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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 265, No. 4, Issue of February 5, pp. 2311-2316,lSSO 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in IJ. S. A.

Localization of Arachidonate 12-Lipoxygenase in Parenchymal Cells of Porcine Anterior Pituitary*

(Received for publication, August 24, 1989)

Natsuo UedaS, Akihiko HiroshimaSB, Kiyoshi NatsuiS, Fukiko ShinjoSn, Tanihiro YoshimotoS, Shozo YamamotoSII, Kunio Ii**, Kiryaki Gerozissis$$., and Fernand Dray*+ From the Departments of SBiochemistry and **Pathology School of Medicine, Tokushima University, Kuramoto-cho, Tokushima 770, Japan and the $$Unite de Radioimmunologie Annlytique, Institut Pasteur, Paris, 75724 France

12-Lipoxygenases oxygenate arachidonic acid pro- ducing its IBS-hydroperoxy derivative and are well known as platelet and leukocyte enzymes. When a peroxidase-linked immunoassay of the enzyme according to the avidin-biotin method was applied to the cytosol fractions from various parts of porcine brain, a consid- erable amount of the enzyme was found in the anterior pituitary. The enzyme level (about 200 ng/mg cytosol protein) corresponded to about 6% of the enzyme content in porcine peripheral leukocytes. Posterior and intermediate lobes showed about one-tenth of the enzyme level of anterior pituitary. Other parts of porcine brain contained the 12-lipoxygenase in amounts below 7 ng/mg cytosol protein. The cytosol fraction (0.7 mg of protein) of anterior pituitary produced l2S-hydroxy-5,8,10,14-eicosatetraenoic acid from 25 FM arachidonic acid in about 34% conversion at 24 “C for 5 min, giving a specific enzyme activity of about 3 nmol/ min/mg protein. Furthermore, various octadecapolyenoic acids were oxygenated almost as fast as the arachidonate 12-oxygenation. When anterior pituitary was investigated immunohistochemically with anti- 12-lipoxygenase antibody, most of the immunostained cells were certain parenchymal cells with granules, which were not blood cells. These biochemical and immunohistochemical results provide a good reason for considering that l%-lipoxygenase does play an impor- tant role in pituitary function.

Arachidonate 12-lipoxygenase has been found in various mammalian tissues, especially in platelets (1,2) and leukocytes (3,4). As pointed out by Brash (5), “the pathways of arachidonic acid metabolism that are understood lead to the formation of potent biological mediators such as the prostaglandins and leukotrienes. However, there is no obvious anal- ogy to these mediators in the 12-lipoxygenase pathway.” Na-

* This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare of Japan, and grants from the Japanese Foundation of Metabolism and Diseases, Takeda Science Foundation, the Mochida Memorial Foundation. the Tokvo Biochemical Research Foundation, and the Uehara Memorial Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§Present address: Kissei Pharmaceutical Company, Research In- stitute, Matsumoto, Nagano, Japan.

(IPresent address: Ontario Cancer Institute, Toronto, Ontario M4X lK9, Canada.

IITo whom correspondences should be addressed.

kao et al. (6) reported an extremely potent activity of 12- HETE’ (but not 5-HETE and 15-HETE), which stimulated the migration of rat aortic smooth muscle cells at a concentration in the order of 10 f~. A more recent work by Piomelli et al. (7) demonstrated that the neurotransmission in the marine mollusk, Aplysia cal~~oornica, was mediated by the 12- lipoxygenase product, 12-HPETE, or its metabolites such as 12-keto-5,8,10,14-eicosatetraenoic acid (8) and 8-hydroxy- 11,12-epoxy-5,9,14-eicosatrienoic acid (9). Furthermore, 12S-HETE (but not 12R-HETE) was proposed to participate in the expression or activation of a tumor cell glycoprotein receptor (10). Generalization of these intriguing observations to many other mammalian tissues has not yet been estab- lished. Thus, the general physiological function of the 12- lipoxygenase pathway is still an open question.

Previously, we prepared monoclonal antibodies against the 12-lipoxygenase of porcine leukocytes and developed a peroxidase-linked immunoassay for determination of the enzyme amount in various porcine tissues (11). However, the occurrence of the 12-lipoxygenase in brain remained unclarified due to the insufficient sensitivity of the assay. As detected by transformation of exogenous arachidonic acid, the 12-lipoxygenase activity was found previously in pituitary (12-16), pineal gland (16), and other parts of the rat brain (17). These previous observations do not necessarily indicate the occurrence of 12-lipoxygenase in the parenchymal cells of these organs rather than contaminating or infiltrating blood cells. Recently, we improved the enzyme immunoassay of 12-lipoxygenase and increased its detectability by introducing the solid-phase and avidin-biotin methods and applied the modi- fied sensitive assay to detect the 12-lipoxygenase in various parts of porcine brain. In addition, we performed immunohistochemical studies on the distribution and localization of 12- lipoxygenase in brain tissues.

EXPERIMENTAL PROCEDURES

Materials-various unsaturated fatty acids were purchased as described previously (18). High molecular weight standard mixture for polyacrylamide gel electrophoresis was obtained from Sigma, Immu- noplate I from Nunc (Roskilde, Denmark), horseradish peroxidase- avidin D and N-hydroxysuccinimidobiotin from Pierce, Vectastain ABC-kit, goat biotinylated anti-rabbit IgG antibody, and normal goat serum from Vector Laboratories (Burlingame, CA), o-phenyle&di- amine from Nakarai (Kvoto), 3,3’-diaminobenzidine 4 HCl from Dojin (Kumamoto), and PD-lb column from Pharmacia (Uppsala).

’ The abbreviations used are: 12-HETE, 12-hydroxy-5Z, 82, lOE, 14Z-eicosatetraenoic acid; 5-HETE, 5-hydroxy-GE,8Z,llZ,14Z-eicosatetraenoic acid; 9-HETE, 9-hydroxy-5,7,11,14-eicosatetraenoic acid; 12-HPETE, 12.hydroxy-5Z,8Z,lOE, 14Z-eicosatetraenoic acid, 15-HETE, 15-hydroxy-5Z,8Z,llZ,l3E-eicosatetraenoic acid; HETE, hydroxy-eicosatetraenoic acid; HPLC, high performance liquid chro- matography.

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2312 12-Lipoxygenase of Porcine Anterior Pituitary

Quetol 812 was supplied from Nisshin EM (Tokyo), Entellan Neu and osmium tetroxide from Merck (Darmstadt), and O.C.T. com- pound from Miles (Elkhart, IN). Monoclonal antibodies (lox-1 and lox-2) against porcine leukocyte 12-lipoxygenase were raised in mice as described previously (11). Both species reacted with 12-lipoxygenase without the loss of enzyme activity. As described in Ref. 19, a polyclonal anti-12-lipoxygenase antibody was raised in rabbits using the immunoaffinity purified enzyme from porcine leukocytes (18) as antigen. In the Western blotting of the porcine leukocyte cytosol, this antibody recognized only one band corresponding to the purified leukocyte 12-lipoxygenase (19). SLipoxygenase, which was also purified from porcine leukocytes (20), did not react with the polyclonal anti-12-lipoxygenase antibody. The cytosol fraction and the purified 12-lipoxygenase (specific enzyme activity, 4 fimol/min/mg protein at 30 “C) were prepared from porcine leukocytes as described in Ref. 18. 5-HETE (21), 12-HETE, SHETE, 13-hydro(pero)xy-9,11-octadecadienoic acid (18), and 15-hydroxy-11,13-eicosadienoic acid (4) were prepared as described previously.

Enzyme Immunoassay of 12-Lipoxygenase-Each part of brain (totally about 1 g wet weight) collected and combined from several pigs was homogenized in 5 volumes of phosphate-buffered saline, pH 7.4, by the use of a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 27,000 X g for 10 min and then at 105,000 X g for 60 min. The supernatant solution was stored at -80 “C as the cytosol fraction. Protein concentration was determined by the method of Lowry et al. (22) with bovine serum albumin as a standard.

Two species of monoclonal anti-lP-lipoxygenase antibody (lox-l and lox-2), which recognized different sites of 12-lipoxygenase (ll), were utilized. lox-2 (1.0 mg) was incubated with 0.1 mg of N-hydroxysuccinimidobiotin at room temperature for 1 h in 1.25 ml of phosphate-buffered saline at pH 7.4 containing 20% N, N-dimethylform- amide. The remaining N-hydroxysuccinimidobiotin was removed by a PD-10 column. The biotinylated antibody was stored at -80 “C in a 50% glycerol solution (125 pg/ml).

Phosphate-buffered saline, pH 7.4, containing 0.1% bovine serum albumin was used for dilution of the purified 12-lipoxygenase, the cytosol fraction of various parts of porcine brain, the biotinylated Zox- 2, and horseradish peroxidase-avidin. When the content in a well of immunoplate was discarded after each incubation described below, the well was washed three or four times with phosphate-buffered saline, pH 7.4, containing 0.01% bovine serum albumin.

As the first step, the other antibody, lox-l (500 ng in 100 ~1 of 50 mM carbonate buffer at pH 9.5), was placed in a well of Immunoplate I (96 wells/plate), and kept at 4 “C overnight for its binding to the well. The second step was the addition of 400 ~1 of 0.5% bovine serum albumin (dissolved-in phosphate-buffered saline at pH 7.4) to the well, followed by incubation at 37 “C for 30 min. At the third step, the sample containing 12-lipoxygenase (100 ~1) was added to the well, and the mixture was kept at 37 “C for 2 h. As the fourth step, biotinvlated lox-2 (50 np in 100 ~1) was added. and the mixture was incubated at 37 “C for-l h. The fifth step was the addition of a peroxidase-avidin conjugate (100 ng in 100 ~1). The mixture was incubated at room temperature for 30 min. At the sixth step, we added 100 ~1 of 50 mM phosphate-citrate buffer, pH 5.0, containing 18.5 mM o-phenylenediamine and 3.5 mM hydrogen peroxide. The peroxidase reaction was carried out at 20 “C for 10 min and stopped by the addition of 50 ~1 of 5 N sulfuric acid. Absorbance at 492 nm was measured by the use of a microplate reader model MTP-32 (Corona Electric). Absorbance at 630 nm was used as a reference. The amount of 12-lipoxygenase in a sample was determined in dupli- cate.

The standard enzyme immunoassay gave an essentially linear calibration curve with the purified 12-lipoxygenase up to 2 ng of protein. The minimum detectability of the 12-lipoxygenase defined as the amount of the enzyme to cause twice as much increase in 492- nm absorbance as the background value, was 0.2 ng of protein. When various amounts of the cytosol fraction of porcine cerebellum were subjected to the standard enzyme immunoassay, an almost linear relationship was observed between the amount of the added cytosol and the amount of 12.lipoxygenase determined by the enzyme immunoassay. In the absence of the immobilized antibody, an increasing amount of the cytosol fraction caused a nonspecific binding of horseradish peroxidase, giving a background value of less than 13%. On the other hand, a known amount of the cerebellum cytosol was mixed with various amounts of the purified 12-lipoxygenase of porcine leukocytes. There was a linearity between the amounts of the added and measured 12-lipoxygenase, and the added enzyme was detected in an average recovery of about 80%. When 10 assays with the same

amount of the cerebellum cytosol were performed, a coefficient of intraassay variation was 5.6%. A coefficient of interassay variation was 33% (n = 9).

Immunostaining for Light and Electron Microscopy-For light microscopy, porcine pituitary was fixed in 4% paraformaldehyde at 4 “C for 6 h, washed successively with cold phosphate-buffered saline, pH 7.4, containing 10, 15, and 20% sucrose, embedded in O.C.T. com- pound, and frozen in isopentane cooled in liquid nitrogen. Frozen sections (6-pm thickness) were prepared with a cryostat, air-dried on glass slides, and rinsed with phosphate-buffered saline, pH 7.4. En- dogenous peroxidase was inactivated by incubation with 0.3% hydrogen peroxide in methanol at room temperature for 30 min. In the following steps phosphate-buffered saline, pH 7.4, was used for wash- ing the specimen. Immunostaining was performed by the avidin- biotin-peroxidase complex method (23). First, specimens were incubated with 1.8% normal goat serum at room temperature for 30 min and then with 50 ~1 of polyclonal anti-12-lipoxygenase antibody raised in rabbit (ammonium sulfate precipitate, 12 pg protein/ml) or non- immunized rabbit IgG (12 pg of protein/ml) at 4 “C overnight in a moist chamber. Then, biotinylated anti-rabbit IgG antibody (6.8 rg/ ml) was allowed to react with the bound anti-12-lipoxygenase antibody at room temperature for 30 min. Furthermore, a horseradish peroxidase-labeled biotin-avidin complex, which was prepared according to the manufacturer’s instruction, was applied to the specimen at room temperature for 1 h. The specimen were subjected to the peroxidase reaction at room temperature for 5 min in 50 mM Tris-HCl buffer, pH 7.6, containing 0.01% hydrogen peroxide and 0.83 mM 3,3’-diaminobenzidine as substrates. The glass slides were counterstained with Mayer’s hematoxylin for 15 s, rinsed in tap water for 10 min, dehydrated with ethanol, cleared with carboxylol and xylol, and mounted with Entellan Neu dissolved in xylene.

Immunostaining for electron microscopy was almost the same as that described above for light microscopy except for the following modifications. After incubation with a peroxidase-labeled biotin- avidin complex, the specimen were refixed for 5 min with 0.1 M phosphate buffer, pH 7.4, containing 1% glutaraldehyde. The glass &ides were immersed in 0.1 M phosphate buffer, pH 7.4, containing 1% gelatin for 2 min. air-dried. and further refixed with the 1% glutaraldehyde for 5 m:n. After the glass slides were preincubated at room temperature for 30 min with 3,3’-diaminobenzidine only, the peroxidase reaction was performed for 5 min at room temperature in the presence of hydrogen peroxide. Then the glass slides were treated with 2% osmium tetroxide dissolved in 0.1 M phosphate buffer, pH 7.4, at 4 “C for 1 h, dehydrated with ethanol, and embedded in Quetol 812. Ultrathin sections were prepared by an LKB model 8800 ultra- microtome and investigated with a Hitachi H-300 electron micro- scope.

RESULTS

Predominant Occurrence of l.%Lipoxygenase in Porcine Pi- tuitary-The standard peroxidase-linked immunoassay was performed to screen the 12-lipoxygenase contents in various parts of porcine brain. As listed in Table I, the highest enzyme content was found in anterior pituitary. The enzyme level was about 6% of that of peripheral leukocytes which were known as the richest source of 12-lipoxygenase in pig (11). About 10% of the enzyme content of anterior pituitary was found in posterior and intermediate lobes of pituitary which were ex- tirpated together. Other parts of porcine brain tested showed a very low content of 12-lipoxygenase (about 0.1% of that in peripheral leukocytes). Although rat pineal gland was reported to have a relatively high 12-lipoxygenase activity (16), it should be noted that only a low enzyme content was found in porcine pineal gland. The cytosol fraction of most regions of porcine brain listed in Table I was also incubated with 25 PM [Ylarachidonic acid. Only anterior and posterior pituitary showed a detectable 12-lipoxygenase activity.

Catalytic Properties of 12-Lipoxygenase of Porcine Anterior Pituitary-The cytosol fraction of anterior pituitary was incubated with arachidonic acid. The hydroperoxy products were reduced with borohydride and analyzed by reverse-phase HPLC. As monitored at 235 nm for a conjugated diene, a major peak appeared with a retention time of 18.3 min (Fig.


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12-Lipoxygenase of Porcine Anterior Pituitary 2313

TABLE I Distribution of 12-lipoxygenase in porcine brain determined by

enzyme immunoassay The cytosol fraction of each part of porcine brain was prepared,

and the 12-lipoxygenase content was determined by the standard enzyme immunoassay. The cytosol fraction of each tissue was included in various amounts in the assay mixture, and the enzyme content was determined from the linear range of the results. The listed values were corrected for the background determined in the absence of lox-I. Values are means f S. D. for n experiments, each with a sample from several animals; low values presented without S.D. were determined with several samples, and the mean values are listed.

Tissues 12-Lipoxygenase content

ng/mg cytosol protein Neocortex -Cl Corpus callosum 6 Olfactory bulb 5 Hippocampus 4 Caudate nucleus 1 Globus pallidus 4 Thalamus 2 Hypothalamus 6 f 3 (n = 4) Pituitary

Anterior lobe 221 + 116 (n = 6) Posterior and middle lobes 22 + 8 (n = 5)

Pineal body 2 f 1 (n = 3) Cerebellum 3 Cerebellar peduncle 4 Superior colliculus 5 Inferior colliculus 4 Pons 3 Medulla oblongata 7 Peripheral leukocytes 3981 f 1645 (n = 4)

lB), and cochromatographed with authentic 12-HETE (Fig. 1A). A minor peak (retention time, 16.4 min), which was coeluted with authentic SHETE, was also found. A peak corresponding to 5-HETE was hardly detected even in the presence of calcium ion (24) and ATP (25), which are known as 5-lipoxygenase activators. When arachidonic acid was incubated with the cytosol fraction of cerebellum, no significant amount of 12-HETE was detected in sharp contrast to the anterior pituitary (Fig. 1C). The specific 12-lipoxygenase activity of the cytosol fraction of anterior pituitary was calcu- lated from the experimental result presented in Fig. 1B and was found to be 5.0 nmol/5 min/mg protein at 24 “C with arachidonic acid as a substrate. This value corresponded to about 7% of the activity of the leukocyte cytosol. The ratio was close to the above-mentioned value of 6% determined by the enzyme immunoassay. The 12-HETE synthesized by the cytosol fraction of anterior pituitary, was purified by reverse- phase HPLC and then derivatized to its methyl ester. Abso- lute configuration of the 12-HETE methyl ester was determined by chiral-phase HPLC as described in Ref. 26. The ratio of the optical isomers (S/R) was approximately 96:4.

As examined by reverse-phase HPLC monitoring at 235 nm for a conjugated diene of oxygenated products, the cytosol fraction of anterior pituitary was active with the following three octadecapolyenoic acids: linoleic acid, 5.5 nmol/5 min/ mg protein at 24 “C (110% of the activity with arachidonic acid); a-linolenic acid, 4.6 (92%); y-linolenic acid, 5.7 (114%). The products from these three fatty acids were chromato- graphically indistinguishable from the oxygenated products obtained by the purified enzyme of porcine leukocytes (18).

Localization of 12-Lipoxygenase in the Parenchymal Cells of Porcine Anterior Pituitary-The specificity of the polyclonal anti-12-lipoxygenase antibody to be utilized in the following immunohistochemical studies was examined by immunoblotting. The cytosol fraction of porcine anterior pituitary showed a major colored band (Fig. 2). The band appeared at the

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FIG. 1. HPLC analyses of l%-lipoxygenase products from arachidonic acid. Arachidonic acid (25 pM) was allowed to react at 24 “C for 5 min with the cytosol fractions of porcine anterior pituitary (3.5 mg of protein) (B) and cerebellum (4.7 mg protein) (C). The reaction mixture (1.0 ml) contained 50 mM Tris-HCl buffer at pH 7.4 and 2 fiM 13-hydroperoxy-9,11-octadecadienoic acid (added to abolish a lag phase of the 12-lipoxygenase reaction). The borohydride-reduced products were analyzed by reverse-phase HPLC on a Tosoh ODS- 120T column equipped with a Waters HPLC system. The solvent system was a mixture of methanol/water/acetic acid (80:20:0.01) at a flow rate of 1.0 ml/min. A, authentic compounds were applied: 13- hydroxy-9,11-octadecadienoic acid (peak I) derived from the 13- hydroperoxy acid, 15-HETE (peak 2), 12-HETE (peak 3), 5-HETE (peak 4), and 15-hydroxy-11,13-eicosadienoic acid (peak 5) included as an internal standard.

position corresponding to a molecular weight of about 68,000, which was slightly lower reproducibly than that of the purified 12-lipoxygenase of porcine leukocytes (72,000) (18). The band was not detected in the absence of the antibody. The anterior pituitary cytosol was incubated with a monoclonal anti-12- lipoxygenase antibody 10x-2, and 12-lipoxygenase was immu- noprecipitated with the aid of protein A-bearing S. aureus. Immunoblotting of the 12-lipoxygenase-free cytosol fraction thus prepared failed to detect the above-mentioned positive band by the use of the polyclonal antibody. Disappearance of the major band in this experiment supported its identity with 12-lipoxygenase protein.

It was possible that the 12-lipoxygenase contained in porcine pituitary was derived from infiltrating or contaminating leukocytes. Therefore, the localization of 12-lipoxygenase in porcine pituitary was investigated immunohistochemically using the polyclonal anti-12-lipoxygenase antibody. As examined by light microscopy, about 7% of anterior pituitary parenchymal cells found in Fig. 3A were positively stained. These cells were distinguishable from granulocytes in blood vessels, which were also positively stained. No stained cells were found in anterior pituitary in a control experiment with non-immunized rabbit IgG at the same concentration (Fig. 3B). For the immunoabsorption test, the IgG fraction of the anti-12-lipoxygenase antiserum was incubated with the purified leukocyte 12-lipoxygenase at 4 “C overnight, and the IgG fraction thus treated failed to stain the parenchymal cells of anterior pituitary. As shown in Fig. 4, immunoelectron mi-


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66K -

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A B FIG. 2. Immunoblotting of 12-lipoxygenase contained in

porcine anterior pituitary. The cytosol fraction of anterior pituitary (58 pg of protein) (lane A) and the purified 12-lipoxygenase of porcine leukocytes (40 ng protein) (/ane B) were subjected to 7.5% polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyi sulfate (l-mm thick) (37). Electrophoretic transfer of protein bands from the gel to a nitrocellulose membrane (38) and immunostaining with polyclonal anti-12-linoxvaenase antibody were performed as described previously (26): - c’

FIG. 3. Light microscopy of porcine anterior pituitary stained with polyclonal anti-12-lipoxygenase antibody (A) or non-immunized rabbit IgG (B). Bar = 50 pm. Arrow, a granulocyte in blood vessel.

croscopy showed that the positive cells of anterior pituitary contained many cytoplasmic granules, and 12-lipoxygenase was mainly localized in the cytoplasm of the stained cells. In the middle lobe a small number of immunostained cells were found, and they were distinguishable from granulocytes. In contrast, the posterior lobe showed no stained parenchymal cells. Only intravascular granulocytes were immunostained.

FIG. 4. Immunoelectron microscopy of a porcine anterior pituitary cell stained with polyclonal anti- 12-lipoxygenase antibody. Bar = 1 Km. N, nucleus.

DISCUSSION

Our improved peroxidase-linked immunoassay of 12-lipoxygenase allowed the determination of 12-lipoxygenase content in porcine brain, which was impossible by the previous less sensitive method (11). The highest enzyme content was found in anterior pituitary. In agreement with this observation, incubation of the cytosol fraction of porcine anterior pituitary demonstrated the 12-lipoxygenase activity with exogenous arachidonic acid as a substrate. Several previous papers described the conversion of exogenous arachidonic acid to 12- HETE in rat pituitary. Pilote et al. (12) incubated 50 PM [‘“C] arachidonic acid for 10 min with rat anterior pituitary as such. 12-HETE was produced as the major product from added radioactive arachidonic acid in a 0.1% conversion rate. When our group reported a 12-lipoxygenase activity in rat pineal gland, we also detected a very low rate of conversion from 10 FM arachidonic acid to 12-HETE (only less than 0.2%) in rat pituitary (16). Vanderhoek et al. (13) prepared a gonadotroph- enriched cell fraction from rat pituitary. Incubation of this cell fraction with radioactive arachidonic acid produced a variety of metabolites of the cyclooxygenase pathway and several lipoxygenase products including 12-HETE as a minor component. The cyclooxygenase products were predominant by at least 3-4-fold over the lipoxygenase products. In all these earlier works the 12-oxygenation of arachidonic acid was observed to a minor extent, and it was unclear whether such a minor conversion was attributed to the parenchymal cells of pituitary or to the contaminating platelets or leukocytes. Indeed, our recent immunohistochemical work demonstrated the distribution of 12-lipoxygenase in porcine alimen- tary tract and lymphatic organs in addition to leukocytes in peripheral blood. Only various types of resident leukocytes were positively stained in the former organs (19). By the use of cloned tumor cells of rat pituitary, Rabier et al. (15) mentioned the production of 12-HETE (in a yield of 14% of all the products) together with 15-, 5-, and 9-HETEs (66, 11, 9%, respectively) from exogenous arachidonic acid. The production of a mixture of HETEs suggests a lack of stereospec- ificity usually associated with non-enzymic oxygenation. Al- ternatively, a small percentage of 12-HETE could arise as a minor product of a 15-lipoxygenase reaction as is known to occur with the reticulocyte enzyme (27).

Thus, these previous papers described a minor extent of 12- HETE production from a quantitative view and a heteroge- neous composition of products from a qualitative view. In contrast, our present work demonstrated clearly the occurrence of 12-lipoxygenase in porcine pituitary on the basis of not only the arachidonate transformation but also the peroxidase-linked immunoassay. Furthermore, we demonstrated


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12-Lipoxygenase of Porcine Anterior Pituitary 2315

the localization of the I2-lipoxygenase in some parenchymal cells of anterior pituitary rather than contaminating or resident blood cells. We attempted to identify the 12-lipoxygenase-containing pituitary cells by the use of antibodies specific for luteinizing hormone, follicle-stimulating hormone, thy- roid-stimulating hormone, and adrenocorticotropic hormone of porcine origin. We have not yet tested antibodies against growth hormone and prolactin. The use of these antibodies has not clarified as yet whether the 12-lipoxygenase-containing cells are composed of several subgroups each secreting different hormone or they are associated with a group of pituitary cells secreting a specific type of hormone.’

Selective production of S-isomer of 12-HPETE is known as a common feature of mammalian 12-lipoxygenase of platelet (1) and leukocyte (26), whereas cytochrome P-450 was reported to produce predominantly 12R-HETE over 12S- epimer in a ratio of 81:19 (28). Since the 12-lipoxygenase of porcine anterior pituitary produced selectively 12S-HPETE, the enzyme is distinguishable from the cytochrome P-450 as a 12R-hydroxylase, which might be contained in the microsome of porcine anterior pituitary. Capdevila and others (14) reported the production of 12-HETE without a mention of stereochemistry as one of various oxygenated products when the microsome of rat anterior pituitary was incubated with arachidonic acid.

It was proposed that there were two types of 12lipoxygen- ase, a leukocyte type and a platelet type (4,26,29). The high reactivities of anterior pituitary I2-lipoxygenase with linoleic and linolenic acids were similar, if not identical, to the activities of porcine leukocyte 12-lipoxygenase with these octadecapolyenoic acids (18). In contrast, platelet 12-lipoxygenase was almost inactive with these octadecapolyenoic acids (2,26,29). Thus, the 12-lipoxygenase of porcine anterior pituitary is a leukocyte-type enzyme in terms of the catalytic properties. Incidentally, it should be noted that 12-lipoxygenase was not detected in porcine platelets by the enzyme immunoassay and the activity assay (11). It cannot be ruled out that the platelet-type 12-lipoxygenase is also present in the cytosol of porcine anterior pituitary in such a small amount that does not significantly affect the results of substrate specificity study. Incubation of arachidonic acid with the cytosol fraction of various parts of porcine brain except for pituitary did not produce 12-HPETE. Thus, in these areas of porcine brain there was no significant amount of the platelet-type 12-lipoxygenase which was not detected by our enzyme immunoassay. A minor peak of 15-HETE observed upon HPLC (Fig. lB), may indicate the presence of 15- lipoxygenase. An alternative interpretation is the l&oxygen- ase activity of 12-lipoxygenase which was clearly demonstrated with 5-HETE and the porcine leukocyte 12-lipoxygenase (18). In our study on the tissue distribution of 12- and 5- lipoxygenase (11, 30), we have not found a 15-lipoxygenase from various porcine tissues.

Several papers have reported stimulatory effects of arachidonate metabolites on the hormone release from anterior pituitary cells of rut: luteinizing hormone by 5,6-epoxyeicos- atrienoic acid (31), leukotriene Cq (32) and 5-HETE (33), and prolactin by 5-HETE (34). The presence of 12-lipoxygenase in porcine anterior pituitary suggests a possible role of 12- HPETE or its metabolites in the hormone release from porcine pituitary cells.

A previous finding that 12-HETE stimulated the release of luteinizing hormone-releasing hormone from rut median eminence (35) prompted us to carefully examine the 12-lipoxygenase content in porcine median eminence. However, only a

* N. Ueda et al., unpublished observation.

very small amount of the enzyme was detected in the porcine hypothalamic area containing median eminence. Earlier we screened lipoxygenase activities in rut brain tissues using l“C- labeled arachidonic acid. The 12-lipoxygenase activity was by far the highest in pineal gland, and less than 5% of the activity was found in pituitary gland and hypothalamus (16). In rela- tion to this observation, 12-HPETE, but not 12-HETE, stimulated melatonin synthesis in rat pineal gland (36). However, in the present work the enzyme immunoassay showed only a low enzyme content in porcine pineal gland. The cytosol of porcine pineal gland did not show the 12-lipoxygenase activity as examined by incubation with arachidonic acid. Thus, porcine pineal gland does not appear to contain significant amount of 12-lipoxygenase of both the leukocyte-type and the platelet-type.

Acknowledgment-We are grateful to Professor Keiichi Watanabe of Tokai University for his discussions on the identification of anterior pituitary cells.

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