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.

Axonin-I is an axon-associated cell adhesion molecule (AxCAM) of the chicken, which promotes neurite outgrowth by interaction with the AxCAM Ll(G4) of the neuritic membrane. Here we report the cloning and sequence determination of a cDNA encoding axonin-I, Peptides generated by enzymatic cleavage showed similarity to the AxCAM F11. Degenerated polymerase chain reaction (PCR) primers were designed and an axonin-I fragment was amplified from mRNA of embryonic retina. Screening of a cDNA library from embryonic brain resulted in the isolation of a 4.0-kb cDNA insert with an open reading frame of 3108 nucleotides. The deduced polypeptide of 1036 amino acids includes a putative hydrophobic N-terminal signal sequence of 23 or 25 amino acids and a C-terminal hydrophobic sequence of 29 amino acids which is suggestive of sequences serving as signal for the attachment of a glycosyl-phosphatidylinositol (glycosyl-PtdIns) anchor. The putative mature form of axonin-1 comprises six immunoglobulin-like repeats, followed by four fibronectin-type I11 repeats.Axonin-1 exhibits 75% amino acid identity with the AxCAM TAG-1 of the rat, suggesting that it is the chicken homologue of TAG-1. Like TAG-1, axonin-I is glycosyl-PtdIns-anchored to the neuronal membrane; in contrast to TAG-1, it does not exhibit an Arg-Gly-Asp sequence.The interconnection of neurons by processes forming synaptic contacts is one of the crucial developmental stages of neurogenesis. In order to span long distances, bundles of axons are formed in a process which is thought to be driven by the tendency of growing axons to adhere specifically to and elongate along preexisting axons. The consistency with which

Neurotransmitter-related messenger RNAs were detected by in situ hybridization in sections of rat and mouse brains by using 35S-radiolabelled RNA probes transcribed from cDNAs cloned in SP6 promoter-containing vectors. The distribution of messenger RNAs for glutamic acid decarboxylase, tachykinins (substance P and K), and tyrosine hydroxylase was examined in the striatum, pallidum, and substantia nigra. Dense clusters of silver grains were observed with the RNA probe complementary of the cellular messenger RNA for glutamic acid decarboxylase (antisense RNA) over most large neurons in the substantia nigra pars reticulata and medium-sized to large neurons in all pallidal subdivisions. A few very densely and numerous lightly labelled medium-sized neurons were present in the striatum. Among the areas examined, only the striatum contained neurons labelled with the antisense tachykinin RNA. Most of these neurons were of medium size, and a few were large. With the antisense tyrosine hydroxylase RNA, silver grains were found over neurons of the substantia nigra pars compacta and adjacent A10 and A8 dopaminergic cell groups. No signal was observed with RNAs identical to the cellular messenger RNA for glutamic acid decarboxylase or tachykinin (sense RNA). These results show a good correlation with immunohistochemical studies, suggesting that documented differences in the distribution and the level of glutamic acid decarboxylase, tyrosine hydroxylase, and substance P immunoreactivities in neurons of the basal ganglia are related to differences in the level of expression of the corresponding genes rather than to translation accessibility, stability, or transport of the gene products.

Neuroendocrine cells release a portion of their stored secretory hormone content when exposed to tissue-specific secretagogues. In the case of the adrenal medulla, catecholamines and enkephalin peptides, as well as other secretory proteins, are secreted in response to acetylcholine, which is released onto cholinergic receptors on chromaffin cells upon splanchnic nerve stimulation in vivo. Secretagogue stimulation thus depletes intracellular stores of exportable hormone. We were interested to know whether the signal for exportable hormone release might also function as a signal for compensatory hormone repletion by enhancing the biosynthesis of the released hormone(s). Accordingly, we have investigated the effect of nicotinic receptor stimulation on Met-enkephalin peptide biosynthesis and expression of proenkephalin messenger RNA in primary cultures of bovine chromaffin cells. Our results, reported here, suggest a model for stimulus-secretion-synthesis coupling in which nicotinic receptor occupancy activates two pathways. One pathway, dependent on calcium and not mimicked by increased intracellular cyclic AMP, leads to exocytotic hormone release; the other, probably via a calcium-dependent increase in intracellular cyclic AMP, leads to a compensatory increase in intracellular enkephalin through activation of transcription of the proenkephalin structural gene.

Exposure of bovine chromaffin cells in primary culture to 5 ,.M reserpine or 25 ,uM forskolin results in an increase in enkephalin peptide levels within 24-48 hr; 25 j!M forskolin (or cholera toxin at 50 ,.g/ml) causes a 1.5-to 2-fold increase in enkephalin peptide levels, which is maximal after 48 hr of exposure and is totally blocked by addition of cycloheximide (0.5 ,Lg/ml). Reserpine (5 ,.M) elicits a 1.5-to 2-fold increase in enkephalin peptide levels within 24 hr, which is only partially blocked by cycloheximide. Chromatographic analysis of cellular extracts shows that forskolin increases levels of both [Metlenkephalin pentapeptide and high molecular weight enkephalin-containing peptides, while reserpine causes an increase in [Metlenkephalin pentapeptide and a concomitant Decrease in high molecular weight enkephalin-containing peptides, suggesting enhanced conversion of enkephalin precursor(s) to the mature polypeptide hormone. Measurement of preproenkephalin messenger RNA (mRNAe k) by RNA blot hybridization witli a cDNA probe for mRNAenk reveals that forskolin and cholera toxin cause a relatively rapid (<17 hr) 3-to 5-fold increase in mRNAenk, while exposure to reserpine elicits a gradual decrease in enkephalin mRNA (a 50%-80% decline) beginning within 24 hr and continuing over a 72-hr peritd. These results suggest that forskolin and reserpine differentially regulate enkephalin biosynthesis in cultured chromaffin cells, the former by increasing, presumably via a cAMP-dependent mechanism, cellular mRNA coding for preproenkephalin and the latter by a post-translational increase in proenkephalin processing.The parenchymal or chromaffin cells of the adrenal medulla have been long studied as a prototype of the biochemistry and cell biology of catecholamine biosynthesis and secretion in diverse neuroendocrine cell types and in neurons (1, 2). The bovine adrenal medulla also contains high levels of enkephalin peptides and has been used as starting material both for the purification and sequencing of preproenkephalin peptides and for cloning and sequencing complementary DNA to preproenkephalin messenger RNA (mRNAenk) (3)(4)(5)(6). In addition, primary cultures of bovine adrenomedullary cells have been used as a model system to examine enkephalin peptide co-release with catecholamines and the regulation of enkephalin biosynthesis by hormones and pharmacological agents (7)(8)(9)(10)(11)(12). In this study, we examined whether the effects of two types of pharmacological agents known to increase enkephalin peptide levels in chromaffin cells (9, 10, 12) could be accounted for by increased enkephalin gene transcription, which would be reflected in increased cellular mRNAenk, or if post-transcriptional events such as mRNA translation or preproenkephalin processing might be regulat- Peptide Radioimmunoassay.[Met]Enkephalin immunoreactivity in culture media and cell extracts (12) was measured by radioimmunoassay, using an enkephalin antiserum (RB-4) provided by Steve Sabol (National Heart, Lung, and Blood Institut...