Catecholamines, opioid peptides, and true opiates in the chromaffin cells of the eel: Immunohistochemical evidence

GENERALANDCOMPARATIVEENDOCRINOLOOY

79,393405(1990)

Catecholamines, Opioid Peptides, and True Opiates in the Chromaffin Cells of the Eel: Immunohistochemical Evidence CHARLES B. HATHAWAY AND AUGUST EPPLE Daniel Baugh Institute of Anatomy, Thomas Jefferson University, Philadelphia,

Pennsylvania 19107

Accepted November 1, 1989 An immunohistological analysis of the chromalXm cell system of the American eel revealed the presence of tyrosine hydroxylase (TH) and dopamine-B-hydroxylase (DBH) in all cells. However, phenylethanolamine-N-methyltransferase (PNMT) was seen only in a fraction of the chromaEm cells. This suggests the presence of both norepinephrine and epinephrine cells and the absence of specific dopamine cells. The chromtim cells are most numerous in the anterior region of the posterior cardinal vein, where they occupy a subendothelial position. Their number decreases caudally, and a relatively small number are present in the larger veins of the opisthonephric kidney. No PNMT-positive cells were identified in this region, although a radioenzymatic assay had previously shown the presence of epinephrine. Methionine-enkephalin immunoreactivity seems to be restricted to the chromaffin cells. However, particularly large amounts of leucine immunoreactivity occur in the interrenal cells, with smaller quantities in the chromaflin cells. The chromaffin cells of the eel also contain morphine immunoreactivity. 8 1990academic PESS. IIIC.

The heterogeneity of secretory granule contents in APUD/paraneuron cells (Pearse, 1969; Fujita, 1976) represents a formidable challenge in the study of chroman cell function given the potential for complex interactions between coreleased messenger substances. In the mammalian adrenal medulla, the enkephalins were the first messenger peptides reported (Schultzberg et al., 1978). However, immunohistochemical evidence now indicates that a variety of peptides (cf. Lundberg and Htikfelt, 1983) are stored in adrenal chromafI?n granules, together with catecholamines (CAs) and other amines (Verhofstad and Jiinsson, 1983; Hlippiila et al., 1985). Recently, endogenous morphine and codeine were discovered in mammalian adrenal extracts (Goldstein et al., 1985; Donnerer et al., 1987), suggesting that true opiates may also be colocalized with CAs. Among nonmammalian vertebrates, the only studies of bioactive substances stored with CAs in chromaffin cells have been in

amphibians (Leboulenger et al., 1983; Kondo and Yui, 1984; Delarue et al., 1988). However, preliminary studies now indicate that mammalian-like quantities of enkepha-

TABLE 1 SUMMARYOFCONTROLSFORDOUBLESTAINING INDIREcTI~~MuN~FL~~REX~~NT COLOCALIZATION STUDIES

and 2” antibody combination Sheep 1" + anti-sheep IgG FITC” Sheep 1" + anti-rabbit IgG RITC* NGSc + anti-sheep [email protected] FITC NGS + anti-rabbit IgG RITC NSG + combined FITURITC Rabbit 1” + anti-sheep I@3 FITC Rabbit 1” + anti-rabbit IgG RITC 1”

Fluorescence Fluorescein? Rbodamine? Yes No No

No

No

No

No

No

No No

No No

No

Yes

a Fluorescein isotbiocyanate conjugate. ’ Rbodamine isotbiocyanate conjugate. c Normal goat serum (2%).

393 0016-6480190 $1.50 Copyri& Q 1990by Academic Press, Inc. AU rights of reproduction in any form reserved.

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AND EPPLE

FIG. 1. ChromaRin cells of the eel, demonstrated with the glyoxylic acid histofluorescence technique of De la Torre and Surgeon (1976). (a) The high concentration of these cells in the subendothelial region of the wall of the posterior cardinal vein (x55, enlarged); (b) the scarcity of these cells in the opisthonephric kidney; in this case, they surround a major tributary of the caudalkardinal vein (x35, enlarged). lu, vessel lumen.

FIG. 2. Catecholamine biosynthetic enzymes in subendothelial chromafSm cells of the eel. Single section of the posterior cardinal vein stained for tyrosine hydroxylase/fluorescein (a) and dopamine-g-hydroxylase/rhodamine (b) immunofluorescence (X 50, enlarged). Note the colocalixation of both enzymes. Single section of a posterior cardinal vein tributary within the head kidney stained for tyrosine hydroxylasekluorescein (c) and phenylethanolamine-N-methyl transferase/rhodamine (d) immunofluorescence (x80, enlarged). Note the absence of phenylethanolamine-N-methyl transferase in tyrosine hydroxylase-positive cells.

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CHROMAFFIN

lins (R. M. Dores, personal communication) and true opiates (S. Spector, personal communication) are present in the cardiovascular chromaffin tissue of a teleost fish, the American eel, Anguilla rostrata. Therefore, we sought to determine whether methionine-enkephalin (ME), leucineenkephalin (LE), and morphine/codeinelike immunoreactivity (LIR) were stored in CA-ergic cells in the eel chromaffin tissue. The bulk of the chromafIin tissue in teleost fishes lies in the walls of the posterior cardinal veins (PCVs) and associated head kidney [HK; (Giacomini, 1908; Oguri, 1960; Nandi, 1%1, 1962; Yaron, 1970; Grove et al., 1972; Abrahamsson and Nilsson, 1976; Mastrolia et al., 1981, 1984; Hathaway et al., 1989)]. In the American eel, the anterior region of this PCV/HK complex has recently been shown to contain the adrenal medulla equivalent (Hathaway and Epple, 1989). While distinct norepinephrine (NE) and epinephrine (E) cells have been demonstrated by histochemical and ultrastructural criteria (Yoakim and Grizzle, I980; Mastrolia et al., 1981, 1984, 1989; Gal10 et al., 1988), immunohistochemical differentiation of CA-producing cells in teleost chromaffin tissue has not been reported. Antibodies to the CA biosynthetic enzymes tyrosine hydroxylase (TH; EC 1.14.16.2), dopamine-p-hydroxylase (DBH; EC 1.14.17.1), and phenylethanolamineiv-methyl transferase (PNMT; EC 2.1.1.28), which distinguish cells capable of converting tyrosine to L-DOPA, dopamine (DA) to NE, and NE to E, respectively, have been used in studies of mammalian (e.g., Goldstein et al., 1971; Verhofstad et al., 1979, 1985; McMillen et al., 1988) and amphibian (Nagatsu et al., 1979) adrenal glands. In nonpathological adrenomedul-

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lary tissue, all cells stain positively for DBH, thus indicating that exclusively DAproducing CA-ergic cells do not exist in a tissue believed to be the principal source of systemic DA (Kuchel et al., 1979; Kvetnansky et al., 1979; Planz and Planz, 1979; Unger et al., 1979). TH, DBH, and PNMT activities have previously been demonstrated in extracts of teleost chromaffin tissue (cf. Nilsson, 1983). In the eel, DA represents 2-3% of the total CA content in chromaffin tissue (Hathaway and Epple, 1989) as compared to 0.3% in the rat adrenal medulla (Dalmaz and Peyrin, 1978), and DA plasma titers in the unstressed eel exceed those of NE and E (Epple et al., 1982; Epple and Nibbio, 1985; Hathaway and Eppie, 1989). Therefore, we felt that the immunohistochemical study of CA-ergic cell types in the American eel may provide important answers to the problem of the origin of plasma DA and of plasma NE and E as well. In addition, we were particularly interested in the cellular origin of both enkephalin- and opiate-LIR. MATERIALS

AND METHODS

Animals. Yellow American eels (average weight 400 g) from U.S. Atlantic coast rivers were obtained from a commercial distributor. Animals were anesthetized in a 3% solution of ethyl carbamate (urethane) for 10 rnin prior to removal of tissues. Adrenal glands from pentobarbital-anesthetized Wistar rats (which were subsequently euthanized) were used as positive controls for the presence of CA biosynthetic enzymes in the procedures described below. Catecholamine histofluorescence. The rapid glyoxylic acid (SPG) technique (De la Torre and Surgeon, 1976) was employed to identify monoamine-containing cells in the eel PCVHK and opisthonephric kidney. Freshly dissected, unperfused tissues were frozen in a cryostat (-25”) and 25urn sections were picked up with untreated glass slides. Following exposure for 3-5 set to a solution of 0.2 M sucrose, 0.24 M potassium

FIG. 3. Tyrosine hydroxylase (a) and phenylethanolarnine-N-methyl transferase (c) immunoreactive subendothelial cells (arrows) in adjacent sections of the posterior cardinal vein stained by the peroxidase-anti-peroxidase method. Normal serum substituted for primary antiserum in control section (b). A comparison of TH (a) and PNMT (c) staining shows that not all chromaflin cells produce epinephrine. x50, enlarged.

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phosphate monobasic, and 1% glyoxylic acid monohydride (SPG solution) and incubation at 80” for 5 mitt, slides were examined for fluorescence using a Zeiss IM3S microscope with filter No. 18. A modification of the SPG technique (Rassmussen and Bunney, 1982) incorporates pretreatment of animals with colchicine as a means of distinguishing between CAs (white-blue fluorescence) and serotonin (yellow fluorescence). Only white-blue fluorescent cells were observed in the present study with or without colchicine pretreatment. Zmmunohistochemistry. Anesthetized eels received a transcardiac perfusion of saline followed by either 4% pamformaldehyde or Bouin’s fixative. Tissues were postfixed for 2-3 hr, dehydrated, cleared, and infiltrated with pa&in. Paraflin sections (6-8 pm) were dried at 37”. The peroxidase-anti-peroxidase (PAP) method (Stemberger er al., 1970) using primary rabbit antisera (Eugene Tech International, Allendale, NJ) against TH, DBH, and PNMT was applied to sections of eel

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PCV, HK, opisthonephric kidney, dorsal aorta, and caudal vein. Rabbit antisera against 3carboxymethylmorphine and N-carboxymethylmorphine were generously donated by Dr. Sidney Spector (Roche Institute of Molecular Biology, Nutley, NJ) and applied to sections of eel PCV. Both of the latter antisera react strongly with morphine; however, that against 3carboxymethylmorphine cross-reacts with codeine (Goldstein et al., 1985; Donnerer et al., 1986). Deparafftnized sections were brought to water, washed in phosphate buffer solutions (PBS) with 0.3% Triton and 1% peroxide, and exposed to 10% normal goat serum (NGS) for 45 min. Primary antisera were applied with incubation for 1620 hr at room temperature in the following dilutions with 2% NGS: anti-TH (1:50), anti-DBH (l:lOO), anti-PNMT (l:lOO), and antimorphine (1600). Following rinses in PBS, 60-min incubations with secondary antiserum (goat anti-rabbit IgG, 1:25; Accurate Scientific, Westbury, NY) and rabbit PAP (1:50; Accurate Scientific), and 5- to 15min exposures to 0.05% diaminobenzidine, sections

FIG. 4. Single section of the posterior cardinal vein stained simultaneously for tyrosine hydroxylase/ fluorescein (a) and leucine-enkephalinkhodamine (b) immunofluorescence. Note the colocalization of TH and LE in the subendothelial chromaflin cells. X 100, enlarged.

CHROMAFFIN

were mounted and examined under bright-field illumination. Immunoreactivity was distinguished as a homogenous dark brown precipitate. Two percent NGS substituted for primary antisera in control sections. The basic technique of indirect immunofluorescence (Coons et al., 1955) with a modification for simultaneous differential staining with two primary antibodies was used to determine whether TH was colocalized with DBH, PNMT, or LE in eel chromaftin cells. Single antibody methods were used to determine whether ME was also stored in these cells. Deparaffinized sections were brought to water, washed in PBSmriton, and exposed to 10% normal donkey serum (NDS) for 45 min. For double staining, colocalization studies, sheep anti-TH (1:12; Pel Freez, Brown Deer, WI) and either rabbit anti-DBH (1:13), rabbit anti-PNMT (l:lO), or rabbit anti-LE (1:20; Amersham, Arlington Heights, IL) diluted in 2% NDS were applied together and sections were incubated for 16-20 hr at room temperature. Following PBS rinses, fluorescein isothiocyanate-labeled donkey anti-sheep IgG (FITC) and rhodamine isothiocyanate-labeled donkey anti-rabbit IgG (RITC) secondary antibodies (1:25; Chemicon International, Temecula, CA) were applied together for 60 min. Rabbit anti-Me primary antiserum (1:20; Amersham, Arlington Heights, IL), rabbit anti-DBH, and LE were used singly with RITC secondary antibodies in an otherwise identical scheme for visualization of opioid- and DBH-LIR on adjacent sections of the eel PCV. Two percent NDS substituting for primary antisera and inappropriate primary + secondary antibody combinations were the basis for a series of controls (Table 1). Slides were mounted in glycerin/PBS and examined for epifluorescence with Zeiss IM35 or Axioskop microscopes equipped with interchangeable filters (NOS. 10 and 14) for maximal and separate excitation of fluorescein and rhodamine. Assuming unidirectionality in the CA biosynthetic pathway (L-DOPA -+ DA + NE + E), TH-, DBH-, and PNMT-LIR were considered as evidence of the capacity for DA, DA + NE, and DA + NE + E production, respectively.

RESULTS

The glyoxylic acid method for CA histofluorescence revealed clusters of whiteblue cells lining the walls of the PCV with the largest concentration of cells occurring in the anterior region (Fig. la). Considerably fewer fluorescent cells were observed in the walls of vessels in the opisthonephric kidney (Fig. lb). The immunohistochemical results indicated that numerous cells and cell clusters containing TH-LIR were located in the

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walls of the PCVs and smaller vessels within the HK (Figs. 2-4). Small numbers of such CA-ergic cells were also observed in the walls of venous tributaries of the PCV located in the opisthonephric kidney. Neither the dorsal aorta nor the caudal vein stained positively for TH. Colocalization studies revealed the presence of DBH-LIR in all TH-positive cells (Fig. 2). However, PNMT-LIR was limited to 90% of TH-positive cells in the PCV wall and only 20% of TH-positive cells in HK vessels (Fig. 2; Table 2). Adjacent sections stained for TH and PNMT with the PAP method confirmed the presence of THpositive/PNMT negative cells (Fig. 3). No TABLE

2

TYROSINE HYDROXYLASE (TH) AND PHENYLETHANOLAMINE-N-METHYL TRANSFERASE (PNMT) IMMUNOREACTIVITY IN CELLS OF THE POSTERIOR CARDINAL VEIN (PCV) AND VESSELS WITHIN THE HEAD KIDNEY (HK) Section Anterior 8-23-l

No. TH + cells

% PNMT

cells

cells

PCV

a-23-3 8-23-2 7-27-l 7-27-2 7-29-l 7-29-2 7-25-l 7-25-2

Posterior 7-27-l 7-27-2 7-29-l 7-25-l 7-25-2 7-25-3

No. PNMT+

344

319

93

121 123 60 57 16 14 97 73

115 104 52 50 14 ii

ii 87 88 88 86 89 82

129 102 22 84 64 36

113 81 19 77 57 36

60

Mean 88 SEM 1.2

PCV 88 79 86 92 a9 100 Mean 89

SEM 2.8 Anterior/posterior 8-23-l 8-23-2 7-25-l 7-25-2 7-25-3 7-29-l

HK 23 14 11 24 6 17

3 3 4 5 1 3

NOW. All sections simultaneously stained (FITC) and PNMT (RITC) immunofluorescence.

for

Mean SEM

13 21 36 21 17 18 21 3.2

both

TH

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CHROMAFFIN

PNMT-LIR was observed in the opisthonephric kidney. LE-LIR was observed to be colocalized with TH-LIR in chromatfin cells in the anterior PCV wall (Fig. 4). In addition, both LE- and ME-LIR were found in the same location in cells whose morphology and subendothelial position are identical to that of DBH-positive cells (Fig. 5). However, the most intense immunofluorescence was observed in interrenal cells in LE-labeled sections (Fig. 5). Morphine-LIR (antiN-carboxymethylmorphine) was found in subendothelial cell clusters (morphologically identical with chromaffin cell accumulations) in PAP sections of the anterior PCV (Fig. 6). Antiserum against 3carboxymethylmorphine failed to yield morphine/ codeine-LIR in similar sections. Rat adrenal control sections showed TH-, DBH-, and PNMT-LIR restricted to the medullary region. Sections of the eel PCV (Figs. 3 and 5) and HK, in which normal serum substituted for primary antiserum, showed no staining. Similarly, inappropriate combinations of primary and secondary antibodies used in double staining for colocalization produced neither FITC nor RITC fluorescence (Table 1). DISCUSSION The distribution of cardiovascular chromaflin cells in the American eel, as determined by the immunohistochemical localization of key CA biosynthetic enzymes, agrees with that described by Giacomini (1908) for the European eel, A. anguilla. The results complement our previous findings (Hathaway and Epple, 1989) by demonstrating that the PCV/HK and opisthonephric kidney, major sites of CA storage

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and release, also contain CA-ergic cells. The complete colocalization of TH and DBH in the eel chromaBin cells indicates the absence of exclusively DA-producing cells. However, NE cells (PNMT-negative) and E cells (PNMT-positive) were found in regionally varying percentages (Table 2). One or both of these cell types are probably the source of plasma DA. The absence of PNMT-LIR in the opisthonephric kidney is difficult to explain given the proven storage and release of E in this region (Hathaway and Epple, 1989). The present results may be due to interference of nonspecific staining of renal tubules with the specific staining and subsequent visualization of sparse subendothelial chromaff’n cells. Nevertheless, the possibility remains that E is stored, but not synthesized from NE, in this region. The high density of chromaflin cells in the anterior region of the PCV/HK complex (Figs. 1 and 5) is consistent with the designation of this region as the adrenal medulla equivalent. In addition, despite the lack of close avian- or mammalian-like association between chromaffin and interrenal cells (Fig. 5), both CA- and steroid-producing tissues find their highest concentrations here (see Butler et al., 1969). Whether chromaffin cells in more posterior regions, without interrenal tissue and fewer chromaffin cells (posterior PCV and opisthonephric kidney), can be considered paraganglia equivalents is unclear. Since the typical mammalian paraganglion is not innervated (Muscholl and Vogt, 1964; Bock, 1982), future studies must establish whether nerves supply chromaffin cells which lie outside the eel adrenal medulla equivalent. A strong, cholinergic-type innervation of subendothelial cells in the eel anterior PCV

FIG. 5. Adjacent sections of the posterior cardinal vein showing dopamine-g-hyroxylase (a), methionine-enkephalin (c), and leucine-enkephaiin (d) rhodamine immunofluorescent cells lining the vein wag. Normal serum substituted for primary antiserum in control section (b). Note the intense staining of the three large interrenal cell clusters only in the LE-treated section (d). x80, enlarged.

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wall has been shown (Hathaway et al., 1989). This is the first demonstration in a fish species of enkephalin-LIR in chromaffin cells. In A. rostrata, both LE- and ME-LIR appear to be colocalized with CAs in cells lining the walls of the PCVs. Surprisingly, LE-LIR was concentrated in the eel inter-

AND EPPLE

renal tissue (adrenocortical equivalent). Substantial quantities of LE have also been measured in extracts of the sheep adrenal cortex (Dunlap et al., 1985). In both cases, the function and actual origin of the interrenal/cortical LE are unclear. Given the greater intensity of LE-specific staining in interrenal tissue than in chromaffin tissue,

FIG. 6. Sections of the posterior cardinal vein treated with antiserum against N-carboxymethylmorphine. Clusters of morphine immunoreactive cells (arrowheads) are seen beneath the endothelium of the vein wall. Several large melanophores deep in the vein walls are shown with arrows. a, X 110, enlarged. b, x 160, enlarged.

CHROMAFFIN

one wonders whether LE staining of the PCV wall (Figs. 4 and 5) is the result of antiserum cross-reactivity with ME. Antibodies against both 3-carboxymethylmorphine and Ncarboxymethylmorphine were used in the immunohistochemical analyses; however, only that directed against N-carboxymethylmorphine revealed specific staining. It is conceivable that the three positions of the endogenous opiate molecules are “protected,” perhaps due to the attachment of conjugating sulfate groups (Donnerer et ul., 1987). The question of the presence of codeine in eel chromaffin cells is therefore unresolved. By topographic and morphological criteria, the morphine immunoreactive subendothelial cells of the anterior PCV wall are identical with chromaffin cells of the eel adrenal medulla equivalent. Being the first report of immunohistochemical localization of a true opiate in vertebrate tissues, the presence of morphine in the eel chromaffin cells suggests that the secretory cocktail contained within the chromaffin ceil granule is more complex than was imagined. Clearly, this mixture of amines, peptides, and alkaloid opiates requires functional analysis. Information on the control of their, possibly differential, release and their ultimate physiological roles will contribute to our overall understanding of adrenomedullary sympathetic responses of vertebrates in general. REFERENCES Abrahamsson, T., and Nilsson, S. (1976). Phenylethanolamine-N-methyl transferase (PNMT) activity and catecholamine content in chromaflin tissue and sympathetic neurons in the cod, Gadus morhua. Acta Physiol. Stand. 96, 94-99. Bock, P. (1982). “The Paraganglia.” Springer-Verlag, Berlin. Butler, D. G., Clarke, W. C., Donaldson, E. M., and Langford, R. W. (1%9). Surgical adrenalectomy of a teleost fish (Anguilla rostrata Le Sueur): Effect on plasma cortisol and tissue electrolyte and carbohydrate concentrations. Gen. Comp. Endocrinol. 12, 503-514.

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Coons, A. H., Leduc, E. H., and Connolly, J. M. (1955). Studies on antibody production. 1. A method for the histochemical demonstration of specific antibody and its application to a study of the hyperimmune rabbit. 1. Exp. Med. 102,49-59. Dalmaz, Y ., and Peyrin, L. (1978). Occurrence of dopamine in the chromatBm tissue of a cartilaginous selachian fish: Scyliorhinus canicula. Comp. Biochem. Physiol. C 59, 135-143. Delarue, C., Leboulenger, F., Morra, M., H&y, F., Verhofstad, A. J., Berod, A., Denoroy, L., Pelletier, G., and Vaudry, H. (1988). Immunohistochemical and biochemical evidence for the presence of serotonin in amphibian adrenal chromaBin cells. Brain Res. 459, 17-26. De la Torre, J. C., and Surgeon, J. W. (1976). A methodological approach to rapid and sensitive monoamine histofluorescence using a modified glyoxylit acid technique: The SPG method. Histochemistry 49, 81-93. Donnerer, J., Cardinale, G., Coffey, J., Lisek, C. A., Jardine, I., and Spector, S. (1987). Chemical characterization and regulation of endogenous morphine and codeine in the rat. J. Pharmacol. Exp. Ther. 242, 583-587. Donnerer, J., Oka, K., Brossi, A., Rice, K. C., and Spector, S. (1986). Presence and formation of codeine and morphine in the rat. Proc. Natl. Acad. Sci. USA 82, 5203-5207. Dunlap, C. E., Sundberg, D. K., and Rose, J. C. (1985). Characterization of opioid peptides from maternal and fetal sheep adrenal glands. Peptides 6, 483489. Epple, A., and Nibbio, B. (1985). Catecholaminotropit effects of catecholamines in a teleost fish, Anguilla rostrata. J. Comp. Physiol. B 155,285-290. Epple, A., Vogel, W., and Nibbio, B. (1982). Catecholamines in head and body blood of eels and rats. Comp. Biochem. Physiol. C 71, 111-118. Gallo, V. P., Civinini, A., and Mastrolia, L. (1988). The adrenal homologue of Gasterosteus aculeatus: Fine cytology of interrenal and chromafhm cells. In “14th Conf. Europ. Sot. Comp. Endocrinol.,” Salzburg, Austria. [Abstract] Giacomini, E. (1908). 11sistema interrenale e il sistema cromaffine (sistema feocromo) nelle anguille adulte, nelle cieche e nei leptocefali. Mem. Accad. Sci. 1st. Bologna Cl. Sci. Fis. Ser. 6 5, 407441.

Goldstein, A., Barrett, R. W., James, I. F., Lowney, L. I., Weitz, C. J., Knipmeyer, L. L., and Rapoport, H. (1985). Morphine and other opiates from beef brain and adrenal. Proc. Natl. Acad. Sci. USA 82, 5203-5207. Goldstein, M., Fuxe, K., H&felt, T., and Joh, T. H. (1971). Immunohistochemical studies on phe-

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Kondo, H., and Yui, R. (1984). Co-existence of enkephalin and adrenaline in the frog adrenal gland. Histochemistry 80, 243-246. Kuchel, 0.. Buu, N. T., and Unger, T. (1979). Free and conjugated dopamine; physiological and clinical implications. In “Peripheral Dopaminergic Receptors” (J.-L. Imbs and J. Schwartz, Eds.), pp. 15-27. Pergamon, New York. Kvetnansky, R., Weise, V. K., Thoa, N. B., and Kopin, I. J. (1979). Effects of chronic guanethidine treatment and adrenal medullectomy on plasma levels of catecholamines and corticosterone in forcibly immobilized rats. J. Pharmacol. Exp. Ther. 209, 287-291. Leboulenger, F., Leroux, P., Tonon, M. C., Coy, D. H., Vat&y, H., and Pelletier, G. (1983). Coexistence of vasoactive intestinal peptide and enkephalins in the adrenal cbromaflin granules of the frog. Neurosci. Lett. 37, 221-225. Lundberg, J. M., and H&felt, T. (1983). Coexistence of peptides and classical neurotransmitters. Trends Neurosci. 6, 325-332. Mastrolia, L., Gallo, V. P., and La Marca, A. (1981). Adrenal homologue in Scardinius erthrophthalrnus (Teleostei, Cyprinidae): Light and electron microscopic observations. Boll. Zool. 48, 127138. Mastrolia, L., GalIo, V. P., and La Marca, A. (1984). The adrenal chromafEn cells of Salmo gairdneri Richardson (Teleostei, Salmonidae). J. Anat. 138, 503-5 11.

Mastrolia, L., Gallo, V. P., and Manelli, H. (1989). On the chromaffin cells in fish adrenals. In “XIth International Symposium on Comparative Endocrinology,” Malaga, Spain. [Abstract] McMillen, I. C., Mulvogue, H. M., Coulter, C. L., Browne, C. A., and Howe, P. R. C. (1988). Ontogeny of catecholamine-synthesizing enzymes

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and enkephalins in the sheep adrenal medulla: An immunocytochemical study. J. Endocrinol. 118, 221-226. Muscholl, E., and Vogt, M. (1964). Secretory responses of extramedullary chromaflin tissue. Brit. J. Pharmacol. 22, 193-203. Nagatsu, I., Karasawa, N., Kondo, Y., and Inagaki, S. (1979). Immunocytochemical localization of tyrosine hydroxylase. dopamine-B-hydroxylase and phenylethanolamine-N-methyl transferase in the adrenal glands of the frog and rat by a peroxidaseantiperoxidase method. Histochemistry 64, 131144. Nandi, J. (1961). New arrangement of inter-renal and chromaffin tissues of teleost fishes. Science 134, 389-390.

Nandi, J. (1%2). The structure of the interrenal gland in teleost fishes. Univ. Calif. Publ. Zool. 65, 129 212. Nilsson, S. (1983). “Autonomic Nerve Function in the Vertebrates.” Springer-Verlag, Berlin. Oguri, M. (1960). Studies on the adrenal glands of teleosts: On the distribution of chromafiin cells and interrenal cells in the head kidneys of fishes. Bull. Japan. Sot. Sci. Fish. 26, 443-447. Planz, G., and Planz, R. (1979). DopamineBhydroxylase, adrenaline, noradrenaline and dopamine in the venous blood of adrenal gland of man: A comparison with levels in the periphery of the circulation. Experientia 35, 207-208. Rassmussen, S. A., and Bunney, B. S. (1982). A modification of the rapid glyoxylic acid technique permits visualization of serotonergic and hypothalamic dopaminergic neurons. J. Neurosci. Methods 6, 139-144. Schultzberg, M., Lundberg, J. M., H&felt, T., Terenius, L., Brandt, J., Elde, R. P., and Goldstein, M. (1978). Enkephalin-like immunoreactivity in gland cells and nerve terminals of the adrenal medulla. Neuroscience 3, 1169-l 186. Stemberger, L. A., Hardy, P. H., Cuculis, J. J., and Meyer, H. G. (1970). The unlabeled antibody enzyme method of immunohistochemistry: Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identilication of spirochetes. J. Histochem. Cytochem. 18, 315-333. Unger, T., Butt, N. T., and Kuchel, 0. (1979). Renal and adrenal dopamine balance: Implications for the role of conjugated dopamine. In “Peripheral Dopaminergic Receptors” (J.-L. Imbs and J. Schwartz, Eds.)., pp. 357-367. Pergamon, New York. Verhofstad, A. A. J., Coupland, R. E., Parker, T. R., and Goldstein, M. (1985). Immunohistochemical and biochemical study on the development of the noradrenaline- and adrenaline-storing cells of the

CHROMAFFIN adrenal medulla of the rat. Cell Tissue Res. 242, 233-243. Verhofstad, A. A. J., Hbkfelt, T., Goldstein, M., Steinbusch, H. W. M., and Joosten, H. W. J. (1979). Appearance of tyrosine hydroxylase, aromatic amino-acid decarboxylase, dopamine-Shydroxylase, and phenylethanolamine N-methyl transferase during the ontogenesis of the adrenal medulla: An immunohistochemical study in the rat. Cell Tissue Res. 200, 1-13. Verhofstad, A. A. J., and Jonsson, G. (1983). Immu-

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