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Endocrinology Vol. 139, No. 3 838-846
Copyright © 1998 by The Endocrine Society

ARTICLES

Regulation of Adrenomedullin Production in Rat Endothelial Cells1

Yoshitaka Isumi, Hiroki Shoji, Shin Sugo, Tomoko Tochimoto, Masanori Yoshioka, Kenji Kangawa, Hisayuki Matsuo and Naoto Minamino1

National Cardiovascular Center Research Institute (Y.I., H.S., S.S., T.T., K.K., H.M., N.M.), Fujishirodai, Suita, Osaka 565, Japan; and Faculty of Pharmaceutical Sciences (Y.I., M.Y.), Setsunan University, Hirakata, Osaka 573–01, Japan

Address all correspondence and requests for reprints to: Naoto Minamino, Ph.D., National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565, Japan. E-mail: minamino{at}ri.ncvc.go.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adrenomedullin (AM) is a potent vasorelaxant peptide recently identified in extracts of pheochromocytoma. We have found that AM is actively secreted from endothelial cell (EC) and vascular smooth muscle cell (VSMC). To elucidate the function of AM secreted from EC, the effects of 43 substances on secretion of AM from cultured rat EC were examined in this study. We first confirmed that synthesized AM was not stored but constitutively secreted from EC, indicating that the amount secreted could be used as an index of AM synthesis in EC. EC secreted AM at a rate 5.8 times higher than VSMC, and AM gene transcription in EC significantly contributed to the total aortic AM messenger RNA. Tumor necrosis factor, interleukin-1, and lipopolysaccharide augmented AM secretion from EC, showing cooperative effects, which suggests that AM secreted from EC participates in the induction of hypotension in septic shock. Transforming growth factor ß1 and FCS suppressed AM secretion but stimulated endothelin-1 (ET-1) secretion. Thrombin potently stimulated AM secretion from EC but suppressed it from VSMC. Thyroid hormone and phorbol ester increased AM and ET-1 secretion but to a lesser extent. Interferon-{gamma} inhibited AM secretion from EC, whereas oxidized LDL stimulated it. Regulation of AM production in EC is found to be similar to that of VSMC with several exceptions, but AM and ET-1 production in EC are deduced to be controlled independently and by different mechanisms. AM stimulates cAMP production in EC, though receptors expressed on cultured rat EC are not specific to AM but to calcitonin gene-related peptide. Based on these findings, AM production in EC is thought to be regulated by a variety of substances coming from blood and neighboring cells, and the secreted AM is deduced to dilate blood vessels as an endothelium-derived relaxing factor competing with ET-1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BLOOD PRESSURE is controlled by a complex network of various regulation systems because its homeostatic balance is crucial to the maintenance of vital activity. Among these systems, regulation of vascular tone has been shown to be a major system controlling blood pressure (1, 2). Vascular tone is actually regulated by many factors coming from the blood stream and perivascular nerves (3, 4, 5). Endothelial cells (ECs) lining blood vessels have long been regarded as a simple semipermeable barrier between blood and vascular smooth muscle cells (VSMCs). The identification of prostacyclin, nitric oxide (NO), endothelin-1 (ET-1), and C-type natriuretic peptide, however, has established that EC serves as a critical interface between blood and VSMC.

Adrenomedullin (AM) is a potent vasorelaxant peptide of 52 residues recently isolated from human pheochromocytoma tissue (6). This peptide structurally belongs to the calcitonin-gene related peptide (CGRP) superfamily and elicits a potent depressor effect comparable to that of CGRP through direct action on vascular cells (7). Another bioactive peptide, PAMP (proadrenomedullin N-terminal 20 peptide), is generated from the N-terminal of the AM precursor protein and induces hypotensive activity through a distinct mechanism inhibiting norepinephrine release from sympathetic nerve endings (8, 9). We have demonstrated that EC and VSMC actively synthesize and secrete AM. Gene transcription levels of AM in cultured rat EC and VSMC are about 20 and 4 times higher than that of adrenal gland, and rat EC secretes AM at a rate comparable with that of ET-1 (10, 11). The secretion rate of AM from EC is about 5 times higher than that of VSMC, and immunoreactive (IR) AM secreted from EC and VSMC has been verified to be chromatographically and biologically indistinguishable from native AM. Furthermore, AM specific receptors coupled with an adenylate cyclase have been shown to be expressed on both EC and VSMC (12, 13, 14, 15). These findings suggest that AM secreted from EC plays an important role in the regulation of vascular tone.

We have reported that interleukin-1 (IL-1), tumor necrosis factor (TNF), and lipopolysaccharide (LPS), which are major factors inducing septic shock, most potently stimulate synthesis and secretion of AM from VSMC (11, 16). Glucocorticoid, thyroid hormone, and many vasoactive substances are found to alter AM synthesis, constituting a complex regulation system of AM production in VSMC (17, 18). Because ECs are recognized to have a critical function in the regulation of vascular tone through modulating and transmitting information coming from the blood stream to VSMC, we systematically surveyed substances stimulating and inhibiting AM production in cultured rat EC and compared them with those regulating AM production in VSMC and ET-1 production in EC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
BSA (RIA grade), bovine thrombin, dexamethasone (DEX), D-aldosterone, T3, reverse T3, (-)-epinephrine, 8-bromo-cAMP (8-Br-cAMP) and 8-bromo-cGMP (8-Br-cGMP) were purchased from Sigma Chemical Co. (St. Louis, MO). Mouse recombinant interleukin-1{alpha} (IL-1{alpha}), IL-2, IL-3 and human TNF-ß were obtained from Genzyme (Cambridge, MA), and mouse recombinant IL-1ß from Intergen (Purchase, NY). Mouse recombinant TNF-{alpha} and bovine basic fibroblast growth factor (FGF) were products of Boehringer Mannheim (Mannheim, Germany), and human recombinant epidermal growth factor (EGF) was from Austral Biologicals (San Ramon, CA). Murine recombinant interferon-{alpha} (IFN-{alpha}), IFN-ß and rat IFN-{gamma} were purchased from Calbiochem (San Diego, CA). Hydrocortisone, testosterone, (-)-(R)-norepinephrine hydrogen tartrate monohydrate, L-phenylephrine hydrochloride, DL-isoproterenol hydrochloride, 12-O-tetradecanolyphorbol-13-acetate (TPA), forskolin, heparin (sodium salt), and mannitol were obtained from Wako Pure Chemicals (Osaka, Japan). Progesterone, 17ß-estradiol, Triton X-100 and Triton X-305 were products of Nacalai Tesque (Kyoto, Japan). Escherichia coli LPS (serotype 026:B6) was purchased from Paesel + Lorei (Frankfurt, Germany). Oxidized low density lipoprotein (LDL) prepared by incubating LDL with EC was donated by Dr. Shimokado of this institute, and lysophosphatidylcholine (lysoPC) was purchased from Avanti Polar-Lipids (Alabaster, AL). NO generator, NOR-4 (3-{(±)-(E)-Ethyl-2'-[(E)-hydroxyimino]-5-nitro-3-hexenecarbamoyl}-pyridine), was obtained from Dojindo Laboratories (Kumamoto, Japan), and NG-nitro-L-arginine methyl ester (L-NAME) was a product of Biomol Research Laboratory (Plymouth Meeting, PA). Human AM(40–52) and its N-Tyr derivative were synthesized by a peptide synthesizer (Applied Biosystems, 431A) and purified by reverse phase HPLC. Human AM(22–52) and human CGRP(8–37) were products of Peptide Institute (Osaka, Japan). Other peptides were of rat origin and were obtained from Peptide Institute, and rat CGRP-I was used as CGRP. All steroids were first dissolved in ethanol, and then diluted with an incubation medium (DMEM containing 0.01% BSA). T3 and reverse T3 were first dissolved in 0.1 M NaOH, and TPA was dissolved in dimethylsulfoxide and then diluted with the incubation medium. Other substances were dissolved according to the producer’s manuals and diluted with the incubation medium.

Animals and preparation of aorta
The experiments were approved by the local committee on animal experiments and care. Eight-week-old Sprague Dawley rats (Charles River Japan, Yokohama, Japan) were maintained under normal conditions for at least 1 week. LPS (5 mg/kg) or saline was injected through the tail vein 30 min after ip administration of pentobarbital (25 mg/kg, Abbott Laboratories, North Chicago, IL). At 3 h after injection of LPS, subjects were injected ip with pentobarbital (50 mg/kg). The heart was exposed, and cold saline containing heparin (5 U/ml) was infused through the left ventricle. Then, the aorta was cut just above the branching point of the renal artery and washed with saline. Whole thoracic aorta was collected, washed well with HBSS, and associated tissue was completely removed. For EC(-) tissue, ECs were swabbed off with cotton buds after longitudinally dissecting the aorta. Total RNA was extracted by the method described below.

Cell culture
Rat ECs were isolated from thoracic aorta by treatment with 0.25% trypsin, cloned, and maintained on collagen-coated dishes in M199 medium containing 20% FCS (Hyclone, Logan, UT) and acidic FGF (5 ng/ml, Wako Pure Chemicals) of bovine brain origin at 37 C in a humidified atmosphere containing 5% CO2 (10). The cloned rat EC was identified by uptake of fluorescent acetylated LDL (Biomedical Technologies, Stoughton, MA), active production of ET-1, and negative immunostaining with monoclonal anti-{alpha} smooth muscle actin antiserum (Clone 1A4, Sigma). In this experiment, the ECs were used at passage 11–20. Rat VSMCs were prepared by the explant and enzyme dispersion method and maintained as reported previously (11).

Preparation and processing of conditioned medium
ECs, grown to confluence in a collagen-coated 24-well plate (Iwaki Glass, Tokyo, Japan), were washed twice with DMEM and incubated in the incubation medium for 2 h. The media were then replaced with incubation media containing the reagents to be tested, and incubated at 37 C for 3–12 h in a CO2 incubator. After being collected into polypropylene test tubes, the culture media were acidified with acetic acid (final concentration, 0.25 M) containing Triton X-100 (final concentration, 0.002%). The acidified media were then heated at 100 C for 10 min, and lyophilized. The lyophilizates were dissolved in a standard RIA buffer and subjected to RIAs for AM and ET-1. Viability of EC after a 12-h incubation was estimated by trypan blue exclusion assay, and more than 98% of ECs were shown to be viable under the present conditions. Longer incubation without FCS decreased viability of ECs and a relatively high ratio of ECs are reported to start apoptosis after a 24-h incubation (19). On the other hand, we could not add FCS to the incubation media because FCS and BSA altered AM production (Fig. 1Go). For measurement of the effect of FCS, BSA and mannitol, the incubation media were desalted and deproteinized with Sep-pak C18 cartridge (Millipore Corp., Waters Division, Millford, MA) (20).



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  		Figure 1. Effects of BSA and FCS on AM and ET-1 secretion from cultured rat EC. Amounts of IR-AM (closed square) and IR-ET-1 (closed circle) secreted into culture medium were measured after a 12-h incubation in the presence of different concentrations of (A) BSA and (B) FCS. IR-AM and IR-ET-1 contents were measured by specific RIAs and expressed as fmol per 105 cells. Zero on the abscissa indicates plain DMEM without BSA or FCS. Each point represents the mean ± SEM of six separate wells. *, P < 0.01 compared with plain DMEM without BSA or FCS.

 
Measurement of cellular content of AM
ECs were washed twice with PBS, scraped in 1 M acetic acid, and then collected in polypropylene test tubes. After heating at 100 C for 15 min, the cell lysates were sonicated and centrifuged, and the resulting supernatants were condensed with Sep-pak C18 cartridges and subjected to RIA for AM.

RIA for AM and ET-1
Details of the RIA system for AM using antiserum #172-CI-7 against human AM(40–52) have been reported previously (21). N-Tyr derivative of human AM(40–52) was radioiodinated by the lactoperoxidase method, and its N-Tyr-monoiodinated form, isolated by reverse phase HPLC, was used as a tracer. Monoclonal antibody against ET-1 was donated by Prof. Nakao (Kyoto University School of Medicine) and RIA was carried out as reported (22).

RNA blot analysis
ECs, grown to confluence in a 10-cm collagen-coated dish, were washed twice with DMEM and incubated with stimulants in the incubation medium for 12 h. Total RNA was extracted by the acid guanidium thiocyanate-phenol-chloroform method (10). Thoracic aortae collected from LPS or saline injected rats were washed with HBSS. ECs were removed by swabbing, washed well with the same solution, and then total RNA was extracted from two aortae in each group by the same method. After denaturation, RNA was electrophoresed and was then transferred to Zeta probe membrane (Bio-Rad, Hercules, CA). Hybridization and washing of the membrane were carried out as reported (10). EcoRI-BglI complementary DNA (cDNA) fragment of rat AM (nucleotide -153–422), PvuII cDNA fragment of rat ET-1 (nucleotide 246–662), EcoRI-BamHI cDNA fragment of rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (nucleotide 492–799), and inducible nitric oxide synthase (iNOS) cDNA fragment (nucleotide 2685–3345) were used as probes (20). Band intensity was estimated by a Bioimage analyzer (BAS 5000, Fuji Photofilm, Tokyo, Japan) and was compared after correction with GAPDH messenger RNA (mRNA) as an internal standard.

Measurement of cAMP
ECs, grown to confluence in a collagen-coated 24-well plate, were washed with DMEM and incubated in HEPES-buffered DMEM (pH 7.4) containing 25 mM HEPES, 0.01% BSA, and 0.5 mM isobutylmethylxanthine (Nacalai Tesque) for 1 h. The media were then replaced with HEPES-buffered DMEM containing various concentrations of AM, CGRP and their antagonists, and further incubated at 37 C for another 1 h. Aliquots of the culture media were succinylated, lyophilized, and then submitted for RIA of cAMP as reported previously (23).

Receptor binding assay
ECs, grown to confluence in a collagen-coated 6-well plate, were washed twice with 1 ml of 20 mM HEPES-buffered DMEM (pH 7.4), and incubated with 1.85 x 10-11 M of 125I-labeled rat AM in 1 ml of DMEM containing 0.05% BSA and 0.01% Triton X-305 at room temperature for 1 h. Nonspecific binding was determined in the presence of 2 µM of unlabeled rat AM. After incubation, the cells were washed once with ice-cold PBS and solubilized with 0.5 M NaOH. Radioactivity was measured with a {gamma}-counter (ARC-1000, Aloka, Tokyo, Japan). Monoiodinated rat AM with biological activity was prepared by the lactoperoxidase method and purified by reverse phase HPLC.

Statistics
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 < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We first measured cellular contents of IR-AM and the IR-AM accumulated in the culture medium of rat EC after 3-, 6-, and 12-h incubation. The IR-AM content in the medium increased almost linearly up to 12 h, and the rate of secretion of IR-AM from the cultured rat ECs used in this study was 1.57 fmol/105 cells·h as an average over a 12-h incubation. On the other hand, the cellular content of IR-AM was not altered during the incubation and was 0.53 fmol/105 cells, which corresponded to 2.8% of total IR-AM in the culture medium after 12-h incubation. These data indicate that AM synthesized in the cultured rat EC was constitutively secreted from the EC without being stored in the cell. IR-AM content in the culture medium was found to be an index of AM synthesis in rat EC, and we measured it in this study (10). Several other cultured rat ECs we have established showed even higher secretion rates than that used in this study. The secretion rates of 5 VSMCs were 0.42–0.12 fmol/105 cells·h with a mean value of 0.27 fmol/105 cells·h. Thus, AM was shown to be secreted from EC at a rate 5.8 times higher than that from VSMC.

Based on data of regulation of AM synthesis and secretion from cultured rat VSMC (16, 17, 18), we subjected 43 substances to the AM production assay of cultured rat EC. We simultaneously monitored concentrations of IR-ET-1 in the culture medium, which was also synthesized and constitutively secreted from EC. As shown in Table 1Go, 26 out of 43 substances significantly influenced IR-AM content in the medium of ECs after 12-h incubation, i.e. 19 substances increased and 7 substances decreased AM secretion. Interestingly, BSA was found to be one of the most stimulatory reagents, whereas FCS was one of the most inhibitory substances of AM secretion from cultured rat EC, as shown in Fig. 1Go. BSA dose dependently stimulated AM secretion, and the secreted IR-AM level was 2.4 times higher than that of the control. BSA elevated IR-ET-1 content in the medium in low concentrations but strongly reduced it at concentrations higher than 0.25%. We examined the effects of BSA up to 2.5%, but 2.5% BSA induced a stimulatory effect on AM secretion only a little stronger than that of 0.5% BSA. To check whether the effect of BSA on AM and ET-1 secretion from EC was derived from osmotic pressure, mannitol was added to the medium. Although we dissolved mannitol up to a concentration corresponding to 5% BSA, it did not affect secretion levels of AM and ET-1 at all. In contrast with BSA, FCS lowered IR-AM content in the medium to 55% and increased IR-ET-1 content up to 132%. Therefore, we employed a serum-free culture medium containing a low concentration (0.01%) of BSA of the same preparation in this study to avoid interference of FCS and BSA.


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  		Table 1. Effects of various substances on IR-AM and IR-ET-1 production in cultured rat EC

 
Among 41 other substances examined, IL-1{alpha}, IL-1ß, TNF-{alpha}, TNF-ß, and LPS augmented AM secretion from rat EC, as in the case of rat VSMC. IL-1{alpha} and TNF-{alpha} increased IR-AM content in the culture medium about 2.2-fold, and IL-1ß, TNF-ß, and LPS elevated it but to a lesser extent. However, these cytokines and LPS did not significantly alter IR-ET-1 content. As shown in Fig. 2Go, IL-1ß did not increase IR-AM content in the medium at concentrations lower than 0.02 ng/ml but elevated it at concentrations higher than 0.2 ng/ml. LPS induced a significant increase of IR-AM content only at a dose of 20 ng/ml. TNF-{alpha} dose dependently augmented IR-AM content in the medium as well as mRNA levels in the cells, as shown in Fig. 3AGo. TNF-ß also increased IR-AM content in the medium, but not as dose dependently as TNF-{alpha}. IL-1{alpha} significantly elevated the IR-AM level at concentrations between 0.02 and 0.2 ng/ml but did not affect it at other concentrations.



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  		Figure 2. Dose-dependent AM and ET-1 secretion from cultured rat EC stimulated with IL-1ß (A), LPS (B), and TPA (C). IR-AM (closed square) and IR-ET-1 (closed circle) contents in the culture medium of rat EC after a 12-h incubation with different concentrations of IL-1ß, LPS and TPA were measured by specific RIAs and expressed as fmol per 105 cells. Each point represents the mean ± SEM of six separate wells. *, P < 0.01 compared with control incubation medium (0.01% BSA/DMEM).

 


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  		Figure 3. Dose-dependent AM and ET-1 secretion and gene transcription in cultured rat EC stimulated with TNF-{alpha} (A), TGF-ß1 (B), and thrombin (C). (Upper) IR-AM (closed square) and IR-ET-1 (closed circle) contents in the culture medium of EC after a 12-h incubation with different concentrations of TNF-{alpha}, TGF-ß1 and thrombin were measured by specific RIAs and expressed as fmol per 105 cells. (Lower) AM and ET-1 mRNA levels in cultured EC were measured after a 12-h incubation with different concentrations of TNF-{alpha}, TGF-ß1 and thrombin. Ten micrograms of total RNA was electrophoresed, transferred to a nylon membrane, and submitted to hybridization to AM probe, ET-1 probe, and then GAPDH probe, successively.

 
Among cytokines and growth factors, TGF-ß1 exerted a dynamic alteration in both AM and ET-1 secretion. TGF-ß1 was the most potent suppressor of AM secretion, decreasing IR-AM content in the medium to 53% of the control, whereas it was the most potent stimulator of ET-1 secretion, increasing IR-ET-1 content to 237%. The effects of TGF-ß1 on AM and ET-1 secretion were dose dependent, and the changes in mRNA levels of AM and ET-1 also paralleled those of IR-AM and IR-ET-1 content in the culture media, as shown in Fig. 3BGo. Basic FGF and EGF showed weak but significant inhibitory effects on AM secretion but no effect on ET-1 secretion. Among the three IFNs, only IFN-{gamma} showed a weak inhibitory effect, decreasing IR-AM content to 80% of control, and none of the IFNs altered IR-ET-1 content.

Thrombin potently elevated both AM and ET-1 concentrations in the medium up to 283% and 155% of the control, respectively. However, no apparent change was observed in the mRNA band intensity of AM and ET-1 after a 12-h stimulation with thrombin (Fig. 3CGo).

We checked the effects of six steroid hormones, including glucocorticoid, mineralocorticoid, and sex steroids, on AM secretion from EC. All of these steroid hormones increased IR-AM contents in the medium of ECs, although they produced, at most, a 35% elevation compared with the control. On ET-1 secretion, only DEX and hydrocortisone showed significant effects. Thyroid hormone, T3, increased both IR-AM and IR-ET-1 contents in the medium by about 20%, and its inactive isomer, reverse T3, did not elicit a significant effect on either IR-AM or IR-ET-1 level.

In the four catecholamines examined, isoproterenol and norepinephrine slightly but significantly increased IR-AM content in the medium, but none of these catecholamines altered IR-ET-1 content. We also administered six vasoactive peptides, vasoactive intestinal polypeptide (VIP), CGRP, ET-1, atrial natriuretic peptide (ANP), angiotensin II, and substance P, to the medium of EC. CGRP and ET-1 decreased the IR-AM level in the media to about 85% of the control, and VIP decreased IR-ET-1 level to 85% of the control.

NO synthase inhibitor (L-NAME) and NO generator (NOR-4) did not alter IR-AM or IR-ET-1 content in culture medium of rat ECs. On the other hand, oxidized LDL and lysoPC increased IR-AM content in the medium about 55% and 22%, whereas these substances did not elicit a significant effect on IR-ET-1 content.

As shown in Fig. 2CGo, TPA elevated both IR-AM and IR-ET-1 contents in the culture medium of rat ECs in lower concentrations, 10-10–10-9 M for AM and 10-12–10-11 M for ET-1. At concentrations higher than 10-8 M, TPA weakly reduced IR-AM content and markedly lowered IR-ET-1 level. We administered forskolin and 8-Br-cAMP to ECs up to 10-5 M and 10-3 M, respectively, but no significant alteration was observed in IR-AM or IR-ET content in the medium. 8-Br-cGMP did not show any effect on either AM or ET-1 secretion.

We checked the cooperativity of TNF-{alpha}, IL-1ß, and LPS on AM synthesis and secretion from cultured rat EC (Fig. 4Go) because these substances often induce a synergistic effect when added simultaneously. Each of the three substances administered alone increased IR-AM content in the culture medium. Coadministration of IL-1ß and LPS or TNF-{alpha} and LPS almost additively elevated IR-AM content in the culture medium. However, administration of IL-1ß with TNF-{alpha} or with TNF-{alpha} and LPS reduced the increase in IR-AM content in the medium.



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  		Figure 4. Effects of coadministration of TNF-{alpha}, IL-1ß, and LPS on AM secretion from cultured rat EC. Stimulants were added and incubated for 12 h in the following concentrations; TNF-{alpha}, 5 ng/ml; IL-1ß, 0.1 ng/ml; LPS, 5 ng/ml. IR-AM content in the culture medium was measured by specific RIA and expressed as fmol per 105 cells. Each value represents the mean ± SEM of six separate wells. In all cases, a significant increase of AM production (P < 0.01) was observed compared with the control incubation medium.

 
Secreted peptide and mRNA levels of AM were further compared after a 12-h stimulation with DEX and IFN-{gamma} at doses inducing maximal effects on AM secretion (data not shown). DEX induced a 2.4-fold increase in the AM mRNA level compared with the control, and IFN-{gamma} slightly enhanced AM mRNA band intensity. In the case of ET-1 mRNA, DEX and IFN-{gamma} produced relatively large increases (3.2-fold and 2.2-fold) in the mRNA band intensity. These results, as well as those shown Table 1Go and Fig. 3Go, indicate that the level of AM secreted from EC is not well correlated with the AM mRNA level as observed in the case of VSMC (11, 16, 17, 18).

Next, we examined properties of AM receptors expressed on the cultured rat EC used for the present study. As shown in Fig. 5Go, rat CGRP dose dependently elevated cAMP production with an ED50 value of 1.2 x 10-10 M, and AM antagonist, human AM(22–52), did not affect the dose-response curve. In the presence of 10-6 M of CGRP antagonist, human CGRP(8–37), the dose-response curve of CGRP was markedly shifted to the high concentration side and the ED50 value was 200 times higher than that without CGRP antagonist. In contrast, the ED50 value of rat AM in the cAMP production was 5.3 x 10-8 M, being 440 times higher than that of rat CGRP. The dose-response curve of AM was not shifted by addition of human AM(22–52) but was shifted with human CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37). We also checked two other lines of rat EC, both of which showed higher affinity for CGRP than AM, as in the case of Fig. 5Go. We also tried to characterize AM receptors by receptor binding assay using a radiolabeled rat AM purified by reverse phase HPLC. No specific binding of AM to cultured rat EC was observed, although we found significant binding of the ligand and a clear displacement curve in the case of bovine EC under the same conditions used in the present study.



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  		Figure 5. Characterization of receptors expressed on cultured rat EC. cAMP concentration in the culture medium of EC was measured after a 1-h stimulation with a series of different concentrations of rat AM (A) and rat CGRP (B) and expressed as pmol per 105 cells. A, Rat ECs were stimulated with increasing concentrations of rat AM in the absence (closed square) or presence of 1 µM of human AM(22–52) (closed circle) or 1 µM of human CGRP(8–37) (closed triangle). B, Rat ECs were stimulated with increasing concentrations of rat CGRP in the absence (open square) or presence of 1 µM of human AM(22–52) (open circle) or 1 µM of human CGRP(8–37) (open triangle). Each point represents the mean ± SEM of four separate wells.

 
Contribution of AM gene transcription in endothelium to the total AM mRNA level in thoracic aorta was evaluated by RNA blot analysis in LPS-injected rats (Fig. 6Go). At 3 h after injection of LPS (5 mg/kg), the AM mRNA level and iNOS mRNA level in the whole aorta were increased 4-fold and 8-fold compared with that of saline-injected control rats. By removal of ECs by swabbing, the AM mRNA level was reduced to 80% of the nontreated aorta, whereas that of iNOS, which was mainly transcribed in VSMC, was increased to 120%.



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  		Figure 6. AM mRNA and iNOS mRNA levels in rat aorta with or without EC. Thoracic aortae of control (saline-injected) and LPS-injected (5 mg/kg) rats were collected 3 h after injection and immediately frozen with liquid nitrogen. In EC(-) group, ECs were swabbed off and washed before freezing. Experiments were performed in two separate groups, Exp 1 and Exp 2. Total RNA was extracted from two aortae, and 10 µg of it was denatured and electrophoresed on each lane. RNA was transferred to a nylon membrane and submitted to hybridization to AM probe, iNOS probe and GAPDH probe, successively. Data are averages of two experiments. Band intensity was measured by BAS 5000 Bioimage analyzer.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cultured rat EC was shown to actively synthesize and secrete AM. The rate of secretion of AM was confirmed to be 5.8 times higher than that of cultured rat VSMC in this study (10, 11). The level of gene transcription of AM in the cultured EC was 20 times higher than that of adrenal gland in the rat, and total weight of ECs in the normal human body is estimated to be more than 1 kg (10, 24). These facts suggest that ECs are the major cells synthesizing and secreting AM. To evaluate the contribution of EC to total AM production in the vascular wall, we measured AM and iNOS mRNA levels in thoracic aorta with or without endothelium obtained from LPS-injected rats. Removal of endothelium clearly reduced the AM mRNA level, but relatively increased the iNOS mRNA level mainly expressed in the VSMC (Fig. 6Go). This result indicates that AM gene transcription in EC significantly contributes to the total gene transcription in the thoracic aorta, although the mass of EC is less than a few percent of the aorta. Based on these findings, EC should be recognized as a major source of AM even in the in vivo system.

In this study, we measured IR-AM content in the culture medium of ECs after a 12-h incubation with various substances as an index of AM synthesis. To elucidate physiological functions of AM in the vascular wall, it is necessary to know how AM production in EC is regulated, even though AM content in the medium is not always altered by direct action of the administered substance during a 12-h incubation. We also compared the regulation of AM production in EC to that in VSMC as well as to the regulation of ET-1 production in EC to help elucidate the functions of AM secreted from EC.

IL-1s, TNFs, and LPS, which are known to be major factors inducing septic shock, significantly increased AM mRNA levels and AM secretion from EC (Figs. 2Go and 3Go), although not as potently as in VSMC (16). ET-1 secretion was not altered by these inflammatory cytokines or by LPS. Coadministration of TNF-{alpha} and LPS or IL-1ß and LPS additively increased AM content in the culture medium of rat ECs, but the effect of TNF-{alpha} was reduced in the presence of IL-1ß (Fig. 4Go). This result indicates that AM synthesis in EC stimulated with LPS is augmented by the addition of TNF-{alpha} or IL-1ß, although their cooperativity in EC is not as high as that observed in VSMC (16). Thus, AM synthesis in the vascular wall is augmented with TNF, IL-1 and LPS, and the secreted AM appears to bind receptors on VSMC and effectively dilate blood vessels.

We have demonstrated that plasma concentration and gene transcription of AM are highly elevated in LPS-injected rat (20). We have also found that plasma AM concentration in patients with septic shock is markedly increased and correlates negatively with systemic vascular resistance index (25). Hirata et al. also reported high levels of plasma AM in septic patients (26). These findings, along with the in vitro data obtained from cultured rat EC and VSMC, indicate that an increase in plasma AM concentration in LPS-injected rats and patients with septic shock results from augmented synthesis and secretion of AM from EC and VSMC. NO is recognized as a major substance inducing hypotension in septic shock (27, 28), but the results mentioned above support the hypothesis that AM generated from EC and VSMC participates in the refractory vasodilation of sepsis.

As shown in Fig. 2CGo, TPA elicits dual regulation of AM synthesis, i.e. stimulation at lower and inhibition at higher concentrations. Because long exposure to a high concentration of TPA is known to inhibit protein kinase C through a feedback mechanism (29), its dual effects are thought to be induced through the protein kinase C pathway. AM synthesis in rat VSMC is also stimulated at low concentrations of TPA, and AP-1 and AP-2 sites are present in the 5'-regulatory region of human AM gene (30). The protein kinase C pathway participates in the regulation of AM gene transcription to some extent. On the other hand, forskolin and cAMP analog failed to alter AM synthesis in EC, in contrast to VSMC where these reagents suppressed AM synthesis (18). Although cAMP responsive element is present in the 5'-upstream region of human AM gene (30), the cAMP-mediated pathway may not contribute to the activating factor for AM gene transcription in the vascular wall cells. CGRP, which also increased intracellular cAMP production, weakly suppressed AM synthesis. This result suggests that the suppressive effect of CGRP is not caused simply by the cAMP-mediated pathway.

TNF-, IL-1-, and LPS-induced activation of gene transcription is considered to be mainly mediated by transcriptional factor NF-{kappa}B (31). In the 5'-upstream region of human AM gene, there are several sites that differ from the 10-base consensus sequence of NF-{kappa}B binding site by one base replacement (30, 32). In the case of VSMC, TNF, IL-1, and LPS exert their effect additively, suggesting that these three substances activate AM gene transcription through the same pathway. TNF-{alpha}-induced AM synthesis is suppressed by IL-1ß in EC, although LPS showed cooperative effects with both TNF-{alpha} and IL-1ß (Fig. 4Go). Because TNF-{alpha}-induced gene transcription is reported to be inhibited by IL-1 in several proteins of rat and human EC (33), AM synthesis stimulated with IL-1ß and TNF-{alpha} in EC might be mediated through different pathways. We administered several different inhibitors of the signal transduction, such as H-7, to identify the pathways used for activation of AM gene transcription stimulated with TPA and cytokines but were not able to observe significant effects on AM content in the medium probably due to cytotoxicity of the inhibitors.

TGF-ß1 was found to be the most potent suppressor of AM synthesis, whereas it is the strongest stimulator of ET-1 synthesis (Fig. 3BGo, Table 1Go). TGF-ß1 has also been reported to increase production of EC-derived vasodilators, prostacyclin and C-type natriuretic peptide (34, 35). These findings indicate that TGF-ß1 is a factor regulating not only growth and differentiation of vascular cells but also vascular tone through alteration of production of vasoactive substances. Because TGF-ß1 is a potent stimulator of ET-1 synthesis and secretion, it is possible that the secreted ET-1 secondarily elevates AM synthesis in EC. However, the concentration of ET-1 was 2 x 10-10 M after a 12-h incubation, which was too low to stimulate AM synthesis.

FCS potently decreased AM content and increased ET-1 content in the culture medium of ECs in a manner similar to that of TGF-ß1 (Fig. 1Go), and no other substances induced comparable effects. Although plasma TGF-ß1 concentration (ca. 0.1 ng/ml) is not sufficient to induce an inhibitory effect, a latent form of TGF-ß1 is expressed on EC (36, 37). Because plasminogen in FCS is activated by plasminogen activator secreted from EC, the activated plasmin can process the latent form to mature TGF-ß1 (37). These findings suggest that the suppression of AM synthesis with FCS is induced by TGF-ß1 generated under the culture conditions. The strong inhibitory effect of FCS partly explains why AM gene transcription is highly suppressed in the in vivo conditions observed in intact aorta (10). On the other hand, no substance has been found to elicit effects comparable to BSA that markedly elevated AM content and reduced ET-1 content in the medium of EC. We also administered higher concentrations of BSA, up to 2.5%, which elicited effects comparable to or a little stronger than those observed for 0.5% BSA. On the other hand, osmotic stress induced with mannitol, which corresponded to 5% BSA, did not alter AM secretion from EC at all. Because serum albumin is reported to inhibit prostacyclin synthesis in EC (38), it is necessary to determine whether the effects are induced by BSA itself or by another associated substance which may alter synthesis of vasoactive peptides.

EGF and basic FGF weakly suppressed AM secretion (Table 1Go). But their effects might be indirect because lower doses of these substances do not induce any effect. IFN-{gamma} showed a suppressive effect on secretion of AM from both EC and VSMC. In the 5'- upstream region of human AM gene, there are many {gamma}-IFN responsive elements (30), but these do not affect AM gene transcription in either EC or VSMC, in contrast with iNOS gene which has {gamma}-IFN responsive elements and is highly activated with IFN-{gamma} in the presence of LPS (39). Thrombin is a potent stimulant of AM and ET-1 synthesis in EC. This is in sharp contrast to VSMC, where thrombin most potently inhibits AM synthesis (18). Thrombin is known to work as a proinflammatory agent by promoting the synthesis of IL-8 and other cytokines (40). Stimulatory effects of thrombin on AM and ET-1 synthesis in EC may be induced not as specific but general reactions of EC against thrombin. Thrombin acts on EC and elicits its effect through the EC-mediated pathway in the normal vascular wall, but it acts on VSMC when the vascular wall is wounded. Thus, the resulting effect of thrombin on AM production in the vascular wall is deduced to be different between normal and wounded blood vessels.

All steroid hormones weakly stimulate AM secretion from EC, although glucocorticoid specifically augments it from VSMC (17). Based on their nonspecific effects, steroid hormones might influence AM synthesis in EC by secondary reaction. In contrast, thyroid hormone is found to increase synthesis and secretion of AM and ET-1 through specific receptors. Imai et al. (41) reported specific and potent effects of glucocorticoid on AM gene transcription in EC but observed no specific stimulation of steroid hormones in this study. Because isoproterenol and norepinephrine slightly stimulated AM secretion, their effects may be mediated by ß-receptor.

In the vasoactive peptides, ET-1 weakly suppressed secretion of AM from EC. Suppression of AM secretion by ET-1 might strengthen the effect of ET-1 secreted from EC. ANP did not alter AM and ET-1 synthesis at all. Vesely et al. (42) reported that ANP infusion elevated plasma AM concentration within 20 min in healthy human subjects, but its time dependency was quite different from that observed when AM synthesis in EC and VSMC was stimulated (16, 17). Their results suggest that AM stored in the endocrine-type cells is secreted into the blood by ANP infusion. Based on the results of regulation of AM synthesis and secretion, EC and VSMC cannot be the source of the transient increase of plasma AM. Gene transcription of iNOS and nitrite production have recently been reported to be stimulated by AM (43, 44). We administered an NO synthase inhibitor and generator to EC vice versa, but no significant effect was observed on basal AM nor on ET-1 production in rat EC. As for ET-1 production in EC, Cao et al. (45) reported that L-NAME increased ET-1 secretion from human umbilical vein EC (45), but we observed no stimulatory effect of L-NAME on ET-1 secretion from human aortic EC (our unpublished observation).

Oxidized LDL increased AM secretion 58%, which was lower than the effect of IL-1{alpha} but higher than that of IL-1ß. LysoPC, a phospholipid fraction of LDL, also stimulated AM secretion. Oxidized LDL is a major substance inducing atherosclerosis and stimulates secretion of growth factors and cytokines that proliferate VSMCs (46). PDGF- or FCS-stimulated growth and migration of VSMC were reported to be inhibited by AM (47, 48), and we also obtained similar results (our unpublished observation). AM secreted from EC by the stimulation of oxidized LDL and cytokines may exert antiproliferative effects on VSMC in the atherosclerotic region.

When regulation of AM production in EC is compared with that in VSMC, most of the substances influence AM production in the same direction, even though their relative potencies are smaller in EC. For example, substances inducing septic shock, such as IL-1, TNF and LPS, potently stimulate AM synthesis and secretion from both EC and VSMC. Steroid and thyroid hormones as well as phorbol ester increase AM synthesis in EC and VSMC, whereas IFN-{gamma} suppresses it. Typical differences in AM synthesis between EC and VSMC are induced by FCS, thrombin, FGF, EGF, and ET-1. FCS as well as FGF, EGF and ET-1 stimulate AM synthesis in VSMCs and inhibit it in ECs, whereas thrombin enhances AM synthesis in ECs and reduces it in VSMCs. Because these substances have proliferative effects on VSMCs in addition to regulating the production of vasoactive peptides, their activity should be evaluated based on short-term vascular tone regulation as well as long term vascular cell growth. The difference in regulation of AM production between EC and VSMC provides a clue to elucidating functions of AM secreted from EC and VSMC.

Among substances which significantly altered either AM or ET-1 synthesis in EC, only thrombin, thyroid hormone and TPA stimulated both AM and ET-1 synthesis. Thyroid hormone receptor-mediated pathway and protein kinase C pathway are probably shared with AM and ET-1 in rat EC. Most of the other substances induce distinct effects on AM and ET-1 synthesis in EC, as typically observed in TGF-ß1 which stimulates ET-1 synthesis but suppresses AM synthesis. Based on these data, AM and ET-1 synthesis in EC is deduced to be generally regulated by independent and different mechanisms. The two vasoactive peptides with opposing biological activity are deduced to be produced in EC by distinct regulation and to participate in the control of vascular tone as competitors.

AM-specific receptors are shown to be expressed on cultured human and bovine ECs (12, 13). As shown in Fig. 5Go, however, presumed AM receptor on rat EC is found to be specific to CGRP but not to AM on the basis of its affinity and behavior to antagonists. Several different rat ECs, which actively synthesize and secrete AM and ET-1, also showed high affinity for CGRP. Because cAMP producing properties of EC receptors are quite similar to those reported for CGRP type-I receptor (49), CGRP receptor is concluded to be a major receptor expressed on cultured rat EC. If this is true in the intact blood vessel, AM is able to function as an EC-derived vasorelaxant but not as an autocrine regulator of EC in rat.

In conclusion, AM production in rat EC is regulated by many cytokines, hormones, and vasoactive substances. Most of the substances regulate AM production in EC and VSMC in the same direction, but AM production and ET-1 production in EC are regulated independently and differently. ECs significantly contribute to total AM production in the vascular wall even in the aorta, and secreted AM participates in the regulation of vascular tone as an endothelium-derived relaxing factor competing with ET-1.


   Acknowledgments
 
The authors are grateful to Dr. K. Kitamura and Prof. T. Eto of Miyazaki Medical College for discussion and kind donation of antisera against AM and cAMP, and Ms. M. Nakatani and M. Higuchi of this institute for technical assistance. We also express our gratitude to Prof. K. Nakao of Kyoto University School of Medicine, Dr. T. Sakurai of University of Tsukuba School of Medicine, Dr. T. Minegishi of Gunma University School of Medicine and Dr. K. Shimokado of this institute for kind donation of antisera against ET-1, rat ET-1 cDNA, rat GAPDH cDNA probe, and oxidized LDL.


   Footnotes
 
1 This work was supported in part by Special Coordination Funds for the Promotion of Science and Technology from the Science and Technology Agency (Encouragement System of C.O.E.), and by research grants from the Ministry of Health and Welfare, the Ministry of Education, Science and Culture, and the Human Science Foundation of Japan. Back

Received July 7, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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