Endothelin

stimulates

protein synthesis in smooth muscle cells

BALVIN H. L. CHUA, CHRISTOPHER CHU CHANG CHUA, AND CLEMENT

J. KREBS, A. DIGLIO

Departments of Pathology and of Immunology and Microbiology, Wayne State University, Detroit, Michigan 48201 Chua, Balvin H. L., Christopher J. Krebs, Chu Chang Chua, and Clement A. Diglio. Endothelin stimulates protein synthesis in smooth muscle cells. Am. J. Physiol. 262 (Endowind. Mktab. 25): E412-E416, 1992.-The present work was

carried out to assess the effect of endothelin on the relative synthesis of protein, RNA, and DNA in confluent rat aortic smooth muscle cells (SMC) derived from Wistar-Kyoto (WKY) rats maintained under serum-free medium in the presence or absence of insulin, transferrin, and selenium. Insulin stimulated protein synthesis by 42%. Endothelin (1 x lo-’ M) rapidly induced protein synthesis by 22% (-insulin) and 30% (+insulin). Prior treatment of SMC for 4 h with endothelin resulted in 50% (-insulin) and 38% (+insulin) increase in protein synthesis. The stimulatory effect of endothelin on protein synthesis could be partially blocked by l-@-isoquinolinylsulfonyl)-2=methylpiperazine, a protein kinase C inhibitor. Atria1 natriuretic factor had no effect on either the basal protein synthesis or protein synthesis stimulated by endothelin. Furthermore, endothelin stimulated RNA synthesis by twofold but had no effect on DNA synthesis in SMC derived from WKY rats. In contrast, SMC derived from spontaneously hypertensive rats showed increased DNA synthesis and cell growth after endothelin stimulation. These studies show that this hormone may play a pivotal role in the development of vascular hypertrophy in hypertension. vascular hypertrophy ELEVATED PERIPHERAL RESISTANCE and structuralvascular changes are general features in chronic hypertension in both animals and humans. These changes in the vessel wall can be attributed largely to an increase in vessel thickness and smooth muscle mass. An increase in wall thickness in both large and small arteries of hypertensive animals has been demonstrated by morphometric studies. Olivetti et al. (26) and Owens and Schwartz (27) demonstrated that the mass increase of the smooth muscle in the aorta of spontaneously hypertensive rats or a two-kidney Goldblatt hypertensive model could be attributed to a great extent to smooth muscle hypertrophy. In experimental hypertension, the thickening of the blood vessel wall is associated with an increase in vascular wall protein synthesis (33). An increase in DNA ploidy has been demonstrated in the smooth muscle of a variety of experimental hypertensive rats (28) and in cultured smooth muscle cells (SMC) (14) For a long time the causes of vascular hypertrophy in hypertension were attributed to the presence of a number of circulating vasoactive agents in blood. Angiotensin II, catecholamines, vasopressin, and serotonin have been shown to stimulate protein synthesis or cellular proliferation (3, 6, 25). Our data (9) and that of others (2, 13) indicated that angiotensin II stimulated protein synthesis in growth-arrested SMC. Recently, a novel peptide termed endothelin was isolated from endothelial cells E412

0193-1849/92

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Copyright

and was shown to be an extremely potent agonist for vascular smooth muscle contraction with a long duration of action (34). Endothelin has the characteristics of a local hormone that constricts the underlying vascular smooth muscle. The present work shows that endothelin is a potent stimulator of protein synthesis in cultured SMC. Because RNA synthesis is an early event in the hypertrophic process, the effect of endothelin on RNA synthesis was examined. To assess the role of protein kinase C, a protein kinase C inhibitor, l-(&isoquinolinylsulfonyl) -2methylpiperazine (H-7)) was included to study its effect on endothelin-induced protein synthesis. METHODS AND MATERIALS Materials. [35S]methionine (800 Ci/mmol) and [ methyZ-3H] thymidine (2 Ci/mmol) were obtained from Du Pont-New England Nuclear. [5-3H]cytidine (15 Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO). Human endothelin-1 was supplied by Peptide International (Louisville, KY). H-7 was obtained from Sigma Chemical. H-7 was dissolved in methanol to obtain a stock solution of 10 mg/ ml and then was diluted with ethanol before use. Insulin, transferrin, and selenium (ITS) were purchased from Collaborative Research. Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s Medium were products of GIBCO. Culturing of aortic SMC. Age-matched (6-wk-old; 300 g) male Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were killed by decapitation, and the thoracic aortas were removed under sterile conditions. Aortas were stripped of adventitia, cut into l- to 1.5-mm “ring” segments and cultured by explant method, as described by Diglio et al. (11). Aortic SMC were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO) supplemented with 10% FBS, 2 mM L-glutamine, and 25 pg/ml gentamycin at 37°C in a humidified atmosphere of 95% oxygen-5% COa. Cells were serially subcultured by treatment with trypsin [0.25% in phosphate-buffered saline (PBS)] and used for experimentation between passages 5 and 15. Measurements of relative protein synthesis. SMC (WKY) were cultivated in DMEM containing 10% FBS in 35-mm dishes. After 3-4 days the confluent cells were incubated with serum-free medium in the presence or absence of ITS (1 PM insulin, 5 pg/ml transferrin, and 5 rig/ml selenous acid). Two days later, endothelin and/or H-7 at appropriate concentrations was added directly to the medium for a period up to 15 h. After the removal of media, SMC were washed two times with 1 ml serum-free medium supplemented with 1 mM L-methionine. Labeling with [35S]methionine was initiated by the addition of 1 ml methionine-free and serum-free medium supplemented with 1 mM L-methionine and 25 &i/ml [36S]methionine. After 1 to 4 h at 37”C, media were removed for the determination of specific activity of [35S]methionine [disintegrations. min-’ (dpm) . nmol methionine-‘1 by the dansylation method (29). SMC in dishes were lysed by 1 ml 0.5N NaOH and transferred to 1.5-ml Eppendorf tubes. Ice-cold 4 N perchloric acid (0.5 ml) was added. After 15 min at 4”C, proteins were pelleted by centrifugation for 10 min at 12,000 g in an Eppendorf microfuge at 4°C. The pellets were washed four times with 1 ml 5%

0 1992 the American

Physiological

Society

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ENDOTHELIUM

AND PROTEIN

trichloroacetic acid (TCA) containing 1 mM methionine and centrifuged. Protein in the pellet was dissolved in 0.2 ml 0.5N NaOH. An aliquot was assayed for protein concentration by Bradford’s method using bovine serum albumin as standard (5). Another aliquot was counted in 10 ml ACS (Amersham) by a liquid scintillation counter. Because it is very difficult to determine the specific activity of methionine-tRNA in cultured cells, the relative rates of protein synthesis were based on the specific activity of extracellular [36S]methionine and the rates of incorporation of [36S]methionine into protein. At 1 mM methionine, the specific activity of methionine in the medium is equal to that of intracellular methionine. The specific activity of intracellular methionine was determined by the method described by Airhart et al. (1). The specific activity remained constant during the 3 h of the labeling period. Endothelin had no effect on these specific activities (unpublished data). Measurement of relative RNA synthesis. Relative RNA synthesis was measured by following the incorporation of [!G3H]cytidine to total [3H]RNA. Confluent SMC (WKY) were growth arrested for 48 h and pretreated with endothelin at indicated doses for 3 h. Cells were then labeled with [5-3H]cytidine (5 &i/ml) for 3 h. RNA extraction was carried out by guanidinium isothiocyanate-phenol-CHCl3 method (7). The amount of [3H]cytidine incorporated into total RNA was determined (dpm/pg RNA). Measurement of relative DNA synthesis. Relative DNA synthesis was measured by following the incorporation of [3H]thymidine to DNA. Confluent SMC (WKY or SHR) were maintained in serum-free media (+ITS) for 48 h and pretreated with endothelin (1 x lo-’ M) for 24 h. Cells were then labeled with [‘H]thymidine (1 &i/ml) for 3 h. After washing three times with PBS, cells were fixed by ice-cold 10% TCA for 10 min, washed one time with ether-alcohol (2:l) and dissolved in 0.5N NaOH. An aliquot was neutralized with 1N HCl for the determination of DNA by a rapid and sensitive binding of DNA to Hoechst 33258 and quantitated by TKO 100 DNA fluorometer (Hoefer Scientific Instrument) using calf thymus DNA as standard (22). Another aliquot was counted in Aquasol (Du Pont-New England Nuclear). The amount of [3H]thymidine incorporated into DNA was determined (dpm+g DNA-l. 3 h-l). Measurement of cell growth. Subconfluent SMC that were maintained in serum-free media (+ITS) for 48 h were treated with endothelin (1 x lo-’ M) for 3 days, with daily change of fresh media and addition of endothelin. Cells were harvested after trypsinization. Cell number was determined by counting in a hemacytometer, in triplicates. Statistical analysis. Statistical analysis was made by analysis of variance followed by the Student-Newman-Keuls multiplerange test for comparison of any two sample means. When a single comparison was made between two sample means, Student’s t test was employed. P < 0.05 was considered to be statistically significant. RESULTS

Present experiments demonstrate that endothelin and insulin are potent regulators of protein synthesis in confluent SMC (derived from WKY rats) maintained in serum-free media. Insulin stimulated protein synthesis by 42% over basal protein synthesis (Table 1). Transferrin had no effect on basal protein synthesis. Addition of endothelin accelerated protein synthesis by 22% in the absence of insulin. Interestingly, endothelin was able to stimulate protein synthesis by 30% in the presence of 1 PM insulin. Pretreatment with endothelin for 4-5 h was enough to maximally acce lerate rates of protein synthe-

E413

SYNTHESIS

Table 1. Effect of endothelin pretreatment on protein synthesis Duration,

h

Protein nmol methionine

Synthesis, . mg protein-‘.

%Change

+ITS

-ITS

-Endothelin +Endothelin 0 1 2 4 5 15

6.92kO.

18

8.44,tO.43* 8.60+0.21* 8.97+0.29* 10.40*0.20* 11.74+0.17*

3 h-l %Change

100

9.84kO.40

100

122 124 129 150 169

12.83+0.80f 12.99k0.62t 12.27*0.17t 13.56k0.357 14.06k0.497 14.15+0.31t

130 132 125 138 143 144

ND

Table 2. Effect of varying dose of endothelin on protein synthesis Protein nmol methionine

Dose, M

0 lo-” lo-lo 1o-g 1o-8 lo-’ lO+j

-ITS

%Change

5.68&O. 19 6.35t0.42 7.07t0.45” 7.00~0.05’ 6.93*0.17* 7.62kO.07’ 7.28~kO.03~

100 112 125 123 122 134 128

Synthesis, . mg protein-‘. +ITS 9.59+0.28 10.5t3+0.25t 11.41+0.22t 11.42kO.527 12.83-tO.65-t 12.54-to.527 14.11+0.38t

3 h-l %Change 100 110 119 119 134 131 147

Values represent means k SE of mean of 3-5 wells of SMC. Confluent cells were used for measuring relative rates of protein synthesis in serum-free media in presence or absence of ITS. Endothelin at dose indicated was added 4 h before initiation of protein synthesis. * P < 0.05 vs. control, -ITS. t P < 0.05 vs. control, +ITS.

sis, since SMC exposed to endothelin for 15 h had no additional effect (Table 1). The next series of experiments was carried out in SMC exposed to various doses of endothelin in the presence or absence of ITS. Endothelin at 1 PM stimulated protein synthesis by 28% (-ITS) and 47% (+ITS, Table 2). The minimal dose necessary to produce significant change in protein synthesis was 1 x lo-l1 M in the presence of insulin or 1 X 10-l’ M in the absence of insulin. To gain insight into the mechanism involved in the induction of protein synthesis by endothelin, H-7, a potent inhibitor for protein kinase C, was included. The addition of H-7 partially inhibited protein synthesis induced by endothelin. However, endothelin still stimulated protein synthesis in H-7-treated cells (Table 3, expt 1). These data suggest that protein kinase C and other pathways may be involved in the endothelin effect. Because atria1 natriuretic factor (ANF) could stimulate guanosine 3’,5’-cyclic monophosphate (cGMP) formation via guanylate cyclase (8, 19) and relax the smooth muscle constricted by endothelin, it was included in this study. ANF alone had no effect on protein synthesis and could not block the stimulation of protein synthesis elicited by endothelin (Table 3, expt 2).

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E414

ENDOTHELIUM

AND PROTEIN

SYNTHESIS

Table 3. Effect of H-7 and ANF on protein synthesis

Table 5. Effect of endothelin

stimulated

incorporation

by endothelin Protein Synthesis, nmol . mg protein-’ 3 h-’

Parameter

l

Expt 1 None Endothelin H-7 Endothelin

+ H-7

9.54*0.38 13.80*0.34*t 8.39k0.35 10.45+0.13t

Expt 2 None 11.10~0.20 Endothelin 14.91*0.23* ANF 11.87t0.32 Endothelin + ANF 13.8151tO.51" Values are means k SE for 6 wells of SMC. Confluent cells were used for measuring relative rates of protein synthesis in growth-arrested serum-free media supplemented with ITS. Endothelin (10 nM), l-(5isoquinolinylsulfonyl)-2-methylpiperazine (H-7; 7 PM) and atria1 natriuretic factor (ANF; 1 PM) were added 4 h before initiation of protein synthesis. Protein synthesis in SMC treated with solvent (2 ~1 alcohol) used to dissolve H-7 was same as control (expt 1). * P C 0.05 vs. none. t P < 0.05 vs. H-7.

Table 4. Effect of endothelin incorporation Endothelin,

on [3H]cytidine

into RNA M

(‘H Jcytidine Incorporation, dpm . pg RNA-‘. 3 h-’

% Control

0 20,158+1,822 100 10'" 20,983&2,154 104 1O"O 37,706&2,551* 187 lo-* 38,058*1,719* 189 10" 40,480+2,532* 200 Values represent means k SE of 4 wells of SMC. Confluent cells were maintained in serum-free medium (+ITS) for 48 h. Cells were treated with endothelin at concentration indicated for 3 h before labeling with [3H]cytidine (5 &i/ml). After 3 h, media were removed, and incorporation of [3H]cytidine into [3H]RNA was determined, as dpm, disintegrations/min. described in METHODS AND MATERIALS. + P < 0.05 vs. no addition.

In addition to its effect on protein synthesis, endothelin had a potent effect on the incorporation of [3H]cytidine into RNA in SMC maintained in serum-free medium supplemented with ITS. Endothelin (1 x 10ml’ M) led to approximately a twofold increase in the incorporation of [3H]cytidine into [3H]RNA (Table 4). Finally, the effect of endothelin on the incorporation of [3H]thymidine into DNA was examined. The changes in relative protein synthesis and RNA synthesis were not accompanied by a change in relative DNA synthesis in SMC derived from WKY rats under our experimental conditions (Table 5). Endothelin pretreatment for 1, 2, and 3 days had no effect on either [3H]thymidine incorporation into DNA or on the total cellular DNA content (data not shown). However, endothelin led to a twofold increase in relative DNA synthesis and cell numbers in SMC derived from age-matched and male SHR. These results clearly demonstrate that the responses of WKY-derived and SHR-derived SMC to endothelin are different. DISCUSSION

The present study clearly shows that endothelin stimulates relative protein and RNA synthesis without af-

SMC Source

on [3H]thymidine into DNA and cell number

Endothelin

[‘H ] thymidine, (dpm x 10W6) pg DNA-’ l

l

3 h”

Cell Number, 1 X lO+/well

2.71kO.03 WKY 7.8*0.4 WKY + 2.84kO.22 7.0k0.2 4.04kO.02 SHR 9.8k0.4 SHR + 7.56kO.12” 19.6k2.0’ Values represent means k SE of 3-6 wells of SMC. SMC were derived from age-matched male Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Determination of effect of endothelin (1 X lo-’ M) on [3H]thymidine incorporation into DNA and cell number was performed as described in METHODS AND MATERIALS. * P C 0.05 vs. SHR, -endothelin.

fecting DNA synthesis in confluent SMC maintained in serum-free media supplemented with ITS. The pathway by which endothelin accelerates protein synthesis was explored. It has been demonstrated that the signaling events in the endothelin action involve the production of inositol trisphosphate and diacylglycerol via a phosphoinositide pathway (16,23). These two second messengers mobilize Ca2+ from intracellular store sites and activate protein kinase C, respectively. Our data suggest that a protein kinase C-dependent pathway may be involved since the endothelin-stimulated protein synthesis could be partially inhibited by a protein kinase C inhibitor, H-7. However, H-7 is also known to inhibit cGMP and adenosine 3’,5’-cyclic monophosphate (CAMP)-dependent protein kinases (17). These two protein kinases are probably not involved since ANF had no effect on protein synthesis (Table 3) and since endothelin reduced the CAMP content in Swiss 3T3 fibroblast (31). Presently, the molecular mechanism that leads to the activation of protein synthesis by endothelin is not known. It is possible that activation of protein kinase C in SMC leads to phosphorylation of the ribosomal protein S6 and other key proteins involved in the protein synthetic machinery. In recent years, considerable evidence has accumulated concerning ribosomal protein phosphorylation as one of the mechanisms, possibly controlling the rate of protein synthesis in various cell systems (15). A variety of mitogenic factors, phorbol ester, and synthetic diacylglycerol induced phosphorylation of the 40s ribosomal S6 protein. Stimulation of S6 phosphorylation, catalyzed by specific protein kinase( s), is often associated with an increase in the rate of protein synthesis. The 40s subunits highly phosphorylated in S6 were shown to be present mainly in polysomes (32), suggesting that S6 phosphorylation is involved in stimulating protein synthesis by facilitating some step(s) in the initiation process. The effect of endothelin on the phosphorylation of S6 remains to be determined. Earlier studies demonstrated that endothelin was mitogenic and could stimulate the expression of c-fos and c-myc oncogenes and DNA synthesis in SMC when insulin was present (18,20,24). Our data show that relative RNA synthesis was induced twofold by endothelin, but relative DNA synthesis was not altered by either varying the dose or duration of pretreatment with endothelin in confluent WKY SMC supplemented with ITS. The disparity between our data and others may reflect differ-

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ENDOTHELIUM

AND PROTEIN

ences in species, differentiation status, or confluency of the SMC employed. In agreement with Bobik et al. (4) endothelin is clearly mitogenic in SMC from SHR aorta under our experimental conditions. The stimulatory effect of endothelin on protein synthesis in cells derived from SHR was greater than that in WKY-derived SMC (data not shown). In previous studies, SMC derived from SHR have been shown to exhibit enhanced growth rates in response to angiotensin II and epidermal growth factor (30) Because the half-life of endothelin in circulation is extremely short (lo), it is conceivable that endothelin secreted by endothelial cells could play P a pivotal and strategic role in regulating the protein content of the underlying SMC present in the blood vessel wall in normal and pathological conditions. Angiotensin II, phenylephrine, vasopressin (12), transforming growth factor-p (21), and shear force (34) have been shown to stimulate endothelin secretion. Some of these parameters are known to be elevated in hypertension. Our results suggest that endothelin may play a role in the development of vascular hypertrophy in WKY rats and hyperplasia of SMC in SHR. We thank Dr. Allen Samarel for providing [3H]dansyl chloride and J. Kostuchowski for typing the manuscript. This work was supported by the American Heart Association (AHA) of Michigan, by National Institutes of Health Grants HL-37011 (to B. H. L. Chua) and CA-45056 (to C. C. Chua), and by AHA Grant-in-Aid 890750 (to C. A. Diglio). Address for reprint requests: B. H. L. Chua, Dept. of Pathology, Wayne State Univ., Detroit, MI 48201. Received 12 November 1990; accepted in final form 4 November 1991. REFERENCES 1. Airhart, J., J. Kelley, J. E. Brayden, and R. B. Low. An ultramicro method of amino acid analysis: application to studies of protein metabolism in cultured cells. Anal. Biochem. 96: 45-55, 1979. 2. Berk,

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1989.

Endothelin stimulates protein synthesis in smooth muscle cells.

The present work was carried out to assess the effect of endothelin on the relative synthesis of protein, RNA, and DNA in confluent rat aortic smooth ...
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