J. clin. Path., 33, Suppl. (Ass. Clin. Path.), 8, 1-6

The endocrine versatility of the gut: general andevolutionary aspects of the active peptides of thegastrointestinal tractG. J. DOCKRAY

From the Physiological Laboratory, University ofLiverpool, Brownlow Hill, PO Box 147, Liverpool L69 3BX

Recent years have seen an unprecedented expansionof interest in the gastrointestinal endocrine systemthat shows no signs of abating. In large measure thisawakening can be attributed to the chemical studiesthat have resulted in the isolation and elucidation ofstructure of a wide variety of biologically active gutpeptides (Gregory and Tracy, 1975; Mutt, 1976).The availability of highly purified preparations ofthese peptides has made possible detailed studies oftheir effects and mode of action at the cellular level.In addition, it has become possible to apply immuno-chemical methods of analysis that have helped toreveal the cellular origins of the peptides and haveallowed their estimation in blood and tissue extracts.Several unexpected findings have emerged from thesestudies. For example, it now seems possible thatsome of the peptides produced by gut endocrinecells are not secreted into the blood stream, butrather act locally by diffusion to their targets throughthe extracellular space (paracrine effects). Further-more, it is now clear that many of the active peptidesin gut extracts originate not just in gut endocrinecells but also in nerve fibres. Peptides of the en-teric plexuses are also found in the central nervous

system, and other peptides previously identified inbrain have since been found in the gut. Thesedevelopments raise questions of fundamental im-portance about the interrelationships of the brain-gutpeptides and their roles in health and disease that,taken together, point to the need for a re-evaluationof the system of peptide messengers as a whole. Animportant aspect of such an analysis is the extent towhich the chemical and functional relationships ofthese peptides can be accounted for in evolutionaryterms. The relevance of this approach is emphasisedby the similarity in structure of groups of brain-gutpeptides that suggest a shared ancestry, both of themolecules in question, and of the entire system ofneuronal and hormonal peptides.

Interrelationships of nerves and endocrine cells

At least eight peptides have been reported to occurin mammalian gut endocrine cells and in central or

peripheral nerve fibres (Table 1). Only substance Pand neurotensin have been isolated from bothtissues (Leeman et al., 1977); much of the evidencefor the distribution of the other peptides rests on

Peptide Gut Gut Brain Amphibian Other tissuesnerves endocrine cells skin glands

Cholecystokn + + + Cacrulein (Phe-Met-Arg-Phe-NH2 in mollusc nerves)Gastrin + + + fVIP + + + _Glucagon - + - - Vertebrate pancreasGIP - + - -

Secretin - + - -

Motilin - + - -Somatostatin + + + - Protochordate gut endocrine cellsTRF ? ? + + Insect gangliaBombesin + + + +Substance P + + + Physaelemin Eledoisin in cephalopod salivary glandNeurotensin - + + XenopsinEnkephalin + + +Chymodenin ? ?ACTH - + + - Vertebrate pituitary

Table 1 Distribution ofpeptides found in vertebrate gut extracts+ Identified by isolation and/or immunochemical methods; - absent, or not yet reported; ? present in extracts but cellular origin uncertain.

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radioimmunoassay or immunocytochemistry, andthe possibility cannot be excluded that these sub-stances exist as distinct but cross-reacting moleculesin the brain and gut. As yet, cellular aspects ofsynthesis and secretion have been studied for only a

few peptides, but the balance of evidence favoursthe view that both hormonal and neuronal peptidesare synthesised initially as large precursors whichare sequestered into secretory granules or vesicles,converted by proteolytic and other enzymes toyield the active peptide, and are then released byexocytosis or a related process. There may also besimilarities in the electrical excitability of nerves andendocrine cells (Tischler et al., 1977). The differencesbetween neuronal and hormonal peptides can there-fore be seen to lie largely in the mode of their deliveryto target cells: on the one hand classical neurotrans-mitters diffuse across a synapse to act at a post-synaptic site, and on the other hand hormones are

released into the blood stream and so transportedto their targets. However, between these two ex-

tremes there are a variety of intermediate situations.For example, some nerves, notably those associatedwith the hypothalamus and neurohypophysis,secrete hormonal peptides directly into the bloodstream. Other neuronal peptides are apparently notreleased either at synapses or capillaries, but may

nevertheless mediate nerve-nerve interactions withslower onset and over longer periods than is gener-ally associated with classical neurotransmitters(Barker, 1977). Similarly, it is thought that in thegut some endocrine cells might release peptideswhich diffuse to, and act on, adjacent mucosal cellsin a local or paracrine mode of regulation (Pearseet al., 1977). Thus, there is no straightforwarddistinction to be made between the mode of actionof neuronal and hormonal peptides; instead thereis an almost continuous spectrum between truehormones on the one hand and true neurotrans-mitters on the other.The evolutionary origins of peptides that function

as extracellular molecular messengers are stilluncertain, and are in any case part of a largerproblem that includes other aspects of cell-cellinteraction, such as the origins of the regulation ofgrowth and differentiation in multicellular organisms.There is evidence that nerves specialised for thesecretion of peptides, so called neurosecretoryneurones, are present in the most primitive of meta-zoans (the coelenterates) and so can be consideredan ancient feature established early in the evolutionof nervous systems (Scharrer, 1978). The coelenter-ate neuropeptides are thought to control growth anddevelopment, and since there is no circulatorysystem in these animals they must presumablydiffuse to their targets. Paracrine-like regulation is

G. J. Dockray

thus one of the earliest forms of extracellularcontrol to be mediated by peptides.On present evidence, peptide-secreting glandular

endocrine cells appear to be absent from the lowermetazoans, so that, in a sense, peptidergic neuronescan be considered ancestral to the endocrine cellsof higher species. This need not imply directevolutionary descent of endocrine cells frompeptidergic neurones, although Pearse (1975) haspresented the evidence for this case. The ability toproduce and secrete active polypeptides occurswidely throughout both vertebrates and inverte-brates. For example, substance P is present inmammalian nerves and gut enterochromaffin cells;in some amphibian species there are also highconcentrations of substance P-like peptides (physa-laemin) in skin, and a related peptide (eledoisin) hasbeen isolated from the salivary gland of the cepha-lopod mollusc, Eledone. The wide distribution ofsubstance P-like peptides is not unique, and othergroups of peptides, such as those related to brady-kinin, also exhibit a wide distribution. In mammals,bradykinin is generated in the peripheral circulationby the action of proteolytic enzymes on a precursorprotein produced in the liver, but related peptidesagain occur in high concentrations in the skin ofsome amphibia, as well as in the venom secretion ofcertain wasps (Bertaccini, 1976). Both examplesserve to illustrate the fact that related active peptidesor their precursors can be found in a variety ofquite distinct systems with no obvious phylogeneticor functional relationships. Conceivably, the re-lated peptides of these diverse systems may havearisen by a process of convergent evolution. Itseems more reasonable to suppose, however, thatthese and probably other peptides were establishedearly in evolution and have since been stronglyconserved. Obviously, active peptides form only onelink in a chain of communication that necessarilyincludes other elements, such as appropriate targetorgan receptors and postreceptor transducingmechanisms. Once established, the capacity toemploy a peptide as a molecular messenger might bedrawn upon independently by systems as differentas brain and gut by virtue of changes in the patternof gene expression in these tissues. This is notparticularly surprising for there are obvious ad-vantages to be gained by deploying in more than onebiological context a single system of extracellularcommunication molecules, for which the necessarygenetic information is already available. Set in thislight the dual distribution of peptides in gut andbrain can be seen as an act of biological economyor conservation.The dual function of a molecule as both hormone

and neurotransmitter is not particularly novel, the

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The endocrine versatility of the gut

catecholamines being a well known case in point.The successful application of the same messenger intwo different systems depends on the adequateseparation of these systems. Segregation can beachieved both functionally and morphologically.Thus, the blood-brain barrier presents an obviousobstacle to the penetration of circulating peptidesinto the nervous system. Within the CNS the releaseof peptides at specific nerve endings must inevitablyrestrict their sites of action. In addition there mightbe differences in the requirements for concentrationsof the same peptide at different targets such that onlylocally released peptide can achieve concentrationssufficient to evoke a response.

Phylogeny of the vertebrate brain-gut peptides

The dual distribution of peptides in nerves and gutendocrine cells was probably established early in theevolution of the vertebrates. The point is wellillustrated by cholecystokinin-like peptides (Table2). We have recently purified from sheep brain apeptide with the sequence of the C terminal octa-peptide of porcine cholecystokinin (CCK 8), and wehave also isolated a second octapeptide which isslightly less acidic but otherwise has identical bio-logical and immunochemical properties to CCK 8(Dockray et al., 1978). CCK 8 has been identifiedimmunochemically in extracts of hog intestinetogether with the cholecystokinins of 33 (CCK 33)and 39 residues isolated by Mutt (1976), and acomponent which is probably of intermediate size(Dockray, 1977a). In extracts of the brain and gutof the lamprey we have found factors which haveimmunochemical and chromatographic propertiesand biological actions resembling, although notidentical to, those ofCCK 8 (Holmquist et al., 1979).Lampreys are of special phylogenetic interest sincethey are living representatives of the earliest of thevertebrate groups, the Agnatha, and have beenseparated from the rest of the vertebrate line

H or RPorcine gastrin,antral mucosa -Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2

Porcine/sheep RCCK, brain andintestine -Arg-Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2

Caerulein, Ramphibianskin gland Glp-Gln-Asp-Tyr-Thr-Gly-Trp-Met-Asp-Phe-NH2

Molluscan neuropeptide Phe-Met-Arg-Phe-NH2

R = SO3H

Table 2 Origins and structures ofgastrin andcholecystokinins

(Gnathostomata) by over 500 million years ofevolution. The distribution and structure of CCK-like peptides have therefore changed relatively littlethroughout the course of vertebrate evolution,suggesting a strongly conserved biological role forthese molecules in brain and gut. This conservationis even more striking when one considers thecellular origins of these peptides in the gastro-intestinal tract. Recent morphological studiesindicate that in the lamprey the gut endocrine cellsthat contain CCK-like factors resemble in ultra-structure and histochemical properties the gutendocrine cells of mammals (Van Noorden andPearse, 1974). Similar cells have also been found inprotochordates, like amphioxus (Van Noorden andPearse, 1976). Thus the organisation of cells pro-ducing CCK-like peptides, and probably other gutpeptides, appears remarkably constant throughoutthe vertebrates, in spite of profound evolutionarychanges in their target organs, for example develop-ment of stomach and pancreas. It would seem thenthat these cells are uniquely suited to responding tothe presence of food in the gut lumen by the secretionof peptides which regulate digestive activity.

Molecular evolution of active peptides

Insight into the evolutionary history of the mam-malian gut hormones and related peptides can beobtained by consideration of their amino-acidsequences. The identical C terminal pentapeptidesequences in porcine gastrin and cholecystokininsuggests that the two hormones evolved from acommon ancestor (Table 2). Likewise, the relatedsequences in glucagon, secretin, vasoactive intestinalpolypeptide (VIP), gastric inhibitory polypeptide(GIP), and possibly bombesin and chymodeninsuggests a shared history (Barrington and Dockray,1976; Dockray, 1977b). The existence of these molec-ular families can be accounted for by the sequentialoperation of two distinct processes: in the first in-stance gene duplication must occur to produce twodaughter genes each coding for the peptide; in thesecond instance point mutations in the structuralgene lead to amino-acid substitution and hencedivergence of the peptides. Only one of the daughtergenes is required to fulfil the role of the original geneso the other is relatively free to diverge in structure.Not all amino-acid substitutions are of equalimportance. Substitutions in the functionally im-portant parts of the molecule will cause markedchanges in biological activity, usually decreasingactivity. Such mutations will therefore face strongselective pressure and are unlikely to survive. Con-sequently, amino-acid substitutions in the bio-logically important parts of a molecule will be less

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frequent than elsewhere, which explains why theconserved regions of gastrin and CCK include theminimum sequences essential for biological activity.However, gene duplication and divergence are not

the only types of mutation that can alter the structureof peptides. Track (1973) has proposed that secretin,glucagon, and gastrin may have arisen from a frame-shift mutation in the insulin gene preceded by geneduplication. Frameshift mutations occur where thereis a shift by one or two bases in the reading frameof DNA codon triplets. Such mutations are unlikelyto have been responsible for the origins of manypeptide hormones since they lead to the productionof a completely new peptide that in the absence ofappropriate target organ receptors will presumablybe biologically inactive. Other possible types ofmutation include those affecting splicing of mRNAat the post-transcriptional level. Recent workindicates that several structural genes (for examplethose for ovalbumin, B globin, etc) are composed ofregions that are not translated interspersed betweenthose that are. The regions that are not expressedare believed to be enzymically spliced from themRNA before translation. Mutations in the splicingregions could lead to translation of differentsequences of the gene either instead of, or in additionto, the usual sequence. Finally, there is goodevidence that many secretory peptides are theproduct of post-translational processing of largeprecursor molecules that are the initial product ofmRNA translation. Several types of processingenzymes are likely to be involved. Thus, proteolyticenzymes cleave the precursor peptide to producesmaller active peptides, while other enzymes areresponsible for modifications such as the C terminalamidation and sulphation of tyrosine that occurs ingastrin and CCK. Mutation of the processingenzymes will inevitably lead to differences in thestructure of the final peptide. Several pieces ofindirect evidence support the idea of differences inprocessing pathways that might be the result ofnatural selection. For example, CCK 8 and CCK 33have been isolated from brain or gut in the formof molecules possessing a sulphated tyrosine residuein the seventh position from the C terminus (Mutt,1976; Dockray et al., 1978), and unsulphated formshave not so far been identified. The sulphate groupis known to be essential for the full biologicalpotency of the peptide and there will therefore bestrong selective pressure to maintain the efficiency ofthe sulphation system. In contrast, antral gastrinoccurs in about equal amounts of sulphated andunsulphated forms, and sulphation is known to havelittle or no effect on the potency of the hormone instimulating acid secretion. The enzyme systemrequired for sulphation of gastrin is therefore under

G. J. Dockray

less pressure than that for CCK, which mightexplain why it is less efficient.There might also be differences in the processing

pathways for a particular peptide in differenttissues. For example, there has recently been con-troversy over the presence of VIP in nerves andendocrine cells. Early reports suggested that VIP-like immunoreactivity was present in endocrine cellsin all parts of the gut (Polak et al., 1974). Morerecently, several studies have failed to confirm thisobservation and have suggested, instead, that VIPis present in the nerve plexuses (Larsson et al., 1976).The discrepancy might be explained by the presencein nerves and endocrine cells of different immuno-reactive forms of VIP that varied in their crossreactivity with different antisera. In keeping withthis idea, we recently showed that in extracts ofhuman colonic mucosa, which contains both nervefibres and endocrine cells, there were, in addition toa form similar to or compatible with the peptideoriginally purified from hog intestine, at least twoother immunoreactive forms of VIP (Fig. 1). Incontrast, in human colonic muscle, which containsnerves but not endocrine cells, there was the singleform similar to the original porcine peptide (Dimalineand Dockray, 1978). On this evidence it seems thatoctacosapeptide VIP has a neuronal origin in the gut,

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Fig. 1 Elution profile of acetic acid extract of humancolonic mucosa (upper panel) and human colonicmuscle (lower panel) after fractionation on CM-Sephadex.Immunoreactive VIP in the column eluates wasestimated by radioimmunoassay using an antiserumspecific for the NH2 terminal region ofporcine VIP.(Reproducedfrom Dimaline and Dockray, 1978.)

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while the molecular variants are present in endocrinecells. This observation could be explained bydifferent processing pathways for VIP in nerves andin endocrine cells (Fig. 1).

Receptors

It is clear that during the course of evolution,molecules which are identical or closely related tothe mammalian gut hormones have developedphysiological roles not just in the control ofdigestion but also in the regulation of metabolismand, in the central nervous system, as neurotrans-mitters or neuromodulators. The evolution of thesepeptides cannot therefore be considered solely interms of changes in their molecular structure andcells of origin, but must also take into account theevolution of their target cells. The importance ofcell surface receptors in mediating the action ofhormones and neurotransmitters is now wellestablished. In particular, considerable progress hasbeen made in the identification and characterisationof the receptors for some mammalian peptides.Theresults of these studies provide a basis for under-standing in molecular terms the evolutionaryrelationships between peptides and their target cells.

Thus, in mammals, cell surface receptors bindingsecretin, VIP, and glucagon have been identified onliver cells and fat cells (Bataille et al., 1974). On bothcells there are receptors with high affinity for VIPand low affinity for secretin that do not bindglucagon; the receptors binding glucagon do notbind VIP or secretin. Thus, in addition to thedivergence of glucagon, VIP, and secretin, there havealso developed different receptors specific for thesepeptides. It is tempting to speculate that these mighthave arisen by an analogous process of duplicationand divergence. In this context recent studies on theexocrine pancreas are particularly revealing. Gardnerand co-workers (1978) have described a populationof receptors on guinea pig pancreatic acinar cellsthat have high affinity for VIP and low affinity forsecretin, while a second population of receptors havehigh affinity for secretin and low affinity for VIP.These results are based on experiments usingdispersed pancreatic acinar cells in vitro and thepossibility cannot yet be completely dismissed thatthe two types of receptor are present on differentcell types. There are estimated to be about 135 000receptors per cell with high affinity for secretin andlow affinity for VIP and about 9000 per cell withhigh affinity for VIP and low affinity for secretin(Gardner et al., 1978). In mammals such as dog andrat, VIP is a weak stimulant of pancreatic exocrinesecretion, whereas secretin is a strong stimulant.However, we have found that in birds (turkey)

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Fig. 2 Response of the pancreas in urethaneanaesthetised turkeys to pure natural porcine secretin (a)and pure natural porcine VIP (0). Basal secretion ismarked by X. Note that the range of doses of secretinwas about 10 times higher than that of VIP. On amolar basis VIP is 25-30 times more potent thansecretin in stimulating the flow ofpancreatic juice.(Reproducedfrom Experientia, 29, 1510, 1973.)

porcine secretin is a weak stimulant of the flow ofpancreatic juice (Fig. 2). In addition, the secretin-likepeptide isolated from chicken intestine by Nilsson(1974) also has low potency on the avian pancreas(R. Dimaline and G. J. Dockray, unpublishedobservations). In sharp contrast, VIP (porcine orchicken) is a strong stimulant of the flow of pancreaticjuice from the avian pancreas (Dockray, 1978). Thethreshold dose is about 10 ng/kg which is comparableto the dose of porcine secretin needed to stimulatethe pancreas in mammals. Although the regulationof the bird exocrine pancreas is still poorly under-stood, the available data would be consistent with arole for VIP analogous to that of secretin in mam-mals. The different responses of the pancreas inbirds and mammals could be explained by differenceseither in the relative numbers, or in the specificities,of pancreatic cell receptors for secretin and VIP.Confirmation of this is now needed by direct studies

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of the avian pancreatic receptors. It is significant,however, that the affinity of mammalian hepatocytesfor secretin is about 1 % of that for VIP, whereas theaffinity of chicken hepatocytes for secretin (porcine)is less than 0-1 % of that for VIP (Bataille et al.,1977). VIP and its liver receptors are presumably acommon inheritance of birds and mammals fromtheir reptilian ancestors, and since the separation ofthese two lines there would seem to have been evolu-tionary changes in the affinity of this receptor forsecretin. In a general sense, these studies reveal thescope for variability of target organ receptors, bothin terms of numbers of receptors per cell andspecificity for different ligands. In the evolution ofnew target-hormone relationships, such as must haveaccompanied the development of the stomach andpancreas, the existence of a pool of variability inboth peptides and receptors must have been ofcrucial importance.

References

Barker, J. L. (1977). Physiological roles of peptides inthe nervous system. In Peptides in Neurobiology, pp.295-343, ed H. Gainer. Plenum Press, New York andLondon.

Barrington, E. J. W., and Dockray, G. J. (1976). Gastro-intestinal hormones. Journal of Endocrinology, 69,299-325.

Bataille, D., Besson, J., Bastard, C., Laburthe, M., andRosselin, G. (1977). Specificity in hormone-receptorinteraction: studies with insulin, glucagon and vaso-active intestinal peptide. In Hormonal Receptors inDigestive Tract Ph.Ysiology, pp. 113-125, ed S. Bonfils,P. Fromageot, and G. Rosselin. Elsevier/NorthHolland, Amsterdam.

Bataille, D., Freychet, P., and Rosselin, G. (1974). Inter-actions of glucagon, gut glucagon, vasoactive intestinalpolypeptide and secretin with liver and fat cell plasmamembranes: binding to specific sites and stimulation ofadenylate cyclase. Endocrinology, 95, 713-721.

Bertaccini, G. (1976). Active polypeptides of non-mammalian origin. Pharmacological Reviews, 28, 127-177.

Dimaline, R., and Dockray, G. J. (1978). Multipleimmunoreactive forms of vasoactive intestinal peptidein human colonic mucosa. Gastroenterology, 75,387-392.

Dockray, G. J. (1977a). Immunoreactive componentresembling cholecystokinin octapeptide in intestine.Nature (London), 270, 359-361.

Dockray, G. J. (1977b). Molecular evolution of guthormones: application of comparative studies on theregulation of digestion. Gastroenterology, 72, 344-358.

Dockray, G. J. (1978). Evolution of secretin-like hor-mones. In Gut Hormones, pp. 64-67, ed S. R. Bloom.Churchill Livingstone, Edinburgh.

Dockray, G. J., Gregory, R. A., Hutchinson, J. B.,Harris, J. I., and Runswick, M. (1978). Isolation,

structure and biological activity of two cholecystokininoctapeptides from sheep brain. Nature (London), 274,711-713.

Gardner, J. D., Long, B. W., Uhlemann, E. R., andPeikin, S. R. (1978). Membrane receptors for VIP andsecretin. In Gut Hormones, pp. 92-96, ed S. R. Bloom.Churchill Livingstone, Edinburgh.

Gregory, R. A., and Tracy, H. J. (1975). The chemistry ofthe gastrins: some recent advances. In GastrointestinalHormones, pp. 13-24, ed J. C. Thompson. Universityof Texas Press, Austin, Texas.

Holmquist, A. L., Dockray, G. J., Rosenquist, G. L., andWalsh, J. H. (1979). Immunochemical characterisationof cholecystokinin-like peptides in lamprey gut andbrain. General and Comparative Endrocrinology, 37,474-481.

Larsson, L. I., Fahrenkrug, J., Schaffalitzky de Muckadell,0. B., Sundler, F., HAkanson, R., and Rehfeld, J. F.(1976). Localisation of vasoactive intestinal polypep-tide (VIP) to central and peripheral neurones. Pro-ceedings of the National Academy of Sciences of theUSA, 73, 3197-3200.

Leeman, S. E., Mroz, E. A., and Carraway, R. E. (1977).Substance P and neurotensin. In Peptides in Neuro-biology, pp. 99-144, ed H. Gainer. Plenum Press, NewYork and London.

Mutt, V. (1976). Further investigations on intestinalhormonal polypeptides. Clinical Endocrinology, 5,(Suppl.), 175s-183s.

Nilsson, A. (1974). Isolation, aminoacid composition,and terminal amino acid residues of the vasoactiveoctacosapeptide from chicken intestine. Partial puri-fication of chicken secretin. FEBS Letters, 47, 284-289.

Pearse, A. G. E. (1975). Neurocristopathy, neuroendo-crine pathology and the APUD concept. Zeitschrift furKrebsforschung und Klinische Onkologie, 84, 1-18.

Pearse, A. G. E., Polak, J. M., and Bloom, S. R. (1977).The newer gut hormones. Cellular sources, physiology,pathology and clinical aspects. Gastroenterology, 72,746-761.

Polak, J. M., Pearse, A. G. E., Garaud, J. C., andBloom, S. R. (1974). Cellular localization of a vaso-active intestinal peptide in the mammalian and aviangastrointestinal tract. Gut, 15, 720-724.

Scharrer, B. (1978). Peptidergic neurons: facts andtrends. General and Comparative Endocrinology, 34,50-62.

Tischler, A. S., Dichter, M. A., Biales, B., and Greene,L. A. (1977). Neuroendocrine neoplasms and theircells of origin. New England Journal of Medicine, 296,919-925.

Track, N. S. (1973). Evolutionary aspects of the gastro-intestinal hormones. Comparative Biochemistry andPhysiology, 45B, 291-301.

Van Noorden, S., and Pearse, A. G. E. (1974). Immuno-reactive polypeptide hormones in the pancreas in thegut of the lamprey. General and Comparative Endo-crinology, 23, 311-324.

Van Noorden, S., and Pearse, A. G. E. (1976). TheEvolution ofPancreatic Islets, pp. 163-178, ed T. A. I.Grillo, L. G. Liebson, and A. Epple. Pergamon Press,Oxford.

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