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Regional distribution of immunoreactive dynorphin A in the human gastrointestinal tract
Regional Distribution of Immunoreactive A in the Human Gastrointestinal Tract S. SPAMPINATO, G.-L. FERRI*, S. CANDELETTI, G. LAE3t)t and S. FERRI
P. ROMUALDI,
Dynorphin
E. CAVICCHINI,
L. SOIMEROt,
Institute of Pharmacology, University of Bologna, lmerio 48, 40126 Bologna, Wepartment of Medicine I, University of Bologna and *Department of Anatomy, “Tor Vergata” University, Roma, Italy (Reprint request to S.S.)
Abstract-lmmunoreactive dynorphin A (ir-Dyn A) was detected throughout the human gastrointestinal tract by a validated radioimmunoassay. Moreover, the stability of ‘251-Dyn A during extraction procedures was confirmed by high performance liquid chromatography. Levels of ir-Dyn A were higher in the stomach and in the small bowel. In tissue samples separated into the main layers composing the gut wall (muscularis externa, submucosa and mucosa) ir-Dyn A was uniformly distributed. An exception was the colon, where concentrations were higher in the muscular portion. Gel permeation chromatography on samples of mucosa and muscalaris externa extracts of ileum and gastric fundus, showed immunoreactive material eluting in several forms of apparently higher molecular weight than Dyn A, while only a minor peak was found to coelute with authentic Dyn A.
Introduction Dynorphin
A (Dyn A) and the other peptides
deriving from the common precursor prodynorphin have been isolated in brain and pituitary of many species including man (1,2,3,4). Prodynorphin-derived peptides have also been detected in numerous peripheral tissues of the rat with higher concentrations in the gastrointestinal (GI) tract (2, 3, 5). However, their presence in the human gut has been little explored. In the present study, we investigated the quantitative distribution of immunoreactive Dyn A (ir-Dyn A) in the human GI tract using a validated Date received 22 December 1987 Date accepted 24 December 1987
radioimmunoassay (RIA) and characterized its molecular forms by gel permeation chromatography. To clarify the intramural distribution of ir-Dyn A tissue samples were also separated into the main layers composing the gut wall: muscularis mucosa. submucosa and mucosa.
Materials and Methods Substances
Synthetic Dyn A and Dyn A-(1-13) were purchased from Peninsula (San Carlos, CA) and purity was checked by high performance liquid chromatography (HPLC). ‘* I-Dyn A and *‘IDyn A-(1-13) were prepared and purified on HPLC as previously described (3). 101
NEUROPEPTIDES
102 Tissue collection and processing
Tissue samples (gastric fundic area, n = 6; gastric antrum, n = 5; proximal duodenum, n = 6; proximal jejunum, n = 5; distal ileum, n = 7; ascending colon, n = 6; sigmoid colon, n = 5; rectum, n = 5) were obtained fresh at surgery from specimens of gut resected for carcinoma and taken at least 8cm away from the tumor. Only those samples found histologically normal were included in the present study. Tissue samples were divided into two portions: one was microdissected into the main layers composing the gut wall as elsewhere reported (6, 7), and the other was taken as a full-thickness control. The first one was separated into mucosa (epithelium and lamina propria together), submucosa and muscularis externa. For colonic samples, the muscularis externa was further divided into “taeniae” region (comprising “taeniae”, subjacent circular muscle and intervening myenteric plexus) and the “inter-taeniae” area (comprising the remainder of the muscle and plexus). In 3 jejunal samples the mucosal epithelium was separated from the remainder of the wall (by treatment with EDTA (7)), in order to separately assess the ir-Dyn content of endocrine cells and gut nerves. Tissue samples were extracted in OSM acetic acid (lOml/g of tissue) as previously reported (6). Extracts were frozen at -70°C until assayed. Recovery of synthetic Dyn A and of 1251-Dyn A, added to the extraction solution. exceeded 85%.
HPLC analysis of the recovered radioactivity indicated that the peptide was not broken down during extraction.
RIA procedures
Ir-Dyn was measured by a previously described method (3) using the “Lucia” antiserum raised against Dyn A-(1-13) which displays full crossreactivity to Dyn A-(1-17) and recognizes larger molecular weight putative precursor forms of Dyn A (8, 9). Routinely, determinations were carried out in triplicate across three dilutions made in O.lM HCI containing 0.1% Triton X-100. For the serially diluted extracts the tracer followed curves parallel to the standard curve of Dyn A-(1-13) (Fig 1). Intra-assay and inter-assay variations were below 5%.
Stability of radiolabeled Dyn A-(I-I3) incubation mixture
in the RIA
The stability of ‘251-Dyn A-(1-13) used as radiolabeled tracer, in the presence of tissue extracts for 24h at 4°C in the RIA incubation mixture was checked in two ways: (1) prolonged incubation as described by Ghazarossian et al. (9). Displacement of radioligand after 48 h of incubation was no different from that after 24h. Delayed addition of 1251-Dyn A-(1-13) and antiserum after 24h incu-
.
\ .
Dyn A 1-13 Fig 1 Parallel displacement radioimmunoassay.
( fmo~/ tuba )
ANTRUM
ILEUM
by the standard dynorphin A-(1-13) and serial dilutions of extracts from antrum and ileum in
IMMUNOREACTIVE
DYNORPHIN
A IN THE HUMAN
GASTROINTESTINAL
bation of tissue extracts alone at 4°C resulted in the same displacement of the radioligand. (2) HPCL analysis of ‘251-Dyn A-(1-13) after 24 h incubation in the RIA mixture. Adopting a procedure described in a previous paper (3), the radioligand was found to be stable under RIA conditions. Gel permeation chromatography Gel permeation chromatography was performed at 4” C on Sephadex G-50 superfine columns (1.5 x 9Ocm). The eluant consisted of 0.15M acetic acid containing 0.1% Triton X-100 and 0.15M NaCl. Pooled samples were lyophilized, redissolved in 2ml of column eluant and applied to the column. Flow rate was 4mVh and 2ml fractions were lyophilized, reconstituted in 100~1 of O.lN HCl containing 0.1% Triton X-100 and assayed. Recovery of Dyn A, and of ‘*“I-Dyn A. approached 85% under these conditions.
Results
Ir-Dyn A was measurable in acetic acid extracts of all human gut specimens. As shown in the Table, the highest concentrations were found in the stomach and small bowel. Table Concentrations of Immunoreactive Dynorphin A in Extracts of Human Gastrointestinal Tract Tissue Gastric fundic area Gastric antrum Duodenum Jejunum Ileum Ascending colon Sigmoid colon Rectum
ir-Dyn A (pmollg) Mean + Sem 3.01 4.07 5.71 4.84 4.77 1.49 2.02 3.47
+ 0.3 + 0.9 + 0.5 zk 2.0 f 1.2 + 0.1 + 0.9 +_0.3
The distribution of ir-Dyn A (expressed as % of the total immunoreactivity) in the separate layers of the wall is reported in Figure 2. The percentages of immunoreactivity were similar in the mucosa, submucosa and muscle layer of all samples examined, except the ascending and sigmoid colon. The muscle layer of these tissues, in fact, contained 51.9 + 2.41% and 56.4 + 8.9% respectively, of the total immunoreactive material. In the ascending colon higher concentrations were present in the “taeniae” than the “inter-taeniae” region (Fig 3).
103
TRACT
ANTRUH
DUCOEM
JEJUNW
SIGMCCUN
RECTUM
+L100 ._
1
-.
z_
ILEUM
ASCCOUN
Fig 2 Percentage distribution of ir-Dyn A in each of the three layers of the human gut (% of total content; means It: SEM)
q mucosa:
n muscle
N submucosa;
layer.
When the endocrine cell-containing mucosal epithelium was separated from the remainder of the wall, the former layer was found to contain 32.0 + 6.1 of the total content of Dyn A immunoreactivity. Gel permeation chromatography studies on extracts of mucosa and muscle layer of the ileum revealed ir-Dyn A peaks in several molecularweight regions (Fig 4). In both extracts two forms with an apparently higher molecular weight than authentic Dyn A were predominant. The first peak emerged near the position of the void volume and the second in the same region as Dyn A-(1-32). Only a small percentage of the total immunoreactivity eluted in the same position as Dyn A.
P&
10
1
ascending
Cl
taeniae
sigmoid colon
l
inter-taeniae
Fig 3 Distribution of ir-Dyn A in the “taeniae” and “intertaeniae” regions of the colonic muscularis externa (means f SEM).
NEUROPEPTIDES
a
50-
MUSCULARlS EXTERNA
h
c 1
b
5oJ
d
e
1
1
MUCOSA
E 0 ._ Y g
40-
c
t u c
2o
s I .‘-
lo-
/
0-,
20
30
40 FRACTION
50
60
10
NUMBER
20
30
40 FRACTION
50
60
;0
NUMBER
Fig 4 Representative elution profile of ir-Dyn A in extracts of mucosa and muscular layer of the proximal ileum after gel permeation chromatography. Markers indicated by arrows are: a = Vo (blue dextran); b = cytochrome c; c = Dyn A-( l-32); d = DynA; e = Vt (CoCl,).
Similar profiles were obtained after gel permeation chromatography of extracts of mucosa and muscularis externa of the stomach specimens (data not shown). Discussion These findings show that ir-Dyn A occurs throughout the human GI tract. The stability of i*‘I-Dyn A during extraction procedures and of ‘*‘I-Dyn A-(1-13), used as radiolabeled tracer in RIA conditions, was confirmed by HPLC. Higher levels of ir-Dyn A were detected in the stomach, duodenum, jejunum and ileum. Intramural distribution of ir-Dyn A was uniform in all layers of the gut were except the colon, where concentrations higher in the muscular portion. Gel permeation chromatography studies showed ir-Dyn A eluting in several forms with an apparently higher molecular weight than Dyn A, which seemed to be present only in a small amount. These data confirm our previous report (3) of the heterogeneity of ir-Dyn A in rat GI tract. Moreover, several molecular forms contribute to the total immunoreactivity in different neural tissues (8). It is known that in the gut biologically active peptides may be of neural origin and/or contained
in edocrine cells (10, 11). The presence of ir-Dyn A in the muscular layer, as well as in the mucosa and submucosa, suggests that it may be located in nerve endings and endocrine cells. The observation that in the jejunum almost all the mucosal immunoreactivity was found in the endocrine cell-containing epithelium, further supports the occurrence of ir-Dyn A in both structures. The detection of different molecular weight forms of ir-Dyn A, showed by gel chromatography, could suggest an intramural pattern of distribution similar to that reported for somatostatin (12), with a predominance of larger molecular weight forms in the epithelial cells. As regards the localization of pro dynorphinderived peptides within neural structures, Wolter (5) reported the presence of Dyn B in neural cell bodies of the rat duodenum. Moreover, immunocytochemical staining of guinea pig ileum showed a heavy concentration of Dyn-positive fibers in the sub-mucosus plexus (13). The finding of ir-Dyn A in the mucosa confirms previous observations by Hokfelt et al. (14) who detected Dyn-positive cell bodies in lamina propria of the guinea pig colon, by immunohistochemistry. In contrast, other opioid peptides, such as enkephalins, tend preferentially to concentrate in neural structures (11, 14, 15, 16).
IMMUNOREACTIVE
DYNORPHIN
A IN THE HUMAN
GASTROINTESTINAL
Whether the presence of prodynorphin-derived peptides throughout the human GI tract is indicative of a physiological role of the dynorphins is still unknown. Besides the enkephalins, these peptides too may be involved in controlling intestinal motility (17. 18) or secretory activity of several digestive glands (19, 20). It has been shown, in fact, that Dyn A is the most potent endogenous opioid in depressing muscle twitch in the guinea pig ileum myenteric plexus-longitudinal muscle bioassay (21) and it is released from vascularly perfused guinea pig small intestine during peristalsis (22, 23). Acknowledgements The authors wish to thank Prof. A. Goldstein for the kind gift of “Lucia” antiserum used in this study. This research was supported in part, by a grant from the Regione Emilia Romagna “Ricerca Sanita-ia Finalizzata - Malattie Disendocrine, Dismetaboliche e dell’Apparato Digerente”, by a grant from MPI and by a CNR Research Contract (86.01781.56).
References I. Goldstein.
7.
3.
4.
5.
6.
7.
8. 9.
A, and Ghazarossian, V. E. (1980). Immunoreactive dynorphin in pituitary and brain. Proc. Natl. Acad. Sci. USA 77: 6207-6210. Maysinger, D.. Hollt, V., Seizinger, B. R., Mehraeinm A.. Pasi. A. and Herz, A. (1982). Parallel distribution of immunoreactive a-neo-endorphin and dynorphin in rat and human tissues. Neuropeptides 2: 211-225. Spampinato. S. and Goldstein, A. (1983). Immunoreactive dynorphin in rat tissues and plasma. Neuropeptides 3: 193-212. Gramsch, C., Hollt. V.. Pasi. A., Mehraein, P. and Herz. A. (1982). Immunoreactive dynorphin in human brain and pituitary. Brain Res. 233: 65-74. Walter. H. J. (1986). Identification of the tridecapeptide dynorphin B (rimorphin) within perikarya of rat duodenum. Life Sci. 39: 727-730. Ferri. G.-L.. Adrian, T. E., Ghatei. M.A., O’Shaughnessy. D. J.. Proberts, L., Lee, Y. C., Buchan, A. M. J., Polak, J. M. and Bloom. S. R. (1983). Tissue localization and relative distribution of regulatory peptides in separated layers from the human bowel. Gastroenterology 84: 777-786. Fcrri. G. L., Marangos, P. J., Bloom, S. R. and Polak. J. M. (19X3). Intramural distribution of neuron specific cnolase (NSE) in the human gastrointestinal tract. Experientia 39: 622-623. .Xie. G.-X. and Goldstein. A. (1987). Characterization of big dynorphins from rat brain and spinal cord. J. Neurosci. 7: 204Y-2055. Ghazarossian, V. E.. Chavkin, C. and Goldstein, A. I lY80). A specific radioimmunoassay for the novel opioid peptide dynorphin. Life. Sci. 27: 75-86.
TRACT
10.5
10. Keast, J. R.. Furness, J. B. and Costa, M. (1985). Distribution of certain peptide-containing nerve fibers and endocrine cells in the gastrointestinal mucosa in five mammalian species. J. Comp. Neurol. 236: 403-422. 11. Schultzberg. M., Hiikfelt, T.. Nilsson, G.. Terenius, L., Rehfeld. J. F., Brown, M., Elde, R.. Goldstein. M. and Said. S. I. (1980). Distribution of peptide- and catecholamine-containing neurons in the gastrointestinal tract of rat and guinea pig: immunohistochemical studies with antisera to substance P.. vasoactive intestinal polypeptide. enkephalins, somastatin, gastrin/cholecystokinin, neurotensin and dopamine 8-hydroxylase. Neuroscience 5: 689-774. 12. Vinik, A. I., Gaginella, T. S., O‘Dorisio. T. M., Shapiro. B and Wagner L. (1981). The distribution and characterization of somatostatin-like immunoreactivity in epithelial cells. submucosa and muscle of the rat stomach and intestine. Endocrinology 109: 1921-1926. 13. Watson. S. J., Akil, H.. Ghazarossian, V. E. and Goldstein, A. (1981). Dynorphin immunocytochemical localization in brain and peripheral nervous system: preliminary studies. Proc. Natl. Acad. Sci. USA 78: 1260-1263. 14. Hokfelt. T.. Vincent, S. R.. Dalsgaard, C. J.. HerreraMarschitz. Ungerstedt, U.. Schultzberg, M., Christensson. I. and Terenius. L. (1984). Some aspects on distribution and role of opioid peptides in the central and peripheral nervous system. In: Muller. E. E. and Genazzani. A. R. (eds) Central and Peripheral Endorphins: Basic and Clinical Aspects. Raven Press. New York, p. l-16. IS. Furness. J. B., Costa, M. and Miller. R. J. (1983). Distribution and projections of nerves with enkephalinlike immunoreactivity in the guinea pig small intestine. Neuroscience 8: 653-664. 16. Ferri, G.-L., Morreale, R. A. and Dockray, G. J. (1986). Met”-enkephalin-Arg”-Gly7-LeuX immunoreactivity in the human gut. Peptides 7: 735-739. 17. North, R. A. and Egan, T. M. (1983). Actions and distribution of opioid peptides in peripheral tissues. Br. Med. Bull. 39: 71-75. 18. Manara. L. and Bianchetti, A. (1985). The central and peripheral influences of opioids on gastrointestinal propulsion. Ann. Rev. Pharmacol. Toxicol. 25: 249-273. 19. Flemstrom, G., Jedstedt. G. and Nylander, 0. (1986). B-endorphin and enkephalins stimulate duodenal mucosal alkaline secretion in the rat “in vivo”. Gastroenterology 90: 368-372. 20. Coupar. I. M. (1987). Opioid action on the intestine: the importance of the intestinal mucosa. Life Sci. 41: 917-925. 21 Goldstein, A.. Fischli, W.. Lowney. L. I.. Hunkapiller, M. and Hood. L. (1981). Porcine pituitary dynorphin: complete amino acid sequence of the biologically active heptadecapeptide. Proc. Natl. Acad. Sci. USA 78: 72197223, 22 Kromer. W., Hollt. V.. Schmidt, H. and Herz. A. (1981). Release of immunoreactive-dynorphin from the isolated guinea pig small intestine is reduced during peristaltic activity. Neurosci. Lett. 25: 53-56. 23 Donnerer. J.. Holzer, P. and Lembeck, F. (1984). Release of dynorphin. somatostatin and substance P from the vascularly perfused small intestine of the guinea pig during peristalsis. Br. J. Pharmacol. 83: 919-925.
P. ROMUALDI,
Dynorphin
E. CAVICCHINI,
L. SOIMEROt,
Institute of Pharmacology, University of Bologna, lmerio 48, 40126 Bologna, Wepartment of Medicine I, University of Bologna and *Department of Anatomy, “Tor Vergata” University, Roma, Italy (Reprint request to S.S.)
Abstract-lmmunoreactive dynorphin A (ir-Dyn A) was detected throughout the human gastrointestinal tract by a validated radioimmunoassay. Moreover, the stability of ‘251-Dyn A during extraction procedures was confirmed by high performance liquid chromatography. Levels of ir-Dyn A were higher in the stomach and in the small bowel. In tissue samples separated into the main layers composing the gut wall (muscularis externa, submucosa and mucosa) ir-Dyn A was uniformly distributed. An exception was the colon, where concentrations were higher in the muscular portion. Gel permeation chromatography on samples of mucosa and muscalaris externa extracts of ileum and gastric fundus, showed immunoreactive material eluting in several forms of apparently higher molecular weight than Dyn A, while only a minor peak was found to coelute with authentic Dyn A.
Introduction Dynorphin
A (Dyn A) and the other peptides
deriving from the common precursor prodynorphin have been isolated in brain and pituitary of many species including man (1,2,3,4). Prodynorphin-derived peptides have also been detected in numerous peripheral tissues of the rat with higher concentrations in the gastrointestinal (GI) tract (2, 3, 5). However, their presence in the human gut has been little explored. In the present study, we investigated the quantitative distribution of immunoreactive Dyn A (ir-Dyn A) in the human GI tract using a validated Date received 22 December 1987 Date accepted 24 December 1987
radioimmunoassay (RIA) and characterized its molecular forms by gel permeation chromatography. To clarify the intramural distribution of ir-Dyn A tissue samples were also separated into the main layers composing the gut wall: muscularis mucosa. submucosa and mucosa.
Materials and Methods Substances
Synthetic Dyn A and Dyn A-(1-13) were purchased from Peninsula (San Carlos, CA) and purity was checked by high performance liquid chromatography (HPLC). ‘* I-Dyn A and *‘IDyn A-(1-13) were prepared and purified on HPLC as previously described (3). 101
NEUROPEPTIDES
102 Tissue collection and processing
Tissue samples (gastric fundic area, n = 6; gastric antrum, n = 5; proximal duodenum, n = 6; proximal jejunum, n = 5; distal ileum, n = 7; ascending colon, n = 6; sigmoid colon, n = 5; rectum, n = 5) were obtained fresh at surgery from specimens of gut resected for carcinoma and taken at least 8cm away from the tumor. Only those samples found histologically normal were included in the present study. Tissue samples were divided into two portions: one was microdissected into the main layers composing the gut wall as elsewhere reported (6, 7), and the other was taken as a full-thickness control. The first one was separated into mucosa (epithelium and lamina propria together), submucosa and muscularis externa. For colonic samples, the muscularis externa was further divided into “taeniae” region (comprising “taeniae”, subjacent circular muscle and intervening myenteric plexus) and the “inter-taeniae” area (comprising the remainder of the muscle and plexus). In 3 jejunal samples the mucosal epithelium was separated from the remainder of the wall (by treatment with EDTA (7)), in order to separately assess the ir-Dyn content of endocrine cells and gut nerves. Tissue samples were extracted in OSM acetic acid (lOml/g of tissue) as previously reported (6). Extracts were frozen at -70°C until assayed. Recovery of synthetic Dyn A and of 1251-Dyn A, added to the extraction solution. exceeded 85%.
HPLC analysis of the recovered radioactivity indicated that the peptide was not broken down during extraction.
RIA procedures
Ir-Dyn was measured by a previously described method (3) using the “Lucia” antiserum raised against Dyn A-(1-13) which displays full crossreactivity to Dyn A-(1-17) and recognizes larger molecular weight putative precursor forms of Dyn A (8, 9). Routinely, determinations were carried out in triplicate across three dilutions made in O.lM HCI containing 0.1% Triton X-100. For the serially diluted extracts the tracer followed curves parallel to the standard curve of Dyn A-(1-13) (Fig 1). Intra-assay and inter-assay variations were below 5%.
Stability of radiolabeled Dyn A-(I-I3) incubation mixture
in the RIA
The stability of ‘251-Dyn A-(1-13) used as radiolabeled tracer, in the presence of tissue extracts for 24h at 4°C in the RIA incubation mixture was checked in two ways: (1) prolonged incubation as described by Ghazarossian et al. (9). Displacement of radioligand after 48 h of incubation was no different from that after 24h. Delayed addition of 1251-Dyn A-(1-13) and antiserum after 24h incu-
.
\ .
Dyn A 1-13 Fig 1 Parallel displacement radioimmunoassay.
( fmo~/ tuba )
ANTRUM
ILEUM
by the standard dynorphin A-(1-13) and serial dilutions of extracts from antrum and ileum in
IMMUNOREACTIVE
DYNORPHIN
A IN THE HUMAN
GASTROINTESTINAL
bation of tissue extracts alone at 4°C resulted in the same displacement of the radioligand. (2) HPCL analysis of ‘251-Dyn A-(1-13) after 24 h incubation in the RIA mixture. Adopting a procedure described in a previous paper (3), the radioligand was found to be stable under RIA conditions. Gel permeation chromatography Gel permeation chromatography was performed at 4” C on Sephadex G-50 superfine columns (1.5 x 9Ocm). The eluant consisted of 0.15M acetic acid containing 0.1% Triton X-100 and 0.15M NaCl. Pooled samples were lyophilized, redissolved in 2ml of column eluant and applied to the column. Flow rate was 4mVh and 2ml fractions were lyophilized, reconstituted in 100~1 of O.lN HCl containing 0.1% Triton X-100 and assayed. Recovery of Dyn A, and of ‘*“I-Dyn A. approached 85% under these conditions.
Results
Ir-Dyn A was measurable in acetic acid extracts of all human gut specimens. As shown in the Table, the highest concentrations were found in the stomach and small bowel. Table Concentrations of Immunoreactive Dynorphin A in Extracts of Human Gastrointestinal Tract Tissue Gastric fundic area Gastric antrum Duodenum Jejunum Ileum Ascending colon Sigmoid colon Rectum
ir-Dyn A (pmollg) Mean + Sem 3.01 4.07 5.71 4.84 4.77 1.49 2.02 3.47
+ 0.3 + 0.9 + 0.5 zk 2.0 f 1.2 + 0.1 + 0.9 +_0.3
The distribution of ir-Dyn A (expressed as % of the total immunoreactivity) in the separate layers of the wall is reported in Figure 2. The percentages of immunoreactivity were similar in the mucosa, submucosa and muscle layer of all samples examined, except the ascending and sigmoid colon. The muscle layer of these tissues, in fact, contained 51.9 + 2.41% and 56.4 + 8.9% respectively, of the total immunoreactive material. In the ascending colon higher concentrations were present in the “taeniae” than the “inter-taeniae” region (Fig 3).
103
TRACT
ANTRUH
DUCOEM
JEJUNW
SIGMCCUN
RECTUM
+L100 ._
1
-.
z_
ILEUM
ASCCOUN
Fig 2 Percentage distribution of ir-Dyn A in each of the three layers of the human gut (% of total content; means It: SEM)
q mucosa:
n muscle
N submucosa;
layer.
When the endocrine cell-containing mucosal epithelium was separated from the remainder of the wall, the former layer was found to contain 32.0 + 6.1 of the total content of Dyn A immunoreactivity. Gel permeation chromatography studies on extracts of mucosa and muscle layer of the ileum revealed ir-Dyn A peaks in several molecularweight regions (Fig 4). In both extracts two forms with an apparently higher molecular weight than authentic Dyn A were predominant. The first peak emerged near the position of the void volume and the second in the same region as Dyn A-(1-32). Only a small percentage of the total immunoreactivity eluted in the same position as Dyn A.
P&
10
1
ascending
Cl
taeniae
sigmoid colon
l
inter-taeniae
Fig 3 Distribution of ir-Dyn A in the “taeniae” and “intertaeniae” regions of the colonic muscularis externa (means f SEM).
NEUROPEPTIDES
a
50-
MUSCULARlS EXTERNA
h
c 1
b
5oJ
d
e
1
1
MUCOSA
E 0 ._ Y g
40-
c
t u c
2o
s I .‘-
lo-
/
0-,
20
30
40 FRACTION
50
60
10
NUMBER
20
30
40 FRACTION
50
60
;0
NUMBER
Fig 4 Representative elution profile of ir-Dyn A in extracts of mucosa and muscular layer of the proximal ileum after gel permeation chromatography. Markers indicated by arrows are: a = Vo (blue dextran); b = cytochrome c; c = Dyn A-( l-32); d = DynA; e = Vt (CoCl,).
Similar profiles were obtained after gel permeation chromatography of extracts of mucosa and muscularis externa of the stomach specimens (data not shown). Discussion These findings show that ir-Dyn A occurs throughout the human GI tract. The stability of i*‘I-Dyn A during extraction procedures and of ‘*‘I-Dyn A-(1-13), used as radiolabeled tracer in RIA conditions, was confirmed by HPLC. Higher levels of ir-Dyn A were detected in the stomach, duodenum, jejunum and ileum. Intramural distribution of ir-Dyn A was uniform in all layers of the gut were except the colon, where concentrations higher in the muscular portion. Gel permeation chromatography studies showed ir-Dyn A eluting in several forms with an apparently higher molecular weight than Dyn A, which seemed to be present only in a small amount. These data confirm our previous report (3) of the heterogeneity of ir-Dyn A in rat GI tract. Moreover, several molecular forms contribute to the total immunoreactivity in different neural tissues (8). It is known that in the gut biologically active peptides may be of neural origin and/or contained
in edocrine cells (10, 11). The presence of ir-Dyn A in the muscular layer, as well as in the mucosa and submucosa, suggests that it may be located in nerve endings and endocrine cells. The observation that in the jejunum almost all the mucosal immunoreactivity was found in the endocrine cell-containing epithelium, further supports the occurrence of ir-Dyn A in both structures. The detection of different molecular weight forms of ir-Dyn A, showed by gel chromatography, could suggest an intramural pattern of distribution similar to that reported for somatostatin (12), with a predominance of larger molecular weight forms in the epithelial cells. As regards the localization of pro dynorphinderived peptides within neural structures, Wolter (5) reported the presence of Dyn B in neural cell bodies of the rat duodenum. Moreover, immunocytochemical staining of guinea pig ileum showed a heavy concentration of Dyn-positive fibers in the sub-mucosus plexus (13). The finding of ir-Dyn A in the mucosa confirms previous observations by Hokfelt et al. (14) who detected Dyn-positive cell bodies in lamina propria of the guinea pig colon, by immunohistochemistry. In contrast, other opioid peptides, such as enkephalins, tend preferentially to concentrate in neural structures (11, 14, 15, 16).
IMMUNOREACTIVE
DYNORPHIN
A IN THE HUMAN
GASTROINTESTINAL
Whether the presence of prodynorphin-derived peptides throughout the human GI tract is indicative of a physiological role of the dynorphins is still unknown. Besides the enkephalins, these peptides too may be involved in controlling intestinal motility (17. 18) or secretory activity of several digestive glands (19, 20). It has been shown, in fact, that Dyn A is the most potent endogenous opioid in depressing muscle twitch in the guinea pig ileum myenteric plexus-longitudinal muscle bioassay (21) and it is released from vascularly perfused guinea pig small intestine during peristalsis (22, 23). Acknowledgements The authors wish to thank Prof. A. Goldstein for the kind gift of “Lucia” antiserum used in this study. This research was supported in part, by a grant from the Regione Emilia Romagna “Ricerca Sanita-ia Finalizzata - Malattie Disendocrine, Dismetaboliche e dell’Apparato Digerente”, by a grant from MPI and by a CNR Research Contract (86.01781.56).
References I. Goldstein.
7.
3.
4.
5.
6.
7.
8. 9.
A, and Ghazarossian, V. E. (1980). Immunoreactive dynorphin in pituitary and brain. Proc. Natl. Acad. Sci. USA 77: 6207-6210. Maysinger, D.. Hollt, V., Seizinger, B. R., Mehraeinm A.. Pasi. A. and Herz, A. (1982). Parallel distribution of immunoreactive a-neo-endorphin and dynorphin in rat and human tissues. Neuropeptides 2: 211-225. Spampinato. S. and Goldstein, A. (1983). Immunoreactive dynorphin in rat tissues and plasma. Neuropeptides 3: 193-212. Gramsch, C., Hollt. V.. Pasi. A., Mehraein, P. and Herz. A. (1982). Immunoreactive dynorphin in human brain and pituitary. Brain Res. 233: 65-74. Walter. H. J. (1986). Identification of the tridecapeptide dynorphin B (rimorphin) within perikarya of rat duodenum. Life Sci. 39: 727-730. Ferri. G.-L.. Adrian, T. E., Ghatei. M.A., O’Shaughnessy. D. J.. Proberts, L., Lee, Y. C., Buchan, A. M. J., Polak, J. M. and Bloom. S. R. (1983). Tissue localization and relative distribution of regulatory peptides in separated layers from the human bowel. Gastroenterology 84: 777-786. Fcrri. G. L., Marangos, P. J., Bloom, S. R. and Polak. J. M. (19X3). Intramural distribution of neuron specific cnolase (NSE) in the human gastrointestinal tract. Experientia 39: 622-623. .Xie. G.-X. and Goldstein. A. (1987). Characterization of big dynorphins from rat brain and spinal cord. J. Neurosci. 7: 204Y-2055. Ghazarossian, V. E.. Chavkin, C. and Goldstein, A. I lY80). A specific radioimmunoassay for the novel opioid peptide dynorphin. Life. Sci. 27: 75-86.
TRACT
10.5
10. Keast, J. R.. Furness, J. B. and Costa, M. (1985). Distribution of certain peptide-containing nerve fibers and endocrine cells in the gastrointestinal mucosa in five mammalian species. J. Comp. Neurol. 236: 403-422. 11. Schultzberg. M., Hiikfelt, T.. Nilsson, G.. Terenius, L., Rehfeld. J. F., Brown, M., Elde, R.. Goldstein. M. and Said. S. I. (1980). Distribution of peptide- and catecholamine-containing neurons in the gastrointestinal tract of rat and guinea pig: immunohistochemical studies with antisera to substance P.. vasoactive intestinal polypeptide. enkephalins, somastatin, gastrin/cholecystokinin, neurotensin and dopamine 8-hydroxylase. Neuroscience 5: 689-774. 12. Vinik, A. I., Gaginella, T. S., O‘Dorisio. T. M., Shapiro. B and Wagner L. (1981). The distribution and characterization of somatostatin-like immunoreactivity in epithelial cells. submucosa and muscle of the rat stomach and intestine. Endocrinology 109: 1921-1926. 13. Watson. S. J., Akil, H.. Ghazarossian, V. E. and Goldstein, A. (1981). Dynorphin immunocytochemical localization in brain and peripheral nervous system: preliminary studies. Proc. Natl. Acad. Sci. USA 78: 1260-1263. 14. Hokfelt. T.. Vincent, S. R.. Dalsgaard, C. J.. HerreraMarschitz. Ungerstedt, U.. Schultzberg, M., Christensson. I. and Terenius. L. (1984). Some aspects on distribution and role of opioid peptides in the central and peripheral nervous system. In: Muller. E. E. and Genazzani. A. R. (eds) Central and Peripheral Endorphins: Basic and Clinical Aspects. Raven Press. New York, p. l-16. IS. Furness. J. B., Costa, M. and Miller. R. J. (1983). Distribution and projections of nerves with enkephalinlike immunoreactivity in the guinea pig small intestine. Neuroscience 8: 653-664. 16. Ferri, G.-L., Morreale, R. A. and Dockray, G. J. (1986). Met”-enkephalin-Arg”-Gly7-LeuX immunoreactivity in the human gut. Peptides 7: 735-739. 17. North, R. A. and Egan, T. M. (1983). Actions and distribution of opioid peptides in peripheral tissues. Br. Med. Bull. 39: 71-75. 18. Manara. L. and Bianchetti, A. (1985). The central and peripheral influences of opioids on gastrointestinal propulsion. Ann. Rev. Pharmacol. Toxicol. 25: 249-273. 19. Flemstrom, G., Jedstedt. G. and Nylander, 0. (1986). B-endorphin and enkephalins stimulate duodenal mucosal alkaline secretion in the rat “in vivo”. Gastroenterology 90: 368-372. 20. Coupar. I. M. (1987). Opioid action on the intestine: the importance of the intestinal mucosa. Life Sci. 41: 917-925. 21 Goldstein, A.. Fischli, W.. Lowney. L. I.. Hunkapiller, M. and Hood. L. (1981). Porcine pituitary dynorphin: complete amino acid sequence of the biologically active heptadecapeptide. Proc. Natl. Acad. Sci. USA 78: 72197223, 22 Kromer. W., Hollt. V.. Schmidt, H. and Herz. A. (1981). Release of immunoreactive-dynorphin from the isolated guinea pig small intestine is reduced during peristaltic activity. Neurosci. Lett. 25: 53-56. 23 Donnerer. J.. Holzer, P. and Lembeck, F. (1984). Release of dynorphin. somatostatin and substance P from the vascularly perfused small intestine of the guinea pig during peristalsis. Br. J. Pharmacol. 83: 919-925.