Heat Shock Protein Content Research Articles

Adaptation to oxidative stress in ageing.

The oxygen uptake by skeletal muscle may increase by two orders of magnitude during contraction and a number of reactive oxygen species are released into the muscle extracellular space during contractile activity. The release of specific free radical species has been studied using a number of different model systems, including detection in the venous effluent from contracting muscles in vivo [4,5], the perfusate of contracting strips of muscle in vitro [6] and the medium surrounding contracting myotubes in culture [3]. Skeletal muscle cells appear to release greatly increased amounts of superoxide anion into the external medium contraction (Fig. 1 and [3]) together with nitric oxide (NO). In addition studies with in vivo models indicate that contraction induces an increase in the hydroxyl radical activity in the venous effluent from skeletal muscle [4], an increase that appears to be dependant upon the presence of iron in the extracellular space [4]. This contraction-induced rise in oxidant production appears to lead to an increase in indicators of lipid, DNA or protein oxidation in skeletal muscle where the amount of contractile activity is unaccustomed or excessive. There is however little evidence that repeated exposure of skeletal muscle to these various oxidants generated during contractile activity leads to sustained damage to the tissue or to accelerated ageing of the tissue ([1] for a review). The most likely explanation for this is that the skeletal muscle cells adapt to the oxidative stress of contractions to reduce the risk of damage to the tissue by any subsequent increases in free radical activity. In both animals and humans, an acute bout of exercise or contractile activity leads to an upregulation of the activity of antioxidant enzymes, such as superoxide dismutase or catalase [2,3]. In addition, other cytoprotective proteins, such as the heat shock proteins, are important additional components of the cellular protective response against reactive oxygen species and an increase in the muscle content of heat shock proteins occurs following contractile activity of muscle in mice [3] or exercise in man [2]. The above adaptations only appear to represent part of the overall responses of skeletal muscle to contraction-induced oxidative stress and, in recent studies, we have attempted to delineate the mechanisms by which skeletal muscle adapts to the oxidative stress component of contraction using a combination of biochemical analyses, mRNA differential display and cDNA microarray techniques. These data appear to indicate a co-ordinated response to oxidative stress involving a reduction in potential enzymatic sources of generation of reactive oxygen species and an upregulation of protective proteins. There is evidence to indicate that this ability to adapt to unaccustomed oxidative stress is attenuated during ageing. A failure to adapt to environmental change is a general characteristic of the ageing process and this has been explored in some detail for the heat shock response. Cells and tissues from

Role of thyroid hormones in stress-induced synthesis of heat-shock proteins in the myocardium

Thyroxine in near-physiological doses increased the content of heat-shock proteins in the myocardium and stimulated their accumulation during immobilization stress. Blockade of thyroid functions with methimazole decreased the content of heat-shock proteins in rat myocardium during stress and heat shock and prevented their accumulation during adaptation to short-term immobilizations.

Cytoprotective Mechanism of Heat Shock Protein 70 against Hypoxia/Reoxygenation Injury

The role of heat shock protein 70 (HSP70) in the cytoprotection against hypoxia/reoxygenation injury was examined. Adult rat cardiomyocytes were isolated, subjected to hyperthermia at 42°C for 15 min (heat shock treatment), and then incubated at 37°C for 3 to 24 h (HSP production process). Heat shock treatment increased HSP70 production (80–260% increase); the peak increase was seen after 9 h of HSP production process. Thereafter, the cells were subjected to 120-min hypoxia and 15-min reoxygenation. Heat shock treatment increased the survival of the cells subjected to hypoxia/reoxygenation (1.5–2.5-fold); the maximal cytoprotection was observed after 12 h of HSP production process. Heat shock treatment increased HSP70 content in the nucleus when cells were subjected to 12 h of HSP production process. To examine the role of HSP70 accumulation in the nuclear fraction, the activity of poly(ADP-ribose) synthetase (PARS), which functions in the nucleus and consumes high-energy phosphates excessively in the reoxygenated state, were measured in the cells with heat shock and 12 h of HSP production process. Heat shock treatment attenuated the hypoxia/reoxygenation-induced increase in the PARS activity (50% decrease). Treatment of the cells with 3-aminobenzamide, an inhibitor of PARS, exerted the effects similar to those of heat shock treatment. These results suggest that attenuation of the PARS activity in the nucleus may play an important role in the cytoprotective effect of HSP70 on hypoxia/reoxygenation injury.

Endurance training increases the expression of mitochondrial and nuclear encoded cytochrome c oxidase subunits and heat shock proteins in rat skeletal muscle.

Cytochrome c oxidase (CCO) is an enzyme complex found on the inner mitochondrial membrane and serves as the final electron acceptor in mitochondrial electron transport. Heat shock proteins (HSPs) are involved in the import of nuclear encoded protein subunits into the mitochondria and induce conformational changes to form active enzyme complexes. As both the nuclear and mitochondrial encoded subunits of CCO have been shown to increase in activity and expression in muscle subsequent to artificial loading, and as exercise has been shown to induce HSPs, we sought to determine whether 16-20 weeks of treadmill exercise would result in enhanced CCO subunit expression, and to determine if there was a relationship between this expression and HSP content in medial gastrocnemius muscle of Fischer 344 rats. Our results indicated that endurance training resulted in a 53%, 87% and 80% increase (P<0.05) in the levels of HSP 60, CCO subunit II and CCO subunit VI, respectively. Enzymatic activity of CCO was 84% greater (P<0.05) after endurance training. Mann Whitney U analyses showed that CCO subunit II and VI increased to the same extent as HSP 60 after endurance training. It appears that 16-20 weeks of endurance training leads to uniform increases in CCO subunits and parts of the transport and assembly mechanisms required for CCO enzyme assembly. The similarity among the increases in CCO subunits II and VI protein levels and the increase in CCO enzyme activity suggest that this increase in activity is due to an increase in the amount of CCO enzyme.

Ischemic tolerance conferred to cultured rat neurons by heat shock is not mediated by opening of adenosine triphosphate-sensitive potassium channels

The effect of sublethal heat shock on the capacity of neurons to resist subsequent ischemia-reperfusion-induced cell injury, was studied in a model of primary rat neuronal cultures, subjected to chemical ischemia. Exposure of the cultures to sublethal heat shock (42°C; 20 min) resulted in elevation in cellular content of heat shock protein (HSP)-70, at 4 h following the shock, and in acquisition of a 15 h ‘time window of protection’ against ischemia-reperfusion insult, with maximum protection at 4 h. Presence in the culture medium of glibenclamide (2 μM), a blocker of ATP sensitive potassium (KATP) channels, did not abolish the acquisition of protection throughout the entire duration of the acquired ‘time window of protection’. The results demonstrate that heat shock induces in neurons a protective mechanism against ischemia-reperfusion insult, probably associated with enhanced expression of HSPs, which does not depend on opening of KATP channels. In this respect, the neuronal ‘heat-shock mechanism’ for the acquisition of ischemic tolerance differs from the neuronal ‘adenosine mechanism’ and probably also from the heart ‘heat shock mechanism’ for the acquisition of protection.

Heat-shock protein 72/73 and impaired wound healing in diabetic and hypercortisolemic states

Background: Impaired wound healing is a well-documented phenomenon in experimental and clinical diabetes. Emerging evidence favors the involvement of glucocorticoids (GCs) in the pathogenesis of this diabetic complication. Recent data indicated that a heat-shock protein (HSP) with a molecular weight of about 70 kd is expressed in wound healing and it is under the control of the hypothalamic-pituitary-adrenal axis. In view of these findings, the current study was designed to examine the influence of diabetes and the hypercortisolemic state on the expression of HSP 72/73 during wound healing. Methods: Induction of diabetes was achieved by the intravenous injection of streptozotocin at a dose of 55 mg/kg. Subcutaneously implanted polyvinyl alcohol (PVA) sponges were used as a wound healing model. Control and diabetic animals received, respectively, subcutaneous 30-day timed-release pellets of GC (200 mg) and RU 486 (25 mg). Corresponding animals received placebo pellets. Expression of HSP 72/73 within the PVA sponges was assayed with use of Western blotting and immunohistochemical techniques. Results: GCs caused a Cushing-like syndrome with weight loss and adrenal atrophy. A pronounced accumulation of constitutive HSP 72/73 was observed in the cytoplasm of various cell types including fibroblasts, macrophages, and endothelium of nondiabetic controls. The PVA sponge contents of HSP 72/73 were decreased as a function of diabetes. A similar phenomenon was seen in control animals receiving high doses of GCs. Partial normalization of the associated hyperglycemic and hypercortisolemic states of diabetes with insulin (hyperglycemia) and the GC receptor block RU 486 (hypercortisolemia) ameliorated the diabetes-related decrease in PVA sponge contents of HSP 72/73. Conclusions: The current study provides evidence that both diabetes and the hypercortisolemic state are associated with a reduction in PVA sponge content of HSP 72/73. An amelioration of these changes was achieved by the institution of RU 486 therapy. Although our data may point to the possibility that the diabetes-related decrease in HSP 72/73 is mediated at least in part by GCs, a confirmation regarding this premise awaits further investigation. (Surgery 1999;125:594-601.)

Acute exercise can improve cardioprotection without increasing heat shock protein content.

The aim of this study was to determine the effects of acute bouts of exercise on myocardial recovery after ischemia and heat shock protein expression. Adult female Sprague-Dawley rats were divided into five groups: 1) 1-day run (1DR; n = 6) and 2) 3-day run (3DR; n = 7), in which rats ran for 100 min at a speed of 20 m/min up a 6 degrees grade for 1 or 3 consecutive days; 3) 1-day cold run (1CR), in which rats ran the same as 1DR but with wet fur at 8 degrees C, which prevented an elevation of core temperature (n = 8); 4) heat shock sedentary (HS), in which rats had their core temperatures raised to 42 degrees C one time for 15 min (n = 5); and 5) sedentary control (n=15). Cardiac function was analyzed 24 h after the last treatment using an isolated, working heart model. Nonpaced hearts were initially perfused under normoxic conditions, then underwent 17 min of global, normothermic (37 degrees C) ischemia, and, finally, were allowed to recover for 30 min under normoxic conditions. The concentration of the 72-kDa heat shock protein (HSP 72) was measured in each left ventricle. Compared with that in the sedentary group, recovery of cardiac output x systolic pressure (CO x SP) was enhanced (P < 0.05) in all treatment groups when the postischemic value was covaried with the preischemic value. No differences in CO x SP were found (P > 0.05) between the following groups: 1DR vs. 3DR, 1DR vs. HS, and 1DR vs. 1CR. Heat shock protein concentration was significantly greater (P < 0.05) than that in the sedentary controls in HS, 1DR, and 3DR groups, but not for 1CR. The concentration of HSP 72 was not significantly correlated with postischemic CO x SP (R2 = 0.197, P > 0.05). We conclude that acute bouts of exercise can produce cardioprotective effects without an elevation of HSP 72.

Inhibition of NaCl-induced heat shock protein 72 expression renders MDCK cells susceptible to high urea concentrations.

Exposure of Madin-Darby canine kidney (MDCK) cells to elevated extracellular NaCl concentrations is associated with increased heat shock protein 72 (HSP72) expression and improved survival of these pretreated cells upon exposure to an additional 600 mM urea in the medium. To establish a causal relationship between HSP72 expression and cell protection against high urea concentrations, two approaches to inhibit NaCl-induced HSP72 synthesis prior to exposure to 600 mM urea were employed. First, the highly specific p38 kinase inhibitor SB203580 was added (100 microM) to the hypertonic medium (600 mosm/kg H2O by NaCl addition, 2 days of exposure), which significantly reduced HSP72 mRNA abundance and HSP72 content. Survival of these cells after a 24-h urea treatment (600 mM) was markedly curtailed compared with appropriate controls. Second, a pcDNA3-based construct, containing 322 bases of the HSP72 open reading frame in antisense orientation and the geneticine resistance gene, was transfected into MDCK cells. Clones with strong inhibition of HSP72 synthesis and others which express the protein at normal levels (comparable to nontransfected MDCK cells) after heat shock treatment or hypertonic stress were established. When these transformants were subjected to hypertonic stress for 2 days prior to exposure to an additional 600 mM urea for 24 h, cell survival was significantly reduced in those clones in which HSP72 expression was strongly inhibited. These results provide further evidence for the protective function of HSP72 against high urea concentrations in renal epithelial cells.

Different constitutive heat shock protein 70 expression during proliferation and differentiation of rat C6 glioma cells.

Analysis of constitutive heat shock protein 70 (HSC70) concentration in unstressed proliferating and differentiated rat C6 glioma cells revealed a striking reduction in the amount of HSC70 in differentiated cells. Proliferating cells showed a significantly higher HSC70 concentration, particularly observable during S phase in synchronous cultures. The activity of the cAMP/PKA signaling pathway was enhanced in differentiated cells. cAMP-elevating treatments both inhibited growth and reduced HSC70 concentration. Inactivation of PKA by H-89 upregulated the reduced HSC70 expression in differentiated cells and stimulated proliferation. Treatment with an inhibitor of MAP kinase activation (PD98059) reduced the HSC70 concentration. We assume that cAMP does not directly inhibit HSC70 expression by transcriptional repression, but by its inhibitory effect on the MAP kinase pathway.

Induction of heat shock protein 27 by hydroxyurea and its relationship to experimental metastasis.

Treatment of tumor cells with hydroxyurea (HU) has been shown to increase the experimental metastatic potential of these cells. We have previously described the induction of stress proteins (antioxidants) by HU in B16 murine melanoma cells and their relationship to the metastatic process. We have now investigated the induction by HU of another set of stress proteins, the heat shock proteins, and their role in experimental metastasis. HU markedly increased the cellular content of heat shock protein (hsp) 27 but not of hsp 90, 72/73, or 60 as measured by immunoblotting. The induction of hsp27 protein was preceded by a specific increase in hsp27 mRNA. Furthermore, HU-treated cells were more thermotolerant. To investigate the functional role of hsp27, human hsp27 cDNA was constitutively overexpressed in B16 cells at levels seen in HU-treated cells. In separate experiments, we induced a global increase in hsps by heat shock. Neither the hsp27 transfectants nor the heat-shocked cells demonstrated an increase in their experimental metastatic capacity. We conclude that hsp27 protein is increased by HU by the specific induction of hsp27 mRNA in B16 melanoma cells but increased hsp27 protein is not responsible for the increase in experimental metastasis. Since high levels of hsp27 are associated with metastatic disease in breast and ovarian cancers, but not in our experimental system, the functional role of hsp27 in metastasis requires further study.

The constitutive heat shock protein-70 is required for optimal expression of myelin basic protein during differentiation of oligodendrocytes.

To further elucidate the role of the constitutive heat shock protein-70 (HSC70) as a chaperone for the synthesis of myelin basic protein (MBP), HSC70 content was decreased in oligodendrocyte precursor cells prior to MBP expression either by transfection with an antisense oligonucleotide specific for HSC70, or by exposure to low levels of quercetin, a bioflavonoid known to decrease synthesis of HSC70. As these cells underwent differentiation in vitro, antisense treatment decreased HSC70 levels to 66% of controls. At the same time, a sharp induction resulted in the stress-inducible heat shock protein-70 (HSP70). Levels of two other stress proteins increased as well, namely, the 25-kDa heat shock protein (HSP25) and the 78-kDa glucose regulated protein (GRP78). MBP synthesis proceeded over a normal time course, but at only 50% of control values. As HSC70 content returned to normal, MBP synthesis was also restored to normal levels. Quercetin reduced the expression of HSC70 to an even greater extent than transfection, and prevented the induction of HSP70. In contrast to antisense-treated cells, MBP synthesis was essentially blocked in quercetin-treated cells even though levels of HSP25 and GRP78 increased. Taken together, these observations (a) indicate that HSP70 partially compensates for decreased chaperoning of nascent MBP by HSC70 (HSC70 and HSP70 are closely related and perform similar functions); (b) preclude the involvement of HSP25 and GRP78 in MBP synthesis; and (c) emphasize the requirement of HSC70 for optimal synthesis of MBP.

Heat‐Shock Protein 70 and Dough‐Quality Changes Resulting from Heat Stress During Grain Filling in Wheat

ABSTRACTIn an attempt to further elucidate the molecular mechanisms that determine the loss of dough strength associated with heat stress of growing wheat, the roles of heat‐shock proteins (HSP) and heat‐shock elements upstream of glutenin genes were investigated. A range of genotypes differed in the extent of synthesis of high molecular weight glutenin subunits (HMW‐GS) and HSP during heat stress. The concentration of HSP 70 remaining in mature grain increased as a result of a few days' heat stress of wheat plants. The amount of HSP 70 in mature grain samples from heat‐stressed plants of 45 genotypes was not strongly correlated with loss of dough strength. There was much less evidence for this mechanism than for other molecular hypotheses from the literature, particularly, changes in glutenin‐to‐gliadin ratio, size distribution of the glutenin polymer, and the involvement of HSP and chaperones during grain‐protein synthesis. HSP 70 was purified from heat‐stressed grain, and was added to (or incorporated into) dough in the direct‐drive mixograph. The HSP behaved similarly to several other hydrophilic proteins when added at a level of 2 mg/2 g of flour. It showed no dramatic effects on dough properties that could constitute a major explanation for the dough‐weakening effects of heat stress, even though the level of addition was well above the maximum levels that might be encountered in field‐grown, mature grain. Furthermore, sequencing of the genes (upstream of the coding region) for HMW‐GS failed to show the presence of heat‐shock promoters, even for genotypes that differed considerably in their reactions to heat stress. The findings simplify the range of possibilities that cause heat‐related loss of dough strength, focusing attention on the degree of polymerization of the glutenin chains, and on the roles of HSP and chaperones in the developing grain.

Relative induction of heat shock protein in coronary endothelial cells and cardiomyocytes: Implications for myocardial protection

Objectives: Induction of the 70 kd heat shock protein in the heart is known to exert a protective effect against postischemic mechanical and endothelial dysfunction. However, the exact site of induction and the mechanisms involved remain unknown. The aim of this study was to investigate the relative capacity of endothelial and myocardial cells to express the 70 kd heat shock protein in response to heat stress, as well as their significance. Methods: (1) Postischemic recovery of cardiac mechanical and endothelial function was studied in isolated rat hearts with and without endothelial denudation with saponin. (2) Semiquantitative determination of induction of 70 kd heat shock protein by Western immunoblotting was performed in the whole cardiac homogenate, in isolated cardiac myocytes, and in coronary endothelial cells. (3) Immunocytochemistry was used to visualize the distribution of induction of 70 kd heat shock protein in both cell types. Results: Postischemic recovery (percent preischemic value ± standard error of the mean) of cardiac output in hearts from heat-stressed animals was significantly improved (66.7 ± 6.9 vs 44.5 ± 4.5 in the control group, p < 0.01). In heat-stressed hearts treated with saponin no improvement in the recovery of cardiac output was noted (44.7 ± 6.9 in heat-stressed hearts vs 38.0 ± 4.0 in heat-stressed, saponin-treated hearts, p = not significant). Endothelial function (as assessed by the vasodilatory response to the endothelium-dependent vasodilator 5-hydroxytryptamine) improved from 31.0 ± 5.2 in the control group to 65.8 ± 7.1 in heat-stressed hearts ( p < 0.02 vs control) and dropped to –1.9 ± 3.8 in heat-stressed hearts treated with saponin. Immunocytochemistry showed that only sections of hearts from heat-treated rats showed a strong specific reaction with heat shock protein antibody. The positive staining was seen in endothelial cells. Induction of 70 kd heat shock protein content in the whole cardiac homogenate from heat-stressed rats as measured by Western immunoblotting was 5.2 ± 1.9 (vs 0.0 in non-heat–stressed rats, p < 0.0001) and dropped to 0.0 in heat-stressed hearts treated with saponin. The tentative amount of 70 kd heat shock protein was 18.1 ± 7.8 in isolated endothelial cells from heat-stressed hearts and 2.3 ± 2.3 in isolated cardiac myocytes ( p < 0.01 vs endothelial cells). Conclusions: Coronary endothelial cells are the main site of induction of 70 kd heat shock protein in the heart and appear to contribute to the protective effects of heat stress on the recovery of mechanical and endothelial function. (J Thorac Cardiovasc Surg 1998;115:200–9)

Solute composition and heat shock proteins in rat renal medulla.

The high content of heat shock proteins (HSPs) 25 and 72 in the hyperosmotic inner medulla of the concentrating kidney has been ascribed to the high NaCl and urea concentrations in this kidney zone. To assess the effects of variations in the composition of solutes in the renal medulla on the intrarenal distribution of HSPs, rats were fed either a high- or low-Na diet for 3 weeks. These diets result in greatly differing urine and inner medullary solute composition. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blot techniques were used to analyse HSP25 and HSP72 in the cortex, outer medulla and inner medulla. In addition, the amounts of organic osmolytes (sorbitol, myo-inositol, betaine and glycerophosphorylcholine) and urea in the tissue were determined by high-performance liquid chromatography. Intra- and extracellular electrolyte concentrations at the papillary tip were measured by electron microprobe analysis. In the high-Na group, urine osmolality was about 1000 mosmol/kg lower than in rats fed a low-Na diet, due to lower urea concentrations. The sum of urine sodium and potassium concentrations, however, did not differ between the two groups. Neither in the outer nor in the inner medulla was the sum of the concentrations of organic osmolytes affected by the dietary treatment. The sum of sodium, potassium and chloride concentrations did not differ between the two experimental groups, neither in the interstitial nor in the intracellular compartments. However, the urea content and the amounts of HSP25 and HSP72 were significantly lower in the inner medulla of the group of rats fed a high-Na diet. Our results suggest that urea participates in the regulation of the medullary levels of the HSPs and that both HSP25 and HSP72 are components of mechanisms protecting medullary cells against the deleterious effects of high urea concentrations.

Compensatory up-regulation of cardiac SR Ca2+-pump by heat-shock counteracts SR Ca2+-channel activation by ischemia/reperfusion.

We tested the hypothesis that heat-shock protected myocardial Ca2+-cycling by sarcoplasmic reticulum from ischemia and reperfusion (I/R) injury. Twenty-four hours after increasing body temperature to 42 degrees C for 15 min, rat hearts were isolated, Langendorff-perfused, and subjected to 30 min ischemia then 30 min reperfusion. Left ventricles were homogenized and their ionized Ca2+ concentration monitored with indo- during Ca2+-uptake in the presence and absence of the Ca2+-release channel (CRC) modulator ryanodine. Tissue content of heat-shock protein 72 (HSP 72) was analyzed. Exposure to I/R resulted in a 37% enhancement of CRC activity but no effect on Ca2+-pumping activity, resulting in 25% decreased net Ca2+-uptake activity. Pre-exposure to heat-shock resulted in a 10-fold increase in HSP 72, and a 25% enhancement of maximal Ca2+-pumping activity which counteracted the effect of I/R on CRC and net Ca2+-uptake activities. This protection of SR Ca2+-cycling was associated with partial protection of myocardial physiological performance. Net Ca2+-uptake activity was correlated with the left ventricular developed pressure and its rate of change. We conclude that one of the mechanisms by which heat-shock protects myocardium from I/R injury is to upregulate SR Ca2+-pumping activity to counteract the enhanced SR Ca2+-release produced by I/R.

Induction of Tolerance to Hypothermia by Previous Heat Shock Using Human Fibroblasts in Culture

The technique of organ preservation is limited by the amount of time which organs can be hypothermically stored. A potential method to effectively extend reliable storage times involves the conditioning of cells to better withstand hypothermia by previous exposure to a less severe stress. Using human fibroblasts in culture, we have demonstrated that such an approach may be feasible. Subjecting human diploid IMR-90 fibroblasts to 5 h 42.5°C heat shock was found to improve cell survival more than 10-fold to subsequent 4°C hypothermic exposure. The prior heat shock resulted in the increased synthesis of heat shock proteins (HSPs), the absolute concentrations of which were measured by an assay which utilized sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blotting techniques. Both the degree of cold tolerance conferred upon IMR-90 cells and the levels of HSP70 and HSP27 were dependent upon initial heat shock duration. Induced cold tolerance was found to be reversible; longer recovery times at 37°C following heat shock resulted in a loss of this cold-tolerant state as well as a disappearance of HSPs. The fact that the degree of cold tolerance and HSP concentrations showed similar trends with respect to both heat shock time at 42.5°C and subsequent recovery time at 37°C suggests that these proteins may be intimately involved in the induction of cold tolerance.

Nitrogen Availability and Vegetative Development Influence the Response of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase, Phosphoenolpyruvate Carboxylase, and Heat-Shock Protein Content to Heat Stress in Zea mays L.

We examined the influence of plant nitrogen (N) status and vegetative development on the accumulation of ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit (rubisco), phosphoenolpyruvate carboxylase (pepcase), and the heat-shock proteins Hsp24 and Hsp60 during and after heat stress in corn (Zea mays L.) to explore the possibility that much HSP-N may originate directly or indirectly from photosynthetic proteins in leaves. In general, rubisco and pepcase content decreased in response to a 45⚬C heat stress compared with unstressed controls (28⚬C). Rubisco and pepcase declined relative to total detergent-soluble protein, which was unaffected by heat stress. Plants provided with lower levels of available N during growth and that had lower total protein content (low-N plants) allocated a greater fraction of total protein to rubisco and pepcase in recently expanded leaves and exhibited greater relative decreases in rubisco and pepcase than high-N plants. The decreases in low-N plants became evident earlier during the 20-h heat stress, whereas decreases in high-N plants occurred later during heat stress or during recovery from stress, when levels of some HSPs (e.g., Hsp60) were still increasing. Decreases in rubisco and pepcase were greater in adult, compared with juvenile, vegetative plants that had greater protein content, but less rubisco and pepcase, than adults. Heat stress appeared to delay ontogenetic changes in rubisco and pepcase content in leaves in some instances. These results, consistent with our previous evidence indicating that HSP production may be N costly, indicate that abundant soluble photosynthetic enzymes such as rubisco and pepcase may supply much of the N required for the heat-stress response in plants, particularly in mature leaves.

Heat shock proteins and circadian rhythms.

Significant circadian rhythms in heat shock gene expression were observed in a prokaryotic species (Synechocystis). In eukaryotes, in contrast, several heat shock genes (constitutive and inducible) were shown to be constantly expressed. A few cases of circadian expression of heat shock proteins (HSPs), however, have been reported. Significant circadian changes of thermotolerance were observed in yeast and several plant species. Higher thermotolerance can be attributed to a higher abundance of HSPs, but also to other adaptive mechanisms. Zeitgeber effects of temperature changes can be explained on the basis of their direct effects on the state variables of the clock gene (per,frq) expression and its negative feedback loop. Effects of increased HSP concentrations, as observed after heat shock, but also after light and serotonin (5HT), appear possible, in particular with respect to nuclear localization of the clock (PER) protein, but these effects have not been documented yet. Thus, the role of HSPs in the circadian clock system is little understood and, from our point of view, deserves more attention.

Depressed expression of the inducible form of HSP 70 (HSP 72) in brain and heart after in vivo heat shock.

The heat shock gene expression plays a role in the protection of cells from injury. In the present study, we have analyzed the expression of heat shock protein (HSP) 72 (the major inducible form of the HSP 70 family) in different rat organs after a total body hyperthermia. The content of HSP 72 was greatest in liver and colon. In contrast, accumulation of HSP 72 was low in heart and brain (3-5% and < 1% of the amount in liver, respectively). This low expression of HSP 72 in heart and brain could not be explained by a difference in the actual temperature within these organs. Analysis of cells in culture that resemble hepatocytes, myoblast, and neurons showed a pattern of HSP 72 expression similar to that observed in liver, heart, and brain in vivo after heat shock. These results suggest that this disparate expression of HSP 72 is due to intrinsic characteristics of the cell types rather than to physiological or environmental conditions. The differential expression of HSP 72 among different cell lines could be correlated with the different levels of protein synthesis protection.

Differential Distribution of 70-kD Heat Shock Protein in Atherosclerosis

Smooth muscle cell death may contribute to necrotic plaque rupture and subsequent thromboembolus. Stress-induced synthesis of heat-shock proteins (HSPs) normally protects cells from death, but vascular HSPs may become insufficient as cytotoxicity increases in advanced plaques. To determine whether vascular HSP content is altered near necrosis, we compared 70-kD HSP (HSP70) distribution between fibrotic and necrotic plagues in immunostained carotid endarterectomy specimens. Average levels of HSP70 immunoreactivity were compared by video densitometry between fibrotic and necrotic plaques or between their underlying media. Both necrotic plaques and their underlying media contained significantly more HSP70 staining than did fibrotic tissues. To test whether cellular HSP70 correlated with resistance to toxicity in vitro, aortic smooth muscle cells (aSMCs) were heat shocked to induce endogenous HSPs or given 2 to 50 micrograms/mL purified HSP70. Cells were then serum deprived or exposed to 12 to 96 mumol/L cholestanetriol (C3ol) or 25-hydroxycholesterol, and survival was determined. Cellular HSP70 content was assayed by immunoblotting, and protein synthesis was monitored by 35S radiolabeling. Serum deprivation inhibited general protein synthesis but induced HSP70; C3ol exposure inhibited both overall protein and HSP70 synthesis, including post-heat shock. Induction of endogenous HSPs or 10 micrograms/mL exogenous HSP70 improved viability of serum-deprived cells (P < .05 and P < .01, respectively), while only exogenous HSP70 protected against C3ol (P < .002). The results suggest that insufficient HSP70 accumulates in aSMCs residing near necrosis to protect against plaque toxicity; aSMC death might then occur, allowing resident macrophages to degrade and destabilize the matrix, leading to rupture.