Thrombin stimulates the production of immunoreactive endothelin-1 in cultured human umbilical vein endothelial cells

Metabolism Clinical and Experimental OCTOBER

VOL 39, NO 10

PRELIMINARY

Thrombin

Masakazu

1990

REPORT

Stimulates

the Production of Immunoreactive Human Umbilical Vein Endothelial

Kohno, Kenichi Yasunari,

Koji Yokokawa,

Miiko Fuzisawa,

Koh-ichi Murakawa,

Takatoshi

Endothelin-1 Cells

in Cultured

Takeshi Horio, Yoshiharu Kanayama,

Inoue, and Tadanao

Takeda

The present study was designed to examine the effect of thrombin on endothelin production by cultured human umbilical vein endothelial cells (HIJVEC) by radioimmunoassay. Cultured HUVEC released immunoreactive endothelin-1 (irendothelin-1) into the medium in a time-dependent manner. This release was inhibited completely by 10 pg/mL cycloheximide and is, therefore, directly related to de novo protein synthesis. Further endothelin release was stimulated by the addition of thrombin to the culture medium. Thrombin-induced ir-endothelin-1 release also was inhibited completely by 10 /.rg/rnL cycloheximide. These results suggest that HUVEC in culture produce ir-endothelin-1 slowly, but continuously, and that this production is stimulated by thrombin. @ 1990 by W.B. Saunders Company.

HE ROLE OF THE vascular endothelium in the regulation of vascular smooth muscle tonus has been the focus of much interest in recent years. Previous studies have described protease-sensitive vasoconstrictor activity in the supernatant of cultured endothelial cells (EC).‘.’ Recently, Yanagisawa et al isolated an endothelium-derived, 21residue, vasoconstrictive peptide, endothelin, from the culture supernatant of porcine aortic EC, determined its amino acid sequence, and molecularly cloned the peptide precursor.4 This peptide has potent and long-lasting vasoconstrictor activity and is closely associated with Ca2+ influx.435 Recently, we have shown that the intravenous infusion of synthetic endothelin- 1 causes a characteristically long-lasting elevation in blood pressure and a marked reduction in renal blood flow in rats.6 These observations strongly suggest that endothelin plays a role in the regulation of vascular tonus. In the present study, we investigated the effects of thrombin on immunoreactive endothelin- 1 (ir-endothelin- 1) production by cultured human umbilical vein endothelial cells (HUVEC) using a radioimmunoassay.’

T

MATERIALS AND METHODS EC Culture

HUVEC were obtained in culture by the collagenase treatment of umbilical veins and were grown to confluence in medium 199 containing 20% fetal calf serum (FCS), EC growth supplement (20 pg/mL), penicillin (I 00 pg/mL), and streptomycin (50 pg/mL). EC were identified by typical phase contrast “cobblestone” morphology Metabolism, Vol39,

No 10 (October), 1990: pp 1003-1005

and by immunofluorescence to factor VIII antigen. Cultures were maintained at 37°C under 95% air and 5% CO,, and the subcultures were performed after treatment with Versene followed by trypsin.

Materials

and Supplies

Cycloheximide, angiotensin II, vasopressin, and EC growth supplement were purchased from Sigma Chemical (St Louis, MO). Medium 199, trypsin, Versine, and FCS were purchased from Gibco Laboratories (Grand Island, NY). Flasks were purchased from Becton Dickinson (Oxnard, CA). A gift of purified human thrombin was provided by Sankyo (Tokyo, Japan). Synthetic endothelin-1, antibody to endothelin-1, somatostatin, @-endorphin, human secretin, and synthetic a-human atrial natriuretic peptide were purchased from Peptide Institute (Osaka, Japan). All other materials were obtained as described previously.’

Pharmacologic

Treatment

Tissue culture flasks (25~cm* style) that contained confluent EC culture (1.5 to 2.0 x lo4 cells/cm’) in passages five to eight were

From the First Department of Internal Medicine, Osaka City University Medical School, Asahimachi, Abeno-ku, Osaka, Japan. Supported by a Grant-in-Aid for Scientific Research (No. 6148210) from the Ministry of Education, Science and Culture. Japan. Address reprint requests to Masakazu Kohno, MD, The First Department of Internal Medicine, Osaka City University Medical School, i-5-7, Asahimachi, Abeno-ku, Osaka 545, Japan. @ 1990 by W.B. Saunders Company. 0026-0495/90/391 O-0001 $3.00/O

1003

1004

KOHNO ET AL

used in this study. The culture medium was removed, and the cell monolayers were washed twice with serum-free medium 199. Drugs were added to the medium in a volume of less than 0.2% of the medium, and both control and treated cultures were incubated for 3, 10, or 20 hours at 37OC.All experiments were performed in 5 mL of serum-free medium 199. After incubation, the medium was aspirated and the residua centrifuged at 1,000x g for 10 minutes. Then the supernatant was collected and stored at -8OOC until radioimmunoassay.

Extraction of ir-Endothelin-I Four milliliters from each sample were diluted with 4 mL of 4% acetic acid. After centrifugation, the solution was pumped at a rate of 1 mL/min through a Sep-Pak C,, cartridge (Water Associates, Milford, MA). After washing with 5 mL of water, the adsorbed peptides were eluted with 86% ethanol in 4% acetic acid and evaporated by a centrifugal evaporator (Model RD-31, Yamato Scientific, Tokyo, Japan). The dry residue was redissolved in assay buffer. The recovery rate was calculated by adding four different quantities of cold endothelin-1 (25 pg/mL, 50 pg/mL, 100 pg/mL, and 500 pg/mL) to serum-free medium 199. The recovery rate was 61.7% t 6.5%. Radioimmunoassay The ir-endothelin-1 concentration was determined using antibodies against synthetic endothelin-1 and ‘ZSI-endothelin-l as the tracer. This peptide did not cross-react with somatostain, &endor-

3hours

lOhours

2Ohours

phin, human secretin, angiotensin II, vasopressin, or a-human atria1 natriuretic peptide. Endothelin-1 was radioiodinated by the chloramine T method. Briefly, 5 ag of endothelin-1 was iodinated using 0.5 mCi Na “‘1 in the presence of 10 g chloramine-T in 40 L phosphate buffer for 15 seconds, followed by the addition of 20 pg sodium metabisulfite in 10 rL buffer. The mixture was loaded into a column of Sephadex G25. Labeled endothelin-1 was repurified with a column of CM Sephadex C-25 and eluted with Tris/HCl buffer. The radioimmunoassay was performed in assay buffer of 0.01 mol/L sodium phosphate, pH 7.4, containing 0.05 mol/L NaCl, 0.1% bovine serum albumin (BSA), 0.1% Nonidet NP-40 and 0.01% NaN,. Rehydrated antiserum (100 pL) was added to 100 rL of the sample or to 100 ,uL of the endothelin- 1 standard prepared in assay buffer and incubated for 24 hours at 4OC. Approximately 15,000 cpm ‘r51-endothelin-1 was added to each reaction, and incubation was continued for an additional 24 hours. After the second 24-hour incubation, 100 pL of diluted normal rabbit serum and 100 NL of diluted goat anti-rabbit IgG serum were added and incubated for another 24 hours. After the third 24-hour incubation, the precipitate was collected by centrifugation at 1,700 g for 30 minutes. The supernatant was removed by aspiration, and the pellet was counted for ‘*‘I using a gamma counter. The effective range of the standard curve was between 20 and 2,000 pg of endothelin-1 per assay tube-50% intercept, 350 pg of endothelin-1. The interassay variation was 13.1%, and the intraassay variation was 6.3%. After the extraction, medium 199 with or without thrombin or cycloheximide did not interfere with the ratioimmunoassay.

20hours p< 0.05

lOhours I

p< 0.05 1 r pco.05 pco.05 1 I n p
CON THR THR THR THR O.llJ/d lu/ml IOU/d + CYC

p
CON THR THR THR 0.1lJ/ml W/ml Mu/ml

Fig 1. (A) Release of ir-endothelin-1 by cultured HUVEC. Confluent monolayers were washed twice with serum-free medium 199 and incubated with g ml serum-free medium 199 alone (CON) or serum-free medium 199 containing 10 pg/mL cycloheximide (CYC). The number of tissue culture flasks is shown in perenthesis. and the bar shows the SD. SDL, below detection limit (cl6 pg/mLI. (61 Effect of thrombin (10 U/mL, 1 U/mL, and 0.1 U/mL) on the release of ir-endothelin by cultured HUVEC. EC were incubated with 6 mL serum-free medium 199 alone (CON), serum-free medium 199 containing 10 U/mL thrombin (THR, 10 U/mL), 1 U/mL thrombin (THR, 1 U/mL) 0.1 U/mL thrombin (THR, 0.1 U/mL). or serum-free medium 199 containing 10 U/mL thrombin and 10 gg/mL cycloheximide (THR + CYC). Incubation was for 10 or 20 hours. Each column represents the mean of seven flasks; the bar shows the SD. SDL. below detection limit (<16 pg/mL).

ENDOTHELIN PRODUCTION BY HUMAN

1005

ENDOTHELIUM

Calculations and Statistical Analysis

The statistical significance of the results was evaluated by an ANOVA, and Pvalues were determined by Sheffe’smethod.‘Values are expressed as the mean + SD. RESULTS

ir-Endothelin-1 Release by Cultured HUVEC

Confluent cultures of HUVEC released ir-endothelin into the culture medium in a time-dependent manner, although the amount of additional ir-endothelin-1 released after 3 hours of incubation was below the detectable Iimit (~15 pg/mL) (Fig IA). This release was inhibited completely when the cells were incubated in the presence of 10 rg/mL cycloheximide. Effect of Thrombin on ir-Endothelin-I Release

The addition of thrombin resulted in a significant increase in the accumulation rate of ir-endothelin-1 in the culture medium (Fig 1B). The response to thrombin stimulation was essentially dose-dependent. Incubation of HUVEC with 10 U/mL thrombin in combination with 10 wg/mL cycloheximide did not result in the release of ir-endothelin into the culture medium above the level of detection (~15 pg/mL). DISCUSSION

The present study showed that cultured HUVEC release ir-endothelin-1 into the culture medium in a time-dependent manner. This release was completely inhibited by the protein synthesis inhibitor cycioheximide, and therefore, is related to do novo protein synthesis. These findings are consistent with the previous report by O’Brien et al3 that bioassayable constrictor activity increased progressively over 12 hours of incubation with serum-free medium, and cycloheximide abolished the production of constrictor activity of EC. More

recently, Schini et al” have shown that cultured porcine EC release endothelial-1 in a time-dependent manner. These observations suggest that cultured EC release endothelin- 1 continuously. However, this finding does not necessarily mean that produced endothelin is released into the general circulation and acts as a circulating hormone in vivo. In fact, cultured EC may more closely approximate a state of “injury.“” Our experiment showed that thrombin stimulates the production and/or release of ir-endothelin-1 from cultured HUVEC. This finding involving human tissue confirms data obtained using cultured porcine and bovine EC.“+” These observations seem compatible with the recent report4 that the level of preproendothelin mRNA increases significantly within 1 hour of the addition of 2 U/mL thrombin. Stimulated release of endothelin-1 by the clotting enzyme thrombin from EC may play a role in the pathogenesis of vasospasm associated with the activation of the coagulation cascade. On the other hand, thrombin is known to stimulate the synthesis of the potent vasodilatory prostaglandin, prostacyclin,‘3*‘4 and endothelium-derived relaxing factor (EDRF).15 Therefore, endothelin and prostacyclin or EDRF release may interact to maintain vascular homeostasis. Although the exact mechanism of endothelin production and release by human endothelium remains to be clarified, our experiment shows clearly that human EC can generate vasoconstrictor signals via endothelin production. Further studies will be necessary to establish the nature of in vivo production in the endothelium and to determine whether this peptide is released into the general circulation and acts as a circulating hormone, or acts as a local autacoid on the subjacent smooth muscle cells. ACKNOWLEDGMENT We wish to express our gratitude to Machiko Johchi for her

technical assistance.

REFERENCES

1. Hickey KA, Rubanyi Cl, Paul RJ, et al: Characterization of a coronary vasoconstrictor produced by cultured endothelial cells. Am J Physiol248:C550-556, 1985 2. Gillespie MN, Owasoyo JO, Mcmurtry IF, et al: Sustained coronary vasoconstriction provoked by a peptidergic substance released from endothelial cells in culture. J Pharmacol Exp Ther 236:336-343, 1986 3. O’Brien RF, Robbins RJ, McMurtry IR: Endothelial cells in culture produce a vasoconstrictor substance. J Cell Physiol 132:263270.1987 4. Yanagisawa M, Kurihara H, Kimura S, et al: A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332:41 l-415, 1988 5. Lippton H, Goff J, Hyman A: Effects of endothelin in the systemic and renal vascular beds in vivo. Eur J Pharmacol 155:197199,1988 6. Yokokawa K, Kohno M, Murakawa K, et al: Acute effects of endothelin on renal hemodynamics and blood pressure in anesthetized rats. Am J Hypertens 2715-171, 1989 7. Kohno M, Murakawa K, Yasunari K, et al: Prolonged blood pressure elevation after endothelin administration in bilaterally nephrectomized rats. Metabolism 38:712-713, 1989

8. Yasunari K, Kohno M, Balmforth A, et al: Effect of glucocorticoid on DA-l dopamine receptors on vascular smooth muscle cells. J Hypertens 6:1023-1028, 1988 9. Wallenstein S, Zucker CI, Fleiss JL: Some statistical methods useful in circulation research. Circ Res 47: 1-9, 1980 10. Schini VB, Hendrickson H, Heublein DM, et al: Thrombin enhances the release of endothelin from cultured porcine aortic endothelial rats. Eur J Pharmacol 165:333-334, 1989 11. Emori T, Hirata Y, Ohta K, et al: Secretory mechanism of immunoreactive endothelin in cultured bovine endothelial cells. Biochem Biophys Res Commun 160:93-loo,1989 12. Ross R: The pathogenesis of atherosclerosis-An update. N Engl J Med 314:488-500, 1986 13. Weksler BB, Ley SC, Jaffe EA: Stimulation of endothelial cell prostacyclin production by thrombin, trypsin, and the ionophore A23 187. J Clin Invest 62:923-930, 1978 14. Hong SL: Effect of bradykinin and thrombin on prostasyclin synthesis in endothelial cells from calf and pig aorta and human umbilical vein. Thromb Res 18:787-795, 1980 15. Furchgott RF: Role of endothelium in response of vascular smooth muscle. Circ Res 52:557-573, 1983