Regulation of Endothelin-1 Production in Cultured Rat Vascular Smooth Muscle Cells

Abstract

Endothelin-1 (ET-1) is secreted from all rat vascular smooth muscle cells (VSMCs) examined, in addition to endothelial cells (ECs). An average secretion rate of ET-1 from rat VSMCs was determined to be 10% that excreted from ECs. We examined the effects of 22 substances on ET-1 secretion from VSMCs and compared them with those from ECs. Transforming growth factor-β₁ (TGF-β), acidic and basic fibroblast growth factors, epidermal growth factor, angiotensin II, and adrenaline stimulated ET-1 secretion from VSMCs, whereas forskolin, thrombin, and platelet-derived growth factor-BB reduced it. Only TGF-β and phorbol ester elicited consistent effects on ET-1 secretion from VSMCs and ECs. Regulation of ET-1 and adrenomedullin secretion from VSMCs was distinctly different. These data suggest that ET-1 production in VSMCs is regulated by a mechanism separate from that in ECs and from adrenomedullin production in VSMCs. Chromatographic analysis showed immunoreactive ET-1 secreted from VSMCs was mainly composed of big ET-1, whereas approximately 90% of that from ECs was ET-1. By TGF-β stimulation of VSMCs, the ratio of big ET-1 to ET-1 was further increased. Because big ET-1 is converted into ET-1 only on the surface of the ECs in the culture system, big ET-1 secreted from the VSMCs may function as a mediator transmitting a signal from VSMCs to ECs in vivo.

Vascular tone is mainly balanced between constriction and relaxation of vascular smooth muscle cells (VSMCs). VSMCs constituting the bulk of blood vessel tissue have long been recognized only as target cells of vasoactive substances coming from the blood stream and perivascular nerves, as VSMCs are passively regulated by these substances. However, recent studies have demonstrated that VSMCs produce interleukin-1 (IL-1), tumor necrosis factors (TNF), and colony stimulating factor ^(1-3). This evidence suggests that VSMCs have an ability to produce biologically active substances. Because receptors for IL-1, TNF, and colony stimulating factor are expressed on VSMCs and neighboring cells ^(1-5), the substances secreted from VSMCs can function as autocrine and paracrine factors.

Endothelin-1 (ET-1) is the most potent vasoconstrictor peptide isolated from the culture medium of porcine endothelial cells (ECs) ⁽⁶⁾. Two other endothelin isopeptides-ET-2 and ET-3-have also been identified ⁽⁷⁾. Two ET receptors, ETA and ETB, have been cloned and shown to be expressed on a variety of cells ^(8,9). In the vascular system, the ETA receptor that has a higher affinity for ET-1 and ET-2 than ET-3 is mainly expressed on the VSMCs, and the ETB receptor with no selectivity is predominantly expressed on the ECs ⁽¹⁰⁾. In addition to ECs, VSMCs have recently been shown to synthesize and secrete ET-1 ^(11-15). Adrenomedullin (AM) is a potent vasorelaxant peptide identified in pheochromocytoma tissue ⁽¹⁶⁾. We have demonstrated that like ET-1, AM is actively synthesized and secreted from VSMCs and ECs, and that AM receptors are expressed on VSMCs and ECs ^(17-20).

Synthesis and secretion of ET-1 and AM from VSMCs provides evidence that VSMCs have the potential to regulate the functions of the vascular wall cells, because receptors for both peptides are expressed on ECs and VSMCs. Although ET-1 secretion from VSMCs has been reported, its regulation is still not well studied compared with that of ECs in the same species ^(11-14). We recently investigated the regulation of AM production in rat VSMCs ^(21-23). During these studies, it was noted that ET-1 and AM, which have opposing effects, are produced in VSMCs at almost comparable rates. To elucidate the physiologic role of ET-1 secreted from VSMCs, we examined the regulation of ET-1 production in VSMCs and compared it to that in ECs and to the regulation of AM production in VSMCs. We performed these experiments on cultured VSMCs and ECs prepared in our laboratory from the same rat strain. We further characterized immunoreactive (IR) ET-1 accumulated in culture media of VSMCs and ECs to help understand its function.

METHODS

Materials

Rat ET-1, rat big ET-1, [Arg⁸]-vasopressin (AVP), angiotensin II (Ang II), atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP), AM, leupeptin, pepstatin, and phosphoramidon were purchased from Peptide Institute, Inc. (Osaka, Japan). Human AM[40-52] and its N-Tyr derivative were synthesized on a peptide synthesizer (PE Biosystems, Foster City, CA, U.S.A.) and purified by reverse phase high-performance liquid chromatography (HPLC). Mouse recombinant IL-1β was purchased from Intergen (Purchase, NY, U.S.A.). Mouse recombinant TNF-α and bovine recombinant basic fibroblast growth factor (FGF) were obtained from Hoffmann-La Roche (Basel, Switzerland). Human recombinant transforming growth factor-β₁ (TGF-β), bovine brain acidic FGF, adrenaline, noradrenaline, aldosterone, hydrocortisone, forskolin, 12-O-tetradecanoylphorbol-13-acetate (TPA) and 4-(2-aminoethyl)benzenesulfonyl-fluoride (p-APSF) were purchased from Wako Pure Chemicals (Osaka, Japan). Human recombinant epidermal growth factor (EGF), platelet-derived growth factor-AA homodimer (PDGF-AA), PDGF-BB and fetal calf serum (FCS) were obtained from Biomedical Technologies (Stoughton, MA, U.S.A.), Genzyme-Techne (Cambridge, MA, U.S.A.), Austral Biologicals (San Ramon, CA, U.S.A.) and Life Technologies (Gaithesburg, MD, U.S.A.), respectively. Thrombin was purchased from Sigma (St. Louis, MO, U.S.A.). Lyophilized substances were dissolved according to the producers' manuals and diluted with Dulbecco's modified Eagle's medium (DMEM) containing 0.1% bovine serum albumin (BSA). Hydrocortisone, aldosterone, TPA, and forskolin were dissolved in ethanol and diluted with DMEM containing 0.1% BSA. Escherichia coli lipopolysaccharide (LPS) (serotype 026:B6) was purchased from Paesel+Lorei (Frankfurt, Germany) and dissolved in 0.9% NaCl solution.

Animals

The experiments were approved by the local ethical committee on animal experimentation and care. Eight-week-old Sprague-Dawley (SD) rats (Charles River Japan, Yokohama, Japan) were maintained under normal conditions for at least 2 weeks before collecting tissues. Tissues were obtained after the animals were killed by CO₂ gas.

Cell culture

Cultured rat VSMCs and ECs were prepared, identified, and maintained as reported previously ^(17,18). VSMC-1 and VSMC-2 were isolated from the thoracic aorta of a SD rat by the explant method, and VSMC-3 and VSMC-4 were isolated from SD rat aortas by the enzyme dispersion method. Wistar rat VSMC and bovine VSMC, prepared by the enzyme dispersion method, were donated by Dr. T. Iwamoto of this institute and Dr. Y. Morishita of Kyowa Hakko Kogyo (Tokyo, Japan). Rat ECs were prepared by the enzyme dispersion method and cloned ⁽²⁴⁾. VSMCs and ECs were used at passages 8 to 20 in the present study.

Preparation of conditioned medium

VSMCs and ECs were maintained under culture conditions reported previously ^(17,18). VSMCs and ECs, grown to confluence in culture dishes or six-well plates, were washed twice with serum-free DMEM and preincubated in DMEM containing 0.1% BSA for VSMCs or 0.01% BSA for ECs for 2 h. The medium were then replaced with DMEM containing 0.1% BSA or 0.01% BSA with stimulants, and incubated at 37°C for 0.25 to 48 h. Culture media (0.5 ml/well from six-well plates and 5 ml from 10-cm dishes) were collected and immediately acidified with acetic acid (final concentration: 0.5 M). Triton X-100 was added (final concentration: 0.002%) and the media were heated at 100°C for 10 min to inactivate proteases and lyophilized. Culture media from VSMCs and ECs incubated with adrenaline, noradrenaline, or FCS were loaded onto Sep-Pak C₁₈ cartridges (Waters, Millford, MA, U.S.A.) soon after collection and acidification. The cartridges were washed twice with 0.1% trifluoroacetic acid (TFA), and adsorbed material was eluted with 60% CH₃CN in 0.1% TFA. The eluate was then evaporated and lyophilized. The lyophilizates were dissolved in a radioimmunoassay (RIA) buffer and submitted to RIAs for ET-1 and AM. For measurements of intracellular ET-1, VSMCs were washed twice with phosphate-buffered saline (PBS), scraped in 1 M acetic acid, and then collected. After heating at 100°C for 10 min, the cell lysates were sonicated and centrifuged, and the resulting supernatants were extracted with Sep-Pak C₁₈ cartridges as described above and submitted to RIAs. Viability of VSMCs was estimated to be more than 95% by the trypan blue exclusion assay.

Radioimmunoassays for ET-1 and adrenomedullin

Monoclonal antibody (KY-ET-1-I) against ET-1 recognizes ET-1, ET-2, ET-3, and big ET-1 with comparable affinity ⁽²⁵⁾. The ET-1 RIA was the same as that for AM[40-52] except that 100 μl of antibody and 100 μl of tracer solution were added to each tube instead of 50 μl each in the RIA for AM[40-52] ⁽²⁶⁾. Antiserum #172CI-7 against human AM[40-52], which strictly recognizes the C-terminal region of human and rat AM with the same avidity, was donated by Dr. Kitamura of Miyazaki Medical College and was used for RIA of AM. ET-1 and the N-Tyr derivative of human AM[40-52] were radioiodinated by the lactoperoxidase method and the mono-radioiodinated peptides were isolated by reverse-phase HPLC and used as tracers.

Characterization of IR-ET-1 in culture medium of rat VSMCs

Culture medium (120 ml) collected from rat VSMCs after 24-h incubation was acidified with acetic acid (final concentration: 1M), and boiled for 10 min. After cooling, the medium was extracted with Sep-Pak C₁₈ ENV cartridge as described above. The concentrated material was subjected to gel filtration on a Sephadex G-50 column (fine, 1.5 × 100 cm, Amersham Pharmacia Biotech, Upsalla, Sweden) in 2 M acetic acid. Peak fractions of IR-ET-1 were pooled and then separated by reverse-phase HPLC on a μ-Bondasphere 5μ C₁₈ column (300Å, 3.9 × 150 mm, Waters) using a linear gradient of CH₃CN from 10% to 60% in 0.1% TFA over 200 min. Oxidation of methionine residues in ET-1 or big ET-1 was performed with 0.05% H₂O₂ in 1 M formic acid for 30 min ⁽²⁷⁾.

In addition to the control culture medium, conditioned medium (60 ml) of the VSMCs stimulated with 20 ng/ml of TGF-β for 12 h was prepared. IR-ET-1 in the TGF-β-stimulated culture medium was then processed and characterized by gel filtration and reverse-phase HPLC as described above.

RNA blot analysis

Tissues were collected from male SD rats (11-week-old), immediately frozen in liquid nitrogen and stored at −85°C. Total RNA was extracted by the acid guanidium thiocyanate-phenol-chloroform method ⁽²⁸⁾ and purified by repeated ethanol precipitation. To cultured VSMCs and ECs, 4 M guanidinium thiocyanate was directly added, and total RNA was extracted by the same method. Poly(A)⁺RNA was purified by using Oligotex dT-30 Super (Daiichi Pure Chemicals, Tokyo, Japan). Poly(A)⁺ RNA (5 μg) was denatured with formaldehyde and formamide, and electrophoresed on a 1% agarose gel containing formaldehyde, except for thoracic aorta, where total RNA (25 μg) was used because of the small amount of tissue available and its low RNA content. RNA was then transferred to a Zeta probe membrane (Bio-Rad, Hercules, CA, U.S.A.) and fixed with ultraviolet irradiation. Hybridization and washing of the membrane was carried out as previously reported ⁽¹⁷⁾. Rat ET-1 cDNA was donated by Dr. T. Sakurai (Institute of Basic Medical Sciences, University of Tsukuba, Ibaraki, Japan), and its Pvu II fragment (nucleotide 246-662) was radiolabeled by the random primed method and used for hybridization ⁽²⁹⁾. For comparison of mRNA content, the membrane was stripped and rehybridized to rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe (Eco RI-Bam HI fragment; 492-799) donated by Dr. T. Minegishi (Gunma University School of Medicine, Maebashi, Japan) ⁽³⁰⁾. Band intensity was estimated with a Bioimage analyzer (BAS 2000 Fuji Photofilm, Tokyo, Japan).

Conversion of big ET-1 to ET-1 by endothelial cells and VSMCs

Vascular smooth muscle cells and ECs, grown to confluence in six-well plates, were washed twice with PBS containing 0.01% BSA, and then 500 μl of the same solution containing either three protease inhibitors (11.7 μM leupeptin, 7.3 μM pepstatin, and 100 μM p-ABSF) or the three inhibitors plus 10 μM phosphoramidon was added to each well. Immediately after replacing the incubation media, 68 pmol of rat big ET-1 in 100 μl of PBS containing 0.01% BSA was added to each well, mixed well, and then incubated at 37°C for 0.5 to 4 h. After incubation, the media were collected, chilled on ice, and then centrifuged to remove cells. Hydrochloric acid and Triton X-100 were added to the supernatant to give a final concentration of 40 mM and 0.002%, and immediately loaded onto a Sep-Pak C₁₈ cartridge. The adsorbed material was eluted as described above and injected into reverse phase HPLC. ET-1, big ET-1 and other fragments were separated on a Chemcosorb 5ODS-H column (4.6 × 250 mm, Chemco, Osaka, Japan) using a shallow CH₃CN gradient from 30% to 40% in 0.1% TFA. An aliquot of each fraction was lyophilized, dissolved in RIA buffer, and submitted to RIA for ET-1. To confirm their elution profiles, synthetic ET-1 and big ET-1 were added to the incubation solution, processed by the same procedures, and analyzed by reverse-phase HPLC. Net amounts of ET-1 generated by enzymatic conversion were estimated based on differences between amounts of IR-ET-1 in the presence and absence of phosphoramidon observed at the retention time of ET-1 on reverse-phase HPLC.

Statistical analysis

Values were expressed as mean ± SEM. Statistical analysis of the results was performed with a one-way ANOVA, followed by a multiple comparison test (Dunnett's test), and p < 0.01 was considered statistically significant.

RESULTS

Secretion of ET-1 from cultured VSMCs

We first characterized VSMCs used in this study by the three different methods. All VSMCs were positively immunostained with monoclonal anti-α smooth muscle actin antibody, while they did not uptake acetylated low-density lipoprotein at all. These VSMCs also showed hill-valley features when they reached confluence. Thus the VSMCs used in this study were identified as VSMCs ^(17,18). All of these VSMCs were found to secrete IR-ET-1 into the culture medium, as shown in Figure 1. The secretion rate of IR-ET-1 from SD rat VSMC-1 was determined to be 10.18 fmol/10⁵ cells/24 h. The IR-ET-1 levels in the culture medium of other rat VSMCs were in a range of 2.53-28.29 fmol/10⁵ cells after 24 h incubation, and the average secretion rate of IR-ET-1 from the five different VSMCs was 11.31 fmol/10⁵ cells/24 h. Bovine VSMCs secreted IR-ET-1 at a low rate of 0.25 fmol/10⁵ cells/24 h. Under the same conditions, rat aortic ECs secreted IR-ET-1 at the rate of 109.15 fmol/10⁵ cells/24 h, which was 10 times higher than the average secretion rate of VSMCs. VSMC-1 was used in the following studies.

FIG. 1: Secretion of immunoreactive endothelin-1 (IR-ET-1) from cultured rat vascular smooth muscle cells (VSMC), bovine VSMC, and rat endothelial cells (EC) under non-stimulated conditions. Rat VSMC were prepared from thoracic aorta, and bovine VSMC from carotid artery. Sprague-Dawley (SD) rat VSMC-1 and VSMC-2 were prepared by the explant method, and SD rat VSMC-3, VSMC-4, Wistar rat VSMC, and bovine VSMC were prepared by the enzyme dispersion method. Each cell line was preincubated in serum-free medium for 2 h, and IR-ET-1 accumulated in the culture medium during the succeeding 24 h was measured by specific radioimmunoassay. Each value represents the mean ± SEM of four or six separate wells.

Endothelin-1 mRNA levels in various rat tissues, cultured VSMCs, and ECs were estimated by RNA blot analysis. As shown in Figure 2, strong bands corresponding to 2.3 kb of ET-1 mRNA were observed in lanes of lung, cultured ECs, and VSMCs, and positive bands were also observed in lanes of brain, eye, and atrium. In the lane of intact aorta, a weak but significant band was observed, although only 25 μg of total RNA was loaded. The band intensity of ET-1 mRNA from cultured VSMCs was estimated to be approximately 30% that of ECs.

FIG. 2: RNA blot analysis of endothelin (ET-1) gene transcripts in rat tissue, cultured rat endothelial cells (EC) and vascular smooth muscle cells (VSMC). Five micrograms of poly(A)⁺ RNA was denatured and electrophoresed on each lane, except a lane for thoracic aorta (total RNA, 25 μg). Lanes: 1) brain, 2) eye, 3) lung, 4) liver, 5) jejunum/ileum, 6) kidney, 7) cardiac atrium, 8) cardiac ventricle, 9) testis, 10) thoracic aorta, 11) cultured EC, and 12) cultured VSMC (Sprague-Dawley rat VSMC-1 shown in Fig. 1). RNA molecular size standards are shown at left in kilobase. Autoradiography was −80°C for 24 h. Lower panel indicates RNA blot analysis of glyceraldehyde-3-phosphate dehydrogenase transcripts.

As shown in Figure 3, a secretion rate of IR-ET-1 from rat VSMCs was linear in the first 6 h (0.69 fmol/10⁵ cells/h) and then gradually decreased to 0.16 fmol/10⁵ cells/h during 24-48 h. The transcript level of the ET-1 gene slightly increased in the first 4 h and then gradually decreased. Compared with the secreted IR-ET-1, the cellular contents of IR-ET-1 were constant over 48 h and corresponded to 5% of total IR-ET-1 accumulated in the medium after 24 h incubation. These results indicate that ET-1 is secreted from cultured VSMCs through the constitutive secretion pathway without being stored in the cells.

FIG. 3: Time-dependency of endothelin-1 (ET-1) secretion and gene transcription in vascular smooth muscle cells (VSMC) stimulated with transforming growth factor (TGF)-β. A. Immunoreactive endothelin-1 (IR-ET-1) accumulated in culture medium of Sprague-Dawley (SD) rat VSMC-1 was measured at the indicated periods under the conditions without stimulation (control, open circles) and stimulated with TGF-β (20 ng/ml, closed circles). Each point represents the mean ± SEM of four separate wells. B. ET-1 mRNA level in cultured SD rat VSMC-1 was measured at the indicated periods under conditions without stimulation (control, open circles) and stimulated with TGF-β (20 ng/ml, closed circles). C. RNA blot analysis of ET-1 gene transcripts and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene transcripts in cultured SD rat VSMC-1 was performed at the indicated periods under the conditions without stimulation (control, right) and stimulated with TGF-β (20 ng/ml, left). Twenty-five micrograms of total RNA were loaded and electrophoresed on each lane.

To compare ET-1 production levels between growth and quiescent stages of VSMCs, we applied a 24 h or 48 h serum-free preincubation to stop proliferation of VSMCs, and then measured IR-ET-1 secretion and ET-1 gene transcript level after a subsequent 24 h incubation. IR-ET-1 was secreted at a rate of 11.17 and 8.17 fmol/10⁵ cells/24 h in the 24 h and 48 h serum-starved group, respectively. Significant levels of ET-1 gene transcript were found in VSMCs from both the 24 h and 48 h serum-starved groups at an intensity slightly weaker than that obtained with the standard 2 h preincubation used in this study (data not shown).

Characterization of IR-ET-1 secreted from cultured VSMCs

On gel filtration of the VSMC-1 culture medium, approximately 80% of total IR-ET-1 was eluted at the position corresponding to big ET-1, and the remaining IR-ET-1 was eluted at the position of ET-1 (Fig. 4A). On reverse-phase HPLC of the major IR-ET-1 peak from gel filtration (fractions 28-30), the IR-ET-1 emerged at retention times identical to that of big ET-1 and its methionine sulfoxide form, and the IR-ET-1 from the minor peak (fractions 34-36) was eluted at the same retention times as that of ET-1 and its methionine sulf-oxide form (Figs. 4B and 4C). Conversely, immunoreactivity corresponding to ET-2, ET-3, big ET-2, and big ET-3, which could be detected by the antibody used, were not observed on reverse-phase HPLC. Using the same chromatographic procedures, IR-ET-1 in the culture medium of rat ECs was characterized to be a mixture of big ET-1 and ET-1 in the ratio of 1:9 (data not shown).

FIG. 4: Characterization of immunoreactive endothelin-1 (IR-ET-1) in culture medium of rat vascular smooth muscle cells (VSMC) by gel filtration and reverse phase high-performance liquid chromatography (HPLC). A. Gel filtration of IR-ET-1 secreted from VSMC. Sample: 120-ml equivalents of culture medium of Sprague-Dawley rat VSMC-1, extracted with Sep-pak C₁₈ ENV cartridge. Column: Sephadex G-50, 1.5 × 100 cm. Solvent: 2 M acetic acid. Fraction size: 4.0 ml/tube. Flow rate: 5.0 ml/h. B and C. Reverse-phase HPLC of IR-ET-1 in fractions from the Sephadex G-50 gel filtration above. Sample: 1/20 of fractions 28-30 (B) or 1/20 of fractions 34-36 (C) from graph A was injected. Column: μ-Bondasphere C₁₈ column (3.9 × 150 mm). Flow rate: 1.0 ml/min. Fraction size: 0.5 ml/tube. Solvent system: linear gradient elution from 10% to 60% CH₃CN in 0.1% TFA over 200 min. Arrows indicate elution positions or times of 1) bovine serum albumin, 2) ribonuclease A, 3) big ET-1, 4) ET-1, 5) NaCl, 6) methionine sulfoxide form of big ET-1, and 7) methionine sulfoxide form of ET-1.

Substances regulating ET-1 and adrenomedullin secretion from VSMCs and endothelial cells

We examined the effects of 22 substances on ET-1 and AM secretion from cultured rat VSMCs and ECs. IR-ET-1 and IR-AM in culture media were measured as indexes of their synthesis, because ET-1 and AM were shown to be not stored in the cells but constitutively secreted after biosynthesis ^(6,18). To establish incubation conditions, the effects of FCS and BSA were first examined. FCS (1%) was shown to increase IR-ET-1 content in the medium of VSMCs and ECs to 238% and 207% of the control, and to slightly elevate IR-AM content in the medium of VSMCs. A maximal effect of FCS on IR-ET-1 content in the medium of VSMCs was observed at a concentration of 0.3%, increasing the IR-ET-1 content to 247% of the control, while 5% and 10% FCS elevated it to 200% and 188%. On the other hand, BSA did not alter IR-ET-1 and IR-AM content in the medium of VSMCs. To avoid the effects of FCS, we employed a serum-free medium containing 0.1% BSA in this study.

As shown in Table 1, most of the cytokines and growth factors as well as LPS altered ET-1 and AM secretion from either VSMCs or ECs. TGF-β most potently stimulated ET-1 secretion from both VSMCs and ECs, elevating the IR-ET-1 content in the medium to 336% and 271% of the control. Acidic FGF, basic FGF, and EGF increased the IR-ET-1 content in the medium of VSMCs, whereas PDGF-BB suppressed it in VSMCs. Conversely, these growth factors induced no effect on ET-1 secretion from ECs.

TABLE 1: Effect of various stimulants on production of ET-1 and AM in cultured rat VSMC or EC

Thrombin and forskolin diminished the IR-ET-1 content in the medium of VSMCs to 59% and 25% of the control, whereas in ECs, thrombin augmented and forskolin did not alter it. These two substances potently reduced IR-AM in the medium of VSMCs. Ang II elevated IR-ET-1 and IR-AM levels in the medium of VSMCs, but did not alter ET-1 secretion from ECs, whereas AM significantly increased ET-1 secretion from ECs. Adrenaline elevated both IR-ET-1 and IR-AM levels in the medium of VSMCs. At a high concentration (0.1 mM), noradrenaline decreased IR-ET-1 secretion from ECs, but this was omitted from Table 1, as it reduced the viability of ECs. Aldosterone and hydrocortisone slightly elevated IR-ET-1 secretion from ECs but did not alter it from VSMCs, while these steroids highly augmented IR-AM secretion from VSMCs. TPA reduced IR-ET-1 content in the medium of VSMCs and ECs to 57% and 41% of the control at 1 μM. PDGF-AA, AVP, ANP, and CNP did not alter IR-ET-1 from ECs and VSMCs.

To examine whether the stimulation mentioned above altered the ratio of big ET-1 to ET-1 secreted from VSMCs, we as an example characterized the IR-ET-1 after incubation of VSMCs with the most potent stimulant, TGF-β. Compared with the control incubation, TGF-β augmented IR-ET-1 secretion 2.7-fold after 12-h incubation and the ratio of big ET-1 to ET-1 was also increased 1.7-fold by estimating from the results of gel filtration. The IR-ET-1 was further confirmed to be derived from ET-1 and big ET-1 by reverse-phase HPLC (data not shown).

Time-dependency of ET-1 secretion from VSMCs stimulated with TGF-β

We examined in detail the effects of TGF-β, which showed the strongest stimulatory effects on ET-1 secretion and gene transcription in VSMCs (Fig. 3). When stimulated with TGF-β, the secretion rate of IR-ET-1 was comparable to that of the control in the first 3 h, and then reached a maximal rate (2.19 fmol/10⁵ cells/h) between 6- and 12-h incubation. After 12 h, the secretion rate decreased and then returned to the control level (0.27 fmol/10⁵ cells/h) between 36 and 48 h. The ET-1 mRNA level in control VSMCs increased to 150% around 3-6 h, and then decreased to the starting level. TGF-β stimulated ET-1 gene transcription in VSMCs and increased the ET-1 mRNA level 2.2-fold after 2-h stimulation. The level of ET-1 mRNA was retained up to 12 h, and then gradually decreased to the control level. TGF-β did not alter GAPDH mRNA level during 48-h incubation.

Dose-dependent effects of substances stimulating or inhibiting ET-1 secretion from VSMCs

Basic FGF and TGF-β dose-dependently augmented ET-1 secretion and the ET-1 mRNA level in VSMCs with a half-maximal effective dose (ED₅₀) of 20 ng/ml and 1-2 ng/ml (Fig. 5). After 11-h incubation, basic FGF and TGF-β increased IR-ET-1 content in the medium of VSMCs to 250% and 280% of the control, and ET-1 mRNA level to 181% and 193%. Conversely, forskolin and thrombin dose-dependently decreased IR-ET-1 content in VSMCs to 47% and 60%, with ED₅₀ of about 0.2 μM and 1 NIH unit/ml (Fig. 6). Forskolin dose-dependently reduced the ET-1 mRNA level in VSMCs to 45% of the control with an ED₅₀ of about 0.3 μM, while thrombin greatly suppressed it but not dose-dependently to 22% after 16-h incubation. Basic FGF, TGF-β and thrombin also increased the GAPDH mRNA level, but forskolin did not alter it.

FIG. 5: Dose-dependent endothelin-1 (ET-1) secretion from vascular smooth muscle cells (VSMC) and ET-1 gene transcription in VSMC stimulated with transforming growth factor (TGF)-β and basic fibroblast growth factor (FGF). A. Immunoreactive ET-1 (IR-ET-1) content in culture medium of Sprague-Dawley (SD) rat VSMC-1 was measured after 11-h incubation with the indicated concentrations of TGF-β (closed circles) and basic FGF (open circles). Each point represents the mean ± SEM of six separate wells. Asterisks indicate p < 0.01 compared with control (without stimulation). B. ET-1 mRNA level in SD rat VSMC-1 was measured after 16-h incubation with the indicated concentrations of TGF-β (closed circles) and basic FGF (open circles). C. RNA blot analysis of ET-1 gene transcripts and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene transcripts in SD rat VSMC-1 after 16-h stimulation with TGF-β (left) or basic FGF (right). Conc, concentration.

FIG. 6: Dose-dependent endothelin-1 (ET-1) secretion and ET-1 gene transcription in vascular smooth muscle cells (VSMC) stimulated with forskolin and thrombin. A. Immunoreactive ET-1 (IR-ET-1) content in culture medium of Sprague-Dawley (SD) rat VSMC-1 after 16-h incubation with the indicated concentrations of forskolin (open triangles) and thrombin (closed triangles). Each point represents the mean ± SEM of six separate wells. Asterisks indicate p < 0.01 compared with control (without stimulation). B. ET-1 mRNA level in SD rat VSMC-1 was measured after 16-h incubation with the indicated concentrations of forskolin (open triangles) and thrombin (closed triangles). C. RNA blot analysis of ET-1 gene transcripts and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene transcripts in SD rat VSMC-1 after 16-h stimulation with forskolin (left) or thrombin (right). Conc. indicates concentration and thrombin is expressed by NIH units.

Adrenaline dose-dependently elevated the IR-ET-1 content in the culture medium of VSMCs to 176% of the control. PDGF-BB suppressed ET-1 secretion only at a concentration of 20 ng/ml. TPA augmented ET-1 content in the medium of VSMCs to 165% of the control at 10 nM, but suppressed it at concentrations lower than 0.1 nM and higher than 1 μM.

Conversion of big ET-1 to ET-1 by endothelial cells and VSMCs

As most of IR-ET-1 secreted from VSMCs was found to be composed of big ET-1, an almost inactive intermediate form generated in the processing of the pro-ET-1, we examined whether or not big ET-1 could be converted to ET-1 by ECs or VSMCs. To exclude nonspecific degradation of big ET-1 by enzymes other than endothelin-converting enzyme (ECE), we measured amounts of IR-ET-1 eluting at the retention time of ET-1 on reverse-phase HPLC. Then, in the total amount of IR-ET-1 eluted at the retention time of ET-1, only the portion of IR-ET-1, which was reduced by addition of phosphoramidon to the incubation solution, was considered to be specific. ET-1 and big ET-1 spiked into the incubation solution were eluted at their authentic retention times with a high recovery after extraction on Sep-pak C₁₈ cartridges (Fig. 7A). Apparent IR-ET-1 levels (the total IR-ET-1 observed at the retention time of ET-1) increased linearly up to 2 h on incubation with ECs. Specific ET-1 generation from big ET-1 also increased almost linearly up to 2 h on incubation with ECs (Fig. 7B). When big ET-1 was incubated with VSMCs, the generation of true ET-1 was not observed, although the apparent IR-ET-1 levels was elevated during the incubation. These results indicate that specific and significant conversion of big ET-1 to ET-1 takes place only on the surface of ECs in the present culture system.

FIG. 7: Conversion of big endothelin-1 (ET-1) into ET-1 by endothelial cells (EC) and vascular smooth muscle cells (VSMC). A. Reverse-phase high-performance liquid chromatography (HPLC) of synthetic big ET-1 and ET-1 added to the incubation solution followed by Sep-pak C₁₈ cartridge extraction. Sample: rat big ET-1 (200 pmol) and ET-1 (200 pmol). Column: Chemcosorb 5ODS-H (4.6 × 250 mm). Flow rate: 1 ml/min. Fraction size: 0.5 ml/tube. Solvent system: linear gradient elution of CH₃CN from 30% to 40% in 0.1% trifluoroacetic acid over 30 min. Peaks of absorbance eluted at 24.1 min and 25.8 min were big ET-1 and ET-1, respectively. B. Conversion of big ET-1 to ET-1 by EC (closed squares) and VSMC (closed circles). Rat big ET-1 (68 pmol) was incubated at 37°C for 0.5, 1, and 2 h with confluent rat VSMC or EC in a six-well plate in 600 μl of incubation solution (PBS containing 0.01% BSA, 11.7 μM leupeptin, 7.3 μM pepstatin, and 100 μM p-ABSF) in the presence and absence of 10 μM phosphoramidon. The incubation solution was processed as described in the text and separated by reverse-phase HPLC as shown in A. Net amounts of IR-ET-1 generated by specific conversion were calculated based on differences between the amounts of IR-ET-1 in the presence and absence of phosphoramidon observed at the retention time of ET-1 on reverse-phase HPLC.

DISCUSSION

We assessed ET-1 secretion from six cultured VSMCs of rat and bovine origin. All VSMCs examined synthesized and secreted IR-ET-1, the highest secretion rate being 30% of that from rat ECs (Fig. 1). The ET-1 mRNA level in rat VSMC-1 was estimated to be approximately one third of that in rat ECs by RNA blot analysis (Fig. 2). By chromatographic analysis, IR-ET-1 secreted from VSMC-1 was shown to be exclusively composed of ET-1 and big ET-1 (Fig. 4). These data indicate that VSMCs actively transcribe the ET-1 gene and secrete ET-1 and big ET-1. As VSMCs constitute the bulk of tissue in blood vessels, relatively high rates of ET-1 secretion from VSMCs suggest its contribution to the regulation of the vascular wall cell function.

Resink et al. reported that human and rat VSMCs did not significantly transcribe the ET-1 gene in the quiescent stage prepared by 48-h incubation in serum-free medium ^(11,12). In the present study, however, we detected a significant level of ET-1 mRNA in rat VSMC-1 even after 48-h serum-free incubation (Figs. 2B and 2C), and the ET-1 level secreted in the next 24 h after 48-h starvation corresponded to 80% that of the 2-h starved VSMCs. As all VSMCs used in this study were identified as VSMCs, rat VSMCs were found to synthesize and secrete ET-1 even in the quiescent state.

Immunoreactive ET-1 was constitutively secreted from VSMCs soon after its synthesis, so the IR-ET-1 content in the culture medium was shown to be a usable index of ET-1 synthesis. Thus we examined the regulation of ET-1 synthesis in rat VSMCs by measuring the IR-ET-1 content in the medium. We compared the regulation of ET-1 synthesis in the VSMCs to that in the ECs established from thoracic aortae of the same rat strain, because the reported data were obtained from the cells of different species and blood vessels ^{(12-14,31-33)}.

Among the cytokines and growth factors examined, TGF-β markedly stimulated ET-1 synthesis in both VSMCs and ECs, whereas acidic and basic FGF enhanced ET-1 synthesis in VSMCs but not in ECs. The ED₅₀ for basic FGF in VSMCs was 10 times higher than that of TGF-β, and the maximal stimulation level achieved with basic FGF was lower than that with TGF-β. These results suggest that TGF-β and basic FGF stimulate ET-1 synthesis through different pathways. Because the stimulatory effect of TGF-β on ET-1 synthesis has been reported in porcine aortic ECs, rat aortic VSMCs, and human omental VSMCs ^(11,12,31), we conclude that TGF-β is a general stimulant of ET-1 synthesis in VSMCs and ECs. TGF-β did not significantly increase ET-1 secretion from VSMCs up to 3 h (Fig. 3), suggesting ET-1 induction with TGF-β is slow in onset or mediated by an indirect mechanism. EGF stimulated ET-1 synthesis and PDGF-BB suppressed it in VSMCs, but they had no effect on ET-1 synthesis in ECs. These findings indicate that ET-1 synthesis in VSMCs is regulated by a mechanism distinct from that of growth regulation.

Forskolin, an activator of adenylate cyclase, lowered ET-1 synthesis and the mRNA level in rat VSMCs (Fig. 6). Suppressive effects of forskolin and cAMP analogs on ET-1 synthesis in rat and rabbit VSMCs have also been reported by Kanse et al. ⁽¹⁴⁾. These data suggest that ET-1 synthesis in VSMCs is downregulated by the cAMP-mediated pathway. AM did not alter ET-1 synthesis in VSMCs used in this study, although AM receptors on VSMCs coupled with the adenylate cyclase. This is possibly owing to the low maximal level of cAMP stimulated with AM (data not shown). Conversely, AM increased ET-1 synthesis in ECs, whereas forskolin did not influence it (Table 1). In human ECs, however, ET-1 synthesis was reported to be suppressed with forskolin and cAMP analogs ⁽³³⁾. As AM receptors on ECs mediate two signal transduction pathways increasing cAMP concentration and Ca²⁺ mobilization ^{(19, 20)}, the different effects of AM on ET-1 production in ECs may be caused by distinct signal transduction systems.

Atrial natriuretic peptide and CNP elevating intracellular cGMP showed no effects on ET-1 synthesis in VSMCs and ECs. The effect of ANP is consistent with the data on the ET-1 secretion from human and rat ECs reported by Kohno et al. ⁽³⁴⁾. These data indicate that ET-1 synthesis in VSMCs and ECs is not regulated by the cGMP-mediated pathway.

Thrombin and forskolin suppressed ET-1 synthesis and gene transcription in VSMCs. The suppression of ET-1 synthesis by thrombin was weaker than that by forskolin, but thrombin diminished ET-1 mRNA level more potently than forskolin after 16 h (Fig. 6). Because thrombin is thought to induce its effects mainly via a protein kinase C (PKC) pathway ^(35,36), their different signal transduction mechanisms may result in distinct suppression of ET-1 synthesis and gene transcription in VSMCs. TPA, an activator of PKC, diminished ET-1 synthesis in VSMCs at 1 μM by feedback inhibition due to excessive stimulation of PKC (Table 1), but it increased ET-1 synthesis at 10 nM. Ang II, which mainly uses the PKC pathway, elicited a weak increase of ET-1 synthesis in VSMCs. Based on these results, the PKC pathway contributes to the regulation of ET-1 synthesis in VSMCs.

Adrenaline showed weakly stimulatory effects on ET-1 synthesis in VSMCs, but it did not alter ET-1 synthesis in ECs. Although the effect of adrenaline is weaker than that expected from its contribution to the vascular tone regulation, its effects on ET-1 synthesis in VSMCs should be taken into account. FCS increased ET-1 synthesis in cultured rat VSMCs and ECs. TGF-β may be a candidate for the substance directly eliciting this effect, because FCS and TGF-β showed similar dose-response curves. We deduce that the effect of FCS is a consequence of activation of a latent form of TGF-β on the surface of the cells ⁽³⁷⁾.

Aldosterone and hydrocortisone induced no effect on ET-1 synthesis in VSMCs, but they stimulated it in ECs (Table 1). We confirmed the results in several rat ECs and VSMCs (data not shown). Kanse et al. have reported that glucocorticoids enhance ET-1 release from rat and rabbit VSMCs but not from bovine ECs ⁽¹³⁾. The same result was obtained only for bovine ECs (data not shown). The high basal secretion rate of ET-1 from our rat VSMCs may alter response to glucocorticoids. In the report of Kanse et al., hydrocortisone is more potent in ET-1 secretion than dexamethasone, which is unusual based on their affinity for the glucocorticoid receptor ⁽³⁸⁾. Because steroids are the important factors in the basal regulation of vascular tone ⁽³⁹⁾, their effects on ET-1 synthesis should be investigated in more different VSMCs.

Endothelin-1 synthesis in the vascular wall cells is regulated by a variety of substances, as discussed above. Among them, TGF-β markedly stimulates ET-1 synthesis in both rat VSMCs and ECs. Phorbol ester stimulates ET-1 synthesis at low concentrations and inhibits at high concentrations in VSMCs and ECs. Other substances elicited different effects on ET-1 synthesis in VSMCs and ECs. For example, thrombin suppresses ET-1 synthesis in VSMCs but stimulates it in ECs. Based on these findings, ET-1 synthesis in VSMCs and ECs is found to be differently regulated except for TGF-β and phorbol ester, suggesting that ET-1 secreted from VSMCs and ECs may have distinct functions from each other.

As shown in Table 1, only five substances altered ET-1 and AM synthesis in VSMCs in the same direction. Synthesis of AM, a potent vasodilator peptide, in VSMCs was mainly stimulated with TNF-α, IL-1, and LPS ^(40,41), but these substances did not alter ET-1 synthesis. Glucocorticoid augmented AM synthesis in VSMCs, but it did not alter ET-1 synthesis. On the other hand, AM did not alter ET-1 synthesis in VSMCs but slightly enhanced it in ECs, whereas ET-1 weakly stimulated AM synthesis in VSMCs ⁽²²⁾. Thus syntheses of the two peptides exerting opposite activities on the control of vascular tone and cell growth of VSMCs are found to be regulated via different pathways ^(42,43), and these peptides affect their production with each other. The active production and dynamic regulation of ET-1 and AM in VSMCs suggests that the mediators secreted from VSMCs significantly contribute to the regulation of the vascular cell function.

Rat VSMCs secreted IR-ET-1 as a mixture of ET-1 and big ET-1 in a ratio of 1:4, while the ratio of ET-1 to big ET-1 in the medium of rat ECs was 9:1. As big ET-1 has only 1/100 of the biological activity of ET-1 ⁽⁴⁴⁾, most of IR-ET-1 secreted from VSMCs cannot induce significant effects in its secreted form. The ratio of big ET-1 to ET-1 in rat VSMCs and ECs is consistent with that in human VSMCs and ECs ⁽¹⁵⁾, and IR-ET-1 secreted from bovine ECs is mainly composed of ET-1 ⁽⁴⁵⁾. Thus the ratio of ET-1 to big ET-1 secreted from VSMCs or ECs is regulated by the similar system regardless of their origins. We also estimated the ratio of big ET-1 to ET-1 after stimulation with TGF-β. TGF-β increased IR-ET-1 secretion from rat VSMCs and elevated the ratio of big ET-1 to ET-1 about 1.7-fold. This result indicates that the high ratio of big ET-1 in the total IR-ET-1 secreted from the VSMCs is maintained even under the presence of the substances regulating ET-1 production. Taken together, VSMCs are confirmed to secrete IR-ET-1 mainly as an inactive intermediate form.

Big ET-1 is converted into the active ET-1 by ECE, which cleaves between Trp-21 and Val-22. It is possible that big ET-1 secreted from VSMCs diffuses into the EC-VSMC space and is converted into ET-1 by ECE mainly expressed on ECs ^(46,47). In the present culture experiment, big ET-1 was converted into ET-1 only on the surface of ECs (Fig. 7). These findings suggest the possibility that big ET-1 secreted from the VSMCs is converted into ET-1 on the surface of the ECs and then acts on ET receptors of the ECs. If this is the case in vivo, big ET-1 secreted from VSMCs by the different regulatory mechanism may act as a mediator transmitting a signal from VSMCs to ECs. In the future study, the expression levels of ECE in ECs and VSMCs should also be examined to elucidate the physiologic role of the VSMC-derived big ET-1.

In normal blood vessels, a low level of ET-1 is reported to be synthesized in VSMCs ⁽⁴⁸⁾. Once ECs are damaged, however, macrophages and platelets start to aggregate on the surface of the vascular wall and secrete cytokines, which in turn stimulate production of ET-1 in VSMCs. In fact, ET-1 was shown to be actively produced in medial VSMCs of atherosclerotic plaque ⁽⁴⁹⁾, in which VSMCs are transformed into a synthetic phenotype similar to cultured VSMCs, expressing ET receptors ^(10,50). Under the pathophysiologic conditions that growth and differentiation factors are secreted, therefore, big ET-1 and ET-1 secreted from the VSMC are expected to show more contribution to the regulation of vascular remodeling as well as vascular tone.

Endothelin-1 is synthesized and secreted from VSMCs at a lower rate than ECs, but its synthesis in the VSMCs is dynamically regulated by cytokines, growth factors, and vasoactive substances in a manner different from that in the ECs. These findings, supported by differences in the molecular forms of ET-1 secreted from VSMCs and ECs, suggest that ET-1 secreted from VSMCs elicits physiologic functions distinct from those from ECs.

Acknowledgment: The authors are grateful to Dr. K. Kitamura of Miyazaki Medical College for donation of antibody against AM, Dr. Y. Morishita of Kyowa Hakko Kogyo and Dr. T. Iwamoto of this institute for donation of bovine VSMC and rat VSMC, and Dr. T. Sakurai of University of Tsukuba and Dr. T Minegishi of Gunma University for donation of rat ET-1 cDNA and GAPDH cDNA. The authors also express deep gratitude to Ms. M. Nakatani and M. Higuchi for technical assistance. This work was supported in part by the Special Coordination Funds for the Promotion of Science and Technology from the Science and Technology Agency, and by research grants from the Ministry of Education, Science and Culture, and the Ministry of Health and Welfare of Japan.