INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THERE ARE A VARIETY OF PREDISPOSING factors that affect thermal tolerance. Among these, only two adaptations are directly invoked to combat heat stress: 1) the rapid heat shock response (HSR); and 2) heat acclimation (10, 26, 35). Heat acclimation is a long-term developing process leading to an expanded dynamic body temperature regulatory range due to left and right shifts in the temperature thresholds for heat dissipation and thermal injury, respectively (7, 11). In contrast, the HSR is a rapid molecular cytoprotective mechanism and involves the production of heat shock proteins (HSP). Under normothermic conditions, the resting cellular 72-kDa HSP (HSP72) level is low. However, a rise in body temperature increases transcription of the heat shock genes, leading to rapid augmentation of their expression. Their binding to denatured or nascent polypeptides in the cells protects vital structural components and, in turn, physiological functions, from thermal damage. This facilitates survival and recovery after removal of the stressor. Prior induction of the HSP72 by mild stress should, therefore, be protective against subsequent, more severe stress (20).

Recently, our laboratory showed (22) that, in rats, heat acclimation leads to a marked upregulation of the basal level of HSP72, an inducible member of the HSP72 family that is considered the most responsive to heat stress, and to a faster HSR. Genetic manipulations to overexpress this protein enhance thermal tolerance in cell cultures and in several animal species (5, 18, 19, 28). Thus preexisting, large HSP72 reserves allow the organism to deal with abrupt changes in core temperature in a hot environment without the need for de novo synthesis of HSP72 (5, 22, 26). This enhanced cytoprotection may be the underlying mechanism involved in the delayed temperature threshold for thermal injury on heat acclimation, implying that the HSP72 defense pathway plays an integral role in the heat-acclimation repertoire (10, 11). It fits with the finding that a variety of species genetically adapted to high ambient temperatures, including ethnic human populations, are characterized by a constitutively higher level of 70-kDa HSP-like proteins, compared with their related species inhabiting temperate or cold environments (40). This may indicate that heat acclimation recapitulates evolutionary adaptation (10, 26).

The mechanisms leading to enhanced heat-acclimation-induced HSP72-related cytoprotection in mammalian species are intriguing for the following reasons. 1) In homeotherms, heat acclimation does not involve a marked elevation in body temperature. 2) No correlation was found between heat strain and rectal temperature (T_re) per se and hsp72 transcription. This may suggest that hsp72 transcription is mobilized via intermediate messenger(s) (22). Moseley (26) hypothesized that the evoked cytokines act as a trigger. This could be applicable to whole body severe hyperthermia, although, so far, it has not been evident on acclimation provoked by moderate heat under sedentary conditions. Considering the initial phase of heat acclimation, during which body temperature is regulated by increased excitability of the autonomic nervous system (10), likely mediator candidates are the accelerated sympathetic system and the release of catecholamines. Sympathetically induced acceleration of hsp72 transcription, via -adrenergic receptors, has already been reported in brown adipose tissue after cold stress or cold acclimation and in blood vessels on cold stress and surgical stress, whereas -adrenergic signaling mediates HSP72 induction after exercise stress (23, 24, 32, 38, 39).

Sustained low-plasma thyroxine, characterizing acclimatory homeostasis, plays a pivotal role in the development of several important acclimatory responses (3, 14), including alterations in the density and affinity of the adrenergic receptors, in turn leading to altered responsiveness to sympathetic signaling (3, 4, 13). The interdependence between sympathetic activity and plasma thyroxine level has been documented, including under conditions of cold acclimation (23, 24). Taken together, we hypothesize an effect of thyroxine on the HSP72 level and HSR occurring on acclimation. The influence of thyroxine on the cytosolic and mitochondrial 70-kDa HSP levels (36), and on the HSR, has been defined in cardiac and skeletal muscles (31), thus supporting our hypothesis.

Given the involvement of 1) -adrenoreceptor activation through sympathetic stimulation or catecholamine release and 2) thyroxine level in many physiological responses to heat acclimation, we hypothesize that -adrenergic signaling and/or plasma thyroxine level plays a role in establishing the heat-acclimation-induced HSP72 elevation and the altered HSR. To prove this hypothesis, the hsp72 steady-state transcript level and the subsequently encoded HSP72 were measured in hearts of rats subjected to -adrenoreceptor blockade or to pharmacological manipulations influencing the level of plasma thyroxine during the acclimation regimen, before and on evocation of the HSR. -Adrenergic blockade during heat acclimation abolished HSP72 accumulation without disrupting the HSR, whereas sustained low-thyroxine level diminished the magnitude of the HSR at the posttranscriptional level. In contrast, long-term thyroxine administration (to both normothermic and acclimating rats) arrested the heat stress-evoked hsp72 transcription. Cumulatively, thyroxine has two opposing effects on hsp72 transcription: inhibitory and, indirectly, stimulatory via -adrenergic signaling. Thus the cross talk between adrenergic signaling and low-thyroxine level is influential in upregulation of the basal HSP72 level on heat acclimation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Male 3-wk-old Rattus norvegicus (Zabar strain, albino var), initially weighing 80-90 g, fed on Ambar laboratory chow and water ad libitum, were randomly assigned to long- (30 days) and short-term (2 days) heat-acclimated (AC) and normothermic groups (Fig. 1). The AC groups were divided into 1) AC rats; 2) AC rats with blockade of -adrenergic receptors [AC propranolol treated (APROP)]; and 3) AC euthyroid rats [AC thyroxine (ATHY)]. AC and APROP included long-term, fully acclimated rats and those that had undergone short-term heat acclimation (AC and APROP vs. 2d-AC and 2d-PROP, respectively). This allowed us to study both the autonomically mediated short-term and the sustained long-term acclimatory responses (10). ATHY rats underwent only long-term heat acclimation to blunt the sustained, low-thyroxine-mediated responses developing in our acclimation experimental model. The normothermic groups included 1) untreated animals, which served as controls (C); 2) hypothyroid [control 6-n-propyl-2-thiouracil-treated (CPTU)] rats; and 3) hyperthyroid [control thyroxine-administered (CTHY)] rats.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Protocol bar illustrating the experimental plan. C, controls; 2d-AC, 2-day heat-acclimated rats; AC, long-term (30 days) heat-acclimated rats; 2d-PROP, 2-day heat-acclimated rats administered propranolol; APROP, long-term (30 days) heat-acclimated rats administered propranolol; ATHY, long-term (30 days) heat-acclimated euthyroid rats obtained by administering L-thyroxine in the drinking water; CTHY, hyperthyroid rats, obtained by administering L-thyroxine in the drinking water; CPTU, hypothyroid rats obtained by administering 6-n-propyl-2-thiouracil (PTU) in the drinking water; HS, heat stress; HSR, heat shock response. For further details, see text.

HSR was characterized, in the present investigation, by the magnitude of elevation of steady-state hsp transcript [peak-to-basal ratio (peak/basal)] in response to heat stress and the subsequent encoded protein. Hence, to characterize the effect of -adrenergic blockade and thyroxine levels on the HSR, all groups were subdivided into groups of rats that received no additional treatment and those that were subjected to heat stress. The levels of the transcripted hsp72 mRNA and the expression of the HSP72 protein were measured in these groups before the heat stress session and post-heat stress, after their subjection to several recovery periods at room temperature, as described below (see Experimental protocols). The heart was chosen as the organ model. It is a major effector of the cardiovascular system, responding to acute, as well as chronic, changes in ambient temperature. A large body of data on gene expression in this organ during heat acclimation, including HSP72 and HSP73 (heat stress control) profiles from previous studies, are already available (22). All experimental protocols were approved by the Ethics Committee for Animal Experimentation of the Hebrew University.

Experimental conditions. The C group was held at an ambient temperature of 24 ± 1°C; heat acclimation was attained by continuous exposure to 34 ± 1°C and 30-40% relative humidity in a light-cycled room (12:12 h) for the required time as stated above. This acclimation model was previously characterized at our laboratory for young and old rats for several thermoregulatory physiological parameters, including "classic criteria" for heat acclimation, such as growth rate, heart rate, body temperature, and metabolic rate (7, 9, 12, 13, 15, 16, 25). In the present study, successful acclimation was assessed by body weight. AC euthyroid and hyperthyroid rats were obtained by administering 3 ng/ml L-thyroxine (Sigma Chemical) in the drinking water for 1 mo, whereas hypothyroid rats were obtained by administrating 0.02% PTU in their drinking water for 1 mo (4). -Adrenergic blockade during acclimation (APROP rats) was achieved by twice daily administration of propranolol (1 mg/100 g body wt, Sigma Chemical). The efficacy of this treatment was validated previously on heart rate (13). Heat stress was attained by subjecting the rat to 41°C for 2 h. During the heat stress session, T_re was monitored on-line, by using a YL 402 thermistor, inserted 6 cm beyond the anal sphincter and attached to a computerized data-acquisition system (22). On termination of the heat stress, the animals were returned to room temperature for different time intervals as described below.

Experimental protocols. All rats were killed by cervical dislocation. For mRNA analysis, the rats were killed before and 20, 40, and 60 min after the given heat stress; to determine HSP72 expression, the animals, except for those treated with propranolol, were killed 1, 4, 24, and 48 h after the given stress (22). The 2d-PROP and APROP rats were killed 1 and 4 h after the heat stress, respectively. The hearts were rapidly excised, mounted on a Langendorff perfusion apparatus, retrogradely perfused (for 2 min) to wash out all remaining blood with Krebs-Henseleit buffer containing (in mM) 120 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 1.25 CaCl₂, 25 NaHCO₃, and 11 glucose, at pH 7.4, and aerated with a mixture of 95% O₂ and 5% CO₂ at 37°C (3, 4, 22). The left ventricle was carefully excised, frozen, and stored at 70°C until analysis.

Semiquantitative detection of mRNA by RT-PCR. To measure hsp72 mRNA, semiquantitative RT-PCR was performed as previously described (22). Briefly, total RNA was extracted with TRI-Reagent (Molecular Research Center, Cincinnati, OH) from the left ventricle homogenate. Total RNA (10 µg) was reverse transcribed in a 50-µl reaction mixture containing 0.5 µg of oligo(dT₁₅) as primer, together with 400 units of Moloney murine leukemia virus reverse transcriptase, according to the manufacturer's instructions (United States Biochemical, Cleveland, OH). For the PCR, 5 µl of the cDNA mixture were added to 50 µl of a master mix containing 200 µM of each 2-deoxynucleotide 5'-triphosphate, 100 pM of each specific primer, and 1.5 units of Vent polymerase (United States Biochemical). We synthesized DNA oligonucleotide primers for HSP72 selected from the published hsp72 gene nucleotide sequence (21). The sense primer was 5'-GCT-GAC-CAA-GAT-GAA-GGA-GAT-C-3' (corresponding to sequence 546-567), and the antisense primer was 5'-GAG-TCG-ATC-TCC-AGG-CTG-GC-3' (corresponding to sequence 1017-1038). Amplification was carried out for 40 cycles with denaturation at 94°C for 30 min, annealing at 64°C for 45 min, and extension at 72°C for 1 min. To ensure equal amounts of initial mRNA, parallel actin amplification was performed (annealing temperature: 62°C, 35 cycles) (30). The PCR products were resolved on 1.5% agarose gel, stained with ethidium bromide, and visualized under ultraviolet light. The density of the bands was computer analyzed by using Tina software (Raytest, Straubenhardt, Germany).

Western blot analysis. The left ventricles were homogenized with 20 mM HEPES (pH 7.5), 1.5 mM MgCl₂, 0.2 mM EDTA, 0.1 M NaCl, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 1.2 mM Na₃VO₄. NaCl was added to a final concentration of 0.45 M (43). The homogenate was centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was mixed with an equal volume of buffer solution, as above, together with 40% (vol/vol) glycerol (43). The protein concentration of the myocardial specimens was quantified by using the Bradford reagent (Bio-Rad Laboratories, Richmond, CA). Total protein (50 µg/lane) was run on 12.5% polyacrylamide gels under denaturing conditions (17). After separation by electrophoresis (50 mA for 2 h), the proteins were transferred onto nitrocellulose (190 mA, 4°C, 1 h). The nitrocellulose membranes were then blocked for 2 h in PBS containing 0.1% dried skimmed milk powder and probed overnight, at 4°C, with monoclonal IgG cross-reactive to HSP72 (Stressgen, Victoria, BC) diluted 1:1,000. After repeated washings, the membranes were incubated at room temperature for 1 h with horseradish peroxidase-conjugated rabbit anti-mouse IgG (Jackson) diluted 1:1,000. Specific antibody binding was detected by using enhanced chemiluminescence (Amersham) and visualized by exposing X-ray film to the membrane (for further details, see Refs. 3 and 22). The density of the scanned HSP72 band was calculated with Tina software.

Calculations and statistics. The heating rate (°C/min) was calculated from the regression lines fitted to the T_re points, starting from normothermic temperatures until the onset of the hyperthermic plateau. The area below the T_re change (T_re) curves during the entire period of heat exposure was used to calculate heat storage ( T_re/min × 0.83 × body wt) and was compatible with the cumulative heat strain (22). All data were normalized to 100 g body wt.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Body weight, body temperature, and heating rate. Body weight, basal T_re on termination of heat stress, and the actual heat strain of all of the experimental animals are presented in Table 1; Fig. 2 illustrates changes in T_re during the course of the heat stress. As previously published (e.g., Refs. 9, 34), the AC rats grew at a slower rate than the normothermic ones. Neither propranolol nor thyroxine affected growth rate. Thus AC, APROP, and ATHY rats had similar body weight and were significantly smaller than the normothermic or the 2d-AC groups, except for the CPTU rats, which were markedly smaller. Taken together, all experimental groups were age matched but only partially weight matched. The basal T_re of the AC euthyroid rats (ATHY) was significantly higher than that of the matched C rats. In contrast, the hypothyroid state (CPTU) resulted in a marked drop in T_re. -Adrenergic blockade during long-term acclimation (APROP group) also resulted in a significantly lower T_re than that of the C rats.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Effect of ambient HS (at 41°C) on rectal temperature change in the course of heating in CTHY or CPTU rats, and 2-day and 30-day heat-acclimated rats at 34°C and 35% relative humidity with no further treatment (C, AC) or treated with propranolol (2d-PROP, APROP) or thyroxine (ATHY). Data were derived from representative groups of 5 or 6 animals for each treatment. For statistics, see Table 1.

Steady-state hsp72 mRNA and HSP72 levels. The steady-state levels of hsp72 mRNA and the encoded protein HSP72 in C and AC hearts before and after heat stress at 41°C are presented in Fig. 3. Under resting conditions, hsp72 mRNA in the AC group was almost undetectable, whereas HSP72 was pronouncedly elevated with respect to the C group (P < 0.005). After being subjected to heat stress, the magnitude of the hsp72 mRNA elevation in AC rats was greater (10- vs. 2.3-fold), and mRNA and the encoded HSP72 peaked earlier than in the matched C rats (mRNA: 40 vs. >60 min; HSP72: 1 vs. 4 h; Fig. 3). HSP72 was then maintained at the attained peak level 24 and 48 h after the given heat stress. 2d-AC rats were characterized by a marked upregulation of the basal hsp72 transcript (Fig. 4A), with a gradual decline after termination of the superimposed heat stress (41°C). These data are in agreement with our laboratory's previously published findings (22) and provided us with baseline data for comparison with the hsp72 mRNA and HSP72 levels obtained after the pharmacological manipulations.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   A: quantification of 72-kDa heat shock protein (HSP72) levels in left ventricle of hearts excised from C and AC rats before and 1-4 h after HS at 41°C for 2 h. Top: representative sets of blots (ECL detection). Bottom: HSP72 protein level, normalized to commercial-positive HSP72 control (Stressgen). B: semiquantitative RT-PCR analysis of hsp72 mRNA steady-state level in left ventricle of hearts excised from C and AC rats before and 20, 40, and 60 min after HS at 41°C for 2 h. Top: representative sets of bands of transcripts. Bottom: bar graph. Data are means ± SE. * Significant difference from the matched control at each time point, 0.001 < P < 0.005. # Significant difference from time point 0 within each experimental treatment, P < 0.05.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   A: basal steady-state hsp72 mRNA content in C, 2d-AC, and 2d-PROP rats. B: semiquantitative RT-PCR analysis of hsp72 mRNA steady-state level in left ventricle of hearts excised from 2d-PROP and APROP rats before and 20, 40, and 60 min after HS at 41°C for 2 h. Top: representative set of bands (10 µl/lane). For comparison, representative set of bands of 2d-AC rats is also presented. Bottom: bar graph showing relative amounts of hsp72 mRNA, normalized to -actin. Values are means ± SE. * Significant difference from the matched APROP, 0.01 < P < 0.03; & Significantly lower than the matched 2d-AC group, P < 0.009 or P < 0.04. C: HSP72 protein accumulation before and 1-4 h after HS at 41°C for 2 h. Top: representative sets of blots (ECL detection). Bottom: bar graph showing HSP72 protein level, normalized to commercial-positive HSP72 control (Stressgen). Values are means ± SE (n = 5-6).

Effects of -adrenergic-receptor blockade on hsp72 mRNA and HSP72 levels. Blockade of -adrenergic receptors during the acclimation regimen abolished upregulation of the steady-state hsp72 transcript level characterizing the 2d-AC group (Fig. 4A) and attenuated the post-heat stress elevation (Fig. 4B). On long-term heat acclimation, the hsp72 transcript level of the APROP rats was similar to that observed in the 2d-PROP group, both under basal conditions and after heat stress (Fig. 4B), and there was no significant elevation in basal HSP72 from that measured for C rats (Fig. 4C vs. Fig. 3). In the APROP rats, the evoked HSP72 synthesis 1 and 4 h post-heat stress resembled that observed for the short-term acclimated 2d-PROP rats (peak/basal of 1.4 and 1.3 for APROP and 2d-PROP, respectively). It is noteworthy that acute propranolol administration (data not shown) failed to block the HSR.

Effects of plasma thyroxine level on hsp72 mRNA and HSP72 levels. Both hypothyroidism and hyperthyroidism alone (CPTU and CTHY groups, respectively) did not significantly affect the basal HSP72 level characterizing the nonacclimated state. The hsp72 transcript in the CTHY group was barely detectable (not shown). The hyperthyroid state also abolished the induction of HSR, and HSP72 failed to increase significantly above control level until 48 h post-heat stress (Fig. 5). In contrast, in the CPTU group (Fig. 6A), heat stress induced a rapid elevation in hsp72 mRNA (mRNA peak/basal of 1.9). No posttranscriptional changes, however, were observed in this group, and the HSP72 level remained at the basal constitutive level, despite the heat stress episode (Fig. 6B). In the ATHY rats (Fig. 5), similar to the AC rats, HSP72 accumulation, characterizing the acclimated state, took place. Basal HSP72 level at the end of the acclimation regimen in this group was 14.2 ± 0.4 vs. 5.6 ± 0.5 in the nonacclimated (P < 0.001). Exposure of the ATHY rats to heat stress, as in the CTHY rats, did not increase hsp72 transcription or HSP72 production, characteristic of the HSR response.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Quantification of HSP72 levels in left ventricle of hearts excised from CTHY and ATHY rats before and 1-48 h after HS at 41°C for 2 h. Top: representative sets of blots (ECL detection). Bottom: graph shows HSP72 protein level, normalized to commercial-positive HSP72 control (Stressgen). There was no expression of hsp72 mRNA in these experimental groups. Values are means ± SE (n = 5-6). § Significant difference from AC group, P < 0.05. ¥ Significant difference from C group, 0.0001 < P < 0.05.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   A: semiquantitative RT-PCR analysis of hsp72 mRNA steady-state level in left ventricle of hearts excised from C and CPTU rats before and 20, 40, and 60 min after HS at 41°C for 2 h. Top: representative set of bands (10 µl/lane). Bottom: bar graph showing relative amounts of hsp72 mRNA, normalized to -actin. Values are means ± SE. # Significant difference from time point 0 within each experimental treatment, 0.001 < P < 0.005. B: quantification of HSP72 levels in left ventricle of hearts excised from C and CPTU rats before and 1-48 h after HS at 41°C for 2 h. Top: representative sets of blots (ECL detection). Bottom: graph shows HSP72 protein level, normalized to commercial positive HSP72 control (Stressgen). Values are means ± SE (n = 5-6). * Significant difference from the matched control at each time point, 0.001 < P < 0.005.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Our laboratory's previous studies (22) indicated that alterations in HSP72 signaling constitute an integral part of the heat-acclimation repertoire. This is evident from the high levels of HSP72 expression without the need for de novo protein synthesis to confer protection and from the faster response to heat stress. In the present investigation, using a nonspecific -adrenergic antagonist, we provide causal evidence for the significant influence of -adrenergic signaling on the buildup of the cellular HSP72 reserves and on the magnitude of the HSR. The sustained low-thyroxine level, occurring on acclimation, diminishes the magnitude of the HSR, possibly via its influence on adrenergic signaling. A novel finding in this study is the interference of a high-thyroid level with the HSR. The mechanism leading to this effect is not clear.

-Adrenergic signaling and HSP72 responsiveness on acclimation. Acclimation is a biphasic process. The initial phase, 2d-AC, is characterized by several significant alterations in sympathetic activity, -adrenergic signaling, and catecholamine turnover (10, 11). In the heart, this is also reflected by a marked decrease in the affinity of the adrenergic receptors and by impaired chronotropic and inotropic responses (3). Thus accelerated sympathetic activity compensates for these detriments. HSP72 also shows a biphasic profile of acclimation dynamics, with hsp72 mRNA upregulated and downregulated during short- and long-term heat acclimation, respectively. A nearly reciprocal biphasic profile is exhibited by the protein: slight downregulation during the short-term phase of heat acclimation, with pronounced upregulation characterizing acclimatory homeostasis (22). In the propranolol-administered rats, the biphasic acclimation kinetics were blunted (Fig. 7). Sustained propranolol treatment resulted in stabilized steady-state hsp72 transcription throughout the entire heat-acclimation regimen, with the hsp72 mRNA level being essentially the same as in the preacclimation state. Subsequently, the rise in HSP72 level to that characterizing the long-term heat-acclimation phase was attenuated. Mobilization of the molecular machinery to heighten hsp72 transcription after the superimposed heat stress was also abolished, implying the involvement of adrenergic signaling in both processes. Based on the results obtained for the 2d-PROP group (compared with the 2d-AC group), it is likely that the accelerated sympathetic flow, together with the surge in circulating catecholamines occurring during the short-term acclimation via -adrenergic signaling, contributes to the elevated hsp72 transcription observed at that acclimatory phase and the subsequently sustained high HSP72.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Effect of propranolol administration on basal HSP72 content and steady-state hsp72 mRNA in 2d-PROP and APROP rat hearts. For comparison, the matched values for C, 2d-AC, and AC rats are presented. A: basal HSP72 protein level. B: basal hsp72 mRNA steady-state level measured by RT-PCR. Values are means ± SE (n = 5-6). NS, nonsignificant. * Significant difference from control, 0.001 < P < 0.005; # Significant difference from AC group and from 2d-AC, P < 0.005.

Thyroxine level and HSP72 responsiveness on acclimation. In the present investigation, the effects of 1-mo-long thyroxine manipulations are compatible with the time-dependent, thyroxine-induced acclimatory metabolic influences (14). Our data show that, in the thyroxine-administered groups CTHY and ATHY, the HSR was arrested. This finding seemingly disagrees with the results of Pantos et al. (31), showing that hearts from long-term hyperthyroid rats overexpress hsp72 mRNA in response to ischemic stress. However, HSP72 synthesis was evoked only in the presence of cardiac hypertrophy, suggesting that the development of hypertrophy rather than increased thyroxine level played a role in the induction of transcripted hsp. Our finding may be compatible with that of Dillmann et al. (2), that 3,5,3'-triiodothyronine administration to hypothyroid rats does not enhance either the level or the translational activity of several mRNA species (including 70- to 75-kDa proteins), despite the general increase in total mRNA. The primary effect of thyroxine is on the transcriptional regulation of target genes, via its binding to nuclear receptors (42). We can thus conclude that, in our experiments, indirect cellular thyroxine mediation of the rapid HSR was not brought into play. In contrast, thyroxine administration did not abolish basal HSP72 upregulation in the course of the acclimation. This may imply more than one pathway for HSP72 induction.

View larger version (12K): [in this window] [in a new window]   Fig. 8.   Proposed model of the effect of thyroxine on hsp72 transcription. The data obtained in this investigation imply dual, direct and indirect, effects of thyroxine on hsp72 transcription. Long-term elevation of thyroxine arrests hsp72 transcription during HS (1). In parallel, the sustained elevation of thyroxine increases the density of the -adrenergic receptors (1), leading, in turn, via -adrenergic stimulation, to the induction of hsp72 transcription (2). In contrast, sustained low-thyroxine level diminishes the thyroxine inhibitory effects (via pathway 1), and thus the -adrenergic facilitatory pathway (2) predominates and enhances hsp72 transcription (based on Figs. 3 and 4 and Refs. 8, 37).

Rate of heating and HSP72. The various pharmacological manipulations used in this investigation affected resting body temperature, rate of heating, and the cumulative heat strain of the rats assigned to each group. When the experimental manipulation did not blunt hsp72 transcription, it was possible to establish a correlation between the extent of the heating effects and HSP72 synthesis. Our data show a high positive correlation between the rate of heating and HSP72 synthesis but not with heat strain or the hyperthermic plateau temperature.