Hyperthermia Induces the Synthesis of a Heat Shock Protein by Polysomes Isolated from the Fetal and Neonatal Mammalian Brain

Abstract: Analysis of the cell-free translation products of polysomes isolated from fetal brain and other organs indicates that elevation of maternal body temperature induces the synthesis of a heat shock protein of molecular weight 74,000 (74K). The newborn mammal is particularly sensitive to induction of the 74K protein. As early postnatal development proceeds, higher body temperatures are required to induce synthesis of the 74K heat shock protein.     

Effects of p,p''-DDT on the Rat Brain Concentrations of Biogenic Amine and Amino Acid Neurotransmitters and Their Association with p,p''-DDT-Induced Tremor and Hyperthermia

Male, Fischer strain 344 adult rats were given various doses (25-100 mg/kg) of p,p'-DDT by oral gavage, and levels of biogenic amines, their metabolites, and amino acid neurotransmitters, tremor activity, and rectal temperature were measured at several intervals (2, 5, 12, and 24 h) after dosing. Dose-related increases in rectal temperature and in tremor activity were observed at 50-100 mg/kg 12 h after dosing. Tremorigenic doses of DDT increased the 5-hydroxyindoleacetic acid (5-HIAA) level in hypothalamus, brainstem, and striatum, whereas doses of 75 and 100 mg/kg increased the 3-methoxy-4-hydroxyphenylglycol (MHPG) level in hypothalamus and brainstem and the 3,4-dihydroxyphenylacetic acid level in striatum. Six amino acids were assayed in the brainstem, hypothalamus, and striatum; aspartate and glutamate levels were increased only in brainstem at 25-100 mg/kg. No consistent changes in concentrations of taurine, glutamine, glycine, or gamma-aminobutyric acid were observed in any of the regions assayed. Time-related increases in rectal temperature were seen 2-12 h after dosing, and the presence of tremor was observed 5-12 h after dosing; for both the time of peak effect was at 12 h. The DDT-induced hyperthermia and tremor were associated with dose- and time-related increases in levels of 5-HIAA, MHPG, aspartate, and glutamate. It is suggested that an increase in the turnover rate of 5-hydroxytryptamine (5-HT) may be responsible for the DDT-induced hyperthermia, whereas increases in the metabolism of 5-HT and norepinephrine may be involved in the tremor.     

Heat Shock RNA Levels in Brain and Other Tissues After Hyperthermia and Transient Ischemia

A number of studies have demonstrated increased synthesis of heat shock proteins in brain following hyperthermia or transient ischemia. In the present experiments we have characterized the time course of heat shock RNA induction in gerbil brain after ischemia, and in several mouse tissues after hyperthermia, using probes for RNAs of the 70-kilodalton heat shock protein (hsp70) family, as well as ubiquitin. A synthetic oligonucleotide selective for inducible hsp70 sequences proved to be the most sensitive indicator of the stress response whereas a related rat cDNA detected both induced RNAs and constitutively expressed sequences that were not strongly inducible in brain. Considerable polymorphism of ubiquitin sequences was evident in the outbred mouse and gerbil strains used in these studies when probed with a chicken ubiquitin cDNA. Brief hyperthermic exposure resulted in striking induction of hsp70 and several-fold increases in ubiquitin RNAs in mouse liver and kidney peaking 3 h after return to room temperature. The oligonucleotide selective for hsp70 showed equivalent induction in brain that was more rapid and transient than observed in liver, whereas minimal induction was seen with the ubiquitin and hsp70-related cDNA probes. Transient ischemia resulted in 5- to 10-fold increases in hsp70 sequences in gerbil brain which peaked at 6 h recirculation and remained above control levels at 24 h, whereas a modest 70% increase in ubiquitin sequences was noted at 6 h. These results demonstrate significant temporal and quantitative differences in heat shock RNA expression between brain and other tissues following hyperthermia in vivo, and indicate that hsp70 provides a more sensitive index of the stress response in brain than does ubiquitin after both hyperthermia and ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of hyperthermic killing cultured mammalian cells by treatment with anisotonic NaCl or medium solutions

The exposure of cultured Chinese hamster cells (CHO) to anisotonic medium increased the cellular sensitivity to heat treatment at 42.3°C. A greater potentiation of heat killing is observed when the anisotonic solution consists of pure NaCl in water compared to growth medium made anisoltonic by dilution or by addition of NaCl. Hypertonic treatment caused greater heat sensitization than hypotonic treatment. Thermal tolerance observed in the control cells after 4–6 hours of heating in medium was also observed for cells exposed to anisotonic media during heating if the heating period was greater than 4 h. The exposure of cells to anisotonic media during heating if the heating period was greater than 4 h. The exposure of cells to anisotonic NaCl solutions during heating removed the shoulder from the heat survival curve, while the curves for cells heated in medium made anisotonic retained their shoulders. These studies suggest: (1) that either the plasma membrane is a primary target for heat inactivation of mammalian cells, or (2) that changes in intracellular ion concentrations enhance thermal damage occurring in critical intracellular structures.     

Effects of Hyperthermic Conditions on the Reactivity of Oxygen Radicals

Generation and reactivity of superoxide (0₂^?) and hydroxyl (OH') radicals in enzymatic and radiolytic systems were investigated over the temperature range from 20^o-50^oC. The generation rate and reaction kinetics of both enzymatically and radiolytically produced superoxide radicals were determined by a cytochrome c reduction assay. For OH' radical reaction studies the degradation of hyaluronic acid was assayed. An increase in temperature leads to a greater reactivity of both radicals, but in the case of an enzymatic source a disproportionate increase in the rate of generation is observed. In the pulse radiolysis system, the reactivity of superoxide radicals was found to be stimulated 15-fold over the temperature range from 20^oC to 60^oC, although the activity of superoxide dismutase was only minimally increased (about 1.6-fold). The results are discussed with respect to the possible importance of active oxygen species to the biological effects of hyperthermia.     

Deep body and surface temperature responses to hot and cold environments in the zebra finch

Global warming increasingly challenges thermoregulation in endothermic animals, particularly in hot and dry environments where low water availability and high temperature increase the risk of hyperthermia. In birds, un-feathered body parts such as the head and bill work as ‘thermal windows’, because heat flux is higher compared to more insulated body regions. We studied how such structures were used in different thermal environments, and if heat flux properties change with time in a given temperature. We acclimated zebra finches (Taeniopygia guttata) to two different ambient temperatures, ‘cold’ (5 °C) and ‘hot’ (35 °C), and measured the response in core body temperature using a thermometer, and head surface temperature using thermal imaging. Birds in the hot treatment had 10.3 °C higher head temperature than those in the cold treatment. Thermal acclimation also resulted in heat storage in the hot group: core body temperature was 1.1 °C higher in the 35 °C group compared to the 5 °C group. Hence, the thermal gradient from core to shell was 9.03 °C smaller in the hot treatment. Dry heat transfer rate from the head was significantly lower in the hot compared to the cold treatment after four weeks of thermal acclimation. This reflects constraints on changes to peripheral circulation and maximum body temperature. Heat dissipation capacity from the head region increased with acclimation time in the hot treatment, perhaps because angiogenesis was required to reach peak heat transfer rate. We have shown that zebra finches meet high environmental temperature by heat storage, which saves water and energy, and by peripheral vasodilation in the head, which facilitates dry heat loss. These responses will not exclude the need for evaporative cooling, but will lessen the amount of energy expend on body temperature reduction in hot environments.     

Extremity cooling for heat stress mitigation in military and occupational settings

Physical work, high ambient temperature and wearing protective clothing can elevate body temperature and cardiovascular strain sufficiently to degrade performance and induce heat-related illnesses. We have recently developed an Arm Immersion Cooling System (AICS) for use in military training environments and this paper will review literature supporting such an approach and provide details regarding its construction. Extremity cooling in cool or cold water can accelerate body (core temperature) cooling from 0.2 to 1.0 °C/10 min vs. control conditions, depending on the size/surface area of the extremity immersed. Arm immersion up to the elbow results in greater heat loss than hand- or foot-only immersion and may reduce cardiovascular strain by lowering heart rate by 10–25 beats/min and increase work tolerance time by up to 60%. The findings from studies in this paper support the use of AICS prototypes, which have been incorporated as part of the heat stress mitigation procedures employed in US Army Ranger Training and may have great application for sports and occupational use.     

N-terminal and Central Segments of the Type 1 Ryanodine Receptor Mediate Its Interaction with FK506-binding Proteins

We used site-directed labeling of the type 1 ryanodine receptor (RyR1) and fluorescence resonance energy transfer (FRET) measurements to map RyR1 sequence elements forming the binding site of the 12-kDa binding protein for the immunosuppressant drug, FK506. This protein, FKBP12, promotes the RyR1 closed state, thereby inhibiting Ca²⁺ leakage in resting muscle. Although FKBP12 function is well established, its binding determinants within the RyR1 protein sequence remain unresolved. To identify these sequence determinants using FRET, we created five single-Cys FKBP variants labeled with Alexa Fluor 488 (denoted D-FKBP) and then targeted these D-FKBPs to full-length RyR1 constructs containing decahistidine (His₁₀) “tags” placed within N-terminal (amino acid residues 76–619) or central (residues 2157–2777) regions of RyR1. The FRET acceptor Cy3NTA bound specifically and saturably to these His tags, allowing distance analysis of FRET measured from each D-FKBP variant to Cy3NTA bound to each His tag. Results indicate that D-FKBP binds proximal to both N-terminal and central domains of RyR1, thus suggesting that the FKBP binding site is composed of determinants from both regions. These findings further imply that the RyR1 N-terminal and central domains are proximal to one another, a core premise of the domain-switch hypothesis of RyR function. We observed FRET from GFP fused at position 620 within the N-terminal domain to central domain His-tagged sites, thus further supporting this hypothesis. Taken together, these results support the conclusion that N-terminal and central domain elements are closely apposed near the FKBP binding site within the RyR1 three-dimensional structure.     

Ocular blood flow decreases during passive heat stress in resting humans

Background

Heat stress induces various physiological changes and so could influence ocular circulation. This study examined the effect of heat stress on ocular blood flow.

Findings

Ocular blood flow, end-tidal carbon dioxide (P_ETCO₂) and blood pressure were measured for 12 healthy subjects wearing water-perfused tube-lined suits under two conditions of water circulation: (1) at 35°C (normothermia) for 30 min and (2) at 50°C for 90 min (passive heat stress). The blood-flow velocities in the superior temporal retinal arteriole (STRA), superior nasal retinal arteriole (SNRA), and the retinal and choroidal vessels (RCV) were measured using laser-speckle flowgraphy. Blood flow in the STRA and SNRA was calculated from the integral of a cross-sectional map of blood velocity. P_ETCO₂ was clamped at the normothermia level by adding 5% CO₂ to the inspired gas. Passive heat stress had no effect on the subjects’ blood pressures. The blood-flow velocity in the RCV was significantly lower after 30, 60 and 90 min of passive heat stress than the normothermic level, with a peak decrease of 18 ± 3% (mean ± SE) at 90 min. Blood flow in the STRA and SNRA decreased significantly after 90 min of passive heat stress conditions, with peak decreases of 14 ± 3% and 14 ± 4%, respectively.

Conclusion

The findings of this study suggest that passive heat stress decreases ocular blood flow irrespective of the blood pressure or arterial partial pressure of CO₂.     

Overexpression of manganese superoxide dismutase promotes the survival of prostate cancer cells exposed to hyperthermia

It has been hypothesized that exposure of cells to hyperthermia results in an increased flux of reactive oxygen species (ROS), primarily superoxide anion radicals, and that increasing antioxidant enzyme levels will result in protection of cells from the toxicity of these ROS. In this study, the prostate cancer cell line, PC-3, and its manganese superoxide dismutase (MnSOD)-overexpressing clones were subjected to hyperthermia (43°C, 1 h). Increased expression of MnSOD increased the mitochondrial membrane potential (MMP). Hyperthermic exposure of PC-3 cells resulted in increased ROS production, as determined by aconitase inactivation, lipid peroxidation, and H₂O₂ formation with a reduction in cell survival. In contrast, PC-3 cells overexpressing MnSOD had less ROS production, less lipid peroxidation, and greater cell survival compared to PC-3 Wt cells. Since MnSOD removes superoxide, these results suggest that superoxide free radical or its reaction products are responsible for part of the cytotoxicity associated with hyperthermia and that MnSOD can reduce cellular injury and thereby enhance heat tolerance.