Immunocytochemical localisation of the icosapeptide fragment of the PP precursor: a marker for ‘true’ PP cells?

Regulatory Peptides, 8 (1984) 217-224

217

Elsevier RPT 00276

Immunocytochemical localisation of the icosapeptide fragment of the PP precursor: a marker for'true' PP cells? F. Sundler, G. B6ttcher, R. H ~ k a n s o n a n d T . W . S c h w a r t z Departments of Histology and Pharmacology, University of Lund, Lund, Sweden, and Laboratory of Molecular Endocrinology, Department of Clinical Chemistry, Rigshospitalet, Copenhagen, Denmark (Received 19 December 1983; revised manuscript received 29 February 1984; accepted for publication 29 February 1984)

Summaff Antisera were raised against the icosapeptide fragment of the pancreatic polypeptide (PP) isolated from the canine pancreas. They were used for the immunocytochemical study of the cellular localisation and distribution of the icosapeptide in the gut and pancreas of various mammals. The results indicate that PP and the icosapeptide coexist in the majority of the PP-immunoreactive cells in the pancreas of cat, dog, pig, monkey and man and in all the PP-immunoreactive cells in the stomach of the cat and dog. The icosapeptide does not seem to occur in cells or nerves containing PP-related peptides, such as peptide YY or neuropeptide Y. PP-immunoreactive cells devoid of the icosapeptide could be demonstrated in the large intestine. These cells are probably distinct from the pancreatic PP cell type, and the PP-immunoreactive material probably represents the homologous peptide YY rather than PP. The present findings support the view that the icosapeptide is part of the PP precursor and hence, only the cells containing immunoreactive icosapeptide in addition to immunoreactive PP are to be considered 'true' PP cells. The icosapeptide antisera did not stain PP cells in mouse, rat and guinea-pig, suggesting marked species variation in the amino acid sequence of the icosapeptide portion of the PP precursor. pancreatic polypeptide (PP); pancreatic islets; PP precursor; PP cell marker; immunocytochemistry

Address for correspondence: Dr. Frank Sundler, Department of Histology, Biskopsgatan 5, S-223 62 Lund, Sweden. 0167-0115/84/$03.00 © 1984 Elsevier Science Publishers B.V.

218 Introduction

Pancreatic polypeptide (PP), a 36 amino acid hormone, is located in a special type of pancreatic islet ceils, the so-called PP cells [1]. PP-immunoreactive endocrine cells occur also in the gastrointestinal tract [1]. In the dog and cat PP-immunoreactive ceils are quite numerous in the antropyloric mucosa of the stomach. On the whole, PP-immunoreactive cells are few in the small intestine. In the large intestine, however, they are fairly numerous [1,2]. In the latter location they constitute a subpopulation of the glucagon/glicentin cells [3]. Recently, two PP-related peptides have been isolated from the porcine brain and gut, respectively. The gut peptide, peptide YY (PYY) [4], occurs in endocrine cells in the distal intestine [5]. The brain peptide, neuropeptide Y (NPY) [6], occurs in central and peripheral neurones [7-9]. NPY-immunoreactive nerve fibres are numerous in the gut and pancreas [9]. Available data thus suggest the existence of a family of PP-related peptides [10]. Recently, Schwartz and Tager [11] have isolated a 20 amino acid peptide from the canine pancreas and provided evidence that this icosapeptide represents a COOHterminal fragment of the PP precursor [11,12]. In the present report two antisera raised against the icosapeptide fragment of the PP precursor were used in order to study its cellular localisation in the dog and in some other mammals. The results suggest (1) that the icosapeptide occurs only in the cells storing 'true' PP and not in cells and nerves containing PP-related peptides such as PYY or NPY, and (2) that the icosapeptide sequence differs more between species than the PP sequence.

Material and Methods

Tissues from adult mice, rats, guinea-pigs, cats, dogs, pigs, monkeys (Macaca

macaca) and man were examined (at least 3 individuals of each species). Cats, dogs, pigs and monkeys were killed by sodium pentobarbitone (Brietal~). Tissue specimens were collected from various regions of the gastrointestinal tract and from the duodenal and splenic portions of the pancreas. Fresh human specimens were obtained at abdominal surgery (Department of Surgery, University of Lund). Fetal (age 15-20 weeks) and neonatal human pancreas was obtained at legal abortions (Department of Obstetrics and Gynaecology, University of Lund) and at autopsies. Some specimens were frozen in a mixture of propane and propylene at the liquid nitrogen temperature and freeze-dried. They were vapour-fixed in formaldehyde [13] (1 h at 80°C) or diethylpyrocarbonate [14] (3 h at 55°C) and embedded in paraffin or Araldite. Other specimens were immersed in ice-cold buffered formaldehyde solution (4% in 0.1 M sodium phosphate buffer, pH 7.2) overnight and rinsed thoroughly in 10% sucrose-enriched buffer. These specimens were frozen in solid CO 2 for cryostat sectioning. Paraffin sections (6 #m thickness), plastic sections (1 t~m) and cryostat sections (10-20 t~m) were processed for the immunocytochemical demonstration of PP, NPY, PYY or the icosapeptide using the indirect immunofluorescence [15] or immunoperoxidase [16] techniques. The peptide antisera

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used are listed in Table I. Sequential staining of the same section for two different peptides was performed according to Tramu et al. [17]. Control sections were incubated with peptide antiserum inactivated by prior addition of antigen in excess (10 #g of peptide per ml diluted antiserum, added at least 24 h before use). Possible cross-reactivity of the icosapeptide antisera with PP-related peptides at the immunocytochemical level was excluded by adding bovine PP, PYY or NPY (100 gg per ml diluted antiserum). The NPY antiserum has previously been shown to contain antibodies that cross-react with PP (and PYY) [9]. Therefore, the NPY antiserum was preincubated with bovine PP (100 #g/ml diluted antiserum) before use. After such preabsorption, the NPY antiserum stained only nervous elements in the gut and pancreas. The peptides were obtained from Peninsula Inc., Belmont, California, with the.exception of the icosapeptide which was isolated from dog pancreas as described elsewhere [11].

Results

Immunoreactive icosapeptide was demonstrated in endocrine cells in the pancreas of the cat, dog, pig, monkey and man (Fig. 1). Absorption of the antisera with icosapeptide abolished staining, whereas absorption with bovine PP, PYY and NPY did not. The icosapeptide cells were moderate in number (man) or numerous in the duodenal portion and few in the splenic portion. Sequential staining of the same paraffin section or staining of consecutive semithin plastic sections revealed coexistence of PP and the icosapeptide (Fig. 2). A minority population of PP-immunoreactive cells in the pancreas did not contain the icosapeptide. The proportion of such cells varied from approx. 25% in the primate pancreas to a few percent in the feline pancreas. The pancreas of dog and pig contained an intermediary number of such cells. TABLE I Details and sources of peptide antisera used Antigen

Code No.

Dilution

Source

Canine icosapeptide Canine icosapeptide Bovine PP

3202

1 : 80

own

3204 a

1 : 80

own

BPP

1 : 160

H u m a n PP Porcine neuropeptide Y (NPY) Porcine peptide YY (PYY)

HPP NPYY/2

1 : 640 1 : 400

366

1 : 400

R.E. Chance, Eli Lilly, Minneapolis, MN, U.S.A. " P.C. Emson, MRC, Cambridge, U.K. L. Terenius, Dept. Pharm., Uppsala, Sweden

This antiserum recognizes the C-terminal region of the icosapeptide [12].

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Fig. 1. Immunofluorescence staining of icosapeptide-containing islet cells in the duodenal portion of the pancreas, (a) cat, (b) pig, (c) monkey, (d) man. Paraffin sections, formaldehyde fixation. Antiserum No. 3204. 120 x .

Fig. 2. Coexistence of PP and the icosapeptide m pancreatic endocrine cells of the dog. Immunofluorescence staining of PP-containing (a) and icosapeptide-containing (b) cells in consecutive semithin plastic sections. 160 x .

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a

b

Fig. 3. Coexistence of PP- and icosapeptide-immunoreactive material in endocrine cells in the antrum of the dog. Immunofluorescence staining for PP (a) and the icosapeptide (b) in consecutive semithin plastic sections. 250 × .

Fig. 4. Partial coexistence of PP (a) and the icosapeptide (b) in pancreatic endocrine cells of a human neonate as revealed by immunofluorescence staining of consecutive semithin plastic sections. The PP cells greatly outnumber the icosapeptide-containing cells. 135 x .

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Fig. 5. PYY-immunoreactivecells (a) in the human rectum do not contain the icosapeptide(b) as revealed by immunofluorescencestaining of consecutivesemithin plastic sections. 160 x.

In the cat and dog the icosapeptide antisera stained fairly numerous cells in the antrum (Fig. 3) and few scattered cells in the body of the stomach and in the large intestine. Staining of consecutive plastic sections showed that the icosapeptide-immunoreactive cells in the digestive tract also displayed PP immunoreactivity (Fig. 3). The icosapeptide antisera did not stain nerve fibres in the gut or pancreas. In man only one of the icosapeptide antisera (No. 3204) stained endocrine cells. These cells were restricted to the pancreas and were identical with PP-immunoreactive cells. However, the PP-immunoreactive cells were clearly more numerous than those immunostained with the icosapeptide antiserum, particularly in the fetal and neonatal pancreas (Fig. 4). A rich number of cells in the ileum, colon and rectum displayed PYY immunoreactivity; these cells did not stain with the icosapeptide antiserum (Fig. 5). In the mouse, rat and guinea-pig neither of the two icosapeptide antisera revealed endocrine cells in the gut or pancreas. However, the PP antisera demonstrated PP cells in these species as previously reported [1]. The NPY antiserum demonstrated a rich supply of nerve fibres, but no endocrine cells, in the pancreas and gut wall (as studied in the cat, dog and pig).

Discussion PP and the pancreatic icosapeptide were found to coexist in the majority of the PP-immunoreactive cells in the pancreas of cat, dog, pig, monkey and man and in all the PP-immunoreactive cells in the stomach of the cat and dog. There is much evidence to indicate that the pancreatic icosapeptide is generated from the COOH-

223 terminal part of the PP precursor. This view is based on peptide mapping of biosynthetically labelled peptides [12], kinetic behaviour during pulse-chase labelling experiments [12], and most importantly, the fact that the PP precursor is specifically immunoprecipitated by antibodies against the icosapeptide [18]. It may thus be argued that the presence of immunoreactivity representing b6th the icosapeptide sequence and the PP sequence is characteristic of 'true' PP cells. The PP-immunoreactive cells in the large intestine, lacking the icosapeptide, are probably identical with the glicentin cells and seem to contain PYY rather than PP [20]. The PP-immunoreactive cells in the pancreas, which could not be stained with icosapeptide antiserum, do not store PYY (Sundler et al., unpublished results), and the nature of these cells remains unknown. It cannot be excluded that they represent true PP cells containing predominantly large forms of the precursor, poorly recognized by the icosapeptide antisera. The icosapeptide does not seem to occur in cells or nerves containing PP-related peptides, such as PYY or NPY, and it may therefore be a useful marker for 'true' PP cells. The inability of the two icosapeptide antisera to demonstrate PP cells in the mouse, rat and guinea-pig and the fact that only one of the antisera demonstrated PP cells in man suggest that the icosapeptide sequence differs from one species to another. Recent chemical analyses of the icosapeptide from man, dog and sheep show that the PP part of the common precursor is better preserved than the icosapeptide sequence [18,19].

Note added in proof (Received 12 April 1984) The notion that pancreatic icosapeptide is synthetized together with PP on a common precursor has recently been confirmed through sequence analysis of a c D N A complementary to human PP m R N A which also codes for the pancreatic icosapeptide (study performed in collaboration with E. Boel and coworkers at N O V O Research Institute, Copenhagen, Denmark) (EMBO Journal, 1984, in press).

Acknowledgements Supported by grants from the Swedish Medical Research Council (4499, 1007), the Danish Medical Research Council, P~hlsson's Foundation and the Swedish Diabetes Foundation.

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224 3 Sj61und, K., Sand6n, G., Hfikanson, R. and Sundler, F., Endocrine cells in human intestine: an immunocytochemical study, Gastroenterology, 85 (1983) 1120-1130. 4 Tatemoto, K., Isolation and characterization of peptide YY (PYY), a candidate gut hormone that inhibits pancreatic exocrine secretion, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 2514-2518. 5 Lundberg, J.M., Tat~moto, K., Terenius, L., Hellstr6m, Mutt, V., HOkfelt, T. and Hamberger, B., Localization of peptide YY, PYY, in gastrointestinal endocrine cells and effects on intestinal blood flow and motility, Proc. Natl. Acad. Sci. U.S.A. 79 (1982) 4471-4475. 6 Tatemoto, K., Carlquist, M. and Mutt, V., Neuropeptide Y - a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide, Nature, 296 (1982) 659-660• 7 Lor6n, I., Alumets, J., Hhkanson, R. and Sundler, F., Immunoreactive pancreatic polypeptide (PP) occurs in the central and peripheral nervous system: preliminary immunocytochemical observations, Cell Tiss. Res., 200 (1979) 179-186. 8 Lundberg, J.M., Terenius, L., HOkfelt, T., Martling, C.R., Tatemoto, K., Mutt, V., Polak, J. and Goldstein, M., Neuropeptide Y (NPY)-like immunoreactivity in peripheral noradrenergic neurons and effects of NPY on sympathetic function, Acta Physiol. Scand., 116 (1982) 447-480. 9 Sundler, F., Moghimzadeh, E., Hhkanson, R., Ekelund, M. and Emson, P., Nerve fibres in the gut and pancreas of the rat displaying neuropeptide-Y immunoreactivity, Cell Tiss. Res., 230 (1983) 487-493. 10 Tatemoto, K., New peptides, their structures and functions. In S.R. Bloom, J.M. Polak and E. Linderlaub (Eds.), Systemic Role of Regulatory Peptides, Symposia Medica Hoechst 18, F.K. Schattauer, Stuttgart, 1982, pp. 507-531. 11 Schwartz, T.W. and Tager, H.S., Isolation and biogenesis of a new peptide from pancreatic islets, Nature, 294 (1981) 589-591. 12 Schwartz, T.W., Gingerich, R.L. and Tager, H.S., Biosynthesis of pancreatic polypeptide: Identification of a precursor and a cosynthesized product, J. Biol. Chem., 225 (1980) 494-498. 13 BjOrklund, A., Falck, B. and Owman, Ch., Fluorescence microscopic and microspectrofluorometric techniques for the cellular localization and characterization of biogenic amines. In J.E. Rall and I.J. Kopin (Eds.), Methods of Investigative and Diagnostic Endocrinology, vol. 1: The Thyroid and Biogenic Amines, North-Holland, Amsterdam, 1972, pp. 318-368. 14 Pearse, A.G.E., Polak, J.M., Adams, C. and Kendall, P.A., Diethylpyrocarbonate, a vapour-phase fixative for immunofluorescence studies on polypeptide hormones, Histochem. J., 6 (1974) 347-352. 15 Coons, A.H., Leduc, E.H. and Conolly, J.M., Studies on antibody production. I. A method for the histochemical demonstration of specific antibody and its application to a study of the hyperimmune rabbit, J. Exp. Med., 102 (1955) 49-60. 16 Sternberger, L.A., Immunocytochemistry, 2nd edn. John Wiley & Sons, New York, 1979, pp. 1-354. 17 Tramu, G., Pillez, A. and Leonardelli, J., An efficient method of antibody elution for the successive or simultaneous location of two antigens by immunocytochemistry, J. Histochem. Cytochem., 26 (1978) 322-324. 18 Schwartz, T.W. and Hansen, H.F., Isolation of ovine pancreatic icosapeptide: a peptide product containing one cysteine residue, FEBS Lett., 168 (1984) 293-298. 19 Schwartz, T.W., Hansen, H.F., H~kanson, R., Sundler, F. and Tager, H., Human pancreatic icosapeptide - isolation, sequence and immunocytochemical localization of the COOH-terminal fragment of the pancreatic polypeptide precursor, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 708-712. 20 B6ttcher, G., Sj61und, K., Ekblad, E., Hhkanson, R., Schwartz, T.W. and Sundler, F., Coexistence of peptide YY and glicentin immunoreactivity in endocrine cells of the gut, Regul. Peptides, 8 (1984) in press.