Histamine release from Weibel-Palade bodies of toad aortas induced by endothelin-1 and sarafotoxin-S6b.код для вставкиСкачать
THE ANATOMICAL RECORD 242:374-382 (1995) Histamine Release From Weibel-Palade Bodies of Toad Aortas Induced by Endothelin-1 and Sarafotoxin-S6b YOSHIAKI DOI, TAKATOSHI OZAKA, MASATSUGU KATSUKI, HIROSHI FUKUSHIGE, EIICHIRO TOYAMA, YOSUKE KANAZAWA, KEIICHI ARASHIDANI, AND SUNAO FUJIMOTO Departments of Anatomy (Y.D., T.O., M.K., H.F., Y.K., S.F.) and Surgery (E.T.) and Section of Occupational Hygiene, School of Nursing and Medical Technology (K.A.), University of Occupational and Environmental Health, School of Medicine, Kitakyushu, Japan ABSTRACT Background: Endothelin-1 (ET-1) and sarafotoxin-S6b (STX) induce a remarkable degranulation of Weibel-Palade (WP) bodies prior to the vasocontraction of toad aortas. As WP bodies play the role of a reservoir site of the histamine in the endothelial cells, there is the possibility that ET-1 and STX evoke the release of histamine from W P bodies of this vessel. Methods: Histamine concentrations were assayed by high-performance liquid chromatography (HPLC) from the perfusate after being perfused with a solution containing ET-1 and STX. Each vessel was fixed and embedded for conventional electron microscopy and immunoelectron microscopy using antihistamine sera. Results: The appreciable concentrations of histamine were assayed by HPLC from the perfusate after the toad aortas were perfused with a solution containing ET-1 and STX. The immunoelectron microscopy revealed that histamine immunoreactive gold particles in the W P bodies remarkably decreased in number in the treated samples when compared to the control ones. Our immunoelectron micrographs indicated that the release of histamine from the endothelial cells occurred in association with the degranulation and the exocytosis of the W P bodies after treatment with ET-1 and STX. Conclusions: The present study clearly shows that ET-1 and STX induce the histamine release from W P bodies of the toad aortas by means of HPLC and immunoelectron microscopy. Histamine discharged from the W P bodies may be involved in the vasocontraction evoked by ET-1 and STX. 0 1995 Wiley-Liss, Inc. Key words: Chromatography, Endothelial cell, Endothelin, Histamine, Immunoelectron microscopy, Sarafotoxin, Toad aorta, WeibelPalade body Since Weibel and Palade (1964) first described the membrane-bound osmiophilic granules in the rat small artery, which are now called Weibel-Palade (WP) bodies, their nature and function have been analysed by biochemical, physiological, and morphological approaches. Burri and Weibel (1968) have suggested that WP bodies may contain a “procoagulative substance,” and it has also been revealed that WP bodies are a storage site of the von Willebrand factor (vWf), which has an important role in the adhesion of blood platelets to the endothelium after vascular injuries (Wagner et al., 1982; Kagawa and Fujimoto, 1987). GMP-140, an intracellular granule membrane protein first identified in the platelet a-granules (Stenberg et al., 1985),is also localized on the limiting membrane of WP bodies (Bon0 1995 WILEY-LISS, INC. fanti et al., 1989; McEber et al., 1989). Furthermore, Hattori et al. (1989) reported the co-existence of vWf and GMP-140 on WP bodies by immunofluorescent microscopy. In contrast, the involvement of WP bodies in hypertensive activities has been proposed by Bertini and Santolaya (1970). Our previous chromatographic studies suggested the presence of histamine in WP bodies in the endothelial cells of both the toad aortas (Fujimoto, 1982a; Fujimoto et al., 1984) and the rabbit umbilical Received June 21, 1994; accepted December 18, 1994. Address reprint requests to Dr. Yoshiaki Doi, Department of Anatomy, University of Occupational and Environmental Health, School of Medicine, Kitakyushu 807, Japan. HISTAMINE RELEASE FROM WP BODIES 375 Fig. 1. The endothelial cells (EC) contain an abundance of WP bodies (arrows), and the perfusion with an OR-2 solution only does not result in any significant ultrastructural changes such as swelling and degranulation of WP bodies. X 15,000. vein (Fujimoto e t al., 1982b). After treatment with compound 48/80, a liberator of histamine, WP bodies of the toad aortas show a remarkable degranulation, and a n appreciable concentration of histamine was assayed from the perfusate (Fujimoto et al., 1984). This suggests that WP bodies are a storage site of histamine. Moreover, Ueda et al. (1992) revealed a simultaneous localization of histamine and vWf on WP bodies of the human umbilical vein using double-labeling immunoelectron microscopy. Recently, endothelin (ET) was purified from the medium of cultured endothelial cells and has been reported to possess a potent vasoconstrictive activity (Yanagisawa et al., 1988). ET consists of three isopeptides, ET-1, ET-2, and ET-3 (Inoue et al., 1989), and these peptides have a close similarity in amino acid residues and in biological actions to sarafotoxins (STX) purified from snake venom (Takasaki et al., 1988; Kloog et al., 1988; Hirata et al., 1989). Among these peptides, ET-1 and STX-S6b induce vasocontraction to both endothelium-preserved and denuded toad aortas and evoke a remarkable degranulation of the WP bodies before the onset of the endothelium-dependent amplification of the vasocontraction (Doi and Fujimoto, 1993). This indicates that some vasocontractive components, such as histamine, may be released from the WP bodies in association with degranulation of the WP bodies induced by ET-1 and STX-S6b and that in turn may amplify the vasocontraction. But to date, there have been no available biochemical or morphological studies describing the release of histamine from WP bodies induced by these peptides. On these grounds, the present study was designed to clarify by means of high performance liquid chromatography (HPLC) and immunoelectron microscopy the question of whether ET-1 and STX-S6b actually induce the release of histamine from WP bodies of the toad aortas. MATERIALS AND METHODS Tissue Preparation Isolated toad abdominal aortas, 0.8 mm-1.0 mm in diameter, were cleaned of all surrounding connective tissue and cut into 15-mm-long specimens. A 22G (diameter: 0.7 mm) stainless-steel canule was inserted into the proximal end of the vascular lumen and then connected to a minipump (Perista SJ-121S, Atto, Tokyo, Japan). A balanced salt solution for frogs (OR-2 solution, composition in millimolar concentration: NaCl 82.5, KC1 2.5, CaC1,.2H,O 1.0, MgC1,.6H20 1.0, Na,HP04-2H,0 1.0, Hepes 5.0, pH: 7.6) was passed through each vessel at a constant flow (1.0 ml/min) by the minipump for 5 min in order to remove the blood cells and plasma. After being perfused with the solution, each vessel was divided into three groups: the control group was perfused with a n OR-2 solution only, the ET-1 perfused group was given a solution containing ET-1 (Sigma Chemical Co., St. Louis, MO) (10-'M), and the STX perfused group was given a solution containing STX-S6b (Peptide Institute, Osaka, Japan) 376 Y. DO1 ET AL. Fiq 2. In the vessels perfused with a solution containing ET-1 (10- M, 10 m i d , a marked indentation of the internal elastic lamina (IL) is observed. Swollen WP bodies with a wide peripheral halo and decreasing electron density (WP), large intracellular vacuoles (LV), and extrusion of their contents in a manner of exocytosis both to the apical and basal sides of the endothelial cells (arrows) are observed. x 13,000. (lO-'M). Each group was perfused with a 10 ml of the above mentioned solutions with a constant flow (1.0 ml/min), and the perfusate was collected from the other end. For immunoelectron microscopy, the vessels of each group were fixed in a periodate-lysine-paraformaldehyde solution (composition: 0.01 M NI04-0.075 M lysine-2% paraformaldehyde in 0.0375 M phosphate buffer, pH: 6.2) (Mclean and Nakane, 1974) for 6 h r at 4°C. After dehydration in graded concentrations of ethanol, specimens were embedded in Lowicryl K4M (Polaron Equipment, Watford, UK), and polymerized at 4°C with a n ultraviolet polymerizer (Dosaka EM, Kyoto, Japan). For conventional electron microscopy, the vessels were fixed in 2% paraformaldehyde-2.5% glutaraldehyde in 0.1 M phosphate buffer for 2 h r a t 4°C and postfixed in 1% osmium tetroxide in the same buffer for 2 h r a t 4°C. After dehydration in graded concentrations of acetone, the specimens were embedded in epoxy resin. Ultrathin sections were stained with 5% uranyl acetate for 6 min and lead citrate for 4 min and examined in a JEM 1200 EX electron microscope. lution, and the histamine was extracted by n-butanol. Then the n-butanol fraction was added to benzene, and the histamine in this fraction was extracted by 0.1 M HC1. HC1-extracted histamine fraction was added to 1 M NaOH and o-phthalaldehyde (OPT). To stop this reaction, 0.45 M H,S04 was added after 4 min. The OPT-histamine complex was separated and determined by a reversed phase high performance liquid chromatograph (Hitachi 650-LC) equipped with a spectrofluorometer. The peak of OPT-histamine of each sample solution was identified by comparing the retention time of the standard OPT-histamine, and the histamine concentration was determined by a calibration curve from the peak area of the standard OPT-histamine. The recovery of histamine by this method was 103.1 +- 4.9% (mean S.D., n = 4 ) . Histamine Determination For histamine determination, 4 ml of each perfusate was used; 5 M NaOH and NaCl were added to the so- * lmmunoelectron Microscopy All procedures were done at room temperature. U1trathin sections of Lowicryl-embedded specimens were collected on uncoated 150-mesh nickel grids and placed for 15 min on drops of 1.0%egg albumin (EA) in phosphate-buffered saline (PBS) to absorb nonspecific proteins. Sections were incubated for 2 h r with rabbit antihistamine serum (Chemicon International, Los Angeles, CAI, which consisted of whole rabbit serum, diluted a t HISTAMINE RELEASE FROM WP BODIES 377 F i t 3.In the vessels perfused with a solution containing STX-S6b (10- M, 10 rnin), some WP bodies are also altered in a similar manner to ET-1.Swollen WP bodies with a wide peripheral halo and decreasing electron density (WP), large intracellular vacuoles (LV), and the extrusion of their contents to the apical sides of the endothelial cells (arrows) are observed. A marked indentation of the internal elastic lamina (IL) is also seen. x 14,000. 1:400 in 0.1% EA-PBS. After rinsing them in PBS, the grids were reacted to goat antirabbit IgG-coated 15 nm colloidal gold (Ultra Biosols, Liverpool, UK), diluted at 1 : l O O in 0.1% EA-PBS for 1 hr, washed in PBS, and washed again in distilled water. The sections were finally stained with 5% uranyl acetate for 3 min and examined in a JEM 1200EX electron microscope. The specificity of the immunolabelings was confirmed by replacing the antisera with either normal rabbit sera diluted a t 1:400 in 0.1% EA-PBS or PBS. RESULTS Cytological Observations Quantitative Analysis of Immuno-gold Particles Quantitative analyses as to the mean Weibel-Palade (WP) body area, the number of gold particles per mean WP body area, and the number of gold particles per 1.0 pm2 WP body area were carried out with a n imageanalysing device (Nikon Cosmosome IS) from immunoelectron micrographs of randomly selected endothelial cells of the control group (12 cells from 4 vessels), the ET-1 perfused group (7 cells from 3 vessels), and the STX perfused group (11cells from 4 vessels). Statistical comparisons between data from the control group and the drug perfused group were made using Student's t-test for unpaired comparisons. The endothelial cells contain a n abundance of WP bodies, and the perfusion with a n OR-2 solution only does not result in any significant ultrastructural changes such as swelling and degranulation of the WP bodies (Fig. 1). In the vessels perfused with a solution containing ET-1 (1OP8M,10 rnin), some of the WP bodies are altered. These alterations include a decrease in electron density, swelling with a wide peripheral halo between a n osmiophilic dense core and the limiting membrane, formation of large intracellular vacuoles, contacts of the altered WP bodies with the plasma membrane, and extrusion of their contents in a manner suggestive of exocytosis both to the apical and basal sides of the endothelial cells (Fig. 2). However, there are considerable variations in these changes from cell to cell: in some endothelial cells, most WP bodies are remarkably altered, whereas almost all WP bodies remain intact in the other cells (Fig. 2). The internal elastic lamina covering the squeezed endothelium occasionally becomes deeply infolded (Fig. 2). In the vessels perfused with a solution containing STX-S6b (lO-'M, 10 rnin), some WP bodies are also 378 Y. DO1 ET AL. Fig. 4. In the vessels perfused with a solution containing STX-S6b (lO-*M, 10 min), extrusions of WP bodies contents into both the subendothelial connective tissue and vascular lumen are frequently observed (arrows). x 15,000. altered in a similar manner to ET-1 (Fig. 3). Extrusions of their contents from the altered WP bodies into both the subendothelial connective tissue and vascular lumen are frequently observed (Fig. 4). Histamine Quantification All the sample solutions containing ET-1 (n = 5) and STX-S6b (n = 5) showed fluorescence peaks, which had the same retention time as that of the standard histamine solution (Fig. 5), but those containing an OR-2 solution only (n = 5) did not show any peaks. The histamine concentration from a solution con20.1 (pg/ml, expressed as taining ET-1 was 151.0 S.E.M.) and that from a solution containing means STX-S6b was 316.2 t 111.6. * * lmmunoelectron Microscopy Control specimens perfused with an OR-2 solution only in which the antihistamine sera were replaced by normal rabbit sera (Fig. 6) or PBS for the negative staining show few or no gold particles on the endothelial cells. By immunoelectron microscopy of the endothelial cells of control specimens, histamine immunoreactive gold particles are preferentially localized on WP bodies (Fig. 7 ) .The immunoreactions are also seen on the apical plasma membrane of the endothelial cells (Fig. 71, but rarely seen on other endothelial cell organelles, on the nonendothelial structures and on the nontissue areas of the grid. In endothelial cells perfused with a solution containing ET-1, the immunoreactive gold particles of the WP bodies decrease in number when compared to those perfused with an OR-2 solution only, but the immunoreactions are often observed in the cytoplasm near the limiting membrane of degranulated WP bodies (Fig. 8A,B). The immunoreactions are seen in the vascular lumen, especially near the degranulation sites of WP bodies (Fig. 8C). In endothelial cells perfused with a solution containing STX-SGb, the gold particles decrease in number inside WP bodies as in case of the ET-1 perfusion. Some swollen W P bodies are in contact with both apical and basal membrane of endothelial cells where immunoreactions of histamine are often found (Fig. 9A,B). The gold particles are seen on the limiting membrane of the swollen WP bodies (Fig. 9A) and the subendothelial space near the degranulated WP bodies (Fig. 9B). Quantitative Analysis of Immuno-gold Particles The data from quantitative analyses are illustrated in Table 1. As shown, the mean area of WP bodies of the ET-1 perfused group and STX perfused group significantly increase when compared to that of the control group. However, the number of gold particles labeling histamine per mean WP area and per 1.0 pm2 HISTAMINE RELEASE FROM WP BODIES 379 A ET-1 B STX-S6b .- 0 Standard Histamine 2 4 6 8 (rnin] Retention time Fig. 5. The standard histamine solution (C) shows a histamine peak (H). All the sample solutions containing ET-1 (A) and STX-S6b (B) show fluorescence peaks (HI, which have the same retention time as that of the standard histamine solution. Chromatographic conditions: Column, LiChrosorb RP18, 5 pm (150 X 4 mm, I.D.). Mobile phase; 0.2M NaCl: CH,OH: CH,CN (60:20:20, viv, pH 3). Flow rate; 0.5 mlimin. Fluorescence detection; excitation 350 nm, emission 450 nm. WP areas of the ET-1 perfused group and STX perfused group significantly decrease in comparison with those of the control group. DISCUSSION In our previous studies, WP bodies of the toad aortas were remarkably degranulated after perfusion with compound 48/80, a liberator of histamine, and an appreciable concentration of histamine was assayed from Fig. 6. Few or no gold particles are seen in the endothelial cell (EC) perfused with a n OR-2 solution only in which normal antihistamine sera were replaced by normal rabbit sera for the negative staining. x 23,000. Fig. 7. Histamine immunoreactive gold particles are preferentially localized on WP bodies (WP) of the endothelial cells of toad aortas perfused with a n OR-2 solution only. The immunoreactions are also seen on the apical plasma membrane of the endothelial cell (arrows). x 45,000. the perfusate using HPLC (Fujimoto, 1982; Fujimoto et al., 1984). Also, we reported that both ET-1 and STXS6b induce ultrastructural changes of the WP bodies. The altered WP bodies showed a decrease in electron density, formation of the peripheral halo, fusion to each other, which results in the formation of large intracellular vacuoles, and expulsion of their contents in a manner of exocytosis. We found no basic inconsistencies in these changes of the altered W P bodies with those treated with compound 48/80. 380 Y. DO1 ET AL. toad aorta. Immunoelectron microscopy showed the release of histamine from the WP bodies in association with their degranulation. After being perfused with ET-1 and STX-SGb, the immunoreactive gold particles that were localized in the WP bodies remarkably decreased in number when compared to the control group and are often localized on and outside the limiting membrane of the swollen WP bodies. They also exist not only in the vascular lumen but also in the subendothelial space, especially in regions where the extracellular release of the WP bodies occurs. These immunocytochemical findings suggest that histamine stored in WP bodies may permeate into the cytoplasm through the limiting membrane and are then discharged extracellularly in association with exocytosis of the WP bodies after treatment with ET-1 and STX-S6b. ET-1 has been recently reported to provoke the release of histamine from pulmonary mast cells (Uchida et al., 1992) and to induce bronchoconstriction by its indirect action through the production of some chemical mediators such as histamine in pulmonary mast cells a s well as its direct action on the smooth muscle cells (Ninomiya et al., 1992). Our previous data (Doi and Fujimoto, 1993) indicate the existence of some vasocontractive substances in WP bodies of the toad aortas, and the present data suggest that histamine included in WP bodies and released from them is one of the candidates. It is therefore reasonable to consider that indirect action after the release of histamine may be involved in the vasocontraction of the toad aorta induced by ET-1 and STX-S6b a s in the case of the bronchoconstriction induced by ET-1. However, there still remains the question whether histamine amplifies the vasocontraction induced by ET-1 and STX-S6b. To answer this question, it should be confirmed that histamine and these peptides show a synergistic action. More detailed studies, now in progress in our laboratory, are necessary to solve this problem. ACKNOWLEDGMENTS We thank Ms. Tomoko Nishino for her technical assistance, and Ms. Toyono Nobukuni for typing the manuscript. LITERATURE CITED Fig. 8. In endothelial cells (ECs) perfused with a solution containing ET-1, histamine immunoreactive gold particles of the WP bodies decrease in number when compared to those perfused with an OR-2 solution only (Fig. 71, but the immunoreactions are often observed in the cytoplasm near the limiting membrane of degranulated WP bodies (WP) in B (high magnification view of the box in A), and in the vascular lumen near the degranulation sites of WP bodies (arrow) in C. A, x 23,000; B, $st 45,000; C, 35,000. Our chromatographic study revealed that both ET-1 and STX-S6b induce the release of histamine from the Bertini, F., and R. Santolaya 1970 A novel type of granules observed in toad endothelial cells and their relationship with blood pressure active factors. Experientia, 26t522-523. Bonfanti, R., B.C. Furie, B. Furie, and D.D. Wagner 1989 PADGEM (GMP140) is a component of Weibel-Palade bodies of human endothelial cells. Blood, 73:1109-1112. Burri, P.H., and E.R. Weibel 1968 Beeinflussung einer spezifischen cytoplasmatischen Organelle von Endothelzellen durch Adrenalin. 2. Zellforsch., 88:426-440. Doi, Y., and S. Fujimoto 1993 Vasocontractions of the in-vitro toad aortas induced by endothelin-1 and sarafotoxin-S6b. Anat. Rec., 235:253-260. Fujimoto, S. 1982a Degranulation of endothelial specific granules of the toad aorta after treatment with compound 48/80. Anat. Rec., 203t197-204. Fujimoto, S., K. Yamamoto, K. Arashidani, I. Hayabuchi, M. Yoshizuka, and T. Nomiyama 198213 Endothelial specific granules in the umbilical veins of the postnatal rabbit. Cell Tissue Res., 227: 509-518. Fujimoto, S., K. Yamamoto, H. Ueda, K. Hamasaki, and T. Nomiyama 1984 Histamine disposition in endothelial specific granules of the toad aorta. Acta Anat., 118t38-41. 381 HISTAMINE RELEASE FROM WP BODIES Fig. 9. In endothelial cells perfused with a solution containing STXS6b, the gold particles decrease in number inside WP bodies as in case of the ET-1 perfusion. Some swollen WP bodies are in contact with both the apical and basal membrane of the endothelial cells where immunoreactions for histamine are often found (arrowheads). The gold particles are seen on the limiting membrane of the swollen WP bodies (arrows in A), and the subendothelial space near the degranulated WP bodies (arrow in B, high magnification view of the box in A). A, X 23,000; B, X 45,000. TABLE 1. Number of gold particles labeling histamine within Weibel-Palade bodies (mean Control group (12 cells from 4 vessels) ET-1 perfused group (7 cells from 3 vessels) STX perfused group (11 cells from 4 vessels) WP body profile area (urn2) 0.075 0.003 0.101 0.006** 0.110 -t 0.011** * * Number of gold varticles/WP bodv 1.355 -t 0.139 0.335 0.033** 0.422 & 0.058** f S.E.M.) Number of gold aarticles/um2 19.417 2 1.782 3.754 t 0.412** 4.037 2 0.525** **P<O.Ol, Asterisks show a statistically significant difference between the control group and the drug perfused group. Hattori, R., K.K. Hamilton, R.D. Fugate, R.P. McEver, and P.J. Sims 1989 Stimulated secretion of endothelial von Willebrand factor is accompanied by rapid redistribution to the cell surface of the intracellular granule membrane protein GMP-140. J. Biol. Chem., 264t7768-7771. Hirata, Y., H. Yoshimi, F. Marumo, T.X. Watanabe, S. Kumagaye, K. Nakajima, T. Kimura, and S. Sakakibara 1989 Interaction of synthetic sarafotoxin with rat vascular endothelin receptors. Biochem. Biophys. Res. Commun., 162t441-447. Inoue, A,, M. Yanagisawa, S. Kimura, Y. Kasuya, T. Miyauchi, K. Goto, and T. Masaki 1989 The human endothelin family: Three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc. Natl. Acad. Sci. USA, 86:28632867. Kagawa, H., and S. Fujimoto 1987 Electron-microscopic and immunocytochemical analyses of Weibel-Palade bodies in the human umbilical vein during pregnancy. Cell Tissue Res., 249t557-563. Kloog, Y., I. Ambar, M. Sokolovsky, E. Kochva, Z. Wollberg, and A. Bdolah 1988 Sarafotoxin, a novel vasoconstrictor peptide: Phosphoinositide hydrolysis in rat heart and brain. Science 242r268270. McEver, R.P., J.H. Beckstead, K.L. Moore, L. Marshall-Carlson, and D.F. Bainton 1989 GMP-140, a platelet a-granule membrane protein, is also synthesized by vascular endothelial cells and is localized in Weibel-Palade bodies. J . Clin. Invest., 8 4 3 - 9 9 . Mclean, I.W. and P.K. Nakane 1974 Periodate-lysine-paraformaldehyde fixative: A new fixative for immunoelectron microscopy. J . Histochem. Cytochem., 22t1077-1083. Ninomiya, H., Y. Uchida, M. Saotome, A. Nomura, H. Ohse, H. Matsumoto, F. Hirata, and s. Hasegawa 1992 Endothelins constrict guinea pig tracheas by multiple mechanisms. J . Pharmacol. Exp. Therap., 262t570-576. Stenberg, P.E., R.P. McEver, M.A. Shuman, Y.V. Jacques, and D.F. Bainton 1985 A platelet alpha-granule membrane protein (GMP140) is expressed on the plasma membrane after activation. J. Cell Biol., 101t880-886. Takasaki,C., M. Yanagisawa, S. Kimura, K. Goto, andT. Masaki 1988 Similarity of endothelin to snake venum toxin. Nature, 335t303. Uchida, Y., H. Ninomiya, T. Sakamoto, J.Y. Lee, T. Endo, A. Nomura, S. Hasegawa, and F. Hirata 1992 ET-1 released histamine from guinea pig pulmonary but not peritoneal mast cells. Biochem. Biophys. Res. Commun., 189:1196-1201. 382 Y. DO1 ET AL. Ueda, H., Y . Doi, Y. Sakamoto, K. Hamasaki, and S. Fujimoto 1992 Simultaneous localization of histamine and factor VIII-related antigen in the endothelium of the human umbilical vein. Anat. Rec., 232:257-261. Wagner, D.D., J.B. Olmsted, and V.J. Marder 1982 Immunolocalization of von Willebrand protein in Weibel-Palade bodies of human endothelial cells. J. Cell Biol., 95:355-360. Weibel, E.R., and G.E. Palade 1964 New cytoplasmic components in arterial endothelia. J. Cell Biol., 23:lOl-112. Yanagisawa, M., H. Kurihara, S. Kimura, Y. Tomoboe, M. Kobayashi, Y. Mitsui, Y. Yazaki, K. Goto, and T. Masaki 1988 A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature, 332t411-415.