Degranulation of endothelial specific granules of the toad aorta after treatment with compound 4880.код для вставкиСкачать
T H E ANATOMICAL RECORD 203:197-204 (1982) Degranulation of Endothelial Specific Granules of the Toad Aorta After Treatment With Compound 48/80 SUNAO FUJIMOTO Department of Anatomy. Uniiieriity of Occiipational and Enuirunmental Health, Sthool of Medicin<,.Kitukyu\hu, Japan 8 O ï ABSTRACT Incubation of isolated toad aortas in Ringer solution containing compound 48/80, a histamine liberator, resulted in marked degranulation of endothelial specific granules. Since incubation of these vessels in Ringer solution only did not show significant morphologic changes in these granules, these findings suggest that the degranulation was induced by histamine release from the granules, as in the case for mast cel1 degranulation, and that endothelial specific granules are a storage site of histamine in the toad aorta. The present morphologic data were supported by preliminary chromatography, which showed appreciable concentrations of histamine in the granule-containing pellets of subcellular fractions of homogenized toad aortas. Since the first description of endothelial specific granules by Weibel and Palade (1964),the existence of similar cytoplasmic components in endothelial cells of various blood and lymphatic vessels has been described by many workers (Zelickson, 1964; Fuchs and Weibel, 1966; Kühnel, 1966; Fujimoto, 1967; Oehmke, 1968; Burri and Weibel, 1968; Piezzi e t al., 1969; Lemeunier et al., 1969; Bertini and Santolaya, 1970; Matsuda and Sugiura, 1970; Steinsiepe and Weibel, 1970; Tabuchi and Yamamoto, 1974). Several histochemical and biochemica1 approaches have been made on these granules to elucidate their exact nature. Lemeunier e t al. (1969) have denied the possibility that these granules might be lysosomal bodies, since they did not show a positive reaction for acid phosphatase activities. Burri and Weibel (1968) have suggested that these granules might contain "a procoagulative substance," since epinephrine-perfused rabbit aortas released a coagulation-activating substance int0 t h e perfusate concomitantly with a marked decrease in number of the endothelial specific granules. Recently, we observed a remarkable increase in number of endothelial specific granules similar in ultrastructure to those described by others in the postnatal rabbit umbilical veins between 2 and 10 days after birth (Takeshige and Fujimoto, 1977;Fujimoto et al., 1978).The 0003-276X/82/2032-0197$02.50 C 1982 ALAN R. LISS. INC. increase was much more pronounced in regions where vascular obliteration was actively progressing. Since concentrations of these granules were not found in the late prenatal vessels except for a few immature forms near Golgi regions and since almost al1 granules have disappeared around 30 postnatal days, such a temporary increase in number of granules in a limited postnatal stage suggests that they play an important role in the postnatal obliteration of this vessel. I t is wel1 known that endothelial contraction can be induced by biogenic amines such as histamine, serotonin, and bradykinin in some vertebrates, including frogs (Majno et al., 1967, 1969), and roles of histamine in the regulation of vascular tonus have been proposed by many workers. Furthermore, appreciable concentrations of histamine were assayed in the homogenates of rat and rabbit blood vessels by chromatographic techniques (Howland and Spector, 1972). On these grounds, we tried incubation of toad aortas in Ringer solution containing compound 48180. The purpose of this experiment was to examine the ultrastructural effects of this histamine liberator on the morphology of endothelial specific granules and to throw some light on our hypothesis that endothelial . Keceived July 20. 19x1; accepted Fehruary 2 4 . 1982. 198 S. FUJIMOTO specific granules in various vertebrates might be histamine storage particles. MATERIALS A N D ME‘I’HODS Abdominal aortas of the toads, Rana catesbiana, were isolated and incubated in Ringer solution containing compound 48/80 (1, 2, 10, 100, and 500 pgiml Ringer) for 5 min. For control experiments, the vessels were incubated in Ringer solution without the drug. After incubation, materials were fixed in 4% glutaraldehyde in 0.1 M cacodylate buffer for 2 hr, postfixed in 1%osmium tetroxide in the same buffer for 1 hr, dehydrated in graded concentrations of acetone, and embedded in epoxy resins. Sections were made on a Sorvall MT-1 ultramicrotome, stained both with uranyl acetate and lead acetate, and observed either with a Hitachi 12-A or a Hitachi 500 electron microscope. The preliminary high-performance liquid chromatography (HPLC)was done to correlate with the present morphologic studies. The toad aortas in 0.01 M tris-HC1 buffer containing 0.25 M sucrose (pH 7.4) were homogenized and then centrifuged a t 80,000g for 10 min. One-half of the precipitates was prepared for electron microscopic samples t o confirm that they were rich in the intact endothelial specific granules, together with contaminant mitochondria, and the other half for chromatographic analyses was homogenized again in 0.4 M perchloric acid and centrifuged at 10,000g for 10 min. After basic amines were extracted from the supernatants by a column chromatograph (Amberlite CG 50, 10mm X 10 mm), they were condensed by a freeze-drying method and reacted with o-phthalaldehyde and 2-mercaptoethanol (OPT-2ME)for the formation of histamine fluorescence. The sample solution (pH 5.0) was injected int0 a Hitachi 638-50 HPLC (a separation column was filled with Unisil P H ) equipped with a Hitachi 650-10 LC fluorometric detector (excitation 355 nm, emission 440 nm). RESULTS Endothelia incubated in Ringer solution only Incubation of the isolated vessels resulted in a marked vascular contraction, and nuclear regions of the endothelial cells were protruded int0 the vascular lumen (Fig. 1). However, we found basically no inconsistencies with the neneral cvtodasmic features of toad arteries reported by &hers (Stehbens, 1965; Piezzi e t al., 1969; Steinsiepe and Weibel, 1970) in our materials. Each endothelial cell contained abundant osmiophilic granules, which were round or rodlike in shape and bounded with a single limiting membrane (Fig. 2). Concentrations of these granules greatly varied from cell to cell. The cytoplasm of some endothelial cells contained a large number of the specific granules but others contained only a few. Most granules had a fine granular matrix (Fig. 2), but a few clearly showed microtubular structures approximately of i00Ä inner diameter in their interiors. Incubation only in Ringer solution did not result in significant ultrastructural changes in these granules. Endothelia incubated i n Ringer solution containing compound 48/80 In specimens treated with lower doses of the drug (1pg and 2 pg), the outline of the apical endothelial cell surface became irregular owing to the occurrence of many short cytoplasmic projections (Fig. 3). The ultrastructure of almost al1 granules was not much different from that of the controls described above. With a few exceptions, altered granules with a somewhat swollen appearance and decreased electron density were observed (Fig. 3). In these granules, microtubular structures were frequently present. However, such altered granules were always “intracellular” and we did not find any contact with or opening to the apical cell membrane in materials treated with low doses of compound 48/80. In specimens treated with 10 pg of the drug, the increase in number of altered intracellular granules as described above was apparent (Fig. 4). Many granules contained fibrillar masses of low electron density with a widened space separating the granular matrix from the limiting membrane (peripheral halo). With progressing degranulation, fusion of the adjacent altered granules and contact with or close apposition to the apical cell membrane were seen (Fig. 5). One end of such fused granules was often opened t o the extracellular space with the extrusion of the granular contents. In specimens treated with 100 pg and 500 pg of the drug, the general morphology of the degranulation process was fundamentally the same as t hat of specimens treated with 10 pg, but the degree of alteration was more pronounced. In an extreme case of a successive fusion of adjacent altered granules and their extrusion int0 the extracellular space, a greater part of the cell cytoplasm was occupied by . - large - membrane-bound anastomosing I DEGRANULATION O F ENDOTHELIAL SPECIFIC GRANULES 199 Fig. 1. The endothelium contains abundant specific grandes. Incubation in Ringer solution only does not result in significant degranulation processes of the granules a s shown in this figure. X 6,000. cavities containing remnants of altered granular contents (Fig. 6). In addition, fusion of the altered grandes with the basal cel1 membrane of the endothelial cells and extrusion of the granular masses int0 the subendothelial connective tissue were observed (Fig. 7). Chromatographic separation of histamine in the endothelial grandes A half of the precipitate was observed with the electron microscope and it was confirmed that they were rich in the endothelial specific granules. After the other half of these granule-containing pellets were reacted with OPT-ZME for the formation of histamine fluorescence, the sample solution was injected int0 the HPLC equipped with the fluorometric detector. Chromatographic separation revealed a fluorescent peak Fig. 2. The grandes are bounded with a single iimiting membrane and have eiectron-dense fine granuiar matrix. Incubated in Ringer soiution oniy. X 32.000. 200 S. FUJIMOTO Fig. 3. A swollen g r a n d e ( S G )with a wide peripheral halo and decreasing electron density is observed. Microtuhular structures exist in the matrix of this altered grande. ï r e a t e d with 2 pg of compound 48/80. X 19,000. that had the Same retention time as that of the standard histamine solution (Fig. 8). By light microscopy of a greater sampling of the nonincubated vessels, we seldom encountered mast cells in the adventitia (Fig. 9). DISCUSSION Since Weibel and Palade (1964) described membrane-bound endotheliai specific granules in dog arteries, similar cytoplasmic components have been found in various blood and lymphatic vessels. Although detailed descriptions of their ultrastructural features have been made by previous morphologists, their exact nature and functional significance Fig. 5 . Successive fusions of adjacent altered granules result in the formation of many large vacuoles (FG)in the cytoplasm. One end of such fused g a n u l e s is opened to the extracellular space with theextrusionof thegranular contents (arrow).‘ïreated with 10 pg of compound 48/80. X 26.000. Fin. 4. Altered manules a s shown in Firnre 3 increase in Fig. 6. A greater part of the cytoplasm is occupied hy large memhrane-hounded anastomosing cavities containing remnants of altered granules. Treated with 500 /cg of com- DEGRANULATION O F ENDOTHELIAL SPECIFIC GRANULES 20 1 202 S. FUJIMOTO O 8 2 4 MINUTES I I 6 8 DEGRANULATION OF ENDOTHELIAL SPECIFIC GRANULES remain uncertain to the present time. The possibility that they contain acid phosphatase has been denied by Lemeunier et al. (1969),and the absence of catalase activity with the diaminobenzidine reaction suggests that they are different in nature from such organelles as peroxisomes in liver cells (Tabuchi and Yamamoto, 1974). Burri and Weibel (1968) observed that epinephrine-perfused rabbit aortas deliver “a coagulation activating substance” in the perfusate with a marked loss of the specific granules and suggested that they might contain some biologically active substances that have an important role in the regulation of vascular tonus. A biochemica1 approach to elucidate the nature of endothelial specific granules was tried by Bertini and Santolaya (1970). They revealed that subcellular fractions of these granules together with contaminant mitochondria from homogenized toad aortas are rich in “hypertensive activity.” These data may indicate that endothelial specific granules play some role in the regulation of blood pressure. In our previous studies (Takeshige and Fujimoto, 1977; Fujimoto et al., 1978), postnatal rabbit umbilical veins showed remarkable increase in number of endothelial specific granules originating from the Golgi apparatus. Since concentrations of these granules were extremely rare in prenatal vessels, this finding suggests that the increase might be related to physiologic obliteration of this fetal circulation after birth. I t is wel1 known that vascular endothelial and medial cells in some vertebrates contract under the influence of histamine-type mediators (Majno et al., 1969). In dogs, intravenous injection of compound 48/80 causes a rise in Fig. 7. The contents of altered granules ( S G )are exocytosed from the basal part of the endothelial cells (arrow). Treated with 100 pg of compound 48/80. X 17,000. Fig. 8. A chromatographic separation of OF‘T-2ME-histamine complex is shown. The vertical and horizontal axes represent the fluorescent intensity and retention time, respectively. H. Histamine fluorescent peak of the sample solution with the same retention time a s that of standard histamine solution. Fig. 9. A light micrograph of 1 pm section of the toad aorta stained with toluidine blue. Mast cells were not ob. served in this section. although two profiles of pigment cells (P)were observed in the adventitia (A). I, Intima: M, Media. x 200. 203 the pressure of the portal vein (Paton, 1951; Rowley, 1964).If histamine-type mediators are important in the regulation of vascular tonus or blood pressure, knowledge of its disposition in various vessels might be of special significance. Compound 48/80, a condensation product of p-methoxyphenylethylmethylamine and formaldehyde, is the most specific and least toxic agent in various histamine liberators, and ultrastructural effects of this drug on mast cell degranulation have been observed by several workers (Bloom and Haegermark, 1965; Horsfield, 1965; Singleton and Clark, 1965; Yamasaki et al., 1970; Röhlich et al., 1971). I t is of interest that endothelial specific granules of toad aortas showed several dynamic changes after treatment with this drug in the present experiments. The formation of the peripheral halo with a decreased electron density, the fusion of such altered granules with adjacent ones or with the apical cell membrane, and the extracellular release of the granular contents were findings similar to those observed in mast cell degranulation processes accompanying histamine release by the Same drug (Bloom and Haegermark, 1965; Horsfield, 1965; Röhlich et al., 1971). In the case of mast cells, degranulation without accompanying histamine release can als0 be induced by sonic oscillation, lysis in distilled water, and incubation in the buffer solution (Bloom and Haegermark, 1965). However, in the present experiments, morphologic changes of the endothelial cell granules were extremely rare in specimens incubated only in Ringer solution or those treated with smaller doses of the drug, although a few granules appeared slightly swollen. On the contrary, in the cells treated with higher doses of the drug, the degranulation became more pronounced. These findings strongly suggest that the degranulation may represent the liberation of histamine from the endothelial specific granules. Considerably high concentrations of histamine in the granule-containing pellets of the homogenized toad aortas were determined by the present chromatographic analyses. One question that wil1 arise is whether this value might be affected by contamination of mast cell granules in the pellets. However, our chromatography has als0 demonstrated an appreciable concentration of histamine in the perfusate from compound 48180 - perfused toad aortas (unpublished data). This would give a validity to the tentative conclusion that the histamine concentration of the pellets came mainly from endothelial specific granules. Al- 204 S. FUJIMOTO though detailed descriptions of these chromatographic studies including those of the postnatal rabbit umbilical veins wil1 appear in a separate article, the present preliminary chromatographic data may give further weight to the morphologic data that suggest that endothelial specific granules may be a storage site of histamine. More detailed morphologic and biochemica1 studies are necessary to elucidate the nature and functional role of endothelial specific granules from the standpoint of comparative anatomy; such studies are now in progress in our laboratory. mine in mammalian blood vessels. J. Pharmacol. Exp. Ther., ZX2239-245. Kuhnel. W. (1966) Elektronenmikroskopische Befunde am Ductus thoracicus. Z. Zellforsch., 70:519-531. Lemeunier, A., P.H. Burri. and E.R. Weibel (1969) Absence of acid phosphatase activity in specific endothelial organelles. Histochemie, 20:143-149. Majno. G.. V. Gilmore. and M . Levental (1967)On the mechanism of vascular leakage caused by histamine-type mediators. Circulation Ites.. 212333-847. Majno. G.. S.M. Shea. and M . Leventhal (1969)Endothelial contraction induced hy histamine-type mediators. An electron microscopie study. J. Cell Biol., 42647-672. Matsuda, H.. and S. Sugiura (1970) Ultrastructure of “tubular body” in the endothelial cells of the ocular blood vessels. Invest. Ophthalmol.. 9:919-925. Oehmke, H.J. (1968)Periphere Lymphgefasse des Menschen ACKNOWIXDGMENTS und ihre funktionelle Struktur: Licht und elektronenmikroskopische Studien. Z.Zellforsch. 0:320-352. The author thanks Dr. K. Arashidani for his Paton. W.D.M. (1951)Compound 48/80: A potent histamine chromatographic advice, Dr. K. Yamamoto for liberator. Hr. J. I’harmacol.. ö:499-508. his photographic assistance, and Miss R. Piezzi, 1t.S.. R.C. Santolaya. and F. Bertini (1969)Fine structure of endothelial cells of toad arteries. Anat. Rec.. 165; Masaki for her typewriting of the manuscript. 229-236. This work was supported in part by grant Hohlich. P.,P.Andersom and í3. Unvas (1971) Electron mi448087 from the Ministry of Education of croscope observations on compound 48/80 induced degranulation in rat mast cells. J . Cell Biol.. 51.465-483. Japan. Rowley. D.A. (1964) Venous constriction as the case of increased vascular permeability produced hy 5-hydroxytryptamine, histamine. hradykinin and 48/80 in the rat. Bertini, F., and K. Santolaya (1970)A novel type of granules Br. J. Exp. Pathol., 4,5:56-67. observed in toad endothelial cells and their relationship Singleton, E.M.. and S.L. Clark, J r . (1965) The response of with blood pressure active factors. Experimentia, 2ö:522mast cells t o compound 48180 studied with the electron 523. microscope. Lab. Invest.. 14:1744-1763. Bloom, G.D., and O. Haegermark (1965)A study on morpho- Stehbens. W.E. (1965) Ultrastructure of vascular endothellogica1 changes and histamine release induced hy comium in the frog. Q. J.Exp. Physiol., 50:375-384. pound 48/80 in r a t peritonea] mast cells. Exp. Cell Res., Steinsiepe, K.F.. and E.R. Weibel (1970) Elektronenmikro40:637-654. skopische Untersuchungen an spezifischen Organelles Burri, P.H.. and E.R. 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Yamamoto, and Y. ïakeshige (1978) Hisponents in arterial endothelia. J. Cell Riol.. %3:101-112. tochemical and autoradiographic findings on specific Yamasaki, €1.. T. Fujita, Y. Ohara, and S. Komoto (1970) granules of the endothelial cells. In: ölectron Microscopy. Electron microscope studies on the release of histamine J.M. Sturgess, ed. Electron Microscopy Society of from rat peritonea] mast cells. Arch. Histol. Jpn., 31: Canada, Toronto, Vol. 2. pp. 466-467. 393-408. Horsfield. G . I . (1965)The effect of compound 48/80 on the Zelickson. A.S. (1964) A tuhular structure in the endothelrat mast cell. J. Pathol. Racteriol.. S0:599-605. ia1 cells and pericytes of human capillaries. J . Invest. €iowland. H.D.. and S. Spector (1972) Disposition of histaIlermatol.. 46:167-171.