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.

Phylogenetic distribution of peptides related to chromogranins A and B.

The presence of chromogranin-related peptides in a wide range of species was investigated by one and two-dimensional electrophoresis followed by immunoblotting. Antisera against bovine chromogranins A and B and the peptide WE-14 (chromogranin A316-329) were used. Chromogranins were identified by their heat stability, by their electrophoretic behavior, and by immunological cross-reaction with antisera.

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.

Calcium requirements for barium stimulation of enkephalin and vasoactive intestinal peptide biosynthesis in adrenomedullary chromaffin cells.

The divalent cation barium was used to study the role of calcium in coupling neuropeptide secretion and biosynthesis following secretagogue stimulation of bovine chromaffin cells. Barium chloride (0.1-2.

The enkephalin-containing cell: strategies for polypeptide synthesis and secretion throughout the neuroendocrine system.

1. Enkephalinergic cells are found throughout the diffuse neuroendocrine system, in the adrenal medulla, brain, spinal cord, peripheral and enteric nervous systems, and endocrine pancreas. 2.

Barium distinguishes separate calcium targets for synthesis and secretion of peptides in neuroendocrine cells.

The effect of barium and potassium on the secretion and biosynthesis of enkephalin in bovine chromaffin cells, and prolactin and beta-endorphin in rat anterior pituitary cells, was examined to determine whether calcium-dependent secretion and biosynthesis are mediated by the same or by different calcium targets within the neuroendocrine cell. In the presence of 1.8 mM calcium, barium and potassium stimulated the secretion of all three peptides over 30 min, and increased the levels of proenkephalin and prolactin mRNA in 24 hr.

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.

Measurement of mRNA specific for preprocholecystokinin in rat caudatoputamen and areas projecting to it.

The concentrations of mRNA specific for preprocholecystokinin were measured with a hybridisation procedure after its extraction from rat brain samples. mRNA specific for preprocholecystokinin was not consistently found in the caudatoputamen. In contrast, its concentrations were quite high in the cingulate, subcallosal and parietal cortical areas, which are assumed to send cholecystokinin-containing axons to the caudatoputamen.

Calcium-independent and calcium-dependent mechanisms regulate corticotropin-releasing factor-stimulated proopiomelanocortin peptide secretion and messenger ribonucleic acid production.

We have evaluated the role of calcium in basal and secretagogue-stimulated release of beta-endorphin and PRL and the levels of their respective mRNAs in primary cultured rat anterior pituitary cells. Treatment of anterior pituitary cells with the calcium channel blocker methoxyverapamil (D600; 10 microM) or with calcium-free medium for 1 h did not alter the basal release of beta-endorphin and only partially blocked CRF (10 nM)-stimulated beta-endorphin release. In contrast to these effects of D600 or calcium-free medium on corticotrophs, both of these test conditions decreased basal secretion of PRL from lactotrophs by 50-70% and completely blocked forskolin (10 microM)-stimulated PRL secretion.

Regulation of enkephalin, VIP, and chromogranin biosynthesis in actively secreting chromaffin cells. Multiple strategies for multiple peptides.

Enkephalins, vasoactive intestinal polypeptide, and chromogranin A are all contained in the secretory vesicles of chromaffin cells in culture, and are all released from this compartment by secretagogues in a calcium-dependent way. The biosynthesis of each of these peptides, however, is under quite independent regulation. The synthesis and secretion of enkephalin is tightly coupled to acetylcholine and elevated potassium stimulation by calcium influx.

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.

Metorphamide levels in chromaffin cells increase after treatment with reserpine.

Exposure of bovine chromaffin cells in primary culture to 0.01-1 microM reserpine caused a dose- and time-dependent increase in intracellular levels of the amidated enkephalin peptide metorphamide. Maximal levels (approximately 800% of control) were obtained at 0.

Enkephalin and neuropeptide Y: two colocalized neuropeptides are independently regulated in primary cultures of bovine chromaffin cells.

We have found that Neuropeptide Y is colocalized with enkephalin in bovine adrenal chromaffin cells. The two peptides can be found in the same granules in those cells where they coexist. These cells correspond to the adrenergic subpopulation of chromaffin cells since they contain the epinephrine synthetic enzyme, phenylethanolamine N-methyltransferase.

Vasoactive intestinal peptide and electrical activity influence neuronal survival.

Blockade of electrical activity in dissociated spinal cord cultures results in a significant loss of neurons during a critical period in development. Decreases in neuronal cell numbers and 125I-labeled tetanus toxin fixation produced by electrical blockade with tetrodotoxin (TTX) were prevented by addition of vasoactive intestinal peptide (VIP) to the nutrient medium. The most effective concentration of VIP was 0.

[Regulation of enkephalin biosynthesis in chromaffin cells].

Enkephalin peptides (ENK) are co-released with catecholamines from bovine chromaffin cells in culture. Drugs mimicking the effects of c-AMP increase ENK biosynthesis by increasing ENK mRNA, but are uneffective on ENK secretion. Nicotine, which causes a rapid release of ENK from these cells, also induces an increase in ENK biosynthesis and ENK mRNA.

Ethanol exposure decreases pituitary corticotropin-releasing factor binding, adenylate cyclase activity, proopiomelanocortin biosynthesis, and plasma beta-endorphin levels in the rat.

Animals exposed continuously for 14 days to ethanol vapor in an inhalation chamber at sufficient ethanol vapor concentration to maintain blood ethanol levels from 100-250 mg/100 ml exhibited approximately 36% lower corticotropin-releasing factor binding and 24% lower adenylate cyclase activity in anterior (AL) and neurointermediate lobe (NIL) membranes of the pituitary gland compared to controls not treated with ethanol. To determine the effect of chronic ethanol exposure on proopiomelanocortin (POMC) biosynthesis, the levels of POMC mRNA in the AL and NIL were quantified by Northern blot and slot blot techniques. Ethanol treatment for 1, 7, or 14 days produced a time-related decrease in POMC mRNA levels, relative to total RNA levels, in both the AL and NIL.

Elevated potassium stimulates enkephalin biosynthesis in bovine chromaffin cells.

Exposure of bovine adrenal medullary cells in culture to a depolarizing concentration of potassium (50 mM), causes a rapid rise in both cellular and secreted Met-enkephalin peptide. The induction of peptide is preceded by the appearance of a nuclear preproenkephalin transcript and subsequent increases in cytoplasmic preproenkephalin mRNA. These data suggest that the depolarizing medium acts by enhancing enkephalin gene transcription.

Specific regulation of vasoactive intestinal polypeptide biosynthesis by phorbol ester in bovine chromaffin cells.

Two neuropeptides, enkephalin and vasoactive intestinal polypeptide (VIP), are simultaneously increased in cultures of bovine chromaffin cells after diverse treatments including elevation of cAMP, application of nicotine, or chronic depolarization. We now show that phorbol esters can specifically elevate VIP in cultured chromaffin cells without changing the amount of enkephalin. Peptide histidine isoleucine, a VIP-related peptide, is also expressed concomitantly with VIP after treatment with phorbol ester.

Vasoactive intestinal polypeptide afferents to the bed nucleus of the stria terminalis in the rat: an immunohistochemical and biochemical study.

Vasoactive intestinal polypeptide (VIP) has a markedly heterogeneous distribution in the rat bed nucleus of the stria terminalis. The dorsal bed nucleus contains the highest concentration of VIP in the rat brain, with the exception of the suprachiasmatic nucleus, 4-fold higher than the VIP concentration in the frontal cortex. These biochemical findings agree well with the immunohistochemical analysis of this area.

Neuropeptide Y and peptide YY neuronal and endocrine systems.

An extensive system of neuropeptide Y (NPY) containing neurons has recently been identified in the central and peripheral nervous system. In addition, NPY and a structurally related peptide, peptide YY (PYY), containing endocrine cells have been identified in the periphery. The NPY system is of particular interest as the peptide coexists with catecholamines in the central and sympathetic nervous system and adrenal medulla.

Corticotropin-releasing factor binding to peripheral tissue and activation of the adenylate cyclase-adenosine 3',5'-monophosphate system.

Specific binding sites for rat corticotropin-releasing factor (rCRF) are present in rat adrenal medulla, ventral prostate, spleen, liver, kidney, and testis and bovine chromaffin cells in culture. Maximal binding of [125I]rCRF occurred within 25 min at 4 C and was saturable. Scatchard analysis of rCRF binding to rat adrenal membranes and bovine chromaffin cells revealed the existence of two classes of binding sites.