When rats are treated daily with reserpine or maintained at 4 degrees C, the level of a specific RNA coding for tyrosine hydroxylase is elevated in the adrenal gland. The increase in this specific RNA temporally precedes and is quantitatively equal to the increase in adrenal tyrosine hydroxylase enzyme activity elicited by these treatments. These results suggest that prolonged stress may lead to changes in the levels of specific RNA species in the adrenal gland.
Tyrosine hydroxylase (TH) gene expression in the adrenal medulla is regulated by numerous stimuli via transsynaptic mechanisms. The adrenal chromaffin cell receptors that mediate this transsynaptic response remain unidentified. In this report we demonstrate that the muscarinic acetylcholine receptor agonist bethanechol stimulates the TH gene transcription rate in both innervated and denervated adrenal glands. Hence, this muscarinic response is not dependent on transsynaptic influences, suggesting that agonist occupation of adrenal chromaffin cell muscarinic receptors is sufficient to activate intracellular signaling pathways that stimulate the TH gene. When bethanechol is administered repeatedly over a 3-h interval (four injections spaced 1 h apart), TH mRNA levels are increased two-to threefold at 6 and 12 h after the initial injection of drug. It is surprising that this induction of TH mRNA does not lead to increases in TH activity or TH protein level. These results are consistent with the hypothesis that both transcriptional and posttranscriptional mechanisms must be regulated to induce TH protein and that muscarinic agonists activate only a subset of these mechanisms.
Incubation of rat pheochromocytoma PC12 cells with dibutyryl cyclic AMP or 56 mM K+ is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase in situ. Following incubation of the PC12 cells with 32Pi, rapid isolation of the tyrosine hydroxylase, and tryptic digestion of the enzyme, two distinct 32P-peptides can be identified after paper electrophoresis. 56 mM K+ increases 32Pi incorporation into both of these peptides, whereas dibutyryl cyclic AMP increases 32Pi incorporation into only one of these peptides. The rate of increase in the incorporation of 32Pi into these two peptides in cells treated with 56 mM K+ is similar. The phosphorylation of tyrosine hydroxylase in PC12 cells occurs exclusively on serine residues. These results suggest that tyrosine hydroxylase in PC12 cells is phosphorylated on serine residues at two or more distinct sites after 56 mM K+ -induced depolarization. Since only one of these sites is phosphorylated by cyclic AMP-dependent protein kinase, activation of tyrosine hydroxylase by 56 mM K+ may involve phosphorylation by multiple protein kinases in rat pheochromocytoma PC12 cells.
Cell aggregation is one of several environmental cues that influence the expression of neurotransmitter phenotype during development. The expression of the catecholaminergic phenotype is increased in rat pheochromocytoma cells cultured at high density. In the present study we have investigated whether this cell density‐mediated effect on the catecholaminergic phenotype is due to the stimulation of the tyrosine hydroxylase gene. When rat pheochromocytoma PC 18 cells are cultured at high density (2 ± 105 cells/cm2), tyrosine hydroxylase enzymatic activity and tyrosine hydroxylase protein increase two‐to threefold over that observed in cells cultured at low density (1 ± 104 cells/cm2). This increase in tyrosine hydroxylase protein observed in high‐density cultures is fully accounted for by a preceding increase in tyrosine hydroxylase mRNA levels. The relative transcription rate of the tyrosine hydroxylase gene, measured using a nuclear run‐on assay, is two‐to threefold greater in PC 18 cells cultured at high density than in cells cultured at low density. Using flow cytometry, we have determined that in high‐density cultures, there are approximately twice as many cells in the G0‐G1 phases of the cell cycle compared with the number of G0‐G1 cells observed in low‐density cultures. However, when G0‐G1 cells are isolated by cellular elutriation, tyrosine hydroxylase gene transcription rate remains two‐to threefold greater in G0‐G1 cells from high‐density cultures than in G0‐G1 cells from low‐density cultures. These results indicate that increased cell‐cell contact stimulates the transcription rate of the tyrosine hydroxylase gene, resulting in the subsequent increased expression of tyrosine hydroxylase mRNA and protein.
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