GENERAL
AND
COMPARATIVE
25, 203-210 (1975)
ENDOCRINOLOGY
Comparative
Studies
on Secretinl
GRAHAM J. DOCKRAY Physiological
Laboratory, Liverpool,
University
of Liverpool,
England
Received July 20, 1974 The mammalian intestinal hormone secretin is related in structure to the pancreatic hormone glucagon, and to two peptides recently isolated from hog duodenum, vasoactive intestinal peptide (VIP) and gastric inhibitory peptide (GIP). Because of their structural similarities it has been suggested that these peptides evolved from a common ancester, and comparative studies may be expected to shed light on this question. In the present series of experiments pure porcine secretin was found to be a weak stimulant of the flow of pancreatic juice in a bird (the turkey) although it was a strong stimulant in mammals such as cat and rat. In contrast, pure porcine VIP had weak actions on the flow of pancreatic juice in mammals but was a potent stimulant in birds, while porcine glucagon and GIP showed no stimulatory effects on exocrine pancreatic secretion in either birds or mammals. Like VIP, extracts of the intestine of birds (chicken) and teleost fish (pike and cod) had weak secretinlike actions on the pancreas in mammals but strong actions in birds. The results indicate that in the intestines of birds and fish there are factors with biological properties similar, but not identical, to those of mammalian secretin and VIP, and it is suggested that the secretin group of hormones has a long evolutionary history.
In mammals the presence of acid in the small intestine stimulates the release of the hormone secretin, which in turn evokes a flow of pancreatic juice rich in bicarbonate and poor in enzymes. The secretin mechanism of mammals was discovered by Bayliss and Starling in 1902; indeed, it was with secretin in mind that Starling first introduced the word hormone (Starling, 1905). Soon after their discovery of secretin, Bayliss and Starling (1903) described secretin-like activity in intestinal extracts of many vertebrate species including salmon, dogfish, ray, frog, goose, tortoise, and chicken. However, in spite of this promising start, comparative studies on secretin have been slow to progress. An unexpected insight into the comparative endocrinology of secretin came when the amino acid sequence of porcine secretin was elucidated and it was found that i Submitted to the 7th International Symposium on Comparative Endocrinology, Kenya, 1974.
no fewer than 14 of the 27 amino acid residues in this molecule were identical to those in the pancreatic hormone, glucagon (Mutt and Jorpes, 1967). Recently, two more peptides have been isolated from hog intestine and shown to have amino acid sequences similar to those of secretin and glucagon (Fig. 1). The new molecules are gastric inhibitory peptide (GIP) purified by Brown, Mutt, and Pederson (1970), and vasoactive intestinal peptide (VIP) isolated by Said and Mutt (1972); although the names of the new peptides reflect biological actions their physiological roles and hormonal status have not yet been fully proved. On the basis of their structural similarities, it has been suggested that these four molecules arose from a common evolutionary ancestory by a process of gene duplication and divergence (Adeleson, 197 1; Weinstein, 1972). Comparative studies may be expected to give further insight into the relationships between these molecules. In the present series of 203
Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
204
GRAHAM
SECRETIN
1 HIS
2 3 4 5 6 7 8 9 10 l! 12 SER ASP GLY THR PtiE THR SER GLU LEU SEC AK
GLUCAGON GIP
GLN TYR ALA ELU
VIP
ILE AtA
SECRETIN
VAL
13 14 LEU ARG
ASP TYR
LYS TYR LEU
ASP TYR
ILE ALP riET
ASP ASN TYR THR rl
15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 ASP SER At.4 AR6 LEU GLN AAG LEU LEU GLN GLV LEU VAL NH2
GLUCAGON GIP
L"s
G':;
;I;
ALA f
L,~~,LE~;~
fi
VIP
FIG. 1. Amino acid sequence of mammalian peptides belonging to the secretin-glucagon family. Identities with secretin are indicated by a blank space; other identities are indicated by lines around them. Number of identities: VIP-GIP 4, VIP-glucagon 5, VIP-secretin 9, secretin-GIP 9, secretin-glucagon 14, glucagon-GIP 15. GIP has 43 residues, only 29 of which are shown. All peptides of porcine’origin. Taken from Grossman (1974).
experiments particular attention has been given to the secretin-like actions of the mammalian peptides and extracts of bird and fish intestine on pancreatic exocrine secretion in birds and mammals. MATERIALS
AND METHODS
Pure porcine secretin and VIP, a purified extract of chicken duodenum and an extract of hog duodenum containing 20% cholecystokinin (CCK) were gener-
J. DOCKRAY ous gifts of Dr. V. Mutt. An extract of hog duodenum (prepared by Dr. J. Brown) containing about 40% GIP and trace quantities of CCK was kindly made available by Dr. L. Gerring. Synthetic C-terminal octapeptide of CCK was a gift from Squibb Institute. Extracts of the intestine of pike (&ox Irrcius) and cod (Gadus morhua) were prepared by methods already described (Dockray, 1974). The action of peptides and extracts on pancreatic exocrine secretion was examined in urethaneanesthetized rats and turkeys (Dockray, 1972a,b).
RESULTS Secretin-like Action of Mammalian Peptides in Birds and Mammals
Of the mammalian peptides belonging to the secretin-glucagon family, only secretin and VIP stimulated the flow of pancreatic juice in birds and mammals (Fig. 2). Cholecystokinin also stimulated the flow of juice, but this hormone, unlike secretin and VIP, was a strong stimulant of pancreatic enzyme secretion as well. It seems reasonable to ask whether the four mammalian peptides are the only members of the secretin-glucagon family
FLOW PROTEIN -RAT
TURKEY
FIG. 2. The action of mammalian peptides on the rate of flow of juice and rate of protein secretion from pancreas in rats and turkeys. Protein concentration in the pancreatic juice was used as an index of enzyme secretion, and was estimated from OD at 280 nm; it is expressed in terms of a standard of bovine serum albumin. Peptides were given by rapid iv shot, and responses are peak rates of secretion occurring immediately after injection. Vertical bars represent one SE of mean. Responses are mean of six rats and six turkeys.
COMPARATIVE
STUDIES
or whether there are other members waiting to be discovered. One piece of indirect evidence that suggests there may be another secretin-like molecule in the mammalian intestine comes from studies with CCK-containing extracts of hog duodenum. Cholecystokinin is the hormone which strongly &imulates gallbladder contractions and pancreatic enzyme secretion, and weakly stimulates the bicarbonate concentration of pancreatic juice. The Cterminal octapeptide fragment of CCK (OP-CCK) possesses all the known biological actions of the whole molecule. In dogs OP-CCK and an extract of hog duodenum containing 20% CCK gave similar stimulation of the flow of pancreatic juice relative to pancreatic enzyme secretion, indicating that the CCK extract was essentially free of contamination by secretin (Debas and Grossman, 1973). However, in the turkey 20% CCK was a stronger stimulant of the rate of flow of pancreatic juice, relative to the rate of protein secretion, than was OP-CCK (Fig. 3). Of course, it is possible TURKEY 50
FIG. 3. Relationship between rate of flow of juice and rate of protein secretion from pancreas in urethane-anesthetized turkeys in response to an extract of hog duodenum containing 20% CCK (doses 0.06, 0.25, 1.O, 4.0 ID units/kg) and OP-CCK (doses 0.015, 0.06, 0.25, 1.0 pg/kg). Responses were taken as the peak rate of pancreatic secretion which occurred in a IO-min period after injections, minus the basal rate of secretion. Each point is mean of responses in six animals. Regression for OP-CCK: y = 2.46.x + 2.52, r = 0.992. Regression for CCK: y = 4.05x + 3.02, r = 0.997. Slopes significantly different (P < 0.05).
ON
205
SECRETIN
that there is a genuine difference in the biological actions of CCK and its active fragment in the turkey, and further studies with pure CCK (not yet widely available) may be expected to clarify this situation. However, an alternative explanation for these results is that the 20% CCK preparation contains a factor, in addition to CCK, which stimulates the flow of pancreatic juice in birds. As already mentioned, it is most unlikely that the CCK extract contained secretin, and porcine secretin is in any case a weak stimulant of the avian pancreas. Moreover, the steps involved in the preparation of 20% CCK were also likely to have removed contaminating traces of VIP, which could have accounted for the stronger secretin-like action of the CCK preparation. It would appear then, that partially purified CCK preparations may contain a hitherto unrecognized secretin-like factor. Avian Secretin-like
Factors
For many years it has been known that crude extracts of the intestines of birds stimulate the flow of pancreatic juice in mammals (Bayliss and Starling, 1903; Koschtojanz et al., 1933). However, these early experiments tell us little about avian secretin, since the crude extracts which were studied almost certainly contained CCK, which is known to have weak secretin-like action on the mammalian pancreas. Furthermore, CCK and secretin potentiate each other’s action on pancreatic secretion (Brown et al., 1967; Henriksen and Worning, 1967; Way and Grossman, 1970), so that it is difficult to make a precise estimate of the biological actions of an extract which contains both hormones. Recently an extract of chicken intestine was prepared which had no CCK-like actions on the pancreas or gallbladder, but which stimulated the flow of pancreatic juice in the cat and turkey at similar doses (Dockray, 1972b). Figure 4 shows the results of further
206
GRAHAM
d-i-
r
0.125 2.0
J. DOCKRAY
0.25 4.0
0.50 8.0
ID PORCINE SECRETIN 16.0 CWXEN EXTRACT
2.5 -0 0.125
DOSE
0.25
L bg/kg) 0.5 bgfg)
DOSE
FIG. 4. Action of pure natural porcine secretin and partially purified extract of chicken duodenum on pancreatic secretion in urethane-anesthetized rats and turkeys. Responses expressed as the peak rates of flow and protein secretion from the pancreas which occurred immediately after the injections. Doses were administered in random order. Points are mean responses of either eight rats or six turkeys, and vertical bars represent standard errors of the mean. x Basal rate of secretion; l porcine secretin; 0 chicken extract.
experiments in which the actions of a highly purified extract of chicken intestine were compared with those of porcine secretin in the rat and turkey. In both species the chicken extract stimulated dose-related increases in the flow of pancreatic juice but had little effect on pancreatic protein secretion, and these responses were qualitatively similar to those evoked by pure natural porcine secretion. A quantitative estimate of the relative potencies of mammalian secretin and the chicken extract in turkey and rat was obtained by comparing doses calculated to stimulate half-maximal responses (ED,,) in the two species (Grossman, 1973). Thus, the dose of chicken extract required to stimulate half maximal rate of flow of juice in the turkey was 0.07 pg/kg, compared with 1.7 rug/kg in the rat, so the chicken extract may be said to be 24 times more potent in stimulating pancreatic secretion in the bird than in the mammal. In contrast, porcine secretin was a much weaker stimulant of pancreatic secretion in the
bird than in the mammal; the EDS, in the turkey was 3.5 pg/kg which was 50 times greater than that in the rat (0.07 pg/kg). Secretin-like
Factors in Teleost Fish
Early reports of secretin-like activity in the intestine in teleosts, e.g., salmon (Bayliss and Starling, 1903), are difficult to evaluate, because the crude extracts used probably contained CCK as well (see above). Barrington and Dockray (1972) showed that the responses of the pancreas in the rat to crude extracts of the intestine of eel (Anguifla anguifla) could be accounted for by the presence of a CCK-like factor and concluded that if a secretin-like factor occurred in the eel it was either present in low concentrations, or, had little activity in the rat. Partially purified extracts of pike intestine (Esox Lucius), from which the CCK-like factor had been separated, have also been found to have weak secretin-like properties in the rat. In contrast, these extracts were relatively strong stimulants of the flow of pancreatic juice in the turkey (Dockray, 1974). In the present
COMPARATIVE
STUDIES
ON
207
SECRETIN
RAT
DOSE
DOSE
5. Comparison of the pancreatic responses in turkeys and rats to porcine secretin and extracts of the intestine of cod and pike. Doses of porcine secretin are expressed in pg/kg, and doses of cod and pike extracts given in mg/kg. Experimental conditions were the same as those for experiments shown in Fig. 4. Points are the mean responses of six rats and six turkeys. 0 Porcine secretin; 0 cod extract; A pike extract. FIG.
study these observations have been extended to include the secretin-like properties of intestinal extracts of another species of teleost fish, the cod (Gadus WZOd2UU). Figure 5 shows the results of experiments in which a comparison was made of pancreatic responses in rat and turkey to pure porcine secretin and partially purified extracts of intestine of pike and cod. Both fish preparations stimulated the rate of flow of pancreatic juice in rats and turkeys, but had relatively little effect on the rate of pancreatic protein secretion indicating that they were essentially free of CCK. However, the range of doses of the fish extracts which stimulated the flow of pancreatic juice in the turkey were about twenty times less than those required to stimulate the rat pancreas. In contrast, porcine secretin stimulated the flow of pancreatic juice in the bird at doses about lo-20 times greater than those which stimulated the rat pancreas. In the rat the maximum responses evoked by the cod extract were significantly less than those stimulated by porcine secretin (p < 0.05). Moreover, the dose-response lines for the flow of pan-
creatic juice evoked by the pike extract and porcine secretin in the rat were not parallel, so that it is not possible to make a precise estimate of the relative potency of these preparations. DISCUSSION
In the present study it has been shown that purified extracts of the intestine of birds and fish have actions on pancreatic exocrine secretion which are qualitatively similar to those of porcine secretin. However, there were marked differences in the relative potencies of mammalian secretin and the bird and fish extracts in different species (Table 1). Thus, extracts of the intestine of chicken, cod, and pike were all relatively stronger stimulants of pancreatic secretion in the turkey than in the rat, whereas porcine secretin was a weak stimulant of the flow of pancreatic juice in birds but a strong stimulant in mammals. Secretin is one member of a group of four structurally related peptides which have been isolated from the intestine or pancreas in mammals. It is, of course, possible that the mammalian gastrointestinal tract contains still more peptides related in
208
GRAHAM
J. DOCKRAY
basis of these observations it is tempting to speculate that porcine VIP has some of the features of the secretin-like factors in bird and fish intestine. Further studies on the chemical and biological properties of the active factors in lower vertebrates are Mammals Birds now needed to evaluate this possibility. The similarities in the amino acid Stimulant Rat Cat Turkey Chicken sequences of secretin, glucagon, VIP, and Porcineghtcagon NE NE GIP suggest that these molecules have Porcine GIP NE NE evolved from a common ancester (BarPorcine secretin +++ +++b + NEC rington and Dockray, 1970; Adeleson, 1971; Weinstein, 1972; Track, 1973). Porcine VIP -!-d 4-e +-l--P Extract chicken Since glucagon-like molecules occur in the ++ ++b +++ +++’ intestine pancreas of both birds and fish (HazelExtract cod wood, 1973; Noe and Bauer, 1971) and + NE’ +++ intestine these animals also possess secretin-like Extract pike factors in their intestines, it would appear intestine + +++ that glucagon and secretin have had a long a Data were taken from the present study and from and independent evolution. In this context the literature. + indicates strength of action; NE no it is of interest that cells resembling the effect; and - not tested. glucagon-secreting cells of higher verb Dockray (1972b). c Angelucci et al. (1970). tebrates appear to be absent from agd Dockray (1973). nathans, indicating that glucagon may have e Said and Mutt (1972). originated within the gnathostomes (Barf Unpublished observations. rington, 1969). In contrast, there is evidence of a secretin-like factor in the intesstructure to secretin, and in this study indirect evidence has been presented that a tine of both of the surviving groups of partially purified preparation of CCK may agnathans, the lampreys (Barrington and be contaminated with just such a secretinDockray, 1970) and the hagfish (Nilsson like factor. Two of the peptides related to and Fange, 1970). The physiological role of the active secretin, GIP and glucagon, were shown factors in the bird and fish extracts has not to have no secretin-like actions on the exyet been established. Experiments to ocrine pancreas in rat and in turkey. Preanswer this question in birds could be vious studies have established that in dog made using preparations such as the acute (Dyck et al., 1969) and in man also (Dyck turkey employed here and the chronic panet al., 1970) glucagon has no action on creatic fistula chicken (Kokue and secretion from the resting pancreas. The Hayama, 1972). Unfortunately, it is more remaining peptide, VIP, had weak secretin-like action on the pancreas in difficult to study action of hormones on mammals and was a strong stimulant of the pancreatic secretion in teleost fish, for in most teleosts the exocrine pancreas is difflow of pancreatic juice in the bird (Said fuse and empties by way of many small and Mutt, 1972; Dockray, 1973). The patducts into the intestine, so that pure pantern of activity of VIP on exocrine pancreatic secretion in birds and mammals, creatic juice cannot easily be collected. therefore, resembles that of the bird and However, it should be possible to obtain teleost intestinal extracts (Table 1). On the direct evidence of secretin mechanisms in TABLE 1 RELATIVEPOTENCIESOFMAMMALIANPEPTIDES AND EXTRACTS OF FISH AND BIRD INTESTINE ON THE RATE OF FLOW OF PANCREA~C JUICE IN MAMMALS (RAT AND CAT) AND BIRDS (TURKEY AND CHICKEN)
COMPARATIVE
STUDIES
other lower vertebrate groups such as the elasmobranchs, which do possess a pancreatic duct sufficiently large to permit cannulation (Babkin, 1929; Ledrut and Alechinsky, 1933). The possibility that a secretin mechanism exists in chondrichthean fish has already been indicated by the report of secretin-like activity in the intestine of Chimaera monstrosa (Nilsson, 1970). REFERENCES ADELESON, J. W. (197 1). Enterosecretory proteins. Nature (London) 229, 321-325. ANGELUCCI, L., BALDIERI, M., AND LINARI, G. (1970). The action of caerulein on pancreatic and biliary secretion of the chicken. Eur. J. Pharmacol.
11, 217-232.
BABKIN, B. P. (1929). Studies on the pancreatic secretion in skates. Biol. Bull. 57, 272-29 1. BARRINGTON, E. J. W. (1969). Unity and diversity in comparative endocrinology. Gen. Comp. Endocrinol.
13, 482-488.
BARRINGTON, E. J. W., AND DOCKRAY, G. J. (1970). The effect of intestinal extracts of lampreys (Lampetra JIuviatilis and Petromyzon marinus) on pancreatic secretion in the rat. Gen. Comp. Endocrinol.
14, 170- 177.
BARRINGTON, E. J. W., AND DOCKRAY, G. J. (1972). Cholecystokinin-pancreozymin-like activity in the eel (Anguilla anguilla). Gen. Comp. Endocrinol.
19, 80-87.
BAYLISS, W. M., AND STARLING, E. J. (1902). Mechanism of pancreatic secretion. J. Physiol. 28, 352-353. BAYLISS, W. J., AND STARLING, E. H. (1903). On the uniformity of the pancreatic mechanism in vertebrata. J. Physiol. 29, 174-180. BROWN, J. D., HARPER, A. A., AND SCRATCHERD,T. (1967). Potentiation of secretin stimulation of the pancreas. J. Physiol. 190, 519-530. BROWN, J. D., MUTT, V., AND PEDERSON, R. A. (1970). Further purification of a polypeptide demonstrating enterogastrone activity. J. Physiol. 2Q9, 57-66. DEBAS, H. T., AND GROSSMAN, M. I. (1973). Pure cholecystokinin: pancreatic protein and bicarbonate response. Digestion 9, 469-48 1. DOCKRAY, G. J. (1972a). The action of secretin, cholecystokinin-pancreozymin and caerulein on pancreatic secretion in the rat. J. Physiol. 225, 679-692. DOCKRAY, G. J. (1972b). Pancreatic secretion in the turkey. J. Physiol. 227, 49-50 P.
ON
209
SECRETIN
DOCKRAY, G. J. (1973). Vasoactive intestinal peptide: secretin-like action on the avian pancreas. Experientia 29, 15 lo-151 1. DOCKRAY, G. J. (1974), Extraction of a secretin-like factor from intestines of pike (Esox lucius). Gen. Comp.
Endocrinol.
23, 340-347.
DYKE, W. P., RUDICK, J., HOEXTER, B., AND JANOWITZ, H. D. (1969). Influence of glucagon on pancreatic exocrine secretion. Gastroenterology 56, 531-534. DYKE, W. P., TEXTER, E. C. JR., LASATER, J. M., AND HIGHTOWER, N. C. (1970). Influence of glucagon on pancreatic exocrine secretion in man. Gastroenterology 58, 532-539. GROSSMAN, M. I. (1973). What do you do with basal in dose-response studies? A suggested answer. Gastroenterology
65, 341-344.
GROSSMAN, M. 1. (1974). Gastrointestinal hormones. In “Symposium on Peptide Hormones,” MacMillan, in press. HAZELWOOD, R. L. (1973). The avian endocrine pancreas. Amer. 2001. 13, 699-709. HENRIKSEN, F. W., AND WORNING, H. (1967). The interaction of secretin and pancreozymin on the external pancreatic secretion in dogs. Acta Physiol. Stand.
70, 241-249.
KOKUE, E., AND HAYAMA, T. (1972). Chronic pancreatic fistula in chicken. Jap. J. Zootech. Sci. 43, 340-341. KOSCHTOJANZ, I. I., MIRIEFF, M., KORINIEFF, P., AND OTARSCHAKOWSKAIA, S. (1933). Zurfrage des Spezifitat des Sekretins: vergleichende physiologische Untersuchung. Z. Vergl. Physiol. 18, 112-117. LEDRUT, J., AND ALECHINSKY, G. (1933). Absence de specificit& des &cretin&s de raie et de port. Arch.
Int Physiol.
37, 329-335.
MUTT, V., AND JORPES, E. (1967). developments in the biochemistry testinal hormones. Recent Progr. 683-695. NILSSON, A. (1970). Gastrointestinal holocephalian fish Chimaera Camp.
Biochem.
Physiol.
Contemporary of the gastroinHorm.
Res. 23,
hormones in the (L).
monstrosa 32, 387-390.
NILSSON, A., AND FANGE, R. (1970). Digestion proteases in the cyclostome Myxine glutinosa (L). Comp.
Biochem.
Physiol.
32, 237-250.
NOE, B. T., AND BAUER, G. E. (197 1). Evidence for glucagon biosynthesis involving a protein intermediate in islets of the Angle&h (Lophius americanus). Endocrinology 59, 642-65 1. SAID, S. I., AND MUTT, V. (1972). Isolation from pertine intestinal wall of a vasoactive octacosapeptide related to secretin and glucagon. Eur. J. Biochem.
28, 199-204.
STARLING, E. H. (1905). The chemical correlation of
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the functions of the body. The chemical control of the functions of the body. Lancer 2, 338-341. TRACK, N. W. (1973). Evolutionary aspects of the gastrointestinal hormones. Comp. Biochem. PhySol. 45B, 291-301. WAY, L., AND GROSSMAN, M. 1. (1970). Pancreatic
.I. DOCKRAY stimulation by duodenal and exogenous mones in conscious cats. Amer. J. Physiol. 449-454. WEINSTEIN, B. (1972). A generalized homology relation for various hormones and proteins. perientia 28, 1517-1522.
hor219, corEx-
AND
COMPARATIVE
25, 203-210 (1975)
ENDOCRINOLOGY
Comparative
Studies
on Secretinl
GRAHAM J. DOCKRAY Physiological
Laboratory, Liverpool,
University
of Liverpool,
England
Received July 20, 1974 The mammalian intestinal hormone secretin is related in structure to the pancreatic hormone glucagon, and to two peptides recently isolated from hog duodenum, vasoactive intestinal peptide (VIP) and gastric inhibitory peptide (GIP). Because of their structural similarities it has been suggested that these peptides evolved from a common ancester, and comparative studies may be expected to shed light on this question. In the present series of experiments pure porcine secretin was found to be a weak stimulant of the flow of pancreatic juice in a bird (the turkey) although it was a strong stimulant in mammals such as cat and rat. In contrast, pure porcine VIP had weak actions on the flow of pancreatic juice in mammals but was a potent stimulant in birds, while porcine glucagon and GIP showed no stimulatory effects on exocrine pancreatic secretion in either birds or mammals. Like VIP, extracts of the intestine of birds (chicken) and teleost fish (pike and cod) had weak secretinlike actions on the pancreas in mammals but strong actions in birds. The results indicate that in the intestines of birds and fish there are factors with biological properties similar, but not identical, to those of mammalian secretin and VIP, and it is suggested that the secretin group of hormones has a long evolutionary history.
In mammals the presence of acid in the small intestine stimulates the release of the hormone secretin, which in turn evokes a flow of pancreatic juice rich in bicarbonate and poor in enzymes. The secretin mechanism of mammals was discovered by Bayliss and Starling in 1902; indeed, it was with secretin in mind that Starling first introduced the word hormone (Starling, 1905). Soon after their discovery of secretin, Bayliss and Starling (1903) described secretin-like activity in intestinal extracts of many vertebrate species including salmon, dogfish, ray, frog, goose, tortoise, and chicken. However, in spite of this promising start, comparative studies on secretin have been slow to progress. An unexpected insight into the comparative endocrinology of secretin came when the amino acid sequence of porcine secretin was elucidated and it was found that i Submitted to the 7th International Symposium on Comparative Endocrinology, Kenya, 1974.
no fewer than 14 of the 27 amino acid residues in this molecule were identical to those in the pancreatic hormone, glucagon (Mutt and Jorpes, 1967). Recently, two more peptides have been isolated from hog intestine and shown to have amino acid sequences similar to those of secretin and glucagon (Fig. 1). The new molecules are gastric inhibitory peptide (GIP) purified by Brown, Mutt, and Pederson (1970), and vasoactive intestinal peptide (VIP) isolated by Said and Mutt (1972); although the names of the new peptides reflect biological actions their physiological roles and hormonal status have not yet been fully proved. On the basis of their structural similarities, it has been suggested that these four molecules arose from a common evolutionary ancestory by a process of gene duplication and divergence (Adeleson, 197 1; Weinstein, 1972). Comparative studies may be expected to give further insight into the relationships between these molecules. In the present series of 203
Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
204
GRAHAM
SECRETIN
1 HIS
2 3 4 5 6 7 8 9 10 l! 12 SER ASP GLY THR PtiE THR SER GLU LEU SEC AK
GLUCAGON GIP
GLN TYR ALA ELU
VIP
ILE AtA
SECRETIN
VAL
13 14 LEU ARG
ASP TYR
LYS TYR LEU
ASP TYR
ILE ALP riET
ASP ASN TYR THR rl
15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 ASP SER At.4 AR6 LEU GLN AAG LEU LEU GLN GLV LEU VAL NH2
GLUCAGON GIP
L"s
G':;
;I;
ALA f
L,~~,LE~;~
fi
VIP
FIG. 1. Amino acid sequence of mammalian peptides belonging to the secretin-glucagon family. Identities with secretin are indicated by a blank space; other identities are indicated by lines around them. Number of identities: VIP-GIP 4, VIP-glucagon 5, VIP-secretin 9, secretin-GIP 9, secretin-glucagon 14, glucagon-GIP 15. GIP has 43 residues, only 29 of which are shown. All peptides of porcine’origin. Taken from Grossman (1974).
experiments particular attention has been given to the secretin-like actions of the mammalian peptides and extracts of bird and fish intestine on pancreatic exocrine secretion in birds and mammals. MATERIALS
AND METHODS
Pure porcine secretin and VIP, a purified extract of chicken duodenum and an extract of hog duodenum containing 20% cholecystokinin (CCK) were gener-
J. DOCKRAY ous gifts of Dr. V. Mutt. An extract of hog duodenum (prepared by Dr. J. Brown) containing about 40% GIP and trace quantities of CCK was kindly made available by Dr. L. Gerring. Synthetic C-terminal octapeptide of CCK was a gift from Squibb Institute. Extracts of the intestine of pike (&ox Irrcius) and cod (Gadus morhua) were prepared by methods already described (Dockray, 1974). The action of peptides and extracts on pancreatic exocrine secretion was examined in urethaneanesthetized rats and turkeys (Dockray, 1972a,b).
RESULTS Secretin-like Action of Mammalian Peptides in Birds and Mammals
Of the mammalian peptides belonging to the secretin-glucagon family, only secretin and VIP stimulated the flow of pancreatic juice in birds and mammals (Fig. 2). Cholecystokinin also stimulated the flow of juice, but this hormone, unlike secretin and VIP, was a strong stimulant of pancreatic enzyme secretion as well. It seems reasonable to ask whether the four mammalian peptides are the only members of the secretin-glucagon family
FLOW PROTEIN -RAT
TURKEY
FIG. 2. The action of mammalian peptides on the rate of flow of juice and rate of protein secretion from pancreas in rats and turkeys. Protein concentration in the pancreatic juice was used as an index of enzyme secretion, and was estimated from OD at 280 nm; it is expressed in terms of a standard of bovine serum albumin. Peptides were given by rapid iv shot, and responses are peak rates of secretion occurring immediately after injection. Vertical bars represent one SE of mean. Responses are mean of six rats and six turkeys.
COMPARATIVE
STUDIES
or whether there are other members waiting to be discovered. One piece of indirect evidence that suggests there may be another secretin-like molecule in the mammalian intestine comes from studies with CCK-containing extracts of hog duodenum. Cholecystokinin is the hormone which strongly &imulates gallbladder contractions and pancreatic enzyme secretion, and weakly stimulates the bicarbonate concentration of pancreatic juice. The Cterminal octapeptide fragment of CCK (OP-CCK) possesses all the known biological actions of the whole molecule. In dogs OP-CCK and an extract of hog duodenum containing 20% CCK gave similar stimulation of the flow of pancreatic juice relative to pancreatic enzyme secretion, indicating that the CCK extract was essentially free of contamination by secretin (Debas and Grossman, 1973). However, in the turkey 20% CCK was a stronger stimulant of the rate of flow of pancreatic juice, relative to the rate of protein secretion, than was OP-CCK (Fig. 3). Of course, it is possible TURKEY 50
FIG. 3. Relationship between rate of flow of juice and rate of protein secretion from pancreas in urethane-anesthetized turkeys in response to an extract of hog duodenum containing 20% CCK (doses 0.06, 0.25, 1.O, 4.0 ID units/kg) and OP-CCK (doses 0.015, 0.06, 0.25, 1.0 pg/kg). Responses were taken as the peak rate of pancreatic secretion which occurred in a IO-min period after injections, minus the basal rate of secretion. Each point is mean of responses in six animals. Regression for OP-CCK: y = 2.46.x + 2.52, r = 0.992. Regression for CCK: y = 4.05x + 3.02, r = 0.997. Slopes significantly different (P < 0.05).
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that there is a genuine difference in the biological actions of CCK and its active fragment in the turkey, and further studies with pure CCK (not yet widely available) may be expected to clarify this situation. However, an alternative explanation for these results is that the 20% CCK preparation contains a factor, in addition to CCK, which stimulates the flow of pancreatic juice in birds. As already mentioned, it is most unlikely that the CCK extract contained secretin, and porcine secretin is in any case a weak stimulant of the avian pancreas. Moreover, the steps involved in the preparation of 20% CCK were also likely to have removed contaminating traces of VIP, which could have accounted for the stronger secretin-like action of the CCK preparation. It would appear then, that partially purified CCK preparations may contain a hitherto unrecognized secretin-like factor. Avian Secretin-like
Factors
For many years it has been known that crude extracts of the intestines of birds stimulate the flow of pancreatic juice in mammals (Bayliss and Starling, 1903; Koschtojanz et al., 1933). However, these early experiments tell us little about avian secretin, since the crude extracts which were studied almost certainly contained CCK, which is known to have weak secretin-like action on the mammalian pancreas. Furthermore, CCK and secretin potentiate each other’s action on pancreatic secretion (Brown et al., 1967; Henriksen and Worning, 1967; Way and Grossman, 1970), so that it is difficult to make a precise estimate of the biological actions of an extract which contains both hormones. Recently an extract of chicken intestine was prepared which had no CCK-like actions on the pancreas or gallbladder, but which stimulated the flow of pancreatic juice in the cat and turkey at similar doses (Dockray, 1972b). Figure 4 shows the results of further
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d-i-
r
0.125 2.0
J. DOCKRAY
0.25 4.0
0.50 8.0
ID PORCINE SECRETIN 16.0 CWXEN EXTRACT
2.5 -0 0.125
DOSE
0.25
L bg/kg) 0.5 bgfg)
DOSE
FIG. 4. Action of pure natural porcine secretin and partially purified extract of chicken duodenum on pancreatic secretion in urethane-anesthetized rats and turkeys. Responses expressed as the peak rates of flow and protein secretion from the pancreas which occurred immediately after the injections. Doses were administered in random order. Points are mean responses of either eight rats or six turkeys, and vertical bars represent standard errors of the mean. x Basal rate of secretion; l porcine secretin; 0 chicken extract.
experiments in which the actions of a highly purified extract of chicken intestine were compared with those of porcine secretin in the rat and turkey. In both species the chicken extract stimulated dose-related increases in the flow of pancreatic juice but had little effect on pancreatic protein secretion, and these responses were qualitatively similar to those evoked by pure natural porcine secretion. A quantitative estimate of the relative potencies of mammalian secretin and the chicken extract in turkey and rat was obtained by comparing doses calculated to stimulate half-maximal responses (ED,,) in the two species (Grossman, 1973). Thus, the dose of chicken extract required to stimulate half maximal rate of flow of juice in the turkey was 0.07 pg/kg, compared with 1.7 rug/kg in the rat, so the chicken extract may be said to be 24 times more potent in stimulating pancreatic secretion in the bird than in the mammal. In contrast, porcine secretin was a much weaker stimulant of pancreatic secretion in the
bird than in the mammal; the EDS, in the turkey was 3.5 pg/kg which was 50 times greater than that in the rat (0.07 pg/kg). Secretin-like
Factors in Teleost Fish
Early reports of secretin-like activity in the intestine in teleosts, e.g., salmon (Bayliss and Starling, 1903), are difficult to evaluate, because the crude extracts used probably contained CCK as well (see above). Barrington and Dockray (1972) showed that the responses of the pancreas in the rat to crude extracts of the intestine of eel (Anguifla anguifla) could be accounted for by the presence of a CCK-like factor and concluded that if a secretin-like factor occurred in the eel it was either present in low concentrations, or, had little activity in the rat. Partially purified extracts of pike intestine (Esox Lucius), from which the CCK-like factor had been separated, have also been found to have weak secretin-like properties in the rat. In contrast, these extracts were relatively strong stimulants of the flow of pancreatic juice in the turkey (Dockray, 1974). In the present
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5. Comparison of the pancreatic responses in turkeys and rats to porcine secretin and extracts of the intestine of cod and pike. Doses of porcine secretin are expressed in pg/kg, and doses of cod and pike extracts given in mg/kg. Experimental conditions were the same as those for experiments shown in Fig. 4. Points are the mean responses of six rats and six turkeys. 0 Porcine secretin; 0 cod extract; A pike extract. FIG.
study these observations have been extended to include the secretin-like properties of intestinal extracts of another species of teleost fish, the cod (Gadus WZOd2UU). Figure 5 shows the results of experiments in which a comparison was made of pancreatic responses in rat and turkey to pure porcine secretin and partially purified extracts of intestine of pike and cod. Both fish preparations stimulated the rate of flow of pancreatic juice in rats and turkeys, but had relatively little effect on the rate of pancreatic protein secretion indicating that they were essentially free of CCK. However, the range of doses of the fish extracts which stimulated the flow of pancreatic juice in the turkey were about twenty times less than those required to stimulate the rat pancreas. In contrast, porcine secretin stimulated the flow of pancreatic juice in the bird at doses about lo-20 times greater than those which stimulated the rat pancreas. In the rat the maximum responses evoked by the cod extract were significantly less than those stimulated by porcine secretin (p < 0.05). Moreover, the dose-response lines for the flow of pan-
creatic juice evoked by the pike extract and porcine secretin in the rat were not parallel, so that it is not possible to make a precise estimate of the relative potency of these preparations. DISCUSSION
In the present study it has been shown that purified extracts of the intestine of birds and fish have actions on pancreatic exocrine secretion which are qualitatively similar to those of porcine secretin. However, there were marked differences in the relative potencies of mammalian secretin and the bird and fish extracts in different species (Table 1). Thus, extracts of the intestine of chicken, cod, and pike were all relatively stronger stimulants of pancreatic secretion in the turkey than in the rat, whereas porcine secretin was a weak stimulant of the flow of pancreatic juice in birds but a strong stimulant in mammals. Secretin is one member of a group of four structurally related peptides which have been isolated from the intestine or pancreas in mammals. It is, of course, possible that the mammalian gastrointestinal tract contains still more peptides related in
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basis of these observations it is tempting to speculate that porcine VIP has some of the features of the secretin-like factors in bird and fish intestine. Further studies on the chemical and biological properties of the active factors in lower vertebrates are Mammals Birds now needed to evaluate this possibility. The similarities in the amino acid Stimulant Rat Cat Turkey Chicken sequences of secretin, glucagon, VIP, and Porcineghtcagon NE NE GIP suggest that these molecules have Porcine GIP NE NE evolved from a common ancester (BarPorcine secretin +++ +++b + NEC rington and Dockray, 1970; Adeleson, 1971; Weinstein, 1972; Track, 1973). Porcine VIP -!-d 4-e +-l--P Extract chicken Since glucagon-like molecules occur in the ++ ++b +++ +++’ intestine pancreas of both birds and fish (HazelExtract cod wood, 1973; Noe and Bauer, 1971) and + NE’ +++ intestine these animals also possess secretin-like Extract pike factors in their intestines, it would appear intestine + +++ that glucagon and secretin have had a long a Data were taken from the present study and from and independent evolution. In this context the literature. + indicates strength of action; NE no it is of interest that cells resembling the effect; and - not tested. glucagon-secreting cells of higher verb Dockray (1972b). c Angelucci et al. (1970). tebrates appear to be absent from agd Dockray (1973). nathans, indicating that glucagon may have e Said and Mutt (1972). originated within the gnathostomes (Barf Unpublished observations. rington, 1969). In contrast, there is evidence of a secretin-like factor in the intesstructure to secretin, and in this study indirect evidence has been presented that a tine of both of the surviving groups of partially purified preparation of CCK may agnathans, the lampreys (Barrington and be contaminated with just such a secretinDockray, 1970) and the hagfish (Nilsson like factor. Two of the peptides related to and Fange, 1970). The physiological role of the active secretin, GIP and glucagon, were shown factors in the bird and fish extracts has not to have no secretin-like actions on the exyet been established. Experiments to ocrine pancreas in rat and in turkey. Preanswer this question in birds could be vious studies have established that in dog made using preparations such as the acute (Dyck et al., 1969) and in man also (Dyck turkey employed here and the chronic panet al., 1970) glucagon has no action on creatic fistula chicken (Kokue and secretion from the resting pancreas. The Hayama, 1972). Unfortunately, it is more remaining peptide, VIP, had weak secretin-like action on the pancreas in difficult to study action of hormones on mammals and was a strong stimulant of the pancreatic secretion in teleost fish, for in most teleosts the exocrine pancreas is difflow of pancreatic juice in the bird (Said fuse and empties by way of many small and Mutt, 1972; Dockray, 1973). The patducts into the intestine, so that pure pantern of activity of VIP on exocrine pancreatic secretion in birds and mammals, creatic juice cannot easily be collected. therefore, resembles that of the bird and However, it should be possible to obtain teleost intestinal extracts (Table 1). On the direct evidence of secretin mechanisms in TABLE 1 RELATIVEPOTENCIESOFMAMMALIANPEPTIDES AND EXTRACTS OF FISH AND BIRD INTESTINE ON THE RATE OF FLOW OF PANCREA~C JUICE IN MAMMALS (RAT AND CAT) AND BIRDS (TURKEY AND CHICKEN)
COMPARATIVE
STUDIES
other lower vertebrate groups such as the elasmobranchs, which do possess a pancreatic duct sufficiently large to permit cannulation (Babkin, 1929; Ledrut and Alechinsky, 1933). The possibility that a secretin mechanism exists in chondrichthean fish has already been indicated by the report of secretin-like activity in the intestine of Chimaera monstrosa (Nilsson, 1970). REFERENCES ADELESON, J. W. (197 1). Enterosecretory proteins. Nature (London) 229, 321-325. ANGELUCCI, L., BALDIERI, M., AND LINARI, G. (1970). The action of caerulein on pancreatic and biliary secretion of the chicken. Eur. J. Pharmacol.
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BABKIN, B. P. (1929). Studies on the pancreatic secretion in skates. Biol. Bull. 57, 272-29 1. BARRINGTON, E. J. W. (1969). Unity and diversity in comparative endocrinology. Gen. Comp. Endocrinol.
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BARRINGTON, E. J. W., AND DOCKRAY, G. J. (1970). The effect of intestinal extracts of lampreys (Lampetra JIuviatilis and Petromyzon marinus) on pancreatic secretion in the rat. Gen. Comp. Endocrinol.
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BARRINGTON, E. J. W., AND DOCKRAY, G. J. (1972). Cholecystokinin-pancreozymin-like activity in the eel (Anguilla anguilla). Gen. Comp. Endocrinol.
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BAYLISS, W. M., AND STARLING, E. J. (1902). Mechanism of pancreatic secretion. J. Physiol. 28, 352-353. BAYLISS, W. J., AND STARLING, E. H. (1903). On the uniformity of the pancreatic mechanism in vertebrata. J. Physiol. 29, 174-180. BROWN, J. D., HARPER, A. A., AND SCRATCHERD,T. (1967). Potentiation of secretin stimulation of the pancreas. J. Physiol. 190, 519-530. BROWN, J. D., MUTT, V., AND PEDERSON, R. A. (1970). Further purification of a polypeptide demonstrating enterogastrone activity. J. Physiol. 2Q9, 57-66. DEBAS, H. T., AND GROSSMAN, M. I. (1973). Pure cholecystokinin: pancreatic protein and bicarbonate response. Digestion 9, 469-48 1. DOCKRAY, G. J. (1972a). The action of secretin, cholecystokinin-pancreozymin and caerulein on pancreatic secretion in the rat. J. Physiol. 225, 679-692. DOCKRAY, G. J. (1972b). Pancreatic secretion in the turkey. J. Physiol. 227, 49-50 P.
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DOCKRAY, G. J. (1973). Vasoactive intestinal peptide: secretin-like action on the avian pancreas. Experientia 29, 15 lo-151 1. DOCKRAY, G. J. (1974), Extraction of a secretin-like factor from intestines of pike (Esox lucius). Gen. Comp.
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DYKE, W. P., RUDICK, J., HOEXTER, B., AND JANOWITZ, H. D. (1969). Influence of glucagon on pancreatic exocrine secretion. Gastroenterology 56, 531-534. DYKE, W. P., TEXTER, E. C. JR., LASATER, J. M., AND HIGHTOWER, N. C. (1970). Influence of glucagon on pancreatic exocrine secretion in man. Gastroenterology 58, 532-539. GROSSMAN, M. I. (1973). What do you do with basal in dose-response studies? A suggested answer. Gastroenterology
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GROSSMAN, M. 1. (1974). Gastrointestinal hormones. In “Symposium on Peptide Hormones,” MacMillan, in press. HAZELWOOD, R. L. (1973). The avian endocrine pancreas. Amer. 2001. 13, 699-709. HENRIKSEN, F. W., AND WORNING, H. (1967). The interaction of secretin and pancreozymin on the external pancreatic secretion in dogs. Acta Physiol. Stand.
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KOKUE, E., AND HAYAMA, T. (1972). Chronic pancreatic fistula in chicken. Jap. J. Zootech. Sci. 43, 340-341. KOSCHTOJANZ, I. I., MIRIEFF, M., KORINIEFF, P., AND OTARSCHAKOWSKAIA, S. (1933). Zurfrage des Spezifitat des Sekretins: vergleichende physiologische Untersuchung. Z. Vergl. Physiol. 18, 112-117. LEDRUT, J., AND ALECHINSKY, G. (1933). Absence de specificit& des &cretin&s de raie et de port. Arch.
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MUTT, V., AND JORPES, E. (1967). developments in the biochemistry testinal hormones. Recent Progr. 683-695. NILSSON, A. (1970). Gastrointestinal holocephalian fish Chimaera Camp.
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NILSSON, A., AND FANGE, R. (1970). Digestion proteases in the cyclostome Myxine glutinosa (L). Comp.
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NOE, B. T., AND BAUER, G. E. (197 1). Evidence for glucagon biosynthesis involving a protein intermediate in islets of the Angle&h (Lophius americanus). Endocrinology 59, 642-65 1. SAID, S. I., AND MUTT, V. (1972). Isolation from pertine intestinal wall of a vasoactive octacosapeptide related to secretin and glucagon. Eur. J. Biochem.
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STARLING, E. H. (1905). The chemical correlation of
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the functions of the body. The chemical control of the functions of the body. Lancer 2, 338-341. TRACK, N. W. (1973). Evolutionary aspects of the gastrointestinal hormones. Comp. Biochem. PhySol. 45B, 291-301. WAY, L., AND GROSSMAN, M. 1. (1970). Pancreatic
.I. DOCKRAY stimulation by duodenal and exogenous mones in conscious cats. Amer. J. Physiol. 449-454. WEINSTEIN, B. (1972). A generalized homology relation for various hormones and proteins. perientia 28, 1517-1522.
hor219, corEx-
