Inhibition of heat shock protein synthesis and thermotolerance by cycloheximide

Chinese hamster ovary (CHO) cells were exposed to a 43 degrees C, 15-min heat shock to study the relationship between protein synthesis and the development of thermotolerance. The 43 degrees C heat shock triggered the synthesis of three protein families having molecular weights of 110,000, 90,000, and 65,000 (HSP). These proteins were synthesized at 37 and 46 degrees C. This heat shock also induced the development of thermotolerance, which was measured by incubating the cells at 46 degrees C 4 h after the 43 degrees C heat treatment. CHO cells were also exposed to 20 micrograms/ml of cycloheximide for 30 min at 37 degrees C, 15 min at 43 degrees C, and 4 h at 37 degrees C. This treatment inhibited the enhanced synthesis of the Mr 110,000, 90,000, and 65,000 proteins. The cycloheximide was then washed out and the cells were incubated at 46 degrees C. HSP synthesis did not recover during the 46 degrees C incubation. This cycloheximide treatment also partially inhibited the development of thermotolerance. These results suggest that for CHO cells to express thermotolerance when exposed to the supralethal temperature of 46 degrees C protein synthesis is necessary.     

Low-temperature pausing of cultivated mammalian cells

There are currently two methods for maintaining cultured mammalian cells, continuous passage at 37 degrees C and freezing in small batches. We investigated a third approach, the "pausing" of cells for days or weeks at temperatures below 37 degrees C in a variety of cultivation vessels. High cell viability and exponential growth were observed after pausing a recombinant Chinese hamster ovary cell line (CHO-Clone 161) in a temperature range of 6-24 degrees C in microcentrifuge tubes for up to 3 weeks. After pausing in T-flasks at 4 degrees C for 9 days, adherent cultures of CHO-DG44 and human embryonic kidney (HEK293 EBNA) cells resumed exponential growth when incubated at 37 degrees C. Adherent cultures of CHO-DG44 cells paused for 2 days at 4 degrees C in T-flasks and suspension cultures of HEK293 EBNA cells paused for 3 days at either 4 degrees C or 24 degrees C in spinner flasks were efficiently transfected by the calcium phosphate-DNA coprecipitation method, yielding reporter protein levels comparable to those from nonpaused cultures. Finally, cultures of a recombinant CHO cell line (CHO-YIgG3) paused for 3 days at 4 degrees C, 12 degrees C, or 24 degrees C in bioreactors achieved the same cell mass and recombinant protein productivity levels as nonpaused cultures. The success of this approach to cell storage with rodent and human cell lines points to a general biological phenomenon which may have a wide range of applications for cultivated mammalian cells.     

Comparison of formation, activation, and nuclear translocation of receptor . estradiol (R . E2) complex at 0 degrees C and 37 degrees C in intact uterine cells.

The present study was undertaken to establish whether molecular events leading to binding, transformation-activation, and nuclear translocation of cytoplasmic uterine estrogen receptor described for cell-free systems also occur in intact uterine cells. Cell suspensions were incubated at 0 degrees C or 37 degrees C with estradiol (E2) and specific binding to intracellular receptors was measured. The data demonstrate that saturation of specific estrogen binding sites occurs within 60 min at 37 degrees C and within 22 h at 0 degrees C, with a total of approximately 24,000 to 30,000 receptor sites per cell. At equilibrium, the total number and subcellular distribution of receptor . estradiol (R . E2) complexes formed in cells incubated at 0 degrees C or 37 degrees C were identical. Scatchard analysis of the equilibrium binding data yielded the same association constants for cytoplasmic and nuclear R . E2 formed in intact cells incubated at either temperature. Sucrose density gradient analysis of nuclear and cytoplasmic R . E2 formed in intact cells at 0 degrees C or 37 degrees C showed that at both temperatures, the nuclear R . E2 had a 5 S sedimentation coefficient; at both temperatures, a 5 S cytosol R . E2 was detected; only in the 0 degrees C incubation, an additional 4 S cytosol R . E2 was found. These results suggest that the molecular interactions regulating the dynamics of estrogen binding in the intact cell are similar at both physiological and low temperatures.     

Cryptococcus neoformans mitochondrial superoxide dismutase: an essential link between antioxidant function and high-temperature growth

Manganese superoxide dismutase is an essential component of the mitochondrial antioxidant defense system of most eukaryotes. In the present study, we used a reverse-genetics approach to assess the contribution of the Cryptococcus neoformans manganese superoxide dismutase (Sod2) for antioxidant defense. Strains with mutations in the SOD2 gene exhibited increased susceptibility to oxidative stress as well as poor growth at elevated temperatures compared to isogenic wild-type strains. The sod2Delta mutants were also avirulent in a murine model of inhaled cryptococcosis. Reconstitution of a sod2Delta mutant restored Sod2 activity, eliminated the oxidative stress and temperature-sensitive (ts) phenotypes, and complemented the virulence phenotype. Characterization of the ts phenotype revealed a dependency between Sod2 antioxidant activity and the ability of C. neoformans cells to adapt to growth at elevated temperatures. The ts phenotype could be suppressed by the addition of either ascorbic acid (10 mM) or Mn salen (200 muM) at 30 degrees C, but not at 37 degrees C. Furthermore, sod2Delta mutant cells that were incubated for 24 h at 37 degrees C under anaerobic, but not aerobic, conditions were viable when shifted to the permissive conditions of 25 degrees C in the presence of air. These data suggest that the C. neoformans Sod2 is a major component of the antioxidant defense system in this human fungal pathogen and that adaptation to growth at elevated temperatures is also dependent on Sod2 activity.     

Recovery from potentially lethal damage of irradiated L5178Y-R and L5178Y-S cells: the effect of temperature and benzamide

Mouse lymphoma L5178 Y-S and Y-R cells differing in radiosensitivity by 1.5 times were treated with benzamide, an inhibitor of poly(ADP-ribosylation), for 24 h before and 18 h after X-irradiation, and incubated after irradiation at 25 degrees C and 37 degrees C. Clonogenic capacity of LY-S cells incubated at 25 degrees C exceeded that of the same cells incubated at 37 degrees C; the clonogenic capacity of LY-R cells did not vary with the postirradiation incubation temperature. Benzamide increased equally the radiosensitivity of LY-R cells incubated at both temperatures, whereas that of LY-S cells was only increased at 37 degrees C. Repair of potentially lethal damages to LY-S cells incubated at 25 degrees C was independent of the effectiveness of poly(ADP-ribosylation).     

T lymphocyte stress response. II. Protection of translation and DNA replication against some forms of stress by prior hyperthermic stress

We have compared the effects of a mild heat shock and febrile temperatures on heat-shock protein (hsp) synthesis and development of stress tolerance in T lymphocytes. Our previous studies demonstrated that febrile temperatures (less than or equal to 41 degrees C) induced the synthesis of hsp110, hsp90, and the constitutive or cognate form of hsp70 (hscp70; a weak induction of the strongly stress-induced hsp70 was also observed. In the studies reported herein, we demonstrate that a mild heat shock (42.5 degrees C) reverses this ratio; that is, hsp70 and not hscp70 is the predominate member of this family synthesized at this temperature. Modest heat shock also enhanced the synthesis of hsp110 and hsp90. In order to assess the relationship between hsp synthesis and the acquisition of thermotolerance, purified T cells were first incubated at 42.5 degrees C (induction temperature) and then subsequently subjected to a severe heat-shock challenge (45 degrees C, 30 min). T cells first incubated at a mild heat-shock temperature were capable of total protein synthesis at a more rapid rate following a severe heat shock than control cells (induction temperature 37 degrees C). This phenomenon, which has been previously termed translational tolerance, did not develop in cells incubated at the febrile temperature (induction temperature 41 degrees C). Protection of translation also extended to immunologically relevant proteins such as interleukin-2 and the interleukin-2 receptor. Because clonal expansion is a critical event during an immune response, the effects of hyperthermic stress on DNA replication (mitogen-induced T cell proliferation) was also evaluated in thermotolerant T cells. DNA synthesis in control cells (induction temperature 37 degrees C) was severely inhibited following heat-shock challenge at 44 degrees C or 45 degrees C; in contrast, T cells preincubated at 42.5 degrees C rapidly recovered their DNA synthetic capacity. T cells preincubated at a febrile temperature were moderately protected against hyperthermic stress. The acquisition of thermotolerance was also associated with enhanced resistance to chemical (ethanol)-induced stress but not to heavy metal toxicity (cadmium) or dexamethasone-induced immunosuppression. These studies suggest that prior hsp synthesis may protect immune function against some forms of stress (e.g., febrile episode) but would be ineffective against others such as elevated glucocorticoid levels which normally occur during an immune response.     

Changes in bleb formation following hyperthermia treatment of Chinese hamster ovary cells

Chinese hamster ovary cells in suspension cultures were heated for various times at 41.5, 43.5, and 45.5 degrees C, and quantitative determinations of microblebbing and macroblebbing of the cell membrane were performed for cells maintained at 4, 25, and 37 degrees C after hyperthermia. The percentage of cells with blebs following heating at 45.5 degrees C was dependent upon the duration of heating with increases from 40% for 5 min to 90% for 30 min. Cells exposed to lower temperatures exhibited less blebbing which was not quantifiable. The changes in bleb formation following 45.5 degrees C were dependent upon the posthyperthermia temperature: a slight decrease of macroblebbing at 25 degrees C, a decrease to 50% by 2 h at 37 degrees C, and a sharp decrease of macroblebbing to less than 10% by 1 h at 4 degrees C. Microblebbing increased slightly at 37 degrees C. When cells were transferred rapidly from the 4 degrees C posthyperthermia incubation to 37 degrees C, the bleb formation percentages returned rapidly to the higher levels which existed before posthyperthermia incubation at the lower temperatures. Gamma irradiation of 20 and 50 Gy produced only a small increase in microblebbing at longer periods (5 to 6 h) but no increase in macroblebbing. The survival of cells heated for 20 min at 45.5 degrees C was decreased 40% for suspension cells maintained at 4 degrees C for 2 to 3 h before incubation at 37 degrees C for colony formation compared to cells immediately incubated at 37 degrees C after heating. The survival of cells maintained at 25 degrees C after heating was not altered in comparison.     

Temperature dependence of cell-cell fusion induced by the envelope glycoprotein of human immunodeficiency virus type 1.

We investigated cell-cell fusion induced by the envelope glycoprotein of human immunodeficiency virus type 1 strain IIIB expressed on the surface of CHO cells. These cells formed syncytia when incubated together with CD4-positive human lymphoblastoid SupT1 cells or HeLa-CD4 cells but not when incubated with CD4-negative cell lines. A new assay for binding and fusion was developed by using fluorescent phospholipid analogs that were produced in SupT1 cells by metabolic incorporation of BODIPY-labeled fatty acids. Fusion occurred as early as 10 min after mixing of labeled SupT1 cells with unlabeled CHO-gp160 cells at 37 degrees C. When both the fluorescence assay and formation of syncytia were used, fusion of SupT1 and HeLa-CD4 cells with CHO-gp160 cells was observed only at temperatures above 25 degrees C, confirming recent observations (Y.-K. Fu, T.K. Hart, Z.L. Jonak, and P.J. Bugelski, J. Virol. 67:3818-3825, 1993). This temperature dependence was not observed with influenza virus-induced cell-cell fusion, which was quantitatively similar at both 20 and 37 degrees C, indicating that cell-cell fusion in general is not temperature dependent in this range. gp120-CD4-specific cell-cell binding was found over the entire 0 to 37 degrees C range but increased markedly above 25 degrees C. The enhanced binding and fusion were reduced by cytochalasins B and D. Binding of soluble gp120 to CD4-expressing cells was equivalent at 37 and 16 degrees C. Together, these data indicate that during gp120-gp41-induced syncytium formation, initial cell-cell binding is followed by a cytoskeleton-dependent increase in the number of gp120-CD4 complexes, leading to an increase in the avidity of cell-cell binding. The increased number of gp120-CD4 complexes is required for fusion, which suggests that the formation of a fusion complex consisting of multiple CD4 and gp120-gp41 molecules is a step in the fusion mechanism.     

Effects of hyperthermia on DNA interstrand crosslinking after treatment with BCNU in 9L rat brain tumor cells

The effects of hyperthermia (42 degrees C) on 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-mediated DNA interstrand crosslink formation were investigated in 9L rat brain tumor cells using the technique of alkaline elution. When cells were treated with 60 microM BCNU for 1 hr at 37 degrees C and incubated for 6 hr in drug-free medium at 42 degrees C, there was a 50% increase in crosslinking; and when cells were treated at 42 degrees C and incubated at 37 degrees C, there was a 45% increase in crosslinking compared with the results for cells treated and incubated at 37 degrees C. When cells were treated and incubated at 42 degrees C, there was a 129% increase in DNA crosslinking. The same relative order of results was found for cell survival. These results suggest that hyperthermia can increase DNA interstrand crosslink formation and the consequent cell death through two independent mechanisms: an increase in the amount of initial alkylation because of the increased rate of hydrolysis of BCNU at higher temperatures, and the effect of heat on DNA structure that leads to an increase in the number of crosslinks formed.     

Temperature-dependent biotransformation of 2,4'-dichlorobiphenyl by psychrotolerant Hydrogenophaga strain IA3-A: higher temperatures prevent excess accumulation of problematic meta-cleavage products

AIMS: The present work investigates the possibility that temperature could regulate the pattern of transformation of 2,4'-chlorobiphenyl (2,4'-CB) by psychrotolerant Hydrogenophaga sp. IA3-A. Methods AND RESULTS: Transformation of 2,4'-chlorobiphenyl to 2- and 4-chlorobenzoic acid (2- and 4-CBA), and meta-cleavage products by cells of strain IA3-A incubated at 10 degrees C, 25 degrees C, 37 degrees C or 45 degrees C were monitored by UV spectrometry, HPLC and GC-MS analyses. Cultures incubated at 10 degrees C, 25 degrees C or 37 degrees C produced low amounts of CBAs and excess levels of meta-cleavage products from 2,4'-CB. Cultures incubated at 45 degrees C transformed most of the degraded 2,4'-CB to CBAs and low level of meta-cleavage product. Culture extracts contained unusual varieties of isomeric hydroxylated metabolic products. CONCLUSIONS: Efficient transformation of 2,4'-CB to CBAs was possible in cultures incubated at 45 degrees C. Evidence for the involvement of multiple pathways in the transformation of 2,4'-CB in strain IA3-A suggests that differential regulation of the pathways at different temperatures was likely responsible for the change in the pattern of transformation of 2,4'-CB in cultures incubated at 45 degrees C. Significance AND IMPACT OF THE STUDY: It may be possible to condition cells to transform chlorinated biphenyls more efficiently without accumulating excess level of toxic intermediates.     

Degradation of asialoglycoproteins mediated by the galactosyl receptor system in isolated hepatocytes. Evidence for two parallel pathways

The ability of rat hepatocytes to degrade internalized surface-bound 125I-asialoorosomucoid (ASOR) was determined by measuring the appearance of acid-soluble radioactivity at 37 degrees C. The degradation kinetics were biphasic in cells previously equilibrated at 37 degrees C for 1 h or cultured for 24 h. Degradation began immediately and was linear for at least 20 min after which the rate increased to a steady state value 3-4 times greater than the initial rate. We previously showed that hepatocytes have two functionally distinct populations of galactosyl receptors that mediate ligand dissociation by two kinetically different pathways (Weigel, P. H., Clarke, B. L., and Oka, J. A. (1986) Biochem. Biophys. Res. Commun. 140, 43-50). The activity of one receptor population, designated State 2 galactosyl receptors, can be reversibly modulated by incubating cells between 22 and 37 degrees C and is not expressed on the surface of freshly isolated cells. When 125I-ASOR was prebound to freshly isolated cells at 4 degrees C and degradation was assessed subsequently at 37 degrees C, the kinetics were monophasic, not biphasic. Degradation of the surface-bound 125I-ASOR began immediately and was greater than 90% complete by 6 h. Freshly isolated cells were incubated at temperatures between 22 and 37 degrees C, chilled to 4 degrees C, allowed to pre-bind 125I-ASOR, and then incubated at 37 degrees C. As the State 2 galactosyl receptor population increased, the kinetics of degradation became progressively more biphasic and the rate of the delayed degradation process increased. This effect could be reversed in cells in culture or in suspension by down-modulating surface receptor activity at temperatures below 37 degrees C; only the degradation process appearing after a 20-min lag was affected. Degradation in both pathways is an apparent first order process with identical rate constants (kappa = 0.006 min-1, t1/2 = 116 min). We conclude that there are two separate pathways by which asialoglycoproteins are degraded. The major "classic" pathway mediated by State 2 galactosyl receptors occurs after a 20-min lag and the minor pathway mediated by State 1 galactosyl receptors begins immediately with no detectable lag.     

Organ culture of mammalian testis. III. Inhibin secretion.

Mouse testes were cultured for 19--20 days at either 31 or 37 degrees C with a change of medium every 4 days. After treatment with charcoal and dextran T, the recovered testis media were incubated with rat anterior pituitary cells, and secretions of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were estimated by radioimmunoassay 3 days later. FSH release was significantly lowered when pituitary cells were grown with media of testes cultured 31 degrees C compared to cultures grown with fresh medium or with media of testes cultured at 37 degrees C for more than 4 days. LH secretion was normal in one experiment and reduced in the other with the media of testes cultured at 31 degrees C. Treatment of testicular media by heat or trypsin reduced the inhibiting activity. After 8 days at 37 degrees C, both germinal and Sertoli cells were damaged in the testis cultures, while at 31 degrees germinal cells alone were destroyed, Sertoli cells remained normal. These studies suggest that (1) a substance which responds to the definition of inhibition (protein--preferentially acting on FSH) is secreted in the medium of testis culture; (2) inhibin is produced by Sertoli cells; (3) inhibin is secreted only if the temperature is inferior to 37 degrees C.     

Sperm membrane functional integrity and response of frozen-thawed bovine spermatozoa during the hypoosmotic swelling test incubation at varying temperatures

The objective of this study was to assess the sperm membrane integrity and permeability of frozen-thawed bovine spermatozoa, processed at varying temperatures during and after thawing, by exposing the spermatozoa to standardized hypoosmotic conditions. The hypoosmotic swelling (HOS) test was employed to measure changes in sperm membrane functional status and permeability. Frozen specimens (from 5 bulls) were thawed at 37h degrees C for 10 sec and transferred to a water bath at 37 (Aliquot 1), 21 (Aliquot 2) or 5 degrees C (Aliquot 3) to complete thawing (1 to 2 min). The specimens were maintained and processed at these temperatures for additional 5 to 10 min. Specimens were slowly diluted 1:1 (v/v) and washed with Ham's F-10 media containing 3% (w/v) BSA. The HOS test was performed by adding 0.1 ml of the sperm specimen to 1.0 ml of a 100 mOsm/L HOS diluent. The following treatments were performed: 1) Aliquot 1 (control), specimens were incubated in HOS solutions at 37 degrees C for 5 min; 2) Aliquot 2, specimens were incubated in HOS solutions at 21 or 37 degrees C for 5 min; and 3) Aliquot 3, specimens were incubated in HOS solutions at 5 or 37 degrees C for 5 min. Samples were obtained from the sperm specimen-HOS diluent mixtures at 1 min intervals (during the 5 min incubation period), fixed and assessed for sperm swelling patterns. The sperm response to the HOS test for specimens processed at temperatures below 37 degrees C was higher when samples were incubated in HOS diluents at 37 degrees C. This finding indicates that the potential for sperm swelling (measurement of sperm membrane functional status) can be maintained when spermatozoa are processed at temperatures below 37 degrees C. The highest response to the HOS test was observed in spermatozoa processed at 21 degrees C and incubated in a HOS solution at 37 degrees C. The response to the HOS test was superior to the one observed in specimens maintained and processed at 37 degrees C throughout. Thawing of spermatozoa at 37 degrees C, followed by processing at 21 degrees C seems to reduce the negative effects associated with osmotic shock and results in the preservation of the sperm membrane functional status during the in vitro handling of frozen-thawed bovine spermatozoa.     

Rapid cell surface appearance of endocytic membrane proteins in Chinese hamster ovary cells.

Lactoperoxidase was used to selectively radiolabel endocytic membrane. CHO cells were incubated with enzyme at 37 degrees C for 10 min to permit lactoperoxidase internalization. Radioiodination was done at 4 degrees C. About 90% of the radioiodinated products pelleted at 100,000 X g. From 12 to 15 different electrophoretic species were detected by one-dimensional gel electrophoresis. When cells labeled by internalized lactoperoxidase were warmed to 37 degrees C, the incorporated radioactivity was lost in a biphasic manner with an overall t1/2 of approximately 20 h. Upon warming cells to 37 degrees C, the labeled species became sensitive to pronase or trypsin digestion. The increase in protease sensitivity was progressive over a 10- to 20-min period. Maximally 45% of the initially intracellular radiolabel could be released. A digest of exterior-radioiodinated cells released 50% of the incorporated radioiodine. These observations strongly suggest a rapid shuttling of approximately 90% of the radioiodinated membrane species initially present within the cell to the cell surface.     

Retinoic acid alters subcellular compartmentalization of ATP pools in 3T3 cells but not in HeLa cells

From the Chinese hamster ovary (CHO) cell, genetic variants (MonR-31 and MonR-32) relatively resistant to monensin, an ionophoric antibiotic, have been isolated. Growth of both MonR-31 and MonR-32 clones required higher doses of serum than CHO. Addition of insulin to media containing a low dose of serum restored full colony formation, but growth of MonR-31 or MonR-32 cells required more insulin than CHO cells. Specific binding of [125I]insulin was observed in these cell lines. The two MonR clones bound about one-half or less the [125I]insulin bound by CHO cells. Scatchard analysis for [125I]insulin binding at 4 degrees C and 37 degrees C showed altered number of binding sites, but not insulin affinity: The number of binding sites in the MonR cell was about a half or less that of the parental CHO cell. Down-regulation of insulin receptor was assayed when both CHO and MonR cells were incubated with 1 microgram/ml insulin. A 50-60% decrease in levels of insulin surface binding capacities was observed in CHO after exposure to insulin, whereas there was no decrease in MonR cell. The cellular uptake of 2-[3H]deoxyglucose into CHO cells was significantly enhanced in the presence of insulin, but only slight, if any, increase was observed in MonR cells.     

Effect of dose rate on cell killing and DNA strand break repair in CHO cells exposed to internal beta-rays from incorporated [3H]thymidine

Survival as well as repair of DNA strand breaks were studied in CHO cells after exposure to internal beta-rays from incorporated [3H]thymidine at 4 degrees C (equivalent to an exposure at 'infinitely high' dose rate) and at 37 degrees C (low dose rate). DNA strand breaks were determined by the alkaline unwinding technique. In cells exposed at 4 degrees C cell killing was five times higher (Do = 250 decays per cell) than in cells exposed at 37 degrees C (Do = 1280 decays per cell). Strand breaks induced by 3H decay at 37 degrees C were repaired with the same kinetics as those generated at 4 degrees C. Therefore the different degrees of cell killing at 4 degrees C and 37 degrees C cannot be attributed to a difference in the repair kinetics for DNA strand breaks.     

Alterations in the nuclear matrix protein mass correlate with heat-induced inhibition of DNA single-strand-break repair

The total protein mass co-isolating with the nuclear matrix or nucleoid from Chinese hamster ovary (CHO) cells was observed to increase in heated cells as a function of increasing exposure temperature between 43 degrees C and 45 degrees C or of exposure time at any temperature. The sedimentation distance of the CHO cell nucleoid in sucrose gradients increased with increasing exposure time at 45 degrees C. Both these nuclear alterations correlated in a log-linear manner with heat-induced inhibition of DNA strand break repair. A two-fold threshold increase in nuclear matrix protein mass preceded any substantial inhibition of repair of DNA single-strand breaks. When preheated cells (45 degrees C for 15 min) were incubated at 37 degrees C the nuclear matrix protein mass and nucleoid sedimentation recovered with a half-time of about 5 h, while DNA single-strand-break repair recovered with a half-time of about 2 h. When preheated cells were placed at 41 degrees C (step-down heating; SDH) a further increase was observed in the nuclear matrix protein mass and the half-time of DNA strand break repair, while nucleoid sedimentation recovered toward control values. These results implicate alterations in the protein mass of the nuclear matrix in heat-induced inhibition of repair of DNA single-strand breaks.     

Effects of temperature on water diffusion in human erythrocytes and ghosts--nuclear magnetic resonance studies

The temperature-dependence of water diffusion across human erythrocyte membrane was studied on isolated erythrocytes and resealed ghosts by a doping nuclear magnetic resonance technique. The conclusions are the following: (1) The storage of suspended erythrocytes at 2 degrees C up to 24 h or at 37 degrees C for 30 min did not change the water exchange time significantly, even if Mn2+ was present in the medium. This indicates that no significant penetration of Mn2+ is taking place under such conditions. (2) In case of cells previously incubated at 37 degrees C for longer than 30 min with concentrations of p-chloromercuribenzene sulfonate (PCMBS) greater than 0.5 mM, the water-exchange time gradually decreased if the cells were stored in the presence of Mn2+ for more than 10 min at 37 degrees C. (3) When the Arrhenius plot of the water-exchange time was calculated on the basis of measurements performed in such a way as to avoid a prolonged exposure of erythrocytes to Mn2+ no discontinuity occurred, regardless of the treatment with PCMBS. (4) No significant differences between erythrocytes and resealed ghosts regarding their permeability and the activation energy of water diffusion (Ea,d) were noticed. The mean value of Ea,d obtained on erythrocytes from 35 donors was 24.5 kJ/mol. (5) The value of Ea,d increased after treatment with PCMBS, in parallel with the percentage inhibition of water diffusion. A mean value of 41.3 kJ/mol was obtained for Ea,d of erythrocytes incubated with 1 mM PCMBS for 60 min at 37 degrees C and 28.3 kJ/mol for ghosts incubated with 0.1 mM PCMBS for 15 min, the values of inhibition being 46% and 21% respectively.