Stimulation of rat adrenal medulla can induce differential changes in the peptide and mRNA levels of chromogranins, neuropeptides and other constituents of chromaffin granules

The levels of various components of chromaffin granules were determined in rat adrenals after treatment with several stimulants. After reserpine the levels of calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and chromogranin B but not those of chromogranin A and secretogranin II were elevated. On the other hand, the mRNA of chromogranins A, B and secretogranin II were significantly increased. Treatment with oxotremorine or nicotine (multiple injections for 2 or 3 days) induced analogous changes for peptide and mRNA levels, however, the increases were smaller and for the mRNA less consistent. A single injection of oxotremorine or nicotine raised only the levels of CGRP and NPY and of the NPY mRNA whereas those of the chromogranins and their respective mRNAs remained unaltered. Amongst the membrane proteins only the levels of dopamine beta-hydroxylase are increased after prolonged stimulation, whereas those of cytochrome b-561, carboxypeptidase H and synaptin/synaptophysin (SYN) remain unaltered. Thus, the biosynthesis of chromaffin granules can be regulated in quite sophisticated patterns.     

Topology of chromogranins in secretory granules of endocrine cells

Summary Chromogranins A and B are glycoproteins originally detected in the adrenal medulla. These proteins are also present in a variety of neuroendocrine cells. The subcellular distribution of the chromogranins, and particularly their intra-granular topology are of special interest with respect to their putative functions.Endocrine cells of the guinea pig adrenal medulla, pancreas and gastric mucosa were investigated immunoelectron microscopically for the subcellular distribution of both chromogranins. Out of 13 established endocrine cell types in all locations, only two endocrine cell types showed immunoreactivity for both chromogranin A and B, and eight endocrine cell types showed immunoreactivities only for chromogranin A. These immunoreactivities varied inter-cellularly. Three endocrine cell types were unreactive for the chromogranins. Moreover, some hormonally non-identified endocrine cells in the pancreas and the gastric mucosa also contained chromogranin A immunoreactivities.Subcellularly, chromogranin A or B were confined to secretory granules. In most endocrine cells, the secretory granules showed chromogranin immunoreactivities of varying densities. Furthermore, the intra-granular topology of chromogranin A or B in the secretory granules varied considerably: in some endocrine cell types, i.e. chromaffin-, gastrin- and enterochromaffin-like-cells, chromogranin A immunoreactivity was localized in the perigranular and/or dense core region of the secretory granules; in others, i.e. insulin-, pancreatic polypeptide-and bovine adrenal medulla dodecapeptide-cells, it was present preferentially in the electron-opaque centre of the secretory granules; chromogranin B immunoreactivity was localized preferentially in the perigranular region of the secretory granules of chromaffin cells and gastrin-cells. The inter-cellular and inter-granular variations of chromogranin A and B immunoreactivities point to differences in biosynthesis or processing of the chromogranins among endocrine cells and their secretory granules.     

Topology of chromogranins in secretory granules of endocrine cells.

Chromogranins A and B are glycoproteins originally detected in the adrenal medulla. These proteins are also present in a variety of neuroendocrine cells. The subcellular distribution of the chromogranins, and particularly their intra-granular topology are of special interest with respect to their putative functions. Endocrine cells of the guinea pig adrenal medulla, pancreas and gastric mucosa were investigated immunoelectron microscopically for the subcellular distribution of both chromogranins. Out of 13 established endocrine cell types in all locations, only two endocrine cell types showed immunoreactivity for both chromogranin A and B, and eight endocrine cell types showed immunoreactivities only for chromogranin A. These immunoreactivities varied inter-cellularly. Three endocrine cell types were unreactive for the chromogranins. Moreover, some hormonally non-identified endocrine cells in the pancreas and the gastric mucosa also contained chromogranin A immunoreactivities. Subcellularly, chromogranin A or B were confined to secretory granules. In most endocrine cells, the secretory granules showed chromogranin immunoreactivities of varying densities. Furthermore, the intra-granular topology of chromogranin A or B in the secretory granules varied considerably: in some endocrine cell types, i.e. chromaffin-, gastrin- and enterochromaffin-like-cells, chromogranin A immunoreactivity was localized in the perigranular and/or dense core region of the secretory granules; in others, i.e. insulin-, pancreatic polypeptide- and bovine adrenal medulla dodecapeptide-cells, it was present preferentially in the electron-opaque centre of the secretory granules; chromogranin B immunoreactivity was localized preferentially in the perigranular region of the secretory granules of chromaffin cells and gastrin-cells. The inter-cellular and inter-granular variations of chromogranin A and B immunoreactivities point to differences in biosynthesis or processing of the chromogranins among endocrine cells and their secretory granules.     

Co-localization of chromogranin A and B,secretogranin II and neuropeptide Y in chromaffin granules of rat adrenal medulla studied by electron microscopic immunocytochemistry

Summary The co-localization of various antigens in rat chromaffin granules was investigated by the immunogold staining procedure. In ultrathin serial sections staining of chromaffin granules was obtained with antisera against chromogranin A, chromogranin B, secretogranin II and neuropeptide Y. These results indicated that these antigens are costored within chromaffin granules. To further corroborate this point a double immunogold staining procedure was used. This method unequivocally established that chromogranin A, chromogranin B, secretogranin II and neuropeptide Y are co-localized in the same chromaffin granules. These results are relevant for studies demonstrating changes in the level of these peptides in adrenal medulla. The co-localization makes it likely that such changes lead to a different relative composition of the secretory quanta of chromaffin granules.     

Chromogranins A and B are co-localized with atrial natriuretic peptides in secretory granules of rat heart

We investigated the occurrence and subcellular localization of chromogranins A and B in atrial myoendocrine cells of rat heart, using immunological methods. Immunoblotting revealed the presence of both chromogranin A and B in an extract from large granules isolated from this tissue by subcellular fractionation. Immunohistochemistry at the ultrastructural level demonstrated the presence of chromogranin A and B in secretory granules. These organelles also immunostained for atrial natriuretic peptides (ANP). Within a given section, all granules were labeled with immunogold for these three antigens. This apparent co-localization of the three antigens was confirmed by double immunostaining with immunogold particles of different sizes. We conclude that, in agreement with their endocrine nature, the secretory organelles of rat atria contain both chromogranins A and B. Apparently these acidic peptides, which have a widespread distribution in the endocrine system, are co-stored and therefore also co-secreted with ANP.     

Glucoreceptors located in the brain mediate NPY release induced by hypoglycemia in normal men

The NPY secretory pattern after an insulin tolerance test (ITT) (0.15 IU/kg body weight) was evaluated in 8 normal men. They were infused with normal saline (control test), glucose or fructose. Insulin-induced hypoglycemia produced a significant increment in serum NPY in the control test. The infusion of fructose was unable to change the NPY secretory pattern during insulin-induced hypoglycemia. In contrast, the NPY increase during ITT was completely abolished when the concomitant infusion of glucose prevented insulin-induced hypoglycemia. These results exclude a direct role of hyperinsulinemia in the mechanism underlying the stimulation of NPY secretion during ITT. Furthermore, since glucose but not fructose crosses the blood-brain-barrier (BBB), the NPY increase during ITT appears to be generated by low glucose concentrations at the level of glucosensitive areas located inside the brain.     

Inhibitory effect of somatostatin on the NPY response to insulin-induced hypoglycemia and the role of endogenous opioids

The present study was undertaken in order to establish whether somatostatin (SRIH) is able to modify the neuropeptide Y (NPY) response to insulin-induced hypoglycemia during insulin tolerance test (ITT) in man. In addition, the possible involvement of opioid peptides in the mediation of hypoglycemia and/or SRIH action was investigated. Subjects were injected intravenously with 0.15IU/kg insulin alone (control test) or with SRIH (4.1μg/min/90min), naloxone (10mg in an iv bolus) or the combination of the two substances. Plasma NPY concentrations rose significantly during ITT. The NPY response was significantly reduced by the treatment with SRIH. The administration of naloxone did not modify NPY levels whereas when both SRIH and naloxone were given, NPY response to hypoglycemia did not differ from that observed in the control test. These data demonstrate that SRIH inhibits the NPY response to hypoglycemia. Naloxone-sensitive endogenous opiates do not seem to be involved in the control of hypoglycemia-induced NPY release. In contrast, since naloxone reversed the inhibiting effect of SRIH, an involvement of opioid peptides in the SRIH action may be supposed.     

Presence and release of the chromogranin B-derived secretolytin-like peptide KR-11 from the porcine spleen

Chromogranin B (CgB) is a major matrix protein in secretory granules/large dense-cored vesicles and a precursor for smaller peptides. In an earlier study, we have identified a secretolytin-like peptide (KR-11, pCgB(637-647)) from porcine chromaffin granules. Further evidence is presented here to show the processing of chromogranin B to this peptide during axonal transport in the splenic nerve and its release in the spleen upon various conditions of stimulation. Immunohistochemical staining showed that in the porcine spleen chromogranin B and NPY completely colocalize in nerve fibres and varicosities in blood vessel walls and trabeculae, and along the loose network of smooth muscle cells in the red pulp, as identified by their alpha-smooth muscle cell actin content. No antibacterial activity was found for the porcine secretolytin-like peptide, KR-11. The change of one amino acid residue (Thr-->Asn) in the porcine homologous fragment of secretolytin appears to be responsible for its loss of antibacterial activity.     

A large form of secretogranin III functions as a sorting receptor for chromogranin A aggregates in PC12 cells

Granin-family proteins, including chromogranin A and secretogranin III, are sorted to the secretory granules in neuroendocrine cells. We previously demonstrated that secretogranin III binds chromogranin A and targets it to the secretory granules in pituitary corticotrope-derived AtT-20 cells. However, secretogranin III has not been identified in adrenal chromaffin and PC12 cells, where chromogranin A is correctly sorted to the secretory granules. In this study, low levels of a large and noncleaved secretogranin III have been identified in PC12 cells and rat adrenal glands. Although the secretogranin III expression was limited in PC12 cells, when the FLAG-tagged secretogranin III lacking the secretory granule membrane-binding domain was expressed excessively, hemagglutinin-tagged chromogranin A was unable to target to the secretory granules at the tips and shifted to the constitutive secretory pathway. Secretogranin III was able to bind the aggregated form of chromogranin A, suggesting that a small quantity of secretogranin III is enough to carry a large quantity of chromogranin A. Furthermore, secretogranin III bound adrenomedullin, a major peptide hormone in chromaffin cells. Indeed, small interfering RNA-directed secretogranin III depletion impaired intracellular retention of chromogranin A and adrenomedullin, suggesting that they are constitutively released to the medium. We suggest that the sorting function of secretogranin III for chromogranin A is common in PC12 and chromaffin cells as well as in other endocrine cells, and a small amount of secretogranin III is able to sort chromogranin A aggregates together with adrenomedullin to secretory granules.     

Adrenal chromaffin granules and secretory granules from thyroid parafollicular cells have several common antigens

The presence of various antigens in two types of isolated endocrine vesicles (chromaffin granules and secretory vesicles of thyroid parafollicular cells) was investigated by immunoblotting. The two types of vesicles have three common secretory proteins: chromogranin A, chromogranin B and secretogranin II. Furthermore, six common membrane antigens were found: cytochrome b-561, carboxypeptidase H, glycoprotein II, glycoprotein III, synaptin/synaptophysin and SV 2. These results demonstrate that vesicles obtained from neural crest-derived endocrine cells not only share several common secretory peptides and proteins, but also have common properties as far as their membrane antigens are concerned.     

Relationship Between the Regulation of Enkephalin-Containing Peptide and Dopamine β-Hydroxylase Levels in Cultured Adrenal Chromaffin Cells

Adrenal medullary chromaffin cells were maintained under conditions known to increase their cellular levels of enkephalin-containing peptides and the effects of these treatments on another chromaffin vesicle component, dopamine beta-hydroxylase, were examined. Catecholamine-depleting drugs, such as tetrabenazine, and cyclic nucleotide-elevating drugs, including forskolin, 8-bromo-cyclic AMP, and theophylline, increase chromaffin cell enkephalin-containing peptide levels but fail to increase dopamine beta-hydroxylase. In contrast, insulin treatment increases the levels of both enkephalin-containing peptides and dopamine beta-hydroxylase. The increased amounts of enkephalin-containing peptides produced by tetrabenazine and by insulin are stored in subcellular particles with properties identical to chromaffin vesicles on density-gradient centrifugation. These results suggest that following insulin treatment chromaffin cells synthesize new chromaffin vesicles with a full complement of enkephalin-containing peptides, but that after treatment with catecholamine-depleting or cyclic nucleotide-related agents enkephalin-containing peptides can be inserted into preexisting vesicles or that new vesicles, made as a part of the normal turnover of cellular components, contain elevated peptide levels.     

A chromogranin peptide is co-stored with insulin in the human pancreatic islet B-cell granules

Summary The immunoreaction of a rabbit chromogranin A and B antiserum was studied in normal human pancreatic islets. By examination of consecutive light microscopical sections, it was revealed that, at high antiserum concentrations (1:2000 or less), the whole islet area was heavily labelled, although the peripheral glucagon (A)-cells were the most intense in their immunoreaction. At low antiserum concentrations (1:4000 or more) the A-cells still showed the same intense labelling reaction, but the central B-cells were weakly labelled. Electron microscopically, reactivity towards the chromogranin A and B antiserum and the monoclonal insulin antibodies was present in the same central electron-dense core of the B-cell secretory granules, as demonstrated after application of the immunogold technique at different antibody dilutions. In the A-cells, the chromogranin immunoreactivity was concentrated at the peripheral mantle of the secretory granules. The D-cell granules showed a weak immunolabelling. Examination of human islets with the monoclonal chromogranin A antibody LK2H10 revealed immunogold labelling only in the peripheal mantle of the A-cell granules, while the B-cell granules were unlabelled.The present results show that a chromogranin peptide is co-stored with insulin the in normal human B-cell secretory granules. Although the exact composition of this B-cell chromogranin is unknown, it is not identical to that of the chromogranin A present in the A-cell granules.     

Targeted ablation of the chromogranin a (Chga) gene: normal neuroendocrine dense-core secretory granules and increased expression of other granins

Chromogranin A (CgA), originally identified in adrenal chromaffin cells, is a member of the granin family of acidic secretory glycoproteins that are expressed in endocrine cells and neurons. CgA has been proposed to play multiple roles in the secretory process. Intracellularly, CgA may control secretory granule biogenesis and target neurotransmitters and peptide hormones to granules of the regulated pathway. Extracellularly, peptides formed as a result of proteolytic processing of CgA may regulate hormone secretion. To investigate the role of CgA in the whole animal, we created a mouse mutant null for the Chga gene. These mice are viable and fertile and have no obvious developmental abnormalities, and their neural and endocrine functions are not grossly impaired. Their adrenal glands were structurally unremarkable, and morphometric analyses of chromaffin cells showed vesicle size and number to be normal. However, the excretion of epinephrine, norepinephrine, and dopamine was significantly elevated in the Chga null mutants. Adrenal medullary mRNA and protein levels of other dense-core secretory granule proteins including chromogranin B, and secretogranins II to VI were up-regulated 2- to 3-fold in the Chga null mutant mice. Hence, the increased expression of the other granin family members is likely to compensate for the Chga deficiency.     

Detection of low molecular mass GTP-binding proteins in chromaffin granules and other subcellular fractions of chromaffin cells

A homogenate of purified chromaffin cells was fractionated, after removal of the nuclear fraction, by sucrose density gradient ultracentrifugation. The presence and subcellular localization of low molecular mass GTP-binding proteins was explored by incubation of blots of proteins from different subcellular fractions with [alpha-32P]GTP in the presence of Mg2+. The fractions enriched in intact chromaffin granule markers, i.e. catecholamines, chromogranin A, chromogranin B and cytochrome b-561 were also enriched in labelled GTP-binding proteins. Two major labelled components of 23 and 29 kDa were rapidly detected by autoradiography. Traces of 26 and 27 kDa components were also present. These components were detectable in both plasma and granule membranes. In addition to these components, the cytosolic fraction contained another GTP-binding protein of about 20 kDa. Binding of [alpha-32P]GTP was specific and dependent on Mg2+. By analogy to the findings reported in non-mammalian systems, the observations described here suggest the involvement of low molecular mass GTP-binding proteins in the chromaffin cell secretory process.     

Increases of neuropeptide Y-like immunoreactivity in plasma during insulin-induced hypoglycemia in man

Neuropeptide Y-like immunoreactivity (NPY-LI) in plasma during insulin-induced hypoglycemia was measured in 4 healthy male volunteers. Plasma NPY-LI increased from 167 +/- 11 pg/ml to 247 +/- 25 pg/ml 30 min after the administration of insulin (0.1 U/kg body weight IV), reached the maximum (296 +/- 6 pg/ml) 45 min after the insulin, and then decreased. These results suggest that NPY is released into the systemic circulation during insulin-induced hypoglycemia in man.     

Inhibition of insulin secretion by betagranin, an N-terminal chromogranin A fragment

Betagranin, an N-terminal fragment of chromogranin A, results from a proteolytic processing, and is co-secreted with insulin. While other chromogranin A-derived peptides negatively modulate hormone secretion, the role of betagranin in pancreatic beta-cells is so far unknown. We have recently shown that pancreatic islet betagranin levels are down-regulated in obese, leptin-deficient mice. In the present study, we have investigated the distribution of betagranin in primary mouse islets and cells of the MIN6 line and have evaluated its effects on insulin secretion. We showed that betagranin co-localizes with insulin within secretory granules and strongly inhibited insulin secretion in response to both glucose and potassium, by blocking the influx of calcium. The data demonstrated a hitherto unknown inhibitory effect of betagranin on insulin secretion.     

Chromogranin B: isolation from pheochromocytoma, N-terminal sequence, tissue distribution and secretory vesicle processing

The chromogranins/secretogranins are a family of neuroendocrine vesicle secretory proteins. Immunohistology and immunoblotting have suggested that a major soluble protein in human chromaffin granules may be chromogranin B (CgB). We purified from pheochromocytoma chromaffin granules an SDS-PAGE 110-120 kDa protein whose N-terminal sequence matched that previously deduced from a human CgB cDNA. An antibody directed against a synthetic human CgB N-terminal region specifically recognized the CgB N-terminus, though not the chromogranin A (CgA) N-terminus or the CgB C-terminus on immunoblots. An antiserum directed against CgB's C-terminus also visualized CgB but not CgA. By immunoblotting, CgB was a quantitatively major protein in human pheochromocytoma chromaffin granules, but a relatively minor in normal bovine adrenal medullary chromaffin granules. In a variety of normal bovine neuroendocrine tissues, the relative abundance of CgB immunoreactivity on immunoblots was: adrenal medulla greater than anterior pituitary greater than pancreas greater than small intestine, hypothalamus. Immunoblotting of neuroendocrine tissues (or their hormone storage vesicle cores) with both anti N-terminal and anti C-terminal CgB antisera suggested bidirectional cleavage or processing of CgB; in the anterior pituitary, a unique 40 kDa C-terminal fragment was observed. Bidirectional CgB cleavage was also suggested on immunoblots of chromaffin tissue from three species (human, bovine, rat). C-terminal processing of CgB was also confirmed by amino acid sequencing of SDS-PAGE-separated, polyvinylidene difluoride membrane-immobilized CgB fragments from pheochromocytoma chromaffin granules. Whether such fragments possess biological activity remains to be investigated.     

Widespread occurrence of chromogranins/secretogranins in the matrix of secretory granules of endocrinologically silent pituitary adenomas.

To investigate the constituents of the matrix of endocrine secretory granules, we analyzed endocrinoilogically silent ("non-functioning") human pituitary adenomas for the occurrence of the chromogranins/secretogranins (granins), a protein family normally stored together with many different hormones. When five non-functioning pituitary adenomas were analyzed by immunoblotting using polyclonal and monoclonal antibodies specific for individual members of the granin family, chromogranin A was detected in four cases and chromogranin B and secretogranin II were detected in all cases. The cellular distribution of the granins and of various hormones known to be expressed in the anterior pituitary was studied by immunocytochemistry in fixed, frozen tissue sections from five additional adenomas. Of the eight hormones investigated, only thyroid-stimulating hormone, luteinizing hormone, and follicle-stimulating hormone were detected, occurring in only two of the five adenomas. In contrast, granins were found in all five tumors. Chromogranin B and secretogranin II were detected in each of the adenomas in virtually every cell studied, whereas chromogranin A exhibited such a widespread cell distribution in only three adenomas, being focally present in one and absent from the other tumor. The subcellular localization of the granins and the three glycoprotein hormones was investigated by double immunoelectron microscopy. Chromogranin A and chromogranin B were mainly co-localized in secretory granules, whereas secretogranin II was either co-localized with the other two granins or segregated to different secretory granules. When present, glycoprotein hormones were immunodetected in both the secretory granules containing all three granins and those containing mainly secretogranin II. Our data indicate that in non-functioning pituitary adenomas chromogranin A is differentially expressed from chromogranin B and secretogranin II. Moreover, the granins appear to be the most widespread constituents of endocrine secretory granules known, forming the dense-core matrix irrespective of the presence or absence of hormones.     

An immunological study on chromogranin A and B in human endocrine and nervous tissues

Summary Antisera were raised against synthetic peptides derived from the primary amino acid sequence of human chromogranin B. These antisera recognized in one- and two-dimensional immunoblotting a component previously designated as chromogranin B. In human chromaffin granules, the major endogenous processing product of chromogranin B is formed by proteolytic cleavage of the protein near theC-terminus. Immunohistochemical localizations were obtained with antisera against human chromogranins A and B and against a synthetic peptide corresponding to the B sequence. In human tissues, chromogranin B is co-stored with chromogranin A in the adrenal medulla, the anterior pituitary, parafollicular cells of the thyroid, in some cells of the endocrine pancreas and in some enterochromaffin cells, whereas only chromogranin A is found in the parathyroid gland and enterochromaffin cells of the gastric corpus mucosa. In the nervous system, no immunostaining was observed for chromogranin A and only a weak one for chromogranin B in some cells of the spinal cord. However, the Purkinje cells of the cerebellum were strongly positive for chromogranin B.