Stress-induced proteins in aortic smooth muscle cells and aorta of hypertensive rats D. S. KOHANE, R. SARZANI, J. H. SCHWARTZ, A. V. CHOBANIAN, Cardiovascular Institute and Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118
AND
P. BRECHER
KOHANE, D. S., R. SARZANI, J. H. SCHWARTZ, A.V. CHOBANIAN, AND P. BRECHER. Stress-induced proteins in aortic smooth muscle cells and aorta of hypertensive rats. Am. J. Physiol. 258 (Heart Circ. Physiol. 27): Hl699-Hl705, 1990.The current studies were performed to determine whether vascular smooth muscle cells produce stress-induced proteins when subjected to experimental hypertension. Two-dimensional gel electrophoresis was used to analyze labeled proteins in cultured cells in response to either heat shock or arsenite treatment. The major heat shock proteins (HSPs) induced had molecular masses of 70, 90, and 110 kDa. Arsenite treatment produced a similar response with the additional induction of a 30-kDa protein. In vitro translations of total RNA from heatshocked cells, and RNA blot hybridization using HSP 70 cDNA suggested that HSP 70 induction was transcriptionally regulated. Treatment of cells with norepinephrine or angiotensin II induced cellular hypertrophy without eliciting HSPs. In vitro translation of aortic RNA from rats rendered hypertensive by administration of deoxycorticosterone and a high salt intake also did not reveal HSP induction. The absence of HSP induction using an in vivo model of hypertension suggests that those proteins may not be required to mediate the vascular response to experimental hypertension.
these cells including hypertrophy (20) and polyploidy (5, 20) and changes in enzymatic activity (1) and in connective tissue turnover (18). However, relatively little is known about the cellular mechanisms involved in effecting such differences. Cell culture has been used as a model system to define the mechanisms involved in the normal regulation of vascular cell growth and to test the hypothesis that abnormalities in autocrine or paracrine regulation account for the vascular changes that occur either in response to hypertension or during atherogenesis (9). In the present study, we cultured aortic smooth muscle cells (SMC) to document the expression of stress-induced proteins in response to both heat shock and arsenite. We also examined the effect on induction of HSPs of two vasoactive agonists, norepinephrine and angiotensin II, which cause hypertrophy of cultured arterial SMC. Finally, we investigated HSP expression in an in vivo model of hypertension, the deoxycorticosterone (DOC) salt-treated rat.
heat shock proteins;
METHODS
hypertension;
heart; vasculature
have demonstrated the synthesis of specific proteins in response to heat shock. These heat shock proteins (HSPs) typically have molecular masses of ~70,90, and 110 kDa. Several smaller HSPs also have been described. This has been shown to be a general response in many cell types and to be initiated by a wide variety of deleterious agents and conditions (17) leading to the more general designation of such proteins as stress-induced proteins. The diversity of stressescausing this expression suggests that these proteins might be functional in various disease states, In the cardiovascular system, there have been reports that HSP 70 is increased in the mammalian heart secondary to pressure overload (6, 7, 13), cardiac ischemia (8), and hypertrophy (6, 13). The purpose of this study was to determine whether the expression of stress-induced proteins is increased in vascular smooth muscle as a result of factors associated with experimental hypertension. In hypertension, the arterial wall is subjected to increased intraluminal pressure that causes many vascular changes (4). Arterial smooth muscle cells are the most abundant cell type in aortic tissue, and hypertension has been shown to induce functional, metabolic, and morphological changes in PREVIOUS
STUDIES
0363-6135/90 $1.50 Copyright
0
[35S]methionine (sp act ~800 Ci/mmol) and Enhance were purchased from New England Nuclear; sodium arsenite, Triton X-100, and glycerol were from Fisher Scientific; ultrapure urea was from Schwartz-Mann; acrylamide, sodium dodecyl sulfate (SDS), and 2-mercaptoethanol were from Bio-Rad Laboratories; ampholytes were from Pharmacia; RNAsin and in vitro translation kits were from Promega; and norepinephrine and angiotensin II were from Sigma. Human HSP 70 genomic DNA probe, deposited by R. Morimoto (25) was obtained from American Type Culture Collection (DNA no. 57495). Rats were obtained from Charles River Breeding Laboratories (Wilmington, MA); DOC acetate pellets were from Innovative Research of America (Toledo, OH). Dulbecco’s modified Eagle’s medium (DMEM), L-glutamine, penicillin, streptomycin, fetal calf serum, and antibiotic-antimycotic solution were all purchased from GIBCO, and type I collagenase was obtained from Sigma. Preparation and culture of rat aortic smooth muscle cells. Rat aortic SMCs were isolated by enzymatic dispersion using a method adapted from Gunther et al. (12). Sprague-Dawley rats (200-300 g) were anesthetized with pentobarbital sodium. The thoracic aorta from six rats were removed under sterile conditions and placed in 4°C
1990 the American
Physiological
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DMEM containing 25 mM N-Z-hydroxyethylpiperazine2V’-2ethanesulfonic acid (HEPES) buffer, 2 mM L-glutamine, 100 U/ml penicillin, and 100 pg/ml streptomycin. The adventitia was removed with a watchmaker forceps, and the aortas were minced with a razor blade into small segments no larger than 2 mm2. The segments were transferred into a plastic tube containing 10 ml of the following solution: DMEM with 2 mg/ml type I collagenase, 0.5 mg/ml elastase, and 1 mg/ml soybean trypsin inhibitor. The tissue suspension was incubated in a shaker bath for 2 h at 37°C. The dissociated cells were washed twice in DMEM, and the final suspension was plated into a 25cm2 flask and cultured at 37°C in a humidified atmosphere of 5% C02-95% air. After 2 h, the medium was supplemented with 20% fetal calf serum and 1% antibiotic-antimycotic solution. The medium was changed every 3 days, and confluent monolayers were obtained within 15 days. The cells were passaged by rinsing with phosphate-buffered saline followed by brief incubation with 1 ml of trypsin-EDTA. Cells were then washed with DMEM supplemented with 20% fetal calf serum and 1% antibiotic-antimycotic solution. Cells of passages 5-15 were used for these studies. Cells were plated into 2-cm’ wells. At confluence, there were 3 x lo5 cells per 2-cm2 well. During experiments, control and stressed cells were in DMEM with or without methionine. In addition, in selected experiments, cells were incubated with either 20 ,uM norepinephrine or 1 PM angiotensin II for different time periods. Ascorbate (100 PM) was added to prevent oxidation of norepinephrine. Culture medium containing hormones was changed daily. For evaluation of cellular hypertrophy, cells were grown to confluence in six-well dishes, and agonists were added to the incubation medium each day for 3 days before analysis of DNA (15) and protein, the latter via a dyebinding calorimetric assay (BioRad, Richmond, CA). Cytosolic free Ca’+ was determined by a fluorimetric method using the calcium fluorescent probe furaas previously described (24). Heat shock. Culture dishes containing confluent cells were heated for 1 h at 42OC. At the end of this period, the plates were returned to a 37°C incubator and at designated times were washed once with methioninefree DMEM and then incubated in that medium at 37°C for 1 h. Methionine-free DMEM containing 50 &i/ml [35S]methionine was then substituted, and the cells were labeled for 1 h, washed in DMEM, scraped into 800 ~1 of the sample buffer [ 13 mM tris( hydroxymethyl)aminomethane (Tris), pH 6.8, 10% glycerol, 2% 2-mercaptoethanol, 3% SDS], used for SDS-polyacrylamide gel electrophoresis (PAGE), and boiled for 4 min. Arsenite treatment. Arsenite treatment was based on the method of Kim et al. (14). Confluent cells were incubated for 4 h in DMEM containing 100 PM sodium arsenite. After arsenite treatment, cells were washed with arsenite-free DMEM lacking methionine and then labeled with [35S]methionine following the protocol described above for heat shock. Cells were then scraped from the plates and suspended in the lysis buffer used for two-dimensional gel electrophoresis. RNA isolation and analysis. RNA isolation was per-
MUSCLE
CELLS
AND
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formed with minor modifications from established procedures (3). Cells were incubated at 37°C for 90 min after heat shock and scraped from six separate wells into 6 ml of guanidinium thiocyanate lysis buffer. Total RNA was isolated by cesium chloride centrifugation as recently described (22). For tissue RNA, hearts were processed individually while aortas from three animals were pooled. RNA was analyzed by Northern blotting procedures using methodology recently described (2 1). Following electrophoresis through 1% agarose-1 M formaldehyde gels, the RNA was transferred to nylon membranes, cross-linked by ultraviolet (UV) irradiation, and prehybridization was performed as described. A human HSP 70 genomic DNA probe that included the entire coding sequence was labeled by a random hexamer priming procedure (lo), and hybridization was performed at 65°C overnight. After hybridization, the membranes were washed three times at 55, 60, and 65°C using 1 x SSC, 0.5 X SSC, and 0.1 X SSC, respectively. SSC is 0.15 M NaCl and 0.015 M sodium citrate at pH 7.0. Blots were exposed to preflashed X-ray film between two intensifying screens, and the size of the mRNA detected was calculated based on ribosomal RNA migration. In vitro translation. Total RNA from control and heatshocked samples were translated in vitro using a micrococcal nuclease-treated, rabbit reticulocyte lysate kit. Reaction mixtures contained 10 pg total RNA, 50 &i [35S]methionine, and RNAsin (an RNase inhibitor). The lysate was utilized without modification of the Mg2+ or K+ concentrations. Incubations were for 1 h at 30°C. RNA was denatured by heating to 65°C for 4 min immediately before translation. After incubation, aliquots of the lysate reaction mixture were dried under vacuum and resuspended in lysis buffer for subsequent analysis by two-dimensional gel electrophoresis. In vitro translations of aortic and cardiac RNA were performed exactly as for cultured cells. Electrophoresis. SDS-PAGE was performed on 12% acrylamide gels after Laemmli (16). For two-dimensional electrophoresis, samples in lysis buffer [9.5 M urea, 2% Triton-X 100, 5% 2-mercaptoethanol, 2% ampholytes (26)] were separated by isoelectric focusing in the first dimension (ampholytes 1.4% 5-7; 0.6% 3-10) and SDSPAGE in the second dimension on 12% acrylamide gels (19). Gels containing [35S]methionine were treated with Enhance for fluorography. Hypertensive animal model. DOC-salt hypertension was produced in male Wistar rats following the procedures described recently (22). Animals were uninephrectomized at 10 wk of age and a subcutaneous pellet containing 100 mg DOC acetate was implanted 1 wk after uninephrectomy. Animals were given 1% saline as drinking water. Blood pressure was monitored weekly using tail cuff plethysmography. Animals were anesthetized with pentobarbital sodium before aortic dissection. Animals were killed at 3, 7,14,21, and 28 days after implantation of the DOC pellet. Blood pressures of the treated animals increased progressively with time, with average systolic pressures (in mmHg) of 124, 128, 162, 183, and 186, respectively, for the times indicated above. Heart weight-to-body weight ratios (x100) also increased in the
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STRESS-INDUCED
a
bcdefg
PROTEINS
IN SMOOTH
kDa
MUSCLE
CELLS
AND AORTA
H1701
RESULTS
Heat shock response in cultured smooth muscle cells.
Actin
-)
FIG. 1. Time course of heat shock protein (HSP) production by arterial smooth muscle cells. Cells were heat shocked, labeled with [““S]methionine for 1 h at time intervals indicated, and resulting labeled proteins were separated by SDS-PAGE. a: control cells; b: during heat shock; c: 1 h after heat shock; d: 2 h; e: 4 h; fi 6 h; g: 23 h. HSPs 70, 90, and 110 were induced.
treated animals, averaging 0.30, 0.33,0.34, 0.38, and 0.49 for 3, 7, 14, 21, and 28 days of treatment, respectively. Control animals that were uninephrectomized but not given DOC did not exhibit increased blood pressures and did not show signs of cardiac hypertrophy. Additional data regarding this animal model have been reported recently (22).
The time course for the induction of HSPs by vascular smooth muscle cells (VSMCs) was determined by labeling cells with [35S]methionine before and during heat shock as well as 1,2,4,6, and 23 h after the end of heat shock. Figure 1 is an autoradiogram of an SDS-PAGE gel of those samples. Three major proteins were induced by heat shock, with molecular masses of -70, 90, and 110 kDa. These were assumed to correspond to HSPs 70, 90, and 110, respectively. Synthesis of those proteins was greatest between 1 and 2 h after heat shock. Incorporation of label into HSP 70 subsided by 4 h poststress; in contrast, HSPs 90 and 110 synthesis was not appreciably reduced 6 h after heat shock. Each of the HSPs returned to control levels after 23 h. Analysis of the heat shock response of VSMCs using two-dimensional electrophoresis is shown in Fig. 2. The labeled proteins produced by unstressed cells and those treated by heat shock are compared. In this and all subsequent figures, the left is the basic side of the gel (pH 7) and the right is the acidic side (pH 4). It was apparent that HSP 70 included at least two proteins. One occurred both in control and heat-shocked cells but was more apparent in the latter. The other protein, which was more basic and had a slightly lower molecular weight, was only present in heat-shocked cells. These two proteins probably correspond to two forms of the 70-kDa protein (2): an “inducible” HSP 70, which is only present in stressed cells, and a “constitutive” HSP 70, which is increased by heat shock, but is also expressed in normal cells. Two-dimensional gel electrophoresis also showed clearly that HSPs 90 and 110 were present in control cells, although at much lower levels than after heat shock. Arsenite treatment. To further define the capability of VSMCs to produce stress-induced proteins, cells were incubated with sodium arsenite, an agent often used to document a cellular response to chemical stress. As in-
FIG. 2. Two-dimensional gel electrophoresis of heat shock proteins (HSPs) produced in vascular smooth muscle cells in response to heat shock for 1 h at 42°C. [%S]methionine incorporation occurred between 1 and 2 h after heat shock. C, control cells; HS, cells exposed to heat shock. Numbers beside arrows are molecular mass (in kDa). In all two-dimensional gels in this paper, left is basic side of gel (pH 7), and right is acidic side (pH 4). Gels resolve molecular mass in range 12-200 kDa. HSPs 70, 90, and 110 were induced, and HSP 70 was resolved into two proteins.
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FIG. 3. Two-dimensional gel electrophoresis of proteins synthesized by smooth muscle cells in response to arsenite. Cells were exposed to 100 PM sodium arsenite for 4 h and then labeled for 1 h with [%]methionine. C, control cells; As, arsenite treatment. Numbers beside arrows are molecular mass (in kDa). HPSs 70, 90, and 110 were induced as well as a protein sized 30 kDa.
FIG. 4. In vitro translation of total RNA from heat-shocked cells. RNA isolated from control cells (C) and cells heat shocked (HS) for 1 h at 42°C were translated in vitro, and resulting %-labeled proteins were separated by two-dimensional gel electrophoresis. Numbers beside arrows are molecular mass (in kDa). HSPs 70, 90, and 110 were induced as well as a protein sized 23 kDa.
dicated by the two-dimensional gel elecrophoretograms shown in Fig. 3, HSPs 90 (inducible and constitutive), 90, and 110 were clearly induced by arsenite treatment. These proteins had the same sizes and pIs as the corresponding proteins made by heat-shocked cells. In addition, arsenite-treated samples produced a labeled protein with a size of -30 kDa, which was not expressed after heat shock. In vitro translation. Total RNA was isolated from control and heat-shocked cells and translated in vitro with [35S]methionine. The resulting radiolabeled proteins were separated by two-dimensional gel electrophoresis and the autoradiograms were analyzed (Fig. 4). HSPs 70,90, and 110 had the same pattern of induction seen in cell-labeling experiments, suggesting that the
synthesis of these proteins in response to stress was transcriptionally regulated. In addition, a protein having a mass of -23 kDa was detected that was not observed in the labeling experiments using intact cells. Northern blot hybridization analysis. Figure 5 shows representative data for expression of HSP 70 mRNA by arterial SMC as well as cardiac and aortic tissue. Figure 5A shows a major induction of HSP 70 mRNAs after heat shock and also shows that both unshocked SMC and normal aortic tissue have three different detectable mRNAs of 2.2, 2.7, and 3.0 kb. Only the major form at 2.2 kb was seen in heart RNA. With the use of a shorter exposure time (30 min), inducible HSP mRNAs of 3.0 and 3.2 kb could be readily detected in heat-shocked SMC (Fig. 5B).
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H
A
C
HS
:
PROTEINS
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H1703
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FIG. 5. Northern blot analysis of total RNA from rat heart, aorta, and arterial smooth muscle cell (SMC) using a cDNA probe for human HSP 70. Each lane contained 20 pg of total RNA from control heart (H), control aorta (A), control SMC (C), and heat-shocked SMC (HS). Locations of 18s and 28s markers are indicated. Numbers beside arrows are RNA size in kb. A: autoradiogram after a 36-h exposure time. One band at 2.2 kb was seen in heart. There were 3 bands in aorta and control SMC: 2.2, 2.7, and 3.0 kb. A large increase in mRNA was seen in HS SMC. B: 0.5-h exposure time of C and HS from A.
1. Effect of angiotensin II and norepinephrine on protein, DNA, and intracellular Ca2+ in vascular smooth muscle cells
TABLE
Protein, mg/well
Treatment
Control Angiotensin Norepinephrine
II
DNA, dwell
0.40+0.06 0.61kO.05
7.6kO.4 7.2kO.4
0.65&0.04
7.4kO.3
Protein/DNA, PPlPg
53 84 87
CC?+, IlM
127f28 527+81 389+42
Values are means & SE; n = 6. Vascular smooth muscle cells (VSMCs) were grown to confluence in 6-well dishes. In DNA and protein determinations, agonists were added to incubation media each day for 3 days before analysis. For measurement of Ca’+, cells were grown on glass cover slips, and agonists were added during period of calcium determination. Ca” concentration is average signal from lo4 cells that were in optical window of fluorometer (24). Values above are peak values obtained 5-7 min after agonist addition.
FIG. 6. Effect of chronic norepinephrine and angiotensin II treatment on protein synthesis by smooth muscle cells. Cells were exposed to 1 PM angiotensin II (ANG II) or 8 PM norepinephrine (NE) for 4 days or left untreated (C), then labeled with [%]methionine for 1 h before separation of proteins by two-dimensional gel electrophoresis. Arrows indicate positions where HSPs 70, 90, and 110 occur. No induction of these proteins was detected.
Effect of vasoactive substances. Norepinephrine has been used to induce hypertrophy in cardiac myocytes (23), and a model of angiotensin II-induced hypertrophy in VSMCs has recently been described (11). Experiments were performed to determine whether these agonists produced chemical and physiological changes in the arterial SMC under study. Cellular hypertrophy and reactivity with respect to intracellular Ca2+were determined and the results are shown in Table 1. Hypertrophy of the cells exposed to either angiotensin II or norepinephrine for 72 h was documented by measuring DNA and protein content of cells grown to confluency and then exposed to either agonist for 72 h. The DNA content did not significantly differ between wells containing cells given no agonist and those given either angiotensin II or norepinephrine. However, there was a significant increment in the protein content of the cells exposed to either angiotensin II or norepinephrine. Thus the protein-toDNA ratio of the agonist-exposed VSMCs was significantly increased, suggesting that both agonists induced hypertrophy. An acute change in cytosolic Ca2+ also was observed with either agonist, with peak increases of
three- and fourfold for norepinephrine and angiotensin II, respectively. Such changes are consistent with receptor-linked signal transduction in these cultured cells. To test the possibility that these vasoactive agents may elicit a stress-induced reaction in VSMCs, the cells were exposed to norepinephrine or angiotensin II for 4, 24, and 72 h and then labeled for 1 h with [35S]methionine. One-dimensional gel electrophoresis of the labeled samples revealed no induction of HSPs at any time period tested (data not shown). For better resolution, the 72-h samples were further analyzed using two-dimensional gel electrophoresis (Fig. 6). The constitutive HSP 70 and HSPs 90 and 110 were detected in control and experimental samples. However, there was no detectable induction of HSP 70 in either of the agonist-treated samples. Hypertensive rat models. The inability of vasoactive agents such as angiotensin II and norepinephrine to elicit stress-induced proteins in cultured SMCs did not exclude the possibility that such proteins might be induced in vivo in intact aortic tissue from animals exposed to hypertension. To assessthis, rats were made hyperten-
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FIG. 7. Effect of DOC-salt-induced hypertension on expression of stress-induced proteins by intact rat aorta. RNA isolated from aortas of control (C) and 21-day DO&salt hypertensive (D21) rats were translated in vitro with [%3]methionine and separated by two-dimensional electrophoresis. Numbers beside arrows are molecular mass (in kDa) and indicate nositions where HSPs 70. 90, and 110 occur. No induction of these proteins was detected.
FIG. 8. Effect of DOC-salt-induced hypertension on expression of stress-induced proteins by cardiac tissue. RNA was isolated from heart of control animals (C) and 2%day DOC-salt hypertensive rats (D28). RNA was translated in vitro with [%]methionine, and proteins were separated by two-dimensional gel electrophoresis. Numbers beside arrows are molecular mass (in kDa) and indicate positions where HSPs 70, 90, and 110 occur. No induction of these proteins was detected.
sive by DOC-salt treatment, a model in which the development of aortic hypertrophy is well documented. Aortas and hearts were removed at 3,7,14,21, and 28 days after the implantation of the DOC pellet. Total aortic RNA was isolated at each time point, translated in vitro with [35S]methionine, and the labeled proteins were run on two-dimensional gels. RNA from three separate animals was analyzed for each time period studied, and the twodimensional gels were analyzed by visual inspection. Figure 7 is a comparison of 21-day control and hypertensive aortas, which is representative of the data obtained overall. In all samples tested, the constitutive HSP 70 was present as were HSPs 90 and 110. The inducible HSP 70 was present to a small degree in both control and hypertensive aortas, but no enhancement of its expression was evident in hypertension at 21 and 28 days, when blood pressures were highest. Because other studies employing experimental models
to induce cardiac hypertrophy had suggested that HSPs were expressed in vivo, we also performed in vitro translations of RNA isolated from cardiac tissue of normotensive and hypertensive rats and analyzed these samples by two-dimensional gel electrophoresis. Again, three samples from each time period were analyzed. As shown in the representative gels of Fig. 8, we found no evidence for enhanced expression of any HSP in cardiac tissue, although hearts from DOC-salt-treated rats underwent considerable hypertrophy during the treatment period. DISCUSSION
To determine whether stress-induced proteins may have a functional role in influencing the response to injury in vascular tissue, we have examined the expression of HSPs in cultured aortic SMCs and have shown that these cells produce a complement of proteins similar
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to that described in several other cell types. The best indices of the stressed condition appeared to be the induction of HSPs 23 and 70 (inducible component) with heat and the 30-kDa protein with arsenite treatment, since those phenomena were totally absent in control cells. The increase in translatable HSP mRNA demonstrated by in vitro translation, together with the increase in steady-state levels of HSP 70 mRNA documented by Northern blot hybridization analysis, suggests that HSP 70 expression was at least in part transcriptionally regulated, although an alteration in HSP mRNA stability cannot be ruled out. We found no induction of HSPs in cultured SMCs by vasoactive agonists even in the presence of cellular hypertrophy. Although it is possible that the lack of HSP expression was due to an insufficient dose of norepinephrine or angiotensin II, we did show that the cells responded to those agonists with respect to a hypertrophic response and an increase in cellular Ca2+. It is possible that our protocol missed a brief time frame in which HSP expression occurred transiently, but we did examine several time periods within a 72-h time frame. An alternative possibility is that the agonist-induced “stresses” differ from heat and arsenite treatments both in intensity and time frame. Heat shock and arsenite treatment are acute stresses and in the case of arsenite, completely nonphysiological and toxic. Increased HSP 70 expression has been reported in several studies employing relatively acute experimental models to produce cardiac hypertrophy (6-8, 13). The experiments on hypertensive rats described herein showed no detectable enhancement of HSP either in aorta or heart despite the appearance of hypertrophy and well-documented morphological changes in both organs. The onset of hypertension in the DOC-salt rat occurred at -10-14 days of treatment. However, there was considerable individual variability in the exact time at which blood pressure became elevated. Furthermore, it was not clear a priori that hypertension alone would be the event to initiate HSP expression. With these reservations in mind, we obtained samples at several time points after DOC-salt treatment (3, 7, 14, 21, and 28 days). Although it is possible that HSP induction was missed because not every time point in the course of hypertension was sampled, we can say that it did not occur at the time points studied, and that it was not a chronic feature of the hypertrophied state. This work was supported by National Institut,es of Health Grants HL-18318 (Hypertension Specialized Center of Research), HL-31195, and DK-37105. Address for reprint requests: P. Brecher, Cardiovascular Inst., Boston Univ. School of Medicine, 80 E. Concord St., Boston, MA 02118. Received
24 April
1989; accepted
in final
form
18 January
1990.
MUSCLE RUTTER.
sources 1979.
CELLS
Isolation enriched
in
AND
H1705
AORTA
of biologically ribonucleases.
active ribonucleic Biochemistry 18:
acid from 5294-5299,
A. V. The arterial smooth muscle cell in systemic Am. J. Cardiol. 60: 941-981, 1987. CHOBANIAN, A. V., A. H. LICHTENSTEIN, J. H. SCHWARTZ, J. HANSPAL, AND P. BRECHER. Effects of deoxycorticosterone/salt hypertension on cell ploidy in rat smooth muscle cells. Circulation 75, SuppZ. I: 1-102-I-106, 1987. DAVALLI, P., S. FERRARI, AND A. CORTI. Induction of a 70,000 dalton protein in hypertrophic rat heart. Experientia Basel 41: 1459-1460, 1985. DELCAYRE, C., J.-L. SAMUEL, F. MAROTTE, M. BEST-BELPOMME, J. J. MERCADIER, AND L. RAPPAPORT. Synthesis of stress proteins in rat cardiac myocytes 2-4 days after imposition of hemodynamic overload. J. CZin. Inuest. 82: 460-468, 1988. DILLMANN, W. H., H. B. MEHTA, A. BARRIEUX, B. D. GUTH, W. E. NEELEY, AND J. ROSS. Ischemia of the dog heart induces the appearance of a cardiac mRNA coding for a protein with migration characteristics similar to heat-shock/stress protein 71. Circ. Res. 59: 110-114, 1986. DZAU, V. J., AND G. H. GIBBONS. Cell biology of vascular hypertrophy in systemic hypertension. Am. J. Cardiol. 62: 30G-35G, 1988. FEINBERG, A. P., AND B. VOGELSTEIN. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. AnaZ. Biochem. 137: 266-267, 1984. GEISTERFER, A. T. A., M. J. PEACH, AND G. K. OWENS. Angiotensin II induced hypertrophy, no hyperplasia, of cultured rat aortic smooth muscle cells. Circ. Res. 62: 749-756, 1988. GUNTHER, S., R. W. ALEXANDER, W. J. ATKINSON, AND M. A. GIMBRONE, JR. Functional angiotensin II receptors in cultured vascular smooth muscle cells. J. CeZZ BioZ. 92: 289-298, 1982. IZUMO, S., B. NADAL-GINARD, AND V. MAHVADI. Protooncogene induction and reprogramming of cardiac gene expression produced by pressure overload. Proc. NatZ. Acad. Sci. USA 85: 339-343, 1988. KIM, Y., J. SHUMAN, M. SETTE, AND A. PRZYBYLA. Arsenate induces stress proteins in cultured rat myoblasts. J. CeZZ BioZ. 96: 393-400, 1983. LABRACCA, C., AND K. PAIGEN. A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102: 334-352, 1980. LAEMMLI, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T,. Nature Lond. 227: 680-685, 1970. LINDQUIST, S. The heat-shock response. Annu. Rev. Biochem. 55: 1151-1191, 1986. MECHAM, R. P., L. A. WHITEHOUSE, D. S. WRENN, W. C. PARKS, G. L. GRIFFIN, R. M. SENIOR, E. C. CROUCH, K. R. STENMARK, AND N. F. VOELKEL. Smooth muscle-mediated connective tissue remodeling in pulmonary hypertension. Science Wash. DC 237:
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19. O’FARRELL, P. H. High resolution two-dimensional electrophoresis of proteins. J. BioZ. Chem. 250: 4007-4021, 1975. 20. OWENS, G. K. Influence of blood pressure on development of aortic medial smooth muscle hypertrophy in spontaneously hypertensive rats. Hypertension Dallas 9: 178-187, 1987. 21. SARZANI, R. B., P. BRECHER, AND A. V. CHOBANIAN. Growth factor expression in aorta of normotensive and hypertensive rats. J. CZin. Inuest. 83: 1404-1408, 1989. 22. SARZANI, R., K. P. CLAFFEY, A. V. CHOBANIAN, AND P. BRECHER. Hypertension induces tissue-specific gene suppression of a fatty acid binding prtoein in rat aorta. Proc. NatZ. Acad. Sci. USA 85: 23.
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1. BRECHER, P., C. T. CHAN, C. FRANZBLAU, AND A. V. CHOBANIAN. Effects of hypertension and its reversal on aortic metabolism in the rat. Circ. Res. 43: 561-569, 1978. 2. BURDON, R. H. Heat shock and the heat shock proteins. &o&em. J. 240: 313-324, 1986. 3. CHIRGWIN. J. M.. A. E. PRZYBYLA. R. J. MACDONALD. AND W. ,J.
25.
26.
WV, B., C. HUNT, AND R. MORIMOTO. Structure and expression of the human gene encoding major heat shock protein HSP70. MOL. CeZZ. BioZ. 5: 330-341, 1985. YOUNG, D. A., B. P. VORIS, E. V. MAYTIN, AND R. A. COLBERT. Very high resolution two-dimensional electrophoretic separation of proteins on giant gels. Methods Enzymol. 91: 190-214, 1983.
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AND
P. BRECHER
KOHANE, D. S., R. SARZANI, J. H. SCHWARTZ, A.V. CHOBANIAN, AND P. BRECHER. Stress-induced proteins in aortic smooth muscle cells and aorta of hypertensive rats. Am. J. Physiol. 258 (Heart Circ. Physiol. 27): Hl699-Hl705, 1990.The current studies were performed to determine whether vascular smooth muscle cells produce stress-induced proteins when subjected to experimental hypertension. Two-dimensional gel electrophoresis was used to analyze labeled proteins in cultured cells in response to either heat shock or arsenite treatment. The major heat shock proteins (HSPs) induced had molecular masses of 70, 90, and 110 kDa. Arsenite treatment produced a similar response with the additional induction of a 30-kDa protein. In vitro translations of total RNA from heatshocked cells, and RNA blot hybridization using HSP 70 cDNA suggested that HSP 70 induction was transcriptionally regulated. Treatment of cells with norepinephrine or angiotensin II induced cellular hypertrophy without eliciting HSPs. In vitro translation of aortic RNA from rats rendered hypertensive by administration of deoxycorticosterone and a high salt intake also did not reveal HSP induction. The absence of HSP induction using an in vivo model of hypertension suggests that those proteins may not be required to mediate the vascular response to experimental hypertension.
these cells including hypertrophy (20) and polyploidy (5, 20) and changes in enzymatic activity (1) and in connective tissue turnover (18). However, relatively little is known about the cellular mechanisms involved in effecting such differences. Cell culture has been used as a model system to define the mechanisms involved in the normal regulation of vascular cell growth and to test the hypothesis that abnormalities in autocrine or paracrine regulation account for the vascular changes that occur either in response to hypertension or during atherogenesis (9). In the present study, we cultured aortic smooth muscle cells (SMC) to document the expression of stress-induced proteins in response to both heat shock and arsenite. We also examined the effect on induction of HSPs of two vasoactive agonists, norepinephrine and angiotensin II, which cause hypertrophy of cultured arterial SMC. Finally, we investigated HSP expression in an in vivo model of hypertension, the deoxycorticosterone (DOC) salt-treated rat.
heat shock proteins;
METHODS
hypertension;
heart; vasculature
have demonstrated the synthesis of specific proteins in response to heat shock. These heat shock proteins (HSPs) typically have molecular masses of ~70,90, and 110 kDa. Several smaller HSPs also have been described. This has been shown to be a general response in many cell types and to be initiated by a wide variety of deleterious agents and conditions (17) leading to the more general designation of such proteins as stress-induced proteins. The diversity of stressescausing this expression suggests that these proteins might be functional in various disease states, In the cardiovascular system, there have been reports that HSP 70 is increased in the mammalian heart secondary to pressure overload (6, 7, 13), cardiac ischemia (8), and hypertrophy (6, 13). The purpose of this study was to determine whether the expression of stress-induced proteins is increased in vascular smooth muscle as a result of factors associated with experimental hypertension. In hypertension, the arterial wall is subjected to increased intraluminal pressure that causes many vascular changes (4). Arterial smooth muscle cells are the most abundant cell type in aortic tissue, and hypertension has been shown to induce functional, metabolic, and morphological changes in PREVIOUS
STUDIES
0363-6135/90 $1.50 Copyright
0
[35S]methionine (sp act ~800 Ci/mmol) and Enhance were purchased from New England Nuclear; sodium arsenite, Triton X-100, and glycerol were from Fisher Scientific; ultrapure urea was from Schwartz-Mann; acrylamide, sodium dodecyl sulfate (SDS), and 2-mercaptoethanol were from Bio-Rad Laboratories; ampholytes were from Pharmacia; RNAsin and in vitro translation kits were from Promega; and norepinephrine and angiotensin II were from Sigma. Human HSP 70 genomic DNA probe, deposited by R. Morimoto (25) was obtained from American Type Culture Collection (DNA no. 57495). Rats were obtained from Charles River Breeding Laboratories (Wilmington, MA); DOC acetate pellets were from Innovative Research of America (Toledo, OH). Dulbecco’s modified Eagle’s medium (DMEM), L-glutamine, penicillin, streptomycin, fetal calf serum, and antibiotic-antimycotic solution were all purchased from GIBCO, and type I collagenase was obtained from Sigma. Preparation and culture of rat aortic smooth muscle cells. Rat aortic SMCs were isolated by enzymatic dispersion using a method adapted from Gunther et al. (12). Sprague-Dawley rats (200-300 g) were anesthetized with pentobarbital sodium. The thoracic aorta from six rats were removed under sterile conditions and placed in 4°C
1990 the American
Physiological
Society
H1699
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DMEM containing 25 mM N-Z-hydroxyethylpiperazine2V’-2ethanesulfonic acid (HEPES) buffer, 2 mM L-glutamine, 100 U/ml penicillin, and 100 pg/ml streptomycin. The adventitia was removed with a watchmaker forceps, and the aortas were minced with a razor blade into small segments no larger than 2 mm2. The segments were transferred into a plastic tube containing 10 ml of the following solution: DMEM with 2 mg/ml type I collagenase, 0.5 mg/ml elastase, and 1 mg/ml soybean trypsin inhibitor. The tissue suspension was incubated in a shaker bath for 2 h at 37°C. The dissociated cells were washed twice in DMEM, and the final suspension was plated into a 25cm2 flask and cultured at 37°C in a humidified atmosphere of 5% C02-95% air. After 2 h, the medium was supplemented with 20% fetal calf serum and 1% antibiotic-antimycotic solution. The medium was changed every 3 days, and confluent monolayers were obtained within 15 days. The cells were passaged by rinsing with phosphate-buffered saline followed by brief incubation with 1 ml of trypsin-EDTA. Cells were then washed with DMEM supplemented with 20% fetal calf serum and 1% antibiotic-antimycotic solution. Cells of passages 5-15 were used for these studies. Cells were plated into 2-cm’ wells. At confluence, there were 3 x lo5 cells per 2-cm2 well. During experiments, control and stressed cells were in DMEM with or without methionine. In addition, in selected experiments, cells were incubated with either 20 ,uM norepinephrine or 1 PM angiotensin II for different time periods. Ascorbate (100 PM) was added to prevent oxidation of norepinephrine. Culture medium containing hormones was changed daily. For evaluation of cellular hypertrophy, cells were grown to confluence in six-well dishes, and agonists were added to the incubation medium each day for 3 days before analysis of DNA (15) and protein, the latter via a dyebinding calorimetric assay (BioRad, Richmond, CA). Cytosolic free Ca’+ was determined by a fluorimetric method using the calcium fluorescent probe furaas previously described (24). Heat shock. Culture dishes containing confluent cells were heated for 1 h at 42OC. At the end of this period, the plates were returned to a 37°C incubator and at designated times were washed once with methioninefree DMEM and then incubated in that medium at 37°C for 1 h. Methionine-free DMEM containing 50 &i/ml [35S]methionine was then substituted, and the cells were labeled for 1 h, washed in DMEM, scraped into 800 ~1 of the sample buffer [ 13 mM tris( hydroxymethyl)aminomethane (Tris), pH 6.8, 10% glycerol, 2% 2-mercaptoethanol, 3% SDS], used for SDS-polyacrylamide gel electrophoresis (PAGE), and boiled for 4 min. Arsenite treatment. Arsenite treatment was based on the method of Kim et al. (14). Confluent cells were incubated for 4 h in DMEM containing 100 PM sodium arsenite. After arsenite treatment, cells were washed with arsenite-free DMEM lacking methionine and then labeled with [35S]methionine following the protocol described above for heat shock. Cells were then scraped from the plates and suspended in the lysis buffer used for two-dimensional gel electrophoresis. RNA isolation and analysis. RNA isolation was per-
MUSCLE
CELLS
AND
AORTA
formed with minor modifications from established procedures (3). Cells were incubated at 37°C for 90 min after heat shock and scraped from six separate wells into 6 ml of guanidinium thiocyanate lysis buffer. Total RNA was isolated by cesium chloride centrifugation as recently described (22). For tissue RNA, hearts were processed individually while aortas from three animals were pooled. RNA was analyzed by Northern blotting procedures using methodology recently described (2 1). Following electrophoresis through 1% agarose-1 M formaldehyde gels, the RNA was transferred to nylon membranes, cross-linked by ultraviolet (UV) irradiation, and prehybridization was performed as described. A human HSP 70 genomic DNA probe that included the entire coding sequence was labeled by a random hexamer priming procedure (lo), and hybridization was performed at 65°C overnight. After hybridization, the membranes were washed three times at 55, 60, and 65°C using 1 x SSC, 0.5 X SSC, and 0.1 X SSC, respectively. SSC is 0.15 M NaCl and 0.015 M sodium citrate at pH 7.0. Blots were exposed to preflashed X-ray film between two intensifying screens, and the size of the mRNA detected was calculated based on ribosomal RNA migration. In vitro translation. Total RNA from control and heatshocked samples were translated in vitro using a micrococcal nuclease-treated, rabbit reticulocyte lysate kit. Reaction mixtures contained 10 pg total RNA, 50 &i [35S]methionine, and RNAsin (an RNase inhibitor). The lysate was utilized without modification of the Mg2+ or K+ concentrations. Incubations were for 1 h at 30°C. RNA was denatured by heating to 65°C for 4 min immediately before translation. After incubation, aliquots of the lysate reaction mixture were dried under vacuum and resuspended in lysis buffer for subsequent analysis by two-dimensional gel electrophoresis. In vitro translations of aortic and cardiac RNA were performed exactly as for cultured cells. Electrophoresis. SDS-PAGE was performed on 12% acrylamide gels after Laemmli (16). For two-dimensional electrophoresis, samples in lysis buffer [9.5 M urea, 2% Triton-X 100, 5% 2-mercaptoethanol, 2% ampholytes (26)] were separated by isoelectric focusing in the first dimension (ampholytes 1.4% 5-7; 0.6% 3-10) and SDSPAGE in the second dimension on 12% acrylamide gels (19). Gels containing [35S]methionine were treated with Enhance for fluorography. Hypertensive animal model. DOC-salt hypertension was produced in male Wistar rats following the procedures described recently (22). Animals were uninephrectomized at 10 wk of age and a subcutaneous pellet containing 100 mg DOC acetate was implanted 1 wk after uninephrectomy. Animals were given 1% saline as drinking water. Blood pressure was monitored weekly using tail cuff plethysmography. Animals were anesthetized with pentobarbital sodium before aortic dissection. Animals were killed at 3, 7,14,21, and 28 days after implantation of the DOC pellet. Blood pressures of the treated animals increased progressively with time, with average systolic pressures (in mmHg) of 124, 128, 162, 183, and 186, respectively, for the times indicated above. Heart weight-to-body weight ratios (x100) also increased in the
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STRESS-INDUCED
a
bcdefg
PROTEINS
IN SMOOTH
kDa
MUSCLE
CELLS
AND AORTA
H1701
RESULTS
Heat shock response in cultured smooth muscle cells.
Actin
-)
FIG. 1. Time course of heat shock protein (HSP) production by arterial smooth muscle cells. Cells were heat shocked, labeled with [““S]methionine for 1 h at time intervals indicated, and resulting labeled proteins were separated by SDS-PAGE. a: control cells; b: during heat shock; c: 1 h after heat shock; d: 2 h; e: 4 h; fi 6 h; g: 23 h. HSPs 70, 90, and 110 were induced.
treated animals, averaging 0.30, 0.33,0.34, 0.38, and 0.49 for 3, 7, 14, 21, and 28 days of treatment, respectively. Control animals that were uninephrectomized but not given DOC did not exhibit increased blood pressures and did not show signs of cardiac hypertrophy. Additional data regarding this animal model have been reported recently (22).
The time course for the induction of HSPs by vascular smooth muscle cells (VSMCs) was determined by labeling cells with [35S]methionine before and during heat shock as well as 1,2,4,6, and 23 h after the end of heat shock. Figure 1 is an autoradiogram of an SDS-PAGE gel of those samples. Three major proteins were induced by heat shock, with molecular masses of -70, 90, and 110 kDa. These were assumed to correspond to HSPs 70, 90, and 110, respectively. Synthesis of those proteins was greatest between 1 and 2 h after heat shock. Incorporation of label into HSP 70 subsided by 4 h poststress; in contrast, HSPs 90 and 110 synthesis was not appreciably reduced 6 h after heat shock. Each of the HSPs returned to control levels after 23 h. Analysis of the heat shock response of VSMCs using two-dimensional electrophoresis is shown in Fig. 2. The labeled proteins produced by unstressed cells and those treated by heat shock are compared. In this and all subsequent figures, the left is the basic side of the gel (pH 7) and the right is the acidic side (pH 4). It was apparent that HSP 70 included at least two proteins. One occurred both in control and heat-shocked cells but was more apparent in the latter. The other protein, which was more basic and had a slightly lower molecular weight, was only present in heat-shocked cells. These two proteins probably correspond to two forms of the 70-kDa protein (2): an “inducible” HSP 70, which is only present in stressed cells, and a “constitutive” HSP 70, which is increased by heat shock, but is also expressed in normal cells. Two-dimensional gel electrophoresis also showed clearly that HSPs 90 and 110 were present in control cells, although at much lower levels than after heat shock. Arsenite treatment. To further define the capability of VSMCs to produce stress-induced proteins, cells were incubated with sodium arsenite, an agent often used to document a cellular response to chemical stress. As in-
FIG. 2. Two-dimensional gel electrophoresis of heat shock proteins (HSPs) produced in vascular smooth muscle cells in response to heat shock for 1 h at 42°C. [%S]methionine incorporation occurred between 1 and 2 h after heat shock. C, control cells; HS, cells exposed to heat shock. Numbers beside arrows are molecular mass (in kDa). In all two-dimensional gels in this paper, left is basic side of gel (pH 7), and right is acidic side (pH 4). Gels resolve molecular mass in range 12-200 kDa. HSPs 70, 90, and 110 were induced, and HSP 70 was resolved into two proteins.
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H1702
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PROTEINS
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FIG. 3. Two-dimensional gel electrophoresis of proteins synthesized by smooth muscle cells in response to arsenite. Cells were exposed to 100 PM sodium arsenite for 4 h and then labeled for 1 h with [%]methionine. C, control cells; As, arsenite treatment. Numbers beside arrows are molecular mass (in kDa). HPSs 70, 90, and 110 were induced as well as a protein sized 30 kDa.
FIG. 4. In vitro translation of total RNA from heat-shocked cells. RNA isolated from control cells (C) and cells heat shocked (HS) for 1 h at 42°C were translated in vitro, and resulting %-labeled proteins were separated by two-dimensional gel electrophoresis. Numbers beside arrows are molecular mass (in kDa). HSPs 70, 90, and 110 were induced as well as a protein sized 23 kDa.
dicated by the two-dimensional gel elecrophoretograms shown in Fig. 3, HSPs 90 (inducible and constitutive), 90, and 110 were clearly induced by arsenite treatment. These proteins had the same sizes and pIs as the corresponding proteins made by heat-shocked cells. In addition, arsenite-treated samples produced a labeled protein with a size of -30 kDa, which was not expressed after heat shock. In vitro translation. Total RNA was isolated from control and heat-shocked cells and translated in vitro with [35S]methionine. The resulting radiolabeled proteins were separated by two-dimensional gel electrophoresis and the autoradiograms were analyzed (Fig. 4). HSPs 70,90, and 110 had the same pattern of induction seen in cell-labeling experiments, suggesting that the
synthesis of these proteins in response to stress was transcriptionally regulated. In addition, a protein having a mass of -23 kDa was detected that was not observed in the labeling experiments using intact cells. Northern blot hybridization analysis. Figure 5 shows representative data for expression of HSP 70 mRNA by arterial SMC as well as cardiac and aortic tissue. Figure 5A shows a major induction of HSP 70 mRNAs after heat shock and also shows that both unshocked SMC and normal aortic tissue have three different detectable mRNAs of 2.2, 2.7, and 3.0 kb. Only the major form at 2.2 kb was seen in heart RNA. With the use of a shorter exposure time (30 min), inducible HSP mRNAs of 3.0 and 3.2 kb could be readily detected in heat-shocked SMC (Fig. 5B).
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STRESS-INDUCED
H
A
C
HS
:
PROTEINS
IN
SMOOTH
MUSCLE
CELLS
AND
AORTA
H1703
HS
FIG. 5. Northern blot analysis of total RNA from rat heart, aorta, and arterial smooth muscle cell (SMC) using a cDNA probe for human HSP 70. Each lane contained 20 pg of total RNA from control heart (H), control aorta (A), control SMC (C), and heat-shocked SMC (HS). Locations of 18s and 28s markers are indicated. Numbers beside arrows are RNA size in kb. A: autoradiogram after a 36-h exposure time. One band at 2.2 kb was seen in heart. There were 3 bands in aorta and control SMC: 2.2, 2.7, and 3.0 kb. A large increase in mRNA was seen in HS SMC. B: 0.5-h exposure time of C and HS from A.
1. Effect of angiotensin II and norepinephrine on protein, DNA, and intracellular Ca2+ in vascular smooth muscle cells
TABLE
Protein, mg/well
Treatment
Control Angiotensin Norepinephrine
II
DNA, dwell
0.40+0.06 0.61kO.05
7.6kO.4 7.2kO.4
0.65&0.04
7.4kO.3
Protein/DNA, PPlPg
53 84 87
CC?+, IlM
127f28 527+81 389+42
Values are means & SE; n = 6. Vascular smooth muscle cells (VSMCs) were grown to confluence in 6-well dishes. In DNA and protein determinations, agonists were added to incubation media each day for 3 days before analysis. For measurement of Ca’+, cells were grown on glass cover slips, and agonists were added during period of calcium determination. Ca” concentration is average signal from lo4 cells that were in optical window of fluorometer (24). Values above are peak values obtained 5-7 min after agonist addition.
FIG. 6. Effect of chronic norepinephrine and angiotensin II treatment on protein synthesis by smooth muscle cells. Cells were exposed to 1 PM angiotensin II (ANG II) or 8 PM norepinephrine (NE) for 4 days or left untreated (C), then labeled with [%]methionine for 1 h before separation of proteins by two-dimensional gel electrophoresis. Arrows indicate positions where HSPs 70, 90, and 110 occur. No induction of these proteins was detected.
Effect of vasoactive substances. Norepinephrine has been used to induce hypertrophy in cardiac myocytes (23), and a model of angiotensin II-induced hypertrophy in VSMCs has recently been described (11). Experiments were performed to determine whether these agonists produced chemical and physiological changes in the arterial SMC under study. Cellular hypertrophy and reactivity with respect to intracellular Ca2+were determined and the results are shown in Table 1. Hypertrophy of the cells exposed to either angiotensin II or norepinephrine for 72 h was documented by measuring DNA and protein content of cells grown to confluency and then exposed to either agonist for 72 h. The DNA content did not significantly differ between wells containing cells given no agonist and those given either angiotensin II or norepinephrine. However, there was a significant increment in the protein content of the cells exposed to either angiotensin II or norepinephrine. Thus the protein-toDNA ratio of the agonist-exposed VSMCs was significantly increased, suggesting that both agonists induced hypertrophy. An acute change in cytosolic Ca2+ also was observed with either agonist, with peak increases of
three- and fourfold for norepinephrine and angiotensin II, respectively. Such changes are consistent with receptor-linked signal transduction in these cultured cells. To test the possibility that these vasoactive agents may elicit a stress-induced reaction in VSMCs, the cells were exposed to norepinephrine or angiotensin II for 4, 24, and 72 h and then labeled for 1 h with [35S]methionine. One-dimensional gel electrophoresis of the labeled samples revealed no induction of HSPs at any time period tested (data not shown). For better resolution, the 72-h samples were further analyzed using two-dimensional gel electrophoresis (Fig. 6). The constitutive HSP 70 and HSPs 90 and 110 were detected in control and experimental samples. However, there was no detectable induction of HSP 70 in either of the agonist-treated samples. Hypertensive rat models. The inability of vasoactive agents such as angiotensin II and norepinephrine to elicit stress-induced proteins in cultured SMCs did not exclude the possibility that such proteins might be induced in vivo in intact aortic tissue from animals exposed to hypertension. To assessthis, rats were made hyperten-
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H1704
STRESS-INDUCED
PROTEINS
IN
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FIG. 7. Effect of DOC-salt-induced hypertension on expression of stress-induced proteins by intact rat aorta. RNA isolated from aortas of control (C) and 21-day DO&salt hypertensive (D21) rats were translated in vitro with [%3]methionine and separated by two-dimensional electrophoresis. Numbers beside arrows are molecular mass (in kDa) and indicate nositions where HSPs 70. 90, and 110 occur. No induction of these proteins was detected.
FIG. 8. Effect of DOC-salt-induced hypertension on expression of stress-induced proteins by cardiac tissue. RNA was isolated from heart of control animals (C) and 2%day DOC-salt hypertensive rats (D28). RNA was translated in vitro with [%]methionine, and proteins were separated by two-dimensional gel electrophoresis. Numbers beside arrows are molecular mass (in kDa) and indicate positions where HSPs 70, 90, and 110 occur. No induction of these proteins was detected.
sive by DOC-salt treatment, a model in which the development of aortic hypertrophy is well documented. Aortas and hearts were removed at 3,7,14,21, and 28 days after the implantation of the DOC pellet. Total aortic RNA was isolated at each time point, translated in vitro with [35S]methionine, and the labeled proteins were run on two-dimensional gels. RNA from three separate animals was analyzed for each time period studied, and the twodimensional gels were analyzed by visual inspection. Figure 7 is a comparison of 21-day control and hypertensive aortas, which is representative of the data obtained overall. In all samples tested, the constitutive HSP 70 was present as were HSPs 90 and 110. The inducible HSP 70 was present to a small degree in both control and hypertensive aortas, but no enhancement of its expression was evident in hypertension at 21 and 28 days, when blood pressures were highest. Because other studies employing experimental models
to induce cardiac hypertrophy had suggested that HSPs were expressed in vivo, we also performed in vitro translations of RNA isolated from cardiac tissue of normotensive and hypertensive rats and analyzed these samples by two-dimensional gel electrophoresis. Again, three samples from each time period were analyzed. As shown in the representative gels of Fig. 8, we found no evidence for enhanced expression of any HSP in cardiac tissue, although hearts from DOC-salt-treated rats underwent considerable hypertrophy during the treatment period. DISCUSSION
To determine whether stress-induced proteins may have a functional role in influencing the response to injury in vascular tissue, we have examined the expression of HSPs in cultured aortic SMCs and have shown that these cells produce a complement of proteins similar
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to that described in several other cell types. The best indices of the stressed condition appeared to be the induction of HSPs 23 and 70 (inducible component) with heat and the 30-kDa protein with arsenite treatment, since those phenomena were totally absent in control cells. The increase in translatable HSP mRNA demonstrated by in vitro translation, together with the increase in steady-state levels of HSP 70 mRNA documented by Northern blot hybridization analysis, suggests that HSP 70 expression was at least in part transcriptionally regulated, although an alteration in HSP mRNA stability cannot be ruled out. We found no induction of HSPs in cultured SMCs by vasoactive agonists even in the presence of cellular hypertrophy. Although it is possible that the lack of HSP expression was due to an insufficient dose of norepinephrine or angiotensin II, we did show that the cells responded to those agonists with respect to a hypertrophic response and an increase in cellular Ca2+. It is possible that our protocol missed a brief time frame in which HSP expression occurred transiently, but we did examine several time periods within a 72-h time frame. An alternative possibility is that the agonist-induced “stresses” differ from heat and arsenite treatments both in intensity and time frame. Heat shock and arsenite treatment are acute stresses and in the case of arsenite, completely nonphysiological and toxic. Increased HSP 70 expression has been reported in several studies employing relatively acute experimental models to produce cardiac hypertrophy (6-8, 13). The experiments on hypertensive rats described herein showed no detectable enhancement of HSP either in aorta or heart despite the appearance of hypertrophy and well-documented morphological changes in both organs. The onset of hypertension in the DOC-salt rat occurred at -10-14 days of treatment. However, there was considerable individual variability in the exact time at which blood pressure became elevated. Furthermore, it was not clear a priori that hypertension alone would be the event to initiate HSP expression. With these reservations in mind, we obtained samples at several time points after DOC-salt treatment (3, 7, 14, 21, and 28 days). Although it is possible that HSP induction was missed because not every time point in the course of hypertension was sampled, we can say that it did not occur at the time points studied, and that it was not a chronic feature of the hypertrophied state. This work was supported by National Institut,es of Health Grants HL-18318 (Hypertension Specialized Center of Research), HL-31195, and DK-37105. Address for reprint requests: P. Brecher, Cardiovascular Inst., Boston Univ. School of Medicine, 80 E. Concord St., Boston, MA 02118. Received
24 April
1989; accepted
in final
form
18 January
1990.
MUSCLE RUTTER.
sources 1979.
CELLS
Isolation enriched
in
AND
H1705
AORTA
of biologically ribonucleases.
active ribonucleic Biochemistry 18:
acid from 5294-5299,
A. V. The arterial smooth muscle cell in systemic Am. J. Cardiol. 60: 941-981, 1987. CHOBANIAN, A. V., A. H. LICHTENSTEIN, J. H. SCHWARTZ, J. HANSPAL, AND P. BRECHER. Effects of deoxycorticosterone/salt hypertension on cell ploidy in rat smooth muscle cells. Circulation 75, SuppZ. I: 1-102-I-106, 1987. DAVALLI, P., S. FERRARI, AND A. CORTI. Induction of a 70,000 dalton protein in hypertrophic rat heart. Experientia Basel 41: 1459-1460, 1985. DELCAYRE, C., J.-L. SAMUEL, F. MAROTTE, M. BEST-BELPOMME, J. J. MERCADIER, AND L. RAPPAPORT. Synthesis of stress proteins in rat cardiac myocytes 2-4 days after imposition of hemodynamic overload. J. CZin. Inuest. 82: 460-468, 1988. DILLMANN, W. H., H. B. MEHTA, A. BARRIEUX, B. D. GUTH, W. E. NEELEY, AND J. ROSS. Ischemia of the dog heart induces the appearance of a cardiac mRNA coding for a protein with migration characteristics similar to heat-shock/stress protein 71. Circ. Res. 59: 110-114, 1986. DZAU, V. J., AND G. H. GIBBONS. Cell biology of vascular hypertrophy in systemic hypertension. Am. J. Cardiol. 62: 30G-35G, 1988. FEINBERG, A. P., AND B. VOGELSTEIN. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. AnaZ. Biochem. 137: 266-267, 1984. GEISTERFER, A. T. A., M. J. PEACH, AND G. K. OWENS. Angiotensin II induced hypertrophy, no hyperplasia, of cultured rat aortic smooth muscle cells. Circ. Res. 62: 749-756, 1988. GUNTHER, S., R. W. ALEXANDER, W. J. ATKINSON, AND M. A. GIMBRONE, JR. Functional angiotensin II receptors in cultured vascular smooth muscle cells. J. CeZZ BioZ. 92: 289-298, 1982. IZUMO, S., B. NADAL-GINARD, AND V. MAHVADI. Protooncogene induction and reprogramming of cardiac gene expression produced by pressure overload. Proc. NatZ. Acad. Sci. USA 85: 339-343, 1988. KIM, Y., J. SHUMAN, M. SETTE, AND A. PRZYBYLA. Arsenate induces stress proteins in cultured rat myoblasts. J. CeZZ BioZ. 96: 393-400, 1983. LABRACCA, C., AND K. PAIGEN. A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102: 334-352, 1980. LAEMMLI, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T,. Nature Lond. 227: 680-685, 1970. LINDQUIST, S. The heat-shock response. Annu. Rev. Biochem. 55: 1151-1191, 1986. MECHAM, R. P., L. A. WHITEHOUSE, D. S. WRENN, W. C. PARKS, G. L. GRIFFIN, R. M. SENIOR, E. C. CROUCH, K. R. STENMARK, AND N. F. VOELKEL. Smooth muscle-mediated connective tissue remodeling in pulmonary hypertension. Science Wash. DC 237:
4. CHOBANIAN,
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5.
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15. 16.
17. 18.
423-426,1987.
19. O’FARRELL, P. H. High resolution two-dimensional electrophoresis of proteins. J. BioZ. Chem. 250: 4007-4021, 1975. 20. OWENS, G. K. Influence of blood pressure on development of aortic medial smooth muscle hypertrophy in spontaneously hypertensive rats. Hypertension Dallas 9: 178-187, 1987. 21. SARZANI, R. B., P. BRECHER, AND A. V. CHOBANIAN. Growth factor expression in aorta of normotensive and hypertensive rats. J. CZin. Inuest. 83: 1404-1408, 1989. 22. SARZANI, R., K. P. CLAFFEY, A. V. CHOBANIAN, AND P. BRECHER. Hypertension induces tissue-specific gene suppression of a fatty acid binding prtoein in rat aorta. Proc. NatZ. Acad. Sci. USA 85: 23.
24.
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hypertrophy of cultured rat myocardial cells is an alpha, aderenergic response. J. CZin. Invest. 72: 732-738, 1983. SISKIND, M. S., C. E. MCCOY, A. CHOBANIAN, AND J. H. SCHWARTZ. Regulation of intracellular calcium by cell pH in vascular smooth muscle cells. Am. J. Physiol. 256 (Cell Physiol. 25)X234-C240,1989.
1. BRECHER, P., C. T. CHAN, C. FRANZBLAU, AND A. V. CHOBANIAN. Effects of hypertension and its reversal on aortic metabolism in the rat. Circ. Res. 43: 561-569, 1978. 2. BURDON, R. H. Heat shock and the heat shock proteins. &o&em. J. 240: 313-324, 1986. 3. CHIRGWIN. J. M.. A. E. PRZYBYLA. R. J. MACDONALD. AND W. ,J.
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