P29: a novel tyrosine-phosphorylated membrane protein present in small clear vesicles of neurons and endocrine cells.

A novel membrane protein from rat brain synaptic vesicles with an apparent 29,000 Mr (p29) was characterized. Using monospecific polyclonal antibodies, the distribution of p29 was studied in a variety of tissues by light and electron microscopy and immunoblot analysis. Within the nervous system, p29 was present in virtually all nerve terminals. It was selectively associated with small synaptic vesicles and a perinuclear region corresponding to the area of the Golgi complex. P29 was not detected in any other subcellular organelles including large dense-core vesicles. The distribution of p29 in various subcellular fractions from rat brain was very similar to that of synaptophysin and synaptobrevin. The highest enrichment occurred in purified small synaptic vesicles. Outside the nervous system, p29 was found only in endocrine cell types specialized for peptide hormone secretion. In these cells, p29 had a distribution very similar to that of synaptophysin. It was associated with microvesicles of heterogeneous size and shape that are primarily concentrated in the centrosomal-Golgi complex area. Secretory granules were mostly unlabeled, but their membrane occasionally contained small labeled evaginations. Immunoisolation of subcellular organelles from undifferentiated PC12 cells with antisynaptophysin antibodies led to a concomitant enrichment of p29, synaptobrevin, and synaptophysin, further supporting a colocalization of all three proteins. P29 has an isoelectric point of approximately 5.0 and is not N-glycosylated. It is an integral membrane protein and all antibody binding sites are exposed on the cytoplasmic side of the vesicles. Two monoclonal antibodies raised against p29 cross reacted with synaptophysin, indicating the presence of related epitopes. P29, like synaptophysin, was phosphorylated on tyrosine residues by endogenous tyrosine kinase activity in intact vesicles.

Production of the alpha subunit of guanine nucleotide-binding protein GO by neuroendocrine tumors.

We have found that neuroendocrine tumors (including neuroblastoma, ganglioneuroma, gut carcinoid, pheochromocytoma, medullary thyroid carcinoma, insulinoma, glucagonoma, prolactinoma, carotid body tumor, and small cell lung carcinoma) produce considerable amounts (about 1000-80,000 ng/g tissue) of the alpha subunit of guanine nucleotide-binding protein, GO (GO alpha), whereas nonneuroendocrine tumors contain less than 300 ng of GO alpha/g tissue. GO alpha in the neuroendocrine tumors was present both in the soluble fraction, and cholate-extractable membrane-bound fraction of tissues. Immunoblots of membrane fractions of neuroblastoma and carcinoid tissues confirmed that the immunoreactive substance in the tumor tissues was GO alpha. Immunohistochemically, GO alpha was localized consistently in the cell membrane and occasionally in the cytoplasm of neuroendocrine tumors. GO alpha was also detected in sera of 73% patients with neuroblastoma at diagnosis, whereas serum GO alpha concentrations in control children, or patients with nonneuroendocrine tumors were lower than the detection limit of the immunoassay method employed. Serum GO alpha concentrations in patients with neuroblastoma changed with the clinical course; they fell in patients responding to treatment and increased in patients who relapsed. Since GO alpha, a specific protein in the neural and neuroendocrine cells, was found to be produced in considerable amounts by all types of neuroendocrine tumors but not in nonneuroendocrine tumors, GO alpha might be a useful biomarker for neuroendocrine tumors.

Chromogranin A biosynthetic cell populations in bovine endocrine and neuronal tissues: detection by in situ hybridization histochemistry.

Chromogranin A is a highly acidic protein that is found in the secretory granules of many endocrine and neuronal cells. To localize bovine cell populations involved in chromogranin A biosynthesis, the distribution of the mRNA encoding this protein was determined with in situ hybridization histochemistry. In the adrenal gland, the mRNA was found in the chromaffin cells of the medulla but was absent from the cortex. The distribution of the mRNA in the medulla was uneven; cells located at the periphery were more heavily labeled than those in the center of the gland. Because the adrenal medulla is composed of several cell types, the chromogranin A-containing cells were further characterized for the presence of neuropeptide and adrenergic markers. Adjacent sections were examined for the mRNAs encoding enkephalin and phenylethanolamine N-methyltransferase (PNMT), the enzyme that catalyzes the formation of epinephrine from norepinephrine. Both mRNAs were present in a narrow band of cells at the periphery of the medulla. However, in contrast to the distribution of chromogranin A mRNA, the enkephalin and PNMT mRNAs were detected in only a small number of cells in the inner medullary region. The difference in the distribution of the enkephalin and PNMT mRNAs from that of chromogranin A suggests that the expression of these genes is differentially regulated. In addition to the adrenal gland, chromogranin A mRNA is expressed by many other tissues. In the parathyroid gland, which is rich in the mRNA but exhibits little chromogranin A-like immunoreactivity, the message was present in most cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Parvalbumin exists in rat endocrine glands.

Simple and rapid purification procedures for parvalbumin, one of the Ca2+-binding proteins (extracted from rat skeletal muscle), were developed, and its antiserum was produced in rabbits to measure the parvalbumin content of various rat tissues by RIA. The heat treatment, ammonium sulfate fractionation, and trichloroacetic acid precipitation of soluble fraction from rat skeletal muscle followed by single diethylaminoethyl Sephadex A-50 column chromatography yielded a pure 120 mg protein from 150 g skeletal muscle. Amino acid analysis, together with electrophoretic mobility, indicated that the protein was identical to parvalbumin. The antisera to this rat skeletal muscle parvalbumin (raised in rabbits) did not cross-react with calmodulin or S-100 proteins. The RIA for parvalbumin using this antisera and [125I]parvalbumin revealed that skeletal muscle and brain contained high levels of the antigen; the values of which were 69,486 +/- 4,933.1 and 881 +/- 165.6 ng/mg protein, respectively. However, the parvalbumin antigen in the heart, lung, liver, and spleen could not be detected. On the other hand, the contents of the antigen in the endocrine glands (in nanograms per mg protein) were as follows: pituitary (125 +/- 46.6), thyroid (108 +/- 50.0), adrenal (341 +/- 64.3), testes (227 +/- 37.2), and ovaries (218 +/- 10.1). All of these values were comparable to levels of antigen found in the brain sample. These results suggest an important role for parvalbumin in endocrine glands.

Immunohistological evidence for renin in human endocrine tissues.

The peroxidase-labeled antibody method and the avidin-biotin-complex method with antiserum to purified human kidney renin were used to identify renin in human endocrine tissues. Renin immunoreactivity was found in some large cells of the anterior pituitary, the zona glomerulosa and the zona reticularis of the adrenal, the Leydig cells of the testis, and the follicular epithelial cells of the thyroid and prostate glands. The specificity of the immunohistochemical reaction was confirmed by immunoabsorption tests. The specific localization of immunoreactive renin in each tissue suggests a possible role of renin in the function of these tissues.

Immunoreactive human chromogranin A in diverse polypeptide hormone producing human tumors and normal endocrine tissues.

Chromogranin A, the major soluble protein costored and coreleased by exocytosis with catecholamines from the adrenal medulla, has recently been detected in several bovine polypeptide hormone producing tissues. We therefore searched for chromogranin, by immunohistology, in human polypeptide hormone producing tumors as well as normal human endocrine tissues. The chromogranin A antigen was purified from catecholamine storage vesicles of human pheochromocytoma, to which rabbit antisera were developed, allowing immunohistologic studies by the indirect rabbit anti-peroxidase technique. Specific chromogranin staining was noted in all polypeptide hormone producing human tumors studied (pheochromocytoma chromaffin cells, n = 3; medullary thyroid carcinoma parafollicular C cells, n = 2; thyroidal C cell hyperplasia cells, n = 1; parathyroid adenoma chief cells, n = 1; pancreatic islet cell tumor islet cells, n = 1; oat cell carcinoma cell line M-103) as well as in all normal polypeptide hormone producing tissues (adrenal medulla chromaffin cells, parathyroid chief cells, thyroid parafollicular C cells, pancreatic islet cells, gut enteroendocrine cells, and anterior pituitary cells). Chromogranin may have a widespread distribution in human polypeptide hormone producing tissues, and may be a useful histologic marker for peptide producing tumors.

Proteinases and their inhibitors in cells and tissues.

A large body of evidence has been assembled to indicate the substantial importance of proteolytic processes in various physiological functions. It has recently become clear too that endo-acting peptide bond hydrolases provisionally characterized and classified at present as serine, cysteine, aspartic and metallo together with unknown catalytic mechanism proteinases sometimes act in cascades. They are controlled by natural proteinase inhibitors present in cells and body fluids. In the first part of the present monograph the author was concerned to present an overview on the morphological and physiological approach to localization, surveying reaction principles and methods suitable for visualization of proteolytic enzymes and their natural and synthetic inhibitors. In the second part the roles played by proteinases have been summarized from the point of view of cell biology. The selection of earlier and recent data reviewed on the involvement of proteolysis in the behavior of individual cells reveals that enzymes, whether they be exogeneous or intrinsic, can be effective and sensitive modulators of cellular growth and morphology. There exists a close correlation between malignant growth and degradation of cells. It appears likely that as yet unknown or at least so far inadequately characterized factors that influence the survival or the death of cells may turn out to be proteinases. The causal role of extracellular proteolysis in cancer cell metastases, in stopping cancer cell growth and in cytolysis remains for further investigated. Ovulation, fertilization and implantation are basic biological functions in which proteolytic enzymes play a key role. The emergence of new approaches in reproductive biology and a growing factual basis will inevitably necessitate a reevaluation of present knowledge of proteolytic processes involved. The molecular aspects of intracellular protein catabolism have been discussed in terms of the inhibition of lysosomal and/or non-lysosomal protein breakdown. Peptide and protein hormone biosynthesis and inactivation are still at the centre of interest in cell biology, and a number of proteinases have been implicated in both processes. A number of conjectures partly based on the author's own work have been discussed which suggest the possibility of the involvement of proteolysis in exocytosis and endocytosis. The author's optimistic conclusion is that through the common action of biochemists, cell biologists, cytochemists, and pharmacologists the mystery of cellular proteolysis is beginning to be solved.

Qualitative and quantitative distribution of plasminogen activators in organs from healthy adult mice.

Twenty organs from healthy adult mice were tested for plasminogen activator activity. All were positive although specific activities varied 200-fold. Tissues with high activity were lung, uterus, brain and kidney. Endocrine glands were moderately rich in activator activity, and lymphoid tissues were poor. Molecular mass characterization was carried out. Two enzymatic forms were observed in all twenty organs: a 70 kDa form similar to human tissue plasminogen activator and a 48 kDa form analogous to mouse urokinase.

Detection of chromogranins A and B in endocrine tissues with radioactive and biotinylated oligonucleotide probes.

We analyzed the distribution of chromogranins A and B in normal and neoplastic endocrine tissues with secretory granules using 35S-labeled and biotin-labeled oligonucleotide probes by in situ hybridization (ISH). Both radioactive and nonradioactive probes detected messenger RNAs (mRNAs) in frozen and paraffin tissue sections. Endocrine tissues with variable immunoreactivities for chromogranin A protein, such as small-cell lung carcinomas, neuroblastomas, insulinomas, and parathyroid adenomas, expressed the mRNA for chromogranins A and B in most cells. Some technical problems with the biotinylated probes included nonspecific nuclear staining and endogenous alkaline phosphatase, which was not completely abolished by levamisole pretreatment. A differential distribution of chromogranins A and B was seen in pituitary prolactinomas, which expressed abundant chromogranin B but not chromogranin A mRNAs, and in parathyroid adenomas, which expressed abundant chromogranin A but only small amounts of chromogranin B mRNAs. These results indicate that ISH can be used to detect chromogranins A and B in endocrine tissues with radioactive and biotinylated oligonucleotide probes and that the mRNAs for chromogranin A and B are demonstrable in some tumors even when the chromogranin proteins cannot be detected by immunohistochemistry.

Immunological characterization of a membrane glycoprotein of chromaffin granules: its presence in endocrine and exocrine tissues.

A glycoprotein was isolated from detergent solubilized membranes of bovine chromaffin granules by high-performance liquid chromatography. Specific antisera raised against this glycoprotein reacted in one- and two-dimensional immunoblots with a heterogeneous component with a pI of 4.2-4.7 and Mr 100,000. The antiserum against bovine glycoprotein II cross-reacted with an analogous component in several species. The specific localization of glycoprotein II in chromaffin granules was established by density gradient centrifugation followed by immunoblotting. The antiserum, as shown by one- and two-dimensional immunoblotting, reacted with an analogous antigen in the posterior pituitary, in endocrine (anterior pituitary, parathyroid gland) and exocrine (parotid gland, pancreas) organs. In the pancreas the protein reacting with the antiserum was found in the membranes of zymogen granules. The results demonstrate for the first time that secretory vesicles of endocrine and exocrine tissues have at least one common antigen, i.e. the glycoprotein II. It seems likely that this protein is involved in a basic function common to all secretory vesicles.

Occurrence and distribution of glycoconjugates in human tissues as detected by the Erythrina cristagalli lectin.

We applied a horseradish peroxidase-Erythrina cristagalli agglutinin (HRP-ECA) conjugate for histochemical staining of tissue sections from various formalin-fixed, paraffin-embedded human tissue specimens. The HRP-ECA conjugate showed broad reactivity, but there was a distinct distribution of native (not masked by sialic acid) and sialic acid-masked ECA binding sites in the various organs. Free ECA binding sites could be detected on red blood cells, lymphocytes of thymus, tonsil, lymph node, and in mucous substances of different organs. Independent of blood group type, the vascular endothelium exhibited strong ECA reactivity. Free ECA binding sites occurred in the cytoplasm of Kupffer's cells in liver, in histiocytic cells of thymus, lymph node, tonsil, and in bone marrow. Podocytes of kidney glomerulus, syncytiotrophoblasts of placenta, megakaryocytes in bone marrow, myelin sheath of nerve, medullary thymocytes, and hepatocytes, as well as islet cells of pancreas, contained only sialic acid-capped ECA binding sites. Inhibiting studies with galactose, lactose, and N-acetyl-lactosamine, as well as other sugars, revealed that this lectin is specific for galactosyl residues. In comparison to galactose and lactose, N-acetyl-lactosamine exhibited the highest inhibitory activity on lectin binding, supporting the concept that this lectin is most reactive with N-acetyl-lactosamine-type (type 2 chain) glycoconjugates.

Ultrastructural localization of serotonin and polypeptide YY (PYY) in endocrine cells of the human rectum.

This study was performed with the aim of ultrastructurally localizing serotonin and polypeptide YY (PYY) in the endocrine cells of the human rectum. Existing basic methods for immunolocalization of antigenic sites in ultrathin sections were tested and modified to allow reproducible results with distinct localization of marker (colloidal gold probes coupled either to IgG or protein A). Probes signifying presence of serotonin were distinctly localized over all heteromorphous granules in argentaffin cells and, in addition, over some of the more monomorphous, rounded granules in a second cell type whose granules all were covered by probes showing localization of the PYY antigen. The results suggest that serotonin in endocrine cells of the gut is not confined to the enterochromaffin type but may also be present in trace amounts in non-enterochromaffin endocrine cells storing peptide hormones. Since probes marking sites of PYY were deposited over some heteromorphous granules in enterochromaffin cells, the evidence obtained also suggests that PYY may occur in low concentration in these cells. The distribution of probes in the sections indicated that antigenic sites were confined to granules in the cells.

Contributions of immunohistochemical and molecular biological techniques to endocrine pathology.

Histochemistry has played a major role in the development and implementation of new methods for analysis of gene expression at the cellular level. With the techniques of immunocytochemistry and in situ hybridization, the products of RNA translation as well as specific messenger RNAs and genomic DNAs can be demonstrated and can provide highly dynamic analyses of gene transcription and translation in individual cells. In endocrine pathology, these approaches have been particularly effective for correlation of functional abnormalities with the varying manifestations of disease at the cellular level. In addition, these methods have been valuable in the formulation of novel clinical and pathological concepts, and will continue to provide important tools for diagnostic and prognostic assessment of neoplastic and non-neoplastic disorders of the endocrine system.

Localization of Met-enkephalin-Arg6-Gly7-Leu8-like immunoreactivity in the gastrointestinal tract of rat and pig.

One of the opioid precursor molecules, pre-pro-enkephalin A, contains within it, in addition to Leu-enkephalin (Leu-Enk) and Met-enkephalin (Met-Enk), Met-enkephalin-Arg6-Gly7-Leu8 (Met-Enk-8), which is specific to this precursor. This study deals with the localization of Met-Enk-8-like immunoreactivity in the gastrointestinal tract of rat and pig. Immunoreactivity was identified in intramural nerve elements of rat and pig, and in gut endocrine cells of pig. Immunoreactive (IR) nerve fibers were seen mainly in the myenteric plexus of rat and in both the myenteric and submucosal plexuses of pig. Some IR fibers were dispersed throughout the lamina propria mucosae of rat. Porcine IR endocrine cells were dispersed in the epithelium from the pyloric antrum to the ileum, existing concomitantly with enterochromaffin (EC) cells. Specificity tests revealed that immunoreactivity to Met-Enk-8 antiserum was not influenced by preincubation of the antiserum with Leu-Enk and Met-Enk. This suggests the possibility that pre-pro-enkephalin A is contained in the gastroenteric nerves of rat and pig and in a population of porcine EC cells.

Endocrine cells in extrahepatic bile duct carcinoma.

A total of 44 extrahepatic bile duct carcinomas comprising 13 well-differentiated adenocarcinomas, 25 moderately differentiated adenocarcinomas, and 6 poorly differentiated adenocarcinomas were examined histologically and immunohistochemically for somatostatin, gastrin, and glicentin. Argyrophil cells, argentaffin cells, and somatostatin- and gastrin-immunoreactive cells within the tumor were detected in 46.2%, 15.4%, 23.1%, and 15.4% of well-differentiated adenocarcinomas, and in 16.0%, 8.0%, 12.0%, and 4.0% of moderately differentiated adenocarcinomas, respectively. No tumor tissues of poorly differentiated adenocarcinomas contained endocrine cells. A statistically significant difference in the frequency of argyrophil cells was observed between well and poorly differentiated adenocarcinoma. The incidence of argyrophil cells and somatostatin-immunoreactive cells in nonneoplastic mucosa adjacent to well-differentiated adenocarcinoma was higher than in that adjacent to poorly differentiated adenocarcinoma. Glicentin-immunoreactive cells could not be demonstrated either in tumor tissue or in nonneoplastic mucosa of the extrahepatic bile duct. With reference to the histogenesis of extrahepatic bile duct carcinoma, it was assumed from these results that the development of well-differentiated adenocarcinoma might be closely related to the occurrence of endocrine cells and that poorly differentiated adenocarcinoma might develop from ordinary mucosa.

Similarities and differences among neuroendocrine, exocrine, and endocytic vesicles.

Secretory and endocytic vesicles have analogous functions as cyclic carriers between specific cellular compartments. The centrifugally functioning secretory system operates from the Golgi complex, whereas the centripetally functioning endocytic system operates from the cell surface. Further, within polarized epithelial cells the export traffic can be directed to a distinct plasmalemmal domain which distinguishes exocrine from endocrine secretion and import traffic can be directed transcellularly. These shuttle operations involve a special class of lipid-rich, protein-poor membranes that appear to use an inwardly directed H+-translocase activity to varying extents for pH-dependent sorting and for accumulation and concentration of transported molecules. Comparative analyses of purified membrane preparations from exocrine and endocrine sources identify compositional overlap between different types of shuttle membrane. However, the structural elements that specify a particular origin or destination for a given carrier or determine function in storage and stimulus-dependent shuttling remain unknown.

Comparative studies of hamster calcitonin from pulmonary endocrine cells in vitro.

The lung-associated peptide calcitonin (CT) has been localized by immunocytochemical means to discrete pulmonary endocrine (PE) cells. A long-term cell culture of CT-staining PE cells has been established. The molecular configuration of immunoreactive (iCT) from PE cell extracts was determined by gel chromatography, revealing predominantly large molecular weight forms of iCT. The size distribution characteristics of PE Cell iCT were similar to those of intact hamster lung. In contrast, hamster thyroid extracts contain predominantly 4000 dalton iCT (presumed monomer) and apparent iCT fragments. The culture media of the PE cells were found to contain mainly 4000 dalton iCT. We conclude that although the predominant forms of iCT found within cultured PE cells are distinct from those found within thyroidal C-cells, both iCT producing cells release mainly the monomer into the media. Malignant human bronchial carcinoid cells store predominantly monomeric iCT while secreting large molecular weight forms of iCT. Since the PE cell is the putative precursor cell to neuroendocrine malignancies, the disparity noted in the processing of CT may have significant pathobiological implications.

Chromogranin: widespread immunoreactivity in polypeptide hormone producing tissues and in serum.

Chromogranin A is the major soluble protein, co-stored and co-released with catecholamines from storage vesicles of adrenal medulla and sympathetic nerve, and has been used as an index of exocytotic sympathoadrenal neurosecretion. Since some neuropeptides have a rather wide distribution in nerve, gut, and endocrine glands, we used a sensitive chromogranin A radioimmunoassay to probe the occurrence of chromogranin in several polypeptide hormone producing tissues. Immunoreactive chromogranin was ubiquitous in such tissues, in rank order of concentration (microgram/g wet weight): adrenal medulla greater than pituitary gland (anterior greater than intermediate greater than posterior) greater than pancreas greater than small intestine greater than thyroid greater than hypothalamus. In each case, the tissue homogenate displaced 125I-labelled chromogranin A from antibody in parallel with displacement generated by pure unlabeled chromogranin A, and the immunoreactivity was not abolished by boiling or by several protease inhibitors. Quantitatively, the endocrine tissues other than adrenal medulla possessed 0.1-2.8% of the immunoreactivity found in the adrenal medulla. Immunoreactive chromogranin was also present in serum and sympathetic nerve, but contamination of the endocrine tissues by chromogranin from serum or sympathetic innervation could not account for the observed immunoreactivity. Immunoreactive chromogranin was undetectable in platelets. Tissues with predominant exocrine function (salivary glands) had very little chromogranin (0.004-0.005% of that found in the adrenal medulla). Within the cell, differential centrifugation localized the immunoreactive chromogranin to a hormone storage granule fraction in the anterior pituitary gland (60 +/- 6% of total, with 2.0 +/- 0.3-fold enrichment in the granule) and the adrenal medulla (74 +/- 13% of total, with 1.7 +/- 0.2-fold enrichment in granules). Gel filtration suggested a lower effective molecular radius for pituitary and pancreatic immunoreactive chromogranin than for purified 125I-labelled chromogranin A. Thus, chromogranin in the other endocrine glands differed from adrenal medullary chromogranin both quantitatively (less microgram/g tissue) and qualitatively (lower molecular weight). The results suggest a widespread, though as yet undefined, role for chromogranin in the neurosecretory process, and raise the possibility that chromogranin may be co-stored and secreted with a variety of polypeptide hormones.

Coexistence of peptide YY and glicentin immunoreactivity in endocrine cells of the gut.

Endocrine cells containing peptide YY (PYY) were numerous in the rectum, colon and ileum and few in the duodenum and jejunum of rat, pig and man. No immunoreactive cells could be detected in the pancreas and stomach. Coexistence of PYY and glicentin was revealed by sequential staining of the same section and by staining consecutive semi-thin sections. Since the PYY sequence is not contained in the glucagon/glicentin precursor molecule the results suggest that the PYY cell in the gut expresses two different genes coding for regulatory peptides of two different families.

Isolation, localization, and characterization of gastrointestinal peptides.

Increasing numbers of peptides have been found in the gastrointestinal tract. Some of these are localized primarily in endocrine cells in the gastrointestinal epithelium (e.g., gastrin, secretin) or pancreatic islets (e.g., insulin, glucagon), and function chiefly as circulating hormones. Other peptides, e.g., VIP, are principally neuropeptides present mainly in nerve cells, nerve fibres and nerve terminals; they function mainly as neurotransmitters or neuromodulators. The isolation of gut peptides has usually depended on the use of characteristic bioassays. More recently, some peptides have been isolated on the basis of unique terminal amino-acid composition. Certain chemical structures are associated with typical biological actions, and several "families" of related peptides are recognized.