedling damage due to prolonged exposure to a mild freezing stress is due to disruption of key metabolic process in the root while cells within the crown remain viable.     

Ice-Encasement Injury to Microsomal Membranes Isolated from Winter Wheat Crowns : II. Changes in Membrane Lipids during Ice Encasement

The physical properties and chemical composition of microsomal membranes were examined during a 7 day period of ice encasement in crown tissue of winter wheat (Triticum aestivum L. cv Norstar). Membrane damage, detected as an increase in microviscosity and electrolyte leakage, began between 1 and 3 days of icing, and was associated with a reduction in the recovery of microsomal membranes from stressed tissue, an increase in the microsomal free fatty acid:total fatty acid ratio, and a decrease in the phospholipid:total fatty acid ratio. These trends were amplified between 3 and 7 days of ice encasement. Examination of the free and total fatty acid fractions showed there was a slight, but not statistically significant (P = 0.05) reduction in the degree of unsaturation of the total fatty acid fraction. The composition of the free and total fatty acid fractions were very similar during ice encasement. Furthermore, analysis of phospholipid classes revealed no significant change in the relative amounts of phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, or lysophospholipids in microsomal membranes during icing. Membrane injury during ice encasement apparently involves hydrolysis of the ester bond between glycerol and the acyl groups of the phospholipid resulting in loss of the phosphate-containing polar head group and a concomitant accumulation of free fatty acids in the bilayer.     

Acceleration of membrane senescence in cut carnation flowers by treatment with ethylene

The lipid microviscosity of microsomal membranes from senescing cut carnation (Dianthus caryophyllus L. cv. White Sim) flowers rises with advancing senescence. The increase in membrane microviscosity is initiated within 3 to 4 days of cutting the flowers and coincides temporally with petal-inrolling denoting the climacteric-like rise in ethylene production. Treatment of young cut flowers with aminoethoxyvinylglycine prevented the appearance of petal-inrolling and delayed the rise in membrane microviscosity until day 9 after cutting. When freshly cut flowers or aminoethoxyvinylglycine-treated flowers were exposed to exogenous ethylene (1 microliter per liter), the microviscosity of microsomal membranes rose sharply within 24 hours, and inrolling of petals was clearly evident. Thus, treatment with ethylene accelerates membrane rigidification. Silver thiosulphate, a potent anti-ethylene agent, delayed the rise in microsomal membrane microviscosity even when the flowers were exposed to exogenous ethylene. Membrane rigidification in both naturally senescing and ethylene-treated flowers was accompanied by an increased sterol:phospholipid ratio reflecting the selective loss of membrane phospholipid that accompanies senescence. The results collectively indicate that the climacteric-like surge in ethylene production during senescence of carnation flowers facilitates physical changes in membrane lipids that presumably lead to loss of membrane function.     

Fluidity of natural membranes and phosphatidylserine and ganglioside dispersions: Effects of local anesthetics,cholesterol and protein

The microviscosity of artificial lipid membranes and natural membranes was measured by the fluorescence polarization technique employing perylene as the probe. Lipid dispersions composed of brain gangliosides exhibited greater microviscosity than phosphatidylserine (268 cP vs 173 cP, at 25 °C). Incorporation of cholesterol (30–50%) increased the microviscosity of lipid phases by 200–500 cP. Cholesterol's effect on membrane fluidity was completely reversed by digitonin but not by amphotericin B. Incorporation of membrane proteins into lipid vesicles gave varying results. Cytochrome b₅ did not alter membrane fluidity. However, myelin proteolipid produced an apparent increase in microviscosity, but this effect might be due to partitioning of perylene between lipid and protein binding sites since the latter have a higher fluorescence anisotropy than the lipid. The local anesthetics tetracaine and butacaine increased the fluidity of lipid dispersions, natural membranes and intact ascites tumor cell membranes. The effect of the anesthetics appears to be due to an increased disordering of lipid structure. The fluidity of natural membranes at the 25 °C varied as follows:polymorphonuclear leukocytes, 335 cP; bovine brain myelin, 270 cP; human erytherocyte, 180 cP; rat liver microsomes, 95 cP; rat liver mitochondria, 90 cP. In most cases the microviscosity of natural membranes reflects their cholesterol : phospholipid ratio. The natural variations in fluidity of cellular membranes probably reflect important fuctional requirements. Similarly, the effects of some drugs which alter membrane permeability may be the result of their effects on membrane fluidity.     

Ice Encasement Injury to Microsomal Membranes from Winter Wheat Crowns : I. Comparison of Membrane Properties after Lethal Ice Encasement and during a Post-Thaw Period

The functional and physical properties of cellular membranes isolated from Triticum aestivum, cvs Norstar and Fredrick, were altered coincident with changes in composition after a lethal ice-encasement stress and further during a 6 hour post-thaw period. Crowns encased in ice for a duration which inhibited regrowth, exhibited enhanced rates of electrolyte leakage. Furthermore, the recovery of total microsomal protein and phospholipid declined, suggesting that some membrane degradation had been induced during the anoxic stress. The microviscosity of microsomes and liposomes prepared from such membranes increased during stress, and this was correlated with a 2- to 4-fold increase in the free fatty acid levels in the microsomal fraction. There was, however, only a relatively minor change in fatty acid unsaturation during the ice-encasement stress. The process continued during a 6 hour aerobic post-thaw treatment, but the pattern was somewhat different. During this phase, the leakage of electrolytes was further increased and the recovery of microsomal protein and phospholipid continued to decline, indicating general degradation; but, in contrast to the anoxic phase, the degree of fatty acid unsaturation declined markedly, indicating lipid peroxidation.     

Changes in Microsomal Enzymes and Phospholipid during Dehardening in Stem Bark of Black Locust

Upon dehardening of stem bark of black locust (Robinia pseudoacacia), a significant decrease in phospholipid content on a milligram protein basis was observed in various crude particulate cell fractions. To ascertain this with a defined membrane, microsomal preparations were separated into several membrane fractions on a discontinuous sucrose gradient. Based on the distribution of various enzymes on the gradient, Golgi apparatus membranes, tonoplast, and unidentified membranes containing acid protease were separated with less contamination by other membranes. The subfraction, with an apparent density of 1.10 g/cc, which was enriched in fragmented tonoplast, contained the most phospholipid per milligram protein. Dehardening resulted in a significant quantitative reduction in protein and phospholipid in the submicrosomal fractions. Significant decreases in phospholipid content per milligram protein were observed during dehardening in tonoplast, Golgi apparatus, and unidentified membranes containing acid protease as well as other membrane fractions. During dehardening, marked decreases in inosine diphosphatase and NADH cytochrome c reductase activities were observed, suggesting a marked degradation of the membranes containing those enzymes. The transition of cell membranes from a phospholipid-enriched state to a phospholipid depleted state is apparently involved in the dehardening process concomitant with a decrease in tissue hardiness.     

The effect of phosphate deficiency on membrane phospholipid composition of bean (Phaseolus vulgaris L.) roots

Plasma membranes were isolated from roots of bean (Phaseolus vulgaris L.) plants cultured on phosphate sufficient or phosphate deficient medium. The phospholipid composition of plasma membranes was analyzed and compared with that of the microsomal fraction. Phosphate deficiency had no influence on lipid/protein ratio in microsomal as well as plasma membrane fraction. In phosphate deficient roots phospholipid content was lower in the plasma membrane, but did not change in the microsomal fraction. Phosphatidylcholine and phosphatidylethanolamine were two major phospholipids in plasmalemma and microsomal membranes (80 % of the total). After two weeks of phosphate starvation a considerable decrease (about 50 %) in phosphatidylcholine and phosphatidylethanolamine in microsomal membranes was observed. The decline in two major phospholipids was accompanied by an increase in phosphatidic acid and lysophosphatidylcholine content. The effect of alterations in plasma membrane phospholipids on membrane function e.g. nitrate uptake is discussed.     

Lipid alterations in liver and kidney induced by normobaric hyperoxia: correlations with changes in microsomal membrane fluidity

The effects of normobaric hyperoxia on both microsomal membrane fluidity and mechanism of phospholipid synthesis in rabbit liver and kidney have been studied. Hyperoxia induces in both organs an impairment of de novo synthesis of glycerolipids which could be due to an inactivation of acyltransferase activities involved in the initial formation of phosphatidic acid. The ability to replace phospholipid fatty acids by reacylation mechanism decreases slightly in the hyperoxic kidney, while it does not change in the hyperoxic liver. Concerning the effect of high arterial pO2 on microsomal membrane fluidity, the hyperoxic liver shows a more fluid environment within the membrane core of microsomes; however, no difference was shown in that of microsomal membrane core of hyperoxic kidney. An insight into the lipid composition of microsomes indicates that liver microsomal membranes have lower cholesterol content and higher unsaturation degree of phospholipid fatty acids, whereas hyperoxic kidney microsomes become more saturated and did not show any difference in their cholesterol content. In both hyperoxic liver and kidney microsomes, phospholipid content decreases in agreement with the depression of phosphatidic acid biosynthesis. These results are discussed in relation to the values of microsomal membrane microviscosity obtained.     

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A² treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and phospholipid environment.     

Proteins from frost-hardy leaves protect thylakoids against mechanical freeze-thaw damage in vitro

We have isolated protein fractions from cold-acclimated, frost-hardy cabbage (Brassica oleracea L.) and spinach (Spinacia oleracea L.) leaves which protect isolated thylakoids from non-hardy spinach against mechanical membrane rupture during an in-vitro freeze-thaw cycle. No protective activity was found in similar preparations from non-hardy leaves. The proteins protected the membranes from damage by reducing their solute permeability during freezing and by increasing their expandability during thawing. The proteins act by increasing the resistance of the membranes against the osmotic stress to which they are exposed during a freeze-thaw cycle. In the absence of cryoprotectants this stress results in membrane rupture.This investigation was supported by the Deutsche Forschungsge-meinschaft.     

Nonsedimentable microvesicles from senescing bean cotyledons contain gel phase-forming phospholipid degradation products

A mixture of liquid-crystalline and gel-phase lipid domains is detectable by wide angle x-ray diffraction in smooth microsomal membranes isolated from senescent 7-day-old cotyledons, whereas corresponding membranes from young 2-day-old cotyledons are exclusively liquid-crystalline. The gel-phase domains in the senescent membranes comprise phospholipid degradation products including diacylglycerols, free fatty acids, long-chain aldehydes, and long-chain hydrocarbons. The same complement of phospholipid degradation products is also present in nonsedimentable microvesicles isolated from senescent 7-day-old cotyledons by filtration of a 250,000g, 12-hour supernatant through a 300,000 dalton cut-off filter. The phospholipid degradation products in the microvesicles form gel-phase lipid domains when reconstituted into phospholipid liposomes. Nonsedimentable microvesicles of a similar size, which are again enriched in the same gel-phase-forming phospholipid degradation products, are also generated in vitro from smooth microsomal membranes isolated from 2-day-old cotyledons when Ca²⁺ is added to activate membrane-associated lipolytic enzymes. The Ca²⁺-treated membranes do not contain detectable gel-phase domains, suggesting that the phospholipid degradation products are completely removed by microvesiculation. The observations collectively indicate that these nonsedimentable microvesicles serve as a vehicle for moving phospholipid degradation products out of membrane bilayers into the cytosol. As noted previously (Yao K, Paliyath G, Humphrey RW, Hallett FR, Thompson JE [1991] Proc Natl Acad Sci USA 88: 2269-2273), the term “deteriosome” connotes this putative function and would serve to distinguish these microvesicles from other cytoplasmic microvesicles unrelated to deterioration.     

Interactions among Flooding, Freezing, and Ice Encasement in Winter Wheat

Exposure of winter wheat (Triticum aestivum L.) to various combinations of flooding and freezing stresses induces much greater damage than the individual stresses. Cold-hardened plants flooded for 1 week or exposed to −6°C for 1 week show 100% survival, while survival of plants exposed to both stresses simultaneously is reduced by 20 to 30%, and cold hardiness decreases by several degrees. The level of nonstructural carbohydrates increases in crown tissue during cold acclimation, but decreases when the plants are exposed to flooding or to −6°C for 1 week. The respiratory capacity of crown tissue segments declines when the plants are stressed. Uptake of ⁸⁶Rb by the roots of intact seedlings declines after exposure to either freezing or flooding, whereas passive efflux of amino acids is observed after freezing but not following flooding. This study has shown that detectable stress-induced metabolic changes occur in winter wheat before the applied stress is severe enough to reduce survival.     

Isolation and properties of cholesterol esterstorage granules from ovarian tissues

Cholesterol ester-storage granules were isolated from luteinized rat ovary and rabbit ovarian interstitial tissue by centrifugal flotation and were investigated with regard to their structure and function. Cholesterol ester, protein, phospholipid and unesterified cholesterol accounted for the dry weight of granules from luteinized rat ovary. The protein and the phospholipid were resistant to removal by washing. Substrate specificities of nucleotide phosphatase and specific radioactivities of lipid-soluble P (determined after administration of [³²P]P_i in vivo) were the same in granules and in a microsomal fraction from the same tissue. After administration of [³²P]P_i in vivo, luteinizing hormone increased the specific radioactivity of lipid-soluble P in granules, mitochondria and the microsomal fraction. Since granules did not swell in hypo-osmotic media, whereas microsomal particles did, it is suggested that adherent phospholipid and protein in granule suspensions is unlikely to result from contamination with endoplasmic reticulum. Luteinizing hormone administered in vivo increased the phospholipid and unesterified cholesterol contents of isolated granules relative to their cholesterol ester content, and also tended to raise their protein content. This treatment decreased the ability of isolated granules to act as a substrate for cholesterol esterase in vitro and increased the activity of cholesterol esterase. Cycloheximide in vivo also raised the unesterified cholesterol/cholesterol ester ratio of isolated granules, and when administered with luteinizing hormone acted synergistically to bring about a further increase. These results are considered compatible with evidence obtained by microscopy which suggests that granules may be surrounded by a membrane, that they arise by pinching off from the endoplasmic reticulum, and that they shrink on trophic stimulation of the tissue.     

Lipid composition and microviscosity of subcellular fractions from rabbit thymocytes. Differences in the microviscosity of plasma membranes from subclasses of thymocytes

There are indications from freeze-fracture experiments that subclasses of rabbit thymocytes show different mobilities of plasma membrane components. Consequently, one would expect differences in the fluidity of the plasma membrane. For this reason, rabbit thymocytes were separated on a Ficoll/Metrizoate gradient yielding three subclasses representing various levels of cell differentiation. These thymocyte subclasses did not show any significant differences in the degree of fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene. The fluorescence polarization of the plasma membrane may be overshadowed by the contribution of all cellular lipids due to penetration of the fluorescent probe into the cell. Therefore, plasma membranes were isolated from rabbit thymocytes using a cell-disrupting pump, differential centrifugation, and sucrose density gradient centrifugation. As shown by biochemical and electron microscopical analyses, plasma membranes with a high degree of purity were obtained. As expected the plasma membrane fractions showed a higher microviscosity than the other subcellular fractions. This was attributed to a higher cholesterol to phospholipid molar ratio and a higher degree of saturation of phospholipid fatty acid chains.Subsequently, the microviscosity was measured of plasma membrane preparations obtained from two main subclasses of thymocytes representing mature and immature lymphocytes. The immature thymocytes yielded two plasma membrane fractions with higher microviscosity than the mature cells.     

Simulation of dehydration injury to membranes from soybean axes by free radicals

Smooth microsomal membranes were isolated from axes of soybean (Glycine max L. Merr.) seeds at the dehydration-tolerant (6 hours of imbibition) and dehydration-susceptible (36 hours of imbibition) stages of development and were exposed to free radicals in vitro using xanthine-xanthine oxidase as a free radical source. Wide angle x-ray diffraction studies indicated that the lipid phase transition temperature of the microsomal membranes from the dehydration-tolerant axes increased from 7 to 14°C after exposure to free radicals, whereas those from the dehydration-susceptible axes increased from 9 to 40°C by the same free radical dose. The increased phase transition temperature was associated with a decrease in the phospholipid:sterol ratio, and an increase in the free fatty acid:phospholipid ratio. There was no significant change in total fatty acid saturation, which indicated that free radical treatment induced deesterification of membrane phospholipid, and not a change in fatty acid saturation. Similar compositional and structural changes have been previously observed in dehydration-injured soybean axes suggesting that dehydration may induce free radical injury to cellular membranes. Further, these membranes differ in their susceptibility to free radical injury, presumably reflecting compositional differences in the membrane since these membranes were exposed to free radicals in the absence of cytosol.     

Fluidity of natural membranes and phosphatidylserine and ganglioside dispersions. Effect of local anesthetics, cholesterol and protein.

The microviscosity of artificial lipid membranes and natural membranes was measured by the fluorescence polarization technique employing perylene as the probe. Lipid dispersions composed of brain gangliosides exhibited greater microviscosity than phosphatidylserine (268 cP vs 173 cP, at 25 degrees C). Incorporation of cholesterol (30-50%) increased the microviscosity of lipid phases by 200-500 cP. Cholesterol's effect on membrane fluidity was completely reversed by digitonin but not by amphotericin B. Incorporation of membrane proteins into lipid vesicles gave varying results. Cytochrome b5 did not alter membrane fluidity. However, myelin proteolipid produced an apparent increase in microviscosity, but this effect might be due to partitioning of perylene between lipid and protein binding sites since tha latter have a higher fluorescence anisotropy than the lipid. The local anesthetics tetracain and butacaine increased the fluidity of lipid dispersions, natural membranes and intact ascites tumor cell membranes. The effect of anesthetics appears to be due to an increased disordering of lipid structure. The fluidity of natural membranes at 25 degrees C varied as follows: polymorphonuclear leukocytes, 335 cP; bovine brain myelin, 270 cP; human erythrocyte, 180 cP; rat liver microsomes, 95 cP; rat liver mitochondria, 90 cP. In most cases the microviscosity of natural membranes reflects their cholesterol: phospholipid ratio. The natural variations in fluidity of cellular membranes probably reflect important functional requirements. Similarly, the effects of some drugs which alter membrane permeability may be the result of their effects on membrane fluidity.     

Vitamin A and microsomal membranes: the effect of retinol deficiency on lipid microviscosity and phospholipid turnover in rat liver microsomes

Studies with the use of the fluorescent probe pyrene revealed that vitamin A deficiency in maturing male rats results in the increased microviscosity of liver lipids. This effect seems to be due to changes in the lipid composition of microsomal membranes (increased cholesterol/phospholipid ratio and lowered polyunsaturated fatty acid content) as well as to the low level of retinol. Analysis of microsomal phospholipids labeled with [3H]palmitate and [14C]glycerol revealed that vitamin A deficiency accelerates the turnover of the glycerol skeleton but sharply decelerates that of fatty acid residues. It is concluded that the observed effect of retinol on the structural and functional properties of biological membranes is due to its ability to control the microviscosity and turnover of membrane lipids.     

Lipid composition and microviscosity of subcellular fractions from rabbit thymocytes. Differences in the microviscosity of plasma membranes from subclasses of thymocytes.

There are indications from freeze-fracture experiments that subclasses of rabbit thymocytes show different mobilities of plasma membrane components. Consequently, one would expect differences in the fluidity of the plasma membrane. For this reason, rabbit thymocytes were separated on a Ficoll/Metrizoate gradient yielding three subclasses representing various levels of cell differentiation. These thymocyte subclasses did not show any significant differences in the degree of fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene. The fluorescence polarization of the plasma membrane may be overshadowed by the contribution of all cellular lipids due to penetration of the fluorescent probe into the cell. Therefore, plasma membranes were isolated from rabbit thymocytes using a cell-disrupting pump, differential centrifugation, and sucrose density gradient centrifugation. As shown by biochemical and electron microscopical analyses, plasma membranes with a high degree of purity were obtained. As expected the plasma membrane fractions showed a higher microviscosity than the other subcellular fractions. This was attributed to a higher cholesterol to phospholipid molar ratio and a higher degree of saturation of phospholipid fatty acid chains. Subsequently, the microviscosity was measured of plasma membrane preparations obtained from two main subclasses of thymocytes representing mature and immature lymphocytes. The immature thymocytes yielded two plasma membrane fractions with higher microviscosity than the mature cells. These finding is in line with earlier observed differences in the glycerol-induced clustering of intramembranous particles. Furthermore, the results of this study support the view that the fluorescence polarization technique applied to whole cells does not exclusively monitor the plasma membrane.     

Microviscosity of mucosal cellular membranes in toad urinary bladder: Relation to antidiuretic hormone action on water permeability

Summary The microviscosity of cellular membranes (or membrane fluidity) was measured in suspensions of single mucosal cells isolated from the urinary bladder of the toad,Bufo marinus, by the technique of polarized fluorescence emission spectroscopy utilizing the hydrophobic fluorescent probe, perylene. At 23°C, 5mm dibutyryl cyclic 3,5-AMP decreased the apparent microviscosity of the cell membranes from 3.31 to 3.07 P, a minimum decrease of 7.3% (P<0.001) with a physiological time course. Direct visualization of the cell suspension indicated that 98% of the cells were viable, as indicated by Trypan Blue dye exclusion. The fluorescent perylene could be seen only in plasma membranes, suggesting that the measured viscosity was that of plasma membrane with little contribution from the membranes of cellular organelles. Addition of antidiuretic hormone to intact hemibladders stained with perylene produced changes in fluorescence consistent with a similar 7% decrease in apparent microviscosity with a physiological time course. However, finite interpretation of the findings in intact tissue cannot be made because the location and the fluorescent lifetime of the probe could only be conducted on the isolated cells. Comparison with previously determined relationships between water permeability and microviscosity in artificial bilayers suggests that the 7% (a lower limit) decrease in microviscosity would produce only a 6.5% increase in water permeability.