Bioscience Reports, Vol. 13, No. 5, 1993
CCK-Containing Paraneurons in Human
Adenomatous Prostate
A . Cecio, 1'3 T. Cocca, 1 M. T. Masotti, z E. La Mura, ~ and
A . Vittoria ~
Received March 17, 1993; accepted May 18, I993
The presence of CCK-containing neuroendocrine cells in human adenomatous prostates, and the
colocalization of CCK together with serotonin in the same cell, have been demonstrated by means of
an immunohistochemical technique and by a double labeling immunofluorescent staining. CCKcontaining neuroendocrine cells had a focal distribution in the prostates and sometimes showed
dendrite-like cytoplasmic processes. The major part of CCK (96.55%) colocalized with serotonin.
CCK probably stimulates muscle contraction and endocrine/exocrine secretions in the urogenital
tract.
KEY WORDS: cholecystokinin; neuroendocrine cells; prostatic adenomas
INTRODUCTION
The presence of neuroendocrine (NE) cells [1], or paraneurons [2, 3, 4], in the
human urogenital tract was first documented by Pretl [5], and then by Feyrter [6],
using histochemical techniques. A large number of reports followed these works,
which described the ultrastructural and immunohistochemical peculiarities of
these cells throughout the urogenital apparatus of humans [7, 8] and other
mammals [8, 9, 10, 11].
Immunohistochemical studies of normal, hyperplastic or carcinomatous
human prostates showed paraneurons containing serotonin, 5-HT [8, 12, 13],
neuron specific enolase, NSE [8, 14], calcitonin [13, 14, 15, 16, 17, 18], calcitonin
gene-related peptide, CGRP [18], katalcin [18], somatostatin, SS-14 [13, 16, 19],
thyroid stimulating hormone, TSH [13, 20], fi-endorphin, /3-end [17, 21],
bombesin [15] or adrenocorticotropic hormone, ACTH [17, 21].
i Dept. of Structures, Functions and Biological Technology, University of Naples, Federico II. Via F.
Delpino, 1 - 80137, Naples, Italy.
2 III General Surgery, II Faculty of Medicine, University of Naples, Federico II.
3 To whom correspondence should be addressed.
251
0144-8463/93/10(10-0251 $(17.0()/0 9 1993 Plenum Publishing Corporation
Cecio, Cocca, Masotti, La Mura and Vittoria
252
A normal prostatic NE population sometimes participates in neoplastic
proliferations of the prostate and may constitute the major part of the tumor. The
hormonal production of such a prostatic tumor depends on the cellular type(s)
which make it up [13, 20].
In our study, utilizing immunohistochemical techniques, the presence of NE
cells containing CCK is demonstrated in two cases of prostatic adenoma. The
distribution of these cells in the gland and the colocalization of CCK with the
amine 5-HT, or with the peptide chromogranin A, considered to be a NE-marker
[22], are also reported.
MATERIALS
AND
METHODS
Tissue samples were collected from the prostates of two patients, aged 35
and 56, suffering from prostatic adenoma, who had undergone transurethral
resection. Soon after their surgical removal, the specimens were fixed in Bouin's
fuid for 24 h and routinely dehydrated, and then embedded in Paraplast. Serial
sections of 3 ~m thickness were stained using Linder's argyrophil method [23]
(recently applied to the study of NE cells [24, 25]), the immunohistochemical
peroxidase-antiperoxidase (PAP) technique of Sternberger [26], and a double
labeling indirect immunofluorescent technique [27]. The PAP technique was
performed using primary antibodies directed against chromogranin A, CCK and
gastrin. Consecutive sections were stained alternately with the anti-CCK and
anti-chromogranin A antibodies to investigate their eventual colocalization in the
same cell. The site of the immunological reaction was revealed by diaminobenzidine (DAB). All positivities were eliminated by liquid-phase absorption of each
antiserum with an excess of the respective antigen (100 ~g/ml), and they were
present when the anti-CCK antibody was pre-absorbed with an excess of gastrin.
This control was performed because of the possibility of cross-reactions between
the anti-CCK antibody and gastrin.
The colocalization of the peptide hormone CCK with the amine 5-HT was
studied using the double labeling immunofluorescent technique described by
Wessendorf and Elde [27]. The two primary antibodies were raised in two
different species; goat for anti-5-HT and rabbit for anti-CCK. The secondary
antibodies were conjugated with either lissamine-rhodamine isothyocyanate
(LRITC) or fluorescein isothyocyanate (FITC). Control sections were also
obtained using the methods described by Wessendorf and Elde [27]. The
microscope used to observe the slides was equipped with a reflecting illumination
system, and with LRITC and FITC filter sets. The LRITC set was composed of a
546/14 nm bandpass excitation filter, and a 580 nm longpass barrier filter. The
FITC set was composed of a 450-490 nm bandpass excitation filter, and a
525/20 nm bandpass barrier filter.
The studies of colocalization of substances were performed on serial sections
in order to view a single paraneuron in its entirety.
The primary antisera dilutions, incubation times, and characteristics are
reported in Table 1.
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Cecio, Cocca, Masotti, La Mura and Vittoria
RESULTS
Numerous argyrophil paraneurons (Fig. la~ b) were found throughout the
prostatic epithelium. They were contained both in the secretory and excretory
portions of the gland and, usually, were located between the basal m e m b r a n e and
the exocrine cells. With regard to size and shape, two types of N E cells were
observed. The first cell type was small and round while the second was large and
elongated. The latter showed one or more dendrite-like cytoplasmic processes,
orientated towards the glandular lumen, basal membrane or neighbouring cells.
Fig. 1 (a-d). a, b: Argyrophil paraneurons intensely stained by Linder's
method, c, d: CCK-containing NE cells showing dendrite-like cytoplasmic
expansions, a, b, c, d: 1280x.
CCK-containing NE cells in prostatic adenomas
255
Immunohistochemistry gave positive results for the antibodies directed against
chromogranin A, 5 - H T and C C K , whereas no positivity a p p e a r e d when antibodies directed against gastrin were used. CCK-containing p a r a n e u r o n s (Fig. lc,
d) were fairly numerous and clearly showed the granular content of their
cytoplasm at high magnifications. T h e y were distributed in small clusters of a few
to twenty or thirty cells scattered in the exocrine epithelium of adjacent secretory
units. Paraneurons containing chromogranin A (Fig. 2a) and 5 - H T (Fig. 2c)
generally showed the same morphological features and distribution as described
for the C C K - i m m u n o r e a c t i v e cells.
Fig. 2 (a-d).
a, b: A single paraneuron stained in two consecutive sections by chromogranin A (2a)
and CCK (2b) antibodies, c, d: The double labeling immunofluorescent technique shows 5-HT (2c)
and CCK (2d) colocalized in the same neuroendocrine cell. a, b: 1280• ; c, d: 500•
256
Cecio, Cocca, Masotti, La Mura and Vittoria
Careful observation of serial sections alternately stained with the antichromogranin A and anti-CCK antibodies showed that such substances are
frequently colocalized in the same cell (Fig. 2a, b). This colocalization was
observed in 548 paraneurons out of 600 studied, reaching a coexistence
percentage of 91.33%. Thirty-six (36) of the remaining paraneurons contained
only chromogranin A (6%) and 16 only CCK (2.67%).
The colocalization of 5-HT with CCK (Fig. 2c, d) was studied using the
double labeling immunofluorescent technique described above applied to serial
sections. 464 cells were observed and 448 of them contained the amine
colocalized with the hormone (96.55%). 11 of the remaining cells contained only
5-HT (2.37%) and 5 only CCK (1.08%).
DISCUSSION
The number of NE cells in hyperplastic prostates is highly variable.
Compared with a healthy gland both higher [13] and lower [12, 19] numbers have
been reported. The increase can involve the whole neuroendocrine population or
only a particular cytotype. When the whole population is involved, the initial
proportions between cellular types may or may not be altered [13, 15]. In any
case, it is assumed that the differences between the neuroendocrine cells of
normal and hyperplastic prostates are quantitative and not qualitative [13, 20].
Cells containing CCK were never found in normal human prostates. The
finding of this cytotype in the urethro-prostatic complex of cattle, sheep, horses,
pigs and rats [11, 28, 29, 30] led us to hypothesize that they are likely to be
present in a normal human gland, although, perhaps in small numbers.
Hyperplasy may induce the increase of this cytotype, making it detectable by
immunohistochemical techniques [13].
The paraneurons contained in human carcinomatous prostates show quantitative differences if compared with those of a normal gland, for example, the
increase/decrease of their number, and/or the appearance of new cytotypes. New
cytotypes are more frequent in poorly, rather than in well differentiated
carcinomas [17] and may contain either fl-endorphin or leu-enkephalin or ACTH
or glucagon [17, 21]. According to Mains et al. [31], under normal conditions, the
first three of these peptides are synthesized in the same polypeptidic chain by
various types of hypophyseal cells. The presence of these three peptides in
prostatic NE cells has been considered to be an ectopic production of carcinomatous cells which have lost control of their genoma [21]. Further studies are
required to clarify this point.
The role played by CCK in the prostate and elsewhere in the urogenital tract
[32] is not yet known. In the gastrointestinal tract the hormone is contained in
both endocrine and nerve cells [33], and stimulates smooth muscle contraction
[34, 35], pancreatic juice secretion [36, 37] and the activity of the lower
oesophageal, pyloric and Oddi sphincters [34]. It could be argued by analogy that
urogenital CCK plays a similar role in muscle contraction and exocrine/endocrine
secretions in the urogenital tract.
CCK-containing NE cells in prostatic adenomas
257
Notwithstanding the fact that the amine 5-HT has long been known to be
present in mammalian prostates [8, 9, 12, 13, 17] its function in the urogenital
system is still unknown. It has been hypothesized that 5-HT is involved in the
regulation of semen composition and fertilizing capability [38]. It has also been
demonstrated that the amine regulates the contraction of a dog's urethra and
urinary bladder [39] thus contributing to the emission of urogenital fluids.
The functional roles hypothesized for urogenital CCK and 5-HT partially
overlap each other, and their possible synergism is supported by the frequent
colocalization of the two substances in the same paraneuron [11, 40].
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Some Observations on Haemoglobin A