Adrenocorticotropic hormone activation of adenylate cyclase in raphe neurons: Multiple regulatory pathways control serotonergic neuronal differentiation

The RN46A cell line was derived from embryonic day 13 rat medullary raphe cells by infection with a retrovirus encoding the temperature-sensitive mutant of SV 40 large T antigen (tsT-ag). The RN46A cell line is neuronally restricted and constitutively differentiates following a shift to nonpermissive temperature. Differentiated RN46A cells express low levels of tryptophan hydroxylase (TPH) but no detectable levels of serotonin (5-HT). Treatment of cultures with the adrenocorticotrophic hormone peptide ACTH_4–10 up-regulates the expression of TPH immunoreactivity in differentiated RN46A cells, but 5-HT synthesis requires initial treatment with ACTH_4–10, followed by partial membrane depolarizing conditions. Up-regulation of TPH by ACTH_4–10 is apparently due to activation of adenylate cyclase, whereas the increased 5-HT synthesis with membrane depolarization can be blocked with the voltage-sensitive Ca²⁺ -channel blockers nifedipine and ω-conotoxin. ACTH_4–10 treatment also markedly up-regulates the expression of the 5-HT reuptake transporter, as do dibutyryl cyclic AMP and forskolin; chronic membrane depolarization has no effect on 5-HT reuptake. The expression of the high-affinity 5-HT_1A receptor is increased threefold by ACTH_4–10 treatment during differentiation and fivefold by differentiation under partial membrane depolarizing conditions. Combining ACTH_4–10 treatment and membrane depolarization does not increase expression of the 5-HT_1A receptor further. 5-HT release is constitutive in ACTH-treated RN46A cells and linked to spontaneous synaptic vesicle fusion in RN46A cells. Considered with previous results, these data indicate that multiple effectors, ACTH, brain-derived neurotrophic factor, and membrane depolarization, have both distinct and overlapping effects that regulate specific elements of the serotonergic neuronal phenotype during differentiation and maturation. © 1995 John Wiley & Sons, Inc.     

The invertible P-DNA segment in the chromosome of Escherichia coli.

In the chromosome of many strains of Escherichia coli K12 the excisable element e14 is found, which contains an invertible DNA region. This invertible P region, and the gene responsible for the inversion (pin) were cloned, together with other e14 sequences. The element e14 contains a gene which kills the host cell. This can be repressed by a function also coded by e14. The kil and repressor genes as well as the attachment site of the element were mapped in different regions of the element. The invertible segment and pin gene were sequenced. The invertible segment is 1794 bp long, and contains one large internal open reading frame of 879 bp and reading frames which overlap the end pont of the invertible segment. Although pin highly homologous to gin of phage Mu, neither the genetic organization of the P segment nor the sequence of the putative proteins resemble the invertible G segment of phage Mu (which codes for genes involved in tail fiber assembly). The complete DNA sequences of both invertible segments were screened for homology. No resemblance was found. The P segment is flanked by inverted repeat sequences of 16 bp. Comparison of these with related inversion systems points out that the recombination site maps probably within a 2-bp region. This cross-over site is contained within a short palindromic sequence (AAACC AA GGTTT) which is more or less conserved in the recombination sites of all related DNA invertases.     

The effects of serotonin on glucocorticoid receptor binding in rat raphe nuclei and hippocampal cells in culture

The raphe-hippocampal serotonin (5-HT) system is involved in the regulation of the hypothalamus-pituitary-adrenal axis. The purpose of this study was to determine and compare the roles of 5-HT in the regulation of glucocorticoid receptor (GR) binding in the raphe nuclei and in the hippocampus. The effects of 5-HT, 5-HT agonists, and the 5-HT reuptake inhibitor citalopram on GR binding sites were studied in primary cultures of the fetal raphe nuclei and the hippocampus. Exposure of hippocampal cells to 5-HT, (+/-)-2,5-dimethoxy-4-iodoamphetamine (DOI; a 5-HT2 agonist), or citalopram resulted in an increase in number of GR binding sites. The effect of DOI was blocked by ketanserin (a 5-HT2 antagonist). Specific and saturable GR binding was found in raphe cells. Exposure of raphe cells to 5-HT, (+/-)-8 hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; a 5-HT1A agonist), or citalopram induced a significant decrease in number of GR binding sites. The effect of 8-OH-DPAT was reversed by WAY 100135 [N-tert-butyl-3-[1-[1-(2-methoxy)phenyl]piperazinyl]-1-phenylpropiona mide; a 5-HT1A antagonist]. These results show that the regulation of GRs during fetal life is structure-dependent and involves different 5-HT receptor subtypes. Moreover, the regulation of hippocampal GRs by citalopram suggests an action of antidepressants independent of their effects on monoamines.