Cell Tissue Res (1991) 264:461-467

Cell and Tissue Research 9 Springer-Verlag1991

Synaptophysin immunoreactivity in the mammalian endocrine pancreas P. Redecker 1., A. Ji~rns 1, R. Jahn 2, and D. Grube 1 1 AbteilungAnatomic 1, MedizinischeHochschuleHannover, W-3000 Hannover 61, Federal Republic of Germany 2 AbteilungNeurochemie,Max-Planck-lnstitutftir Psychiatric,W-8033 Martinsried, Federal Republic of Germany Accepted January 7, 1991

Summary. Synaptophysin, a major membrane glycoprorein of small presynaptic vesicles in neurons, has also been found in microvesicles of endocrine cells, e.g., of the endocrine pancreas. In the present study, the endocrine pancreas in 9 mammalian species (man, dog, mink, bovine, rabbit, guinea pig, rat, mouse, gerbil) has been investigated immunohistochemically for synaptophysin immunoreactivity. Synaptophysin-positive cells have been identified and localized on semithin plastic sections. Our study demonstrates that, in all species examined, all pancreatic endocrine cell types are consistently synaptophysin-positive independent of their location within the tissue, or the conditions of tissue processing. In addition, a few cells that cannot be hormonally identified show synaptophysin immunoreactivity. Hence, synaptophysin appears to be a regular constituent of all pancreatic endocrine cells in mammals. In several species, a subpopulation of endocrine cells, consisting of glucagoncontaining and/or pancreatic-polypeptide-containing cells, exhibits a significantly higher degree of synaptophysin immunoreactivity. In the gerbil, this heterogeneity can readily be detected from the day of birth onwards. Our findings indicate that closely related endocrine cell types may differ with respect to the content of synaptophysin. Key words: Synaptophysin P38 Membrane proteins - Endocrine pancreas - Islet cells Immunohistochemistry - Human - Dog - Gerbil

Synaptophysin (also referred to as protein p38) is a major integral membrane glycoprotein of small presynaptic vesicles in neurons (Jahn et al. 1985; Wiedenmann and Franke 1985). It is also present in various endocrine cells, where it has been localized on small clear vesicles that may constitute a previously unknown secretory Offprint requests to: P. Redecker

pathway and/or an organelle involved in receptor recycling (Navone etal. 1986; Wiedenmann etal. 1986; Johnston et al. 1989a). The widespread distribution of synaptophysin in gastro-entero-pancreatic (GEP) endocrine cells renders this protein one of the most reliable and specific markers of these cells in both normal and pathologic conditions (Wiedenmann et al. 1988). Synaptophysin has also been detected in normal and neoplastic cells of the endocrine pancreas (Navone et al. 1986; Wiedenmann et al. 1986, 1988; Buffa et al. 1988; Hoog et al. 1988). Although it is generally held that all endocrine cell types of the pancreatic islets display synaptophysin immunoreactivity (as may be inferred from the synaptophysin immunoreactivity of various pancreatic adenomas and the overall immunostaining pattern of normal islets), the precise immunohistochemical identification of synaptophysin-positive pancreatic cell types in the normal mammalian gland of diverse species is lacking. Moreover, Navone et al. (1986) have observed some heterogeneity with respect to the concentration of synaptophysin within unidentified subpopulations of rat islet cells. This study has been performed to evaluate in detail the alleged constant presence of synaptophysin in all pancreatic endocrine cells. For this purpose, the synaptophysin-positive pancreatic cell types have been identified immunohistochemically in various mammalian species, and the degree of intra- and interspecies variations of synaptophysin immunoreactivity has been examined. Taking into account that Hoog et al. (1988) have reported that the immunohistochemical demonstrability of synaptophysin is highly dependent on the type of tissue fixation, different fixation methods have been tested.

Materials and methods Tissues and tissue preparation

Pancreata of 9 mammalian species were examined including man, dog, mink, bovine, rabbit, guinea pig, rat, mouse and Mongolian

462 Table 1. Sources and references of the antisera; working dilutions refer to ABC technique

Antiserum

Source

Working dilution

References

Synaptophysin (G 95) Insulin (LAA) Glucagon (A 565) Somatostatin Bovine PP (bPP) Human PP (hPP) Serotonin (5-HT)

R. Jahn, Martinsried

1:500-1 : 10000

4, 5, 6

NOVO, Denmark

1:1000 1:6000

1, 2

DAKO, Denmark

1 : 3000-1 : 10 000

3

H. Etzrodt, Ulm R.E. Chance, Indianapolis

1 : 10000-1:30000 1 : 10000-1:30000

1, 3 1, 3

R.E. Chance, Indianapolis

1 : 10000-1:30000

1, 3

EURO Diagnostics, Holland

1:1000-1:4000

3

Grube and Bohn 1983; 2 Grube etal. 1983; 3 Grube etal. 1986; 4Jahn etal. 1985; 5 Schilling and Gratzl 1988; 6 Schilling et ai. 1989

gerbil. Tissue specimens were snap-frozen in melting Freon 22 precooled with liquid nitrogen, freeze-dried, fixed by vapor-phase pformaldehyde or di-ethylpyrocarbonate (DEPC) and embedded in epoxy resin (Araldite; see Grube and Kusumoto 1986). Alternatively, some samples from the gerbil pancreas were fixed by immersion for 20 h in a solution (final pH 8) consisting of 2% (w/v) p-formaldehyde, 1% (v/v) acrolein, 0.1% (w/v) picric acid, and 0.05% (v/v) glutaraldehyde in 0.1 M cacodylate buffer, or in a phosphate-buffered (0.1 M, pH 7.3) mixture of 4% (w/v) p-formaldehyde, 0.15% (w/v) picric acid, and 0.05% (v/v) glutaraldehyde. These samples were embedded in epoxy resins (Spurr resin or Araldite) and included specimens from old gerbils (21 months of age). Pancreata from young gerbils from postnatal day 1 (day of birth), 2, 4, 8, 12, 17, 28, 35, 42, 49 and 63 were fixed in Bouin's fluid for 24 h and embedded in epoxy resin or paraffin. Pieces of the gerbil cerebellar and cerebral cortices, median eminence, pituitary, pineal and adrenal medulla served as reference organs for the immunohistochemical detection of synaptophysin.

Immunohistochemistry An antiserum (G 95) against synaptophysin was raised in rabbits. It was generated by immunization with synaptophysin purified by affinity chromatography from rat brain membranes (Jahn et al. 1985; Navone et al. 1986). The specificity of this antiserum has been documented (Schilling and Gratzl 1988; Schilling et al. 1989). As a control, we also used an affinity-purified synaptophysin antiserum (see Navone et al. 1986). Details of polyclonal antisera raised against pancreatic hormones and serotonin are listed in Table 1. Semithin (0.5 gm) plastic sections and 6-gm-thick paraffin sections were immunostained by the peroxidase anti-peroxidase technique (see Sternberger 1986). This method has been modified for the immunostaining of plastic-embedded tissues (Grube 1980; Grube and Kusumoto 1986). Preferentially, the avidin-biotin-peroxidase complex (ABC) method (Hsu et al. 1981) was applied to semithin sections. Briefly, this method included the use of biotinylated goat anti-rabbit IgG (Jackson Immuno Research, USA) as a second layer (diluted 1 : 200), and a streptavidin-biotin-peroxidase complex (Jackson Immuno Research, USA) as a third layer (diluted 1 : 1000). Serial semithin sections were sequentially immunostained for synaptophysin and pancreatic hormones, allowing for the clearcut identification of synaptophysin-positive cells on adjacent sections. Approximately 1500 semithin plastic sections were investigated. The stained paraffin and plastic sections were viewed by brightfield illumination, interference-contrast or phase-contrast optics.

Controls for method and antibody specificities were performed as recommended in the literature (Grube 1980; Grube and Kusumoto 1986; Sternberger 1986).

Results

The polyclonal s y n a p t o p h y s i n antiserum consistently labeled nerve varicosities and endocrine cells o f the pancreas, irrespective o f the fixative and e m b e d d i n g m e d i u m applied. I m m u n o p o s i t i v e endocrine cells were a r r a n g e d either within islets o f L a n g e r h a n s , or were situated at extrainsular sites. Islet cells regularly displayed synaptophysin immunoreactivies in all species investigated (Figs. 1, 2, 3, 4; unless otherwise stated in the legends, figures show immunoreactivities in freeze-dried specimens). Only m i n o r interspecies variations with respect to the densities o f i m m u n o s t a i n i n g were found. G u i n e a pig islets exhibited the weakest immunoreactivities. The systematic investigation o f the synaptophysin-positive cell types revealed that, in each species, all endocrine cell types, i.e., insulin (B-), g l u c a g o n (A-), s o m a t o s t a t i n (D-), pancreatic polypeptide (PP-), a n d e n t e r o c h r o m a f fin (EC-) cells, were i m m u n o r e a c t i v e t o w a r d s the synaptophysin antiserum (Figs. 1 D - G , 2 D - G , 3, 4A). Rare EC-cells, which were stained by the serotonin antiserum, showed only weak s y n a p t o p h y s i n immunoreactivity. Generally, a p u n c t a t e reaction p r o d u c t was often distributed evenly t h r o u g h o u t the c y t o p l a s m o f the cells (Figs. 1 A - C , E, 3A, B, D, F). It extended into long cellular processes, the terminals o f which were frequently m o r e densely stained t h a n the perikarya (Figs. 2 F , 4A). Perinuclear c o n c e n t r a t i o n s o f i m m u n o s t a i n were sometimes evident (Fig. 2 A). Moreover, a c c u m u l a t i o n s o f i m m u n o reactivity were also seen near the cell surface (Fig. 2 B D). Despite differences in the intracellular staining pattern o b t a i n e d with antibodies directed against s y n a p t o physin a n d pancreatic h o r m o n e s , their overall intracellular distribution was often r e m a r k a b l y similar (Fig. 2 D -

G).

463

Fig. 1 A-G. Synaptophysin imrnunoreactivities in islet cells of rabbit (A), canine (B) and human (C) pancreas. Note the even distribution of the punctate reaction product throughout the cytoplasm of the endocrine cells. D-G Immunoreactivity of pancreatic endocrine cells for synaptophysin in a tissue specimen fixed by a solution consisting of 2% p-formaldehyde, 1% acrolein, 0.1% picric acid and 0.05% glutaraldehyde. Four adjacent semithin sections of a

small islet in the gerbil pancreas were immunostained for insulin (D), synaptophysin (E), PP (F), and somatostatin (G). All islet cells are immunoreactive for synaptophysin. PP-cells (E, arrows) are more densely immunostained by the synaptophysin antiserum than insulin-containing or somatostatin-containing cells. Phasecontrast optics. • 400 A-C; x 600 D-G. Bar. 20 gm

Fig. 2A-G. Intracellular distribution patterns of synaptophysin. In a section of the guinea pig pancreas (A), perinuclear accumulations (arrows) of immunoreactivity are conspicuous in the islet cells. In B and C, concentrations of reaction product are visible all along the peripheral cytoplasm of rabbit islet cells (arrows; asterisk capillary). The peripheral accumulation of synaptophysin in an extrainsular insulin-containing ceil of the rabbit pancreas (D, arrows) is paralleled by a similar distribution of insulin immunopositivity (E, arrows). The process of an insulin-containing cell from the cortex of a canine islet is densely immunostained by both synaptophysin (F, arrow) and insulin (G, arrow) antibodies. Phase-contrast optics, x 770 A; x 940 B; x1200C; x t 0 3 0 D - G . Bar: 10gm

A l t h o u g h all endocrine cell types were f o u n d to be synaptophysin-positive, o u r study revealed differences in the densities o f i m m u n o s t a i n i n g a m o n g the various cell types. C o m p a r e d with other cell types, D-cells were weakly synaptophysin-positive in the canine pancreas. In contrast, A- a n d / o r PP-cells were strongly i m m u n o r e active in several species. Either PP-cells (dog, bovine), A-cells (rat) or b o t h A- and PP-cells, including cells that coexpressed g l u c a g o n and PP (mouse, gerbil), m o s t l y exhibited m a r k e d l y higher s y n a p t o p h y s i n i m m u n o r e a c t i -

vities (Figs. 1 E, 3 A, B, D, F) t h a n the other cell types. As a consequence, they could be visualized at higher dilutions o f the s y n a p t o p h y s i n antiserum. This staining feature was i n d e p e n d e n t o f the location o f the A- a n d PP-cells within the islets or at extrainsular sites. Moreover, it was als independent o f the fixation a n d embedding schedules applied. A few densely i m m u n o s t a i n e d PP-cells were also evident in h u m a n and rat pancreas, b u t the small n u m b e r o f these cells precluded a systematic study. The heterogeneity o f the s y n a p t o p h y s i n i m m u -

464

Fig. 3A-F. Synaptophysin immunoreactivity varies among endocrine cell types. The more densely immunostained synaptophysinpositive cells in the rat (A, arrows), bovine (B, arrows) and gerbil (D, arrows) pancreas are identified on adjacent sections as glucagon-containing cells in rat (not shown), PP-cells in bovine (C) and glucagon-containing cells (E) in gerbil islets. This heterogeneity

is readily visible throughout the postnatal development of the gerbil pancreas, as can be seen in a Bouin-fixed, paraffin-embedded specimen from postnatal day 12 (F). Phase-contrast optics (A-E), interference-contrast optics (F). x390 A; x480 B and C; x440 D and E; x 180 F. Bar: 30 gm

nostaining patterns among endocrine cell types was confirmed by computer-assisted analyses of optical densities of synaptophysin immunoreactivities in the bovine pancreas (data not shown). In the Mongolian gerbil, the cellular heterogeneity of synaptophysin immunoreactivity was detectable from the day of birth onwards (when all endocrine cell types were readily synaptophysin-positive) throughout postnatal development (Fig. 3 F); it was still evident in old gerbils. As mentioned before, all endocrine cell types also displayed synaptophysin immunoreactivity at extrainsular sites. Thus, synaptophysin-positive single cells or small cell groups were encountered within the exocrine acini (Figs. 2D, 4A), in the connective tissue septa and among the epithelia of intra- and interlobular ducts

(Fig. 4B). At both intra- and extrainsular sites, a few synaptophysin-positive cells were immunonegative for the antibodies directed against pancreatic hormones or serotonin (Fig. 4B). In addition to endocrine cells, numerous nerve varicosities could be visualized. Distinct punctate labeling, indicative of nerve terminals or varicosities, was distributed in nerve fiber bundles of the connective tissue septa, in the wall of blood vessels, around exocrine acini and in the islets of Langerhans. Apart from the strongly immunoreactive A- and PP-cells, nerve terminals were more densely immunostained than endocrine cells, and, therefore, remained immunopositive even at higher dilutions of the antiserum (around 1:10000) when islet cells could not be immunostained (Fig. 4 C).

465

Fig. 4A-C. Two extrainsular synaptophysin-positive cells of the rabbit pancreas are shown (A and B). In A, a PP-cell lies between exocrine acinar cells. Synaptophysin immunoreactivity extends into two slender processes and accumulates in the process terminal facing the periacinous space (arrow). In B, a synaptophysin-positive cell, which could not be hormonally identified, is situated among

cells of the ductular epithelium (L lumen of duct). As demonstrated in the gerbil pancreas (C), densely immunostained dots that correspond to nerve varicosities stand out clearly against the islet cells if the synaptophysin antiserum is applied at higher dilutions. Phase contrast optics, x720 A and B; x 540 C. Bar: 10 Ixm

Discussion

in vertebrate species (Wiedenmann and Franke 1985; Johnston et al. 1989b; Cowan et al. 1990). We conclude that synaptophysin is a regular constituent of all pancreatic endocrine cells under normal conditions and may, therefore, be regarded as a reliable universal marker of these cells. In this respect, synaptophysin differs from other protein molecules frequently used as markers of endocrine cells, e.g. chromogranins and neuron-specific enolase (NSE). The chromogranins form a family of acidic proteins that are widespread constituents of secretory granules in endocrine cells (for a review, see Wiedenmann and Huttner 1989). However, conflicting results are reported in the literature concerning the cellular source of chromogranin-like immunoreactivity in the pancreas; indeed, it has been shown that every mammalian species has its own pattern of chromogranin-like immunoreactivity in pancreatic endocrine cells (Grube et al. 1986). The high variability and only partial chromogranin immunostaining in the pancreas, in comparison with synaptophysin, may be attributed to different causes, e.g., " m a s k i n g " of the chromogranin epitopes by co-stored secretory peptides or significant post-translational modification of the chromogranins. On the other hand, the value of NSE as a marker of endocrine cells is limited by the expression of this enzyme in various non-endocrine cells (Haimoto et al. 1985). It is of interest that a few extra- and intrainsular synaptophysin-positive cells were negative for all hormones examined. Probably, these cells belong to a minority of endocrine cells that have hitherto only been defined ultrastructurally (see Grube et al. 1983; K16ppel and Lenzen 1984). However, since we have made no attempt to identify synaptophysin-positive cells by immuno-electron microscopy, it cannot be excluded that they are degranulated members (with respect to dense-core secre-

In this study, the endocrine pancreas of 9 mammalian species has been investigated for the presence and cellular localization of synaptophysin, a major membrane protein of presynaptic vesicles. A main outcome of this investigation is the observation that, in all species examined, all pancreatic endocrine cell types are consistently synaptophysin-positive, independent of their location within the tissue. In addition to endocrine cells, nerve terminals and varicosities are readily visualized in the pancreas with synaptophysin antibodies. The fact that nerve terminals display more marked synaptophysin immunoreactivity than endocrine cells has been discussed elsewhere (Hoog et al. 1988; Redecker et al. 1990). As a practical histochemical consequence, nerves are less suitable as positive controls if synaptophysin staining patterns of endocrine cells are evaluated (cf. H o o g et al. 1988). As with our results concerning synaptophysin immunoreactivity in the pineal gland (Redecker et al. 1990), the synaptophysin immunostaining in the pancreas was relatively insensitive to changes of fixation and embedding procedures. The negative effect of various fixatives (especially those containing glutaraldehyde) or of the duration of fixation exceeding 4 h on the immunohistochemical demonstrability of synaptophysin in the pancreas, as reported by H o o g et al. (1988), seems to be largely caused by fixation-induced alterations of the epitope recognized by the respective monoclonal antibody SY 38. Our observation of constant synaptophysin immunostaining in pancreata of diverse mammalian species, and even in the pancreas of cartilaginous fish (own unpublished findings), is consistent with a relatively high degree of evolutionary conservation of synaptophysin

466 tory granules) of the established pancreatic endocrine cell types. Our results clearly demonstrate that synaptophysin is heterogeneously distributed a m o n g the various cell types of the endocrine pancreas. A subpopulation of endocrine cells, i.e. A- and/or PP-cells, is characterized by stronger immunoreactivity than the other cells. These differences were consistently observed in several species, independent o f the m o d e o f tissue processing. It is unlikely that " m a s k i n g " effects, as discussed for chromogranins, account for these heterogeneities. Therefore, we assume that different amounts of synaptophysin are present in the respective cell types. However, it remains to be elucidated whether these heterogeneities are really caused by different cellular contents of synaptophysin. Previous studies have shown that, in endocrine cells, synaptophysin is localized within a population of small clear vesicles (Navone et al. 1986) that communicate by vesicular m e m b r a n e flow with the p l a s m a l e m m a (Johnston et al. 1989a) and that have a biogenesis independent of secretory vesicles (Cutler and Cramer 1990). In addition, a minor pool of synaptophysin also seems to be present in the membranes of secretory dense-core granules (Lowe et al. 1988; O b e n d o r f et al. 1988; Schilling and Gratzt 1988), although most o f it has recently been shown to be the result of contamination by microvesicles (Fischer yon Mollard et al. 1990). At present, it is uncertain whether the heterogeneity in synaptophysin content reflects differences in the n u m b e r of synaptophysin-conraining microvesicles or in the a m o u n t of synaptophysin per vesicle. According to our observations in the gerbil pancreas, the heterogeneities are a constant feature t h r o u g h o u t the lifetime o f the animal. However, they seem to be species-dependent and m a y vary with the functional state of the cells. The intracellular distribution patterns of synaptophysin immunoreactivities at the light-microscope level, as disclosed in the present investigation, extend previous findings. It has been stated that, in endocrine cells, synaptophysin characteristically accumulates in the perinuclear area of the Golgi complex (Navone et al. 1986). Our immunostaining reveals that, in the endocrine pancreas, synaptophysin immunoreactivity is distributed m o r e frequently throughout the cytoplasm and also fills cellular processes where it m a y accumulate (see also Redecker et al. 1990). Moreover, we have found reaction product in the periphery of the cytoplasm close to the cell surface. For the time being, synaptophysin-positive microvesicles in endocrine cells are thought to constitute a new secretory p a t h w a y that involves recycling of the synaptophysin-positive organelles from the cell periphery, probably to some extent via the Golgi complex (cf. De Camilli and N a v o n e 1987; Johnston et al. 1989a). Interestingly, we have noted that the overall intracellular distribution of synaptophysin and markers of secretory granules (i.e., pancreatic hormones) is often similar, although not completely identical. This m a y reflect a close spatial association of synaptophysin-containing microvesicles with secretory granules in some cases (cf. Navone et al. 1986, for similar ultrastructural observations

in endocrine cells of the adenohypophysis), but the functional significance is not clear.

Acknowledgements. For expert technical assistance, we thank Mrs. H. Peesel, Mrs. D. von Mayersbach, Mr. S. Gudat and Mr. W. Mfiller. In addition, we are indebted to Dr. P.R. Maycox for critically reading the manuscript.

References Buffa R, Rindi G, Sessa F, Gini A, Capella C, Jahn R, Navone F, De Camilli P, Solcia E (1988) Synaptophysin immunoreactivity and small clear vesicles in neuroendocrine cells and related tumours. Mol Cell Probes 2:367-381 Cowan D, Linial M, Scheller RH (1990) Torpedo synaptophysin: evolution of a synaptic vesicle protein. Brain Res 509:1-7 Cutler DF, Cramer LP (1990) Sorting during transport to the surface of PC12 cells: divergence of synaptic vesicle and secretory granule proteins. J Cell Biol 110:721-730 De Camilli P, Navone F (1987) Regulated secretory pathways of neurons and their relation to the secretory pathway of endocrine cells. Ann NY Acad Sci 493:461-479 Fischer von Mollard G, Mignery GA, Baumert M, Perin MS, Hanson TJ, Burger PM, Jahn R, S/idhof TC (1990) Rab3 is a small GTP-binding protein exclusively localized to synaptic vesicles. Proc Natl Acad Sci USA 87:1988-1992 Grube D (1980) Immunoreactivities of gastrin (G-) cells. II. Nonspecific binding ofimmunoglobulins to G-cells by ionic interactions. Histochemistry 66 : 149-167 Grube D, Bohn R (1983) The microanatomy of human islets of Langerhans with special reference to somatostatin (D-) cells. Arch Histol Jpn 46:327-353 Grube D, Kusumoto Y (1986) Serial semithin sections in immunohistochemistry: techniques and applications. Arch Histol Jpn 49: 391-410 Grube D, Eckert I, Speck PT, Wagner H-J (1983) Immunohistochemistry and microanatomy of the islets of Langerhans. Biomed Res 4 [Suppl] :25-36 Grube D, Aunis D, Bader F, Cetin Y, J6rns A, Yoshie S (1986) Chromogranin A (CGA) in the gastro-entero-pancreatic (GEP) endocrine system. I. CGA in the mammalian endocrine pancreas. Histochemistry 85: 441-452 Haimoto H, Takahashi Y, Koshikawa T, Nagura H, Kato K (1985) Immunohistochemical localization of ?,-enolase in normal human tissues other than nervous and neuroendocrine tissues. Lab Invest 52:257-263 Hoog A, Gould VE, Grimelius L, Franke WW, Falkmer S, Chejfec G (1988) Tissue fixation methods alter the immunohistochemical demonstrability of synaptophysin. Ultrastruct Pathol 12 : 673-678 Hsu SM, Raine L, Fanger H (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques. A comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29 : 577 580 Jahn R, Schiebler W, Ouimet C, Greengard P (1985) A 38000dalton membrane protein (i038) present in synaptic vesicles. Proc Natl Acad Sci USA 82:4137-4141 Johnston PA, Cameron PL, Stukenbrok H, Jahn R, DeCamilli P, S/idhof TC (1989a) Synaptophysin is targeted to similar microvesicles in CHO and PC12 cells. EMBO J 8:2863-2872 Johnston PA, Jahn R, Sfidhof TC (1989b) Transmembrane topography and evolutionary conservation of synaptophysin. J Biol Chem 264:1268-1273 K16ppel G, Lenzen S (1984) Anatomy and physiology of the endocrine pancreas. In: K16ppel G, Heitz PU (eds) Pancreatic pathology. Churchill Livingstone, Edinburg, pp 133-153

467 Lowe AW, Madeddu L, Kelly RB (1988) Endocrine secretory granules and neuronal synaptic vesicles have three integral membrane proteins in common. J Cell Biol 106:51-59 Navone F, Jahn R, DiGioia G, Stukenbrok H, Greengard P, DeCamilli P (1986) Protein p38: an integral membrane protein specific for small vesicles of neurons and neuroendocrine cells. J Ceil Biol 103:2511 2527 Obendorf D, Schwarzenbrunner U, Fischer-Colbrie R, Laslop A, Winkler H (1988) In adrenal medulla synaptophysin (protein p38) is present in chromaffin granules and in a special vesicle population. J Neurochem 51 : 1573-1580 Redecker P, Grube D, Jahn R (1990) Immunohistochemical localization of synaptophysin (p38) in the pineal gland of the Mongolian gerbil (Meriones unguiculatus). Anat Embryol 181:433 440 Schilling K, Gratzl M (1988) Quantification of p38/synaptophysin in highly purified adrenal medullary chromaffin vesicles. FEBS Lett 233 : 22-24 Schilling K, Blanco Barco R, Rhinehart D, Pilgrim C (1989) Expression of synaptophysin and neuron-specific enolase during

neuronal differentiation in vitro : effects of dimethyl sulfoxide. J Neurosci Res 24:347-354 Sternberger LA (1986) Immunocytochemistry, 3rd edn. Wiley, New York Wiedenmann B, Franke WW (1985) Identification and localization of synaptophysin, an integral membrane glycoprotein of M 38000 characteristic of presynaptic vesicles. Cell 41:1017-1028 Wiedenmann B, Huttner WB (1989) Synaptophysin and chromogranins/secretogranins - widespread constituents of distinct types of neuroendocrine vesicles and new tools in tumor diagnosis. Virchows Arch [B] 58:95 121 Wiedenmann B, Franke WW, Kuhn C, Moll R, Gould VE (1986) Synaptophysin: a marker protein for neuroendocrine cells and neoplasms. Proc Natl Acad Sci USA 83 : 3500-3504 Wiedenmann B, Waldherr R, Buhr H, Hille A, Rosa P, Hnttner WB (1988) identification of gastroenteropancreatic neuroendocrine cells in normal and neoplastic human tissue with antibodies against synaptophysin, chromogranin A, secretogranin I (chromogranin B), and secretogranin II. Gastroenterology 95:1364-1374