Fish Physiology and Biochemistry vol. 8 no. 5 pp 399-408 (1990) Kugler Publications, Amsterdam/Berkeley
Gut hormones in cyclostomes Susan Van Noorden Histopathology Department, Royal PostgraduateMedical School, Du Cane Road, London, W12 ONN, England Keywords: cyclostome, lamprey, hagfish, regulatory peptides, neuropeptides, pancreas, intestine, endocrine cells, hormones
Abstract The current state of knowledge about regulatory peptides in endocrine cells and nerves of the alimentary canal of lampreys and hagfishes is reviewed. Cyclostomes have a wide range of peptides similar immunochemically to those of higher vertebrates. They include, in the endocrine cells of the intestine, peptides resembling glucagon, gastrin/cholecystokinin, peptide YY (pancreatic polypeptide/neuropeptide Y), substance P, vasoactive intestinal peptide, somatostatin and, in the larval stages at least, insulin. The enteric nerves of some lamprey species contain peptides resembling bombesin (gastrin-releasing peptide) and calcitonin gene-related peptide, as well as serotonin. The occurrence of other peptides is less well documented. Little is known of the molecular structure or the biological roles of the enteric peptides in cyclostomes. Extraction, purification, sequencing and physiological experiments are greatly needed.
Introduction Cyclostome gut endocrinology was comprehensively reviewed in 1982 (Hardisty and Baker 1982). Findings up to that time will be, therefore, summarised briefly in this review which will address the regrettably small amount of new information that has accrued since then, leaving aside discussion of insulin and somatostatin which are covered elsewhere in this volume (Plisetskaya 1990; Youson 1990).
Structure of the alimentary canal This short and generalised account is given for orientation of the reader. For a detailed description see Youson (1981). In both lampreys and hagfishes the gut is a simple
tube. Behind the pharynx the short oesophagus leads into the anterior intestine at the junction of the bile duct with the gut. The oesophagus is lined with ciliated columnar epithelium and mucous cells. In some species, the anterior intestine is expanded into one or two blind-ending diverticula. The internal surface of the intestine is increased by a spiral fold (typhlosole) which extends along the length of the intestine and in the larva contains submucosal haematopoietic tissue. In addition, the wall of the intestine is folded longitudinally. The wall of the anterior part of the intestine contains mainly absorptive cells with microvilli at the luminal surface, ciliated cells, enzyme-secreting zymogen cells and endocrine cells. The posterior intestine is lined mainly with absorptive cells, mucous cells and endocrine cells. The zymogen cells are concentrated in the foremost part of the intestine, and in some species are confined to the diverticula. They
400 have been equated with the exocrine pancreas of vertebrates, in which this organ is a separate entity. Larval and adult cyclostomes feed in different ways - the larvae are microphagous filter feeders while the adults are non-feeding, parasitic, predatory or scavengers according to species. In lampreys, at metamorphosis the gall bladder and bile duct degenerate, while they are retained in hagfishes. As they become sexually mature, adult lampreys cease to feed and the gut gradually degenerates. The endocrine pancreas consists of an islet organ which develops from the anterior part of the gut in larval lampreys, at the junction of the oesophagus, bile duct and intestine. In hagfishes, it is derived mainly from the wall of the bile duct. In lampreys, the islet tissue may be present in two or three parts, a cranial accumulation round the junction of the bile duct and intestine, a caudal or hepatic area between the intestine and the liver, often almost surrounded by hepatic tissue, at the level of the hepatic portal vein, and cords of islet cells in the intestinal wall between the two main accumulations. Some of the interspecific morphological variations have recently been described by Youson et al. (1988).
Regulatory peptides of the gut and pancreas Immunological methods have played a large part in the discovery of the peptides of the gut and islets of cyclostomes. The main investigative tools have been immunocytochemical localisation along with radioimmunoassay and chromatography of extracts, inevitably using antisera raised to peptides of other vertebrates, mainly mammals. Bioassays have been used occasionally and recently more sophisticated sequencing techniques have come into play. Unfortunately we can still only guess at the functions of the peptides that have been identified.
Pancreaticislets By 1982 it was established that the islet cells of lampreys and hagfishes secreted insulin- and somatostatin-like substances with molecular structure
related to that of their mammalian counterparts. Lamprey islets consist of groups of cells in lobules, the cells of one lobule secreting either insulin or somatostatin, and the two types of lobule being present in approximately equal numbers. In hagfishes, the islet peptide is predominantly insulin with rather few scattered somatostatin-containing cells, at least in Myxine glutinosa(Van Noorden et al. 1977). There may be a higher proportion of somatostatin cells in Eptatretus stouti (Seino et al. 1979). In some mammals, pancreatic and gastric somatostatin cells have long cytoplasmic processes that terminate on or near endocrine cells as well as capillaries. It has been suggested (Larsson et al. 1979) that somatostatin may diffuse from these processes to act as a local or paracrine regulator of insulin and glucagon secretion, and of antral gastrin secretion, as well as acting as a circulating hormone. Although an in vitro inhibition of insulin secretion from E. stouti islets has been recorded (Stewart et al. 1979), it is difficult to imagine how that process could take place in the cyclostome islet where relatively large groups of cells produce the same peptide (lampreys) or the somatostatin-producing cells are few and scattered in relation to the insulin-producing cells (Myxine). For further discussion of cyclostome insulin- and somatostatinlike peptides see Plisetskaya (1989). A rare third type of islet cell has been identified in the sea lamprey, Petromyzon marinusand there is now some evidence that this cell contains a peptide of the pancreatic polypeptide family (Youson 1990), thus bringing the cyclostomes a little closer to their vertebrate relatives.
Intestine Gastrin/CCK-like peptides Early bioassay experiments (Barrington and Dockray 1970; Nilsson 1973; Dockray 1977) had indicated that extracts of lamprey and hagfish intestine contained biological activity capable of stimulating pancreatic enzyme secretion and gall bladder contraction (cholecystokinin (CCK)-like activity) and pancreatic fluid secretion (secretin-like activity) from mammalian organ preparations. Immuno-
401 cytochemical investigations (Van Noorden and Pearse 1974) showed the presence of typical granulated endocrine cells along the length of the intestine of Lampetra fluviatilis, immunostained with antibodies reactive to the C-terminal part of the gastrin/CCK molecule and also with antibodies to glucagon. These cells were present in both the larva and the adult, and in the hagfish M. glutinosa (Ostberg et al. 1976b). It was suggested that the CCKlike activity was due to the gastrin/CCK-immunoreactive substance and the secretin-like activity to the glucagon-immunoreactive substance, in the absence of immunoreactivity for secretin and in view of the molecular similarities between these two mammalian peptides. The finding of immunoreactivity for two structurally unrelated peptides in the same cell was then unknown and it was suggested that a single large peptide might be present with both gastrin/CCK- and glucagon-like amino acid sequences. Latterly, the coexistence of two or more structurally and functionally different peptides in one cell or nerve has become a common finding (Ali-Rachedi et al. 1984; Llewellyn-Smith 1989) and the demonstration of the transient co-existence of insulin with other pancreatic hormones during the embryonic and post-natal development of the mouse (Alpert et al. 1988) indicates that this may be a normal or perhaps primitive condition. The evocation of a giant molecule is thus not necessary, though large precursors giving rise to two separate peptides through differential posttranslational processing are recognised as, for example, in the case of calcitonin and calcitonin gene-related peptide (Rosenfeld et al. 1983). In a radioimmunoassay of extracts of Lampetra brain and gut Holmquist et al. (1979) identified the presence of two peptides immunoreactive towards the C-terminal of CCK, the larger resembling CCK8, and the anterior and mid-intestine containing considerably more than the posterior intestine (6.1 pmol/g: 0.5 pmol/g). A recent radioimmunoassay (Conlon and Falkmer 1989) confirmed the presence of very small amounts (0.056 pmol/g) of C-terminal-immunoreactive CCK/gastrin, as well as glucagon (see below), in extracts of the entire intestine of M. glutinosa. Vigna (1979) and Vigna and Gorbman (1979)
showed that an extract of E. stouti intestine had CCK-like biological activity rather than gastrin-like activity, since it resembled CCK rather than gastrin in its effect on the contraction of guinea pig gall bladder muscle in vitro. The gall bladder of Eptatretusitself, however, was not responsive to porcine CCK although the hormone was able to stimulate lipase secretion from the zymogen cells of the intestinal wall. This action probably corresponds to the earlier finding of pancreatic enzyme secretion stimulated by a lamprey intestinal extract (Barrington and Dockray 1970) and was interpreted as suggesting that receptors for CCK-like substances on the digestive enzyme-secreting cells probably evolved before the receptors on the gall bladder. As Hardisty and Baker (1982) pointed out, in lampreys the gall bladder and bile duct disappear at metamorphosis so that bile is probably not an important component of the digestive process in cyclostomes. However, the experiments of testing the Eptatretusextract on its own gall bladder or of testing a lamprey gut CCK-like extract on larval lampreys which possess a gall bladder have not been carried out. Thus, it seems to be established that the cyclostome intestine contains a CCK-like peptide or peptides and here the matter has rested. Glucagon-like peptides With regard to the glucagon-like immunoreactivity, present in a very large number of endocrine cells in the lamprey gut and rather fewer in hagfishes, little further work has been done. Cyclostome islets, unlike those of all other chordates, contain no glucagon-immunoreactive cells. Perhaps corresponding with this finding, no suitable function has yet been found for glucagon in the field of blood sugar regulation, in experiments using mammalian pancreatic glucagon. The molecular structure of the cyclostome peptide has not been studied and until recently, there was but a single mention of immunoreactivity in a glucagon radioimmunoassay (Zelnick et al. 1976). The extract assayed was from the cranial pancreas, which contains no immunocytochemically detectable glucagon-like peptide, so it is probable that the glucagon immunoreactivity (which was significantly so high as to be off the scale of the assay) was due to contamination from intestinal tissue. No
402 measurable pancreatic glucagon-like immunoreactivity was found in E. stouti plasma (Stewart et al. 1979). However, a single peak of a glucagon-like peptide of the same molecular size as mammalian pancreatic glucagon has been shown by radioimmunoassay and chromatography in extracts of Myxine gut, confirming previous immunocytochemical evidence (Conlon and Falkmer 1989). In view of the close association between the cyclostome gut zymogen cells (equivalent to the exocrine pancreas) and the glucagon-immunoreactive cells, and the absence of any other agent resembling secretin (except perhaps vasoactive intestinal polypeptide (VIP)- see below) which might be responsible for the promotion of pancreatic fluid secretion recorded by Barrington and Dockray (1970), nothing has yet been discovered to refute the idea (Van Noorden and Pearse 1974) that this effect could be caused by the glucagon-like substance(s). In mammals, gut glucagon differs from pancreatic glucagon in molecular form (being part of the larger prohormone molecule and containing glucagon within its structure) and in biological activity. Pancreatic glucagon is mainly concerned with glycogenolysis to raise blood sugar levels while gut glucagon may be trophic to the gut (Bloom and Polak 1976). Possibly, cyclostome glucagon also exists in several forms and it may be that the biologically active component is similar to the glucagon-like peptide 7-37 of some fishes (Plisetskaya et al. 1987). There is an urgent need for isolation and sequencing of the cyclostome gut glucagon(s) and for testing its actions both on mammalian tissue and in the species of origin. Pancreatic polypeptide-like peptides As mentioned above, there are no pancreatic polypeptide(PP)-immunoreactive cells in cyclostome islets, but in 1981 (Falkmer et al. 1985) PP-like immunoreactivity was reported to be present, colocalised with glucagon-(and gastrin/CCK-) like immunoreactivity in the endocrine cells of the intestine, as was then thought to be the case for gut glucagon cells of mammals. This mammalian gut PP was, however, suspect, since no PP could be extracted from the gut, and it was later shown that the mammalian gut PP-like immunoreactivity was due
to cross-reactivity of the antibody to bovine PP with a newly discovered peptide of the same family, peptide tyrosine tyrosine (PYY). This was also the case for the cyclostome gut PP-like immunoreactivity (Ali-Rachedi et al. 1984). A third member of the PP family, neuropeptide Y (NPY) is present in the nervous system in mammals, mainly in adrenergic nerves, and in some endocrine cells, for example in the adrenal medulla. Antibodies to PYY do not cross-react with NPY in mammalian tissue, and antibodies to NPY do not label PYY-immunoreactive cells. However, in lamprey gut the endocrine cells are labelled by antibodies to bPP, PYY and NPY. Thus, the cyclostome peptide(s) of this family cannot yet be said to be PP-like, PYY-like or NPY-like, and it is not known if the rare third cell type of the islet tissue, which Youson (1990) has found to be immunostained by anti-NPY, contains the same peptide(s) as the gut cells. Extraction and sequencing are urgent requirements here, followed by functional studies. In mammals, peptides of the PP family are probably concerned with feeding behaviour and lipid metabolism (see Hazelwood 1989, for review). Other peptides of the gut endocrine cells Immunoreactivity for some other peptides has been established in gut endocrine cells. Reinecke (1981) reported VIP-immunoreactive cells in Myxine gut, using a C-terminally immunoreactive VIP antibody. I have found similar cells in Lampetra. Although VIP and secretin have some common amino acid sequences, the cells are of such infrequent occurrence that the available peptide would probably be insufficient to account for the secretinlike bioactivity mentioned above. Substance P-like immunoreactivity has also been localised to gut cells in Myxine, particularly at the caudal end (Reinecke 1987), and I have found similar immunoreactivity in Lampetra and Petromyzon. Several tachykinin-like peptides, different from mammalian substance P, were identified in a Myxine gut extract (Conlon and Falkmer 1989). Since substance P could cause contraction of a Myxine heart preparation, Reinecke (1987) made the interesting speculation that the substance P-like substance(s) of Myxine gut endocrine cells could be
403 released under the influence of autonomic nerve stimulation and enter the circulation to regulate muscle tone and gut motility. This suggestion has not been further tested. Insulin may be considered as a gut peptide, at least in the larval lamprey. Insulin-immunoreactive cells are localised at the junction of the oesophagus, bile duct and intestine in larval lampreys whence they bud off to form islets. (Van Noorden and Pearse 1974; Elliott and Youson 1987). In Myxine, islet cells bud from the bile duct and this process continues in adult life (Ostberg et al. 1976a). Myxine insulin has been well studied and shown to differ little from pig insulin. The question of whether insulin acts as a regulatory hormone of the gut in the larval stage has not been adressed. Somatostatin, too, may be counted as a gut hormone. In lampreys, somatostatin-immunoreactive cells appear in the gut as the larva approaches metamorphosis, at which stage they migrate into the forming islet lobules (Elliott and Youson 1987). Adult lamprey gut contains cells producing a large molecular form of somatostatin, somatostatin 34 (Youson 1990, this volume). Conlon and Falkmer (1989) have confirmed that there are two molecular forms of somatostatin, somatostatin 14 and 34, in Myxine gut as in the islet organ. Among other gut peptides, gastric inhibitory peptide (glucose-dependent insulin-releasing peptide, GIP) has been found by radioimmunoassay in extracts of Myxine gut (Falkmer et al. 1980) but has never been localised by immunocytochemistry, and neurotensin-immunoreactive cells have been identified by immunocytochemistry alone, again in Myxine (Reinecke et al. 1980). A recent study (Yui et al. 1988) indicated differences in the distribution of gut peptides in the larval and adult forms of L. japonica. In the larva, three separately immunoreactive cell types were found, identified by antisera to somatostatin, gastrin/ CCK (mostly in the anterior intestine) and a third type immunoreactive for three peptides, glucagon (N-terminal and C-terminal), pancreatic polypeptide and FMRF amide. In the adult, only the glucagon/PP/FMRF amide cells were found, somatostatin- and gastrin/CCK-positive cells being absent.
Substance P immunoreactivity was not examined. My own findings in adult L. fluviatilis are that there may be a differential distribution of immunoreactive cell types along the intestine. Cterminal gastrin/CCK immunoreactivity was absent anterior to the liver, although glucagon-, PP (NPY/PYY)-, substance P- and FMRF amide-positive cells were present. Glucagon-positive cells were by far the most frequent. Serially immunostained thin (2 /sm) sections showed that all the PYY/ NPY/PP cells and some of the substance P cells were glucagon-positive (Fig. 1). Posterior to the liver, gastrin/CCK-positive cells were present and were mostly also positive for glucagon (Fig. 2). Substance P cells were markedly more frequent than in the anterior part. Some of these were identical with gastrin/CCK cells (Fig. 3) but little identity was found with glucagon cells in this area. The colocalisation of FMRF-amide- and met-enkephalin immunoreactivities, also present in cells of the lamprey intestine, has not yet been examined. So far, only one granulated intestinal endocrine cell type has been identified at ultrastructural level in any cyclostome species, but with hindsight into the number of different peptides produced, more may be discovered. The findings reported above indicate that the gut endocrine cells of cyclostomes are capable of producing several different peptides and their content of immunocytochemically detectable peptides may alter with changes in the physiological state. The proportions of peptides present and the possibility of differential release under different conditions are studies for the future, as is immunocytochemistry at electron microscopical level.
The enteric nervous system In all classes of vertebrate, the peripheral nervous system, including the nerves of the gut, is now known to contain numerous active peptides such as VIP, enkephalin, substance P and bombesin, with profound effects on gut function and occurrence and distribution characteristic of the species and the area of the gut. (Bjenning and Holmgren 1987; Furness and Costa 1987; Llewellyn-Smith 1989) In
404
Fig. 1. Lampetrafluviatilismature adult intestine at the level of the liver, freeze-dried, fixed in diethyl pyrocarbonate vapour and embedded in paraffin. Serial 2 tm sections immunostained by the peroxidase anti-peroxidase method for (A) glucagon and (B) substance P. One cell only (arrowed) is reactive with both antibodies (x 200).
Fig. 2. Lampetrafluviatilismature adult post-hepatic intestine. Tissue prepared as in Fig. 1. (A) immunostained for glucagon, (B) immunostained for gastrin (C-terminal). All the gastrinpositive cells are also positive for glucagon. Some are identified by arrows (x 200).
any modern study of gut peptide endocrinology the neuropeptides of the nervous system must be taken into account, despite our fragmentary knowledge of their properties. The lamprey enteric aminergic nervous system was studied by Baumgarten et al. (1973) using formaldehyde-induced fluorescence. An extensive intrinsic system of gut nerves was found, containing serotonin and probably another indoleamine. Intrinsic neurones and non-terminal sub-epithelial axons were found along the length of the intestine. The anterior and middle parts of the intestine also contained dopamine and noradrenaline cell bodies and scattered chromaffin cells. No serotonin-containing enterochromaffin cells were present, as already established by others using argentaffin methods. Serotonin was able to cause contraction of the gut wall, but only in the posterior part where the
405
Fig. 3. Lampetrafluviatilis, mature adult post-hepatic intestine, prepared as in Fig. 1. (A) immunostained for substance P, (B) immunostained for gastrin (C-terminal). A few cells (some arrowed) are reactive with body antibodies (x 200).
Fig. 4. Petromyzon marinus adult intestine, fixed in Bouin's solution. Paraffin-embedded section (4 m) immunostained for CGRP. Immuno-positive nerves are present in the outer wall of the intestine (arrowed) and in the sub-mucosa of the intestinal folds (x 125).
Fig. 5. Petromyzon marinusadult intestin. Tissue prepared as in Fig. 4. Immunostained with a monoclonal antibody to amphibian bombesin. Positively stained nerves are present in the outer wall of the intestine and smaller nerves are present in the submucosa of the intestinal folds (arrowed) (x 125).
406
Fig. 6. Petromyzon marinus cranial islet. Tissue prepared as in
Fig. 4. Immunostained for CGRP. Fine nerve are present between the lobules and seem to contact the islet cells. Some possible terminals are arrowed (x 200).
wall is well muscularised. This experiment was carried out on non-feeding adults in which the gut was already degenerating and the authors suggested that in the larva this action might be more widespread. Sakharov and Salimova (1980) confirmed the presence of serotonin in an abundant peripheral nervous system in the larval lamprey, but found no catecholamine-containing nerves. Substance P-like peptides, none of them identical to substance P, are present in the cyclostome central nervous system (Van Dongen et al. 1986; Nozaki and Gorbman 1986; Reinecke et al. 1987), as indeed are many other peptides and active substances (Buchanan et al. 1987; Brodin et al. 1988). I have found substance P-immunoreactive nerves in the lamprey gut wall. The relationships between the substance P-like peptide(s) of the central nervous system, the gut nervous system and the gut endocrine cells remain to be established. Yui et al. (1988) have described in L. japonicaenteric nerves immunoreactive for calcitonin generelated peptide (CGRP) and gastrin-releasing peptide (GRP), each peptide being co-localised with serotonin. CGRP nerves were present in the muscle layers and sub-mucosa of the intestine, while GRPimmunoreactive nerves were confined to the muscle layer. My own observations in L. planerilarvae and in L. fluviatilis and P. marinus adults are in fair agreement with this (Figs. 4 and 5), though I have
not examined the co-localisation with serotonin. CGRP seems to be the dominant peptide of the gut nervous system and is also sparsely present in the islets in P. marinus(Fig. 6). In addition to these peptides and the substance P-like peptide(s) mentioned above, I have also found some VIP-immunoreactive nerves in the gut of lampreys. Galanin and metenkephalin immunoreactivity, often present in fish gut nervous systems, were absent. None of the intestinal peptide-containing nerves appeared to contact the gut endocrine cells directly, and the actions of the neuropeptides may therefore be more concerned with muscle and vascular tone than with promotion of gut secretion, but the possibility remains that peptide/serotonin secretion from nerve terminals may diffuse to the base of the mucosal epithelium and influence secretion. In this review I have tried to bring together what is known about the enteric (neuro)endocrine system of cyclostomes. Work on this subject has been sporadic and unsystematic and it is apparent that we know remarkably little. With discoveries of new peptides the complexity of the system is now being revealed. There is clearly much to be done and a need for coordinated biochemical, physiological and morphological studies.
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pase secretion by porcine cholecystokinin in the hagfish, Eptatretus stouti. Gen. Comp. Endocrinol. 38: 356-359. Youson, J.H. 1981. The alimentary canal. In The Biology of Lampreys. Volume 3, pp. 95-189. Edited by M.W. Hardisty and I.C. Potter. Academic Press. London. Youson, J.H. 1990. Morphogenesis of somatostatin and insulin secreting cells in lampreys. Fish Physiol. Biochem. 8: 389397.
Gut hormones in cyclostomes Susan Van Noorden Histopathology Department, Royal PostgraduateMedical School, Du Cane Road, London, W12 ONN, England Keywords: cyclostome, lamprey, hagfish, regulatory peptides, neuropeptides, pancreas, intestine, endocrine cells, hormones
Abstract The current state of knowledge about regulatory peptides in endocrine cells and nerves of the alimentary canal of lampreys and hagfishes is reviewed. Cyclostomes have a wide range of peptides similar immunochemically to those of higher vertebrates. They include, in the endocrine cells of the intestine, peptides resembling glucagon, gastrin/cholecystokinin, peptide YY (pancreatic polypeptide/neuropeptide Y), substance P, vasoactive intestinal peptide, somatostatin and, in the larval stages at least, insulin. The enteric nerves of some lamprey species contain peptides resembling bombesin (gastrin-releasing peptide) and calcitonin gene-related peptide, as well as serotonin. The occurrence of other peptides is less well documented. Little is known of the molecular structure or the biological roles of the enteric peptides in cyclostomes. Extraction, purification, sequencing and physiological experiments are greatly needed.
Introduction Cyclostome gut endocrinology was comprehensively reviewed in 1982 (Hardisty and Baker 1982). Findings up to that time will be, therefore, summarised briefly in this review which will address the regrettably small amount of new information that has accrued since then, leaving aside discussion of insulin and somatostatin which are covered elsewhere in this volume (Plisetskaya 1990; Youson 1990).
Structure of the alimentary canal This short and generalised account is given for orientation of the reader. For a detailed description see Youson (1981). In both lampreys and hagfishes the gut is a simple
tube. Behind the pharynx the short oesophagus leads into the anterior intestine at the junction of the bile duct with the gut. The oesophagus is lined with ciliated columnar epithelium and mucous cells. In some species, the anterior intestine is expanded into one or two blind-ending diverticula. The internal surface of the intestine is increased by a spiral fold (typhlosole) which extends along the length of the intestine and in the larva contains submucosal haematopoietic tissue. In addition, the wall of the intestine is folded longitudinally. The wall of the anterior part of the intestine contains mainly absorptive cells with microvilli at the luminal surface, ciliated cells, enzyme-secreting zymogen cells and endocrine cells. The posterior intestine is lined mainly with absorptive cells, mucous cells and endocrine cells. The zymogen cells are concentrated in the foremost part of the intestine, and in some species are confined to the diverticula. They
400 have been equated with the exocrine pancreas of vertebrates, in which this organ is a separate entity. Larval and adult cyclostomes feed in different ways - the larvae are microphagous filter feeders while the adults are non-feeding, parasitic, predatory or scavengers according to species. In lampreys, at metamorphosis the gall bladder and bile duct degenerate, while they are retained in hagfishes. As they become sexually mature, adult lampreys cease to feed and the gut gradually degenerates. The endocrine pancreas consists of an islet organ which develops from the anterior part of the gut in larval lampreys, at the junction of the oesophagus, bile duct and intestine. In hagfishes, it is derived mainly from the wall of the bile duct. In lampreys, the islet tissue may be present in two or three parts, a cranial accumulation round the junction of the bile duct and intestine, a caudal or hepatic area between the intestine and the liver, often almost surrounded by hepatic tissue, at the level of the hepatic portal vein, and cords of islet cells in the intestinal wall between the two main accumulations. Some of the interspecific morphological variations have recently been described by Youson et al. (1988).
Regulatory peptides of the gut and pancreas Immunological methods have played a large part in the discovery of the peptides of the gut and islets of cyclostomes. The main investigative tools have been immunocytochemical localisation along with radioimmunoassay and chromatography of extracts, inevitably using antisera raised to peptides of other vertebrates, mainly mammals. Bioassays have been used occasionally and recently more sophisticated sequencing techniques have come into play. Unfortunately we can still only guess at the functions of the peptides that have been identified.
Pancreaticislets By 1982 it was established that the islet cells of lampreys and hagfishes secreted insulin- and somatostatin-like substances with molecular structure
related to that of their mammalian counterparts. Lamprey islets consist of groups of cells in lobules, the cells of one lobule secreting either insulin or somatostatin, and the two types of lobule being present in approximately equal numbers. In hagfishes, the islet peptide is predominantly insulin with rather few scattered somatostatin-containing cells, at least in Myxine glutinosa(Van Noorden et al. 1977). There may be a higher proportion of somatostatin cells in Eptatretus stouti (Seino et al. 1979). In some mammals, pancreatic and gastric somatostatin cells have long cytoplasmic processes that terminate on or near endocrine cells as well as capillaries. It has been suggested (Larsson et al. 1979) that somatostatin may diffuse from these processes to act as a local or paracrine regulator of insulin and glucagon secretion, and of antral gastrin secretion, as well as acting as a circulating hormone. Although an in vitro inhibition of insulin secretion from E. stouti islets has been recorded (Stewart et al. 1979), it is difficult to imagine how that process could take place in the cyclostome islet where relatively large groups of cells produce the same peptide (lampreys) or the somatostatin-producing cells are few and scattered in relation to the insulin-producing cells (Myxine). For further discussion of cyclostome insulin- and somatostatinlike peptides see Plisetskaya (1989). A rare third type of islet cell has been identified in the sea lamprey, Petromyzon marinusand there is now some evidence that this cell contains a peptide of the pancreatic polypeptide family (Youson 1990), thus bringing the cyclostomes a little closer to their vertebrate relatives.
Intestine Gastrin/CCK-like peptides Early bioassay experiments (Barrington and Dockray 1970; Nilsson 1973; Dockray 1977) had indicated that extracts of lamprey and hagfish intestine contained biological activity capable of stimulating pancreatic enzyme secretion and gall bladder contraction (cholecystokinin (CCK)-like activity) and pancreatic fluid secretion (secretin-like activity) from mammalian organ preparations. Immuno-
401 cytochemical investigations (Van Noorden and Pearse 1974) showed the presence of typical granulated endocrine cells along the length of the intestine of Lampetra fluviatilis, immunostained with antibodies reactive to the C-terminal part of the gastrin/CCK molecule and also with antibodies to glucagon. These cells were present in both the larva and the adult, and in the hagfish M. glutinosa (Ostberg et al. 1976b). It was suggested that the CCKlike activity was due to the gastrin/CCK-immunoreactive substance and the secretin-like activity to the glucagon-immunoreactive substance, in the absence of immunoreactivity for secretin and in view of the molecular similarities between these two mammalian peptides. The finding of immunoreactivity for two structurally unrelated peptides in the same cell was then unknown and it was suggested that a single large peptide might be present with both gastrin/CCK- and glucagon-like amino acid sequences. Latterly, the coexistence of two or more structurally and functionally different peptides in one cell or nerve has become a common finding (Ali-Rachedi et al. 1984; Llewellyn-Smith 1989) and the demonstration of the transient co-existence of insulin with other pancreatic hormones during the embryonic and post-natal development of the mouse (Alpert et al. 1988) indicates that this may be a normal or perhaps primitive condition. The evocation of a giant molecule is thus not necessary, though large precursors giving rise to two separate peptides through differential posttranslational processing are recognised as, for example, in the case of calcitonin and calcitonin gene-related peptide (Rosenfeld et al. 1983). In a radioimmunoassay of extracts of Lampetra brain and gut Holmquist et al. (1979) identified the presence of two peptides immunoreactive towards the C-terminal of CCK, the larger resembling CCK8, and the anterior and mid-intestine containing considerably more than the posterior intestine (6.1 pmol/g: 0.5 pmol/g). A recent radioimmunoassay (Conlon and Falkmer 1989) confirmed the presence of very small amounts (0.056 pmol/g) of C-terminal-immunoreactive CCK/gastrin, as well as glucagon (see below), in extracts of the entire intestine of M. glutinosa. Vigna (1979) and Vigna and Gorbman (1979)
showed that an extract of E. stouti intestine had CCK-like biological activity rather than gastrin-like activity, since it resembled CCK rather than gastrin in its effect on the contraction of guinea pig gall bladder muscle in vitro. The gall bladder of Eptatretusitself, however, was not responsive to porcine CCK although the hormone was able to stimulate lipase secretion from the zymogen cells of the intestinal wall. This action probably corresponds to the earlier finding of pancreatic enzyme secretion stimulated by a lamprey intestinal extract (Barrington and Dockray 1970) and was interpreted as suggesting that receptors for CCK-like substances on the digestive enzyme-secreting cells probably evolved before the receptors on the gall bladder. As Hardisty and Baker (1982) pointed out, in lampreys the gall bladder and bile duct disappear at metamorphosis so that bile is probably not an important component of the digestive process in cyclostomes. However, the experiments of testing the Eptatretusextract on its own gall bladder or of testing a lamprey gut CCK-like extract on larval lampreys which possess a gall bladder have not been carried out. Thus, it seems to be established that the cyclostome intestine contains a CCK-like peptide or peptides and here the matter has rested. Glucagon-like peptides With regard to the glucagon-like immunoreactivity, present in a very large number of endocrine cells in the lamprey gut and rather fewer in hagfishes, little further work has been done. Cyclostome islets, unlike those of all other chordates, contain no glucagon-immunoreactive cells. Perhaps corresponding with this finding, no suitable function has yet been found for glucagon in the field of blood sugar regulation, in experiments using mammalian pancreatic glucagon. The molecular structure of the cyclostome peptide has not been studied and until recently, there was but a single mention of immunoreactivity in a glucagon radioimmunoassay (Zelnick et al. 1976). The extract assayed was from the cranial pancreas, which contains no immunocytochemically detectable glucagon-like peptide, so it is probable that the glucagon immunoreactivity (which was significantly so high as to be off the scale of the assay) was due to contamination from intestinal tissue. No
402 measurable pancreatic glucagon-like immunoreactivity was found in E. stouti plasma (Stewart et al. 1979). However, a single peak of a glucagon-like peptide of the same molecular size as mammalian pancreatic glucagon has been shown by radioimmunoassay and chromatography in extracts of Myxine gut, confirming previous immunocytochemical evidence (Conlon and Falkmer 1989). In view of the close association between the cyclostome gut zymogen cells (equivalent to the exocrine pancreas) and the glucagon-immunoreactive cells, and the absence of any other agent resembling secretin (except perhaps vasoactive intestinal polypeptide (VIP)- see below) which might be responsible for the promotion of pancreatic fluid secretion recorded by Barrington and Dockray (1970), nothing has yet been discovered to refute the idea (Van Noorden and Pearse 1974) that this effect could be caused by the glucagon-like substance(s). In mammals, gut glucagon differs from pancreatic glucagon in molecular form (being part of the larger prohormone molecule and containing glucagon within its structure) and in biological activity. Pancreatic glucagon is mainly concerned with glycogenolysis to raise blood sugar levels while gut glucagon may be trophic to the gut (Bloom and Polak 1976). Possibly, cyclostome glucagon also exists in several forms and it may be that the biologically active component is similar to the glucagon-like peptide 7-37 of some fishes (Plisetskaya et al. 1987). There is an urgent need for isolation and sequencing of the cyclostome gut glucagon(s) and for testing its actions both on mammalian tissue and in the species of origin. Pancreatic polypeptide-like peptides As mentioned above, there are no pancreatic polypeptide(PP)-immunoreactive cells in cyclostome islets, but in 1981 (Falkmer et al. 1985) PP-like immunoreactivity was reported to be present, colocalised with glucagon-(and gastrin/CCK-) like immunoreactivity in the endocrine cells of the intestine, as was then thought to be the case for gut glucagon cells of mammals. This mammalian gut PP was, however, suspect, since no PP could be extracted from the gut, and it was later shown that the mammalian gut PP-like immunoreactivity was due
to cross-reactivity of the antibody to bovine PP with a newly discovered peptide of the same family, peptide tyrosine tyrosine (PYY). This was also the case for the cyclostome gut PP-like immunoreactivity (Ali-Rachedi et al. 1984). A third member of the PP family, neuropeptide Y (NPY) is present in the nervous system in mammals, mainly in adrenergic nerves, and in some endocrine cells, for example in the adrenal medulla. Antibodies to PYY do not cross-react with NPY in mammalian tissue, and antibodies to NPY do not label PYY-immunoreactive cells. However, in lamprey gut the endocrine cells are labelled by antibodies to bPP, PYY and NPY. Thus, the cyclostome peptide(s) of this family cannot yet be said to be PP-like, PYY-like or NPY-like, and it is not known if the rare third cell type of the islet tissue, which Youson (1990) has found to be immunostained by anti-NPY, contains the same peptide(s) as the gut cells. Extraction and sequencing are urgent requirements here, followed by functional studies. In mammals, peptides of the PP family are probably concerned with feeding behaviour and lipid metabolism (see Hazelwood 1989, for review). Other peptides of the gut endocrine cells Immunoreactivity for some other peptides has been established in gut endocrine cells. Reinecke (1981) reported VIP-immunoreactive cells in Myxine gut, using a C-terminally immunoreactive VIP antibody. I have found similar cells in Lampetra. Although VIP and secretin have some common amino acid sequences, the cells are of such infrequent occurrence that the available peptide would probably be insufficient to account for the secretinlike bioactivity mentioned above. Substance P-like immunoreactivity has also been localised to gut cells in Myxine, particularly at the caudal end (Reinecke 1987), and I have found similar immunoreactivity in Lampetra and Petromyzon. Several tachykinin-like peptides, different from mammalian substance P, were identified in a Myxine gut extract (Conlon and Falkmer 1989). Since substance P could cause contraction of a Myxine heart preparation, Reinecke (1987) made the interesting speculation that the substance P-like substance(s) of Myxine gut endocrine cells could be
403 released under the influence of autonomic nerve stimulation and enter the circulation to regulate muscle tone and gut motility. This suggestion has not been further tested. Insulin may be considered as a gut peptide, at least in the larval lamprey. Insulin-immunoreactive cells are localised at the junction of the oesophagus, bile duct and intestine in larval lampreys whence they bud off to form islets. (Van Noorden and Pearse 1974; Elliott and Youson 1987). In Myxine, islet cells bud from the bile duct and this process continues in adult life (Ostberg et al. 1976a). Myxine insulin has been well studied and shown to differ little from pig insulin. The question of whether insulin acts as a regulatory hormone of the gut in the larval stage has not been adressed. Somatostatin, too, may be counted as a gut hormone. In lampreys, somatostatin-immunoreactive cells appear in the gut as the larva approaches metamorphosis, at which stage they migrate into the forming islet lobules (Elliott and Youson 1987). Adult lamprey gut contains cells producing a large molecular form of somatostatin, somatostatin 34 (Youson 1990, this volume). Conlon and Falkmer (1989) have confirmed that there are two molecular forms of somatostatin, somatostatin 14 and 34, in Myxine gut as in the islet organ. Among other gut peptides, gastric inhibitory peptide (glucose-dependent insulin-releasing peptide, GIP) has been found by radioimmunoassay in extracts of Myxine gut (Falkmer et al. 1980) but has never been localised by immunocytochemistry, and neurotensin-immunoreactive cells have been identified by immunocytochemistry alone, again in Myxine (Reinecke et al. 1980). A recent study (Yui et al. 1988) indicated differences in the distribution of gut peptides in the larval and adult forms of L. japonica. In the larva, three separately immunoreactive cell types were found, identified by antisera to somatostatin, gastrin/ CCK (mostly in the anterior intestine) and a third type immunoreactive for three peptides, glucagon (N-terminal and C-terminal), pancreatic polypeptide and FMRF amide. In the adult, only the glucagon/PP/FMRF amide cells were found, somatostatin- and gastrin/CCK-positive cells being absent.
Substance P immunoreactivity was not examined. My own findings in adult L. fluviatilis are that there may be a differential distribution of immunoreactive cell types along the intestine. Cterminal gastrin/CCK immunoreactivity was absent anterior to the liver, although glucagon-, PP (NPY/PYY)-, substance P- and FMRF amide-positive cells were present. Glucagon-positive cells were by far the most frequent. Serially immunostained thin (2 /sm) sections showed that all the PYY/ NPY/PP cells and some of the substance P cells were glucagon-positive (Fig. 1). Posterior to the liver, gastrin/CCK-positive cells were present and were mostly also positive for glucagon (Fig. 2). Substance P cells were markedly more frequent than in the anterior part. Some of these were identical with gastrin/CCK cells (Fig. 3) but little identity was found with glucagon cells in this area. The colocalisation of FMRF-amide- and met-enkephalin immunoreactivities, also present in cells of the lamprey intestine, has not yet been examined. So far, only one granulated intestinal endocrine cell type has been identified at ultrastructural level in any cyclostome species, but with hindsight into the number of different peptides produced, more may be discovered. The findings reported above indicate that the gut endocrine cells of cyclostomes are capable of producing several different peptides and their content of immunocytochemically detectable peptides may alter with changes in the physiological state. The proportions of peptides present and the possibility of differential release under different conditions are studies for the future, as is immunocytochemistry at electron microscopical level.
The enteric nervous system In all classes of vertebrate, the peripheral nervous system, including the nerves of the gut, is now known to contain numerous active peptides such as VIP, enkephalin, substance P and bombesin, with profound effects on gut function and occurrence and distribution characteristic of the species and the area of the gut. (Bjenning and Holmgren 1987; Furness and Costa 1987; Llewellyn-Smith 1989) In
404
Fig. 1. Lampetrafluviatilismature adult intestine at the level of the liver, freeze-dried, fixed in diethyl pyrocarbonate vapour and embedded in paraffin. Serial 2 tm sections immunostained by the peroxidase anti-peroxidase method for (A) glucagon and (B) substance P. One cell only (arrowed) is reactive with both antibodies (x 200).
Fig. 2. Lampetrafluviatilismature adult post-hepatic intestine. Tissue prepared as in Fig. 1. (A) immunostained for glucagon, (B) immunostained for gastrin (C-terminal). All the gastrinpositive cells are also positive for glucagon. Some are identified by arrows (x 200).
any modern study of gut peptide endocrinology the neuropeptides of the nervous system must be taken into account, despite our fragmentary knowledge of their properties. The lamprey enteric aminergic nervous system was studied by Baumgarten et al. (1973) using formaldehyde-induced fluorescence. An extensive intrinsic system of gut nerves was found, containing serotonin and probably another indoleamine. Intrinsic neurones and non-terminal sub-epithelial axons were found along the length of the intestine. The anterior and middle parts of the intestine also contained dopamine and noradrenaline cell bodies and scattered chromaffin cells. No serotonin-containing enterochromaffin cells were present, as already established by others using argentaffin methods. Serotonin was able to cause contraction of the gut wall, but only in the posterior part where the
405
Fig. 3. Lampetrafluviatilis, mature adult post-hepatic intestine, prepared as in Fig. 1. (A) immunostained for substance P, (B) immunostained for gastrin (C-terminal). A few cells (some arrowed) are reactive with body antibodies (x 200).
Fig. 4. Petromyzon marinus adult intestine, fixed in Bouin's solution. Paraffin-embedded section (4 m) immunostained for CGRP. Immuno-positive nerves are present in the outer wall of the intestine (arrowed) and in the sub-mucosa of the intestinal folds (x 125).
Fig. 5. Petromyzon marinusadult intestin. Tissue prepared as in Fig. 4. Immunostained with a monoclonal antibody to amphibian bombesin. Positively stained nerves are present in the outer wall of the intestine and smaller nerves are present in the submucosa of the intestinal folds (arrowed) (x 125).
406
Fig. 6. Petromyzon marinus cranial islet. Tissue prepared as in
Fig. 4. Immunostained for CGRP. Fine nerve are present between the lobules and seem to contact the islet cells. Some possible terminals are arrowed (x 200).
wall is well muscularised. This experiment was carried out on non-feeding adults in which the gut was already degenerating and the authors suggested that in the larva this action might be more widespread. Sakharov and Salimova (1980) confirmed the presence of serotonin in an abundant peripheral nervous system in the larval lamprey, but found no catecholamine-containing nerves. Substance P-like peptides, none of them identical to substance P, are present in the cyclostome central nervous system (Van Dongen et al. 1986; Nozaki and Gorbman 1986; Reinecke et al. 1987), as indeed are many other peptides and active substances (Buchanan et al. 1987; Brodin et al. 1988). I have found substance P-immunoreactive nerves in the lamprey gut wall. The relationships between the substance P-like peptide(s) of the central nervous system, the gut nervous system and the gut endocrine cells remain to be established. Yui et al. (1988) have described in L. japonicaenteric nerves immunoreactive for calcitonin generelated peptide (CGRP) and gastrin-releasing peptide (GRP), each peptide being co-localised with serotonin. CGRP nerves were present in the muscle layers and sub-mucosa of the intestine, while GRPimmunoreactive nerves were confined to the muscle layer. My own observations in L. planerilarvae and in L. fluviatilis and P. marinus adults are in fair agreement with this (Figs. 4 and 5), though I have
not examined the co-localisation with serotonin. CGRP seems to be the dominant peptide of the gut nervous system and is also sparsely present in the islets in P. marinus(Fig. 6). In addition to these peptides and the substance P-like peptide(s) mentioned above, I have also found some VIP-immunoreactive nerves in the gut of lampreys. Galanin and metenkephalin immunoreactivity, often present in fish gut nervous systems, were absent. None of the intestinal peptide-containing nerves appeared to contact the gut endocrine cells directly, and the actions of the neuropeptides may therefore be more concerned with muscle and vascular tone than with promotion of gut secretion, but the possibility remains that peptide/serotonin secretion from nerve terminals may diffuse to the base of the mucosal epithelium and influence secretion. In this review I have tried to bring together what is known about the enteric (neuro)endocrine system of cyclostomes. Work on this subject has been sporadic and unsystematic and it is apparent that we know remarkably little. With discoveries of new peptides the complexity of the system is now being revealed. There is clearly much to be done and a need for coordinated biochemical, physiological and morphological studies.
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408 Van Noorden, S., Ostberg, Y.and Pearse, A.G.E. 1977. Localization of somatostatin-like immunoreactivity in the pancreatic islets of the hagfish, Myxine glutinosa and the lamprey, Lampetrafluviatilis.Cell. Tiss. Res. 177: 281-285. Vigna, S.R. 1979. Distinction between cholecystokinin-like and gastrin-like biological activities extracted from gastrointestinal tissues of some lower vertebrates. Gen. Comp. Endocrinol. 39: 512-520. Vigna, S.R. and Gorbman, A. 1979. Stimulation of intestinal li-
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