Effects of Inhibitors of Catecholamine-Synthesizing Enzymes on the Mouse Adrenal Medulla

ACTA HISTOCHEM. CYTOCHEM. Vol. 5 No. 2, 1972

EFFECTS OF INHIBITORS OF CATECHOLAMINE- SYNTHESIZING ENZYMES ON THE MOUSE ADRENAL MEDULLA

IKUKO NAGATSU, YUMIKO SOTOKAWA* and MASAO SANO *

Department of Anatomy and Physiology, Aichi Prefectural Collegeof Nursing, ands Department of Anatomy, School of Dentistry, Aichi-Gakuin University, Nagoya

Received for publication March 16, 1972

Oudenone (a tyrosine hydroxylase inhibitor) and fusaric acid (a dopamine- β-hydroxylase inhibitor) had some effects on fine structures of the adrenal medulla. The Golgi apparatus was not seen to be well developed, and the formation of epinephrine and norepinephrine granules appeared to be decreased. The limiting membrane of norepinephrine granules showed a tendency of fusing each other, and the density of epinephrine granules was seen to be decreased. Both epinephrine and norepinephrine granules showed a reduction in size of their dense core. It is concluded that oudenone and fusaric acid may have some inhibjtory effects on epinephrine and norepinephrine biosynthesis in the mouse adrenal medulla. These results agree well with the biochemical data presented previously.

Specific and potent inhibitors of the enzymes involved in catecholamine biosynthesis have been recently discovered from the culture media of microorganisms by Umezawa et al (7, 11, 12, 13, 17). Oudenone ( (S)-2-[4, 5-dihydro- 5-propyl-2 (3H)-furylidene]-1, 3-cyclopentanedione) (12, 13, 17) is an inhibitor of tyrosine hydroxylase which was isolated from the culture filtrate of Oudenansiella radieata and shown to inhibit the enzyme in vivo to reduce the endogenous level of catecholamines. Fusaric acid (5-butylpicolinic acid) is an inhibtor of dopamine- ,3-hydroxylase which was isolated from the culture filtrate of a fungus of Fusarium species and shown to be a potent inhibitor of the enzyme in vitro and in vivo (7, 11). Both oudenone and fusaric acid were specific and competitive-type inhibitors. Since these inhibitors were active in vivo to reduce the endogenous level of tissue catecholamines, it was of our interest to examine their effects on fine structures of the mouse adrenal medulla.

MATERIALS AND METHODS

Twenty adult male mice (SMA strain) were divided into 3 groups : A) control, B) oudenone-administered, and C) fusaric acid-administered mice. Animals of B and C groups were injected intraperitoneally with 3 mg of oudenone and fusaric acid dissolved in 0.3 ml of distilled water and were sacrificed at 6 and 3 hours after

71 72 NAGATSU, SOTOKAWA AND SANO the injection, respectively. Perfusion fixation was made with 4% paraformaldehyde and 2.5% glutaraldehyde mixture in 0.1 M phosphate buffer (pH 7.4) (8) from the left ventricle for 10 min. Then adrenals were taken out and cut into small blocks and fixed in the same mixture for 3 hours at room temperature. The specimens were washed overnight at 4°C with 0.1 M' phosphate buffer (pH 7.4) containing 7% sucrose. Postfixation was carried out with 1% osmium tetroxide in Palade's buffer (14) including 7% sucrose for 2 hours at 4°C, and then the specimens were stained en block with uranyl acetate (8). The fixed tissues were dehydrated in graded series of acetone, and embedded in Epon 812. Thin sections were cut on a Porter-Blum ultramicrotome, stained with uranyl acetate and lead citrate (15), and examined with a JEM-T7 electron microscope.

RESULTS

By the double fixation method of glutaraldehyde and osmium tetroxide, it is possible to distinguish the differences in the morphology and stainability between epinephrine cells and norepinephrine cells (1, 2, 16, 18). Following these criteria, adrenal medullary cells of a control mouse were shown in Figs. 1 and 2. Epinephrine cells have round or oval granules tightly packed with electron dense material showing a fine internal granularity. Many granules are tightly sur- rounded by the limiting membrane while some are loosely covered showing a wide clear halo under the membrane. Norepinephrine cells have granules containing homogeneously electron dense substance. In many cases the dense core is ec- centrically located within the vesicle and these granules show an asymmetrically vacuolated appearance. As shown in the previous reports (3, 19), both types of cells have a round or oval nucleus containing a prominent nucleolus. Mitochondria are scattered at random. The endoplasmic reticulum studded with ribosomes

FiG. 1. Adrenal medullary cells of a control mouse. Epinephrine cells (E) and norepinephrine cells (N) are shown. X 5,500. EFFECTS OF INHIBITORS OF CATECHOLAMINE-SYNTHESIZING ENZYMES 73

is in a parallel array or in an irregular fashion. Somewhat wide space of the cytoplasm is occupied with the Golgi complex. The cells of the adrenal medulla from the mice which were injected with oudenone or fusaric acid showed some morphological changes. 1. Golgi apparatus There were flattened sacs, large vacuoles, and small and abundant vesicles in the Golgi complex of cells of the control adrenal medulla (Fig. 2, 4). Some of these vesicles contained dense substance. In the region of the Golgi complex all stages of development of the catecholamine droplet were found. The mature catecholamine granules were rich in the peripheral region of the cytoplasm and some were localized in the close vicinity of the cell membrane.

FIG. 2. Golgi complex of the adrenal medulla of a control mouse. Flattened sacs, large vacuoles, and small and abundant vesicles are observed. Some of these vesicles have dense substance. The mature catecholamine granules are localized in the close vicinity of the cell membrane. X 16,000. On the other hand, many adrenal medullary cells in animals administered oudenone had a light appearing Golgi area with flattened sacs, large vacuoles and a few vesicles (Fig. 3) which had no dense content. Near the Golgi region was found few immature granules. In the case of fusaric acid (Fig. 4), the change of Golgi area was similar in general to that in the case of oudenone. 2. Catecholaminegranules After the treatment with oudenone or fusaric acid, the membrane of the catecholamine granules tended to make fusion with each other and the content of the granules decreased in density. The clear space below the membrane considerably increased in width and the dense core of the granule decreased in size, and very frequently there were seen empty droplets in the cytoplasm (Fig. 5, 6). Such a fusion was striking after fusaric acid injection (Fig. 7). To measure the diameters of catecholamine granules they were picked up at 5,000 A interval along the scanning lines on the electron micrographs. The 74 NAGATSU, SOTOKAWA AND SANO

FIG. 3. Golgi complex of epinephrine cells after the administration of oudenone. A light appearing Golgi area is seen with flattened sacs, large vacuoles and a few vesicles. .x 16,000.

FIG. 4. Golgi complex of a norepinephrine cell after the administration of fusaric acid. x 16,000.

FIG. 5. Adrenal medulla of the mouse after the administration of fusaric acid. The clear space below the limiting membrane of catecholamine granules is increased considerably. X 5,500. EFFECTS OF INHIBITORS OF CATECHOLAMINE-SYNTHESIZING ENZYMES 75

Fso. 6. Epinephrine granules of the adrenal medulla of a mouse after the administration of oudenone. The dense core of granules is seen to be decreased in size. A nerve terminal is seen under right corner. x 12,000.

Fm. 7. Epinephrine granules of the adrenal medulla of a mouse after the administration of fusaric acid. The membranes of epinephrine granules tend to make fusion with each other and the content of the droplets is decreased in density. x 12,000. size of a catecholamine granule was expressed by an outer diameter (between granular membranes) and inner diameter (diameter of a dense substance). The results were summarized in Table 1. In control animals mean outer diameter of 743 catecholamine granules in 6 epinephrine cells were 2553.20 A. In the case of norepinephrine cells, mean outer diameter of 787 granules in 7 cells were 2127.22 A. The inner diameter of 743 epinephrine granules was 1445.13 A in average and that of 787 norepinephrine granules was 1033.84A in average. When oudenone was administered, the outer and inner diameters of 1848 epinephrine granules of 10 cells were 2667.29 A and 1205.61 A in average, respectively. In the case of 406 norepinephrine granules of 6 cells, the outer and inner diameters were 2268.17 A and 899.28 A in average, respectively. These results show that the inner diameters of the granules in either epinephrine or norepinephrine cells were decreased, while their outer diameters did not change. In the case of fusaric acid treatment, mean outer and inner diameters of 684 epinephrine granules of 8 cells were 2254.18 A and 961.65 A, respectively. Those of 497 norepinephrine granules of 6 cells were 2622.64 A and 861.86 A, respectively. These results were similar to those by oudenone, and the inner dense material had a tendency to be decreased. In norepinephrine cells, there were seen some differences between the results of oudenone and fusaric acid treatments. In the case of oudenone, outer diameter was not changed so much as in control, but in the case of fusaric acid, outer diameter had some tendency to be increased. 76 NAGATSU, SOTOKAWA AND SANO

3. Other observations In the control adrenal medulla , nerve terminals were seen occasionally on epinephrine or norepinephrine cells (Fig. 6). In a case, a large, intricately lobulated nerve terminal was found on epinephrine cells (Fig. 8). These terminals had almost spherical synaptic vesicles about 400 A in diameter , as well as a few of the cored vesicnles about 800 A in diameter. Following such perfusion studies of adrenal glands which indicated the excretion of the contents of the catecholamine granules into the perfusate , the excretion mechanism of catecholamine granules from adrenal medulla is thought to be due to exocytosis (4). Therefore, efforts were taken to find exocytotic figures, but it was very difficult to find typical figures so far. Fig. 9 appears to show a kind of exocytosis, although it is not a typical one.

FIG. 8. A large, lobulated nerve terminal is found between epinephrine cells. It has almost spherical synaptic vesicles about 400A in diameter, as well as a few of the cored vesicles about 800 A in diameter. x 16,000. FIG. 9. Exocytotic figure of an epinephrine granule. An epinephrine granule (arrow) is located just close to the cell membrane and a small pore was open between the membrane of the catecholamine granule and the cell membrane. The content of the catecholamine granule is still left in the granule. The other empty vesicle (double arrow) is thought to have excreted the content of the granule. x 16,000.

Centriol and cilia were found in either epinephrine or norepinephrine cells near the Golgi area. The pattern of these cilia was 9+0 type (cf. Fawcett (6) ). Desmosomes were sometimes found between two epinephrine cells or between two norepinephrine cells. Usually desmosomes were not present between epinephrine and norepinephrine cells. The swelling of mitochondria was usually found in such cells where the catecholamine granules were enlarged and the inner cores were small. In such cells EFFECTS OF INHIBITORS OF CATECHOLAMINE-SYNTHESIZING ENZYMES 77 lysosomal bodies were noted occansionally in epinephrine cells and norepinephrine cells.

DISCUSSION

Both oudenone and fusaric acid were proved to be specific and potent inhibitors of catecholamine biosynthesis. Both compounds reduced the endogenous level of tissue catecholamines and blood pressure of animals (11, 12). The site of inhibition of catecholamine biosynthesis by oudenone and fusaric acid are different. Oudenone is a specific inhibitor of tyrosine hydroxylase, while fusaric acid as a specific inhibitor of dopamine-/fI-hydroxylase. Tyrosine hydroxylase is thought to be the rate-limiting step in the biosynthesis of catecholamines (10), but dopamine- /1-hydroxylase can be also the rate-limiting step in certain circumstances. It was reported that when a dose of oudenone equivalent of 100 mg/Kg body weight was injected into a rat intraperitoneally tyrosine hydroxylase activity and catecholamine contents in adrenal glands were decreased up to 67% and 69% at hours after the injection, respectively (12). It was also shown that at 3 hours after the intraperitoneal injection of fusaric acid (100 mg/Kg body weight) dopamine-,9-hydroxylase activity and catecholamine content in adrenal medulla were decreased up to 19% and 2700, respectively (11). Therefore, the amount of oudenone or fusaric acid applied in the present experiment is expected to be enough to inhibit the biosynthesis and to reduce the endogenous level of catecholamines. The morphological findings reported in this study also indicated the reduction of tissue catecholamines probably due to the inhibition of synthesis. After the administration of either oudenone or fusaric acid the density of epinephrine or norepinephrine granules was seen to be decreased. It was of interest that the effects of oudenone and fusaric acid were different from each other. Changes in the diameters of the inner dense material and the outer membrane of catecholamine granules after the administration of oudenone and fusaric acid were examined statistically (Table 1). The inner dense material

TABLE 1 Diameter of catecholamine granules

* The differences were statistically significant in comparison with the control. 78 NAGATSU, SOTOKAWA AND SANO of catecholamine granules was found to be decreased by both oudenone and fusaric acid, but the outer diameter (size) of norepinephrine granules appeared to be increased by fusaric acid. Dopamine-9-hydroxylase was found to be localized in the granules (9), and the inhibition to this enzyme by fusaric acid may have some effects on the membranes of the granules. The finding that the Golgi apparatus was not seen to be well developed after the administration of oudenone or fusaric acid suggests an inhibition of the formation of epinephrine or norepinephrine granules, but the mechanism is not clear. This problem remains for further investigation.

ACKNOWLEDGEMENTS

The authors are grateful to Prof. Hamao Umezawa (Institute of Microbial Chemistry, Tokyo) for his generous supply of oudenone and fusaric acid. The valuable assistance of Miss Shigeko Iida (Department of Anatomy and Physiology, Aichi Prefectural College of Nursing) is gratefully acknowledged.

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