Chromogranin A, chromogranin B and secretogranin II mRNAs in the pituitary and adrenal glands of various mammals. Regulation of chromogranin A, chromogranin B and secretogranin II mRNA levels by estrogen.

BACKGROUND: The chromogranin/secretogranin (Cg/Sg) acidic proteins are widely distributed in vertebrate species. They are thought to play a role in hormone packaging within secretory granules, in hormone secretion, and serve as prohormones for various proteolytic cleavage products. The genes for most members of the Cg/Sg family have been cloned, so hybridization analysis can be used to analyze the distribution and regulation of Cg/Sg mRNAs in various vertebrate species. EXPERIMENTAL DESIGN: The method of in situ hybridization was used to localize chromogranin A, chromogranin B, and secretogranin II in adrenal and pituitary tissues from laboratory animals and from humans in order to analyze the distribution of various Cg/Sg mRNAs in these tissues. To gain some insight into the regulation and possible functions of specific Cg/Sg members, female rats were ovariectomized for different periods with and without estrogen replacement and the pituitaries were subsequently analyzed by in situ hybridization and Northern hybridization analyses. Combined ISH and immunohistochemistry were used to localize the specific cell types in normal rat pituitary that expressed the mRNA for chromogranin A, chromogranin B, and secretogranin II. RESULTS: All three Cg/Sg mRNAs were detected in pituitary and adrenal tissues of rats, mice, dogs, monkeys, and humans. Combined in situ hybridization and immunohistochemistry using rat pituitary revealed that the glycoprotein hormone-secreting cells expressed all three Cg/Sg mRNAs in approximately equal amounts. Ovariectomy followed by estrogen replacement resulted in decreased levels of CgA and SgII mRNAs. In contrast, the level of CgB mRNA, that was not changed by ovariectomy, was increased after estrogen treatment, probably secondary to prolactin cell hyperplasia. CONCLUSIONS: The three principal Cg/Sg mRNAs are present in the adrenal and pituitary of various vertebrates. Estrogen plays a significant role in regulating the mRNA levels of different Cgs/Sgs suggesting functional and regulatory differences in Cg/Sg proteins.     

Immunohistochemistry of chromogranin A and B, and secretogranin II in the canine endocrine pancreas

The chromogranins are large acidic proteins contained in secretion granules of adrenal medullary cells. These proteins are also presumed to be regular constituents of other endocrine cells, e.g., pancreatic endocrine cells. In our previous immunohistochemical studies performed in the endocrine pancreas of 10 mammalian species, only canine endocrine cells lacked any immunoreactivity for chromogranin A. Therefore, this problem was reinvestigated in the present study. Antisera against bovine and rat chromogranin A, B, and C (= secretogranin II) were applied under various conditions of the immunohistochemical protocol. Upon meeting certain requirements, chromogranin-immunoreactivities were found also in canine pancreatic B- (insulin), PP- (pancreatic polypeptide), and EC- (enterochromaffin) cells. The immunohistochemical data suggest that canine chromogranins partially differ structurally or immunologically from bovine chromogranins and completely from rat chromogranins. In addition, the present findings confirm our previous findings about both interspecies differences in the cellular localization of chromogranins in the endocrine pancreas and the peculiar localization of secretogranin II to pancreatic PP-cells. Finally, the present methodological studies have shown that even the buffer used in the immunohistochemical protocol may be decisive for a false-positive or false-negative immunostaining.     

Polysialylated N-CAM, chromogranin A and B, and secretogranin II in neuroendocrine tumours of the lung

 Highly α2–8-sialylated N-CAM (neural cell adhesion molecule) impairs N-CAM-mediated cell adhesion. We investigated polysiaN-CAM immunoreactivity in a range of neuroendocrine lung tumours: 15 typical carcinoids, 21 atypical carcinoids, 2 large cell neuroendocrine carcinomas and 12 small cell lung carcinomas were selected on a morphological basis and by their immunoreactivity for chromogranin A and B and secretogranin II. A progressive loss of chromogranin expression, particularly of chromogranin B, was paralleled by the up-regulation of polysiaN-CAM in histologically more aggressive tumours (P = 0.001). These data support the hypothesis that loss of cell–cell adhesion properties might be a relevant factor in the origin of the aggressivity of lung neuroendocrine tumours. Received: 17 October 1996 / Accepted: 7 January 1997     

Aspects of the neuroendocrine cerebellum: expression of secretogranin II,chromogranin A and chromogranin B in mouse cerebellar unipolar brush cells

Morphologically distinct neuron classes can be subdivided in sublineages by differential chemical phenotypes that correlate with functional diversity. Here we show by immunocytochemistry that chromogranin A (CgA) chromogranin B (CgB) and secretogranin II (SgII), the principal granins situated in neuronal secretory granules and large dense-core vesicles, are widely but differentially expressed in cells of the mouse cerebellum and terminals of cerebellar afferents. While CgA and CgB were nearly panneuronal, SgII was more restricted in distribution. The cells most intensely immunoreactive for SgII were a class of small, excitatory interneurons enriched in the granular layer of the vestibulocerebellum, the unipolar brush cells (UBCs), although larger neurons likely to be a subset of the Golgi–Lugaro–globular cell population were also distinctly immunopositive; by contrast, Purkinje cells and granule cells were, at best, faintly stained and, stellate, basket cells were unstained. SgII was also present in subsets of mossy fibers, climbing fibers and varicose fibers. Neurons in the cerebellar nuclei and inferior olive were distinctly positive for the three granins. Double-labeling with subset-specific cell class markers indicated that, while both CgA and CgB were present in most UBCs, SgII immunoreactivity was present in the calretinin (CR)-expressing subset, but lacked in metabotropic glutamate receptor 1alpha (mGluR1α)-expressing UBCs. Thus, we have identified an additional cell class marker, SgII, which serves to study subtype properties in the UBC population. The abundance of SgII in only one of the two known subsets of UBCs is remarkable, as its expression in other neurons of the cortex was moderate or altogether lacking. The data suggest that the CR-positive UBCs represent a unique neuroendocrine component of the mammalian cerebellar cortex, presumably endowed with transynaptically regulated autocrine or paracrine action/s. Because of the well-known organization of the cerebellar system, several of its neuron classes may represent valuable cellular models to analyze granin functions in situ, in acute slices and in dissociated cell and organotypic slice cultures.     

Distribution of chromogranin A and secretogranin I (chromogranin B) in neuroendocrine cells and tumors.

The distribution of chromogranin A and secretogranin I (chromogranin B) in normal and neoplastic human endocrine tissues was analyzed with two human monoclonal antibodies against chromogranin A, anti-bovine antiserum against chromogranin A, and an anti-rat antiserum against secretogranin I. Western blotting analyses showed both chromogranin A and secretogranin I in normal adrenals, pheochromocytomas, a pituitary adenoma, and in normal pituitary glands, but not in a bladder carcinoma. Rat adrenal medullary and anterior pituitary tissues reacted with the polyclonal chromogranin A and secretogranin I antisera, but not with the two monoclonal chromogranin A antibodies. All antibodies reacted with most of the neuroendocrine cells and tumors examined. Pituitary prolactinomas contained immunoreactive secretogranin I, but not chromogranin A. Analysis of the distribution of chromogranin A and secretogranin I in pancreatic islet cells showed that chromogranin A was found predominantly in the glucagon-producing A cells, whereas secretogranin I was present in less than 5% of islet cells. These results indicate that chromogranin A and secretogranin I are both useful in the characterization of some neuroendocrine cells and neoplasms.     

Effects of nerve growth factor on the biosynthesis of chromogranin A and B, secretogranin II and carboxypeptidase H in rat PC12 cells.

We investigated the biosynthesis of various constituents (chromogranins A and B, secretogranin II, carboxypeptidase H and synaptin/synaptophysin) of large dense core and small vesicles in PC12 cells. These cells were treated for up to 18 days with nerve growth factor. Peptide levels were determined by quantitative immunoblotting, their mRNAs by Northern blotting. Nerve growth factor treatment changed the levels of the various peptides investigated and their mRNAs in three patterns. Peptide and mRNA levels for chromogranin A and chromogranin B were increased on day 1 and then declined. Synaptin/synaptophysin levels slightly decreased from day 1 onwards. On the other hand secretogranin II increased steadily up to 217% for peptide levels and 257% for mRNA levels. For carboxypeptidase H for which only the mRNA could be determined an analogous behaviour was seen. Its mRNA after 14 days of nerve growth factor treatment was 459% of controls. These results establish that the biosynthesis of the secretory proteins chromogranin A, chromogranin B and secretogranin II is regulated differentially during nerve growth factor treatment. We suggest that neuronal differentiation is accompanied by an increased biosynthesis of secretogranin II. For carboxypeptidase H, the marked increase in mRNA levels after nerve growth factor treatment is the first example that the biosynthesis of this peptide is significantly up-regulated. Synaptin/synaptophysin biosynthesis is not increased although this peptide is a major constituent of small vesicles which increase in number during nerve growth factor treatment.     

The prevalence and clinical significance of chromogranin A and secretogranin II immunoreactivity in colorectal adenocarcinomas

Colorectal adenocarcinomas may display features of endocrine differentiation, shown by argyrophil stains and by the expression of endocrine markers such as chromogranin A. We investigated chromogranin A and secretogranin II immunoreactivity in a series of 208 carcinomas of the large bowel to assess the prevalence and clinical significance of endocrine differentiation. Tumours expressing endocrine markers were classified as low expressors (< than 1 immunoreactive tumour cell/mm²) and high expressors (> than 1 immunoreactive tumour cell/mm²). There were 33 (16%) carcinomas showing both chromogranin A and secretogranin II immunoreactivity: 11 tumours (5%) were high expressors. Endocrine differentiation was not related to the disease stage, tumour location, grade, DNA ploidy and p53 protein accumulation. In the entire series chromogranin A immunoreactivity did not provide prognostic information using univariate and multivariate analysis. A worse overall survival (P=0.048) was demonstrated for the stage III patients with high expressor tumours, but there were only five patients in this group. The results of our investigation suggest that chromogranin A immunoreactivity is not a useful variable in the prognostic assessment of colorectal adenocarcinomas.     

Comparison of chromogranins A, B, and secretogranin II in human adrenal medulla and pheochromocytoma

Human phenochromocytomas were analyzed for the presence of chromogranins A and B and secretogranin II (chromogranin C). In immunohistochemistry, a positive staining of the tumor cells was observed for all three antigens. For immunoblotting, a method had to be developed which prevented proteolytic degradation of the antigens. Boiling homogenates of freeze-dried tissues achieved this and in addition, led to a significant enrichment of these secretory proteins. All three antigens could be identified in the tumor by immunoblotting. Chromogranin A and B were the major components present in about equal amounts. The relative concentration of all these antigens in the tumors was similar to those in adrenal medulla. A method involving high pressure liquid chromatography is presented which allows the separation of the human chromogranins/secretogranins.     

Chromogranin A and B and secretogranin II in bronchial and intestinal carcinoids

Summary Carcinoid tumours (bronchial and intestinal) were analyzed by immunoblotting for the presence of chromogranin A, B and secretogranin II. In all tumours an antigen corresponding in electrophoretic behaviour to adrenal chromogranin A was present. Lung carcinoids (3 out of 5) contained a relatively high concentration of a proteoglycan form of this antigen in addition. Chromogranin B was found in all tumours. In one and two dimensional immunoblotting it appeared identical to the corresponding adrenal antigen. Secretogranin II was also present, however concentrations (especially in intestinal carcinoids) were low and variable. Furthermore, in intestinal tumours it differed from the adrenal antigen by having a slightly higher molecular size and a more alkaline pI. Immunohistochemistry revealed that the tumour tissues stained positively for all three antigens. For secretogranin II the staining in intestinal tumours was relatively weak and quite variable. These results should provide a defined basis for immunohistochemical screening of carcinoids for the chromogranin/secretogranin antigens.     

Chromogranins A and B and secretogranin II: evolutionary and functional aspects

Chromogranins/secretogranins or granins are a class of acidic, secretory proteins that occur in endocrine, neuroendocrine, and neuronal cells. Granins are the precursors of several bioactive peptides and may be involved in secretory granule formation and neurotransmitter/hormone release. Characterization and analysis of chromogranin A (CgA), chromogranin B (CgB), and secretogranin II (SgII) in distant vertebrate species confirmed that CgA and CgB belong to related monophyletic groups, probably evolving from a common ancestral precursor, while SgII sequences constitute a distinct monophyletic group. In particular, selective sequences within these proteins, bounded by potential processing sites, have been remarkably conserved during evolution. Peptides named vasostatin, secretolytin and secretoneurin, which occur in these regions, have been shown to exert various biological activities. These conserved domains may also be involved in the formation of secretory granules in different vertebrates. Other peptides such as catestatin and pancreastatin may have appeared late during evolution. The function of granins as propeptide precursors and granulogenic factors is discussed in the light of recent data obtained in various model species and using knockout mice strains.     

Immunohistochemical localization of secretogranin II in the rat cerebellum

Secretogranin II (chromogranin C) is a peptide related to chromogranin A and secretogranin I (chromogranin B) which is secreted by a regulated pathway from both neurons and endocrine cells. In the present study we have determined by light microscopic immunocytochemistry its distribution in the cerebellum and in adjacent brain stem regions. Secretogranin II was found to be widely distributed throughout the gray matter of these regions. Highly immunoreactive structures in the cerebellar cortex included the majority of climbing fibers, a large number of mossy fibers, sparse varicose fibers in the molecular layer and a subpopulation of neuronal perikarya in the granule cell layer. The location and shape of these neurons are very similar to those of a novel type of cerebellar neurons which has been recently described. A moderate level of immunoreactivity was observed on fibers travelling among Purkinje cells and parallel to the pial surface in the Purkinje cell layer. A variable, but in general low, degree of immunoreactivity was also detectable in the perikarya of Purkinje cells. In the deep cerebellar nuclei a loose network of secretogranin II-positive fibers was visible. Neurons of the nuclei, however, were non-immunoreactive. A dense network of highly immunoreactive fibers was found throughout the brain stem regions adjacent to the cerebellum. Our results indicate that secretogranin II has in the cerebellum and adjacent regions a distribution more widespread than that of known regulatory peptides and suggest that the peptide-mediated signaling in the cerebellum plays a role more important that has been acknowledged so far.     

Immunohistochemical localization of chromogranins A and B and secretogranin II in normal, hyperplastic and neoplastic prostate

Routinely processed normal, hyperplastic and neoplastic prostatic tissue was immunohistochemically investigated with antibodies against chromogranin A and B and secretogranin II. In normal and hyperplastic prostates all three peptides were immunolocalized in scattered neuroendocrine cells situated within the glandular epithelium. In 17 prostatic carcinomas with pronounced neuroendocrine differentiation and in a case of prostatic carcinoid, chromogranin B was the major component whereas chromogranin A and secretogranin II were virtually absent in poorly differentiated (grade III) tumours. Neuroendocrine differentiation in prostatic cancer is most likely to be associated with a poor clinical outcome; thus, chromogranin B appears to be a useful marker in the histopathological diagnosis of these neoplasms.     

Differential regulation of beta-arrestin 1 and beta-arrestin 2 gene expression in rat brain by morphine

Beta-arrestins are a family of regulatory and scaffold proteins functioning in signal transduction of G protein-coupled receptors including opioid receptors. Upon agonist stimulation, beta-arrestins bind to opioid receptors phosphorylated by G protein-coupled receptor kinases and promote receptor internalization and desensitization. Studies indicated that beta-arrestins are required in the development of morphine tolerance in mice. In the current study, we investigated the potential regulatory effects of morphine administration on beta-arrestin 1 and beta-arrestin 2 mRNA levels in different brain regions in rat using in situ hybridization method. Our results showed that the acute morphine administration (10 mg/kg) resulted in approximately 30% reduction in both beta-arrestin 1 and beta-arrestin 2 mRNA levels in hippocampus while the chronic morphine treatment (10 mg/kg, b.i.d., for 9 days) caused no significant change in level of either beta-arrestin mRNA. In locus coeruleus, both acute and chronic morphine treatments resulted in significant decreases (over 50%) in beta-arrestin 1 mRNA level but failed to induce any change in the level of beta-arrestin 2 gene expression. The acute morphine administration had no significant effect on beta-arrestin 1 or beta-arrestin 2 mRNA level in periaqueductal gray and cerebral cortex. However, after chronic morphine treatment, beta-arrestin 2 mRNA level decreased by 40% in periaqueductal gray and increased by 25% in cerebral cortex, in strong contrast to the unchanged beta-arrestin 1 mRNA level in these two brain regions. Furthermore, spontaneous or naloxone-precipitated withdrawal of morphine that did not affect the level of beta-arrestin 1 mRNA resulted in an aberrant increase (100% over control) in beta-arrestin 2 mRNA level in hippocampus. Our results thus demonstrated for the first time that opiate administration regulates level of beta-arrestin mRNAs in brain and the expression of beta-arrestin 1 and beta-arrestin 2 subtypes is differentially regulated in locus coeruleus, periaqueductal gray, and cerebral cortex by morphine. These data suggest that beta-arrestin 1 and beta-arrestin 2 may play different roles in the development of opioid tolerance and dependence.     

Granin proteins (chromogranin A and secretogranin II C23-3 and C26-3) in the intestine of reptiles

The occurrence, distribution and the possible cellular co-localizations of chromogranin A (CgA) and of two synthetic secretogranin II-peptides (SgIIC23-3 and SgIIC26-3) with several enteric neuropeptides and serotonin have been investigated immunohistochemically in turtles, lizards and snakes. The distribution of CgA-immunoreactivity was restricted only to the enteroendocrine cells in all the reptiles studied. SgII-immunoreactivity--absent in turtle--revealed nerve cells and fibers, besides enteroendocrine cells in lizard and snake guts. Moreover, the two antisera (C23-3 and C26-3) raised against the different regions of the SgII-molecule yielded distinct distribution patterns of immunoreactivity both in the lizard and snake organs. Small amounts of enteric serotonin cells co-stored CgA or SgIIC23-3 in lizards and snakes and only SgIIC26-3-peptide in snakes. CgA was found co-stored with somatostatin in a few enterocytes of the turtle duodenum. In the same gut segment of lizards and throughout the snake organ, neurotensin and the SgIIC23-3-peptide co-existed in a small number of endocrine cells. The pancreatic polypeptide-containing cells were devoid of immunoreactivity both for CgA and SgII. Bombesin immunopositive cells were absent throughout the intestines of the reptiles investigated. The above findings entirely support the heterogenous distribution of granins in neuroendocrine organs and tissues and also within the same neuroendocrine cell population. They further support the concept of a good conservation of granins during phylogeny.     

Pancreastatin secretion by pituitary adenomas and regulation of chromogranin B mRNA expression.

Pancreastatin, a carboxyl-terminal amidated peptide derived from chromogranin (Cg)A, inhibits secretion of insulin and parathyroid hormone. Our recent studies found significant amounts of immunoreactive pancreastatin in all pituitary adenomas except prolactin adenomas. To analyze the effects of pancreastatin on pituitary cell function, 17 cultured pituitary adenomas were examined for immunoreactive pancreastatin and pancreastatin secretion by the tumors. The effects of pancreastatin on pituitary hormone secretion and on pituitary hormone (follicle-stimulating hormone and prolactin), CgA, and CgB mRNA levels were also examined. Immunoreactive pancreastatin and CgA were present diffusely in gonadotroph and null cell adenomas, but only a few prolactin adenoma cells expressed pancreastatin or CgA. When cells were treated with hypothalamic peptides, gonadotroph adenomas were the only group that released increased amounts of pancreastatin in response to gonadotropin-releasing hormone (10(-7) mol/L). Pancreastatin (10(-7) mol/L) treatment did not stimulate pituitary hormone secretion significantly. In situ hybridization analyses showed that gonadotropin-releasing hormone and pancreastatin treatment led to significant increases in CgB and follicle-stimulating hormone mRNAs in gonadotroph adenomas, whereas CgA mRNA levels did not change significantly. These results show that there is a differential distribution of pancreastatin secretion in pituitary adenomas and that the hypothalamic hormone gonadotropin-releasing hormone and the CgA-derived peptide pancreastatin can regulate CgB mRNA in gonadotroph adenomas, suggesting an autocrine effect of pancreastatin on pituitary tumor function.     

Secretoneurin in bronchopulmonary carcinoids—immunohistochemical comparison with chromogranins A and B and secretogranin II

Ninety-nine classical and 11 atypical bronchopulmonary carcinoids were investigated immunohistochemically with an antibody against secretoneurin, a peptide proteolytically processed from secretogranin II (chromogranin C), as well as antibodies against chromogranin A and B and secretogranin II. Secretoneurin was immunolocalized in 86 tumours (78 classical and eight atypical carcinoids); secretogranin II was found in the same tumours in a similar distribution, whereas chromogranin A was present in all 100 and chromogranin B in 106 tumours investigated. Bronchopulmonary carcinoids are usually not associated with clinically or biochemically distinct syndromes. Although we found bronchial carcinoids with different immunohistochemical chromogranins/secretogranin patterns, no correlation with the biological behaviour of these tumours could be demonstrated.     

Immunohistochemical demonstration of chromogranin A,chromogranin B,and secretoneurin in primary non-small-cell carcinomas of the lung

Fifty-three primary non-small-cell lung carcinomas (NSCLC) were immunohistochemically investigated with antibodies against chromogranin A, chromogranin B, and ecretoneurin. All 3 peptides were focally immunolocalized in 3 of 25 adenocarcinomas and in 2 of 6 large-cell anaplastic carcinomas in more than 20% of tumor cells. Two of 15 squamous-cell carcinomas showed chromogranin B reactivity in more than 20% of tumor cells. Neuroendocrine (NE) differentiation was also demonstrated in lymphnode metastases of large-cell anaplastic carcinomas, in 1 adenocarcinoma, and in 1 squamous-cell carcinoma, with NE differentiation of the respective primary tumors. All tumors with NE differentiation exhibited (large cell) anaplastic tumor areas. We conclude that NE differentiation should be immunohistochemically proven or excluded, particularly in NSCLC with anaplastic components. Chromogranin B and secretoneurin are proposed as useful additional neuroendocrine markers for demonstration of NE differentiation in lung carcinomas.     

Neonatal rat brain astroglial dipeptidyl peptidase II activity regulation by cations and anions

Astrocytic glial cells from neonatal rat brains were grown in primary culture. Dipeptidyl peptidase II (DPP-II) enzyme activity was measured in cells disrupted by nonionic detergent. The rate of enzyme activity was measured in the presence of various ions, under isosmotic conditions adjusted using mannitol and NaCl. DPP-II activity was not affected by the candidate metal co-factors (2 mM) Co2+, Mg2+, and Mn2+, nor by the metal chelators EDTA and o-phenanthroline. However, selected cations (50 mM Cl- salts) significantly inhibited DPP-II activity compared to Na+ control; the relative inhibition ranking was Rb+ less than K+ less than Zn2+ less than Hg2+. Many test anions (50 mM Na+ salts) also inhibited DPP-II activity compared to Cl- control: SO4(2-) less than NO3- less than F- less than SO3(2-). Surprisingly, the anion S2O3(2-) was the only test agent which strongly stimulated activity. The data are consistent with the concept that specific ion species interact with the glial DPP-II enzyme to affect catalytic activity.     

Granin proteins (chromogranin A and secretogranin II C23-3 and C26-3) in the endocrine pancreas of amphibians

The occurrence and cellular distribution of chromogranin A (CgA) and of two synthetic secretogranin II (SgII)-fragments (termed C23-3 and C26-3) has been investigated immunohistochemically in the endocrine pancreas of five amphibian species. Immunoreactivity for CgA was detected only in specimens of the genus Rana, whereas for SgII it was found in all the urodeles and anurans studied. Either CgA or the SgII-fragment displayed its own cellular distribution patterns in the endocrine pancreas of a given species. Moreover, immunoreactivity for both regions (C23-3 and C26-3) of the SgII-molecule exhibited by the same endocrine cell population have been encountered in newt and frog organs. Besides the interspecific heterogeneous distribution of CgA and of the two SgII-fragments in relation to the insular cell types, a striking heterogeneity of their immunostaining density among the endocrine cells of the same type was also revealed. The above findings entirely support the concept of a good conservation of granins during phylogeny; they do not support, however, the previously ascribed usefulness of these anionic glycoproteins as markers for all neuro-endocrine cells.     

Granin proteins (chromogranin A and secretogranin II C23-3 and C26-3) in the endocrine pancreas of reptiles.

The endocrine pancreas of four reptile species belonging to the turtles, lizards and snakes was investigated immunohistochemically for the occurrence and cellular distribution of chromogranin A (CgA) and of two synthetic secretonin II (SgII)-peptides (C23-3 and C26-3). CgA-immunoreactivity was found only in the turtle pancreas, whereas that for SGIIC23-3 appeared both in the turtle and snake. None of the species studied displayed immunoreactivity for SgIIC26-3. The two detected granins showed different distributions in relation to the endocrine cell types. Conspicuous variations of the immunostaining density for either granin in the same endocrine cell population and even complete lack of the immunoreaction were recorded. The findings suggest that, despite the restricted presence in the endocrine pancreas of the reptiles investigated, the granins are relatively well conserved during phylogeny; they do not confirm, however, the previously accepted usefulness of the granin protein family as common markers of neuroendocrine cells.