IMMUNOHISTOCHEMICAL LOCALIZATION OF CATECHOLAMINE SYNTHESIZING ENZYMES IN BULLFROG ADRENALS1 IKUKO NAGATSU, HIDEKI KOJIMA*, SHINOBU INAGAKI,

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1 ACTA HISTOCHEM. CYTOCHEM. Vol. 12, No. 5, 1979 IMMUNOHISTOCHEMICAL LOCALIZATION OF CATECHOLAMINE SYNTHESIZING ENZYMES IN BULLFROG ADRENALS1 IKUKO NAGATSU, HIDEKI KOJIMA*, SHINOBU INAGAKI, YUKARI KONDO AND TOSHIHARU NAGATSU** Department of Anatomy. School of Medicine, Fujita-Gakuen University, Toyoake, Aichi , Institute of Brain Diseases*, Kurume University School of Medicine, Kurume 830, Laboratory of Cell Physiology**, Department of Life Chemistry, Graduate School at Nagatsuta, Tokyo Institute of Technology, Yokohama 227 Received for publication March 5, 1979 Frog adrenal chromaffin cells have very long processes. These chromaffin cells are scattered in cortical lipid cells and sometimes in summer cells. By immunofluorescent method, we first confirmed tyrosine hydroxylase (TH)- positive, dopamine-Ĉ-hydroxylase (DBH)-positive, phenylethanolamine-nmethyltransferase (PNMT)-positive adrenaline cells, and TH-positive, DBHpositive, PNMT-negative noradrenaline cells in frog adrenal glands. The activities of TH, DBH and PNMT in the adrenals of the bullfrogs were 215, and 302 pmoles/min/mg protein, respectively. Acidophilic summer cells have neither TH, DBH nor PNMT. These results were reconfirmed by fluorescence microspectrophotometry. It is well known that amphibian adrenals consist of four types of cells: chromaffin cells, lipid cells, acidophilic summer cells and occasional ganglion cells. In the past, two types of chromaffin cells in the adrenal medulla of various vertebrate species (8) were correlated with two different types of hormones (adrenaline and noradrenaline) secreted from the adrenal medulla (1). Using the glutaraldehyde-osmium method, Coupland and Hopwood (3) proposed that adrenaline and noradrenaline were secreted by separate cells in the frog adrenal (Rana esculenta). In the adrenals of the toad and bullfrog, two types of chromaffin cells were reported by Piezzi (18) and Takaya (22). Nakamura (17) and Suzuki (21) reported that the acidophilic summer cells (20) may originate from the lipid cells. More recently, the cellular localization of catecholamines in bullfrog adrenals was clarified by using the Falck-Hillarp fluorescence histochemical method for the demonstration of monoamines (7). This report suggested that there were two types of chromaffin cells containing catecholamines (noradrenaline and adrenaline), acidophilic summer cells and sympathetic ganglion cells containing adrenaline in the adrenals of bullfrogs. It was therefore expected that the distribution of enzymes 1 This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture (No ). 416

2 LOCALIZATION OF TN, DBH AND PNMT IN BULLFROG ADRENALS 417 involved in catecholamine biosynthesis would correspond to that of catecholamines in bullfrog adrenals. The present study was carried out in order to demonstrate the cellular localization of three catecholamine synthesizing enzymes, tyrosine hydroxylase (TH), dopamine-ƒà-hydroxylase (DBH) and phenylethanolamine-n-methyltransferase (PNMT), in bullfrog adrenals by means of immunofluorescence histochemistry, in combination with biochemical assay of these three enzymes and with fluorescence microspectrophotometry (2). Preliminary data of this work were reported earlier (13). MATERIAL AND METHODS Immunofluorescent techniques: About 10 bullfrogs (Rana catesbeiana, g body weight) of both sexes were used for the experiment. Adrenals were freshly obtained, immersed in 2 or 4% paraformaldehyde in phosphate buffer (0.1 M, ph 7.2) for 4 or 1 hr, respectively. After washing in the buffer overnight, 10ƒÊm sections were cut on a cryostat. Some adrenals were freshly frozen, and after cutting sections were fixed in chloroformmethanol (2:1). 10ƒÊm sections were used for indirect immunofluorescence staining (12) using either specific antisera to bovine adrenal TH, DBH and PNMT or preimmune serum taken from rabbits (13, 15, 16). Photographs were taken as previously described (10). Fluorescence microspectrophotometry (2) was performed with a Zeiss MPMOI. Biochemical assays: The bullfrog adrenals were homogenized in 100 times of 0.1 M phosphate buffer (ph 7.4) containing0.1% Cutscum (isooctyl phenoxypolyethoxyethanolcontaining detergent, Fisher Scientific Co.) and centrifuged at 1,000 g for 20min, and the supernatant was used as the enzyme preparation. TH activity was assayed by measuring the formation of [14C]-DOPA from L-[14C-tyrosine (11). The incubation mixture (14, 16) (total volume, 250,ƒÊl) contained:200mm Tris-acetate buffer (to give a final ph of 6.0), 1 mm FeSO4, 50ƒÊl of enzyme solution, 100 mm mercaptoethanol, 1 mm 6-methyltetrahydropterin, and 100ƒÊM L-[14C]-tyrosine (0.07, ƒêci). Incubation was carried out at 30 Ž for 15 min in air with constant shaking. DBH activity was assayed by measuring the formation of octopamine from tyramine by sensitive dual-wavelength spectrophotometry (5). The incubation mixture (16) (total volume, 1.0 ml) contained: 400ƒÊl of the diluted enzyme solution, 0.2 M acetate buffer (ph 5.0), 10mM N-ethylmaleimide, 1, ƒêm CuSO4, 10mM sodium fumarate, 10 mm ascorbic acid, 1,500 units (50ƒÊg) of catalase, and 20mM tyramine hydrochloride. 400,ƒÊ1 of the diluted enzyme solution containing 0.1mM of fusaric acid (as a final concentration) served for the blank (6). Incubation was carried out for 45 min at 37 Ž in air with constant shaking. PNMT activity was assayed by the method of Saavedra et al. (19). 40ƒÊl of enzyme were incubated at 37 Ž for 30 min in a total volume of 200ƒÊl containing: 125ƒÊl of 0.2M Tris-HCL buffer, ph 8.6, 1.25 mm phenylethanolamine (Regis Chemical Co.), and 5 nmole (0.2,ƒÊCi) of S-adenosyl-L-[methyl-3H] methionine

3 418 NAGATSU ET AL. (specific radioactivity, 10.5 Ci/mmol, New England Nuclear Corp.). Blanks were prepared by omitting phenylethanolamine from the incubation mixture or by adding phenylethylamine instead of phenylethanolamine. Protein was measured by the method of Lowry et al. (9), as modified by Hartree (4). Student's t-test was used to determine the significance of the difference between means. The measure of variation used throughout was the standard error of the mean. RESULTS When the sections of bullfrog adrenals were incubated with antibody to TH, specific immunofluorescence reaction products were observed in the adrenal medullary gland cells (Fig.1). The diffuse reaction products with antibody to TH were evenly distributed in their cytoplasm. TH-positive chromafin cells were seen solitarily and some cells tended to make big clusters. The cell bodies and their long processes of TH-positive chromaffin cells were noticed sometimes adjacent to blood vessels. However, specific reaction products were found neither in the lipid cells nor in acidophilic summer cells. Furthermore, cellular localization of DBH in the adrenals of the bullfrog was examined by using antibody to DBH. Distribution of DBH-positive reaction products (Fig.2) coincided with that of TH. On the other hand, using antibody to PNMT, bullfrog adrenal chromaffin cells were divided into two types: one type showing PNMT-positive (presumably the adrenaline-producing cells), and the other type showing PNMT-negative (the noradrenaline-producing cells), as shown in Fig. 3. PNMT-positive chromaffin cells with processes were observed, but PNMT-positive chromaffin cell clusters could not be found. No specific reaction product was found in bullfrog adrenal sections incubated with preimmune rabbit serum (Fig. 4). Although a more intense background was observed when paraformaldehyde TABLE1. Fluorescence intensity (arbitrary units) of immunofluorescence measured by fluorescence microspectrophotometry (Mean }SEM) FIGS Immunofluorescence photomicrographs of bullfrog adrenals. ~340. Specific immunofluorescence to TH (Fig. 1) or to DBH (Fig.2) was seen in solitary and clustered chromaffin cells and their processes. Some chromaffin cells were localized adjacent to blood vessels. The arrow shows a chromaffin cell which has PNMT even in a long process, and the arrow head shows a cluster of chromaffin cells which do not have PNMT immunofluorescence (Fig. 3). When preimmune serum was used instead of antisera, non-specific fluorescence was barely seen (Fig. 4).

4 LOCALIZATION OF TH, DBH AND PNMT IN BULLFROG ADRENALS

5 420 NAGATSU ET AL. 3 4

6 LOCALIZATION OF TH, DBH AND PNMT IN BULLFROG ADRENALS 421 fixed tissues were measured, fluorescence microspectrophotometry indicated that catecholamine-synthesizing enzymes were localized only in chromaffin cells, not in summer cells and cortex cells (Table 1). Activities (mean }SEM) of TH, DBH and PNMT in the adrenals of the bullfrog were 215 }30, }1194 and 302 }34 pmoles/min/mg protein, respectively. DISCUSSION Cellular localizations of three catecholamine synthesizing enzymes, TH, DBH and PNMT, in bullfrog adrenals were observed in the present study using specific antisera to bovine adrenal TH, DBH and PNMT. Adrenal chromaffin cells of the bullfrog were markedly positive when stained to TH and DBH antisera. In addition, some chromaffin cells showed PNMT-positive staining and others negative. These results support the view (7, 18, 22) that there are two distinctly different types of chromaffin cells containing noradrenaline and adrenaline in the adrenals of the bullfrog. According to our previous report (13), the cellular characteristics of catecholamine synthesizing enzymes in adrenal chromaffin cells appear to be identical to those in small intensely fluorescent cells in the bullfrog sympathetic ganglia. On the other hand, the absence of TH-, DBH-, and PNMT-containing acidophilic summer cells observed in the present study cannot support the fluorescence histochemical data (7) that acidophilic summer cells contain adrenaline. This discrepancy may be explained by the possibility that summer cells take up and store catecholamines from intercellular space. This, acidophilic summer cells (20) are confirmed to originate from the cortical lipid cells, based on the absence of catecholamine synthesizing enzymes. It was very difficult to observe ganglion cells in the immunofluorescence photographs. Therefore, the possibility of the existence of ganglion cells which contain adrenaline (7) in bullfrog adrenals remains for further investigation. ACKNOWLEDGEMENT The authors wish to thank Mr. N. Karasawa, Mr. S. Kitta and Miss T. Ohno for their technical assistance. REFERENCES 1. Bander, A.: Uber zwei verschiedener chromaffine Zellen in Nebennierenmark und ihre Bezichung zum Adrenalin- und Arterenolgehalt. Verh. Deut. Anal. Ges. 48; , Caspersson, T., Hillarp, N.-A. and Ritzen, M.: Fluorescence microspectrophotometry of cellular catecholamines and 5-hydroxytryptamine. Exp. Cell Res. 42; , Coupland, R. E. and Hopwood, D.: The mechanism of differential staining reaction for adrenaline- and noradrenaline-storing granules in tissues fixed in glutaraldehyde. J. Anat. (London) 100; , Hartree, E. F.: Determination of protein: A modification of the Lowry method that gives a linear photometric response. Anal. Biochem. 48; , Kato, T., Kuzuya, H. and Nagatsu,T.: A simple and sensitive assay for dopamine-Ĉ-hydroxylase activity by dual-wavelength spectrophotometry. Biochem. Med. 10; , 1974.

7 422 NAGATSU ET AL. 6. Kato, T., Wakui, Y. and Nagatsu, T.: An improved dual-wavelength spectrophotometric assay for dopamine-Ĉ-hydroxylase. Biochem. Pharmacol. 27; , Kojima, H., Kuwahara, M., Anraku, S., Onogi, K. and Ito, R.: Fluorescence histochemical and chemical analyses of catecholamines in bullfrog adrenals. Acta histochem. cytochem. 10; , Kolmer, W.: Zur vergleichenden Histologie, Zytologie und Entwicklungsgeschichte der Saugernebenniere. Arch. mikrosk. Anat. 91; 1-139, Lowry, O.H., Rosebrough, N.J., Farr, A. L. and Randall, R.J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193; , Nagatsu, I.: Localization of dopamine-Ĉhydroxylase in bovine adrenal gland and rat sciatic nerves by the improved enzyme-immunocytochemical and enzyme-immunofluorescent methods. Acta histochem. cytochem. 7; , Nagatsu, T.: Assay of tyrosine hydroxylase activity. In Biochemistry of Catecholamines. The Biochemical Method, University Park Press, Baltimore, 1973, pp Nagatsu, I., Hartman, B. K. and Udenfriend, S.: The anatomical characteristics of dopamineĈ -hydroxylase accumulation accumulation in ligated sciatic nerve. J. Histochem. Cytochem. 22; , Nagatsu, I., Kojima, H., Kondo, Y., Inagaki, S. and Nagatsu, T.: Histochemical and biochemical characterization of the bullfrog lumbar sympathetic ganglia and sciatic nerves. In Catecholamines: Basic and Clinical Frontiers, ed. by E. Usdin et al., Pergamon Press LTD, Oxford, 1979, p Nagatsu, I. and Kondo, Y.: Studies on axonal flow of dopamineĈ-hydroxylase and tyrosine hydroxylase in sciatic nerves by immunofluorescent and biochemical methods. Acta histochem. cytochem. 8; , Nagatsu, I., Kondo, Y., Inagaki, S., Karasawa, N., Kato, T. and Nagatsu, T.: Immunofluorescent studies on tyrosine hydroxylase: Application for its axoplasmic transport. Acta histochem. cytochem. 10; , Nagatsu, I., Kondo, Y., Inagaki, S., Kojima, H. and Nagatsu, T.: Immunofluorescent and biochemical studies on tyrosine hydroxylase and dopamine-Ĉ-hydroxylase of the bullfrog sciatic nerves. Histochemistry 61; , Nakamura, M.: The seasonal variations in the adrenal cortex cells of bullfrog, with special remark to the origination of the summer cell. Endocrinol. Japon. 14; 47-59, Piezzi, R. S.: Chromaffin tissue in the adrenal gland of the toad, Bufo arenarum Hensel. Gen. comp. Endocrinol. 9; , Saavedra, J. M., Coyle, J. T. and Axelrod, J.: The distribution and properties of the nonspecific N-methyltransferase in brain. J. Neurochem. 20; , Stilling, H.: Zur Anatomie der Nebennieren. Archiv.f. mikroskop. Anatomie. 52; , Suzuki, N.: Changes in the adrenocortical cells of bullfrogs following hormonal stimulation. Endocrinol. Japon. 15; 82-93, Takaya, K.: Two types of chromaffin cells in the adrenals of the bullfrog, Rana catesbiana. A comparative study. Arch. histol. jap. 32; 69-80, 1970.