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Hyperthermic stress affects the thermal modulation of glucocorticoid–receptor affinity
Journal of Thermal Biology 26 (2001) 575–584
Hyperthermic stress affects the thermal modulation of glucocorticoid–receptor affinity Dragoslava Zˇivadinovic´a,*, Stojko Vidovic´b, Gordana Matic´a, Radoslav K. Andjusc a
b
Institute for Biological Research, 29 Novembra 142, 11060 Belgrade, Yugoslavia Department of Biology and Human Genetics, Faculty of Medicine, University of Banja Luka, Sime Mrkalja 10, 78000 Banja Luka, Republic of Srpska, Bosnia and Herzegovina c Center for Multidisciplinary Studies, University of Belgrade, 29 Novembra 142, 11060 Belgrade, Yugoslavia Received 6 September 2000; received in revised form 31 October 2000; accepted 5 December 2000
Abstract In vitro binding of a steroid hormone (triamcinolone acetonide) to hepatic glucocorticoid receptors was studied in liver cytosols prepared from untreated control rats and rats sacrificed after being exposed in vivo to whole body hyperthermia (41 or 428C). Positive temperature modulation of glucocorticoid–receptor affinity (decrease of affinity with increasing temperature) was well expressed in all preparations. In preparations from hyperthermic rats, alterations of a possible functional (adaptive) significance have been recorded: the amplitude of positive thermal modulation was reduced, and its lower-temperature limit shifted towards higher temperatures. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Glucocorticoid receptor; Hormone–receptor affinity; Hyperthermia
1. Introduction It was previously shown that short-term whole-body hyperthermia induces serious alterations of the rat liver glucocorticoid-receptor system (reviewed by Matic´ et al., 1998). Hyperthermia affects the rate of dissociation of the hormone–receptor complex and results in a reduction of the receptor’s binding capacity (Matic´ et al., 1989, 1998), in the stimulation of its nuclear import (Matic´ et al., 1995), and in changes of its association with heat-shock proteins (Cˇvoro et al., 1998a, b). In the latter study, the HT-41 stress was shown to stimulate the association of the glucocorticoid receptor (GR) with a heat-shock protein, Hsp70. After HT-41, the GR:Hsp70
*Corresponding author. Present address: NICHD, ERRB, NIH, Building 49, Room 6B/23, 49 Convent Drive, MSC 4510, Bethesda, MD 208924510, USA. E-mail address: [email protected] (D. Zˇivadinovic´).
ratio in receptor complexes amounted to 1:3 as compared to 1:1 in the unstressed controls. It was reasonable to assume that such alterations might also influence mechanisms underlying the socalled ‘‘positive thermal modulation’’ of hormone– receptor affinity (increase of the dissociation constant with increasing temperature). Positive thermal modulation was shown by another line of our studies to characterize the temperature dependence of glucocorticoid–receptor affinity in hepatic cytosol preparations from rats and ground squirrels (Zˇivadinovic´ and Andjus, 1986, 1987; Zˇivadinovic´ et al., 2000). Positive thermal modulation described by us in these mammals was analogous to the thermal modulation of affinity in enzyme–substrate systems from ectotherms. The term was coined to stress the similarity of this effect of temperature to the effect of positive allosteric modulators on the affinity of an enzyme for its substrate (Hochachka and Somero, 1973). In ectotherms, positive thermal modulation of enzymes was described as one of the strategies of biochemical adaptation to short-term
0306-4565/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 6 - 4 5 6 5 ( 0 1 ) 0 0 0 0 3 - 1
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variations of body temperature, since it compensates for excessive temperature-induced changes of kinetic energy on reaction rates and provides the animal with means for immediate compensation of its metabolic rate when confronted with changing ambient temperature (Doumen et al., 1986; Hochachka and Somero, 1973). By analogy, an adaptive role has been envisaged of the positive thermal modulation of hormone–receptor affinity observed by us in the rat and the ground squirrel, the increase of affinity with cooling compensating for an endangering decrease of kinetic energy involved in hormone–receptor interactions. By analogy again with enzyme–substrate systems, it was suggested that the observed shift of the lower limit of positive thermal modulation towards lower temperatures in the more cold-tolerant ground squirrel as compared to the less cold-tolerant rat, had an adaptive value. The relevance of analogies between findings in ecthothermic vertebrates and homeotherms may be questioned, since temperature swings in the latter are comparatively negligible. However, it is well known that many homeotherms, the rat in particular, can tolerate changes in body temperature as broad as those in ectotherms. In rats, unassisted recovery occurs after lowering their body temperature from 37 to 158C. They can be revived by special resuscitation techniques after cooling the body temperature to 08C and even after supercooling below 08C (Andjus, 1969). Comparisons of the dependence of biochemical events on body temperature in the rat and ectotherms are thus legitimate and potentially meaningful. Such comparisons were made in discussing and interpreting the results of the present study as well. In the past, kinetic modeling of glucocorticoid– receptor interactions predicted KD-versus-temperature profiles typical of positive thermal modulation (Dzˇakula et al., 1988; Dzˇakula, 1990). This indicated that such profiles, observed experimentally, might be the consequence of intrinsic kinetic properties of the receptor– ligand system. The model of Dzˇakula (1990) was presently used in analyzing kinetic data on binding of triamcinolone acetonide (a synthetic glucocorticoid) to the hepatic glucocorticoid receptor of rats exposed to whole body hyperthermic stresses of different intensity. As already pointed out, our preceding experiments showed that hyperthermia induces serious alterations of the rat liver glucocorticoid-receptor system. Applying the model of Dzˇakula, we investigated these time effects of hyperthermic exposures on the positive thermal modulation of glucocorticoid–receptor affinity. As expected, marked alterations of this phenomenon have been found as a result of sublethal hyperthermia. 2. Materials and methods Materials: [3H] Triamcinolone acetonide ([3H] TA; specific activity 20 Ci/mmol) was purchased from
Amersham International (Amersham, UK), unlabeled triamcinolone acetonide (TA) from Sigma Chemical Co. (St. Louis, MO, USA), sucrose, Tris base and charcoal from Merck Co. (Darmstadt, Germany) and Dextran T-500 from Pharmacia Fine Chemicals (London, UK). All chemicals used were analytically pure. Animals: Rats of the Wistar strain (2.5 months old, 250–300 g body weight) were reared under standard laboratory conditions (20–228C, 12:12 h light:dark cycle, food and water ad libitum). To free hormone receptors from endogenous glucocorticoids, the animals were bilaterally adrenalectomized. They were subsequently kept on a sodium-rich diet (0.9% NaCl in drinking water) to prevent sodium depletion resulting from lack of mineralocorticoids after adrenalectomy. Rats were exposed to hyperthermic stresses 4 days postoperatively. To induce the ‘‘sublethal hyperthermic stress’’ (termed ‘‘HT-41’’ in the text), animals were anesthetized with Nesdonal (4.6 mg/100 g b.w, i.p.) and placed into a ventilated and humidified chamber preset to 448C (Currie and White, 1981). A digital thermistor thermometer continuously monitored rectal temperature which increased to 418C in about 40 min. A further rise of body temperature was prevented by intermittently ventilating the warm chamber with fresh air. After maintaining in this way the rectal temperature at approximately 418C during 15 min, the animals were sacrificed by rapid decapitation. The more severe ‘‘semilethal hyperthermic stress’’ (termed ‘‘HT-42’’ in the text) was induced by maintaining the animals in the same warm chamber for about 55 min, and by sacrificing them when their rectal temperature reached 428C. The sham-treated controls were Nesdonal-anesthetized, maintained at room temperature, and decapitated at the same time as their heat-stressed counterparts. Cytosol preparation: Methods for preparing cytosol from pooled liver homogenates and for the measurement of specific binding of 3H-labeled triamcinolone acetonide ([3H]TA) were the same as previously described (Zˇivadinovic´ and Andjus, 1995). The livers were perfused in situ with ice-cold 50 mM Tris buffer, pH 7.55 at 208C, containing 0.25 M sucrose, 25 mM KCl, 10 mM MgCl2 and 20 mM Na2MoO4. They were then quickly excised and homogenized in two volumes (w/v) of the same solution. After the first centrifugation of the homogenate (8000 g, 10 min, 48C), the supernatant was re-centrifuged (105, 000 g, 1 h, 48C, Ti50 rotor) to yield the cytosol preparation. The preparation was quickly frozen and stored in liquid nitrogen until use. Time course kinetics of receptor–steroid (RS) complex formation: Cytosol aliquots (100 ml) were incubated with approximately 30 nM [3H]TA. Parallel aliquots, incubated in the presence of a 100-fold molar excess of unlabeled TA, allowed corrections for nonspecific binding. Free steroid was removed from the incubation
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mixture by the addition of a 3.75% charcoal–0.375% Dextran suspension following the procedure described by Atger and Milgrom (1976). The time course of specific [3H]TA binding was studied at 8 temperature levels in the control and the HT-42 groups, and at 12 levels in the HT-41 group (from 0 to 378C; 2–58 apart). At each temperature, concentrations of bound steroid were determined after 8 time intervals following the addition of pre-incubated cytosol aliquots to [3H]TA samples. The time intervals varied with temperature and were considerably shorter during the initial phases of the binding process (ranging from 15 s to 3 h at 378C, and from 2 min to 18 h at 08C). pH changes with temperature were practically the same in cytosols from our three experimental groups (HT-41, HT-42 and controls), so that their influence on the comparative results concerning steroid binding is excluded. Kinetic modeling: The kinetic model used was based on thermodynamic considerations and Eyring’s transition state theory (Dzˇakula et al., 1988; Dzˇakula, 1990). It predicts a number of properties characterizing the formation of steroid–receptor complexes, such as the temperature dependence of the binding process and the U-shaped KD-versus-temperature profile. At its origin, the model has the conceptual kinetic scheme ka
kl
ktr
ki
k1
ktr
Ri þ S . Ra þ S . Ra S . Rtr S: (R}receptor, with subscripts: i}inactive, a}active, tr}transformed; S}steroid). In agreement with empirical data, the model predicts one-maximum time profiles of steroid–receptor binding, with a non-zero equilibrium plateau. The fitting procedure was based on the Nedler–Mead method. Fitting involved simultaneously experimental data obtained at all incubation temperatures and after all time intervals (n=64 for the control and the HT-42 heatstressed group; n=96 for the HT-41 heat-stressed group). Twelve thermodynamic parameters were allowed to vary (six entropies and six enthalpies, concerning receptor activation and inactivation, association, dissociation, complex transformation and reverse transformation). Receptor concentrations were fixed at values close to those experimentally obtained (12.9 nM for control rats; 3.7 and 2.7 nM for the HT-41 and HT-42 heat-stressed groups). KD values for different incubation temperatures were calculated as follows: KD ¼
½So ð½Ro ½RSÞ ð½Ro ½RSÞ: ½RS
([So] } the applied steroid concentration, [Ro] } receptor concentration, [RS] } complex concentration). Values obtained as best-fit requirements, concerning the concentration of receptor molecules and the maximal
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concentrations of the steroid–receptor complex ([RS]max), were introduced into this equation in order to arrive at KD values for each temperature level. [RS]max values for different temperatures were obtained from numerical listings of fitted data-points of the timedependent binding. Protein determination: Protein contents of hepatic cytosols, determined as described by Lowry et al. (1951) and using BSA as a reference, ranged from 25.5 to 33.0 mg/ml. Statistics: The Mann–Whitney and the t-test were applied to differences between sets of data-points. Differences between slopes were evaluated using the Tukey test. Differences between forms of best-fit equations were evaluated using the commercial table curve 2D program (AISN Software, Jandel Scientific). Equations suggested by the program for fitting a given set of experimental data-points are given in explicit form by the program, they are ranked by the goodness of fit criteria and each is identified by an identification number. We refer in the text only to these identification numbers and to the rank of the equation referred to (i.e., Eqs (6122) and (7113), Rank 1).
3. Results Fig. 1 illustrates the time course of [3H]TA binding to the glucocorticoid receptor in cytosols from controls and from the two groups of hyperthermic rats. Experimental data obtained at all incubation temperatures and after all time intervals were fitted simultaneously (n=64 for the control and the HT-42 group, n=96 in the case of the HT-41 group; triplicate analyses for each value). At all incubation temperatures, and in the case of all preparations (from controls and from both groups of hyperthermic rats), the time course of [3H]TA binding was characterized by a biphasic pattern. After rapidly reaching a maximum ([RS]max), the concentration of bound steroid slowly declined, the slopes of the descending branches increasing considerably with temperature. The framed panels in Fig. 1 display profiles obtained by fitting the kinetic model to experimental data concerning control and hyperthermic rats. The lower right-hand panel exemplifies the approximation of experimental data by model-derived curves at incubation temperatures close to Tinv. Differences between families of curves displayed in the four panels of Fig. 1 were fully expressed, although these families did not include temperatures reached in vivo by our hyperthermic rats. Cytosol incubation at 41 and 428C could provoke substantial denaturation and aggregation of proteins and the function of chaperone molecules could have been seriously hampered in vitro. As shown in the inset of Fig. 2, [RS]max values derived from data in Fig. 1, were highly temperature-dependent
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Fig. 1. Temperature dependence of the time course kinetics of specific binding as affected by hyperthermic stresses. Model-derived profiles refer to control data (upper left panel), and to data obtained after the milder HT-41 and the more severe HT-42 stress (upper right and lower left panels, respectively). The lower right-hand panel is an illustration of the adequacy of the approximation of experimental data-points (dots) by model-derived profiles.
and formed bell-shaped [RS]max-versus-temperature profiles. The bell-shaped curves concerning stressed animals were considerably flatter, particularly in the case of animals exposed to the more severe heat stress. They peaked at values of 3.2 and 2.5 nM in case of the HT-41 and HT-42 groups, respectively, as compared to the control peak value of 11.3 nM. In addition, considerable differences were expressed concerning the placement of these peaks along the temperature scale. [RS]max peaks concerning heat-stressed animals were shifted to the right, to temperatures higher by 2–38C: to 17 and 18.58C in the HT-42 and the HT-41 groups,
respectively, as compared to 15.58C in the controls. The shift was thus appreciably greater after the milder than after the more severe hyperthermia. The main panel of Fig. 2 displays theoretical (model derived) KD-versus-temperature profiles. For each temperature level (8–12 temperatures), KD figures were obtained by introducing into the appropriate equation (see Section 2) values concerning the concentration of receptor molecules and the maximal concentration of the steroid–receptor complex ([RS]max), obtained as best-fit requirements. In the case of all preparations, those from the control as well as those from the
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Fig. 2. Main panel: Model-derived KD-versus-temperature profiles. Dots identify values used in Fig. 3 to analyze in a different way the positive thermal modulation branch of the temperature dependence of KD . Inset temperature dependence of the binding maxima ([RS]max-versus-temperature profiles). Verticals in the main panel and inset indicate, respectively, the placement along the temperature scale of the calculated [RS]max maxima and KD minima (15.58C in the controls; 17 and 18.58C in the HT-41 and the HT-42 group, respectively).
Fig. 3. Positive thermal modulation of RS affinity (from 158C upwards). KD values, joined by simple spline fitting, are those identified by dots in the main panel of Fig. 2: (open circles) controls; (closed circles) HT-42; (closed rectangles) HT-41. Upper inset: Amplitude of positive thermal modulation of RS affinity (extent of KD increase from Tinv to T=378C) expressed as percent of the value in the control group.
heat-stressed animals, KD -versus-temperature profiles were U-shaped. They showed a marked decrease of KD values with falling temperature (positive thermal modulation of affinity) and a substantial increase of these values with further cooling below the inversion temperature (negative thermal modulation of affinity). The inversion points matched the temperatures characterized by the maxima of the [RS]max-versus-temperature profiles shown in the inset of the same figure. The inversion temperature, defining the lower limit of positive thermal modulation, was shifted to clearly higher levels in the heat-stressed groups (17 and 18.58C in the HT-42 and the HT-41 groups, respectively) in comparison to the controls (Tinv =15.58C). In the interval between 58 and 298C, the difference between a selected set of data-points (28 apart, n=13) in the HT-41 group and the corresponding set of data-points in the controls, was statistically significant ( p=0.004). In contrast, the difference between the corresponding sets of control and of HT-42 data-points was not significant ( p=0.338). By the Tukey test (regression comparisons), the slope of the KD increases above 318C (7 data-points,
18 apart) was statistically different in the HT-41 group from those in the two other animal groups ( p=0.00001). Dots along curves in the main panel of Fig. 2 identify values used in Fig. 3 to analyze in a different way the phenomenon of positive thermal modulation and accentuate differences between experimental groups within the range of relatively high temperatures (from 15 to 378C). The three sets of KD values in Fig. 3 are joined this time by simple spline fitting. The curve belonging to the HT-41 group (milder hyperthermia) deviates appreciably from the rest of the curves shown: at temperatures below 278C, it runs above, while at temperatures higher than 278C it is displaced below the other two curves. In contrast, differences between curves belonging to the controls and to the HT-42 group (severe hyperthermia) are negligible. The table curve fitting procedure showed that Rank 1 equation for the HT-41 curve (Eq. (7113)) was different from the one satisfying data from both the controls and the HT-42 group (Eq. (6122)). The amplitude of positive thermal modulation, expressed as the distance of KD values at Tinv from those at 378C (inset) was reduced by 29% in
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the HT-41 group, and insignificantly (less than 4%) in the HT-42 group.
4. Discussion The present study confirmed the phenomenon of thermal modulation of RS affinity in the rat, previously described by us in rat and ground squirrel cytosol preparations (Zˇivadinovic´ et al., 2000). This time, however, marked alterations of the thermal modulation of RS affinity have been identified in rats exposed to short-term hyperthermia. The alterations depended to a great extent on the severity of the heat stress applied. According to our experience, rats survive without exception a 15-min exposure to 418C body temperature. If, however, body temperature is raised to 428C, about half of the rats succumb when left to recover at room temperature. The choice of these two hyperthermic exposures (41 and 428C) in the present study was based on our previous experience with their effects on glucocorticoid-receptor’s binding capacity, the rate of receptor–hormone binding, complex dissociation, affinity of steroid binding, complex translocation, and association of receptors with heat-shock proteins (Cˇvoro et al., 1998a, b, 1999, 2000; Matic´ et al., 1989, 1998). The functional significance of the presently observed phenomena and the underlying molecular mechanisms remains to be clarified. 4.1. Functional significance Empirical data on RS affinity agreed with predictions based on kinetic modeling, suggesting again that the observed thermal modulation of affinity might be a direct consequence of intrinsic kinetic properties of the underlying steroid–receptor interactions (Zˇivadinovic´ and Andjus, 1986; Zˇivadinovic´ et al., 2000). The functional meaning of the U-shaped KD-versus-temperature profile in a homeothermic species remains, however, to be elucidated. Forming such profiles, the dissociation constant of RS affinity was temperature-modulated in rats in much the same way as in the case of a number of enzyme– substrate systems of ectotherms (Hochachka and Somero, 1968, 1973, 1984; Klyachko et al., 1995; Andreeva et al., 1996). Both the positive and the negative thermal modulation of affinity were well pronounced. It is of particular interest that the present results were also in agreement with data concerning steroid binding to human glucocorticoid receptors from lymphoblastoid cells (Eliard and Rousseau, 1984). Although the latter authors did not illustrate their results graphically, a U-shaped Km-temperature profile,
with a pronounced positive thermal modulation arm, can easily be derived from their data. As discussed in one of our preceding papers (Zˇivadinovic´ et al., 2000), controversies accompany interpretations of the ‘‘U-shaped curves’’ since 1971 when it was shown that pH might modify the U-shaped response (Hochachka and Lewis, 1971). Nevertheless, U-shaped curves continued to be published by the same authors (Hochachka and Somero, 1984) and by others (Klyachko et al., 1993, 1995; Andreeva et al., 1996). In ectotherms, changes of intracellular pH with temperature were shown to be considerably smaller than those paralleling the pH of water neutrality (Lehoux and Gouderlay, 1996; Marjanovic´ et al., 1998). It was underlined, moreover, that rapidly occurring acute changes in body temperature may not allow for ‘‘correct’’ (temperature-adjusted) pH to be attained (Walsh and Somero, 1982). In mammals, such as our rats, the regulation of the internal milieu is known for a long time now to differ from that in ectotherms (Rahn, 1967). In the rat (and in ground squirrels) such curves have been previously obtained and discussed by us (Andjus et al., 1999; Zˇivadinovic´ et al., 2000). Besides, the fact remains that our theoretical, model derived curves, were U-shaped. They were constructed solely on the basis of a conceptual kinetic scheme based on thermodynamic considerations and Eyring’s transition state theory. The functional meaning of the observed positive thermal modulation of RS affinity in the rat, a homeothermic species, remains unclear. In the rat, positive thermal modulation of RS affinity may simply represent the consequence of intrinsic kinetic properties of steroid–receptor interactions, deprived in the homeotherm of the specific functional role played in its ectothermic ancestors. One could hypothesize, however, that a functional role of the positive thermal modulation might be present in the rat as well. One could assume, for example, that starting from levels of affinity and kinetic energy compatible with hormone action, cooling would tend to induce excessive decreases of kinetic energy, threatening to stop RS interactions. This would be opposed by the concomitant rise of affinity due to its positive thermal modulation (‘‘immediate rate compensation’’). In small mammals such as rats, accidental hypothermia induced by the association of wetting and cold is not a rare phenomenon, and the maintenance of hormone action may be important for a successful rewarming (Andjus, 1969). Positive thermal modulation of RS affinity could therefore contribute to the hypothermic tolerance of the animal. After exposure of rats to hyperthermia, Tinv of RS affinity was shifted to higher incubation temperatures (Fig. 2). This was highly reminiscent of the shift in the same direction of Tinv of ES affinity in ectotherms after their acclimation or evolutionary adaptation to warmer
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environments and consequently higher body temperatures (Hochachka and Somero, 1973, 1984). In our HT-41 group (milder hyperthermia), the increase of KD with temperature was less steep and KD values above 278C were even smaller (affinity greater) than in the controls. The overall amplitude of positive thermal modulation was reduced by almost 30% (Fig. 3 inset). These findings were reminiscent of those concerning ES affinity in ectotherms. Lower Km values (higher affinities) at the majority of incubation temperature are typical of ectothermic species from warmer habitats compared to those adapted to colder environments (Yancey and Siebenaller, 1987). In ectotherms, a reduction of the amplitude of positive thermal modulation of ES affinity has been observed after acclimation/ adaptation to higher environmental/body temperatures. (Hochachka and Lewis, 1971). Km values for fish enzymes were always found to be reduced after adaptation to a higher temperature (Yancey and Siebenaller, 1987). The described reductions in the amplitude of positive thermal modulation of affinity were said to enable the enzyme to function in a broad range of temperatures with comparatively small changes of ES affinity. By minimizing the decrease of affinity with increasing temperature, a relatively high affinity is conserved at all temperatures thus contributing to the eurythermy of the species (Fields and Somero, 1997). By analogy, one could envisage an adaptive role of the modification of positive thermal modulation of RS affinity observed in our experiments with hyperthermic rats, taking into account the following considerations. Positive thermal modulation of RS affinity tends to decrease affinity at high temperature. The association, however, of decreased affinity and increased kinetic energy at elevated body temperature may threaten the stability of the RS complex. This stability is, however, a prerequisite of hormone action, of particular importance for the complex transformation step. An adjustment of positive thermal modulation that would reduce its amplitude and minimize the reduction of affinity at elevated temperatures would thus counteract the potentially harmful effect of the association of reduced affinity and increased kinetic energy on the stability of the RS complex. Such an adjustment is, therefore, suggestive of an adaptive modification, of functional significance to the organism threatened by elevated body temperature. The presently observed effects of the temperature modulation of KD were also reminiscent to some extent of our previous results obtained with glucocorticoid receptors from rats and European ground squirrels (Spermophylus citellus, a hibernator) (Andjus et al., 1982; Zˇivadinovic´ et al., 2000). In the ground squirrel compared to the rat, the whole U-shaped KD-versustemperature curve was shifted upwards, and the KD minimum was displaced to an appreciably lower temperature (lower Tinv than in the rat). As to the
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differences in vertical placement of the U-shaped curve, the ground squirrel differed therefore from the rat in much the same way as the hyperthermic rat from its normothermic controls. The direction of the ‘‘shift’’ of Tinv was, however, opposite, but in agreement with thermal conditions to which the animal is expected to adjust. In the hibernator, adapted to better tolerate low body temperatures, Tinv appeared as displaced to lower temperatures, whereas in the hyperthermic rat, expected to adjust to higher body temperatures, Tinv occurred at higher temperatures. In order to relate our results obtained in vitro to the organismic and whole animal level, known facts should be considered about the capacity of the rat to enhance survival in hyperthermia by metabolic adjustments. Metabolic peculiarities of rodent species adapted to hot environments should also be taken in to consideration. As shown long ago by one of us, the rat is indeed capable of acclimating to extremely hot environments and hyperthermic body temperatures (Andjus and Buzalkov, 1960). When exposed abruptly to ambient temperatures of 38 to 398C, rats inevitably succumb to hyperthermia in a matter of a few hours. If acclimated gradually, over several weeks, to the hot environment by a stepwise increase of environmental temperature (358C followed by 388C), they survive in the same hot environments for months, with hyperthermic levels of body temperature (38–398C). After such acclimation, their metabolic rate (oxygen consumption) is markedly reduced, both as a function of environmental and of body temperature. All changes induced by high-temperature acclimation were shown to be fully reversible. One of the changes observed after acclimation to hyperthermic environments was loss of resistance to cooling. Exposure to moderately cold environments (0–48C), easily tolerated by rats from a thermoneutral environment, provokes in rats adapted to hyperthermia a rapid and continuous fall of body temperature (Andjus and Buzalkov, 1960). The considerable shift of Tinv to higher temperatures, presently observed in our TH-41 group of hyperthermic rats (Fig. 2), may also be regarded as a loss of resistance to cooling. It indicates, namely, that the ability to counteract, by means of positive thermal modulation, the effect of cooling on RS affinity is reduced. The harmful effect of cold on reaction rates is enhanced, since negative thermal modulation makes its appearance earlier, at higher temperatures. This may, therefore, reflect to some extent the loss of cold resistance observed previously in rats adapted to hyperthermic conditions. In general, changes observed after short-term exposures to hyperthermia, such as those presently recorded in our HT-41 group of rats, might be regarded as reflecting the beginning of complex metabolic changes leading eventually to improved hyperthermic tolerance and a loss of resistance to cooling.
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Another line of our studies led us to the identification of a number of metabolic peculiarities in a highly heattolerant desert rodent, Jaculus orientalis (El Hilali et al., 1978). A number of tissues (including brain) showed, namely, higher metabolic rates at ‘‘hyperthermic’’ incubation temperatures (37–448C) than the same tissues of the rat. By analogy, the increase of RS affinity at incubation temperature above 278C, observed presently in the HT-41 group of rats, may also be regarded as beneficial to heat tolerance and thus contributing to an efficient metabolism at elevated temperature. 4.2. Molecular mechanisms Our kinetic modeling suggests that the thermal modulation of RS affinity arises from intrinsic kinetic properties of the underlying steroid–receptor interactions. The mechanisms of the observed modifications induced by the two modalities of heat stress remain, however, to be elucidated. Differences between effects of the milder and the more severe heat stress have been identified recently in our laboratory at the level of the receptor molecule itself. Structural effects were studied using immunopurified receptor preparations. In the HT-42 animals (severe heat stress) steroid-binding assays revealed a significant reduction of specific TA binding both to the immunopurified and the cytosolic glucocorticoid receptor. In the HT-41 animals, in contrast, the reduction involved only binding to the cytosolic receptor (Elez et al., 2000). The data suggested that in the case of the HT-42 stress, the alteration of the RS-binding capacity resulted from the modification of the receptor itself. The HT-41 stress, on the other hand, might have affected the receptor’s binding capacity indirectly, through other cytoplasmic components influencing the receptor function. This explanation was confirmed by the finding that only the HT-42 stress, not the milder one, led to intramolecular disulfide bond(s) formation within the receptor (Elez et al., 2000). It was also supported by the finding that the HT-41 stress stimulates the association of the receptor with heat-shock proteins, Hsp70 in particular (Cˇvoro et al., 1998a, b). It is well known that hyperthermic exposure is accompanied by an accumulation of partially or totally denatured proteins. It is precisely the appearance of structure proteins altered in the cell that is critical for eliciting the cellular response to stress, i.e. the induction of heat shock and other proteins with a protective role. Conformational changes of the receptors and covalent modifications are also possible (Elez et al., 2000), as well as changes of the interaction between receptors and heat-shock proteins (Cˇvoro et al., 1998a, b). All the enumerated changes could have been the cause of the reduction of the receptor’s binding capacity observed in our study. It is known, however, that denaturation may
be reversible and that chaperone molecules (heat-shock proteins), closely associated with receptors, participate in the renaturation process. Control experiments from our previous studies showed that following the HT41type stress the capacity of [3H]TA binding to glucocorticoid receptors returns to normal in about 4 hours (Matic´ et al., 1995).
5. Summary It was assumed that the serious alterations of the rat liver glucocorticoid-receptor system, previously demonstrated in rats exposed to short-term whole-body hyperthermia, might as well influence mechanisms underlying the thermal modulation of hormone–receptor affinity. The latter phenomenon, consisting in a decrease of affinity with increasing incubation temperature, was shown by another line of our previous studies to govern the temperature dependence of the in vitro glucocorticoid-receptor system of rats. In cytosols, from rats made hyperthemic, positive thermal modulation of HR affinity was modified in two main respects: (a) its lower temperature limit (Tinv ) was shifted towards higher temperatures, and (b) the maximal affinity, reached at Tinv , was appreciably reduced (minimal KD increased). Both changes were of a much greater extent in rats exposed to a milder, than to a more severe hyperthermia (41 and 428C, respectively). After milder hyperthermia, in addition, all KD values above 278C were smaller (affinity greater) than in the controls, the overall amplitude of positive thermal modulation being reduced by almost 30%. Empirical data on RS affinity agreed with predictions based on kinetic modeling, suggesting that the observed thermal modulation of affinity might be a direct consequence of intrinsic kinetic properties of the underlying steroid–receptor interactions. By analogy with adaptive modifications of the thermal modulation in other receptor-ligand systems, the results suggest that short-term hyperthermia, if not excessive, may induce changes of HR affinity of functional significance in improving hyperthermic tolerance. Acknowledgements Grant #03E20 of the Ministry for Science and Technology of Serbia and grant F16 of the Serbian Academy of Sciences supported this work. References Andjus, R.K., 1969. Some mechanisms of mammalian tolerance to low body temperatures. Symp. Soc. Exp. Biol. Cambridge 23, 351–394.
D. Zˇivadinovic´ et al. / Journal of Thermal Biology 26 (2001) 575–584 Andjus, R.K., Buzalkov, R., 1960. Thermore´gulation du rat apre`s se´jour prolonge´ aux tempe´ratures supe´rieures a` la neutralite´ thermique. Tendence a l’hypothermie et perte de re´sistance a` la chaleur dans l’e´tat posthypothermique. C. R. Soc. Biol. Paris 154, 709–713. Andjus, R.K., C´irkovic´, T., Rakic´, M., 1982. Kinetics of glucose conversion by synaptosomes and its possible relationship to thermal modulation of enzyme–substrate affinities. Period. Biol. 84, 100–103. Andjus, R.K., Marjanovic´, M., Dzˇakula, Zˇ., Zˇivadinovic´, D., 1999. Cold adaptation mechanisms as revealed by experiments with brain synaptosomes. In: U.S. Army Research Office (Eds.), Hibernation and Adaptations to Cold. Research Triangle Park, North Carolina, pp. 27–39. Andreeva, A.P., Makhrov, A.A., Kuzishchin, K.V., Novikov, G.G., 1996. Adaptive changes of lactate dehydrogenase in white skeletal muscle of the sea trout Salmo trutta L. Biol. Bull. Moscow 21, 331–335. Atger, M., Milgrom, E., 1976. Mechanism and kinetics of the thermal activation of glucocorticoid hormone–receptor complex. J. Biol. Chem. 254, 1758–1762. Currie, R.W., White, F.P., 1981. Trauma-induced protein in rat tissues: a physiological role for a heat-shock protein? Science 214, 72–73. Cˇvoro, A., Dundjerski, J., Trajkovic´, D., Matic´, G., 1998a. Association of the rat liver glucocorticoid receptor with Hsp90 and Hsp70 upon whole body hyperthermic stress. J. Steroid Biochem. Molec. Biol. 67, 319–325. Cˇvoro, A., Dundjerski, J., Trajkovic´, D., Matic´, G., 1998b. Heat stress affects the glucocorticoid receptor interaction with heat shock protein Hsp70 in the rat liver. Biochem. Molec. Biol. Int. 46, 63–70. Cˇvoro, A., Dundjerski, J., Trajkovic´, D., Matic´, G., 1999. The level and phosphorylation of Hsp70 in the rat liver cytosol after adrenalectomy and hyperthermia. Cell Biol. Int. 23, 313–320. Cˇvoro, A., Matic´, G., 2000. Glucocorticoid receptor interaction with Hsp90 remains unaltered after whole body hyperthermia. Stress 3, 257–260. Doumen, C., D’Suze, G., Verheyen, E., Blust, R., 1986. Temperature and pH effects on the total white muscle LDH of Oreochromis niloticus (Pisces: Cichlidae). Comp. Biochem. Physiol. 83B, 441–444. Dzˇakula, Zˇ., 1990. Biophysical modeling of ligand binding to receptor molecules. Ph.D. Thesis, University of Belgrade, Belgrade. Dzˇakula, Zˇ., Zˇivadinovic´, D., Bozˇic´, M., Andjus, R., 1988. Modelling of the kinetics of steroid–receptor complex formation and dissociation. Period. Biol. 90, 119–124. Elez, D., Vidovic´, S., Matic´, G., 2000. The influence of hyperthermic stress on the redox state of glucocorticoid receptor. Stress 3, 247–255. El Hilali, M., Veillat, J.-P., Baddouri, Kh., Bennani, N., Andjus, R.K., 1978. Respiratory rate and thermal sensitivity of brown fat and other tissues from Jaculus orientalis and rat. J. Thermal Biol. 3, 195–201. Eliard, P.H., Rousseau, G.G., 1984. Thermodynamics of steroid binding to the human glucocorticoid receptor. Biochem. J. 218, 395–404. Fields, A.P., Somero, G.N., 1997. Amino acid sequence difference cannot fully explain interspecific variation in
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thermal sensitivies of gobiid fish A4-lactate dehidrogenases (A4-LDHs) J. Exp. Biol. 200, 1838–1850. Hochachka, P.W., Lewis, J.K., 1971. Interacting effects of pH and temperature on the Km values for fish tissue lactate dehydrogenases. Comp. Biochem. Physiol. 39B, 925–933. Hochachka, P.W., Somero, G.N., 1968. The adaptation of enzymes to temperature. Comp. Biochem. Physiol. 27, 659–668. Hochachka, P.W., Somero, G.N., 1973. Strategies of Biochemical Adaptation. W.B. Saunders Co., Philadelphia. Hochachka, P.W., Somero, G.N., 1984. Biochemical Adaptation. Princeton University Press, Princeton, NJ. Klyachko, O.S., Polosukhina, E.S., Ozernyuk, N.D., 1993. Environmental temperature affects the structural and functional properties of lactate dehydrogenase from fish skeletal muscles (in Russian). Biofizika Moscow 38, 596–601. Klyachko, O.S., Polosukhina, E.S., Ozernyuk, N.D., 1995. The effect of temperature and chorionic gonadotropin on the functional properties of lactate dehydrogenase from oocytes of loach Misgurnus fossilis. Comp. Biochem. Physiol. 111b, 517–521. Lehoux, E.A., Gouderlay, H.E., 1996. Thermally induced changes in intracellular pH and modulators of phosphofructokinase in trout white muscle. J. Exp. Biol. 200, 931–939. Lowry, O.H., Rosenbrough, N.J., Farra, A.L., Randall, R.J., 1951. Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Marjanovic´, M., Elliot, A.C., Dawson, M.J., 1998. The temperature dependence of intracellular pH in isolated frog skeletal muscle: lessons concerning the Na+–H+ exchange. J. Membrane Biol. 161, 215–225. Matic´, G., Dundjerski, J., Cˇvoro, A., 1998. Mutually dependent functions of glucocorticoid receptor and heat shock proteins. In: Fotherby, K. et al., (Eds.), Current Topics in Steroid Research, Vol. 1. Research Trends, Trivandrum, India. Matic´, G., Kipic´, J., Ristic´, B., Dundjerski, J., Trajkovic´, D., 1995. Hyperthermic stress modulates the functions of rat liver glucocorticoid receptor. Cell Biol. Int. 19, 203–213. Matic´, G., Trajkovic´, D., Sˇusˇ a, M., Damjanovic´, S., Petrovic´, J., 1989. In vitro evidence for modification of rat liver glucocorticoid receptor binding properties and transformation by hyperthermia. J. Steroid Biochem. 32, 263–270. Rahn, H., 1967. Gas transport from the external environment to the cell. In: De Reuck, A.V.S., Porter, R. (Eds.), Ciba Foundation Symposium on Development of the Lung. Little Brown & Co., Boston, pp. 3–23. Walsh, P.J., Somero, G.N., 1982. Interactions among pyruvate concentration, pH, and Km of pyruvate in determining in vivo Q10 values of lactate dehydrogenase reaction. Can. J. Zool. 60, 1293–1299. Yancey, P.H., Siebenaller, J.F., 1987. Coenzyme binding ability of homologs of M4-lactate dehydrogenase in temperature adaptation. Bioch. Biophys. Acta 924, 483–491. Zˇivadinovic´, D., Andjus, R.K., 1986. Temperature dependence of the equillibrium dissociation constant of a glucocorticoid hormone–receptor complex. Period. Biol. 88, 227–229. Zˇivadinovic´, D., Andjus, R.K., 1987. Thermal modulation of hormone–receptor affinities in a hibernator. Period. Biol. 89, 353–356.
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Zˇivadinovic´, D., Andjus, R.K., 1995. In vitro and in vivo thermal activation of steroid–receptor complexes from rats and ground squirrels (Spermophilus citellus). Comp. Biochem. Physiol. 110B, 451–462. Zˇivadinovic´, D., Dzˇakula, Zˇ., Andjus, R.K., 2000. Temperature modulation of glucocorticoid-receptor affinity in a hiberna-
tor, the European ground squirrel, and a non-hibernator, the rat. In: Heldmaier, G., Klingenstor, M. (Eds.), Proceedings of the Life in the Cold Symposium (Jungholz, Austria). Springer-Verlag, Berlin, pp. 277–284.
Hyperthermic stress affects the thermal modulation of glucocorticoid–receptor affinity Dragoslava Zˇivadinovic´a,*, Stojko Vidovic´b, Gordana Matic´a, Radoslav K. Andjusc a
b
Institute for Biological Research, 29 Novembra 142, 11060 Belgrade, Yugoslavia Department of Biology and Human Genetics, Faculty of Medicine, University of Banja Luka, Sime Mrkalja 10, 78000 Banja Luka, Republic of Srpska, Bosnia and Herzegovina c Center for Multidisciplinary Studies, University of Belgrade, 29 Novembra 142, 11060 Belgrade, Yugoslavia Received 6 September 2000; received in revised form 31 October 2000; accepted 5 December 2000
Abstract In vitro binding of a steroid hormone (triamcinolone acetonide) to hepatic glucocorticoid receptors was studied in liver cytosols prepared from untreated control rats and rats sacrificed after being exposed in vivo to whole body hyperthermia (41 or 428C). Positive temperature modulation of glucocorticoid–receptor affinity (decrease of affinity with increasing temperature) was well expressed in all preparations. In preparations from hyperthermic rats, alterations of a possible functional (adaptive) significance have been recorded: the amplitude of positive thermal modulation was reduced, and its lower-temperature limit shifted towards higher temperatures. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Glucocorticoid receptor; Hormone–receptor affinity; Hyperthermia
1. Introduction It was previously shown that short-term whole-body hyperthermia induces serious alterations of the rat liver glucocorticoid-receptor system (reviewed by Matic´ et al., 1998). Hyperthermia affects the rate of dissociation of the hormone–receptor complex and results in a reduction of the receptor’s binding capacity (Matic´ et al., 1989, 1998), in the stimulation of its nuclear import (Matic´ et al., 1995), and in changes of its association with heat-shock proteins (Cˇvoro et al., 1998a, b). In the latter study, the HT-41 stress was shown to stimulate the association of the glucocorticoid receptor (GR) with a heat-shock protein, Hsp70. After HT-41, the GR:Hsp70
*Corresponding author. Present address: NICHD, ERRB, NIH, Building 49, Room 6B/23, 49 Convent Drive, MSC 4510, Bethesda, MD 208924510, USA. E-mail address: [email protected] (D. Zˇivadinovic´).
ratio in receptor complexes amounted to 1:3 as compared to 1:1 in the unstressed controls. It was reasonable to assume that such alterations might also influence mechanisms underlying the socalled ‘‘positive thermal modulation’’ of hormone– receptor affinity (increase of the dissociation constant with increasing temperature). Positive thermal modulation was shown by another line of our studies to characterize the temperature dependence of glucocorticoid–receptor affinity in hepatic cytosol preparations from rats and ground squirrels (Zˇivadinovic´ and Andjus, 1986, 1987; Zˇivadinovic´ et al., 2000). Positive thermal modulation described by us in these mammals was analogous to the thermal modulation of affinity in enzyme–substrate systems from ectotherms. The term was coined to stress the similarity of this effect of temperature to the effect of positive allosteric modulators on the affinity of an enzyme for its substrate (Hochachka and Somero, 1973). In ectotherms, positive thermal modulation of enzymes was described as one of the strategies of biochemical adaptation to short-term
0306-4565/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 6 - 4 5 6 5 ( 0 1 ) 0 0 0 0 3 - 1
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variations of body temperature, since it compensates for excessive temperature-induced changes of kinetic energy on reaction rates and provides the animal with means for immediate compensation of its metabolic rate when confronted with changing ambient temperature (Doumen et al., 1986; Hochachka and Somero, 1973). By analogy, an adaptive role has been envisaged of the positive thermal modulation of hormone–receptor affinity observed by us in the rat and the ground squirrel, the increase of affinity with cooling compensating for an endangering decrease of kinetic energy involved in hormone–receptor interactions. By analogy again with enzyme–substrate systems, it was suggested that the observed shift of the lower limit of positive thermal modulation towards lower temperatures in the more cold-tolerant ground squirrel as compared to the less cold-tolerant rat, had an adaptive value. The relevance of analogies between findings in ecthothermic vertebrates and homeotherms may be questioned, since temperature swings in the latter are comparatively negligible. However, it is well known that many homeotherms, the rat in particular, can tolerate changes in body temperature as broad as those in ectotherms. In rats, unassisted recovery occurs after lowering their body temperature from 37 to 158C. They can be revived by special resuscitation techniques after cooling the body temperature to 08C and even after supercooling below 08C (Andjus, 1969). Comparisons of the dependence of biochemical events on body temperature in the rat and ectotherms are thus legitimate and potentially meaningful. Such comparisons were made in discussing and interpreting the results of the present study as well. In the past, kinetic modeling of glucocorticoid– receptor interactions predicted KD-versus-temperature profiles typical of positive thermal modulation (Dzˇakula et al., 1988; Dzˇakula, 1990). This indicated that such profiles, observed experimentally, might be the consequence of intrinsic kinetic properties of the receptor– ligand system. The model of Dzˇakula (1990) was presently used in analyzing kinetic data on binding of triamcinolone acetonide (a synthetic glucocorticoid) to the hepatic glucocorticoid receptor of rats exposed to whole body hyperthermic stresses of different intensity. As already pointed out, our preceding experiments showed that hyperthermia induces serious alterations of the rat liver glucocorticoid-receptor system. Applying the model of Dzˇakula, we investigated these time effects of hyperthermic exposures on the positive thermal modulation of glucocorticoid–receptor affinity. As expected, marked alterations of this phenomenon have been found as a result of sublethal hyperthermia. 2. Materials and methods Materials: [3H] Triamcinolone acetonide ([3H] TA; specific activity 20 Ci/mmol) was purchased from
Amersham International (Amersham, UK), unlabeled triamcinolone acetonide (TA) from Sigma Chemical Co. (St. Louis, MO, USA), sucrose, Tris base and charcoal from Merck Co. (Darmstadt, Germany) and Dextran T-500 from Pharmacia Fine Chemicals (London, UK). All chemicals used were analytically pure. Animals: Rats of the Wistar strain (2.5 months old, 250–300 g body weight) were reared under standard laboratory conditions (20–228C, 12:12 h light:dark cycle, food and water ad libitum). To free hormone receptors from endogenous glucocorticoids, the animals were bilaterally adrenalectomized. They were subsequently kept on a sodium-rich diet (0.9% NaCl in drinking water) to prevent sodium depletion resulting from lack of mineralocorticoids after adrenalectomy. Rats were exposed to hyperthermic stresses 4 days postoperatively. To induce the ‘‘sublethal hyperthermic stress’’ (termed ‘‘HT-41’’ in the text), animals were anesthetized with Nesdonal (4.6 mg/100 g b.w, i.p.) and placed into a ventilated and humidified chamber preset to 448C (Currie and White, 1981). A digital thermistor thermometer continuously monitored rectal temperature which increased to 418C in about 40 min. A further rise of body temperature was prevented by intermittently ventilating the warm chamber with fresh air. After maintaining in this way the rectal temperature at approximately 418C during 15 min, the animals were sacrificed by rapid decapitation. The more severe ‘‘semilethal hyperthermic stress’’ (termed ‘‘HT-42’’ in the text) was induced by maintaining the animals in the same warm chamber for about 55 min, and by sacrificing them when their rectal temperature reached 428C. The sham-treated controls were Nesdonal-anesthetized, maintained at room temperature, and decapitated at the same time as their heat-stressed counterparts. Cytosol preparation: Methods for preparing cytosol from pooled liver homogenates and for the measurement of specific binding of 3H-labeled triamcinolone acetonide ([3H]TA) were the same as previously described (Zˇivadinovic´ and Andjus, 1995). The livers were perfused in situ with ice-cold 50 mM Tris buffer, pH 7.55 at 208C, containing 0.25 M sucrose, 25 mM KCl, 10 mM MgCl2 and 20 mM Na2MoO4. They were then quickly excised and homogenized in two volumes (w/v) of the same solution. After the first centrifugation of the homogenate (8000 g, 10 min, 48C), the supernatant was re-centrifuged (105, 000 g, 1 h, 48C, Ti50 rotor) to yield the cytosol preparation. The preparation was quickly frozen and stored in liquid nitrogen until use. Time course kinetics of receptor–steroid (RS) complex formation: Cytosol aliquots (100 ml) were incubated with approximately 30 nM [3H]TA. Parallel aliquots, incubated in the presence of a 100-fold molar excess of unlabeled TA, allowed corrections for nonspecific binding. Free steroid was removed from the incubation
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mixture by the addition of a 3.75% charcoal–0.375% Dextran suspension following the procedure described by Atger and Milgrom (1976). The time course of specific [3H]TA binding was studied at 8 temperature levels in the control and the HT-42 groups, and at 12 levels in the HT-41 group (from 0 to 378C; 2–58 apart). At each temperature, concentrations of bound steroid were determined after 8 time intervals following the addition of pre-incubated cytosol aliquots to [3H]TA samples. The time intervals varied with temperature and were considerably shorter during the initial phases of the binding process (ranging from 15 s to 3 h at 378C, and from 2 min to 18 h at 08C). pH changes with temperature were practically the same in cytosols from our three experimental groups (HT-41, HT-42 and controls), so that their influence on the comparative results concerning steroid binding is excluded. Kinetic modeling: The kinetic model used was based on thermodynamic considerations and Eyring’s transition state theory (Dzˇakula et al., 1988; Dzˇakula, 1990). It predicts a number of properties characterizing the formation of steroid–receptor complexes, such as the temperature dependence of the binding process and the U-shaped KD-versus-temperature profile. At its origin, the model has the conceptual kinetic scheme ka
kl
ktr
ki
k1
ktr
Ri þ S . Ra þ S . Ra S . Rtr S: (R}receptor, with subscripts: i}inactive, a}active, tr}transformed; S}steroid). In agreement with empirical data, the model predicts one-maximum time profiles of steroid–receptor binding, with a non-zero equilibrium plateau. The fitting procedure was based on the Nedler–Mead method. Fitting involved simultaneously experimental data obtained at all incubation temperatures and after all time intervals (n=64 for the control and the HT-42 heatstressed group; n=96 for the HT-41 heat-stressed group). Twelve thermodynamic parameters were allowed to vary (six entropies and six enthalpies, concerning receptor activation and inactivation, association, dissociation, complex transformation and reverse transformation). Receptor concentrations were fixed at values close to those experimentally obtained (12.9 nM for control rats; 3.7 and 2.7 nM for the HT-41 and HT-42 heat-stressed groups). KD values for different incubation temperatures were calculated as follows: KD ¼
½So ð½Ro ½RSÞ ð½Ro ½RSÞ: ½RS
([So] } the applied steroid concentration, [Ro] } receptor concentration, [RS] } complex concentration). Values obtained as best-fit requirements, concerning the concentration of receptor molecules and the maximal
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concentrations of the steroid–receptor complex ([RS]max), were introduced into this equation in order to arrive at KD values for each temperature level. [RS]max values for different temperatures were obtained from numerical listings of fitted data-points of the timedependent binding. Protein determination: Protein contents of hepatic cytosols, determined as described by Lowry et al. (1951) and using BSA as a reference, ranged from 25.5 to 33.0 mg/ml. Statistics: The Mann–Whitney and the t-test were applied to differences between sets of data-points. Differences between slopes were evaluated using the Tukey test. Differences between forms of best-fit equations were evaluated using the commercial table curve 2D program (AISN Software, Jandel Scientific). Equations suggested by the program for fitting a given set of experimental data-points are given in explicit form by the program, they are ranked by the goodness of fit criteria and each is identified by an identification number. We refer in the text only to these identification numbers and to the rank of the equation referred to (i.e., Eqs (6122) and (7113), Rank 1).
3. Results Fig. 1 illustrates the time course of [3H]TA binding to the glucocorticoid receptor in cytosols from controls and from the two groups of hyperthermic rats. Experimental data obtained at all incubation temperatures and after all time intervals were fitted simultaneously (n=64 for the control and the HT-42 group, n=96 in the case of the HT-41 group; triplicate analyses for each value). At all incubation temperatures, and in the case of all preparations (from controls and from both groups of hyperthermic rats), the time course of [3H]TA binding was characterized by a biphasic pattern. After rapidly reaching a maximum ([RS]max), the concentration of bound steroid slowly declined, the slopes of the descending branches increasing considerably with temperature. The framed panels in Fig. 1 display profiles obtained by fitting the kinetic model to experimental data concerning control and hyperthermic rats. The lower right-hand panel exemplifies the approximation of experimental data by model-derived curves at incubation temperatures close to Tinv. Differences between families of curves displayed in the four panels of Fig. 1 were fully expressed, although these families did not include temperatures reached in vivo by our hyperthermic rats. Cytosol incubation at 41 and 428C could provoke substantial denaturation and aggregation of proteins and the function of chaperone molecules could have been seriously hampered in vitro. As shown in the inset of Fig. 2, [RS]max values derived from data in Fig. 1, were highly temperature-dependent
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Fig. 1. Temperature dependence of the time course kinetics of specific binding as affected by hyperthermic stresses. Model-derived profiles refer to control data (upper left panel), and to data obtained after the milder HT-41 and the more severe HT-42 stress (upper right and lower left panels, respectively). The lower right-hand panel is an illustration of the adequacy of the approximation of experimental data-points (dots) by model-derived profiles.
and formed bell-shaped [RS]max-versus-temperature profiles. The bell-shaped curves concerning stressed animals were considerably flatter, particularly in the case of animals exposed to the more severe heat stress. They peaked at values of 3.2 and 2.5 nM in case of the HT-41 and HT-42 groups, respectively, as compared to the control peak value of 11.3 nM. In addition, considerable differences were expressed concerning the placement of these peaks along the temperature scale. [RS]max peaks concerning heat-stressed animals were shifted to the right, to temperatures higher by 2–38C: to 17 and 18.58C in the HT-42 and the HT-41 groups,
respectively, as compared to 15.58C in the controls. The shift was thus appreciably greater after the milder than after the more severe hyperthermia. The main panel of Fig. 2 displays theoretical (model derived) KD-versus-temperature profiles. For each temperature level (8–12 temperatures), KD figures were obtained by introducing into the appropriate equation (see Section 2) values concerning the concentration of receptor molecules and the maximal concentration of the steroid–receptor complex ([RS]max), obtained as best-fit requirements. In the case of all preparations, those from the control as well as those from the
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Fig. 2. Main panel: Model-derived KD-versus-temperature profiles. Dots identify values used in Fig. 3 to analyze in a different way the positive thermal modulation branch of the temperature dependence of KD . Inset temperature dependence of the binding maxima ([RS]max-versus-temperature profiles). Verticals in the main panel and inset indicate, respectively, the placement along the temperature scale of the calculated [RS]max maxima and KD minima (15.58C in the controls; 17 and 18.58C in the HT-41 and the HT-42 group, respectively).
Fig. 3. Positive thermal modulation of RS affinity (from 158C upwards). KD values, joined by simple spline fitting, are those identified by dots in the main panel of Fig. 2: (open circles) controls; (closed circles) HT-42; (closed rectangles) HT-41. Upper inset: Amplitude of positive thermal modulation of RS affinity (extent of KD increase from Tinv to T=378C) expressed as percent of the value in the control group.
heat-stressed animals, KD -versus-temperature profiles were U-shaped. They showed a marked decrease of KD values with falling temperature (positive thermal modulation of affinity) and a substantial increase of these values with further cooling below the inversion temperature (negative thermal modulation of affinity). The inversion points matched the temperatures characterized by the maxima of the [RS]max-versus-temperature profiles shown in the inset of the same figure. The inversion temperature, defining the lower limit of positive thermal modulation, was shifted to clearly higher levels in the heat-stressed groups (17 and 18.58C in the HT-42 and the HT-41 groups, respectively) in comparison to the controls (Tinv =15.58C). In the interval between 58 and 298C, the difference between a selected set of data-points (28 apart, n=13) in the HT-41 group and the corresponding set of data-points in the controls, was statistically significant ( p=0.004). In contrast, the difference between the corresponding sets of control and of HT-42 data-points was not significant ( p=0.338). By the Tukey test (regression comparisons), the slope of the KD increases above 318C (7 data-points,
18 apart) was statistically different in the HT-41 group from those in the two other animal groups ( p=0.00001). Dots along curves in the main panel of Fig. 2 identify values used in Fig. 3 to analyze in a different way the phenomenon of positive thermal modulation and accentuate differences between experimental groups within the range of relatively high temperatures (from 15 to 378C). The three sets of KD values in Fig. 3 are joined this time by simple spline fitting. The curve belonging to the HT-41 group (milder hyperthermia) deviates appreciably from the rest of the curves shown: at temperatures below 278C, it runs above, while at temperatures higher than 278C it is displaced below the other two curves. In contrast, differences between curves belonging to the controls and to the HT-42 group (severe hyperthermia) are negligible. The table curve fitting procedure showed that Rank 1 equation for the HT-41 curve (Eq. (7113)) was different from the one satisfying data from both the controls and the HT-42 group (Eq. (6122)). The amplitude of positive thermal modulation, expressed as the distance of KD values at Tinv from those at 378C (inset) was reduced by 29% in
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the HT-41 group, and insignificantly (less than 4%) in the HT-42 group.
4. Discussion The present study confirmed the phenomenon of thermal modulation of RS affinity in the rat, previously described by us in rat and ground squirrel cytosol preparations (Zˇivadinovic´ et al., 2000). This time, however, marked alterations of the thermal modulation of RS affinity have been identified in rats exposed to short-term hyperthermia. The alterations depended to a great extent on the severity of the heat stress applied. According to our experience, rats survive without exception a 15-min exposure to 418C body temperature. If, however, body temperature is raised to 428C, about half of the rats succumb when left to recover at room temperature. The choice of these two hyperthermic exposures (41 and 428C) in the present study was based on our previous experience with their effects on glucocorticoid-receptor’s binding capacity, the rate of receptor–hormone binding, complex dissociation, affinity of steroid binding, complex translocation, and association of receptors with heat-shock proteins (Cˇvoro et al., 1998a, b, 1999, 2000; Matic´ et al., 1989, 1998). The functional significance of the presently observed phenomena and the underlying molecular mechanisms remains to be clarified. 4.1. Functional significance Empirical data on RS affinity agreed with predictions based on kinetic modeling, suggesting again that the observed thermal modulation of affinity might be a direct consequence of intrinsic kinetic properties of the underlying steroid–receptor interactions (Zˇivadinovic´ and Andjus, 1986; Zˇivadinovic´ et al., 2000). The functional meaning of the U-shaped KD-versus-temperature profile in a homeothermic species remains, however, to be elucidated. Forming such profiles, the dissociation constant of RS affinity was temperature-modulated in rats in much the same way as in the case of a number of enzyme– substrate systems of ectotherms (Hochachka and Somero, 1968, 1973, 1984; Klyachko et al., 1995; Andreeva et al., 1996). Both the positive and the negative thermal modulation of affinity were well pronounced. It is of particular interest that the present results were also in agreement with data concerning steroid binding to human glucocorticoid receptors from lymphoblastoid cells (Eliard and Rousseau, 1984). Although the latter authors did not illustrate their results graphically, a U-shaped Km-temperature profile,
with a pronounced positive thermal modulation arm, can easily be derived from their data. As discussed in one of our preceding papers (Zˇivadinovic´ et al., 2000), controversies accompany interpretations of the ‘‘U-shaped curves’’ since 1971 when it was shown that pH might modify the U-shaped response (Hochachka and Lewis, 1971). Nevertheless, U-shaped curves continued to be published by the same authors (Hochachka and Somero, 1984) and by others (Klyachko et al., 1993, 1995; Andreeva et al., 1996). In ectotherms, changes of intracellular pH with temperature were shown to be considerably smaller than those paralleling the pH of water neutrality (Lehoux and Gouderlay, 1996; Marjanovic´ et al., 1998). It was underlined, moreover, that rapidly occurring acute changes in body temperature may not allow for ‘‘correct’’ (temperature-adjusted) pH to be attained (Walsh and Somero, 1982). In mammals, such as our rats, the regulation of the internal milieu is known for a long time now to differ from that in ectotherms (Rahn, 1967). In the rat (and in ground squirrels) such curves have been previously obtained and discussed by us (Andjus et al., 1999; Zˇivadinovic´ et al., 2000). Besides, the fact remains that our theoretical, model derived curves, were U-shaped. They were constructed solely on the basis of a conceptual kinetic scheme based on thermodynamic considerations and Eyring’s transition state theory. The functional meaning of the observed positive thermal modulation of RS affinity in the rat, a homeothermic species, remains unclear. In the rat, positive thermal modulation of RS affinity may simply represent the consequence of intrinsic kinetic properties of steroid–receptor interactions, deprived in the homeotherm of the specific functional role played in its ectothermic ancestors. One could hypothesize, however, that a functional role of the positive thermal modulation might be present in the rat as well. One could assume, for example, that starting from levels of affinity and kinetic energy compatible with hormone action, cooling would tend to induce excessive decreases of kinetic energy, threatening to stop RS interactions. This would be opposed by the concomitant rise of affinity due to its positive thermal modulation (‘‘immediate rate compensation’’). In small mammals such as rats, accidental hypothermia induced by the association of wetting and cold is not a rare phenomenon, and the maintenance of hormone action may be important for a successful rewarming (Andjus, 1969). Positive thermal modulation of RS affinity could therefore contribute to the hypothermic tolerance of the animal. After exposure of rats to hyperthermia, Tinv of RS affinity was shifted to higher incubation temperatures (Fig. 2). This was highly reminiscent of the shift in the same direction of Tinv of ES affinity in ectotherms after their acclimation or evolutionary adaptation to warmer
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environments and consequently higher body temperatures (Hochachka and Somero, 1973, 1984). In our HT-41 group (milder hyperthermia), the increase of KD with temperature was less steep and KD values above 278C were even smaller (affinity greater) than in the controls. The overall amplitude of positive thermal modulation was reduced by almost 30% (Fig. 3 inset). These findings were reminiscent of those concerning ES affinity in ectotherms. Lower Km values (higher affinities) at the majority of incubation temperature are typical of ectothermic species from warmer habitats compared to those adapted to colder environments (Yancey and Siebenaller, 1987). In ectotherms, a reduction of the amplitude of positive thermal modulation of ES affinity has been observed after acclimation/ adaptation to higher environmental/body temperatures. (Hochachka and Lewis, 1971). Km values for fish enzymes were always found to be reduced after adaptation to a higher temperature (Yancey and Siebenaller, 1987). The described reductions in the amplitude of positive thermal modulation of affinity were said to enable the enzyme to function in a broad range of temperatures with comparatively small changes of ES affinity. By minimizing the decrease of affinity with increasing temperature, a relatively high affinity is conserved at all temperatures thus contributing to the eurythermy of the species (Fields and Somero, 1997). By analogy, one could envisage an adaptive role of the modification of positive thermal modulation of RS affinity observed in our experiments with hyperthermic rats, taking into account the following considerations. Positive thermal modulation of RS affinity tends to decrease affinity at high temperature. The association, however, of decreased affinity and increased kinetic energy at elevated body temperature may threaten the stability of the RS complex. This stability is, however, a prerequisite of hormone action, of particular importance for the complex transformation step. An adjustment of positive thermal modulation that would reduce its amplitude and minimize the reduction of affinity at elevated temperatures would thus counteract the potentially harmful effect of the association of reduced affinity and increased kinetic energy on the stability of the RS complex. Such an adjustment is, therefore, suggestive of an adaptive modification, of functional significance to the organism threatened by elevated body temperature. The presently observed effects of the temperature modulation of KD were also reminiscent to some extent of our previous results obtained with glucocorticoid receptors from rats and European ground squirrels (Spermophylus citellus, a hibernator) (Andjus et al., 1982; Zˇivadinovic´ et al., 2000). In the ground squirrel compared to the rat, the whole U-shaped KD-versustemperature curve was shifted upwards, and the KD minimum was displaced to an appreciably lower temperature (lower Tinv than in the rat). As to the
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differences in vertical placement of the U-shaped curve, the ground squirrel differed therefore from the rat in much the same way as the hyperthermic rat from its normothermic controls. The direction of the ‘‘shift’’ of Tinv was, however, opposite, but in agreement with thermal conditions to which the animal is expected to adjust. In the hibernator, adapted to better tolerate low body temperatures, Tinv appeared as displaced to lower temperatures, whereas in the hyperthermic rat, expected to adjust to higher body temperatures, Tinv occurred at higher temperatures. In order to relate our results obtained in vitro to the organismic and whole animal level, known facts should be considered about the capacity of the rat to enhance survival in hyperthermia by metabolic adjustments. Metabolic peculiarities of rodent species adapted to hot environments should also be taken in to consideration. As shown long ago by one of us, the rat is indeed capable of acclimating to extremely hot environments and hyperthermic body temperatures (Andjus and Buzalkov, 1960). When exposed abruptly to ambient temperatures of 38 to 398C, rats inevitably succumb to hyperthermia in a matter of a few hours. If acclimated gradually, over several weeks, to the hot environment by a stepwise increase of environmental temperature (358C followed by 388C), they survive in the same hot environments for months, with hyperthermic levels of body temperature (38–398C). After such acclimation, their metabolic rate (oxygen consumption) is markedly reduced, both as a function of environmental and of body temperature. All changes induced by high-temperature acclimation were shown to be fully reversible. One of the changes observed after acclimation to hyperthermic environments was loss of resistance to cooling. Exposure to moderately cold environments (0–48C), easily tolerated by rats from a thermoneutral environment, provokes in rats adapted to hyperthermia a rapid and continuous fall of body temperature (Andjus and Buzalkov, 1960). The considerable shift of Tinv to higher temperatures, presently observed in our TH-41 group of hyperthermic rats (Fig. 2), may also be regarded as a loss of resistance to cooling. It indicates, namely, that the ability to counteract, by means of positive thermal modulation, the effect of cooling on RS affinity is reduced. The harmful effect of cold on reaction rates is enhanced, since negative thermal modulation makes its appearance earlier, at higher temperatures. This may, therefore, reflect to some extent the loss of cold resistance observed previously in rats adapted to hyperthermic conditions. In general, changes observed after short-term exposures to hyperthermia, such as those presently recorded in our HT-41 group of rats, might be regarded as reflecting the beginning of complex metabolic changes leading eventually to improved hyperthermic tolerance and a loss of resistance to cooling.
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Another line of our studies led us to the identification of a number of metabolic peculiarities in a highly heattolerant desert rodent, Jaculus orientalis (El Hilali et al., 1978). A number of tissues (including brain) showed, namely, higher metabolic rates at ‘‘hyperthermic’’ incubation temperatures (37–448C) than the same tissues of the rat. By analogy, the increase of RS affinity at incubation temperature above 278C, observed presently in the HT-41 group of rats, may also be regarded as beneficial to heat tolerance and thus contributing to an efficient metabolism at elevated temperature. 4.2. Molecular mechanisms Our kinetic modeling suggests that the thermal modulation of RS affinity arises from intrinsic kinetic properties of the underlying steroid–receptor interactions. The mechanisms of the observed modifications induced by the two modalities of heat stress remain, however, to be elucidated. Differences between effects of the milder and the more severe heat stress have been identified recently in our laboratory at the level of the receptor molecule itself. Structural effects were studied using immunopurified receptor preparations. In the HT-42 animals (severe heat stress) steroid-binding assays revealed a significant reduction of specific TA binding both to the immunopurified and the cytosolic glucocorticoid receptor. In the HT-41 animals, in contrast, the reduction involved only binding to the cytosolic receptor (Elez et al., 2000). The data suggested that in the case of the HT-42 stress, the alteration of the RS-binding capacity resulted from the modification of the receptor itself. The HT-41 stress, on the other hand, might have affected the receptor’s binding capacity indirectly, through other cytoplasmic components influencing the receptor function. This explanation was confirmed by the finding that only the HT-42 stress, not the milder one, led to intramolecular disulfide bond(s) formation within the receptor (Elez et al., 2000). It was also supported by the finding that the HT-41 stress stimulates the association of the receptor with heat-shock proteins, Hsp70 in particular (Cˇvoro et al., 1998a, b). It is well known that hyperthermic exposure is accompanied by an accumulation of partially or totally denatured proteins. It is precisely the appearance of structure proteins altered in the cell that is critical for eliciting the cellular response to stress, i.e. the induction of heat shock and other proteins with a protective role. Conformational changes of the receptors and covalent modifications are also possible (Elez et al., 2000), as well as changes of the interaction between receptors and heat-shock proteins (Cˇvoro et al., 1998a, b). All the enumerated changes could have been the cause of the reduction of the receptor’s binding capacity observed in our study. It is known, however, that denaturation may
be reversible and that chaperone molecules (heat-shock proteins), closely associated with receptors, participate in the renaturation process. Control experiments from our previous studies showed that following the HT41type stress the capacity of [3H]TA binding to glucocorticoid receptors returns to normal in about 4 hours (Matic´ et al., 1995).
5. Summary It was assumed that the serious alterations of the rat liver glucocorticoid-receptor system, previously demonstrated in rats exposed to short-term whole-body hyperthermia, might as well influence mechanisms underlying the thermal modulation of hormone–receptor affinity. The latter phenomenon, consisting in a decrease of affinity with increasing incubation temperature, was shown by another line of our previous studies to govern the temperature dependence of the in vitro glucocorticoid-receptor system of rats. In cytosols, from rats made hyperthemic, positive thermal modulation of HR affinity was modified in two main respects: (a) its lower temperature limit (Tinv ) was shifted towards higher temperatures, and (b) the maximal affinity, reached at Tinv , was appreciably reduced (minimal KD increased). Both changes were of a much greater extent in rats exposed to a milder, than to a more severe hyperthermia (41 and 428C, respectively). After milder hyperthermia, in addition, all KD values above 278C were smaller (affinity greater) than in the controls, the overall amplitude of positive thermal modulation being reduced by almost 30%. Empirical data on RS affinity agreed with predictions based on kinetic modeling, suggesting that the observed thermal modulation of affinity might be a direct consequence of intrinsic kinetic properties of the underlying steroid–receptor interactions. By analogy with adaptive modifications of the thermal modulation in other receptor-ligand systems, the results suggest that short-term hyperthermia, if not excessive, may induce changes of HR affinity of functional significance in improving hyperthermic tolerance. Acknowledgements Grant #03E20 of the Ministry for Science and Technology of Serbia and grant F16 of the Serbian Academy of Sciences supported this work. References Andjus, R.K., 1969. Some mechanisms of mammalian tolerance to low body temperatures. Symp. Soc. Exp. Biol. Cambridge 23, 351–394.
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