Immunofluorescent localization of enteroglucagon cells in the gastrointestinal tract of the dog - Gut
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Gut: first published as 10.1136/gut.12.4.311 on 1 April 1971. Downloaded from http://gut.bmj.com/ on January 30, 2021 by guest. Protected by copyright.
Gut, 1971, 12, 311-318
Immunofluorescent localization of enteroglucagon
cells in the gastrointestinal tract of the dog
JULIA M. POLAK, S. BLOOM, I. COULLING, AND A. G. E. PEARSE
From the Department of Histochemistry, Royal Postgraduate Medical School, and the Institute of Clinical
Research, Middlesex Hospital, London
SUMMARY Localization of the endocrine polypeptide cells responsible for 'glucagon-like immuno-
reactivity' in the gastrointestinal tract of the dog has been achieved with an immunofluorescent
technique using antibodies raised against porcine pancreatic glucagon. The cells, for which we
prefer the term 'enteroglucagon', could only be demonstrated by this technique in tissues fixed in
carbodiimide.
The enteroglucagon cells possess cytological, cytochemical, and ultrastructural characteristics
in common with those of the pancreatic CX2 cell and they are equivalent in the stomach to the A cell
and in the intestine to the L cell of the Wiesbaden terminology. Their distribution, predominantly
in fundus and jejunum, correlates precisely with the distribution of glucagon-like immunoreactivity
by radioimmunoassay and bioassay.
The storage form of enteroglucagon differs in many respects from that of pancreatic glucagon
although there are some close resemblances between the two forms of specific hormone-containing
granule. Elucidation of the role of enteroglucagon should be assisted by the ability to demonstrate
enteroglucagon cells.
Finding that a pancreatectomized dog did not die, (1961) and subsequently in the gastrointestinal tract
but lived to develop symptoms of what came to be of all 12 species examined by Assan, Rosselin, and
recognized, over two centuries later, as experimental Tchobroutsky (1968).
diabetes, Brunner (1683) concluded that the pancreas The existence of two fractions of glucagon-like
was not so vitally important as had been supposed. immunoreactivity (GLI), obtained by filtration of
He suggested that its functions could be taken over acid-ethanol extracts of canine jejunum, was re-
by other glands (eiusdem cum aliarum glandularum). ported by Unger, Ohneda, Valverde, Eisentraut,
A similar conclusion was drawn by Martinotti and Exton (1968) and by Valverde, Rigopoulou,
(1882), namely that dysfunction of the pancreas Exton, Ohneda, Eisentraut, and Unger (1968). The
could be compensated by an augmented action of larger fraction had a molecular weight of approxi-
the glands of Lieberkuhn. mately 7000, double the weight of pancreatic glu-
These observations were clearly supported when cagon, and it was devoid of glycogenolytic, hyper-
Sutherland and de Duve (1948) identified a glyco- glycaemic properties. The lesser fraction was equal
genolytic, hyperglycaemic substance in acid-ethanol in apparent molecular weight to pancreatic glucagon
extracts of dog stomach and duodenum. At a later and it possessed the two properties missing from the
date, extracts of human stomach, duodenum, small larger fraction. Both fractions caused marked insulin
bowel, and colon were reported by Makman and release in dogs during hyperglycaemia and both were
Sutherland (1964) to possess the glucagon-like found to differ immunologically from pancreatic
property of stimulating adenyl cyclase. Glucagon- glucagon. According to Valverde, Rigopoulou,
like immunoreactivity was first identified in acid- Marco, Faloona, and Unger (1970) simple enzy-
ethanol extracts of canine stomach and duodenum matic and chemical procedures could not convert
by Unger, Eisentraut, Sims, McCall, and Madison the heavier fraction of intestinal GLI into the
lighter one.
Received for publication 9 February 1971. In man, rat, and cow, it has been reported that
311Gut: first published as 10.1136/gut.12.4.311 on 1 April 1971. Downloaded from http://gut.bmj.com/ on January 30, 2021 by guest. Protected by copyright.
312 Julia Polak, S. Bloom, L Coulling, and A. G. E. Pearse
the gastric and duodenal content of GLI is rela- intestinal mucosa of the dog contain a hormone
tively low but substantial amounts are present in immunologically similar to pancreatic glucagon;
jejunum and ileum, the more distal segments tending (2) whether there are cytological, cytochemical, or
to have the highest levels. In these species the total ultrastructural resemblances between any endocrine
GLI of the gastrointestinal tract is less than the polypeptide cell and the pancreatic islet a2 cell; and
total pancreatic glucagon. In the dog, however, (3) whether cells reacting with anti-glucagon sera,
there is much more GLI in the stomach and the if present, can be shown to possess a2 cell charac-
total gastrointestinal GLI is equal to total pancreatic teristics.
GLI. Using radioimmunoassay, Unger, Ketterer, We were able to detect immunofluorescent cells
and Eisentraut (1966) found that immunological in both stomach and intestine, which reacted with
glucagon reactivity wa, low in the canine gastric antibodies to pancreatic glucagon, and we decided
antrum and high in the fundus. It was associated to call the substance responsible by its hormonal
with the mucosal layer in both stomach and intestine appellation 'enteroglucagon' rather than to employ
and not, as described by Makman and Sutherland the perhaps more accurate term 'glucagon-like
(1964), with the muscular layer. The proportions immunoreactivity'.
present in the villi and crypts were different in
jejunum and ileum. Materials and Methods
Efforts to demonstrate the localization of GLI in
the gastrointestinal tract by immunofluorescence,
carried out in several laboratories, have hitherto Four puppies (two labrador crosses and two
been unsuccessful, with a single reported exception. mongrels), between 8 and 12 weeks old were
This is the demonstration by Baxter-Grillo (1970) used. Following intraperitoneal injection of
of fluorescence in 'all the cells of the epithelium' of nembutal, samples of mucosa were taken from the
the duodenal villi in a 16-day-old chick embryo, gastric fundus, cardia and pylorus, duodenum,
using an antiserum to porcine pancreatic glucagon. high, mid- and low jejunum, high, mid- and low
Structural evidence for 'glucagon-producing cells' ileum, colon, and pancreas.
in the intestinal mucosa of the rat was produced by Small pieces of mucosa from each of the above
Orci, Pictet, Forssmann, Renold, and Rouiller regions, and similar small pieces of pancreas, were
(1968). Specifically, they claimed that one of the two processed in a variety of different ways, for the
types of non-digestive epithelial cell, as identified various procedures outlined below.
by electron microscopy, was responsible for the
production of enteroglucagon. The cell responsible IMMUNOFLUORESCENCE
contained secretion granules closely resembling Samples were fixed at 40 in formol-calcium,
those of the pancreatic insular a2 cell and it was methanol-free paraformaldehyde (Graham and
therefore described as the 'intestinal A cell' by Karnovsky, 1966) or 2% carbodiimide (Kendall,
Forssmann, Orci, Pictet, Renold, and Rouiller Polak, and Pearse, 1971). To produce this last fixa-
(1969). In the first paper of the series (Orci et al, tive l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
1968) the average diameter of the invariably round hydrochloride (Sigma), or l-cyclohyexyl-3-(2-mor-
granules of the A cell was recorded as 250 nm. In the pholinoethyl)-carbodiimide metho-p-toluene sulph-
second paper (Forssmann et al, 1969) it was de- onate (Aldrich) were dissolved in OO1M phosphate
scribed as varying between 500 and 700 nm. At the buffer saline at pH 7-1 immediately before use.
higher end of this scale granules should be visible Paraffin sections were prepared from tissues fixed in
by optical microscopy. formol-calcium. Other tissues from this fixative were
In the fundus of cat, dog, and rabbit stomach washed and stored in gum sucrose. Tissues fixed in
Vassallo, Solcia, and Capella (1969) found a small paraformaldehyde and in carbodiimide were washed
number of endocrine cells with an ultrastructural for 24 hours in 30 % sucrose phosphate buffer saline
pattern similar to the pancreatic a2 cell, with numer- and stored, if necessary, in the same buffer.
ous dense granules with an average diameter of Selected tissues were quenched in cold (- 165°)
200 nm. Using dark-field microscopy they found Arcton (Freon) 22 and cryostat sections were pre-
cells whose granules gave a silver-white luminiscence, pared. These were used either unfixed or post-fixed
a property exhibited by the granules of the pan- in cold methanol, cold acetone, or cold formol-
creatic a2 cells, in the fundic mucosa of dog and cat calcium.
but not in the pylorus or duodenum. Cryostat sections were prepared from tissues fixed
With these indications in mind, the present work in formol-calcium, paraformaldehyde, and carbo-
was undertaken in order to ascertain (1) whether diimide.
any of the endocrine polypeptide cells in the gastro- Tissue was quenched in cold Arcton 22, freeze-Gut: first published as 10.1136/gut.12.4.311 on 1 April 1971. Downloaded from http://gut.bmj.com/ on January 30, 2021 by guest. Protected by copyright.
Immunofluorescent localization of enteroglucagon cells in the gastrointestinal tract of the dog 313
dried for eight hours at - 40° in a thermoelectric ELECTRON MICROSCOPY
dryer, and embedded in 450 wax. Small pieces of tissue from fundic mucosa, mid-
All sections were cut at 5 ,m and processed by jejunum, and pancreas, were processed immediately
an indirect immunofluorescence technique (Coons, on removal from the animal for electron microscopy.
Leduc, and Connolly, 1955). These areas have been shown, by present assay
techniques, to contain the highest levels of intestinal
Antisera glucagon.
Antibodies were raised in rabbits and guinea pigs Samples were fixed in 3 % glutaraldehyde in
against porcine pancreatic glucagon using the 0 1M phosphate buffer (pH 7.6) for two hours at 4°.
carbodiimide-glucagon polymer technique of Hed- Excess fixative was removed from the blocks by
ding (1969). The globulin fraction was obtained by repeated washing in 0.1M phosphate buffer contain-
cold precipitation with ammonium sulphate (Nairn, ing 0.1M sucrose. Following fixation the blocks were
1969). Fluorescein-labelled goat anti-rabbit and anti- dehydrated in an ascending ethanol series, taken
guinea pig globulin was obtained commercially through epoxy propane, and finally embedded in
(Microbiological Associates Inc). Araldite CY 212. Other samples, with the primary
fixation described above, were post-fixed in Millonig's
Controls (1962) osmium tetroxide at 40 for two hours.
The following controls were used. (1) Anti-glucagon Sections were stained by lead citrate and uranyl
serum with added excess porcine pancreatic glucagon acetate and viewed in an AEI EM6B electron micro-
followed by fluorescein-labelled antiserum. (2) scope.
Normal rabbit or guinea pig globulin, followed by
the second layer. (3) Rabbit anti-human calcitonin Results
and anti-ACTH globulin, followed by the second
layer. (4) Fluorescein-labelled goat anti-rabbit and IMMUNOFLUORESCENCE
anti-guinea pig globulin fraction alone. Immunofluorescent demonstration of entero-
glucagon cells was obtained solely with tissues fixed
OPTICAL MICROSCOPY in carbodiimide (CDI), and cut in the cryostat after
Cytochemical and other techniques characteristically washing. Other methods of preparation gave negative
positive with pancreatic a2 cells were applied. These or, occasionally, weak and uncertain results. The
included dark-field luminescence, argyrophilia, lead glucagon-containing a2 cells of the pancreatic islets,
haematoxylin, oxidized phosphotungstic acid- on the contrary, could be demonstrated by immuno-
haematoxylin (PTAH) the xanthydrol method for fluorescence after most of the preparative procedures
tryptophan, and the o-phthalaldehyde method described.
(Takaya, 1970). All regions of stomach and intestine, with the
Small blocks of mucosa were taken from each of exception of the pylorus and duodenum, contained
the regions already described and treated in the enteroglucagon (EG) cells. These cells were most
following ways. After fixation in 6 % glutaraldehyde numerous in the fundus, and in mid- and terminal
in 0 1M phosphate buffer saline (pH 7.4), and paraffin jejunum. The fundic EG cells were situated mostly in
embedding, 5 ,tm sections were processed by the the middle and deeper portions of the mucosa (Fig. 1)
Masson-Hamperl (argentaffin) method (Pearse, while in the intestine they tended to predominate in
1960); the Lillie xanthydrol method (Solcia, the middle zone. Many of the EG cells appeared to
Sampietro, and Capella, 1969); McConnaill's lead have their long axes along the basement membrane,
haematoxylin (Solcia et al, 1969). and there was no evidence of their reaching the
After fixation in Bouin's fluid, and paraffin em- lumen of the gland. Immunofluorescence was
bedding, 5 gm sections were processed by a modified strongest in the basal portions of the cell giving, at
Grimelius technique (de Grandi, 1970) and by the times, a half-moon appearance. The degree of
oxidized PTAH method. localization in the CDI preparations was precise,
After carbodiimide fixation, and the immuno- and background fluorescence was very low (Fig. 1).
fluorescence procedure, sections were viewed by All the control sections were free from specific
dark-field microscopy or restained by lead haema- immunofluorescence.
toxylin or the Masson-Hamperl silver impregnation.
Fresh frozen cryostat sections were examined by COMPARATIVE CYTOCHEMISTRY
dark-field microscopy and, subsequently, they were The EG cells showed the following characteristics:
stained by the o-phthalaldehyde method. (1) dark-field luminosity with both fresh cryostat
Photomicrographs were taken in Ilford FP4 and CDI-fixed cryostat sections; (2) fluorescence
(immunofluorescence) or Pan F (other methods). with the o-phthalaldehyde method (Fig. 2); (3)'~ ~. .!
314 Julia Polak, S. Bloom, L. Coulling, and A. G. E. Pearse
Gut: first published as 10.1136/gut.12.4.311 on 1 April 1971. Downloaded from http://gut.bmj.com/ on January 30, 2021 by guest. Protected by copyright.
Fig. 2
Fig. 1
........ t sectFig. 1 Fundic mucosa. Carbodiimide-fixed, cryostat
section. Indirect immnunofluorescence technique. Shows
GLI in three large basigranular EG cells situated in the
t, ~.. deeper portion of the glands. (x 950.)
IES|tj
l.l ............... .. ''
Fig. 2 Fundic mucosa. Fresh frozen cryostat section.
The o-phthalaldehyde method shows fluorescence, in four
large
- basigranular cells, in the shape of a half-moon.
Fig. 3 Fundic mucosa. Bouin-fixed. Argyrophilia
demonstrated by Grimelius silver technique. Both EC
and non-EC cells are situated in the lower and middle
third of the glands. Comparison with Figs. 1 and 2
suggests that only a proportion of the non-EC cells are
EG cells. ( x 950.)
Fig. 3Gut: first published as 10.1136/gut.12.4.311 on 1 April 1971. Downloaded from http://gut.bmj.com/ on January 30, 2021 by guest. Protected by copyright.
Immunofluorescent localization of enteroglucagon cells in the gastrointestinal tract of the dog 315
Fig. 4 Jejunal mucosa. a. Specific
immunofuorescence in an EG cell.
b. Dark-field photomicrograph of the
same cell shows refractile granules
like those ofpancreatic % cells.
( x 1,400.)
K .! ]hI .41-
KI,
:.
,,,.!ws .,
.: :.
:: :,.
*:t. + +: KEC
v wr
j . . K:^
w =
w .. \\D
Fig. 5a Fig. 5b
Fig. 5 Fundic mucosa. a. Specific immunofluorescence in three EG cells (and part of a fourth). b. Lead
haematoxylin stain on same preparation shows moderately positive granulation in the EG cells with much stronger
staining in two groups of EC cells. ( x 950.)
moderate tryptophan content, much weaker than gave a weak to moderate positive reaction by con-
the (normal) strong reaction given by the EC cells; trast with the strongly reacting EC cells. Other
(4) positive lead haematoxylin staining; (5) argyro- sections, after immunofluorescence, were stained by
philia blt not argentaffinity (Fig. 3); (6) a weak the Masson-Hamperl procedure. This demonstrated
positive reaction with oxidized PTAH. that the EG cells were non-argentaffin; that is to say
To identify the cells showing specific immuno- they were not EC cells. Cryostat sections viewed by
fluorescence pairs of reactions or tests were applied dark-field microscopy, and subsequently stained
to single sections. A section of the mid-jejunal with o-phthalaldehyde, showed that the cells with
mucosa shows specific immunofluorescence (Fig. 4a) these two characteristics were identical.
and dark-field luminosity in the same cell (Fig. 4b).
Immunofluorescence sections, post-fixed in 6 % ULTRASTRUCTURAL FINDINGS
glutaraldehyde for 30 minutes, were washed and Cells closely resembling pancreatic a2 cells were
stained by the lead haematoxylin method. The found in both fundic and mid-jejunal mucosa (Fig. 6)
results are shown in Figures 5a and b. The EG cells These cells contained round, densely osmiophilicGut: first published as 10.1136/gut.12.4.311 on 1 April 1971. Downloaded from http://gut.bmj.com/ on January 30, 2021 by guest. Protected by copyright.
316 Julia Polak, S. Bloom, l. Coulling, and A. G. E. Pearse
s
lt1i1FtSi ; , ,3~ ~ ~ ~ ~ ~ ~ ~ ~
Fig. 6 Fundic mucosa. Electron micrograph shows one Fig. 7 Fundic mucosa. Electron micrograph shows the
of the two predominant non-EC endocrine polypeptide second predominant non-EC endocrine polypeptide cell
cells of this region. This is an A cell (Wiesbaden of the region. This is an EC-like cell (Wiesbaden
terminology) ( x 9,000.) terminology). ( x 15,000.)
granules with a diameter of between 200 and 300 nm. His observations were confirmed by Bussolati and
In the fundus they were considered to represent the Pearse (1970) for the porcine antrum and by Pearse
A cells of the Wiesbaden terminology (Pearse, and Bussolati (1970) for human stomach. There
Coulling, Weavers, and Friesen, 1970) while in the remained, therefore, three or four endocrine poly-
jejunum they were regarded as the large granule peptide cells in the stomach, and three in the
cells (L) of the same terminology. intestine, which had no established product. We
A second endocrine polypeptide cell was found in have now shown that in dog stomach the fundic A
the fundic mucosa. This was the EC-like cell (ECL) cell is the source of enteroglucagon while in the
shown in Figure 7. Both this cell and the A cell intestine (jejunum) this hormone is localized in the
were clearly distinguishable from the argentaffin L cell (large granule). In the human jfundus Pearse
cell (EC), with its dense polymorphic granules. No et al (1970) were unable to demonstrate A-type cells
small granular cells (S) were found in the mid- and the enteroglucagon demonstrated therein by
jejunal samples examined. Assan et al (1968) must be secreted by either D or
ECL cells, presumably the former. All these cells
Discussion (A, D, EC-like, and L) belong to the APUD series
of endocrine polypeptide cells (Pearse, 1968).
We may presume that at least four polypeptide Following the precedent set by the G cell we propose
hormones (secretin, cholecystokinin-pancreozymin, to call the enteroglucagon producing cell the EG
gastrin, and enteroglucagon) are produced by the cell, although in the case of the pancreatic islets the
eight or more endocrine cells at present recog- old Greek alphabetical notation has not yet been
nizable in the mammalian gastrointestinal tract. superseded.
Gastrin was localized by McGuigan (1968) in the The successful demonstration of the EG cell was
G cells of porcine and human stomach, so named by wholly dependent on prior fixation with carbodi-
Solcia, Vassallo, and Sampietro (1967) on the basis imide (Kendall et al, 1971). Demonstration of glu-
of their distribution and staining characteristics. cagon in the pancreatic a. cell was possible, on theGut: first published as 10.1136/gut.12.4.311 on 1 April 1971. Downloaded from http://gut.bmj.com/ on January 30, 2021 by guest. Protected by copyright.
Immunofluorescent localization of enteroglucagon cells in the gastrointestinal tract of the dog 317
contrary, after a variety of fixatives or even in un- This work was made possible by grants from the
fixed tissues. We postulate that carbodiimides react Wellcome Trust and the Medical Research Council.
with side-chain carboxyl groups, in the polypeptide We are grateful to Dr B. A. L. Hurn (Wellcome
or in its precursor protein, to yield intermediates Research Laboratories) for the gift of anti-ACTH
which then cross-react with adjacent nucleophiles serum and to Professor G. Wolf Heidegger for draw-
(ie, protein or polypeptide NH2 groups) to form ing our attention to the contributions of Brunner
peptide bonds. We do not know the structure of the (1683) and Martinotti (1883).
storage form either of pancreatic glucagon or of References
enteroglucagon. The 29 amino acid sequence of
porcine glucagon is known, and it shares a common Assan, R., Rosselin, G., and Tchobroutsky, G. (1968). Glucagon in
blood and immunological specificity of glucagon molecule
C-terminal octapeptide with secretin. Valverde, protein and polypeptide hormones. Excerpta Med. (Amst.)
Rigopoulou, Marco, Faloona, and Unger (1970) Sect., pp. 87-90.
Baxter-Grillo, D. L. (1970). Enzyme histochemistry and hormones of
indicated that peak II of their gastrointestinal GLI the developing gastrointestinal tract of the chick embryo. III.
was a molecule of similar size, glycogenolytic activity, Enterochromaffin cells-their possible products, glucagon, 5-
and electrophoretic behaviour to pancreatic gluca- hydroxytryptamine and the relation of monoamine oxidase.
Histochemie, 21, 129-135.
gon. It differed, however, with respect to its im- Brunner, J. C. (1683). Experimenta circa Pancreas, accedit diatribe de
munological behaviour with anti-porcine and anti- Lympha et Genuino Pancreatis Usu. Amsterdam.
Bussolati, G., and Pearse, A. G. E. (1970). Immunofluorescent
beef glucagon and its molecular structure was localization of the gastrin-secreting G cells in the pyloric
presumed, therefore, to differ from that of pancreatic antrum of the pig. Histochemie, 21, 1-4.
Coons, A. H., Leduc, E. H., and Connolly, J. M. (1955). Studies on
glucagon. Our observations point to a similar con- antibody production. I. A method for the histochemical
clusion. It is clear, moreover, that the conformation demonstration of specific antibody and its application to a
of carbodiimide-fixed enteroglucagon, but not of study of the hyperimmune rabbit. J. exp. Med., 102, 49-60.
de Grandi, P. (1970). The routine demonstration of C cells in human
the formaldehyde orglutaraldehyde-fixed hormone, is and animal thyroid glands. Virchows Arch. Zellpath., 6, 137-150.
such that the state of the antibody-combining site per- Forssmann, W. G., Orci, L., Pictet, R., Renold, A. E., and Rouiller,
C. (1969). The endocrine cells in the epithelium of the gastro-
mits the formation of the antigen-antibody complex. intestinal mucosa of the rat. J. Cell Biol., 40, 692-715.
Interesting correlations between the pancreatic Graham, R. C., and Karnovsky, M. J. (1966). The early stages of
a2 cell and the EG cell were provided by our staining absorption of injected horseradish peroxidase in the proximal
tubules of mouse kidney: ultrastructural cytochemistry by a
and cytochemical studies although there is no new technique. J. Histochem. Cytochem., 14, 291-302.
explanation, in terms of structure, for the dark-field Hedding, L. G. (1969). The production of glucagon antibodies in
rabbits. Hormone and Metabolic Res., 1, 87-88.
luminescence and o-phthalaldehyde staining of the Kendall, P. A., Polak, Julia M., and Pearse, A. G. E. (1971). Car-
two granule types. Both are clearly argyrophil and bodiimide fixation for immunohistochemistry: Observations on
the fixation of polypeptide hormones. Experientia (Basel), in
both contain reactive indole groups (tryptophan) the press.
although the EG granules react less strongly than McGuigan, J. E. (1968). Gastric mucosal intracellular localization of
the a2 granules. gastrin by immunofluorescence. Gastroenterology, 55, 315-327.
Makman, M. H., and Sutherland, E. W., Jr. (1964). Use of liver adenyl
The ultrastructural similarities between a2 cells cyclase for assay of glucagon in human gastro-intestinal tract
and EG cells were precisely those pointed out for a2 and pancreas. Endocrinology, 75, 127-134.
Martinotti, G. (1888). Sulla estirpazione del pancreas. G. Accad. Med.
and intestinal A cells by Orci et al (1968) and Torino, 36, 348-351.
Vassallo et al (1969). The former authors' suggestion, Millonig, G. (1962). Further observations on a phosphate buffer for
osmium solutions in fixation. In Proceedings of the 5th Inter-
based solely on electron microscopical observations, national Congress for Electronmicroscopy, Philadelphia.
that intestinal A cells produce enteroglucagon, was Edited by Sydney S. Breese, Jr. Academic Press, New York.
thus correct. Nairn, R. C. (1969). Fluorescent Protein Tracing. Livingstone, Edin-
burgh and London.
The distribution of EG cells in the dog stomach Orci, L., Pictet, R., Forssmann, W. G., Renold, A. E., and Rouiller,
and intestine exactly parallels the GLI levels of the C. (1968). Structural evidence for glucagon producing cells in
the intestinal mucosa of the rat. Diabetologia, 4, 56-57.
different regions, as determined by radioimmuno- Pearse, A. G. E. (1960). Histochemistry, Theoretical and Applied.
assay. We have clearly shown that the enterochro- Churchill, London.
Pearse, A. G. E. (1968). Common cytochemical and ultrastructural
maffin cells, which are distributed throughout the characteristics of cells producing polypeptide hormones (The
gastrointestinal tract, contain no enteroglucagon. APUD Series) and their relevance to thyroid and ultimo-
The physiological function of enteroglucagon has branchial C cells and calcitonin. Proc. roy. Soc. B., 170, 71-80.
Pearse, A. G. E., and Bussolati, G. (1970). Immunofluorescence studies
not been elucidated. The ability to demonstrate the of the distribution of gastrin cells in different clinical states.
hormone in its storage form in the EG cell should Gut, 11, 646-648.
Pearse, A. G. E., Coulling, I., Weavers, B., and Friesen, S. (1970).
facilitate studies on the response of the latter to The endocrine polypeptide cells of the human stomach, duo-
different stimuli, and throw some light on its role in denum and jejunum. Gut, 11, 649-658.
Solcia, E., Sampietro, R., and Capella, C. (1969). Differential staining
the gastrointestinal tract in health and disease. of catecholamines, 5-hydroxytryptamine and related com-
It seems likely that the straightforward views of pounds in aldehyde fixed tissues. Histochemie, 17, 273-283.
Brunner and Martinotti are an oversimplification Solcia, E., Vassallo, G., and Sampietro, R. (1967). Endocrine cells in
the antro-pyloric mucosa of the stomach. Z. Zellforsch., 81,
of the true state of affairs. 474-486.Gut: first published as 10.1136/gut.12.4.311 on 1 April 1971. Downloaded from http://gut.bmj.com/ on January 30, 2021 by guest. Protected by copyright.
318 Julia Polak, S. Bloom, I. Coulling, and A. G. E. Pearse
Sutherland, E. W., and de Duve, C. (1948). Origin and distribution of Exton, J. (1968). Characterization of the responses of circulat-
the hyperglycemic-glycogenolitic factor of the pancreas. J. ing glucagon-like immunoreactivity to intraduodenal and jin-
biol. Chem., 175, 663-674. travenous administration of glucose. J. clin. Invest., 47, 48-65.
Takaya, K. (1970). A new fluorescent stain with o-phthalaldehyde for Valverde, I., Rigopoulou, D., Exton, J., Ohneda, A., Eisentraut, A.,
A cells of the pancreatic islets. J. Histochem. Cytochem., 18, and Unger, R. H. (1968). Demonstration and characterization
178-186. of a second fraction of glucagon-like immunoreactivity in
Unger, R. H., Eisentraut, A. M., Sims, K., McCall, M. S., and jejunal extracts. Amer. J. med. Sci., 255, 415-420.
Madison, L. L. (1961). Sites of origin of glucagon in dogs and Valverde, I., Rigopoulou, D., Marco, J., Faloona, G. R., and Unger,
humans. Clin. Res., 9, 53. R. H. (1970). Characterization of glucagon-like immunore-
Unger, R. H., Ketterer, H., and Eisentraut, A. M. (1966). Distribution activity (GLI). Diabetes, 19, 614-623.
of immunoassayable glucagon in gastrointestinal tissues. Vassallo, G., Solcia, E., and Capella, C. (1969). Light and E.M.
Metabolism, 15, 865-867. identification of several types of endocrine cells in the gastro-
Unger, R. H. Ohneda, A., Valverde, I.. Eisentraut, A. M., and intestinal mucosa of the cat. Z. Zellforsch., 98, 333-356.You can also read
