Amphetamine, 3,4-methylenedioxymethamphetamine, lysergic acid diethylamide, and metabolites of the catecholamine neurotransmitters are agonists of a rat trace amine receptor.

The trace amine para-tyramine is structurally and functionally related to the amphetamines and the biogenic amine neurotransmitters. It is currently thought that the biological activities elicited by trace amines such as p-tyramine and the psychostimulant amphetamines are manifestations of their ability to inhibit the clearance of extracellular transmitter and/or stimulate the efflux of transmitter from intracellular stores. Here we report the discovery and pharmacological characterization of a rat G protein-coupled receptor that stimulates the production of cAMP when exposed to the trace amines p-tyramine, beta-phenethylamine, tryptamine, and octopamine. An extensive pharmacological survey revealed that psychostimulant and hallucinogenic amphetamines, numerous ergoline derivatives, adrenergic ligands, and 3-methylated metabolites of the catecholamine neurotransmitters are also good agonists at the rat trace amine receptor 1 (rTAR1). These results suggest that the trace amines and catecholamine metabolites may serve as the endogenous ligands of a novel intercellular signaling system found widely throughout the vertebrate brain and periphery. Furthermore, the discovery that amphetamines, including 3,4-methylenedioxymethamphetamine (MDMA; "ecstasy"), are potent rTAR1 agonists suggests that the effects of these widely used drugs may be mediated in part by this receptor as well as their previously characterized targets, the neurotransmitter transporter proteins.

Behavioral screening for cocaine sensitivity in mutagenized zebrafish.

Understanding the molecular basis of addiction could be greatly aided by using forward genetic manipulation to lengthen the list of candidate genes involved in this complex process. Here, we report that zebrafish exhibit cocaine-induced conditioned place preference. In a pilot screen of 18 F(2) generation families of mutagenized fish, we found three with abnormally low responses to cocaine. This behavior was inherited by the F(3) generation in a manner that suggests the abnormalities were because of dominant mutations in single genes. Performance profiles in secondary behavioral screens measuring visual dark-adaptation and learning suggest that the defects were the result of mutations in distinct genes that affect dopaminergic signaling in the retina and brain.

Orphanin-FQ/nociceptin (OFQ/N) modulates the activity of suprachiasmatic nucleus neurons.

Neurons in the suprachiasmatic nucleus (SCN) constitute the principal circadian pacemaker of mammals. In situ hybridization studies revealed expression of orphanin-FQ/nociceptin (OFQ/N) receptor (NOR) mRNA in the SCN, whereas no expression of mRNA for preproOFQ/N (ppOFQ/N) was detected. The presence of OFQ/N peptide in the SCN was demonstrated by radioimmunoassay. SCN neurons (88%) responded dose-dependently to OFQ/N with an outward current (EC50 = 22.3 nM) that was reduced in amplitude by membrane hyperpolarization and reversed polarity near the theoretical potassium equilibrium potential. [Phe1psi(Ch2-NH)Gly2]OFQ/N(1-13)NH2 (3 microM), a putative NOR antagonist, activated a small outward current and significantly reduced the amplitude of the OFQ/N-stimulated current. OFQ/N reduced the NMDA receptor-mediated increase in intracellular Ca2+. When injected unilaterally into the SCN of Syrian hamsters housed in constant darkness, OFQ/N (1-50 pmol) failed to alter the timing of the hamsters' wheel-running activity. However, injection of OFQ/N (0.1-50 pmol) before a brief exposure to light during the midsubjective night significantly attenuated the light-induced phase advances of the activity rhythm. These data are consistent with the interpretation that OFQ/N acting at specific receptors modulates the activity of SCN neurons and, thereby, the response of the circadian clock to light.

Orphanin FQ/nociceptin: a role in pain and analgesia, but so much more.

The publication of the delta opioid receptor sequence led to the cloning of three homologous receptors: the mu and kappa opioid receptors, and a novel opioid-like orphan receptor. The orphan receptor's endogenous ligand, a 17-amino-acid peptide that resembles dynorphin, was named 'orphanin FQ' and 'nociceptin' (OFQ/N1-17). The OFQ/N1-17 receptor is expressed widely in the nervous system, and it is becoming clear that the peptide is likely to participate in a broad range of physiological and behavioral functions. At the cellular level, OFQ/N1-17 has much in common with the classical opioids; however, functional studies are now revealing distinct actions of this peptide. Identified only two years ago, OFQ/N1-17 has already attracted a great deal of attention. The number and diversity of papers focused on OFQ/N1-17 at the recent meeting of the Society for Neuroscience augur an exciting future for this new peptide.

Orphanin FQ is the major OFQ1-17-containing peptide produced in the rodent and monkey hypothalamus.

In order to investigate the processing of OFQ containing peptides in the hypothalamus we have developed a sensitive and quantitative radioimmunoassay for OFQ. We fractionated rodent and monkey hypothalamic extracts by reversed-phase high performance liquid chromatography and found that the extracts contained multiple peaks of OFQ immunoreactivity with the major peak co-eluting with synthetic OFQ1-17. Mouse hypothalamic extracts were also fractionated by SDS-PAGE to determine the apparent molecular weights of molecules containing the OFQ peptide. Multiple peaks of OFQ immunoreactivity, ranging in size from approximately 1 to 30 kilodaltons, were detected by this method. These results suggest that OFQ1-17 is processed to smaller peptides in mouse and monkey hypothalamic neurons.

Immortalized Hensen's node cells secrete a factor that regulates avian neural crest cell fates in vitro.

The derivatives of the neural crest are regionally specified with respect to the anterior-posterior axis of the avian embryo. We have shown previously that young Hensen's node can act in vitro to regulate the expression of certain region-specific phenotypes in trunk neural crest cells. To study potential factors acting on the neural crest, we have generated an immortalized cell line from young Hensen's node. Here we show that a factor produced by these cells stimulates the expression of two cranial-specific phenotypes (fibronectin and smooth muscle actin) in trunk neural crest cells and decreases their expression of a trunk-specific phenotype (melanin). The active factor is a secreted protein with a molecular weight >30 kDa. Clonal studies suggest that the factor acts by changing the phenotypic fates of individual neural crest cells, rather than by selective effects on cell proliferation or survival. Previous work has shown that TGF-betas can mimic the effects of Hensen's node cells on neural crest differentiation. Results from the present study suggest that the factor in the conditioned medium of the immortalized node cell line is not a TGF-beta isoform. However, the cranial phenotype-inducing activity of the conditioned medium factor requires the presence of neural crest cell-derived TGF-betas.

Role of the transforming growth factor-beta family in the expression of cranial neural crest-specific phenotypes.

Cranial and trunk neural crest cells produce different derivatives in vitro. Cranial neural crest cultures produce large numbers of cells expressing fibronectin (FN) and procollagen I (PCol I) immunoreactivities, two markers expressed by mesenchymal derivatives in vivo. Trunk neural crest cultures produce relatively few FN or PCol I immunoreactive cells, but they produce greater numbers of melanocytes than do cranial cultures. Treatment of trunk neural crest cultures with transforming growth factor-beta 1 (TGF-beta 1) stimulates them to express both FN and PCol I immunoreactivities at levels comparable to those normally seen in cranial cultures and simultaneously decreases their expression of melanin. These observations raised the possibility that endogenous TGF-beta is involved in specifying differences in the phenotypes expressed by cranial and trunk neural crest cells in vitro. Consistent with this idea, we found that treatment of cranial cultures with a function-blocking TGF-beta antiserum inhibits the development of FN immunoreactive cells and stimulates the development of melanocytes. Cranial and trunk neural crest cells express approximately equal levels of TGF-beta mRNA. However, trunk neural crest cells are significantly less sensitive to the FN-inducing effect of TGF-beta 1 than are cranial neural crest cells. These results suggest that: (1) endogenous TGF-beta is required for the expression of mesenchymal phenotypes by cranial neural crest cells, and (2) differences in the phenotypes expressed by cranial and trunk neural crest cells in vitro result in part from differences in the sensitivities of these two cell populations to TGF-beta.

Suppression of v-sis-dependent transformation by the transcription factor, Egr-1.

The transcription factor Egr-1, stimulates the activity of a number of genes and inhibits other genes, by binding to the sequence GCGGGGGCG in 5' enhancer regions. However, the functions of Egr-1 are obscure in spite of its rather ubiquitous expression. Egr-1 may play a role in proliferation in mitogen-stimulated cells but its expression is also correlated with the differentiated state in teratocarcinoma cells. The constitutive expression of Egr-1 appears to have little effect on the growth rate of normal immortalized cell-lines. We show that in NIH3T3 cells that are conditionally transformed by the expression of v-six, the presence of Egr-1 is inhibitory to the production of transformed colonies (foci) and to growth in soft agar. In addition, the first appearance of tumors in nu/nu mice is delayed in tumorigenicity tests with cells that over-express Egr-1 and tumor growth is suppressed compared to control cells. We used a series of fragments of Egr-1 cloned into expression vectors to show that not only full length, but also truncated Egr-1 fragments inhibit colony formation. Using deletion mutants, we observed that this inhibitory activity is dependent on the presence of the DNA-binding 'zinc-finger' region. Wilm's tumor protein, WT1, (known to be a tumor suppressor gene) that exhibits the same DNA binding activity is also inhibitory. In contrast, colony formation is stimulated by an Egr-1 antisense RNA-expressing plasmid, since colonies grow rapidly and the colony-forming frequency is higher than in the presence of v-sis alone. We conclude that proteins containing the Egr-1 'zinc-finger' domain can bind to the regulatory regions of one or more genes that are required for the transformation of fibroblasts by v-sis thus inhibiting transformation. One function for Egr-1 implied by these results is the restraint of transformed growth in mitogen-stimulated cells.

Regulation of epidermal growth factor receptor gene expression in murine embryonal carcinoma cells.

The protooncogene c-erbB1 [epidermal growth factor receptor (EGF-R)] is expressed in a wide variety of cell types and in most adult tissues. The precise roles of the EGF-R in vivo are largely unknown, especially their role in growth and development of embryonic tissues. We reported earlier that EGF-Rs are not expressed on the cell surface of undifferentiated embryonal carcinoma (EC) cells, but intracellular receptor protein is detectable (A. Weller, J. Meek, and E. D. Adamson, Development, 100: 351-363, 1987). We document here that in embryonal carcinoma cells, low levels of both receptor mRNA and protein are observed, but after 4 days of retinoic acid-induced differentiation, large increases are seen. Most notable is the 35-70-fold rise in the levels of EGF-R transcripts during the differentiation of P19 embryonal carcinoma cells to neural and glial cells, and this is paralleled by a 10-fold rise in protein. Measurements of the degradation rates of EGF-R mRNA and receptor protein show that both are rather stable and may partially explain the steady-state increases during differentiation. Run-on transcription assays of the EGF-R gene show very low rates of transcriptional activity at all stages: about 2-fold changes in transcription rate can be detected. It is concluded that transcriptional mechanisms may also partially account for increased levels of gene products. We hypothesize that the appearance of EGF-Rs at the cell surface leads to the slow induction of further receptor levels by EGF/transforming growth factor alpha stimulation, and this contributes to the driving force of differentiation and to the stability of the differentiated state.

The transcription factor, Egr-1, is rapidly modulated in response to retinoic acid in P19 embryonal carcinoma cells.

The pluripotent murine embryonal carcinoma cell line, P19, differentiates along at least three main pathways under the inductive influence of retinoic acid (RA). The events most critical to the establishment of a particular differentiation pathway must occur early since P19 cells are committed to differentiation pathways after 30 min of exposure to RA (M. W. McBurney, personal communication and our unpublished results). We have, therefore, looked for genes that are induced (or repressed) within 30 min of RA addition and find that Egr-1 is one of these genes. Egr-1 is a transcription factor of the zinc-finger class and is known to transactivate genes after binding to specific oligonucleotide sequences. We describe here the extremely rapid and transient increase of Egr-1 transcript and protein levels in P19 cells after RA addition. Stable induction of Egr-1 transcripts occurred in the presence of protein synthesis inhibitors. Simultaneous addition of RA and cycloheximide did not result in an additive effect. The mechanism of induction with either drug appears to involve relief of a block to transcriptional elongation. The response was more rapid at high RA concentrations and this suggests that the Egr-1 transcription factor could play a role in initiation of differentiation pathways of P19 EC cells.

Overexpression of uvomorulin in a compaction-negative F9 mutant cell line.

The mutant F9 cell line F9att-5.51 synthesizes reduced amounts of uvomorulin (UM) protein and we hypothesized earlier (Adamson, Baribault, and Kemler, Dev. Biol. (1990), 138, 338) that this may account for its inability to compact into tightly aggregated balls of cells. Subsequently, when 5.51 cells are treated with retinoic acid to stimulate their differentiation, they are unable to form embryoid bodies as do wild-type cells which form an outer epithelial layer of visceral endoderm cells. We have now examined the possibility that the UM protein made in the mutant line is defective, but find that it is normal in structure and stability. The gene coding for UM appears to be normal as does the mRNA which is synthesized at a normal rate but is severely reduced in steady-state measurements of mutant cells. A rescue experiment was performed by increasing levels of UM in mutant cells by means of transfection with a UM expression vector. The resulting cells expressed abundant UM mRNA and protein but were still unable to form compacted aggregates and did not differentiate into embryoid bodies. Interestingly, the stability of endogenous UM mRNA was improved in the presence of exogenous UM; therefore, a positive feedback mechanism contributes to low mRNA levels in mutant cells. The accumulated data suggest that UM in 5.51 cells is unable to mount a compaction activity because a distal connecting link in the multicomponent process initiated by UM is missing or or aberrant. The missing component is likely to connect UM to actin and the cytoskeleton of the cell.

Regulation of Egr-1 (Zfp-6) and c-fos expression in differentiating embryonal carcinoma cells.

The Egr-1 gene (zfp-6) encodes a 'zinc finger'-type transcription factor that is one of the early growth response genes induced, together with c-fos proto-oncogene, in many cell types. Our earlier work indicated that Egr-1 and c-fos may also play roles in differentiation and we now present data to show some features of their regulation. Transcriptional regulation accounts at least partly for the increased steady-state levels of Egr-1 mRNA in differentiating teratocarcinoma cells; this rate increases threefold over the 7-10 days of differentiation of P19 embryonal carcinoma cells with both 0.5% DMSO (to give predominantly cardiac muscle) and 1 microM retinoic acid (to give nerve and glial cells). The stability of Egr-1 transcripts remains the same (T1/2 = 90 min) in undifferentiated EC and differentiated cell products. In contrast, transcripts for c-fos are barely detectable in EC cells and increase 20-fold during differentiation. The basis for this is a marked increase in stability of c-fos mRNA after differentiation. The protein products of both genes parallel the steady-state levels of their mRNAs, but both proteins become more stable in differentiated cells. This is particularly marked for c-Fos protein, which appears as a distinct 58 kDa species in terminally differentiated P19 cells. Both Egr-1 and c-Fos proteins remain at high constitutive levels in differentiated cells indicating a distinct role for these transcription factors, For instance, it appears that this form of Fos protein may not repress the synthesis of the Egr-1 gene as it does during transient expression of serum-stimulated genes.