Targeting of green fluorescent protein to secretory granules in oxytocin magnocellular neurons and its secretion from neurohypophysial nerve terminals in transgenic mice.

Oxytocin (OT) is a hypothalamic nonapeptide that is synthesized as part of a larger precursor protein that also contains an approximately 10-kDa protein called neurophysin at its C-terminus. This precursor protein is trafficked through the regulated secretory pathway into secretory granules and then axonally transported to and secreted from nerve terminals in the neural lobe of the pituitary. In this paper, we show that the AI-03 transgene that contains enhanced green fluorescent protein (EGFP) fused to the end of the neurophysin at the C-terminus of the OT pre-prohormone, is expressed selectively in OT-magnocellular neurons and is trafficked to secretory granules in transgenic mice. The EGFP-containing secretory granules are then transported to OT-neurosecretory terminals in the neurohypophysis, where the EGFP fluorescence undergoes depolarization-induced calcium-dependent secretion. The endogenous fluorescence in the neural lobes is sufficiently intense to image secretory events in individual OT nerve terminals (neurosecretosomes) isolated from the posterior pituitaries in these transgenic mice.

Transgenic expression of green fluorescent protein in mouse oxytocin neurones.

Routine targeting of neurones for expression of exogenous genes would facilitate our ability to manipulate their internal milieu or functions, providing insight into physiology of neurones. The magnocellular neurones of the paraventricular and supraoptic nuclei of the hypothalamus have been the objects of limited success by this approach. Here we report on the placement of the enhanced green fluorescent protein (eGFP) coding sequence at various locations within an oxytocin transgene. Placement within the first exon yielded little to no expression, whereas placement in the third exon (as an in-frame fusion with the carboxyl terminus of the oxytocin preprohormone) resulted in cell-specific expression of eGFP in oxytocin neurones. Furthermore, placement of the eGFP sequence downstream of a picornavirus internal ribosomal entry site (IRES), also in the third exon, allowed expression of the eGFP as a separate protein. Other coding sequences should now be amenable to expression within oxytocin neurones to study their physiology.

Identification of a TPA-responsive element mediating preferential transactivation of the galanin gene promoter in chromaffin cells.

The gene encoding the neuropeptide galanin is upregulated by second messenger signal transduction pathways in bovine chromaffin cells. To identify its transcriptional regulatory elements, 5'-flanking sequences of the galanin gene were transiently transfected into primary cultures of bovine chromaffin cells within reporter gene constructs. Multiple regions of the galanin 5' flank seem to be necessary for basal activity. The most promoter-proximal of these regions contains a sequence (TGACG) -66 to -62 nucleotides upstream from the transcriptional start site which mediates stimulation by 12-O-tetradecanoylphorbol-13-acetate (TPA), as demonstrated by site-directed mutagenesis and cis-activation experiments. This cis-regulatory element mediates preferential TPA stimulation of transcription from the galanin promoter in chromaffin cells compared with bovine endothelial or HeLa cells. DNA-protein binding assays indicate that an oligonucleotide that includes the galanin TPA-responsive element (GTRE) binds specifically to proteins from nuclear extracts of chromaffin cells. TPA treatment persistently increases this binding activity in chromaffin but not in endothelial cells. Mutation of the galanin promoter within the -66 to -62 region renders it unresponsive to transcriptional stimulation by TPA, and a correspondingly mutated oligonucleotide fails to bind chromaffin cell nuclear proteins in a gel-shift assay. Chromaffin cell nuclear extracts also contain proteins that bind consensus TPA-responsive (TRE) and cyclic AMP-responsive (CRE) elements. GTRE, TRE, and CRE oligonucleotides all compete more efficiently for protein binding to their labeled congeners than for protein binding to either of the other labeled oligonucleotides, suggesting that the GTRE, TRE, and CRE oligonucleotides, suggesting that the GTRE, TRE, and CRE oligonucleotides each bind unique as well as common proteins, likely to be members of the Jun/Fos and cAMP-responsive element-binding protein/activating transcription factors (CREB/ATF) families of transcription factors, in chromaffin cells.

Sites of synthesis of chromogranins A and B in the human brain.

The sites of synthesis of the chromogranins A and B, and their potential processed peptides, were examined by quantitating the levels of chromogranin A and B mRNA in various regions of the human brain by Northern blot analysis. Chromogranin A and B mRNA expression in the brain is region-specific and confined to grey matter. In situ hybridization histochemistry detected chromogranin A and B mRNA in pyramidal neurons of human cerebral cortex. Cell-specific expression in subpopulations of cerebrocortical neurons suggest that chromogranin A and B gene products may play a role in central neuronal function.

Enkephalin biosynthesis is coupled to secretory activity via transcription of the proenkephalin A gene.

The molecular mechanisms regulating neuropeptide and secretory protein biosynthesis in neuroendocrine cells were examined using the prototype neuropeptide and secretory proteins enkephalin and chromogranin A (CGA). Treatment with the secretogogue nicotine results in the calcium-dependent secretion of enkephalin peptides from bovine chromaffin cells in primary culture and a concomitant increase in enkephalin peptide biosynthesis. Both secretion and biosynthesis are also stimulated by cell depolarization with elevated potassium. Elevation of intracellular cyclic AMP, on the other hand, results in stimulation of enkephalin biosynthesis and long-term, but not acute, secretion of enkephalin peptides. Coupling of enkephalin biosynthesis to calcium influx occurs at the level of transcription of the enkephalin gene. Thus, potassium depolarization causes a calcium-dependent elevation of enkephalin mRNA which is preceded by an increase in the rate of transcription of the enkephalin gene in the chromaffin cell. The accumulation of enkephalin message or peptide by potassium depolarization or treatment with nicotine is prevented by D600 or hexamethonium respectively, added 1 h after addition of nicotine or KCl and following acute release, suggesting that calcium acts as a continuous rather than triggering stimulus for enkephalin biosynthesis. Sequence analysis of the bovine enkephalin promoter identified sequence conservation of three enhancers previously reported in the human gene which are required for regulation of the gene by calcium, cAMP, and phorbol ester in vitro. In contrast to the regulation of the enkephalin system, no increase in either CGA or CGB mRNA or gene transcription attended depolarization-induced secretion from chromaffin cells. Since enkephalin and CGA are co-stored in and co-released from the same secretory vesicles in these cells, the results imply that a surplus of CGA is constitutively synthesized in chromaffin cells such that compensatory up-regulation during changes in the secretory state of the cell, such as occurs for enkephalin, is not required for the secretory proteins.

The bovine chromogranin A gene: structural basis for hormone regulation and generation of biologically active peptides.

The structure of the gene encoding bovine chromogranin-A has been determined by characterization of two isolated genomic clones. Chromogranin-A is encoded by eight exons, which organize the coding region into several distinct structural and functional domains. Exons 1-5 represent the highly conserved signal peptide and N-terminal domain, which are separated into regions corresponding to the signal peptide, N-terminal sequence, disulfide-bonded loop, and remainder of the conserved N-terminal domain. Exon 6 represents the variable domain and encodes a region that is identical to the novel chromogranin-A-derived peptide chromostatin. Exon 7 encodes the biologically active peptide pancreastatin as well as most of the conserved C-terminal domain, with the remainder found on exon 8. The mRNA sequence obtained from the gene contains five nucleotide differences from the consensus sequence of four reported bovine chromogranin-A cDNA clones. Two of the differences in the gene result in two amino acid changes in the region encoded by exon 6. The structural organization of the chromogranin-A gene resembles that of the chromogranin-B gene in the exons corresponding to the signal peptide, N-terminal sequence, disulfide loop, and C-terminal sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence analysis, tissue distribution and regulation by cell depolarization, and second messengers of bovine secretogranin II (chromogranin C) mRNA.

Secretogranin II is a very acidic, tyrosine-sulfated protein found in secretory granules of cells belonging to the diffuse neuroendocrine system. It gained more general importance recently as a universal immunohistochemical marker for endocrine neoplasms. Sequence information was obtained from secretogranin II isolated from bovine anterior pituitaries, allowing the isolation of cDNA clones and deduction of its primary structure. Bovine secretogranin II is a 586-amino acid protein of 67,455 Da which is preceded by a signal peptide of 27 residues and contains 9 pairs of basic amino acids in its sequence which are used as potential cleavage sites for generation of physiologically active peptides. Moderately abundant mRNA levels were found in adrenal medulla, pituitary, hippocampus, and caudate. Secretogranin II message was absent from parathyroid gland, adrenal cortex, kidney, liver, and spleen. Depolarization of isolated chromaffin cells by various secretagogues significantly up-regulated secretogranin II mRNA levels by mechanisms distinct from those established for chromogranins and neuropeptides, components maintained along with secretogranin II in neuroendocrine storage vesicles.

Chromogranin A and B messenger ribonucleic acids in pituitary and other normal and neoplastic human endocrine tissues.

The distribution of the messenger ribonucleic acids (mRNAs) for chromogranin A and B was analyzed by in situ hybridization in normal and neoplastic endocrine tissues using frozen and paraffin tissue sections. Combined in situ hybridization and immunochemical staining was also done on tissue sections from the same cases using a monoclonal antibody against chromogranin A (LK2H10). Most endocrine tumors expressed chromogranin A and B mRNAs as well as chromogranin A protein. Normal pituitary expressed chromogranin A and B mRNAs and chromogranin A protein in the anterior pituitary gland. Most of these cells were gonadotropic hormone-producing cells. Prolactinomas (5/5) did not express chromogranin A mRNA or protein, but contained chromogranin B mRNA. Null cell or nonfunctional adenomas (8/8) expressed chromogranin A and B mRNAs and reacted with antibody LK2H10. In some tumors such as Merkel cell carcinomas, insulinomas, and parathyroid adenomas, a stronger signal for chromogranin A mRNA was detected than for the immunoreactive proteins. These results indicate that in situ hybridization complements immunochemical techniques in the analysis of endocrine cells and neoplasms. The gene products for chromogranin A and B are widely distributed in many endocrine cells and tumors, but some neoplasms such as prolactinomas have a differential distribution of chromogranin A and B mRNA and proteins.

Glucocorticoid- and nerve growth factor-induced changes in chromogranin A expression define two different neuronal phenotypes in PC12 cells.

The regulation of chromogranin A mRNA was examined in PC12 cells after treatment with nerve growth factor, dexamethasone, or a combination of the two agents. PC12 cells have low levels of chromogranin A mRNA, and this does not change upon treatment with nerve growth factor. Dexamethasone treatment of these cells results in a 4-fold increase in the amount of chromogranin A mRNA. The dexamethasone-stimulated increase in chromogranin A mRNA is not apparent until at least 16 h after the addition of the drug and is maintained only with continuous culture in the presence of the drug. Dexamethasone and nerve growth factor together increase chromogranin A mRNA to the level seen with dexamethasone alone. Immunohistochemistry shows a similar pattern of protein accumulation within individual cells. Chromogranin B mRNA levels are unaltered by any of the drug treatments described. Treatment with dexamethasone plus NGF seems to be required for full expression of the adrenergic, neuronal phenotype in PC12 cells. Measurement of chromogranin A mRNA provides more specific delineation of neural differentiation and how it is influenced by hormones and growth factors.

Neural and humoral factors separately regulate neuropeptide Y, enkephalin, and chromogranin A and B mRNA levels in rat adrenal medulla.

The influence of neurogenic versus humoral factors on mRNA levels of several secretory proteins of rat adrenal medulla was studied in vivo. Increased splanchnic nerve activity was generated (reflexly) with insulin treatment. Twenty-four hours after insulin injection, levels of mRNAs encoding neuropeptides (enkephalin and neuropeptide Y) were increased 6.5-fold, whereas those of mRNAs for the major secretory proteins (chromogranins A and B) were unchanged. Bilateral transection of the splanchnic nerves completely prevented this increase. Hypophysectomy decreased levels of chromogranin A mRNA to 32% of control, suggesting a dependence on hormones of the pituitary-adrenal axis. Treatment of hypophysectomized rats with dexamethasone restored chromogranin A mRNA to basal levels. Chromogranin B mRNA levels were not changed by either insulin treatment or hypophysectomy. These results demonstrate (i) that different classes of secretory proteins present in chromaffin granules are regulated by different mechanisms, (ii) that this regulation occurs at a pretranslational site, and (iii) that the relative concentration of secretory constituents of chromaffin granules may vary. The significance of an altered composition of secretory-granule constituents, which may be important in hypotension or stress, is discussed.

The sequence of porcine chromogranin A messenger RNA demonstrates chromogranin A can serve as the precursor for the biologically active hormone, pancreastatin.

Specific oligonucleotide priming of double-stranded DNA has been employed to sequence a porcine chromogranin A adrenomedullary cDNA. Porcine chromogranin A is more than 80% identical to human, bovine, and rat chromogranin A at its deduced N- and C-termini. A 49-amino acid region of the porcine molecule is 59-71% homologous to corresponding areas of rat, bovine, and human chromogranin A, and identical to the amino acid sequence of porcine pancreastatin. The sequence is preceded by an arginine at the N-terminus and followed by a GKR sequence at the C-terminus. Thus, porcine chromogranin A can serve as the precursor for pancreastatin, a polypeptide capable of inhibiting insulin release from the endocrine pancreas and acid secretion from parietal cells of the gut.

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)

Primary structure of rat chromogranin A and distribution of its mRNA.

The primary structure of rat chromogranin A has been deduced from a rat adrenal cDNA clone. A comparison of rat and bovine chromogranin A reveals similar features: clusters of polyglutamic acid, similar amino acid composition, position of seven of 10 pairs of basic amino acids, identical placement of the only two cysteine residues, a highly conserved N- and C-terminus, and a sequence homologous to porcine pancreastatin 1-49 [(1986) Nature 324, 476-478]. Unique features of rat chromogranin A are an eicosaglutamine sequence and two potential N-linked glycosylation sites. Chromogranin A mRNA is detectable in adrenal medulla, anterior pituitary, cerebral cortex, and hippocampus, as well as tumor cell lines derived from pancreas, pituitary, and adrenal medulla.

Chromogranin A synthesis and secretion in chromaffin cells.

A sensitive and selective radioimmunoassay for chromogranin A (Chrg A) has been developed to quantitate content, release, and biosynthesis of this secretory protein in neuroendocrine tissues. An antiserum raised against Chrg A from bovine adrenal medulla was found to detect predominantly only the Mr 70-75 kilodalton Chrg A in its native form, allowing the use of this antiserum as a quantitatively specific probe for Chrg A in cell-free extracts of the adrenal medulla and chromaffin cells. Chrg A comprises about 10% of the total protein of the chromaffin cell. It is released in parallel with Met-enkephalin and catecholamines from the bovine chromaffin cell in primary culture in response to nicotine and nicotinic cholinergic agonists. From 14 to 22% of total Chrg A is released from the cell during a 15-min exposure to a maximally stimulatory dose of nicotine (10-100 microM). Chrg A release on nicotinic stimulation is blocked by D-600 and hexamethonium to the same extent as Met-enkephalin and catecholamine release. The parallel time course and percent release of Chrg A and Met-enkephalin indicate that these secretory polypeptides are contained in, and released from, functionally identical cellular compartments. Chrg A and Met-enkephalin pentapeptide sequences are present in the chromaffin cell at a ratio of about 2:1, although Chrg A is far more abundant on a mass basis. Chrg A and Met-enkephalin biosynthesis appear to be differentially regulated within the chromaffin cell, since chronic treatment of cells with nicotine and forskolin causes an elevation of Met-enkephalin pentapeptide without a concomitant elevation of intracellular levels of Chrg A.

Bovine chromogranin A sequence and distribution of its messenger RNA in endocrine tissues.

Chromogranin A is contained in storage vesicles of chromaffin cells of the adrenal medulla and released with catecholamines when the splanchnic nerve is stimulated. Chromogranin A is similar to secretory protein I (SP-I), a major secreted protein of the parathyroid. Chromogranin A/SP-I immunoreactivity is abundant in endocrine cells that secrete peptide hormones from storage vesicles. Chromogranins may act in neuroendocrine secretion by binding intravesicular calcium. Serum levels of chromogranin are raised in hypertension and endocrine neoplasia. We report here the isolation and sequencing of a cDNA encoding bovine chromogranin A, providing the first complete primary structure of a chromogranin protein. Chromogranin A is a highly acidic protein with an apparent relative molecular mass (Mr) of 75,000 on SDS-PAGE, but an actual Mr of 48,000. Adrenal medulla, brain, pituitary and parathyroid are all sites of synthesis of chromogranin A. The primary structure of chromogranin A, and the presence of chromogranin mRNA in the parathyroid, indicate that chromogranin A and SP-I are identical.