Molecular pathways of pain: Fos/Jun-mediated activation of a noncanonical AP-1 site in the prodynorphin gene.

Noxious stimulation provokes the activation of genes that are thought to play a crucial role in the phenomena of stress and pain. Among these is the prodynorphin gene. By double-labeling in situ hybridization/immunohistochemistry, we show that increased prodynorphin gene expression is preceded, in the same neurons, by an early induction of c-fos. Inspection of the prodynorphin promoter region revealed the presence of several AP-1-like sequences. We demonstrate that only one of these sites is a functional AP-1 element. It is constituted by the noncanonical TGACAAACA sequence, in which the palindromic structure is partly conserved by the 3' terminal CA dinucleotide. Transfection experiments in NCB20 neuroblastoma cells indicated that this site is a target of Fos/Jun trans-activation. Our results suggest that Fos/Jun oncoproteins may function as third messengers in the signal transduction mechanisms of stress/pain processes.

Transcriptional response to cAMP in brain: specific distribution and induction of CREM antagonists.

Changes in cAMP levels are often associated with the modulation of neuronal function. The CREM gene encodes both antagonists and activators of the cAMP-dependent transcriptional response by alternative splicing. CREM transcripts in rat brain show a characteristic pattern of expression, being specific for the inner layer of the cerebral cortex, anterior thalamus, hippocampus, and hypothalamus. Strikingly, the CREM transcripts correspond to the antagonist isoforms in these areas, suggesting a down-regulatory role for CREM in brain; in contrast, the expression of CREM tau and CREB activators is more diffuse and generalized. In the supraoptic nucleus, CREM expression is induced after osmotic stimulus. Importantly, this demonstrates physiological inducibility of CREM, which is novel within the CRE/ATF family.

Molecular mechanisms of pain: serotonin1A receptor agonists trigger transactivation by c-fos of the prodynorphin gene in spinal cord neurons.

By using spinal cord neurons cultured in chemically defined medium, a double labeling procedure, and blockage with antisense oligonucleotides, we show that induction of c-fos and the subsequent transactivation of the prodynorphin gene are coupled events, triggered by serotonin1A receptor agonists. Addition of the specific 1A agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) to the culture, at concentrations similar to that needed for transactivation of the prodynorphin gene, also significantly increases cAMP levels. Furthermore, in rats depleted of serotonin by intrathecal administration of 5,7-dihydroxytryptamine, the induction of prodynorphin after noxious stimulation is dramatically decreased compared with the induction in sham-operated rats. These results suggest that the expression of the prodynorphin gene in spinal cord is under the control of the raphe-spinal efferents containing serotonin.

Protein kinase A-dependent derepression of the human prodynorphin gene via differential binding to an intragenic silencer element.

Induction of the prodynorphin gene has been implicated in medium and long-term adaptation during memory acquisition and pain. By 5' deletion mapping and site-directed mutagenesis of the human prodynorphin promoter, we demonstrate that both basal transcription and protein kinase A (PKA)-induced transcription in NB69 and SK-N-MC human neuroblastoma cells are regulated by the GAGTCAAGG sequence centered at position +40 in the 5' untranslated region of the gene (named the DRE, for downstream regulatory element). The DRE repressed basal transcription in an orientation-independent and cell-specific manner when placed downstream from the heterologous thymidine kinase promoter. Southwestern blotting and UV cross-linking experiments with nuclear extracts from human neuroblastoma cells or human brain revealed a protein complex of approximately 110 kDa that specifically bound to the DRE. Forskolin treatment reduced binding to the DRE, and the time course paralleled that for an increase in prodynorphin gene expression. Our results suggest that under basal conditions, expression of the prodynorphin gene is repressed by occupancy of the DRE site. Upon PKA stimulation, binding to the DRE is reduced and transcription increases. We propose a model for human prodynorphin activation through PKA-dependent derepression at the DRE site.

DREAM-alphaCREM interaction via leucine-charged domains derepresses downstream regulatory element-dependent transcription.

Protein kinase A-dependent derepression of the human prodynorphin gene is regulated by the differential occupancy of the Dyn downstream regulatory element (DRE) site. Here, we show that a direct protein-protein interaction between DREAM and the CREM repressor isoform, alphaCREM, prevents binding of DREAM to the DRE and suggests a mechanism for cyclic AMP-dependent derepression of the prodynorphin gene in human neuroblastoma cells. Phosphorylation in the kinase-inducible domain of alphaCREM is not required for the interaction, but phospho-alphaCREM shows higher affinity for DREAM. The interaction with alphaCREM is independent of the Ca(2+)-binding properties of DREAM and is governed by leucine-charged residue-rich domains located in both alphaCREM and DREAM. Thus, our results propose a new mechanism for DREAM-mediated derepression that can operate independently of changes in nuclear Ca(2+).

Mechanisms of Ca(2+)-dependent transcription.

Ca(2+) has a central role in coupling synaptic activity and transcriptional responses. Recent studies have focused on Ca(2+)-dependent nuclear mechanisms that bring to the nucleosomal level cascades of events initiated in the submembranous space at the synapse. In addition, a new Ca(2+)-dependent interaction between a calcium sensor and DNA has been shown to regulate transcription directly.

Differential expression of the jun family members in rat brain.

The transcription factor AP-1 is phorbol ester-regulated and, as such, is considered to be a nuclear target of the signal transduction pathway involving protein kinase C. AP-1 is constituted by the various products of the jun and fos gene family members. These genes belong to the early response class and are inducible in different ways by growth factors, phorbol esters and depolarization. We studied the transcript distribution of c-jun, junB and junD in the rat brain. Our results show that the transcripts for these three genes are differentially distributed in various neuronal tissues. We also provide evidence for developmentally regulated expression of jun genes in post-natal brain. The spatiotemporal pattern of expression of c-jun, junB and junD offers clues to the understanding of the links between gene regulation and neuronal processes.

Co-induction of jun B and c-fos in a subset of neurons in the spinal cord.

Noxious stimulation in vivo provokes the transcriptional activation of several genes which are thought to play an important role in the phenomena of stress and pain. In the rat, the expression of the c-fos proto-oncogene is rapidly induced upon noxious stimulation in defined neurons in the dorsal horn of the spinal cord. Interestingly, expression of the prodynorphin gene, which is thought to be involved in the endogenous mechanisms for pain/stress control, also localizes in the same anatomical area. Fos proteins are known to associate in transcriptional complexes with the products of the jun family constituting nuclear factor AP-1. These considerations prompted us to analyse the expression of the jun gene family members c-jun, jun B and jun D in rats subjected to noxious stimulation. We present data indicating that in unstimulated animals the transcripts of the three genes are differentially expressed and abundant within the various laminas of the lumbar spinal cord. Surprisingly, upon stimulation only the jun B transcript is augmented, being co-localized with Fos in a subset of neurons of the medial dorsal horn.

The DREAM-DRE interaction: key nucleotides and dominant negative mutants.

Transcriptional repressor DREAM, an EF-hand containing calcium-binding protein, blocks basal expression of target genes through specific interaction with DRE sites in the DNA. The sequence GTCA forms the central core of the DRE site, whereas flanking nucleotides contribute notably to the affinity for DREAM. Release of binding of DREAM from the DRE results in derepression, a process that is regulated by Ca(2+). Change of two amino acids within an EF-hand in DREAM blocks Ca(2+)-induced derepression and results in potent dominant negative mutants of endogenous DREAM.

Clomipramine treatment of obsessive-compulsive disorder. II. Biochemical aspects.

Concentrations of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA), the dopamine metabolite homovanillic acid, and the noradrenaline metabolite 4-hydroxy-3-methoxyphenyl glycol were measured in CSF before and after three weeks' treatment of severe obsessive-compulsive disorder with clomipramine hydrochloride. Patients who responded to clomipramine treatment had significantly higher CSF levels of 5-HIAA before treatment. The amelioration of obsessive-compulsive symptoms was positively correlated to the reduction of CSF concentrations of 5-HIAA during clomipramine treatment but negatively correlated to plasma concentrations of clomipramine. Reduction of CSF concentrations of 5-HIAA, which probably reflects drug action on central serotonin neurons, was maximal at a plasma clomipramine concentration of about 300 nmole/L. At higher levels, the reduction of CSF levels of 5-HIAA was smaller. The antiobsessive effect of clomipramine may be connected to its capacity to inhibit serotonin uptake.

Clomipramine treatment of childhood obsessive-compulsive disorder. A double-blind controlled study.

Nineteen children (mean [+/- SD] age, 14.5 +/- 2.3 years) with severe, primary obsessive-compulsive disorder completed a ten-week, double-blind, controlled trial of clomipramine hydrochloride (mean dosage, 141 mg/day) or placebo, each of which was administered for five weeks. Half of the subjects had not responded to previous treatment with other tricyclic antidepressants. There was a significant improvement in observed and self-reported obsessions and compulsions that was independent of the presence of depressive symptoms at baseline. Improvement in obsessive-compulsive symptoms did not correlate significantly with plasma concentrations of the drug or its metabolites. Clomipramine appears to be effective in the treatment of children with obsessive-compulsive disorder and the treatment seems to be independent of an antidepressant effect.

Induction of CREM activator proteins in spermatids: down-stream targets and implications for haploid germ cell differentiation.

The cAMP-responsive element modulator (CREM) gene encodes a family of transcriptional regulators that bind to promoter sequences activated by increased intracellular cAMP levels. Both activators and repressors are generated by alternative splicing and alternative translational initiation. During the development of male germ cells, there is a switch in the transcripts generated by CREM. Specifically, from the prophase of meiosis, there is an increase in the CREM tau activator transcript. Here we present results showing that expression of the CREM activator protein is restricted to postmeiotic germ cells. We show that CREM tau is efficiently phosphorylated at a serine residue at position 117 by the protein kinase-A endogenous to germ cells, indicating that it constitutes a natural target of the adenylyl cyclase pathway during spermatogenesis. Phosphorylation of serine-117 turns CREM tau into a powerful activator. The rise in CREM tau protein coincides with the transcriptional activation of several genes. We show that CREM tau efficiently binds to CREs present in the promoters of these genes, suggesting that they could constitute down-stream targets of CREM. We have analyzed in more detail the regulation of one of these genes, the male germ cell-specific RT7. The RT7 promoter is cAMP inducible and activated by CREM tau in transfection assays. The RT7 promoter is efficiently transcribed in vitro with nuclear extracts from seminiferous tubules. CREM-specific antibodies block RT7 in vitro transcription, implicating a role for CREM tau in a cascade of transcriptional events during spermatogenesis.

Independent expression of the alpha and beta c-erbA genes in developing rat brain.

Thyroid hormone is important for normal brain development. Cellular responses to thyroid hormone are mediated by multiple nuclear receptors, classified into alpha- and beta-subtypes. In the rat, expression of both the alpha and beta genes results in several translation products. By using cRNA probes common to alpha transcripts or specific for alpha-1 and beta-1, we have studied the distribution of these transcripts in rat brain at different stages of development from embryonic day 14 to adult age by using in situ hybridization histochemistry. On embryonic day 14, the alpha-1 mRNA is already widely expressed at a low level in the developing brain. The alpha-1 mRNA is developmentally regulated and showed a peak in expression during the first 3 postnatal weeks in the cerebral cortex, amygdala, hippocampus, and cerebellum. The probe common to the alpha transcripts detected a widespread distribution and high levels of these forms in the same regions throughout postnatal development. The level of beta-1 mRNA before birth was low or undetectable. The beta-1 transcript showed developmental regulation as well, with a high level at birth in the mitral cell layer of the olfactory bulb, accumbens nucleus, caudate, and hippocampal field CA1 and increasing levels in other regions later during development. Complementary expression of the alpha and beta forms was seen in the cerebral cortex and hippocampus. The differential temporal and spatial distribution as well as coexpression at comparable levels in certain brain regions suggest different roles for the c-erbA proteins during brain development and in the mature animal.

Experimental brain glioma: growth arrest and destruction by a blood-group-related tetrasaccharide.

A synthetic tetrasaccharide (TS4), structurally related to blood groups, inhibited the proliferation of the C6 glioma cells in culture and the growth of tumors formed after intracerebral transplantation of C6 cells. TS4-treated tumors were substantially smaller than controls, as expected from TS4 cytostatic action on C6 glioma cells in culture. However, in vivo treatment also caused extensive tumor destruction. This effect appeared to be caused by indirectly, either by activation of natural killer cells, cytotoxic lymphocytes, or by inhibition of tumor vascularization. Enhanced antigenicity of TS4-treated glioma may be related to the increased expression of connexin 43 observed in glioma cell cultures treated with the oligosaccharide. Because concentrations of up to 20 mg/ml of TS4 were not toxic for normal neuronal or glial cells, specific oligosaccharides such as TS4 offer the possibility of selective tumor treatment.

Differential effect of thyroid hormone on NGFI-A gene expression in developing rat brain.

NGFI-A is an immediate early gene that is rapidly activated in quiescent cells by mitogens or in postmitotic neurons after depolarization. We have previously shown that the expression of NGFI-A in the developing rat brain is under the control of thyroid hormone. Now we report, by means of in situ hybridization histochemistry, the differential effect of thyroid hormone on NGFI-A expression in distinct brain regions depending on the developmental stage. NGFI-A messenger RNA (mRNA) content was analyzed in the piriform cortex, striatum, hippocampus, and cerebral cortex of control, hypothyroid, and T3-injected hypothyroid rats at birth and on postnatal days 5 and 15. In the newborn rats, experimental hypothyroidism is associated with reduced levels of NGFI-A mRNA in most of the brain regions studied. On postnatal day 15, the difference in NGFI-A expression between control and hypothyroid rats is less apparent in the striatum or no longer present in the piriform cortex and the hippocampus. In the cerebral cortex, hypothyroidism is associated with reduced levels of NGFI-A mRNA on postnatal day 15. The dentate gyrus is always insensitive to the thyroidal state. Administration of T3 accelerates the recovery of NGFI-A mRNA in 5- and 15-day-old rats. However, in newborn rats, the effect of the hormone is noticeable only in the piriform cortex. We also show that the reduced level of NGFI-A mRNA in hypothyroidism is accompanied by a reduction in the protein level. Convulsions induced by pentylenetetrazole administration resulted in an increased expression of the NGFI-A gene, which is of similar magnitude in control and hypothyroid rats.

Amitriptyline metabolism: association with debrisoquin hydroxylation in nonsmokers.

Eleven healthy nonsmokers with wide variation in the ability to hydroxylate debrisoquin (D) were given single oral doses of amitriptyline and nortriptyline on different occasions. The urinary D/4-hydroxy-D ratio correlated significantly (P less than 0.01) with all three parameters of amitriptyline disposition measured (total plasma clearance, clearance by demethylation, and clearance by pathways other than demethylation), with rs = -0.89, -0.78, and -0.83, respectively. In contrast, we failed to demonstrate such correlations in a previous sample of smokers. Our data suggest that there may be a common regulation of the hydroxylation of D and the oxidative metabolism of amitriptyline in nonsmokers. It is hypothesized that an additional demethylase/hydroxylase is induced in smokers that is not involved in D hydroxylation.

Amitriptyline metabolism: relationship to polymorphic debrisoquine hydroxylation.

Amitriptyline AT demethylation to nortriptyline NT was determined in nine healthy subjects who had been phenotyped with respect to debrisoquine D hydroxylation capacity. AT demethylation was calculated from the ratio between the plasma AUCs of NT after single oral doses of AT and NT. Plasma clearance of AT by demethylation did not correlate with the ratio between D and 4-hydroxy-D in urine (rs = -0.55).

E- and Z-10-hydroxylation of nortriptyline: relationship to polymorphic debrisoquine hydroxylation.

Eight healthy subjects [who were phenotyped with a debrisoquine (D) hydroxylation test] were selected to cover a wide range in the ratio between D and 4-hydroxydebrisoquine (4-OH-D) in the urine. After a single oral dose of nortriptyline (NT) the metabolic clearance by 10-hydroxylation in the E-position, but not in the Z-position, correlated closely to the metabolic ratio D/4-OH-D (rs = -0.88, p less than 0.01). This indicates that common enzymatic mechanisms are involved in the hydroxylation of D and the E- but not the Z-10-hydroxylation of NT. Slow hydroxylators of NT and D excreted less 10-hydroxynortriptyline in urine and had lower plasma clearance of NT than the rapid hydroxylators. The strong correlation (r = 0.96) between the total plasma clearance of NT and the metabolic clearance by E-10-hydroxylation shows that this metabolic reaction is important in the disposition of the drug.

Nortriptyline and debrisoquine hydroxylations in Ghanaian and Swedish subjects.

Eleven Ghanaian and 12 Swedish subjects phenotyped with a debrisoquine (D) hydroxylation test were given a single oral dose of nortriptyline (NT). Much the same percentage of the given NT dose was excreted as 10-hydroxy-NT (10-OH-NT) by Ghanaians (43.1%) and Swedes (49.2%). There was a close correlation between plasma clearance of NT by 10-hydroxylation and the D metabolic ratio (D/4-OH-D in urine) in the Ghanaians (rs = -0.95; P less than 0.01) and Swedes (rs = -0.84; P less than 0.01). The E-isomer of 10-OH-NT is the major isomer in both Ghanaians (76% to 92% of total 10-OH-NT) and Swedes (78% to 95%). It is suggested that the E-10-hydroxylation of NT and the 4-hydroxylation of D are similarly coregulated in Ghanaians and Swedes.

Nortriptyline formation after single oral and intramuscular doses of amitriptyline.

Oral (50 mg) and intramuscular (25 mg) amitriptyline (AT) was given to six normal subjects and the area under the plasma concentration-time curve (AUC) for nortriptyline (NT) formed was calculated. There was no difference between the AUCs (corrected for dose) after the two routes of administration. The ratio between the AUCs (corrected for dose) after the two routes of administration. The ratio between AUCoral and AUCim averaged 0.95 (range 0.69 to 1.13). After intramuscular AT maximum NT plasma concentration was reached after 24 to 48 hr, whereas it was 8 to 24 after oral dosing.