Blood Pressure and Heat Shock Protein Expression in Response to Acute and Chronic Stress
Abstract We previously demonstrated that restraint and pharmacological agents that activate sympathetic nervous system activity induce expression of the 70-kD heat shock protein (HSP70) in major blood vessels. The magnitude and rapidity in which HSP70 is induced in the aorta suggest that it may play a salient role in the mechanical properties of vascular smooth muscle. Other investigators have reported that HSP70 inducibility is increased in genetically hypertensive animals. In this report, we have investigated the effects of acute and chronic (8-week) exposure to restraint and restraint in the presence of a randomized intermittent air jet on the development of hypertension and the induction of HSP70 in the aorta and adrenal glands of normotensive adult male Sprague-Dawley rats. Acute restraint or air jet resulted in a fivefold to sixfold increase in aortic HSP70 mRNA expression. Chronic exposure to restraint reduced the HSP70 response to acute restraint. In contrast, no adaptation of the HSP70 response to acute air jet was observed in aortas of chronically air jet–treated rats. In adrenal glands, HSP70 expression was reduced after chronic restraint and air jet, indicating that in this tissue, adaptation occurs to both stressors. There was no difference in HSP70 expression in unstressed rats that had been chronically exposed to restraint or air jet in either adrenal gland or aorta. A significant increase (P<.05) in systolic blood pressure developed in air jet–treated animals (120±3 mm Hg) but not in restrained rats (107±2 mm Hg) compared with unstressed controls (106±3 mm Hg). Plasma catecholamine concentrations were not indicative of HSP70 expression in the aorta. From these results, we conclude that adaptation to a stressor influences both resting blood pressure and the magnitude of the HSP70 response in aorta to an acute stress. Thus, the ability to induce HSP70 in vascular tissue may contribute to the development of hypertension in chronically stressed animals.
We previously showed that restraint stress induces expression of the 70-kD heat shock protein (HSP70) in the aorta.1 This restraint-induced HSP70 expression could be blocked by specific α1-adrenergic receptor antagonists and could be evoked in unstressed animals administered adrenergic receptor agonists. In subsequent studies, we showed that dopamine agonists2 and catecholamine reuptake inhibitors3 share an ability with restraint to induce HSP70 expression in the aorta. These investigations provided substantial evidence that stress-induced expression of HSP70 in aorta results from catecholamine neurotransmission after activation of the sympathetic nervous system. On the basis of these results, we hypothesized that HSPs may affect the mechanical function of vascular smooth muscle and may be involved in the development of hypertension.
Support for this hypothesis has come from studies demonstrating that the inducibility of HSP70 is greater in hypertensive animals.4 5 These studies have shown that genetically hypertensive animals display a greater increase in heat-induced HSP70 expression than do normotensive control animals. This hypertension-dependent supersensitivity to heat-induced HSP70 expression was demonstrated in vascular smooth muscle cultures,5 fibroblast cultures,6 and several tissues of in vivo heat-stressed animals.4 Further reports showed that this supersensitivity results from enhanced activation of heat-shock transcriptional regulatory factors in genetically hypertensive animals7 and may be linked to a polymorphism of the HSP 70 gene.8 There is even a report that lymphocytes taken from hypertensive human patients display a similar enhanced heat shock response relative to normotensive individuals.9
It is generally accepted that stress contributes to human high blood pressure. This concept is difficult to test in laboratory experiments because not all stressful stimuli are of equal intensity, nor do they produce identical physiological and behavioral effects. Reproducible stressful stimuli are needed that effectively elevate blood pressure in otherwise normal rats that do not have hereditary hypertension. In this report, we compare the effects of acute and chronic restraint and restraint in combination with a randomized air jet on the development of hypertension and the induction of HSP70 in the aorta and adrenal gland. We demonstrate a differential efficacy of restraint and air jet to induce aortic HSP70 mRNA and report a novel relation between HSP70 expression induced by acute and chronic stress and the development of hypertension.
Animals and Treatment Conditions
Adult male Sprague-Dawley rats (250 to 300 g) were used in all experiments described below. Rats were fed Rodent Laboratory Chow (No. 5001, Ralston Purina) and were maintained under previously described conditions.10 Before any treatments, rats were handled gently and frequently to acquaint them with laboratory personnel. All treatment conditions described below were conducted between 9 am and noon.
Animals exposed to restraint stress were placed in plexiglass rodent restraints that restricted their movement but did not completely immobilize them.11 Restrained animals were placed on a rack used for air jet–treated animals in an isolated room with the temperature set at 24°C. Chronically restrained rats were exposed to these treatment conditions for 30 min/d, 5 days per week for 8 weeks.
Air jet–treated animals were restrained as described above. Additionally, rats in this treatment group were exposed to a jet of air (15 psi) from a distance of 50 mm during restraint. Air jets were intermittent, with onset and duration randomized during the 30-minute exposure period. The duration of the jet varied randomly from 0.5 to 10.0 seconds. The interjet interval varied from 10 to 60 seconds. Air jet intervals and durations were regulated by a solenoid valve controlled by a personal computer with custom-designed software. Chronically air jet–treated animals were exposed to these treatment conditions for 30 min/d, 5 days per week for 8 weeks.
Control animals were neither restrained nor exposed to air jet but were moved into the exposure room daily. During the last week of the 8-week period, blood pressure was measured in all animals from each treatment group by the tail-cuff method.11 12 Blood pressure measurements were taken from restrained and air jet–treated animals 24 hours after their last stress exposure to reduce the influence of the acute stress treatment.
On the final day of experimentation, the control and treatment groups were further divided. The control group was divided into three groups receiving no treatment (n=7; CC), an acute exposure to restraint (n=7; CR), or an acute exposure to air jet (n=5; CA). The chronically restrained group was divided into two groups receiving either no treatment (n=5; RC) or a final exposure to restraint (n=5; RR). The chronically air jet–treated rats were also divided into two groups receiving either no treatment (n=5; AC) or a final exposure to air jet (n=5; AA). Immediately after their last treatment, rats were decapitated, trunk blood was collected, and adrenal glands and aortas were removed and frozen in liquid nitrogen. As with the treatment conditions, rats were killed and blood samples and tissues were collected between 9 am and noon.
All procedures involving the manipulation of animals conform to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the animal care and use committees of the US Department of Agriculture, Agricultural Research Service, Grand Forks Human Nutrition Research Center; and the University of North Dakota.
RNA Isolation and Northern Analysis
Total RNA was isolated from tissue samples with RNAzol B (Tel-Test, Inc). RNA was quantified spectrophotometrically before samples were loaded. For Northern analysis, 10 μg of total RNA was denatured in formaldehyde, fractionated on 1.0% agarose gels, and then transferred to Genescreen Plus membranes (DuPont NEN Research Products). Ethidium bromide was added to each sample before gel electrophoresis. Photographic negatives of the ethidium-stained gels were used to eliminate individual samples that were not loaded evenly (see below). Hybridization and wash procedures were conducted with previously published methods.13
Probes and Labeling Reactions
An HSP70 cDNA probe isolated from a Chinese hamster ovary cell line14 and previously shown to recognize at least three HSP70 mRNA transcripts in the rat was used to probe Northern blots.15 16 Isolated inserts were labeled with [32P]dCTP (DuPont NEN Research Products) by use of the random primer method.17
Densitometric Analysis of Northern Blots
Before the autoradiograms were scanned, a photographic negative of the ethidium-stained samples was used to eliminate degraded or unevenly loaded samples. Initially, if any sample appeared degraded as evidenced by a less than 2:1 ratio of 18S to 28S rRNA or noticeable smearing, it was removed from the analysis. The negative of the ethidium-stained samples was densitometrically scanned, along with the resultant autoradiogram. If the density of the 18S or 28S rRNA band of a given sample deviated by more than 10% from the mean density for all samples in a gel, then it also was removed from analysis. Densitometric values were then determined from digitized images of autoradiograms. Values were corrected for background and expressed as a percent of the average signal in naive unstressed animals (CC). Because all gels were prepared so that samples from all treatment groups were present on each gel, expressing values as a percent of the CC group provided a means to standardize sample values between different blots. Corrected values were subjected to ANOVA, with probability values of <.01 considered significant. When appropriate, a Bonferroni simple effects test was conducted to determine significant differences (P<.01) between sample means, which provides a probability value that is adjusted for multiple comparisons.
Serum Collection and Radioimmunoassay
Trunk blood was collected in lavender-topped EDTA tubes and centrifuged (2000g) for 10 minutes at 4°C. Plasma was stored at −80°C until assayed. Radioimmunoassay (RIA) for adrenocorticotropic hormone (ACTH) was conducted by use of a kit obtained from Diagnostic Systems Laboratories Inc, with rat ACTH substituted for standards. The ACTH standards in this kit were calibrated against the US National Pituitary Association Standard Preparation of Human Corticotropin (ACTH) and a preparation of ACTH obtained from the World Health Organization (Preparation No. 74/555). Rat ACTH (Peninsula Laboratories, Inc) was used in parallel with the standards in the kit and was found to produce essentially identical standard curves.
Plasma Catecholamine Determinations
Plasma catecholamines were measured by high-pressure liquid chromatography (HPLC) and electrochemical detection. The HPLC system consisted of one Beckman 110B high-pressure pump (Beckman Instruments), a Beckman 502 automatic injector with a 20-μL injection loop, and a catecholamine HR-80 column (ESA, Inc). Data were collected with the assistance of a Beckman Analog Interface module 406 and a PS2 IBM model 55 SX personal computer. The computer was used to integrate and interpret the resulting chromatogram with the aid of system gold software from Beckman. Plasma samples were extracted according to the instructions accompanying the Catecholamine Analysis Kit from ESA. The samples were injected onto the column in a mobile phase consisting of 50 mmol/L NaH2PO4, 250 mg/L 1-heptanesulfonic acid, 80 mg/L EDTA, and 3% methanol, pH 2.6, at a flow rate of 1 mL/min. The contents of norepinephrine and epinephrine were detected by an ESA model 5100A Coulochem Multi-Electrode Electrochemical Detector and an ESA model 5021 Conditioning Cell in line with an ESA model 5011 Analytical Cell. Hydrodynamic voltammetric curves were run with standard solutions (ESA) to determine optimal detector settings as follows: conditioning cell at +0.3 V, detector 1 at +0.05 V, and detector 2 at −0.35 V; gain 50×10; and response time of 4.0 seconds.
Stress-Induced HSP70 Expression in Aorta and Adrenal Gland
In previous reports, we demonstrated that HSP70 mRNA is elevated in blood vessels and the adrenal gland after exposure to 1 hour of restraint or to other types of stressors.18 19 In this experiment, we sought to determine whether the predictability and/or severity of a stressor would influence the amount of HSP70 expression observed in these tissues and whether repeated exposure to a stressor results in altered HSP70 expression relative to animals exposed to a stressor for the first time. To address these questions, seven groups of rats (n=5 to 7 per group) were exposed to acute or chronic air jet or restraint as described in the “Methods” section. Animals in all treatment groups were killed; then their adrenal glands and aortas were removed. Total RNA was isolated from individual tissues, with HSP70 expression determined by Northern blot analysis. Trunk blood was also collected from each animal for subsequent determination of circulating catecholamines and ACTH.
Fig 1⇓ summarizes the effects of the different treatment conditions on HSP70 induction in the aorta. In naive animals, exposure to restraint (CR) and air jet (CA) resulted in a fivefold to sixfold increase in HSP70 mRNA expression relative to unstressed controls (CC). In rats exposed to 8 weeks of restraint, the final restraint exposure (RR) resulted in significantly less HSP70 expression than in naive animals exposed to restraint for the first time (CR). This reduction in HSP70 inducibility after repeated restraint suggests that an adaptation of the HSP70 response occurs after chronic exposure to restraint. In contrast, HSP70 expression in chronically air jet–treated rats receiving a final air jet exposure (AA) was not different than in naive rats receiving an air jet exposure for the first time (CA). Thus, there was no adaptation of the HSP70 response in the aorta to repeated air jet as observed in restrained animals. The amount of HSP70 mRNA did not differ in unstressed animals regardless of their chronic treatment conditions (RC and AC), indicating a lack of effect of chronic stress on basal HSP70 expression at the RNA level.
Fig 2⇓ shows the effects of acute and chronic restraint and air jet stress on adrenal HSP70 expression. As previously reported, restraining naive animals (CR) resulted in increased HSP70 mRNA expression; however, the magnitude of the induction was less than that observed in the aorta of similarly treated rats. Exposure of naive animals to air jet (CA) also elevated HSP70 mRNA expression, and the magnitude of this induction was significantly greater (P<.01) than that observed with restraint. Thus, air jet is apparently a much more effective inducer of adrenal HSP70 expression than restraint alone. In chronically restrained animals exposed to a final restraint session (RR), HSP70 expression was not different from that in unstressed controls (CC), which indicates that an adaptation to the effects of restraint had occurred, as observed in the aorta. HSP70 expression in chronically air jet–treated rats receiving a final air jet exposure (AA) was greater than in unstressed controls (CC) but less than in naive, air jet–treated animals (CA). Thus, in the adrenal gland, there is an adaptation of the HSP70 response after chronic exposure to both restraint and air jet, while in the aorta, adaptation only to the effects of repeated restraint occurs. As observed in the aorta, there were no differences in adrenal HSP70 induction between any of the unstressed groups (RC and AC).
Stress-Induced Elevation in Blood Pressure
During the final week of stress exposure, blood pressure measurements were performed on all rats in each treatment group. Blood pressure measurements were performed by the tail-cuff method 24 hours after the last stress exposure. Rats in the control group had been handled daily to acclimate them to the blood pressure measurement procedures. A one-way ANOVA indicated significant differences (F[2,42]=6.78; P=.0041) in blood pressure among the three treatment groups. Individual comparisons showed that the air jet group had significantly (P<.01) higher blood pressure than either the restraint or the control group (Fig 3⇓). There was no difference in blood pressure between the control and restraint-stressed groups.
Stress-Induced Elevation in Plasma Catecholamines and ACTH
Trunk blood collected from individual animals in the treatment groups described above was assayed for catecholamines by HPLC and for ACTH by RIA. Consistent with previous reports, ACTH concentrations were significantly (P<.01) greater in naive restraint-treated animals (CR) relative to unstressed controls (the Table⇓). Surprisingly, ACTH concentrations were not elevated in unstressed air jet–treated (CA) or air jet–treated rats that received a final exposure to air jet (AA), even though HSP70 expression in the adrenal glands of these animals was markedly increased.
Circulating epinephrine and norepinephrine were significantly (P<.01) elevated in unstressed air jet–treated (CA) relative to unstressed controls (CC) (the Table⇑). Although catecholamine concentrations fluctuated between the other treatment groups, none were significantly different than those in rats in the CC group.
The significance of the results of the experiments presented in this study address two somewhat disparate aspects of HSP expression in vivo. Results from this report describe, for the first time, a novel relation between the induction of HSPs by behavioral stressors and the development of hypertension. A second aspect of these results addresses adaptation of the HSP response after chronic exposure to repeated stress and a relation between the severity of the stress and the magnitude of HSP expression in aorta and adrenal gland.
Stress-Induced HSP70 Expression and the Development of Hypertension
In this study, hypertension develops in normal rats in response to repeated exposure to restraint in combination with intermittent air jet. No hypertension develops in rats exposed only to repeated restraint. These findings suggest that the combination of the two stressors is effective in generating hypertension, while restraint alone is not. The combination of restraint and air jet may be additive; thus, hypertension develops in air jet–treated animals because the stress is more severe. However, the intermittent delivery of the air jet treatment may also contribute to its efficacy as a stressor. Indeed, it is well known that a subject’s ability to anticipate a stressor greatly influences its perception as a stressor and alters behavioral responses to stress.
Our model of hypertension is greatly different from genetic models. Rather than developing hypertension because of defects in gene structure, previously normotensive rats in this study become hypertensive in response to a repeated exposure to stressful environmental stimuli. Furthermore, in our model system, the stimulus responsible for generating hypertension is the same stimulus that is responsible for inducing HSP70 expression in aorta and adrenal gland. Despite these differences between air jet–induced hypertension and genetic models of hypertension, an increase in the HSP70 response was observed. Apparently, an increased sensitivity of the HSP response occurs concomitant with hypertension, regardless of the underlying mechanism responsible for generating the hypertension.
Heat and a variety of other cellular stressors are known to induce HSP synthesis.20 21 With many of these stressors, cell survival is threatened. There have been several reports that prior induction of HSPs provides protection against the life-threatening effects of a stressor.22 23 24 Thus, one function attributed to HSPs is to protect the cell from death in the presence of a stressor. Neither restraint nor air jet is a sufficiently severe stressor to directly affect the viability of vascular smooth muscle cells. Thus, in this model system, HSPs do not seem to provide a protective response to toxic, life-threatening environmental stimuli. Rather, it appears that HSPs are induced in aorta as part of an integrated response to environmental stressors and may function in the normal physiological homeostasis of this tissue.
It is known that HSPs can function as molecular chaperons within cells.25 26 27 In this regard, it has been demonstrated that HSPs bind to intracellular proteins and facilitate their translocation to different cellular compartments. Perhaps stress-induced HSP70 expression in the aorta has a similar function. During periods of increased vascular activity (ie, during stress), HSP70 may be induced to ensure that newly synthesized contractile proteins are correctly transported and/or assembled into the proper functional configuration. In this way, HSPs may be contributing to the normal maintenance and turnover of the proteins responsible for the functioning of this tissue.
Adaptation of the Heat Shock Response in the Aorta and Adrenal Gland
HSP70 mRNA expression in aorta of naive animals was markedly induced by restraint and air jet stress. After 8 weeks of chronic treatment, restraint could no longer induce HSP70 expression in the aorta. In contrast, the HSP70 response in aortas of chronically air jet–treated rats remained intact. Thus, in the aorta, there is an adaptation to the HSP70-inducing effects of restraint but not to air jet.
In adrenal glands, both restraint and air jet induce HSP70 expression in naive animals. In contrast to the aorta, the magnitude of HSP70 expression induced by restraint was significantly less than that induced by air jet, which suggests that the adrenal gland is more sensitive to the type of stressor invoking the HSP70 response. As with blood pressure, it is possible that differences in HSP70 expression in adrenal gland can be attributed to either the severity or the predictability of the stressor. After 8 weeks of chronic treatment, stress-induced HSP70 expression was attenuated in both restraint- and air jet–treated rats. Thus, unlike HSP70 expression in the aorta, an adaptation to the effects of both restraint and air jet occurs in the adrenal gland. These changes in HSP70 expression in the adrenal gland are believed to be restricted to the adrenal cortex because we have previously reported no HSP70 induction in the medulla in response to restraint or drug administration.2 18
We previously demonstrated that restraint-induced expression in the adrenal gland may depend on release of ACTH after stimulation of the hypothalamic-pituitary-adrenal axis, while HSP70 expression in the aorta may depend on catecholamine release after activation of the sympathetic nervous system.1 18 As in previous reports, ACTH concentrations were elevated in naive, restrained rats (CR) but not in any other group. Although adrenal HSP70 expression was elevated in this group, a similar level of expression was also observed in the AA group, and even greater expression was observed in the CA group. Thus, plasma ACTH levels are not sufficient to explain the differences observed in adrenal HSP70 expression after acute and chronic stress and indicate that other factors may be involved.
Epinephrine and norepinephrine were elevated in plasma samples from naive air jet–treated rats (CA). HSP70 expression was increased in this group but also was elevated in the CR and AA groups. Apparently, the concentrations of circulating catecholamine do not provide an index for predicting HSP70 expression in the aorta. This result is not surprising because we have hypothesized that HSP70 expression in aorta is more closely dependent on sympathetic neural transmission than on increases in circulating catecholamines.
The lack of adaptation of the HSP70 response may be of particular significance in the aorta. The HSP70 response in the aorta adapted to chronic restraint treatment. Chronically restrained animals also did not become hypertensive. These results parallel previous reports that demonstrated that repeated immobilization stress28 or repeated exposure to hyperthermic ambient temperatures29 reduces resting mean blood pressure in spontaneously hypertensive rodent models. There was no adaptation of the HSP response to air jet, and these animals did become hypertensive. Apparently, if HSP70 expression in the aorta in response to a stressor is maintained, then hypertension develops. If the HSP70 response adapts, hypertension does not develop. However, the causal relation between the maintenance HSP response and hypertension remains to be elucidated.
Although we did not measure acute blood pressure responses during our stress conditions, other investigators reported that restraint-induced blood pressure responses are greater in hypertensive rats after repeated immobilization.28 They demonstrated that resting blood pressure in genetically hypertensive rats adapts to repeated stress exposure, while acute blood pressure responses are increased. In contrast, our air jet–treated animals were initially normotensive and became hypertensive with repeated exposure. Whether this relation holds for acute blood pressure responses to air jet treatment after repeated exposures awaits further investigation.
In this study, we measured HSP70 expression at the mRNA level. In previous reports, we demonstrated that maximal HSP70 expression may not be reached for up to 3 hours after exposure.1 Thus, it is likely that the function of the HSP response in aorta is more closely related to adaptive changes in aortic smooth muscle that prepare the tissue for a subsequent challenge than a protective response to an acute stress exposure. Additionally, these results suggest that an animal’s ability to anticipate a stressor may influence whether adaptation occurs and therefore indirectly alters aortic HSP70 mRNA induction and the development of hypertension. Future studies investigating the effects of behavioral stressors on the cellular HSP stress response in vascular tissue may provide novel insights into the mechanisms of cardiovascular homeostasis.
This work was supported in part by the American Heart Association, Dakota Affiliate; the National Institutes of Health (NS-30493); and the National Science Foundation, ASEND Program. We appreciate the help of Orris Johnson and Lawrence Siu for engineering and computer programming, respectively.
Reprint requests to Michael J. Blake, Department of Pharmacology and Toxicology, University of North Dakota School of Medicine, 501 N Columbia Rd, Grand Forks, ND 58202.
- Received July 5, 1994.
- Revision received September 8, 1994.
- Accepted December 9, 1994.
Udelsman R, Blake MJ, Stagg CA, Li D, Putney J, Holbrook NJ. Vascular heat shock protein expression response to stress. J Clin Invest. 1993;91:465-473.
Blake MJ, Buckley AR, Buckley DJ, LaVoi KP, Bartlett T. Neural and endocrine mechanisms of cocaine-induced 70-kDa heat shock protein expression in aorta and adrenal gland. J Pharm Exp Ther. 1994;268:522-529.
Klevay LM. Hypertension in rats due to copper deficiency. Nutr Rep Int. 1987;35:999-1005.
Church GM, Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984;81:1991-1995.
Blake MJ, Gershon D, Fargnoli J, Holbrook NJ. Discordant expression of heat shock protein mRNAs in tissues of heat-stressed rats. J Biol Chem. 1990;265:15275-15279.
Blake MJ, Gershon D, Fargnoli J, Holbrook NJ. Concomitant decline in heat-induced hyperthermia and HSP70 mRNA expression in the aged rat. Am J Physiol. 1991;260:R663-R667.
Blake MJ, Udelsman R, Fuelner GJ, Norton DD, Holbrook NJ. Stress-induced heat shock protein 70 expression in adrenal cortex: an adrenocorticotropic hormone-sensitive, age-dependent response. Proc Natl Acad Sci U S A. 1991;88:9873-9877.
Welch WJ. Mammalian stress response: cell physiology, structure/function of stress proteins, and implications for medicine and disease. Physiol Rev. 1992;72:1063-1081.
Barbe MF, Tytell M, Gower DJ, Welch WJ. Hyperthermia protects against light damage in the rat retina. Science. 1988;241:1817-1820.
Jakob U, Gaestel M, Engel K, Buchner J. Small heat shock proteins are molecular chaperons. J Biol Chem. 1993;268:1517-1520.
Kvetnansky R, McCarty R, Thoa NB, Lake CR, Kopin IJ. Sympatho-adrenal responses of spontaneously hypertensive rats to immobilization stress. Am J Physiol. 1979;236:H457-H462.