Heat-induced increases in endothelial NO synthase expression and activity and endothelial NO release

Endothelial nitric oxide (NO) synthase (eNOS) is regulated by heat shock protein 90 (HSP90), a heat-inducible protein; however, the effect of heat shock on eNOS expression and eNO release is unknown. Bovine aortic endothelial cells were incubated for 1 h at 37 degrees C, 42 degrees C, or 45 degrees C and cell lysates were evaluated with the use of Western blotting. We observed a 2.1 +/- 0.1-fold increase in eNOS protein content, but no change in HSP90 content, HSP70 content, or HSP90/eNOS association, 24 h after heat shock at 42 degrees C. We also observed a 7.7 +/- 1.5-fold increase in HSP70 protein content, but did not observe a change in eNOS or HSP90 24 h after heat shock at 45 degrees C. eNOS activity and maximal bradykinin-stimulated NO release was significantly increased 24 h after heat shock at 42 degrees C. Heat shock in rats (core temperature: 42 degrees C, 15 min) resulted in a significant increase in aortic eNOS, HSP90, and HSP70 protein content. The aorta from heat-shocked rats exhibited a decreased maximal contractile response to phenylephrine, which was abolished by preincubation with NG-nitro-l-arginine. We conclude that prior heat shock is a physical stimulus of increased eNOS expression and is associated with an increase in eNOS activity, agonist-stimulated NO release, and a decreased vasoconstrictor response.     

Translational regulation of the synthesis of a major heat shock protein in HeLa cells

The synthesis of a major heat shock protein (HSP 70) was measured in HeLa cells incubated at 42.5 degrees C and then transferred to 37 degrees C or 30 degrees C. After 90 min, synthesis of HSP 70 decreased by 54 and 85%, respectively, whereas HSP 70 mRNA was reduced at most by 20%. Therefore, the reduced synthesis of HSP 70 could not be accounted for by mRNA turnover. HSP 70 was associated with large polyribosomes (6-10 ribosomes) in cells kept at 42.5 degrees C, but with medium or small polyribosomes in cells transferred to 37 degrees C or 30 degrees C (5-6 or 2-3 ribosomes, respectively). Addition of puromycin to these cells resulted in the release of all ribosomes from HSP 70 mRNA, indicating that they were translationally active. The regulation of HSP 70 synthesis was investigated in cell-free systems prepared from heat-shocked or control cells and incubated at 30 degrees C and 42 degrees C. After 5 min at 42 degrees C, the cell-free system from heat-shocked cells synthesized protein at 3 times the rate of the control cell-free system. This difference was in large part due to synthesis of HSP 70. Addition of HSP mRNA to the control cell-free system stimulated protein synthesis at 42 degrees C, but not at 30 degrees C. These findings suggest that translation of HSP 70 mRNA is specifically promoted at high temperature and repressed during recovery from heat shock by regulatory mechanisms active at the level of initiation.     

Effect of hyperthermia on protein synthesis in monolayer and suspension cultures of mouse L cells

Changes in protein synthesis that occurred under the influence of heat shock (HS) in monolayer (L929) and suspension (LS) mouse cell cultures were studied. The rates of protein synthesis determined as 35S-methionine incorporations were seen reduced from the initial level up to 40-60 and 6-13% after HS at 42 and 44 degrees C, respectively. Simultaneously the rate of actin and tubulin syntheses decreased, the decrease being more pronounced in LS cells. According to electrophoresis and autoradiography data, after hyperthermia both the cell cultures were able to synthesize heat shock proteins (HSP), primarily HSP70. After a 40 min HS towards L929 and LS cells at 43 degrees C, the shares of their HSP70 bands in the total label loaded on the gel constituted, resp., 8.8 and 5.4%. The data suggest that L929 cells, with their synthetic activity lower than in LS cells, appear more resistant to HS and are able eventually to synthesize larger amounts of HSP70, compared to the latter.     

Expression of heat shock protein70 in pig oocytes: heat shock response during oocyte growth

The heat shock response of growing and fully-grown pig oocytes was analyzed in vitro by determining heat shock protein70 (HSP70) synthesis under both normal conditions (39 degrees C; 0 and 6h) and after heat shock (43 degrees C; 1, 4 and 6h). The expression of HSP70 in oocytes was detected by immunoblotting analysis. Growing oocytes measuring 80-99 microm synthesized a high number of HSP70 without heat shock effect, and these were capable of increasing the synthesis of HSP70 after heat shock to a maximum after 1h. Growing oocytes measuring 100-115 microm also synthesized HSP70 without heat shock and after it, but the HSP70 synthesis was not statistically changed by increasing duration of heat shock. In fully-grown oocytes, great amounts of HSP70 were found without heat shock treatment, and the contents of HSP70 significantly decreased after heat shock. These results indicate that growing oocytes are able to synthesize HSP70 after heat shock. This ability declines at the end of the growth period, and fully-grown oocytes are unable to induce HSP70 synthesis after heat shock. HSP70 is synthesized and stored during oocyte growth. The high HSP70 synthesis in non-heat-treated growing oocytes and a great amount of HSP70 in fully-grown oocytes support the hypothesis that HSP70 is important for oocyte growth and maturation.     

Substrate utilization for lactate and energy production by heat-shocked L929 cells

The hypothesis that heat shock protein (HSP) induction depends on inhibition of respiration was tested by examining the effects of heat shock on tricarboxylic acid (TCA) cycle function. In control L929 cell cultures, glucose and exogenous pyruvate were converted primarily to lactate, and glutamine was extensively oxidized, accounting for more than one-half of the calculated ATP production. During heat shock at 42 degrees C, lactate production from all of the labeled substrates and total unlabeled lactate production increased significantly while oxygen consumption increased slightly. TCA cycle oxidation of pyruvate decreased during this period while that of glutamine increased. Uncoupling of oxidative phosphorylation caused large increases in oxygen consumption at both 37 degrees C and 42 degrees C, indicating that the capacity of the respiratory chain is not exceeded during heat shock. The net effect of these alterations in substrate utilization were decreased ATP generation and increased NADH utilization. Both 14CO2 and lactate production declined during the 24-h period after cultures were returned to 37 degrees C. On the basis of these data, we conclude that while inhibition of respiration plays no apparent role, other metabolic consequences of heat shock related to energy metabolism may be involved in HSP induction.     

Patterns of protein synthesis in various cells after extreme heat shock

The analysis of proteins synthesized in rat thymocytes and mouse teratocarcinoma PCC-4 Aza 1 and myeloma Sp2/0 cells after 1 h of treatment at 42 or 44 degrees C was carried out. Shock at 42 degrees C reduced the total synthetic rate of proteins in all three cell lines and induced "classical" heat-shock protein with a mass of 70 kDa (hsp 70). Heat shock at 44 degrees C resulted in almost complete inhibition of protein synthesis; only a small amount of hsp 70 was synthesized. Meanwhile a new 48-kDa polypeptide (pI = 7.5) was found in the cells exposed to severe heat shock. This protein was compared by peptide mapping with other known polypeptides of the same size: heat-shock protein from chicken embryo cells and mitogen-stimulated polypeptide from human lymphoid cells. The peptide maps were not identical. It was also shown that after a shock at 44 degrees C teratocarcinoma cells were able to accumulate anomalous amounts of hsp 70 despite hsp 70 synthesis inhibition. The data show that reaction of various cells to extreme heat shock depends heavily on cell type.     

Expression of heat shock and cold shock proteins in the gorgonian Leptogorgia virgulata

In this study, we analyzed the response of the temperate, shallow-water gorgonian, Leptogorgia virgulata, to temperature stress. Proteins were pulse labeled with (35)S-methionine/cysteine for 1 h to 2 h at 22 degrees C (control), or 38 degrees C, or for 4 h at 12.5 degrees C. Heat shock induced synthesis of unique proteins of 112, 89, and 74 kDa, with 102, 98 and 56 kDa proteins present in the control as well. Cold shock from 22 degrees C-12.5 degrees C induced the synthesis of a 25 kDa protein, with a 44 kDa protein present in the control as well. Control samples expressed unique proteins of 38, and 33 kDa. Non-radioactive proteins expressed under the same conditions as above, as well as natural field conditions, were tested for reactivity with antibodies to heat shock proteins (HSPs). HSP60 was the major protein found in L. virgulata. Although HSP47, HSP60, and HSP104 were present in all samples, the expression of HSP60 was enhanced in heat stressed colonies, while HSP47 and HSP104 expression were greatest in cold shocked samples. Inducible HSP70 was expressed in cold-shocked, heat-shocked, and field samples. Constitutively expressed HSP70 was absent from all samples. The expression of HSP90 was limited to heat shocked colonies. The expression of both HSP70 and HSP104 suggests that the organism may also develop a stress tolerance response.     

玉米胚发育过程中热激蛋白的合成

35S-Met标记玉米胚蛋白合成结果表明,热激处理(42℃)与对照(25℃)的蛋白合成趋势相近,热激抑制16 DAP的蛋白合成,增加22和34 DAP蛋白合成.SDS-PAGE自显影图谱表明,热激诱导16DAP的胚合成86.4、80.0、73.2 kD等3种分子量较高的热激蛋白,22DAP后热激诱导合成86.4、80.0、73.2、24.4、18.2、16.8和13.6 kD等7种分子量的热激蛋白.2D-PAGE自显影图谱进一步显示,热激诱导22和28 DAP的胚合成近20种热激蛋白,其中超过10种为小分子热激蛋白.特异热激蛋白BiP(HsP70)、PDI(HsP60)Western blot表明,这2种热激蛋白在玉米胚发育过程均有高水平的表达,热激对其合成影响不明显.     

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.     

Induction of acquired thermotolerance in Tetrahymena thermophila: effects of protein synthesis inhibitors.

When Tetrahymena thermophila cells growing at 30 degrees C are shifted to either 40 or 43 degrees C, the kinetics and extent of induction of heat shock mRNAs in both cases are virtually indistinguishable. However, the cells shifted to 40 degrees C show a typical induction of heat shock protein (HSP) synthesis and survive indefinitely (100% after 24 h), whereas those at 43 degrees C show an abortive synthesis of HSPs and die (less than 0.01% survivors) within 1 h. Cells treated at 30 degrees C with the drugs cycloheximide or emetine, at concentrations which are initially inhibitory to protein synthesis and cell growth but from which cells can eventually recover and resume growth, are after this recovery able to survive a direct shift from 30 to 43 degrees C (ca. 70% survival after 1 h). This induction of thermotolerance by these drugs is as efficient in providing thermoprotection to cells as is a prior sublethal heat treatment which elicits the synthesis of HSPs. However, during the period when drug-treated cells recover their protein synthesis ability and simultaneously acquire the ability to subsequently survive a shift to 43 degrees C, none of the major HSPs are synthesized. The ability to survive a 1-h, 43 degrees C heat treatment, therefore, does not absolutely require the prior synthesis of HSPs. But, as extended survival at 43 degrees Celsius depends absolutely on the ability of cells to continually synthesize HSPs, it appears that a prior heat shock as well as the recovery from protein synthesis inhibition elicits a change in the protein synthetic machinery which allows the translation of HSP mRNAs at what would otherwise be a nonpermissive temperature for protein synthesis.     

Phosphorylation of HSP27 during development and decay of thermotolerance in Chinese hamster cells

The small molecular weight heat shock protein HSP27 was recently shown to confer a stable thermoresistant phenotype when expressed constitutively in mammalian cells after structural gene transfection. These results suggested that HSP27 may also play an important role in the development of thermotolerance, the transient ability to survive otherwise lethal heat exposure after a mild heat shock. In Chinese hamster O23 cells increased thermoresistance is first detected at 2 h after a triggering treatment of 20 min at 44 degrees C, attains a maximum at 5 hours, and decays thereafter with a half-life of 10 h. We found that the development and decay of transient thermotolerance cannot be solely explained on the basis of changes in the cellular concentration of HSP27. The cellular HSP27 concentration is not increased appreciably at 2 h after heat shock and attains a maximum at 14 h. Similar results were obtained in the case of another heat shock protein, HSP70. HSP70 follows slightly faster kinetics of accumulation (peaks at 10 h) and decays much more rapidly (ti/2 = 4h) than HSP27 (t1/2 = 13h). HSP27 has 3 isoelectric variants A, B, and C of which B and C are phosphorylated. In cells maintained at normal temperature, HSP27A represents more than 90% of all HSP27. Shifting the cell culture temperature from 37 to 44 degrees C induces the incorporation of 32P into the more acidic B and C forms, a process that occurs very rapidly since the reduction in the concentration of the A form and a corresponding increase in the level of B and C is detectable by immunoblot analysis within 2.5 min at 44 degrees C. Analyses performed at various times during development and decay of transient thermotolerance revealed a close relationship between the effect of heat shock on HSP27 phosphorylation and cell ability to survive. For example, fully thermotolerant cells (5 h post-induction) are refractory to induction of HSP27 phosphorylation by a 20-min heat shock. The induction of HSP27 phosphorylation was also studied in a family of clonal cell lines of O23 cells that are thermoresistant as a result of the constitutive expression of a transfected human HSP27 gene. In these thermoresistant cells, phosphorylation of the endogenous hamster HSP27 is induced to a level comparable to that found in the thermosensitive parental cells. However, phosphorylation of the exogenous human protein, which represents more than 80% of total HSP27 in these cells, was much less induced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protection of Chinese hamster ovary cells from heat killing by treatment with cycloheximide or puromycin: involvement of HSPs?

Cycloheximide (CHM) or puromycin (PUR) added for 2 h before heating at 43 degrees C followed by either PUR or CHM during heat greatly protected cells from heat killing. This protection increased with inhibition of protein synthesis. Since treatment with a drug both before and during heating was required for heat protection, and since one drug could be exchanged for the other after the 2-h pretreatment without affecting the heat protection, a common mode of action involving inhibition of protein synthesis is suggested for the two drugs. Drug treatment reduced the synthesis of heat-shock proteins (HSPs) as studied by one-dimensional gel electrophoresis by 80-98% relative to 37 degrees C untreated controls. Synthesis of large molecules (greater than 30 kDa) was preferentially inhibited by PUR but not by CHM. Also for CHM, but not for PUR treatment, a 42 kDa band appeared along with a great reduction in the 43 kDa actin band during CHM treatment at both 37 and 43 degrees C. Furthermore, during CHM or PUR treatment, incorporation of [35S]methionine into HSP families 70, 87, or 110 was not increased relative to incorporation into total protein. However, synthesis of the 70 kDa HSP family was selectively suppressed when cells were incubated at 37 degrees C after CHM treatment, but when cells were incubated at 37 degrees C after treatment at 43 degrees C with CHM, synthesis of the 70 kDa HSP family resumed. When cells were labeled for 3 days, there was no preferential accumulation or turnover of HSP families during heating with or without CHM. Therefore, heat protection caused by treatment with CHM or PUR apparently involves a common mode of action not associated with changes in either total levels or synthesis of HSP families during drug treatment before and during heating. The significance of the changes observed in the synthesis of the HSP 70 family after heat is unknown. As thermotolerance developed during 5 h at 42 degrees C without drugs, synthesis of HSP families 70, 87, and 110, as studied with one-dimensional gels, increased 1.4-fold relative to synthesis of total protein, but compared to HSP families in cells labeled for 5 h at 37 degrees C incorporation was reduced by 40%. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater.(ABSTRACT TRUNCATED AT 400 WORDS)     

Heat shock response during development of the malaria vector Anopheles stephensi (Culicidae: Diptera)

The pattern of synthesis of heat shock proteins (HSP) and thermotolerance to elevated temperatures during the development of the malaria vector Anopheles stephensi normally reared at 28 +/- 2 degrees C was studied using SDS-PAGE. In total twelve heat shock proteins (i.e. 31, 33, 38, 43, 44, 51, 57, 62, 69, 71, 113 and 121 kD were induced by heat shock during various stages of development. Eight polypeptides (HSP during one or other of the instars) appeared during normal development of the adult, which showed very little response towards heat shock. Only two polypeptides (57 and 69 kD) were induced while the 22.5 kD protein disappeared during adult life. The HSP 62 and 71 kD induced during the larval stages showed a sharp decline in quantity in male and female adults upon heat shock. Three HSP (31, 43 and 44 kD) were induced in pupae due to heat shock. The synthesis of HSP in A. stephensi was correlated with the various morphological and physiological events occurring during development.     

Germ cell-specific heat shock protein 105 binds to p53 in a temperature-sensitive manner in rat testis.

Heat shock protein (HSP)105 is a testis-specific and HSP90-related protein. The aim of this study was to explore the functions of HSP105 in the rat testis. Signals of HSP105 were detected immunohistochemically in the germ cells and translocated from the cytoplasm to the nucleus at 2 days after experimental induction of cryptorchidism. In cultured testicular germ cells, a significant increase in the expression of HSP105 in response to heat stress (37 degrees C) was detected in the insoluble protein fractions. Several binding proteins were isolated from rat testis using a HSP105 antibody immunoaffinity column, and p53, the tumor suppressor gene product, was copurified with these. Furthermore, immunoprecipitation using antibodies to p53 led to coprecipitation of HSP105 together with p53 after culturing germ cells at 32.5 degrees C, but not at 37 or 42 degrees C. In conclusion, HSP105 is specifically localized in the germ cells and may translocate into the nucleus after heat shock. HSP105 is suggested to form a complex with p53 at the scrotal temperature, and dissociate from it at suprascrotal temperatures. At scrotal temperature, HSP105 may thus contribute to the stabilization of p53 proteins in the cytoplasm of the germ cells, preventing the potential induction of apoptosis by p53.     

Heat-induced preferential synthesis and redistribution of HSP 70 and 28 families in Chinese hamster ovary cells

We observed that members of two HSP families (70 and 28 kDa) preferentially redistributed into the nucleus after heating at 45.5 degrees C for 10 min. The rates of synthesis and redistribution of these proteins were different for each member of HSP families during incubation period at 37 degrees C after heat shock. The maximum rates of synthesis of HSP 70 and HSP 28 families, except HSP 28c, were 6-9 hr after heat shock, whereas the maximum rates of redistribution were 3-6 hr after heat shock. These results suggest that the rates of redistribution of these proteins may be dependent on the amount of intracellular proteins as well as the alteration of binding affinity of nucleoproteins following heat shock.     

Heat stress protection against mesenteric I/R-induced alterations in intestinal mucosa in rats.

Prior induction of heat shock protein 70 (HSP70) protects against ischemia-reperfusion (I/R) mucosal injury, but the ability of HSP70 to affect I/R-induced alterations in epithelial cell function is unknown. Rats subjected to whole body hyperthermia (41.5-42 degrees C for 6 min) increased HSP70 and heat shock factor 1 mRNA expression, reaching a maximum 2 h after heat stress and declining thereafter. HSP70 production was maximally elevated at 4 h after heat stress and remained elevated until after 12 h. Heat stress alone had no effect on mucosal function except to enhance secretion in response to ACh. Heat stress provided complete morphological protection against I/R-induced mucosal injury but did not confer a similar protection against I/R-induced decreases in mucosal resistance, sodium-linked glucose absorption, or tachykinin-mediated chloride secretion. Heat stress, however, attenuated the I/R-induced suppression of ACh response, and this effect was dependent on enteric nerves. Thus induction of heat shock protein 70 is associated with the preservation of mucosal architecture and attenuation of some specific functional alterations induced by I/R.     

Development of acute thermotolerance in L929 cells: lack of HSP28 synthesis and phosphorylation.

We investigated the correlation between the development of acute thermotolerance and the phosphorylation, synthesis, and expression of the HSP28 family in murine L929 cells. Following heating at 43 degrees C for 30 min, thermotolerance developed rapidly in exponential-phase cells and reached its maximum 4-9 h after heat shock. Maximal thermal resistance was maintained for 24 h and then gradually decayed. However, heat-induced phosphorylation of HSP28 was not detected. Furthermore, HSP28 synthesis during incubation at 37 degrees C for 12 h following heat shock was not detected by [3H]-leucine labeling followed by two-dimensional polyacrylamide gel electrophoresis. In addition, Northern blots failed to demonstrate expression of the HSP28 gene. Unlike HSP28, the expression of constitutive and inducible HSP70 genes, along with the synthesis of their proteins, was observed during incubation at 37 degrees C after heat shock. These results demonstrate that HSP28 synthesis and its phosphorylation are not required to develop acute thermotolerance in L929 cells.     

Effect of temperature and duration of hyperthermia on HSP72 induction in rat tissues

The aim of the present study was to determine whether heat shock protein 72 (HSP72) is induced in a heated rat model at rectal temperatures below 42 degrees C. Rats were divided into a control group and six groups (n = 6) heated to different rectal temperatures: 39 degrees C for 1 h (39), 40.0 degrees C for either 15 min (40S) or 1 h (40L), 41.0 degrees C for either 15 min (41S) or 1 h (41L) and 42.0 degrees C for 15 min (42). Tissues were sampled 4 h after heating. Following 1 h at 40.0 degrees C, HSP72 was significantly elevated in heart (p < 0.005), but not in gut or liver tissue. In all three tissues, HSP72 was significantly elevated under the conditions 41L and 42 compared to control tissue (p < 0.005). Marked differences were found in the amount of HSP72 induced in different tissues in response to the same heat stress. Duration of heating was important in modulating HSP72 induction, with a significantly greater induction of HSP72 following 1 h compared to 15 min at 41 degrees C in all three tissues (p < 0.02). A correlation was found between thermal load and HSP72 content in liver, heart (both p < 0.01) and gut (p < 0.001) for the rats heated to 41 and 42 degrees C. These data show that HSP72 is induced at temperatures below 42 degrees C, with striking differences between tissues.