0013-7227/91 /1292-1113$O3.OO/O Endocrinology Copyright ^ 1991 by The Endocrine Society
Vol. 129, No. 2
Printed in U.S.A.
Ultrastructural Evidence lor Cortico-Chromaffin Hybrid Cells in Rat Adrenals? Steian R. Bornstein, Monika Ehrhart-Bornstein, Werner A. Scherbaum Abtoilung Innere Medizm
I, Medizinische Klinik und
Poliklinik. Universitat Ulm. D-7900 Ulm.
Germany
ABSTRACT: The adrenals of perfusion fixed rats were investigated by electron Microscopy. In 2 out of 12 animals studied, chromaffin cells were detected that contained organelles identical to adrenocortical mitochondria and smooth endoplasmic reticulum. The mitochondria were round-shaped with the vesicular cristae typical for steroid hormone producing cells. They were located within the cytoplasm of adrenomedullary cells neighbouring chromaffin vesicles, rough endoplasmic reticulum and the characteristic elongated mitochondria of chromaffin cells with laminar cristae. The mitochondria-like structures could be clearly differentiated from the extracellularly located synaptic endings. It can therefore be concluded that some rare cortico-chromaffin hybrid cells or chromaffin cells that present a mitochondrial-endoplasmic system of adrenocortical cells exist within the adrenal medulla. This surprising finding may offer an explanation for the long line of inconsistencies in clinico-pathological reports of adrenal morphology.
INTRODUCTION
MATERIALS AND METHODS
IN MAMMALIAN adrenals, nature has combined two
12 five-week-old male Sprague-Dawley rats were used in this
endocrine
tissues
under
catecholamine-producing
a
common
chromaffin
capsule,
cells
and
the
study. The animals were fixed under Nembutal anesthesia by
the
perfusion through the left ventricle (2% paraformaldehyde,
steroid-producing cortical cells. Evidence for both, an in-
2% glutaraldehyde in 0.1 M phosphate, pH 7.3). The
fluence of the adrenal cortex on the medulla (1), as well as
adrenal glands were removed, dissected and fixed for a
an influence of the adrenal medulla on the cortex has been
further 3 hours in the above fixative. The tissue slices were
reported
(2,3). Thus the purpose of this biological
postfixed for 90 min (2% OsO4 in 0.1 M cacodylate, pH
arrangement has been seen in a bidirectional interaction of
7.3), dehydrated in ethanol and embedded in epoxy resin.
chromaffin and cortical cells (2,4). In clinical case reports
Semithin sections (0.5 /xtn) were stained with toluidine blue.
with histopathological investigations of adrenoglandular
Ultrathin sections (70 nm) were stained with uranylacetate
tumors discrepancies between the clinical data and the
and lead citrate and examined at 80 kV in a Phillips EM
histological results have been frequently described. Patients
301.
presenting clinically and by laboratory data with adrenocortical
hyperfunction
were
found
to
have
RESULTS
a
The ultrastructural investigation of chromaffin cells of
pheochromocytoma at histology (5-7). On the other hand, reports of patients with pheochromocytomas mimicking
untreated rats with completely unremarkable histological ap-
adrenocortical tumors have been described (8,9). Electron
pearance led to an unusual observation in two animals. In the
microscopy has been widely accepted as an excellent method
adrenals of these animals, particularly in the transition zone
to differentiate adrenal morphology and adrenal lesions (for
of adrenal medulla and adrenal cortex, we found a number
review: 10,11). The ultrastructural observations of the
of cells which seemed to present characteristics of chromaffin
following
and cortical cells. They contained two types of mitochondria:
study may suggest the rare occurrence of
the laminar form of chromaffin cells as well as the tubulo-
cortico-chromaffin hybrid cells within normal rat adrenals.
vesicular form of the adrenal cortex (Fig. 1). These cells appeared
to be predominantly
chromaffin
cells with
characteristic chromaffin vesicles and rough endoplasmic reticulum Fig. la, c). On the other hand, these cells showed ample smooth endoplasmic reticulum, and organelles that were identical to the mitochondria found in the adrenal cortex. Mitochondria with laminar cristae were found in Received in Iowa C i t y :
May 6, 1991
juxtaposition with mitochondria with vesicular cristae (Fig.
1113
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 19:54 For personal use only. No other uses without permission. . All rights reserved.
RAPID COMMUNICATIONS
1114
Endo • 1991 Voll29«No2
lb). SER was arranged around the outer mitochondrial membrane (Fig.lb, c) To clarify whether these organelles could be small, round-shaped nerve endings, the morphology of these two structures was compared (Fig. 2). The nerve endings that could be seen in the adrenal medulla were clearly extracellular, and they formed synaptic densities (Fig. 2b). In contrast to nerve endings, the mitochondria-like organelles never contained mitochondria (Fig. 1, 2a). In addition, these structures were located intracellularly within the cytosol of chromaffin cells, neighbouring with mitochondria, chromaffin vesicles and smooth endoplasmic reticulum (Fig 1, 2a).
Fig. 2 A, B Comparison of vesicular mitochondria and synaptic ending in the adrenal medulla: Vesicular mitochondria (large arrow) are localized intracellularly; they are round-shaped with the inner membranes branching off the mitochondrial capsule (small arrows in A). Synaptic nerve endings (*) are localized extracellulary with clearly visible plasmaJemm (short arrow). Besides the synaptic vesicles other organelles such as mitochondria (arrow head) as well as synaptic junctions can be discerned (long arrows in B). Rat adrenal medulla, x 21 600. Bar = 0.5 pm
DISCUSSION Most morphologists analyzing the adrenal gland have either concentrated on the adrenal cortex or on the adrenal medulla. Within the adrenal medulla we detected a number of cells Fig. 1 A Chromaffin
cells within the rat adrenal medulla with
demonstrating the characteristic features of both chromaffin
cytoplasm
and cortical cells. So-called mixed cells in the adrenal cortex
mitochondria with vesicular cristae (short arrows) smooth endoplasmic
have been described before in the zona reticularis (12-14).
characteristic
chromaffin
vesicles
(cv).
Within
the
reticulum (ER) and Golgi fields (Golgi); N: nucleus, x 18000. Bar = 0.5
j*m. B Mitochondria
with
laminar cristae neighbouring a
mitochondrium with vesicular cristae within the cytoplasm of a
However, these observations were made in immersion fixed tissue and considered to be cellular detritus of chromaffin
chromaffin cell (arrow head). Inner membranes are branching off the
cells in the sinusoids of the zona reticularis (14). In our
mitochondrial capsule (thin arrows; enlargement off Fig 2A). Smooth
study, these cells were detected in perfusion fixed tissue with
endoplasmic reticulum (ER) is localized around the outer mitochondrial
a well preserved ultrastructure and no indication for an arte-
membrane (arrow), x 23100. Bar = 0.4/xm. C A field of rough
fact whatsoever. A confusion with nerve endings is very un-
endoplasmic reticulum (RER) in neighbourhood with a vesicular mitochondrium (short arrow) in the midst of the cytoplasm of a chromaffin cell, x 18000. Bar = 0.5 /im
likely since the latter are clearly located extracellularly and usually synapses can be seen. Therefore, it seems as if these
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 19:54 For personal use only. No other uses without permission. . All rights reserved.
RAPID COMMUNICATIONS cells could be hybrids or chromaffin cells which present the characteristic mitochondria and SER of steroid cells. The organelles we described here were located within the chromat'fin cells. They were identical in size and shape to adrenocortical vesicular mitochondria. They are in direct apposition with SER as it is characteristic for cortical cells. The occurrence of these cells seems to be a rare phenomenon since we detected these structures within medullary cells of only two animals out of the 12 analysed. It can not be excluded that these cells are mutants of chromaffin cells which express aberrant information, although the histological appearance was unremarkable. Human adrenal pathology revealed the occurrence of mixed corticomedullary tumors (6,7)- There is also a long history of discrepant findings between the clinical presentation suggesting adrenocortical tumors and the pathology revealing an adrenomedullary tumor as well as vice versa (5,6,8,9,15-17). Our observations in supposedly normal, untreated rats may give a clue to these phenomena. It has been demonstrated that chromaffin tissue from pheochromocytomas or from bovine adrenals possesses the capacity to carry out steroid biosynthetic reactions (for review: 17). It is conceivable that such steroidogenic activity of adrenal medullary cells is located in the adrenal cortex-like mitochondria observed in this study. Further studies with immunocytochemical techniques will have to elucidate whether the vesicular mitochondria described within the chromaffin cells contain the cytochrome P450 enzymes relevant for steroidogenesis.
8.
1115
Ramsay JA, Asa SL, van Nostrand AWP, Hassaram ST, deHarven EP 1987 Lipid degeneration in pheochromocytoinas mimicking adrenal conical tumors. Am J Surg Pathol 11:480-486
9.
linger PD, Cohen JM, Thung SN, Gordon R, Pertsemlidis D, Dikman SH 1990 Lipid degeneration in a pheochromocytoma histologically mimicking an adrenaJ cortical tumor. Arch Pathol Lab Med 114:892-894
10.
Nussdorfer GG 1986 Cytophysiology or the adrenal cortex. Int Rev Cytol 98:1-395
11.
Tannenhaum M 1970 Ultrastructural pathology of adrenal medullary tumors. Path Annu 5:145-169
12.
Probst A, Miiller O 1966 Die Zonen der Nebennierenrinde der Ratte. Z Zellforsch Mikrosk Anat 75:404-412
13.
Coupland RE 1965 Electron microscopic observations on the structure of the adrenal medulla. J Anat (Lond) 99:231-237
14. 15.
Idelmann S 1970 Ultrastructure of the mammalian adrenal cortex. Int Rev Cytol 27:181-281 Robinson
PL, Baker-Bates ET
1954 Adrenal cortical
16.
Williams GA, Crockett LC, Butler WWS, Crispell KR 1960 The coexistence of pheochromocytoma and adrenal cortical hyperplasia. J Clin Endocrinol Metabol 20:622-631
17.
Walters G, Wyatt GB, Kelleher J 1962 Carcinoma of the adrenal cortex presenting as a pheochromocytoma: report of a case. J Clin Endocrinol 22:575-580
18.
Carballeira A, Fishman.LM 1980. The adrenal functional Unit: A hypothesis. In: University of Chicago (ed) Perspectives in Biology and Medicine. University of Chicago, Chicago, p 573-596
REFERENCES Axelrod J. Reisine TD 1984 Stress hormones: Their interaction and regulation. Science 224:452-459 Bomstein SR. Ehrhart-Bornstein M, Scherbaum WA, Pfeiffer EF. Hoist JJ 1990 Effects of splanchnic nerve stimulation on the adrenal cortex may be mediated
by chromaffin
cells in a paracrine
manner.
Endocrinology 127:900-906 Hinson JP 1990 Paracrine control of adreno-cortical function: a new role for the medulla?. J Endocrinol 124:7-9 Bornstein
SR.
F.hrhart-Bornstein
M,
Usadel
H.
Bockmann
M.
Scherbaum WA 1991 Morphological evidence for a close interaction of chromaffin cells with cortical cells within the adrenal giand. Cell Tissue Res (In Press) I^Compte
PM
1944
Cushing's
syndrome
with
possible
pheochromocytoma: report of a case. Am J Pathol 20:689-707 Mathison
DA,
hypertensive
Waterhousc CA crisis
and
1969 Cushing's syndrome mixed
adrenal
carcinoma
simulating a pheochromocytoma. Br J Surg 41:399-403
with
cortical
adenoma-pheochromocytoma (cortico-medullary adenoma). Am J Med 47:635-641 Kovacs K, Horvath E 1973 Ultrastructural features of corticomedullary cells in a human adrenal cortical adenoma and in rat adrenal cortex. Anat An/. 134:387-389
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 19:54 For personal use only. No other uses without permission. . All rights reserved.
Vol. 129, No. 2
Printed in U.S.A.
Ultrastructural Evidence lor Cortico-Chromaffin Hybrid Cells in Rat Adrenals? Steian R. Bornstein, Monika Ehrhart-Bornstein, Werner A. Scherbaum Abtoilung Innere Medizm
I, Medizinische Klinik und
Poliklinik. Universitat Ulm. D-7900 Ulm.
Germany
ABSTRACT: The adrenals of perfusion fixed rats were investigated by electron Microscopy. In 2 out of 12 animals studied, chromaffin cells were detected that contained organelles identical to adrenocortical mitochondria and smooth endoplasmic reticulum. The mitochondria were round-shaped with the vesicular cristae typical for steroid hormone producing cells. They were located within the cytoplasm of adrenomedullary cells neighbouring chromaffin vesicles, rough endoplasmic reticulum and the characteristic elongated mitochondria of chromaffin cells with laminar cristae. The mitochondria-like structures could be clearly differentiated from the extracellularly located synaptic endings. It can therefore be concluded that some rare cortico-chromaffin hybrid cells or chromaffin cells that present a mitochondrial-endoplasmic system of adrenocortical cells exist within the adrenal medulla. This surprising finding may offer an explanation for the long line of inconsistencies in clinico-pathological reports of adrenal morphology.
INTRODUCTION
MATERIALS AND METHODS
IN MAMMALIAN adrenals, nature has combined two
12 five-week-old male Sprague-Dawley rats were used in this
endocrine
tissues
under
catecholamine-producing
a
common
chromaffin
capsule,
cells
and
the
study. The animals were fixed under Nembutal anesthesia by
the
perfusion through the left ventricle (2% paraformaldehyde,
steroid-producing cortical cells. Evidence for both, an in-
2% glutaraldehyde in 0.1 M phosphate, pH 7.3). The
fluence of the adrenal cortex on the medulla (1), as well as
adrenal glands were removed, dissected and fixed for a
an influence of the adrenal medulla on the cortex has been
further 3 hours in the above fixative. The tissue slices were
reported
(2,3). Thus the purpose of this biological
postfixed for 90 min (2% OsO4 in 0.1 M cacodylate, pH
arrangement has been seen in a bidirectional interaction of
7.3), dehydrated in ethanol and embedded in epoxy resin.
chromaffin and cortical cells (2,4). In clinical case reports
Semithin sections (0.5 /xtn) were stained with toluidine blue.
with histopathological investigations of adrenoglandular
Ultrathin sections (70 nm) were stained with uranylacetate
tumors discrepancies between the clinical data and the
and lead citrate and examined at 80 kV in a Phillips EM
histological results have been frequently described. Patients
301.
presenting clinically and by laboratory data with adrenocortical
hyperfunction
were
found
to
have
RESULTS
a
The ultrastructural investigation of chromaffin cells of
pheochromocytoma at histology (5-7). On the other hand, reports of patients with pheochromocytomas mimicking
untreated rats with completely unremarkable histological ap-
adrenocortical tumors have been described (8,9). Electron
pearance led to an unusual observation in two animals. In the
microscopy has been widely accepted as an excellent method
adrenals of these animals, particularly in the transition zone
to differentiate adrenal morphology and adrenal lesions (for
of adrenal medulla and adrenal cortex, we found a number
review: 10,11). The ultrastructural observations of the
of cells which seemed to present characteristics of chromaffin
following
and cortical cells. They contained two types of mitochondria:
study may suggest the rare occurrence of
the laminar form of chromaffin cells as well as the tubulo-
cortico-chromaffin hybrid cells within normal rat adrenals.
vesicular form of the adrenal cortex (Fig. 1). These cells appeared
to be predominantly
chromaffin
cells with
characteristic chromaffin vesicles and rough endoplasmic reticulum Fig. la, c). On the other hand, these cells showed ample smooth endoplasmic reticulum, and organelles that were identical to the mitochondria found in the adrenal cortex. Mitochondria with laminar cristae were found in Received in Iowa C i t y :
May 6, 1991
juxtaposition with mitochondria with vesicular cristae (Fig.
1113
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 19:54 For personal use only. No other uses without permission. . All rights reserved.
RAPID COMMUNICATIONS
1114
Endo • 1991 Voll29«No2
lb). SER was arranged around the outer mitochondrial membrane (Fig.lb, c) To clarify whether these organelles could be small, round-shaped nerve endings, the morphology of these two structures was compared (Fig. 2). The nerve endings that could be seen in the adrenal medulla were clearly extracellular, and they formed synaptic densities (Fig. 2b). In contrast to nerve endings, the mitochondria-like organelles never contained mitochondria (Fig. 1, 2a). In addition, these structures were located intracellularly within the cytosol of chromaffin cells, neighbouring with mitochondria, chromaffin vesicles and smooth endoplasmic reticulum (Fig 1, 2a).
Fig. 2 A, B Comparison of vesicular mitochondria and synaptic ending in the adrenal medulla: Vesicular mitochondria (large arrow) are localized intracellularly; they are round-shaped with the inner membranes branching off the mitochondrial capsule (small arrows in A). Synaptic nerve endings (*) are localized extracellulary with clearly visible plasmaJemm (short arrow). Besides the synaptic vesicles other organelles such as mitochondria (arrow head) as well as synaptic junctions can be discerned (long arrows in B). Rat adrenal medulla, x 21 600. Bar = 0.5 pm
DISCUSSION Most morphologists analyzing the adrenal gland have either concentrated on the adrenal cortex or on the adrenal medulla. Within the adrenal medulla we detected a number of cells Fig. 1 A Chromaffin
cells within the rat adrenal medulla with
demonstrating the characteristic features of both chromaffin
cytoplasm
and cortical cells. So-called mixed cells in the adrenal cortex
mitochondria with vesicular cristae (short arrows) smooth endoplasmic
have been described before in the zona reticularis (12-14).
characteristic
chromaffin
vesicles
(cv).
Within
the
reticulum (ER) and Golgi fields (Golgi); N: nucleus, x 18000. Bar = 0.5
j*m. B Mitochondria
with
laminar cristae neighbouring a
mitochondrium with vesicular cristae within the cytoplasm of a
However, these observations were made in immersion fixed tissue and considered to be cellular detritus of chromaffin
chromaffin cell (arrow head). Inner membranes are branching off the
cells in the sinusoids of the zona reticularis (14). In our
mitochondrial capsule (thin arrows; enlargement off Fig 2A). Smooth
study, these cells were detected in perfusion fixed tissue with
endoplasmic reticulum (ER) is localized around the outer mitochondrial
a well preserved ultrastructure and no indication for an arte-
membrane (arrow), x 23100. Bar = 0.4/xm. C A field of rough
fact whatsoever. A confusion with nerve endings is very un-
endoplasmic reticulum (RER) in neighbourhood with a vesicular mitochondrium (short arrow) in the midst of the cytoplasm of a chromaffin cell, x 18000. Bar = 0.5 /im
likely since the latter are clearly located extracellularly and usually synapses can be seen. Therefore, it seems as if these
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 19:54 For personal use only. No other uses without permission. . All rights reserved.
RAPID COMMUNICATIONS cells could be hybrids or chromaffin cells which present the characteristic mitochondria and SER of steroid cells. The organelles we described here were located within the chromat'fin cells. They were identical in size and shape to adrenocortical vesicular mitochondria. They are in direct apposition with SER as it is characteristic for cortical cells. The occurrence of these cells seems to be a rare phenomenon since we detected these structures within medullary cells of only two animals out of the 12 analysed. It can not be excluded that these cells are mutants of chromaffin cells which express aberrant information, although the histological appearance was unremarkable. Human adrenal pathology revealed the occurrence of mixed corticomedullary tumors (6,7)- There is also a long history of discrepant findings between the clinical presentation suggesting adrenocortical tumors and the pathology revealing an adrenomedullary tumor as well as vice versa (5,6,8,9,15-17). Our observations in supposedly normal, untreated rats may give a clue to these phenomena. It has been demonstrated that chromaffin tissue from pheochromocytomas or from bovine adrenals possesses the capacity to carry out steroid biosynthetic reactions (for review: 17). It is conceivable that such steroidogenic activity of adrenal medullary cells is located in the adrenal cortex-like mitochondria observed in this study. Further studies with immunocytochemical techniques will have to elucidate whether the vesicular mitochondria described within the chromaffin cells contain the cytochrome P450 enzymes relevant for steroidogenesis.
8.
1115
Ramsay JA, Asa SL, van Nostrand AWP, Hassaram ST, deHarven EP 1987 Lipid degeneration in pheochromocytoinas mimicking adrenal conical tumors. Am J Surg Pathol 11:480-486
9.
linger PD, Cohen JM, Thung SN, Gordon R, Pertsemlidis D, Dikman SH 1990 Lipid degeneration in a pheochromocytoma histologically mimicking an adrenaJ cortical tumor. Arch Pathol Lab Med 114:892-894
10.
Nussdorfer GG 1986 Cytophysiology or the adrenal cortex. Int Rev Cytol 98:1-395
11.
Tannenhaum M 1970 Ultrastructural pathology of adrenal medullary tumors. Path Annu 5:145-169
12.
Probst A, Miiller O 1966 Die Zonen der Nebennierenrinde der Ratte. Z Zellforsch Mikrosk Anat 75:404-412
13.
Coupland RE 1965 Electron microscopic observations on the structure of the adrenal medulla. J Anat (Lond) 99:231-237
14. 15.
Idelmann S 1970 Ultrastructure of the mammalian adrenal cortex. Int Rev Cytol 27:181-281 Robinson
PL, Baker-Bates ET
1954 Adrenal cortical
16.
Williams GA, Crockett LC, Butler WWS, Crispell KR 1960 The coexistence of pheochromocytoma and adrenal cortical hyperplasia. J Clin Endocrinol Metabol 20:622-631
17.
Walters G, Wyatt GB, Kelleher J 1962 Carcinoma of the adrenal cortex presenting as a pheochromocytoma: report of a case. J Clin Endocrinol 22:575-580
18.
Carballeira A, Fishman.LM 1980. The adrenal functional Unit: A hypothesis. In: University of Chicago (ed) Perspectives in Biology and Medicine. University of Chicago, Chicago, p 573-596
REFERENCES Axelrod J. Reisine TD 1984 Stress hormones: Their interaction and regulation. Science 224:452-459 Bomstein SR. Ehrhart-Bornstein M, Scherbaum WA, Pfeiffer EF. Hoist JJ 1990 Effects of splanchnic nerve stimulation on the adrenal cortex may be mediated
by chromaffin
cells in a paracrine
manner.
Endocrinology 127:900-906 Hinson JP 1990 Paracrine control of adreno-cortical function: a new role for the medulla?. J Endocrinol 124:7-9 Bornstein
SR.
F.hrhart-Bornstein
M,
Usadel
H.
Bockmann
M.
Scherbaum WA 1991 Morphological evidence for a close interaction of chromaffin cells with cortical cells within the adrenal giand. Cell Tissue Res (In Press) I^Compte
PM
1944
Cushing's
syndrome
with
possible
pheochromocytoma: report of a case. Am J Pathol 20:689-707 Mathison
DA,
hypertensive
Waterhousc CA crisis
and
1969 Cushing's syndrome mixed
adrenal
carcinoma
simulating a pheochromocytoma. Br J Surg 41:399-403
with
cortical
adenoma-pheochromocytoma (cortico-medullary adenoma). Am J Med 47:635-641 Kovacs K, Horvath E 1973 Ultrastructural features of corticomedullary cells in a human adrenal cortical adenoma and in rat adrenal cortex. Anat An/. 134:387-389
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 19:54 For personal use only. No other uses without permission. . All rights reserved.
