J Clin Pathol: first published as 10.1136/jcp.s1-8.1.68 on 1 January 1978. Downloaded from

J. clin. Path., 33, Suppl. (Ass. Clin. Path.), 8, 68-75

Brain and gut peptides *

J. M. POLAK From the Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 OHS

The first evidence that the same peptides could be may show a preferential localisation to either cells present in both the brain and the gut was given in or nerves (Fig. 1) no clear-cut distinction can be 1931 with the discovery of (Von Euler seen between the peptides which may be localised and Gaddum, 1931). In spite of this early start, the to one or both of these structures. The classical important revelation that there were a large number division of the autonomic nervous system into of peptides with this dual localisation and also with adrenergic and cholinergic parts, originally proposed powerful and extensive pharmacological actions took by Langley and Anderson (1895), was recently more than 40 years to materialise. After the finding challenged by Baumgarten et al. (1970) who de- of large quantities of the hypothalamic peptide scribed another type of neurosecretory granule with (Arimura et al., 1975) in the gastro- an ultrastructure different from that of the adren- intestinal tract, increasing numbers of peptides have ergic or cholinergic types. Baumgarten's granules been found to be common to the gut and brain. To were much denser and larger than the classical date the list includes somatostatin, substance P, autonomic nervous system vesicles and were termed (CCK), enkephalin, , p-type for their resemblance to the central nervous vasoactive intestinal polypeptide (VIP), and bombe- system peptidergic neurosecretory granules respon-copyright. sin. A number of these peptides, such as somato- sible for the production of oxytocin and vasopressin. statin, neurotensin, and enkephalin, were originally It is now well accepted that among the neuronal found in the brain and later in the gut, whereas peptides, VIP at least is present in this p-type of others, such as VIP, , and , were neurosecretory granule. found first in the gut. There is increasing evidence that outside the brain Technology these peptides are localised in typical endocrine cells, http://jcp.bmj.com/ or in fine efferent nerve fibres, or both, as part of the Information on the distribution, cellular localisation, autonomic innervation. Although some peptides and precise quantities of the peptides in a tissue sample can be given by two immunological pro- cedures, radioimmunoassay (RIA) of tissue extracts * Submitted to Proceedings of the Royal Society of Medicine, November 1978. and fluids to determine the quantities of peptides

The spectrum on September 28, 2021 by guest. Protected

Fig. 1 Diagram showing the 'spectrum' of localisation ofneuro- peptides to both cells and nerves of the gastrointestinal tract. At one end, substance P (Sub P), enkephalin (ENK), and vasoactive intestinal peptide (VIP) are mainly found in nerves, while at the other end somatostatin (SRIF), cholecystokinin (CCK), and neurotensin (NT) are mainly in cells. Bombesin (BN) appears equally in both structures.

Sub P VIP BN SRIF CCK ENK NT 68 J Clin Pathol: first published as 10.1136/jcp.s1-8.1.68 on 1 January 1978. Downloaded from

Brain and gut peptides 69 present, and immunocytochemistry (IC) of tissue are raised to an insoluble complex composed of the sections to establish their location. These two pure peptide coupled covalently to a larger molecule, techniques, especially when used in combination, such as albumin. The resulting antiserum will have provided extremely valuable insights. contain a mixed proportion of desirable specific and Two major problems are frequently encountered unwanted non-specific antibodies. To ensure that by immunocytocheniists. only the specific antibodies participate, it is extremely (1) The are water soluble and important to dilute the antiserum to the maximal therefore easily washed out of a tissue section. It is extent. This is possible with recently developed thus extremely important to render the peptide immunocytochemical procedures, which use high insoluble without altering its antigenic site. This dilutions with long incubation times, particularly cannot b. done by conventional fixation but is best when combined with the peroxidase-antiperoxidase achieved by the use of two cross-linking agents, (PAP) staining procedure (Stemberger, 1974). diethylpyrocarbonate vapour (DEPC) (Pearse et al., It is also extremely important to use a range of 1974), or p-benzoquinone, used either as a vapour or well-characterised antibodies as well as rigorous as an aqueous solution (Pearse and Polak, 1975). controls, the latter including the use of antiserum (2) Specificity. Antibodies to peptide after absorption with the antigen being studied. copyright. http://jcp.bmj.com/

Fig. 2 Section ofhuman gut (muscle wall) fixed in a solution of p-benzoquinone and stained with antibodies to (A) synthetic substance P, and (B) pure porcine VIP. Note the classical beaded appearance of on September 28, 2021 by guest. Protected the nerve fibres. (Original magnifi- cation x 252.) J Clin Pathol: first published as 10.1136/jcp.s1-8.1.68 on 1 January 1978. Downloaded from

70 J. M. Polak Individual peptides which are known to originate from EC cells (Powell et al., 1978), and in extracts of such tumours. SUBSTANCE P The undecapeptide substance P was discovered SOMATOSTATIN incidentally by Von Euler and Gaddum (1931) Somatostatin is a 14-amino-acid peptide which was while they were working on the distribution of originally extracted from the hypothalamus and acetylcholine in the gut. Substance P exerts powerful identified as the factor which inhibits the release of pharmacological actions mostly on growth from the pituitary (Brazeau et al., and the vasculature (Powell et al., 1978). It has been 1973). It was later found that somatostatin is present localised by immunocytochemistry mainly to fine in large quantities in the gastrointestinal tract nerve fibres of the gut wall (Fig. 2A). The production (Arimura et al., 1975). It exerts powerful inhibitory of substance P by ganglion cells in the gut was until actions on the release of a number ofhormones from recently controversial, but has now been fully the pituitary and the gastrointestinal tract (see below). substantiated by the use of tissue culture methods (Schultzberg et al., 1978; Jessen et al., 1979). ACTIONS OF SOMATOSTATIN Substance P is also present in scattered endocrine, Inhibition of: growth hormone, thyroid stimulating argentaffin cells (enterochromaffin or EC cells) of hormone, insulin, , pancreatic polypeptide, the gut mucosa which also simultaneously store gastrin, , insulin-dependent insulinotropic . The largest concentrations of substance hormone, , enteroglucagon, gastric acid, P in the central nervous system are found in the gastric emptying, pancreatic bicarbonate, pancreatic dorsal roots of the spinal cord, in the pons, medulla enzymes, gall bladder contraction, myoelectric oblongata, hypothalamus, mesencephalus, and complexes, coeliac blood flow, xylose absorption. amygdaloid complex (Hokfelt et al., 1978). In the gut, somatostatin is found mostly in the In addition, an important network of substance gastric antrum and the pancreas. Immunocyto-copyright. P-containing nerves has recently been found in chemical methods show it to be localised to endo- many other organs such as the lung (Wharton et al., crine cells (Fig. 3). These have been characterised, 1979), genitourinary tract, and skin. The distribution and recently internationally accepted, as the D cells of these nerve fibres, particularly beneath the ofthe ultrastructural classification (Polak et al., 1975). epithelial layer of areas of high tactile sensitivity, In the brain, somatostatin is found not only in suggests a sensory role for substance P. the hypothalamus but also in many other areas

Substance P has been found, by bioassay, to be including pons, medulla, amygdaloid complex, http://jcp.bmj.com/ absent from the aganglionic segment of the gut in limbic area, and hippocampus (Hokfelt et al., 1978). Hirschsprung's disease (Tafuri et al., 1974) and Ultrastructural and immunocytochemical studies Chagas's disease (Hial et al., 1973). Large quantities have shown that brain somatostatin is present in of substance P have been found by RIA in the neurosecretory granules of a larger size (170 nm circulation of patients with gastrointestinal carcinoids diameter) than those storing acetylcholine and on September 28, 2021 by guest. Protected

Fig. 3 Somatostatin cells in human antrum. Note distribution E ISi ofcells on basal membrane. (Original magnification x 203.) J Clin Pathol: first published as 10.1136/jcp.s1-8.1.68 on 1 January 1978. Downloaded from

Brain and gut peptides 71 adrenaline (Pelletier et al., 1977). Somatostatin has duodenum and found to be a 28 amino-acid peptide also been found in the thyroid gland (Van Noorden closely related to glucagon, secretin, and glucose- et al., 1977), the thymus, and in sympathetic ganglia dependent insulinotropic peptide (GIP). It has been containing catecholamines (Hokfelt et al., 1978). shown to exert powerful pharmacological actions on In spite of the fact that recent reports indicate the smooth muscle and blood vessels, as well as presence of somatostatin in the pancreatic and influencing the secretory activity of the gastro- gastric effluent blood (Harris et al., 1978), its wide intestinal tract (Said, 1978). The role of VIP as a and powerful range of actions, as well as its dis- or hormone is discussed elsewhere tribution throughout the body, suggests that in the Symposium (page 63). somatostatin must act mainly in the capacity of a VIP is very widely distributed. It has been found paracrine (local) hormone. The term paracrine was to be present not only in the brain and gut but also originally introduced by Feyrter (1938) to indicate in the genitourinary tract (Bishop et al., 1979), a 'local' action of the clear cells of his 'diffuse pancreas (Polak et al., 1978c), posterior pituitary '. Feyrter suggested that these clear (Van Noorden et al., 1978), and salivary glands cells acted upon neighbouring non-endocrine epithe- (Bloom et al., 1978a). Immunocytochemical studies lial cells. localise VIP mainly to fine nerve fibres of these Numerous single or mixed somatostatin-contain- organs. Using the technique of serial semithin ing endocrine tumours of the gut and pancreas have (800 nm)/thin (80 nm) sections, with examination by recently been described (Larsson et al., 1976; Ganda immunocytochemistry and electron microscopy, et al., 1977; Alumets etal., 1978; Bloom et al., 1978b). respectively, has shown that VIP is stored in the The powerful inhibitory actions of somatostatin p-type of neurosecretory granules originally de- on the secretion of pancreatic and gastrointestinal scribed by Baumgarten (Polak and Bloom, 1978). hormones suggest that somatostatin may be one of In the gut, fine VIP nerve fibres are found in the the several factors whose excess or deficiency may mucosa, the sub-mucosa, and the two muscle layers copyright. play a part in the development of duodenal ulcer as well as in the plexi of Meissner and Auerbach disease (Polak et al., 1978d). Somatostatin (Fig. 2B). bodies are also occasionally seen deficiency may also be involved in many cases of which contain VIP. severe intractable neonatal hypoglycaemia with The involvement of VIP in the Vemer-Morrison hyperinsulinaemia. Samples of pancreas removed syndrome is discussed elsewhere (page 85). from sick children show a significant decrease of An interesting recent finding is the remarkable extractable and stainable somatostatin (Fig. 4) and increase of VIP nerve fibres in the affected gut http://jcp.bmj.com/ alteration of the normal arrangement of the insulin/ segment in Crohn's disease (Polak et al., 1978b). The somatostatin-secreting cells (Polak et al., 1978a). VIP nerve fibres appear extremely hyperplastic, enlarged, and disorganised. There is twice as much VASOACTIVE INTESTINAL POLYPEPTIDE (VIP) extractable VIP, estimated by RIA, in the abnormal VIP was originally extracted from the porcine area of the gut as in comparable regions of normal on September 28, 2021 by guest. Protected

Fig. 4 (A) Normalpancreas (2-month-old term baby) showing the presence ofnumerous, well stained somatostatin cells. (B) Pancreas from a child with intractable hypoglycaemia showing scattered andpoorly stained somatostatin cells. (Original magnification x 285.)

&.. B .L J Clin Pathol: first published as 10.1136/jcp.s1-8.1.68 on 1 January 1978. Downloaded from

72 J. M. Polak gut wall. In contrast, another inflammatory bowel and during the search for a possible endogenous disease, ulcerative colitis, shows the normal number morphine. and distribution of VIP nerve fibres (Fig. 5A, B). A One of them, met-enkephalin, belongs to a larger possible explanation may be that in Crohn's disease group of endogenous opiates, grouped under the the inflammatory process, which affects all the gut generic name of endorphin (endogenous morphine). layers, acts as a 'stimulus to proliferation' for the A larger peptide containing leu-enkephalin has not scattered intrinsic nerve cell bodies, whereas in yet been found. ulcerative colitis the is only superficial In the brain the enkephalins are found in areas and perhaps fails to 'irritate' the VIP cell bodies. It related to pain transmission (Hokfelt et al., 1978). is possible therefore that estimation of gut VIP may Both met- and leu-enkephalins are found in the become a useful tool for diagnosis in undetermined gastrointestinal tract (Polak et al., 1977). The largest cases of inflammatory bowel disease. quantities are present in the gastric antrum and upper duodenum where they are localised to fine ENKEPHALINS nerve fibres of the gut wall and occasional endocrine The enkephalins are two closely related pentapep- cells of the gut and pancreas. The cell bodies for tides differing only by one amino-acid (methionine or these fine nerves are rarely seen. However, support leucine) at the C terminal end. They were first found for the location of enkephalinergic neurones in the in the brain, after the discovery of optiate receptors, bowel wall comes from the observation that enke- copyright. http://jcp.bmj.com/

Fig. 5 (A) Endoscopic biopsy of human colon from a patient with all the clinical and histopathologicalfeatures of ulcerative colitis. Note the normal appearance of the VIP nerve fibres. (B) on September 28, 2021 by guest. Protected Section ofhuman colon from a patient with Crohn's disease. VIP nerve fibres appear highly stained, thickened, and irregularly distributed. (Original magnification x 2S2.) J Clin Pathol: first published as 10.1136/jcp.s1-8.1.68 on 1 January 1978. Downloaded from

Brain and gut peptides 73 phalin fibres can be demonstrated by immuno- major area of overlap between the classically chemical methods in cultures of Auerbach's plexus separated endocrine and nervous systems and (Schultzberg et al., 1978; Jessen et al., 1979). reconciles the previously opposing views of Pavlov The localisation of enkephalins in the gastro- and Bayliss and Starling. It is now realised that the intestinal tract may provide an explanation for the two control systems, far from being antagonistic, powerful effects of morphia, known from antiquity, are highly synergistic and interdigitated. It also on many functions of the gastrointestinal tract. strongly supports Pearse's hypothesis (1969) of a Furthermore, it has recently been shown that common origin in the embryonic neuroectoderm of enkephalins exert powerful modulating effects on the peptide-producing endocrine cells and the the smooth muscle, blood flow, and secretions of neurones of this significant and powerful diffuse the gut (Konturek, 1978). neuroendocrine system. There are several unexplained effects ofautonomic BOMBESIN nerve stimulation which cannot be altered by Bombesin is a 14 amino-acid peptide originally cholinergic or adrenergic blockade. These include extracted from amphibian skin by Erspamer and vasodilation of the salivary gland (Heidenhain, Melchiorri (1973). It was then shown to have many 1872), pancreatic bicarbonate release (Hickson, potent pharmacological actions in mammals on the 1970), and numerous pulmonary reactions (Richard- gut, the brain, the lung, and the urinary tract. son and B6land, 1976). The mechanism behind these The distribution of bombesin-like material has effects can now be explained by the existence of this been analysed by combined RIA and immuno- additional component of the autonomic nervous cytochemistry. Large quantities of bombesin are system, from which peptides can be released after found in the brain (hypothalamus, thalamus, electrical stimulation. limbic system, and amygdaloid complex), gut Gastrointestinal function as a whole is, generally, (throughout the entire length), and lung (fetal and equally ill-understood. It is quite likely that many copyright. neonatal). Chromatographic analysis of the normal gut functions are the culmination of a well bombesin-like material in mammals indicates the orchestrated sequence of actions by the agonistic presence of two molecular forms, one which elutes and antagonistic modulators of the endocrine, from gel columns in the same position as amphibian nervous, and neuroendocrine systems. bombesin and another larger form (Polak et al., It is clear that the study of a single aspect of gut 1978e). physiology will yield insufficient information to

Immunocytochemical techniques localise bombe- solve any problem. We will have to achieve an overall http://jcp.bmj.com/ sin to fine efferent nerve fibres of the gut wall and to understanding of how the peptides act, where they endocrine cells of the bronchial mucosa, where they are localised, and what factors cause their release. are found singly or in small groups (Wharton et al., Intriguing details are already emerging which 1978). It has recently been shown that bombesin is a promise an exciting outlook for future research into powerful, pH independent, gastrin releaser. It is this new and fascinating discipline of gut neuro- thus possible that bombesin plays a key role in endocrinology.

gastrointestinal disorders associated with abnor- on September 28, 2021 by guest. Protected malities of acid secretion. Neurotensin and cholecystokinin are discussed by References Dr Blackburn (page 12) and Professor Rehfeld Alumets, J., Ekelund, G., Hakanson, R., Ljungberg, 0., (page 26). Ljunqvist, U., Sundler, F., and Tibblin, S. (1978). Jejunal endocrine tumour composed of somatostatin Conclusions and gastrin cells and associated with duodenal ulcer disease. Virchows Archiv A, 378, 17-22. Arimura, A., Sato, H., Dupont, A., Nishi, N., and At the end of the 19th century the control of gut Schally, A. V. (1975). Somatostatin: abundance of functions was considered to be mediated almost immunoreactive hormone in rat stomach and pancreas. entirely by the nervous system. This concept formed Science, 189, 1007-1009. the theory of 'nervism' postulated by Pavlov. The Baumgarten, H. G., Holstein, A. F., and Owman, C. discovery, by Bayliss and Starling, of 'chemical (1970). Auerbach's plexus of mammals and man: messengers', or hormones, which have a powerful electron microscopic identification of three different influence on gut function, caused the replacement of types of neuronal processes in myenteric ganglia of the nervism theory by the new concept of the large intestine from rhesus-monkeys, guinea-pigs endocrine and man. Zeitschrift fur Zellforschung und Mikro- control. skopische Anatomie, 106, 376-397. The latest discovery ofa peptidergic component of Bishop, A. E., Polak, J. M., and Bloom, S. R. (1978). the autonomic nervous system shows that there is a Journal ofEndocrinology, 77, 25. J Clin Pathol: first published as 10.1136/jcp.s1-8.1.68 on 1 January 1978. Downloaded from

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