Alternative modes of enkephalin biosynthesis regulation by reserpine and cyclic AMP in cultured chromaffin cells.

Catecholamines are produced in the medulla of the adrenal gland and may participate in the intraglandular regulation of its cortex. We analyzed the adrenal structure and function of albino tyrosine hydroxylase-null (TH-null) mice that are deficient in adrenal catecholamine production. Adrenal catecholamines were markedly reduced, and catecholamine histofluorescence was abrogated in 15-day-old TH-null mice. Chromaffin cell structure was strikingly altered at the ultrastructural level with a depletion of chromaffin vesicles and an increase in rough endoplasmic reticulum compared with wild-type mice. Remaining chromaffin vesicles lined up proximally to the cell membrane in preparation for exocytosis providing a ''string-ofpearls'' appearance. There was a 5-fold increase in the expression of proenkephalin mRNA (502.8 ؎ 142% vs. 100 ؎ 17.5%, P ‫؍‬ 0.016) and a 2-fold increase in the expression of neuropeptide Y (213.4 ؎ 41.2% vs. 100 ؎ 59.9%, P ‫؍‬ 0.014) in the TH-null animals as determined by quantitative TaqMan (Perkin-Elmer) PCR. Accordingly, immunofluorescence for met-enkephalin and neuropeptide tyrosine in these animals was strongly enhanced. The expression of phenylethanolamine N-methyl transferase and chromogranin B mRNA was similar in TH-null and wild-type mice. In TH-null mice, adrenocortical cells were characterized by an increase in liposomes and by tubular mitochondria with reduced internal membranes, suggesting a hypofunctional state of these steroid-producing cells. In accordance with these findings, plasma corticosterone levels were decreased. Plasma ACTH levels were not significantly different in TH-null mice. In conclusion, both the adrenomedullary and adrenocortical systems demonstrate structural and functional changes in catecholaminedeficient TH-null mice, underscoring the great importance of the functional interdependence of these systems in vivo.

Section: Discussionmentioning

confidence: 66%

Deletion of tyrosine hydroxylase gene reveals functional interdependence of adrenocortical and chromaffin cell system in vivo

Bornstein

Tian

Haidan

et al. 2000

“…Cyclic AMP has been recently demonstrated to increase specific mRNA levels for a number of neuropeptide precursor genes (44)(45)(46). It is of interest to determine whether there is a generalized program by which cyclic AMP can increase production of specific neurotransmitter, and whether this mechanism extends to other genes under transcriptional control by cyclic AMP.…”

Section: Resultsmentioning

confidence: 99%

Transcriptional regulation of the tyrosine hydroxylase gene by glucocorticoid and cyclic AMP.

Lewis

Harrington

Chikaraishi

1987

286

174

Glucocorticoid and cyclic AMP increase tyrosine hydroxylase (TH) activity and mRNA levels in pheochromocytoma cultures. The transcriptional activity of the TH gene, as measured by nuclear run-on assay, is also increased when cultures are treated with the synthetic glucocorticoid dexamethasone or agents that increase intracellular cyclic AMP, such as forskolin and 8-BrcAMP. Both inducers effect transcriptional changes within 10 min after treatment and are maximal after 30 min for forskolin and after 60 min for dexamethasone. The 5' flanking sequences of the TH gene were fused to the bacterial gene chloramphenicol acetyltransferase (CAT), and the hybrid gene was transfected into pheochromocytoma cultures and GH4 pituitary cells. In both cell lines, a region of the TH gene containing bases -272 to +27 conferred induction of CAT by cyclic AMP, but not by glucocorticoid. The same results were found when a region of the TH gene containing -773 to +27 was used. Thus, the sequences required for induction of TH by cyclic AMP are contained within 272 bases of 5' flanking sequence, but sequences sufficient for glucocorticoid regulation are not contained within 773 bases.The rate of biosynthesis of specific neurotransmitters can be modulated by a variety of environmental, neuronal, and hormonal stimuli. In catecholamine biosynthesis, the rate of flow through the pathway is largely dependent on the activity of the initial enzyme, tyrosine hydroxylase (TH), which is expressed in the adrenal medulla, sympathetic ganglia, and certain defined nuclei of the brain. Activity of tyrosine hydroxylase can be modulated both by changes in the synthetic rate of new tyrosine hydroxylase polypeptide and by posttranslational modification of preexisting enzyme molecules.The synthesis of TH is influenced by a variety of factors; in vivo, TH is induced in response to environmental stimuli, such as stress (1), and this induction can be mimicked pharmacologically by treatment of animals with agents that deplete cellular catecholamine stores, such as reserpine (2). In cultures of adrenal chromaffin or pheochromocytoma cells, tyrosine hydroxylase activity is induced by a number of effectors, including glucocorticoid (3, 4), cyclic AMP (5, 6), epidermal growth factor (7), and nerve growth factor (6,8,9).Whether these effectors act through similar mechanisms and whether changes are modulated transcriptionally or posttranscriptionally is not known. Using a cloned cDNA probe for TH, we have previously shown increases in the mRNA for tyrosine hydroxylase following treatment of clonal rat pheochromocytoma cells from an adrenal medullary tumor with analogs of cyclic AMP and glucocorticoid (10). The TH RNA is also increased in adrenal glands and superior cervical ganglia in vivo when animals are subjected to cold stress or reserpine treatment (11-14). In this communication, we have extended those studies by demonstrating that both glucocorticoid and cyclic AMP stimulate the transcriptional activity of the TH gene. In addition, we report that the 5...

“…1) and FT, combination of these drugs. In b-d, cells were plated as described and RNA was extracted by proteinase K digestion (29). In a, RNA from flasks of cells at near confluence was extracted by the guanidinium thiocyanate method (30).…”

Section: Resultsmentioning

confidence: 99%

Lineage-specific regulation of the vasoactive intestinal peptide gene in neuroblastoma cells is conferred by 5.2 kilobases of 5'-flanking sequence.

Waschek

Hsu

Eiden

1988

Self Cite

The expression of a transfected plasmid containing 5.2 kilobases (kb) of 5' regulatory DNA sequence of the human vasoactive intestinal peptide (VIP) gene attached to coding sequences of the reporter gene chloramphenicol acetyltransferase (CAT) was compared with endogenous VIP expression in subclones of the human neuroblastoma cell line SK-N-SH. These subclones vary widely in basal and inducible quantities of VIP and its precursor mRNA and can be interconverted under specified culture conditions. Endogenous VIP immunoreactivity, detectable in all subclones, was lowest in the neuronal subclone SH-SY-5Y, whereas 15-to 25-fold higher levels were observed in the epithelial-appearing SH-EP and intermediate SH-IN subclones. Treatment with 10 nM phorbol 12-myristate 13-acetate (PMA) stimulated VIP peptide levels =5-fold in SH-SY-5Y cells but did not increase appreciably VIP levels in the other subclones. Treatment with 2.5 ,uM forskolin resulted in <50% stimulation of VIP expression in all subclones. Levels of mRNA encoding the VIP precursor generally paralleled these differences in VIP immunoreactivity. In cells transfected with the VIP/CAT fusion gene, CAT activity reflected closely these differences in basal VIP expression and the changes in response to PMA and forskolin. Deletion of 2.7 kb of the most upstream sequences resulted in an 80-90% reduction in basal CAT activity in SH-IN, but not SH-SY-SY cells, and resulted in an 80% reduction in PMA stimulation in SH-SY-5Y cells. Deletion to within 74 nucleotides of the transcription start site resulted in CAT expression in SH-IN cells that was only 3% of that seen with the full 5.2-kb flanking sequences and further diminished the remaining PMA responsiveness in SH-SY-5Y cells. The data indicate that important cell-type-specific transcription regulatory sequences reside >2.5 kb upstream from the VIP transcription start site.Several important eukaryotic DNA regulatory sequences have been identified in recent years as well as some of the proteins that act on them (1-7). DNA sequences responsible for the constitutive and inducible expression of a number of genes have been identified by deletion and/or mutation analysis of transfected DNA (7-10). Analysis of tissue or cell-type-specific expression usually involves comparison of transfected gene activity in a cell line that endogenously expresses the gene of interest with that in an unrelated control cell line that does not express the gene (11-17). The appropriateness of such a comparison relies on the assumption that differences in endogenous transcription rates are controlled actively by regulatory factors present in one or both cell lines.A number of neuroblastoma cell lines display distinct cell subtypes that mimic properties of various neural crest derivatives (18, 19). We show here that SK-N-SH human neuroblastoma subtypes, which have been stably subcloned and characterized (18), display large differences in basal and phorbol ester-stimulated levels of vasoactive intestinal peptide (VIP). Because these subclon...