Case Report Regular immunohistochemical localization of endothelin-1 and endothelin-B receptor in normal, hyperplastic and neoplastic human adrenocortical cells
Hiroyuki Hiraki,1 Nobuo Hoshi,1 Hiroshi Hasegawa,1 Toshitaka Tanigawa,2 Iwao Emura,3 Tsutomu Seito,4 Toshifumi Yamaki,1 Takeaki Fukuda,1 Kazuo Watanabe1 and Toshimitsu Suzuki1
“Department of Pathology, Fukushima Medical College, Fukushima, 2Department of Urology, 3Department of Surgical Pathology, Niigata University School of Medicine, Niigata and ^Immunobiological Laboratories, Gunma, Japan
The localization of endothelin (ET)-1/big ET-1, ET-3/big ET-3, ET-A and ET-B receptor was immunohistochemically exam- ined in human adrenal glands composed of 36 normal cases, nine hyperplasia, 70 adenomas and seven carcinomas of cortical cells. In normal adrenals, ET-1/big ET-1 and ET-B receptor were regularly detected in the cortical cells, espe- cially in the zona fasciculata for ET-1 and zona glomerulosa for ET-B receptor but not in the medulla, while ET-A receptor localized occasionally in endothelial cells or rarely in cortical cells and ET-3/big ET-3 was very limited In the cortical cells. In hyperplasia, adenoma and carcinoma, ET-1/big ET-1 and ET-B receptor showed frequent localization, although focal distribution of the ET-B receptor was rather predominant in these groups. ET-A receptor and ET-3/big ET-3 were very Infrequently expressed. Functioning versus non-functioning and hypertensive versus normotensive cases revealed no significant differences in the frequency of positive cells for ET-1/big ET-1, ET-3/big ET-3, ET-A receptor or ET-B receptor. Alternatively, the frequency of Immunoreactivity to ET-1/big ET-1 or ET-B receptor significantly decreased in hyperplasia, adenoma and carcinoma, when compared with that of normal adrenal cortex.
The present study, therefore, indicates that ET-1/big ET-1 and ET-B receptor are a prevalent ligand-receptor system in normal and hyperplastic/neoplastic adrenocortical cells, even with a malignant profile, and may contribute in maintaining adrenocortical cell function or cell viability but not cell growth or systemic hypertension.
Key words: adrenocortical cells, endothelin, endothelin recep- tor, immunohistochemistry
Correspondence: Toshimitsu Suzuki, MD, Department of Pathology, Fukushima Medical College, 1-Hikariga-oka, Fukushima 960-12, Japan.
Endothelin (ET)-1 is a potent vasoconstrictive peptide com- posed of 21 amino acid residues originally isolated from the supernatant of cultured porcine endothelian cells.1 From subsequent cDNA cloning from human genomic library there have emerged three distinct isopeptides termed ET-1, ET-2 and ET-3.2 In addition, cDNA cloning from murine intestine has revealed a similar peptide named vasoactive intestinal contractor.3 These peptides are a member of the ET family. Similar to other hormones, ET is synthesized in a pre-proform, which is metabolized to an intermediate ‘big endothelin’. It is then cleaved by a specific ET-converting enzyme that yields the mature peptide. Although ET was initially described as endothelial cells from a large vessel, it is now appreciated that ET is widely distributed among different organs and localized in various cell types including human brain, lung, kidney, jejunum and placenta for ET-1 mRNA,4,5 renal medulla and jejunum for ET-2 mRNA, and adrenal, jejunum, brain and renal medulla for ET-3 mRNA.5 Recently human adrenal cortex and adrenocortical adenoma have been reported to contain ET-1, ET-3 and ET receptor subtypes concomitantly at each mRNA level.6 Moreover, ET-1 distribution in normal and neoplastic adrenal glands has been described.7
Two kinds of receptors for ET have been found, namely ET-A receptor and ET-B receptor.8-11 ET-A receptor is ET-1 specific and expressed in lung, aorta, atrium, liver, muscle, cerebral cortex, cerebellum, kidney, adrenal, duodenum, colon and placenta at the mRNA or cDNA level, while ET-B receptor lacks isopeptide specificity and is found in liver, lung, placenta, kidney, heart and brain.8-10,12 Cellular locali- zation of ET-receptors in these organs or tissues, however, has not been elucidated, although stromal cells, but not epithelial cells of the breast, express ET-1 receptor in vitro. 13
The present immunohistochemical study reports frequent localization of immunoreactive ET-1 and ET-B receptors in normal, hyperplastic and neoplastic human adrenocortical cells.
MATERIALS AND METHODS
Tissues
Eighty-six formalin-fixed (10%), paraffin-embedded speci- mens of adrenals with hyperplasia, adenoma or carcinoma, which were obtained by surgery, were employed in the present study. Sex- and age-matched normal 36 adrenals of autopsy cases from normotensive and hypertensive groups were also sampled. The duration of hypertension as esti- mated by clinical records was 23.2 ± 11.5 years in males and 24.8 ± 11.0 years in females. Every specimen was his- tologically re-evaluated for diagnosis. The adrenocortical carcinoma was diagnosed according to the criteria by Weiss14 and reconfirmed thereafter.15 The number of cases exam- ined, endocrinological state and presence or absence of hypertension are summarized in Table 1.
Antibodies
Antibodies against ET-1, ET-3, big ET-1, ET-A receptor and ET-B receptor were raised in rabbits by subcutaneous injec- tions of each immunogen coupled with thyroglobulin (Sigma, St Louis, MO, USA). Each antigen was used as follows: ET-1 itself, sept-mer peptide of ET-1 (positions 15-21 amino acid residues, ET-1 (15-21)), 17-mer peptide of big ET-1 (posi- tions 22-38 amino acid residues, big ET-1 (22-38)), ET-3 itself, 11-mer peptide of human ET-A receptor (positions 59-69 amino acid residues, ET-AR (59-69))ª and 14-mer peptide of human ET-B receptor (positions 420-433 amino acid residues, ET-BR (420-433)).11 Every antiserum obtained was fractionated to IgG through protein A AvideGel (Bio Probe International, Tustin, CA, USA) and specifically purified
| Category (n) | Symptom of endocrinology (n) | Hypertension (n) |
|---|---|---|
| Normal adrenal (36)* Adrenocortical hyperplasia (9) Adrenocortical | None (36) Primary aldosteronism (9) | Present (19) |
| Present (9) | ||
| adenoma (70) | Primary aldosteronism (38) Cushing's syndrome (26)+ None (6) | Present (38) Present (18) Present (1) |
| Adrenocortical carcinoma (7) | Cushing's syndrome (2) Primary aldosteronism (1) None (4) | |
| Present (2) Present (1) Present (1) |
*Autopsy case. +A case with mixed tumor of pheochromocyte and adrenocortical cell is included.
through antigen-bound activated thiol AvideGel F affinity column chromatography (Bio Probe International). Specificity of the antibody except for the receptor antibodies was deter- mined by enzyme-linked immunosorbent assay (ELISA) method in which each antigen, including vasoactive intestinal contractor (Peptide Inst., Osaka, Japan), 50 ng/well in amount, was immunobilized in the ELISA plate. After incubation with the antibody, anti-rabbit IgG coupled with horseradish per- oxidase was applied to the plate, after which peroxidase reaction product was visualized by o-phenylenediamine sub- strate (Sigma). Optical density of the product was detected by Bio Rad immunoreader (Nippon Bio Rad Lab., Tokyo, Japan).
Mouse antityrosine hydroxylase monoclonal antibody (Chemicon International, Temecula, CA, USA) and antichro- mogranin A polycional antibody (Dakopatts, Copenhagen, Denmark) were also used.
Western blotting
Normal adrenal glands obtained at autopsy were subjected to western blotting. Briefly, the adrenal cortices were sonicated for 5 min in lysis buffer (0.1% sucrose monolaurate, 0.01% ethylene diamine tetra-aceticacid, 2 mmol/L phenylmethyl- sulfonyl fluoride and 0.25% sucrose in phosphate-buffered saline (PBS). After sonication, the samples were centrifuged at 15000xg for 30 min and the resultant supernatant was admixed with sample buffer (1 mol/L Tris-hydrochloric acid pH7.8, 18.75 mL; 2-mercaptoethanol, 15mL; glycerol, 30mL; sodium dodecyl sulfate, 6.9 g; 0.1% bromophenol blue, 3 mL; distilled water, to 100 ml) at 2:1 ratio. Protein concentration was determined for the sodium dodecyl extract. Various amounts of protein were loaded on SDS-PAGE and then blotted onto nitrocellulose membranes. After the testing 20 µg protein was finally applied to each lane. The membrane blots were incubated with either anti-ET-AR (59-69) or anti-ET-BR (420-433) antibody followed by anti-rabbit goat IgG conju- gated with horseradish peroxidase. The peroxidase reaction product was visualized by Konika Immunostain HRP1S-50B (Konika, Tokyo, Japan).
Immunohistochemistry
The working dilution of the primary antibody was 2 µg lgG/ mL for ET-1, and 5µg IgG/mL for ET-3, ET-A receptor and ET-B receptor. Antibodies against ET-1 (15-21) and big ET-1 (22-38) were not used in this study because of broad cross- reactivity with other antigens of ET-1 (15-21) antibody (Table
| Antigen | Anti-ET-1 (%) | Anti-ET-1 (15-21) (%) | Anti-ET-1 (22-38) (%) | Anti-ET-3 (%) |
|---|---|---|---|---|
| ET-1 | 100 | 100 | 0 | 0 |
| ET-2 | 1 | 100 | 0 | 0 |
| ET-3 | 0.4 | 100 | 0 | 0 |
| Big ET-1 | 100 | <0.1 | 100 | 0.8 |
| Big ET-2 | 0.2 | <0.1 | <0.1 | 0.8 |
| Big ET-3 | 0.4 | <0.1 | <0.1 | 100 |
| VIC | 0.2 | 100 | 0 | 0.1 |
VIC, vasoactive intestinal contractor; ET, endothelin.
2) or poor avidity of big ET-1 (22-38) antibody to the tissue antigen of the section.
Antityrosine hydroxylase monoclonal antibody and anti- chromogranin A antibody were applied to the sections at working dilutions of 1:100 or 1:500, respectively. Endog- enous peroxidase activity in each tissue section was blocked by methanol-hydrogen peroxide for 20 min and then the slides were treated with 5% skimmed milk (Yukijirushi, Sap- poro, Japan) in PBS for 30 min to avoid non-specific adsorp- tion of IgG. After incubation with each antibody overnight at 4℃, the slides were incubated with biotin-labeled anti-rabbit IgG or anti-mouse Ig (Nichirei, Tokyo, Japan), and then streptavidin-conjugated peroxidase (Nichirei) for 20 min at room temperature. Peroxidase reaction product was visual- ized by Graham-Karnovsky solution16 containing 65 mg NaN,/dL to block endogenous peroxidase activity again. Cellular nuclei were counterstained with 1% methyl green. Specificity of the immunostaining was confirmed as follows: omission of the primary antibody and application of the pre- absorbed antibody with 5 µg homologous antigen in the first step of immunostaining in cases where the antibodies were produced by us. In addition, vascular endothelial cells for ET-1, lung for ET-A receptor, placenta for ET-3 and vascular smooth muscle cells for ET-B receptor were evaluated in the immunohistochemistry as positive control tissues.
Evaluation of the immunohistochemistry
The frequency of the positively immunolabeled cells was scored as follows: diffuse, more than 50%; focal, less than 50% to more than 10%; and scattered, less than 10%.
Statistics
The frequency of positivity expressed by percent was statis- tically analyzed using Chi-squared test or Fisher’s t-test.
RESULTS
Specificity of antibodies produced
Cross-reactivity of each antibody determined by the ELISA method is summarized in Table 2. Anti-ET-1 antibody reacts with ET-1 and big ET-1 but not with ET-2, ET-3, big ET-2, big ET-3 and vasoactive intestinal contractor (VIC), while anti- ET-1 (15-21) antibody binds with ET-1, ET-2, ET-3 and VIC but not with any big ET. Anti-ET-3 antibody shows reactivity with ET-3 and big ET-3 but not with the other ET-related antigens examined. Anti-big ET-1 (22-38) antibody recog- nizes big ET-1 alone. As described previously, anti-ET-1 and anti-ET-3 antibodies were used in this study and the immuno- histochemical results were evaluated as ET-1/big ET-1 or ET- 3/big ET-3 positivity from the cross-reactivity of these two antibodies.
On western blotting, anti-ET-AR (59-69) and anti-ET-BR (420-433) antibodies revealed single broad bands at 49 kDa and 51 kDa in molecular weight, respectively (Fig. 1). These molecular weights are compatible with those of the respec- tive receptors.8.11
Immunohistochemistry
ET-1/big ET-1-positive cells were diffusely detected in normal adrenal cortex mainly in the zona fasciculata to zona reticu- laris (Fig. 2a), while pre-absorbed antibody with the homolo- gous ET-1 antigen failed to label the cortical cells (Fig. 2b).
STANDARD
ET-AR(2)
STANDARD
ET-BR(2)
97.4-+
66.2+
45.0-+
kD
a
b
C
(C)
(c)
C
g
d
e
h
Occasional labeling of endothelial cells with anti-ET-1 anti- body was found in large and/or capillary blood vessels of adrenal glands used (not shown). ET-3/big ET-3-stained cells were rarely and limitedly found in the deep zona fasciculata or zona reticularis (Fig. 2c). Cells with ET-BR (420-433) localized mainly in the zona glomeralosa to upper fasciculata and pre-absorbed ET-BR (420-433) antibody gave a nega- tive result (Fig. 2e). Staining was observed in the cell mem- brane, cytoplasm and/or nucleus of the cortical cells (Fig. 2f). ET-1/big ET-1 or ET-BR (420-433)-labeled cells were rarely distributed in the medulla but these were heterotopic cortical cells as they were negative for tyrosine hydroxylase or chromogranin A (data not shown). In contrast, ET-AR (59-69) was hardly found in the adrenal gland cells (Fig. 2g), although occasional labeling of vascular endothelial cells were en- countered in the deep cortex or in the medulla (Fig. 2h). The frequency and the expression pattern of both ET-1/big ET-1 and ET-BR (420-433) were not different between the normo- tensive and hypertensive group (P<0.5; Table 3).
Adrenocortical cell hyperplasia, adenoma or carcinoma disclosed ET-1/big ET-1 mainly in the diffuse pattern (Fig. 3a,d). In adenoma, clear cells showed dotted, granular posi- tivity, while compact cells revealed dense homogenous prod- ucts in the cytoplasm (Fig. 3b). On the other hand, ET-BR (420-433) was present diffusely in the hyperplasia (Fig. 3e) but mainly focally in the adenoma (Fig. 3f) or carcinoma (Fig. 3g). In adenocortical hyperplasia, the pattern of ET-1/
big ET-1 and ET-BR (420-433) expression seemed to be similar between the diffuse and nodular type. One case with mixed tumor of pheochromocyte and adrenocortical cell revealed ET-1/big ET-1 and ET-B receptor immunoreactivity only in the adrenocortical cell component and lacked immuno- reactivity to ET-3/big ET-3 or ET-A receptor (data not shown). ET-3/big ET-3 and ET-AR (59-69) were infrequently and very limitedly detected in the hyperplasia, adenoma (Fig. 4a,b) or carcinoma (Fig. 4c). One case of carcinoma, however, was focally populated with a number of ET-3/big ET-3-laden tumor cells (Fig. 4d). These data are summarized in Table 4. The frequency of diffuse expression of ET-1/big ET-1 seemed to be higher in functioning adrenocortices, especially in primary aldosteronism, than that in non-functioning ones, but the difference is not significant (P<0.13; Table 5). The fre- quency of diffuse expression of ET-BR (420-433) was almost equal between the functioning and non-functioning groups, and although the frequency was very low, ET-3/big ET-3 and ET-AR (59-69) expression between these two groups also were almost equal (Table 5).
In the hyperplasia, adenoma and carcinoma group, the diffuse distribution pattern of ET-1/big ET-1 was slightly but not significantly higher in the hypertensive cases than in the normotensive cases (P<0.06), the same as for ET-3/big ET-3 and ET-B receptor irrespective of the very low frequency of positivity, while for ET-B receptor the two cases were almost similar (Table 6).
| Blood pressure | ET-1/big ET-1 | ET-3/big ET-3 | ET-A receptor | ET-B receptor | ||||
|---|---|---|---|---|---|---|---|---|
| Diffuse (%) | Focal (%) | Diffuse (%) | Scattered (%) | Diffuse (%) | Scattered (%) | Diffuse (%) | Focal (%) | |
| Normotensive n=17 | 17/17 (100) | 0/17 (0) | 0/17 (0) | 1/17 (6) | 0/17 (0) | 2/17 (12) | 17/17 (100) | 0/17 (0) |
| Hypertensive n=19 | 19/19 (100) | 0/19 (0) | 1/19 (5) | 1/19 (5) | 0/19 (0) | 3/19 (16) | 16/19 (84) | 3/19 (16) |
ET, endothelin.
| Category | ET-1/big ET-1 | ET-3/big ET-3 | ET-A receptor | ET-B receptor | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Diffuse | (%) | Focal (%) | Diffuse (%) | Scattered | (%) | Diffuse | (%) | Scattered | (%) | Diffuse (%) | Focal | (%) | ||
| Adrenocortical hyperplasia n=9* | 7/9 | (78) | 2/9 | (22) | 0/9 | 2/9 | (11) | 0/9 | 3/9 | (33) | 5/9 (56) | 4/9 | (44) | |
| Adenoma | ||||||||||||||
| Primary aldosteronism n=38 | 31/38 | (82) | 7/35 | (18) | 0/38 (0) | 6/38 | (16) | 0/38 | (0) | 5/38 | (13) | 6/38 (16) | 31/38 | (82) |
| Cushing's syndrome n=27+ | 16/27 | (59) | 11/27 | (41) | 0/27 (0) | 10/27 | (37) | 0/27 | (0) | 4/27 | (15) | 16/27 (59) | 11/27 | (41) |
| None n=6 | 3/6 | (50) | 3/6 | (50) | 0/6 (0) | 0/6 | (0) | 0/6 | (0) | 0/6 | ( | 1/6 (17) | 5/6 | (83) |
| Carcinoman=7 | 4/7 | (57) | 3/7 | (43) | 0/7 (0) | 3/7 | (43) | 0/7 | (0) | 1/7 | (14) | 3/7 (43) | 4/7 | (57) |
| Cushing's syndrome n=2 | 2/2 | (100) | 0/2 | (0) | 0/2 (0) | 1/2 | (50) | 0/2 | (0) | 0/2 | (0) | 0/2 (0) | 2/2 | (100) |
| Primary aldosteronism n= 1 | 0/1 | (0) | 1/1 | (100) | 0/1 (0) | 0/1 | (0) | 0/1 | (0) | 0/1 | (0) | 0/1 (0) | 1/1 | (100) |
| None n=4 | 2/4 | (50) | 2/4 | (50) | 0/4 | 2/4 | (50) | 0/4 | (0) | 1/4 | (25) | 3/4 (75) | 1/4 | (25) |
*Aldosterone-producing.
+27 adenomas examined.
ET, endothelin.
C
a
b
6
e
f
g
The frequency of diffuse expression of ET-1/big ET-1 in normal adrenals was significantly higher than that of prolifera- tive adrenocortices (P<0.002), including normal versus hyperplasia (P<0.0038), normal versus adenoma (P<0.0001) and normal versus adrenocortical carcinoma (P<0.0028; Table 7). This was the same or more conspicuous in the
frequency of ET-B receptor between normal and proliferative adrenal cortices (P<0.001), showing normal versus hyper- plasia (P<0.015), normal versus adenoma (P<0.0001) and normal versus carcinoma (P<0.0047; Table 7). The frequency of ET-3/big ET-3 in proliferative adrenal cortices was higher than that of normal adrenal cortices (P<0.0085) indicating
| Category | ET-1/big ET-1 | ET-3/big ET-3 Scattered (%) | ET-B receptor | ET-A receptor Scattered (%) | ||
|---|---|---|---|---|---|---|
| Diffuse (%) | Focal (%) | Diffuse (%) | Focal (%) | |||
| Functioning n=77 | 56/77 (73) | 21/77 (27) | 19/77 (25) | 27/77 (35) | 49/77 (64) | 12/77 (16) |
| Primary aldosteronism n=48 | 38/48 (79) | 10/48 (21) | 8/48 (17) | 11/48 (23) | 36/48 (75) | 8/48 (17) |
| Cushing's syndrome n=29* | 18/29 (62) | 11/29 (38) | 1/29 (38) | 16/29 (55) | 13/29 (45) | 4/29 (14) |
| Non-functioning n=10 | 5/10 (50) | 5/10 (50) | 2/10 (20) | 4/10 (40) | 6/10 (60) | 1/10 (10) |
*29 adrenal glands examined. ET, endothelin.
| Hypertension | ET-1/big ET-1 | ET-3/big ET-3 Scattered (%) | ET-B receptor | ET-A receptor Scattered | ||
|---|---|---|---|---|---|---|
| Diffuse (%) | Focal (%) | Diffuse (%) | Focal (%) | |||
| Present n=69* | 53/69 (77) | 16/69 (23) | 14/69 (20) | 25/69 (36) | 43/69 (62) | 9/60 (15) |
| Absent n=12 | 6/12 (50) | 6/12 (50) | 1/12 (8) | 4/12 (33) | 8/12 (67) | 0/12 (0) |
*One borderline case is omitted. ET, endothelin.
| Category | ET-1/big ET-1 Diffuse (%) | ET-3/big ET-3 Scattered (%) | ET-A receptor Scattered (%) | ET-B receptor Diffuse (%) |
|---|---|---|---|---|
| Normal n=36 | 36/36 (100) | 2/36 (6) | 5/36 (14) | 33/36 (92) |
| Proliferative n= 87 | 61/87 (70) | 21/87 (24) | 13/87 (15) | 34/87 (39) |
| Hyperplasia n=9 | 7/9 (78) | 2/9 (11) | 3/9 (33) | 5/9 (56) |
| Adenoma n=71 | 50/71 (70) | 16/71 (23) | 9/71 (13) | 26/71 (37) |
| Carcinoma n=7 | 4/7 (57) | 3/7 (43) | 1/7 (14) | 3/7 (43) |
ET, endothelin.
C
a
a
b
normal versus adenoma (P<0.016) and normal versus car- cinoma (P<0.015), but that of ET-AR (59-69) remained unchanged in these two categories (Table 7).
DISCUSSION
In the present study the ET-1/big ET-1 and ET-BR were regularly demonstrated in normal and hyperplastic or neo- plastic adrenocortical cells, as expected from the recent report that mentioned concomitant occurrence of mRNA for ET-1, ET-3 and ET receptors in human adrenal cortex.6 Moreover, both of them show similar distribution patterns, while ET-3/big ET-3 and ET-AR are exceptionally and limit- edly detected in the adrenocortical cells in the aforemen- tioned categories. In addition, adrenomedullary cells are negative for ET-1/big ET-1, ET-3/big ET-3 and ET receptors, although Amico et al. have described that some of the normal cells in adrenal medulla were labeled by anti-ET-1 antibody.17 They did not confirm these cells to be adrenomedullary cells by catecholamine or chromogranin-positivity. From results of the present study, these cells seem to be heterotopic cortical cells in the normal adrenal medulla. For ET-1, Li et al. also have described that normal adrenomedullary cells were negative, and they have reported that immunohistochemical products of ET-1 in adrenocortical cells, normal and neoplas- tic, showed the form of vacuoles, grains or cell membranes.7 Our results were almost similar, but diffusely stained cells in the cytoplasm were also frequently found, especially in the compact cells of adenoma. The reason for this minor differ- ence is not clear but the difference of the antibody used and/ or enhancement procedure (glucose oxidase-DAB-nickel method) of the immunoreactive products employed by them may be a contributing factor. Staining fashion of the ET-B receptor in adrenocortical cells, on the other hand, revealed to be membranous and diffuse in the cytoplasm and/or nuclear in both the normal and proliferative state. Nuclear localization of ET-receptor immunoreactivity may be feasible as ET-1 binds both the cell membrane and nuclear fraction of rat liver cells. 18
ET-1/big ET-1 expression seems to be higher, but not significantly, in the group with endocrinological symptoms than that of the silent group with adenoma or carcinoma, although exogenous ET-1 stimulates cortisol and/or aldoster- one genesis and secretion in the cultured rat or human adrenal cortex in vitro.19-21 The hypertensive group with or without adrenocortical diseases showed no differences in frequency in ET-1 expression compared to the normotensive group of the respective category. From these observations, ET-1 may not contribute to the cause and/or maintenance of high blood pressure but to the regulation and/or mainte- nance of the normal state of adrenocortical cells.
In addition to long-lasting vasoconstriction, ET has potent hyperplastic-hypertrophic effects that have been implicated in normal growth and development,22-30 and proliferation associated with neoplasia.31-34 Cell growth by ET-1 has been reported to be mediated through its receptor ET-A.35 In adrenal cortex analysed by Ki-67 labeling, normal zona fas- ciculata and adenoma have been reported to show a similar labeling index, while that of carcinoma is significantly higher.36 ET-1/big ET-1 and ET-BR system in the adrenocortical cells, either normal or hyperplastic/neoplastic, found in the present study may not work as an auto- or paracrine growth stimula- tion system, because normal adrenocortical cells are more intensely and frequently positive than adrenocortical cells in hyperplasia, adenoma and carcinoma. Conversely, carcinoma revealed rather low frequency of labeling with ET-1/ET-B receptor. These observations are basically compatible with the report of Nelson et al.37 They described decreased ET- BR expression in advanced prostatic cancer, while ET-1 and ET-AR expression were retained. In adrenocortical cells, ET- 1 and ET-AR system, however, also seem not to be associ- ated with cell growth, as ET-AR expression was very limitedly detected in normal and hyperplastic/neoplastic adrenocorti- cal cells. ET-3/big ET-3-immunoreactive cells were more frequently detected in diseased adrenocortices than in nor- mal. The contribution of ET-3/big ET-3 to cell growth of the former category seems to be unlikely because the frequency or distribution was very low or very limited.
In conclusion, ET-1/big ET-1 and ET-BR system, of which true biological significance waits for further investigation, is regularly present in normal and neoplastic adrenocortical cells, and these molecules, therefore, are a reliable cell marker of adrenocortical cells, normal or neoplastic, even in formalin-fixed and paraffin-embedded specimens, in addition to vimentin in adrenocortical tumor cells described by Schröder et al. 38
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