GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

81, 51-63 (1991)

lmmunoreactivity to Peptides Belonging to the Pancreatic Polypeptide Family (NPY, aPY, PP, PYY) and to Glucagon-like Peptide in the Endocrine Pancreas and Anterior Intestine of Adult Lampreys, Petromyzon marinus: An lmmunohistochemical Study R. CHEUNG,*

P. C. ANDREW%?

E. M. PLISETSKAYA,$

AND J. H. YOUSON*~’

*Department of Zoology and Scarborough Campus, Universiry of Toronto, West Hill, Ontario, Canada MlC iA4; tDepartment of Biochemistry, Purdue University, West Lafayette, Indiana 47907; and #School of Fisheries, WH-IO, University of Washington, Seattle, Washington 98195 Accepted December 11, 1989 Immunoreactivity of antisera directed against human neuropeptide Y (NPY), anglerfish polypeptide YG (aPY), bovine pancreatic polypeptide (bPP), salmon pancreatic polypeptide (sPP), porcine peptide tyrosine tyrosine (PYY), and salmon glucagon-like peptide (GLP) was investigated in the endocrine pancreas and anterior intestine of adult lampreys, Petromyzon marks, by immunohistochemical analysis. There was no immunoreactivity to antisPP and anti-bPP in any tissue and anti-GLP immunostaining was only present in the anterior intestine. The immunoreactivity to antisera raised against NPY, aPY, and PYY was colocalized within the same small number of cells in the caudal and cranial pancreas of juveniles and the caudal pancreas of upstream migrant adults. These antibodies did not immunostain B- or D-cells and thus, NPY, aPY, and PYY were likely localized in a third cell type (3a) in the lamprey pancreas. Immunostaining of a few cells with only anti-aPY suggested the possibility of a fourth cell type (3b). Immunoreactivity was similar in the cranial and caudal pancreas of male upstream migrants; however, in the female cranial pancreas, a few cells demonstrated intense immunoreaction to anti-aPY, while weaker immunostaining with this antiserum was observed in B-cells. In the intestine of juvenile and upstream migrant lampreys, positive immunostaining to GLP, NPY, aPY, and PYY antibodies was colocalized within the same cell. We believe that this cell may contain PYY/glucagon family peptides. Other intestinal cells immunostained with either GLP or somatostatin-34 antiserum. 0 1991 Academic Press, Inc.

The pancreas of lampreys is of considerable interest due to the fact that the endocrine portion is distinctly separate from the exocrine part which is represented by zymogen cells scattered in the intestinal epithelium (Barrington, 1972; Youson, 1981). When larval lampreys (ammocoetes) undergo metamorphosis to adults, the pancreas consequently becomes modified. Previous studies on the endocrine pancreas of lampreys by immunohistochemistry (Van

Noorden et al., 1977; Elliott and Youson, 1986; Youson et al., 1988), histochemistry (Barrington, 1972; Epple and Brinn, 1975; Hilliard et al., 1985), and electron microscopy (Titlbach and Kern, 1969; Elliott and Youson, 1988) have shown that in larvae, only insulin-secreting B-cells are present but that the adult endocrine pancreas is composed of somatostatin-containing Dcells, B-cells, and an unidentified third cell type (Hilliard et al., 1985; Elliott and Youson, 1988; Youson et al., 1988). Epple and Brinn (1975) showed through hght- microscopy that four different acidophilic cell types (Pi-i,) were dispersed among the Bcells within the pancreas of adult lampreys.

’ To whom reprint requests should be addressed at Division of Life Sciences, Scarborough Campus, University of Toronto, West Hill, Ontario, Canada MlC lA4. 51

00166480191 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

52

CHEUNG ET AL.

Elliott and Youson (1988) used immunocytochemistry and the line structure of cytoplasmic granules in their analysis and suggested that the Pi-cells designated by Brinn and Epple (1976) correspond to somatostatin-containing D-cells and the Pi,-cell may be the third cell type in the adult lamprey pancreas. Hilliard et al. (1985) have described, in addition to B- and D-cells, an argyrophilic cell type in the pancreas of the adult lamprey, Geotria australis. Although Falkmer and Van Noorden (1983) observed immunoreactivity to mammalian glucagon and pancreatic polypeptide (PP) antibodies within the intestinal epithelium of the adult river lamprey, Lampetra fluviatilis, and Yui et al. (1988) reported similar results in the adult Arctic lamprey, Lampetra japonica, there is no evidence of such immunoreactivity in the intestine of the adult lamprey, Petromyzon marinus L. Neuropeptide Y (NPY), pancreatic polypeptide (PP), and peptide tyrosine tyrosine (PYY) belong to the pancreatic polypeptide family. NPY has recently been isolated from porcine brain (Tatemoto, 1982), PP from avian pancreas (Kimmel et al., 1971), and PYY from duodenal extracts (Tatemoto et al., 1982). All of these 36-amino acid polypeptides possess a high degree of sequence homology (Tatemoto, 1982) and it has been shown that there is some crossreactivity between them (Laburthe et al., 1986; Inui et al., 1988). Lundberge et al. (1983) reported the presence of PP and PYY within the endocrine cells of mammalian alimentary canal and NPY in neural tissues. PYY-immunoreactive cells were identified in the mammalian colon and terminal ileum using immunohistochemical and radioimmunological techniques (ElSalhy and Grimelius, 1982; Taylor, 1985). Bottcher et al. (1984, 1986) reported the colocalization of PYY and a gut-type glucagon within distal intestinal endocrine cells of mammals. Andrews et al. (1985) recently identified a polypeptide YG (aPY) in the anglerfish endocrine pancreas (Brockmann

bodies) which possesses a 64% sequence identity with porcine NPY and PYY. Other polypeptides belonging to the same family and having equal or greater homology with NPY were isolated from salmon (Kimmel et al., 1986) sculpin (Conlon et al., 1986), and garfish (Pollock et al., 1987). Multiple forms of pancreatic polypeptide-related compounds were recently localized in the lamprey central nervous system (Brodin et al., 1989).

This present study was initially designed to identify the third cell type within the endocrine pancreas of adult lampreys, P. marinus L., but was broadened to include the distribution of pancreatic and intestinal cells immunoreactive to antisera raised against the pancreatic polypeptide family and glucagon-like peptide (GLP). Immunohistochemical techniques utilizing antisera directed against anglerfish polypeptide (aPY), human NPY, salmon GLP, bovine PP (bPP), salmon PP (sPP>, porcine PYY, lamprey somatostain-34 (SST-34), and lamprey insulin, at the light microscopic level, were employed to aid in identification. MATERIALS

AND METHODS

Animals Anadromous juvenile lampreys, P. marinus L. were removed from trap-netted gaspereau (Alosa pseudoherengus L.) and shad (Alosa sapidissima) in Lake Washademoak, New Brunswick. The lampreys had just commenced feeding and were in their downstream migration in early May, 1987 and 1988. The juveniles were transported to the laboratory at Scarborough Campus, University of Toronto, where they were placed in large “living stream” fibreglass tanks containing dechlorinated, recirculating, aerated tap water adjusted to 8-10°C. They were provided with rainbow trout (Salmo gairdneri Richardson) on which they fed. Prespawning mature adults (upstream migrants) were captured by dip-netting in the Humber River, Toronto, Ontario, during their nontrophic, upstream migration period from late April to early May, 1987 and 1988. They were transported back to the laboratory and maintained in aquaria with dechlorinated, aerated, continuously flowing tap water at 10-15” until their sacrifice in late June to early August.

LAMPREY

INTESTINE

Tissue Preparation All animals were anesthetized in a 0.05% solution of tricaine methanesulfonate (MS-222). prior to sacrifice. The cranial and caudal pancreas and intestine with the surrounding connective tissue were excised from the bodies of 10 juveniles (132-153 mm, 2.2-4.5 g) and 10 upstream migrants (410-510 mm, 160-380 g) and fixed in Bouin’s fluid for 24 to 48 hr. Following fixation, the tissues were stored in 70% ethanol for up to 1 year and then dehydrated in a graded series of ethanols, cleared in Histoclear, and embedded in Tissue-prep or Paraplast paraffin. Adjacent sections 6 pm thick were placed on separate glass slides and immunostained using the unlabeled peroxidase-antiperoxidase (PAP) antibody technique (Stemberger et a/., 1970).

Immunohistochemistvy Deparaffinized tissue sections were hydrated and incubated with phosphate-buffered saline (PBS), pH 7.4, containing 1% heat inactivated goat serum (HIGS) for 20 min at room temperature. Adjacent sections were then incubated for 48 hr at 4” in one of the following primary antisera: (1) rabbit anti-lamprey somatostatin34 antiserum (anti-SST-34) diluted 1:750, (2) rabbit anti-lamprey insulin antiserum diluted 1: 1000, (3) rabbit anti-synthetic glycine-extended anglerfish polypeptide Y antiserum (anti-aPY) diluted l:lOOO, (4) rabbit anti-human neuropeptide Y antiserum (anti-NPY) diluted 1: 1000, (5) rabbit anti-bovine pancreatic polypeptide antiserum (anti-bPP) diluted l:lOOO, (6) rabbit anti-salmon pancreatic polypeptide antiserum (antisPP) diluted 1:750, (7) rabbit anti-synthetic porcine peptide YY (anti-PYY) diluted l:lOOO, (8) rabbit antisalmon glucagon-like peptide antiserum (anti-GLP) diluted 1: 1000. To reduce background endogenous protein staining, equal volumes of PBS and lamprey liver extract were used as the diluent. Following incubation in the primary antisera, the slides were allowed to warm to room temperature for 1 hr. The slides were then incubated in PBS and HIGS for 20 min, goat anti-rabbit antiserum (Sigma Chemicals, St.L) diluted at l/25 for 30 mitt, PBS and HIGS for 20 min, and then rabbit PAP (ICN Immunobiologicals, IL) diluted l/25 for 30 min in the order stated. Tissue sections were then washed with Tris buffer, at pH 7.6, followed by an incubation period of 15 min with 0.003% hydrogen peroxide and 0.0125% 3,3’-diaminobenzidine tetrahydrochloride. Mouse brain, rat pancreas, anglerfish Brockmann bodies, trout pancreas, and juvenile caudal pancreas, were used at various times to determine positive immunoreaction with the antisera. Staining was abolished when controls for antibody specificity were per-

53

AND PANCREAS

formed by replacement of the primary antisera with either (1) PBS and HIGS or (2) preimmune rabbit serum.

RESULTS The results of immunohistochemistry in the lamprey pancreas and intestine are summarized in Table 1. The reader is referred to Youson (1981) for detailed morphology of the pancreas and intestine of adult lampreys and to Elliott and Youson (1986) for immunohistochemistry of B and D cells in the adult pancreas. The NPY antisera showed staining of the mouse telencephalon, and as in all positive immunostaining, the brown deposits were located in the cytoplasm of cells. In juvenile adults, NPY-immunoreactive cells were represented by a small population of cells within the cranial and caudal pancreas. These cells were circular or pyramidal shaped and randomly distributed within the islets. The aPY antisera strongly immunostained anglerfish Brockmann bodies and cells within the juvenile cranial and caudal pancreas which were similar in morphology to NPY-immunoreactive cells. Adjacent sections showed that cells immunoreactive to anti-NPY also immunostained with antiaPY and were termed type 3a cells (Table 1, Fig. la). PYY antibodies also immunostained type 3a cells as well as cells in rat pancreatic islets. However, there were some cells, called type 3b cells, which immunostained with anti-aPY (Fig. lb) but which showed no immunoreaction to antiNPY and anti-PYY. Adjacent sections stained with either anti-lamprey insulin or anti-SST-34 indicated that cells immunoreactive to either anti-NPY or anti-aPY were not B-cells (Fig. lc) or D-cells (Fig. Id). Although antisera directed against bPP and GLP showed positive reactivity in rat pancreas and trout pancreas, respectively, no immunostaining was observed in the juvenile pancreas with these antisera or with SPP. In the intestinal epithelium of juveniles,

54

CHEUNG

numerous columnar or flask-shaped cells demonstrated immunoreactivity to antiNPY. Cells were found either singly or in groups of two or three and appeared to extend from the basement membrane to the apical surface of the epithelium. Observations of adjacent sections demonstrated that cells which immunostained with antiNPY (Fig. 2a) also colocalized aPY (Fig. 2b), anti-PYY, and anti-GLP. Adjacent sections also showed that immunoreactants to anti-SST-34 were localized within distinctly different intestinal endocrine cells. In addition, there were some cells that were immunoreactive to anti-GLP but not to anti-aPY, anti-PYY, or anti-NPY (Table 1). There was no immunoreaction with anti-bPP or anti-sPP. In the caudal pancreas of upstream migrants, there was immunoreactivity to antiSST-34. There were also a few cells that were immunoreactive to both anti-NPY and TABLE DISTRIBUTION

Period Juvenile

AND RELATIVE

Cell

SST-34

GLP

Cranial and caudal pancreas

B D 3a 3b 1 2 3 B D 3a 3b B D 3a 3b B D 3a 3b 1 2 3

+++

-

++

++ ++ -

++ -

-

Cranial pancreas (female) Cranial pancreas (male) Caudal pancreas (male and female) Intestine

anti-aPY (3a cells), and they had shape, size, and distribution similar to those of cells previously described in the juvenile caudal pancreas (Fig. 3). There were some cells which immunostained with anti-aPY (3b cells) but not with anti-NPY, although they had a morphology similar to that of 3a cells. The 3a cells also demonstrated weak immunostaining with anti-PYY. The cranial pancreas of females showed a few, intensely stained anti-aPY cells and also lighter staining follicles and islets. By comparing adjacent sections of cranial pancreas stained with anti-NPY (Fig. 4a), anti-aPY (Fig. 4b), anti-insulin (Figs. 4c), anti-PYY, and anti-SST-34, it was determined that 3a and 3b cells were not B- or D-cells. However, the cells staining lightly with anti-aPY were also immunoreactive with antiinsulin. Staining of the male cranial pancreas was comparable to that seen in the caudal pancreas. There was no evidence of 1

INTENSITY OF PEPTIDES WITHIN PANCREAS AND INTESTINE

Tissue

Intestine Upstream migrant

IMMUNOSTAINING ADULT LAMPREY

ET AL.

NPY

++ +++ -

-

PYY

+ ++ -

+ + ++ -

ENDOCRINE

CELLS OF

bPP

SPP

aPY

Insulin

-

-

++ ++ +++ + ++ ++ ++ ++ ++ ++ ++ -

+++ +++ +++ ++ -

Note. + + + , Intense staining; + + , moderate staining: + . weak staining: - , no staining.

FIG. 1. Adjacent sections of the cranial pancrc :as of juvenile adult immunostained with anti-NPY (a), anti-aPY (b), anti-insulin (c), and anti-SST- 34 (d). Type 3b cells immunostain with anti-aPY (arrow) but not with anti-NPY (arrow). Type 3a cc:Ils (arrowhead) are stained by both antisera. These cells were not B-cells (B) or D-cells (D). x425.

55

56

CHEUNG

ET

AL.

LAMPREY

INTESTINE

anti-bPP, anti-sPP, or anti-GLP immunoreactivity in either the cranial or caudal pancress . The pattern of immunostaining of the intestine of upstream migrants with the various antisera was consistent with that seen in the juvenile adult (Table 1, Figs. 5a-5d). Although it seemed that a greater number of anti-NPY cells were observed scattered within the intestinal epithelium compared to the pancreas of upstream migrants, numbers in the intestine appeared reduced compared to those of juveniles. Results are summarized in Table 1. DISCUSSION The present study illustrates that a small population of cells within the endocrine pancreas of juvenile and upstream migrant adult lampreys reveal positive immunostaining with anti-aPY, an antiserum prepared against a synthetic peptide first isolated and characterized from anglerfish Brockmann bodies (Andrews et al., 1985). Some, but not all, of these aPY-immunoreactive cells also stain with anti-NPY and anti-PYY. The small numbers of these cells in the pancreas suggest they may represent the third type of cell previously characterized by its fine structure and by an absence of immunoreactivity. These cells are different from B- and D-cells in adult lampreys (Elliott and Youson, 1988; Youson et al., 1988). It is also of interest to note that im-

AND PANCREAS

57

munoreactivity to anti-NPY and anti-aPY in the intestine suggest that aPY and NPY may be colocalized in the same cell or at least that the same cytoplasmic peptide is cross-reacting with the different antisera. This result differs from immunostaining patterns seen in the pancreas where immunoreactivity to these antisera did not show consistent colocalization of these peptides and implied that a fourth cell type may be present. For this reason we distinguished between 3a and 3b cells in the pancreas. Previous studies have shown that NPY is a neuropeptide confined to neural tissues or, more specifically, the central and peripheral nervous systems (Lundberge et al., 1983; Ekblad et al., 1984; O’Donohue et al., -1985), while PYY and PP are primarily localized within the endocrine cells of the gut and pancreas, respectively (Schwartz, 1983; O’Donohue et al., 1985; Servin et al., 1989). Noe et al. (1986) reported the localization of NPY-immunostained cells and nerve tibers in the pancreatic islets of angler-fish. Data provided by reverse-phase liquid chromatography (RPLC) and radioimmunoassays (RIA) indicate that pancreatic cells demonstrating immunoreactivity to NPY are likely aPY-containing cells (Noe et al., 1989). A small quantity of peptides from Brockmann body extracts had chromatographic retention times identical to those of human and porcine NPY, suggesting that NPY was localized in the nerve fibers which permeate the islet (Noe et al., 1989). Assuming that the distribution of the

FIG. 2. Adjacent sections ofjuvenile adult anterior intestine immunoreactive to antisera against aPY (a) and NPY (b). Endocrine cells within the intestinal epithelium (I) are immunostained by both antisera (arrows). x 225. FIG. 3. aPY-immunoreactive cells in the cranial pancreas ofjuvenile adult lamprey. Isolated circular cell (arrow) and pyramidal-shaped cells belonging to the islet (arrowheads) are present. x460. FIG. 4. Adjacent sections of the cranial pancreas of female upstream migrant adult lamprey demonstrating immunoreaction to antisera directed against NPY (a) aPY (b) and insulin (c). Cells immunostained with anti-NPY (arrow) were not insulin-containing B-cells (B), SST-3Ccontaining D-cells (D), or aPY-immunoreactive cells (arrowheads). Also note the similar immunostaining pattern of the weakly stained aPY-immunoreactive islets (A) with the adjacent section (b) demonstrating insulinimmunostained B-cells (B). x225.

FIG. 5. Adjacent sections demonstrating immunoreaction to antibodies of NPY (a), aPY (b), GLP (c), and SST-34(d) in upstream migrant adult anterior intestine. NPY, aPY, and GLP immunostain the same endocrine cells (arrows) within the intestinal epithelium (I), in contrast to SST-34-immunoreactive cells (arrowhead), which are different cells. x475. 58

LAMPREY

INTESTINE

above peptides is representative of all vertebrates, this would mean that the NPYimmunoreactive cells in the pancreas of adult lampreys are not NPY-containing cells but possess a derivative or analogue of NPY. No cells that were immunoreactive to mammalian PP were found in the adult lamprey pancreas, a feature previously noted by Yui et al. (1988) in L. juponica. aPY has a 64% degree of sequence homology to both porcine NPY and PYY but only a 47% sequence identity to porcine PP, suggesting that the peptide is more closely related to porcine NPY and PYY than to PP (Table 2). However, it was determined in RIA that anti-aPY has less than 1% crossreactivity to NPY, PYY, and sPP (Milgram et al., 1989). Since NPY and PYY have a high degree of sequence identity and NPY has previously been shown to bind with great specificity to PYY but not PP receptors (Laburthe et al., 1986; Inui et al., 1988), it is tempting to speculate that the antiNPY-like immunoreactive staining observed in the pancreas and intestinal epithelial lining in P. murinus is actually PYY or PYY-like cells. This view is supported by the positive immunostaining of the 3a cells with anti-PYY. Our results indicate that there may be two distinctly separate populations of cells which we have designated as type 3a and 3b cells within the adult cranial and caudal pancreas. Both cell types are immunoreactive with anti-aPY but we are uncertain as to the nature of the peptide with which anti-

COMPARISON

OF THE PRIMARY

aPY NPY PYY SPP bPP Note. aPY, anglerfish pancreatic polypeptide;

STRUCTURES

AND

59

PANCREAS

aPY is reacting. It may be cross-reacting with an aPY-like peptide or some other NPY analogue. Anti-aPY was raised in rabbits against the glycine-extended form and does not recognize the carboxyl-terminally amidated aPY-amide (Balasubramaniam et al., 1989a, b; Noe et al., 1989). Therefore, depending upon the specificity of the NPY antiserum, the difference between the 3a and 3b cells may be that the 3b cell lacks the amidating enzyme. Since bPP and sPP are carboxyl-terminally amidated (Kimmel et al., 1986), aPY has only a 47% sequence identity with bPP, and immunoreactivity to anti-bPP and anti-sPP is absent, it is unlikely that anti-aPY is immunostaining PPcontaining cells. Since it has been shown in fish (Nozaki et al., 1988) and in mammals (Solcia et al., 1985; Mojsov et al., 1986; Qrskov et al., 1986; Vaillant and Lund, 1986) that GLPcontaining cells are also processing glucagon, our findings, which demonstrate a lack of GLP-immunoreactive cells in the pancreas of P. murinus, are reflective of the absence of glucagon or A-cells in lamprey pancreas. Recently, Elliott and Youson (1988) used immunohistochemistry and antisera directed againt mammalian glucagon to show the absence of A-cells in P. murinus. NPY and aPY-immunoreactive cells in the intestine of both juvenile and spawning lampreys demonstrate immunoreactivity with anti-PYY as well as anti-GLP. PYY and a gut-type glucagon have been shown to be colocalized within the same endocrine

TABLE 2 OF aPY, HUMAN

NPY,

PORCINE

PYY,

sPP, AND bPP

YPPKPETPGSNASPEDWASYQAAVRHYVNLITRQRYG-COOH YPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY-NH2 YPAKPEAPGEDASPEELSRYYASLRHYLNLVTRQRY-NH2 YPPKPENPGEDAPPEELAKYYTALRHYINLITRQRY-NH2 APLEPEYPGDDATPEQMAQYAAELRRYINMLTRPRY-NH2 polypeptide; bPP, bovine

NPY, neuropeptide pancreatic polypeptide.

Y; PYY,

peptide

tyrosine

tyrosine;

sPP,

salmon

60

CHEUNG ET AL.

cell in the distal intestine of several mammalian species (Biittcher et al., 1984, 1986; Ali-Rachedi et al., 1984; Barbosa et al., 1987). Yui et al. (1988) have suggested the existence of PYY-like-containing endocrine cells in the gut of adult lamprey. Their conclusions were derived from indirect evidence, that is, the colocalization of PP and glucagon within endocrine gut cells. We have suggested that the NPY-immunoreactive cells in juvenile pancreas are PYYcontaining cells and the same supposition can be made for the intestine. Further studies, including isolation and purification of lamprey-specific peptides from pancreas and intestine, must be carried out before more definitive conclusions can be reached. The number of NPY-immunoreactive cells in the intestine of the upstream migrant appeared reduced compared to that of juveniles. It has been reported that NPY stimulates feeding and inhibits sexual behavior in rats (Clark et al., 1985; Sahu et al., 1988; and Kalra et al., 1988). Juvenile lampreys, P. marinus, feed but are not sexually active. Upstream migration in lampreys is accompanied by sexual maturation and cessation of feeding (Larsen, 1980). Whether these mammalian observations on the effects of NPY have any relevance to explaining lamprey feeding or sexual behavior at any period in the life cycle requires further study. In general, immunostaining of the pancreatic tissue was similar in upstream migrant and juvenile adults. However, results obtained from observations of the cranial pancreas of female upstream migrants also showed a striking difference from juveniles in that fainter staining patches of islets and follicles were immunostained with antiaPY. By immunostaining adjacent sections with anti-aPY and rabbit anti-lamprey insulin, it was determined that anti-aPY was faintly staining B-cells. When comparing the amino acid sequence of both chains of lamprey insulin (Plisetskaya et al., 1988) with the sequence of aPY (Andrews et al.,

1985), it was determined that there is no sequence homology between the two peptides. Furthermore, anti-aPY was produced against synthetic aPY and it is not likely that there will be any contaminants (Andrews, unpublished data). Carillo et al. (1986) and JGrns et al. (1988) have shown that different peptides may be packed within different granules or within the same granule in the same cell. This may provide a possible rationalization for the apparent cross-reactivity between anti-aPY and lamprey insulin-containing cells but does not explain why this result is restricted to only the cranial pancreas of female upstream migrants. Barrington (1972) has suggested that the larval pancreas is formed by the aggregation of follicles exfoliated from the gut epithelium. This type of morphogenesis is also involved in the formation of the cranial pancreas during lamprey metamorphosis (Elliott and Youson, 1987). Preliminary studies performed in our laboratory indicate that an increase in the mass of the cranial pancreas continues in this manner even during the upstream migrant period (Youson and Cheung, 1990). Taking all the evidence into consideration, it would appear that all cranial pancreatic cells may be derived from a common precursor containing aPY-insulin-packed granules and that cross-reactivity shows up in the cranial pancreas due to the continued production of new islets. This is also the first evidence from lampreys that there may be either sexual dimorphism and/or regional variation in pancreatic hormone. Close examination and comparison of the different peptides in the intestine and cranial and caudal pancreas of lampreys are required. ACKNOWLEDGMENTS The authors thank Dr. J. M. Polak, Department of Histochemistry, Royal Postgraduate Medical School, for donating the PYY antisera used in this study. This study was supported by Grant A5945 from the Natural Sciences and Engineering Research Council of Canada

LAMPREY

INTESTINE

to J.H.Y. and by Grant DCB 8615551 from the National Science Foundation of the U.S.A. to E.M.P. P.C.A. was supported by a grant from the American Diabetes Association.

REFERENCES Ah-Rachedi, A., Varndell, I. M., Adrian, T. E., Gapp, D. A., Van Noorden, S., Bloom, S. R., and Polak, J. M. (1984). Peptide YY (PYY) immunoreactivity is co-stored with glucagon-related immunoreactants in endocrine cells of the gut and pancreas. Histochemistry 80, 487-491. Andrews, P. C., Hawke, D., Shively, J. E., and Dixon, J. E. (1985). A nonamidiated peptide homologous to porcine peptide YY and neuropeptide YY. Endocrinofogy 116, 2677-268 I. Balasubramaniam, A., Andrews, P. C., Renugopalakrishnan, V., and Rigel, D. F. (1989a). Glytine-extended anglerfish peptide YG (aPY) a neuropeptide Y (NPY) homologue may be a precursor of a biologically active peptide. Peptides 10, 581-585. Balasubramaniam, A., Knittel, J. J., Gil, C., and Andrews, P. C. (1989b). Detection of conformational isomers of anglerfish peptide YG (aPY) by reversed phase chromatography. ht. J. Pept. Protein Res. 34, 158-160. Barbosa, A. J. A., Nogueira, J. C., Penna, F. J., and Polak, J. M. (1987). Distribution of enteroglucagon- and polypeptide YY-immunoreactive cells in the gastrointestinal tract of the white-belly opossum (Dideiphis albiventris). Histochemistry 88, 37-40. Barrington, E. J. W. (1972). The pancreas and intestine. In “The Biology of Lampreys” (M. W. Hardisty and I. C. Potter, Eds.), Vol. 2, pp. 135-169. Academic Press, London. Bottcher, G., Alumets, J., Hakanson, R., and Sundler, F. (1986). Co-existence of glicentin and peptide YY in colorectal L-cells in cat and man. An electron microscopic study. Regul. Pept. 13,283-291. Bottcher, G., Sjolund, K., Ekblad, E., Hakanson, R., Schwartz, T. W., and Sundler, F. (1984). Coexistence of peptide YY and glicentin immunoreactivity in endocrine cells of the gut. Regul. Pept. 8, 261-266. Brinn, J. E., and Epple, A. (1976). New types of islet cells in a cyclostome, Petromyzon marinus L. Cell Tissue Res. 171, 317-329. Brodin, L., Rawitch, A., Taylor, T., Ohta, Y., Ring, H., Hokfelt, T., Grillner, S., and Terenius, L. (1989). Multiple forms of pancreatic polypeptiderelated compounds in the lamprey CNS: Partial characterization and immunohistochemical localization in the brain stem and spinal cord. J Neurosci.

9, 3428-3442.

AND

PANCREAS

61

Canillo, M., Zanuy, S., Duve, H., and Thorpe, A. (1986). Identification of hormone-producing cells of the endocrine pancreas of the sea bass, Dicentrarchus labrax, by ultrastructural immunocytochemistry. Gen. Camp. Endocrinol. 61, 287-301. Clark, J. T., Kalra, P. S., and Kalra, S. P. (1985). Neuropeptide Y stimulates feeding but inhibits sexual behaviour in rats. Endocrinology 117, 2435-2442, Conlon, J. M., Schmidt, W. E., Gallwitz, B., Falkmer, S., and Thim, L. (1986). Characterization of an amidated form of pancreatic polypeptide from the daddy sculpin (Cottus scorpius). Regul. Pept. 16, 261-268.

Ekblad, E., Edvinsson, L., Wahlestedt, C., Uddman, R., Hakanson, R., and Sundler, F. (1984). Neuropeptide Y co-exists and co-operates with noradrenaline in perivascular nerve tibres. Regul. Pept.

8, 225-235.

Elliott, W. M., and Youson, J. H. (1986). Immunocytochemical localization of insulin and somatostatin in the endocrine pancreas of sea lamprey, Petromyzon marinus L., at various stages of its life cycle. Cell Tissue Res. 243, 629-634. Elliott, W. M., and Youson, J. H. (1987). Immunohistochemical observations of the endocrine pancreas during metamorphosis of the sea lamprey, Petromyzon marinus L. Cell Tissue Res. 247,351357. Elliott, W. M., and Youson, J. H. (1988). Fine structure and immunocytochemistry of cells within the endocrine pancreas of larval and adult sea lampreys, Petromyzon marinus L. Amer. J. Anat. 182, 73-83.

El-Salhy, M., and Grimelius, L. (1983). Immunocytochemical demonstration of polypeptide YY (PYY) in the gastrointestinal tract of the monkey, Macaca rhesus: Light and electron microscopic study. Biomed. Res. 4, 289-294. Epple, A., and Brinn, J. E. (1975). Islet histophysiology: Evolutionary correlations. Gen. Camp. Endocrinol.

27, 320-349.

Falkmer, S., and Van Noorden, S. (1983). Ontogeny and phylogeny of the glucagon cell. In “Handbook of Experimental Pharmacology” (P. J. Lefebvre, Ed.), Vol. 66.0, pp. 81-119. SpringerVerlag, Berlin. Hilliard, R. W., Epple, A., and Potter, I. C. (1985). The morphology and histology of the endocrine pancreas of the southern hemisphere lamprey, Geotria australis Gray. .I. Morphol. 184, 253-262. Inui, A., Oya, M., Okita, M., Inoue, T., Sakatina, N., Morioka, H., Shii, K., Yokomo, K., Mizuno, N., and Baba, S. (1988). Peptide YY receptors in the brain. Biochem. Biophys. Res. Commun. 150,2532. Jams, A., Barklage, E., and Grube, D. (1988). Heter-

62

CHEUNG

ogeneities of the islets in the rabbit pancreas and the problem of “paracrine” regulation of islet cells. Anat.

Embryo/.

178, 297-307.

Kalra, S. P., Clark, J. T., Sah, A., Dube, M. G., and Kalra, P. S. (1988). Control of feeding and sexual behaviors by neuropeptide Y: Physiological implications. Synapse 2, 254-257. Kimmel, J. R., Plisetskaya, E. M., Poilock, H. G., Hamilton, J. W., Rouse, J. B., Ebner, K. E., and Rawitch, A. B. (1986). Structure of a peptide from coho salmon endocrine pancreas with homology to neuropeptide Y. Biochem. Biophys. Res. Commun.

141, 1084-1091.

Kimmel, J. R., Pollock, H. G., and Hazelwood, R. L. (1971). A new pancreatic polypeptide hormone. Fed. Proc. 30, 1318A. Laburthe, M., Chenut, B., Rouyer-Fessard, C., Tatemoto. K., Couvineau, A., Servin, A., and Amiranoff, B. (1986). Interaction of peptide YY with rat intestinal epithelial plasma membranes: Binding of the radioiodinated peptide. Endocrinology 118, 1910-1917.

Larsen, L. 0. (1980). Physiology of adult lampreys, with special regard to natural starvation, reproduction, and death after spawning. Canad. J. Fish.

Aquat.

Sci. 37, 1762-1779.

Lundberge, J. M., Terenius, L., Hokfelt, T., and Goldstein, M. (1983). High levels of neuropeptide Y in peripheral noradrenergic neurons in various mammals including man. Neurosci. Lett. 42, 167172. Milgram, S. L., Balasubramaniam, A., Andrews, P. C., McDonald. J. K., and Noe, B. D. (1989). Characterization of aPY-like peptides in anglerfish brain using a novel radioimmunoassay for aPY-Gly. Peptides 10, 1013-1017. Mojsov, S.. Heinrich, G., Wilson, I. B., Ravazzola, M., Orci, L., and Habener, J. F. (1986). Preproglucagon gene expression in pancreas and intestine diversifies at the level of post-translational processing. 1. Biol. Chem. 261, 1180-l 189. Noe, B. D., McDonald, J. K.. Greiner, F.. and Wood, J. G. (1986). Anglerfish islets contain NPY immunoreactive nerves and produce the NPY analog aPY. Peptides 7, 147-154. Noe, B. D., Milgram, S. L., Balasubramaniam, A., Andrews, P. C., Calka, J., and McDonald, K. (1989). Localization and characterization of NPYlike peptides in the brain and islet organ of the angle&h (Lophius americanus). Cell Tissue Res. 257, 303-3 Il. Nozaki, M., Miyata, K., Oota, Y., Gorbman, A., and Plisetskaya, E. M. (1988). Colocalization of glucagon-like peptide and glucagon immunoreactivities in pancreatic islets and intestine of salmonids. Cell Tissue

Res. 253, 371-375.

ET AL.

O’Donohue, T. L., Chronwall, B. M., Pruss, R. M., Mezey, E., Kiss, J. Z., Eiden, L. E., Massari, V. J., Tessel, R. E., Pickel, V. M., DiMaggio, D. A.. Hotchkiss. A. J., Crowley, W. R., and Zukowska-Grojec, Z. (1985). Neuropeptide Y and peptide YY in neuronal and endocrine systems. Peptides

6, 755-768.

@rskov, C., Holst, J. J., Knuhtsen, S.. Baldissera, F. G. A., Poulsen, S. S., and Nielsen, 0. V. (1986). Glucagon-like peptides GLP-1 and GLP-2 predicted products of the glucagon gene are secreted separately from pig small intestine but not pancreas. Endocrinology 119, 1467-1475. Plisetskaya, E. M., Pollock. H. G., Elliott, W. M., Youson, J. H., and Andrews, P. C. (1988). Isolation and structure of lamprey (Petromyzon marinus) insulin. Gen. Comp. Endocrmof. 69, 46-55. Pollock, H. G.. Kimmel, J. R., Hamilton, J. W., Rouse, J. B., Ebner, K. E.. Lance, V.. and Rawitch, B. (1987). Isolation and structure of alligator gar (Lepisosteus spatula) insulin and pancreatic polypeptide. Gen. Comp. Endocrinol. 67, 375-382. Sahu, A., Kalra, S. P., Crowley, W. R., and Kalra, P. S. (1988). Evidence that NPY-containing neurons in the brainstem project into selected hypothalamic nuclei: Implication in feeding behaviour. Brain

Res. 457, 376-378.

Schwartz, T. W. (1983). Pancreatic polypeptide: A hormone under vagal control. Gastroenrerology 85, 141 I-1425. Servin, A. L., Rouyer-Fessard, C., Balasubramaniam, A., Saint Pierre, S., and Laburthe, M. (1989). Peptide-YY and neuropeptide-Y inhibit vasoactive intestinal peptide-stimulated adenosine 3’S’-monophosphate production in rat small intestine: Structural requirements of peptides for interacting with peptide-YY preferring receptors. Endocrinology

124, 692-700.

Solcia, E.. Fiocca, R.. Capella, C., Usellini, L., Sessa, F.. Rindi, G., Schwartz, T. W., and Yanaihara, N. (1985). Glucagon- and PP-related peptides of intestinal L-cells and pancreatic/gastric A or PPcells: Possible interrelationships of peptides and cells during evolution, fetal development and tumor growth. Peptides 6, (Suppl. 3), 223-229. Stemberger, L. A., Hardy, P. H.. Jr., Cuculis, J. J.. and Meyers, H. G. (1970). The unlabelled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigenantibody complex (horseradish peroxidase antihorseradish peroxidase) and its use in the identification of spirochetes. J. Hisfochem. Cytochem. 18, 315-333.

Tatemoto, K. (1982). Neuropeptide Y: Complete amino acid sequence of the brain peptide. Proc. Natl.

Acad.

Sri.

USA 79, 5485-5489.

LAMPREY

INTESTINE

Tatemoto, K., Carlquist, M., and Mutt, V. (1982). Neuropeptide Y-A novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature (London) 296, 659-660. Taylor, I. L. (1985). Distribution and release of peptide YY in dog measured by specific radioimmunoassay. Gastroenterology 88, 731-737. Titlbach, M., and Kern, H. F. (1969). Licht- und elektronenmikroskopische Untersuchungen am Inselorgan des Bachneunauges Lampetra planeri (Bloch). Z. Zellforsch. 97, 403415. Vaillant, C. R., and Lund, P. K. (1986). Distribution of glucagon-like peptide 1 in canine and feline pancreas and gastrointestinal tract. J. Histochem. Cytochem. 34, 1117-1121. Van Noorden, S., Ostberg, Y., and Pearse, A. G. E. (1977). Localization of somatostatin-like immunoreactivity in the pancreatic islets of hag&h, Myx-

AND

PANCREAS

ine glutinosa and the lamprey, Lampetra tilus. Cell Tissue Res. 177, 281-285.

63 j7uvia-

Youson, J. H. (1981). The alimentary canal. In “The Biology of Lampreys” M. W. Hardisty and I. C. Potter, Eds., Vol. 3, pp. 95-189. Academic, London. Youson, J. H., and Cheung, R. (1990). Morphogenesis of somatostatin- and insulin-secreting cells in the lamprey endocrine pancreas. Fish Physiol. Biothem., in press. Youson, J. H., Elliott, W. M., Beamish, R. J., and Wang, D. W. (1988). A comparison of endocrine pancreatic tissue in adults of four species of lampreys in British Columbia: A morphological and immunohistochemical study. Gen. Comp. Endocrinol. 70, 247-261. Yui, R., Nagata, Y., and Fujita, T. (1988). Immunocytochemical studies on the islet and the gut of the Arctic lamprey, Lampetra japonica. Arch. Histol. Cytol.

51, 109-I 19.