Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction.

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.

Upregulated dynorphin opioid peptides mediate alcohol-induced learning and memory impairment.

The dynorphin opioid peptides control glutamate neurotransmission in the hippocampus. Alcohol-induced dysregulation of this circuit may lead to impairments in spatial learning and memory. This study examines whether changes in the hippocampal dynorphin and glutamate systems are related, and contribute to impairment of spatial learning and memory in a rat model of cognitive deficit associated with alcohol binge drinking. Hippocampal dynorphins (radioimmunoassay) and glutamate (in vivo microdialysis) were analyzed in Wistar rats exposed to repeated moderate-dose ethanol bouts that impair spatial learning and memory in the Water Maze Task (WMT). The highly selective, long-acting κ-opioid receptor (KOR) antagonist nor-binaltorphimine (nor-BNI) was administered systemically or into the hippocampal CA3 region to test a role of dynorphins in alcohol-induced dysregulations in glutamate neurotransmission and behavior in the WMT. The ethanol treatment impaired learning and memory, upregulated dynorphins and increased glutamate overflow in the CA3 region. Administration of nor-BNI after cessation of ethanol exposure reversed ethanol-induced changes in glutamate neurotransmission in animals exposed to ethanol and normalized their performance in the WMT. The findings suggest that impairments of spatial learning and memory by binge-like ethanol exposure are mediated through the KOR activation by upregulated dynorphins resulting in elevation in glutamate levels. Selective KOR antagonists may correct alcohol-induced pathological processes, thus representing a novel pharmacotherapy for treating of ethanol-related cognitive deficits.

Caspase-3 activity is reduced after spinal cord injury in mice lacking dynorphin: differential effects on glia and neurons.

Dynorphins are endogenous opioid peptide products of the prodynorphin gene. An extensive literature suggests that dynorphins have deleterious effects on CNS injury outcome. We thus examined whether a deficiency of dynorphin would protect against tissue damage after spinal cord injury (SCI), and if individual cell types would be specifically affected. Wild-type and prodynorphin(-/-) mice received a moderate contusion injury at 10th thoracic vertebrae (T10). Caspase-3 activity at the injury site was significantly decreased in tissue homogenates from prodynorphin(-/-) mice after 4 h. We examined frozen sections at 4 h post-injury by immunostaining for active caspase-3. At 3-4 mm rostral or caudal to the injury, >90% of all neurons, astrocytes and oligodendrocytes expressed active caspase-3 in both wild-type and knockout mice. At 6-7 mm, there were fewer caspase-3(+) oligodendrocytes and astrocytes than at 3-4 mm. Importantly, caspase-3 activation was significantly lower in prodynorphin(-/-) oligodendrocytes and astrocytes, as compared with wild-type mice. In contrast, while caspase-3 expression in neurons also declined with further distance from the injury, there was no effect of genotype. Radioimmunoassay showed that dynorphin A(1-17) was regionally increased in wild-type injured versus sham-injured tissues, although levels of the prodynorphin processing product Arg(6)-Leu-enkephalin were unchanged. Our results indicate that dynorphin peptides affect the extent of post-injury caspase-3 activation, and that glia are especially sensitive to these effects. By promoting caspase-3 activation, dynorphin peptides likely increase the probability of glial apoptosis after SCI. While normally beneficial, our findings suggest that prodynorphin or its peptide products become maladaptive following SCI and contribute to secondary injury.

Dysregulation of dynorphins in Alzheimer disease.

The opioid peptides dynorphins may be involved in pathogenesis of Alzheimer disease (AD) by inducing neurodegeneration or cognitive impairment. To test this hypothesis, the dynorphin system was analyzed in postmortem samples from AD and control subjects, and subjects with Parkinson or cerebro-vascular diseases for comparison. Dynorphin A, dynorphin B and related neuropeptide nociceptin were determined in the Brodmann area 7 by radioimmunoassay. The precursor protein prodynorphin, processing convertase PC2 and the neuroendocrine pro7B2 and 7B2 proteins required for PC2 maturation were analyzed by Western blot. AD subjects displayed robustly elevated levels of dynorphin A and no differences in dynorphin B and nociceptin compared to controls. Subjects with Parkinson or cerebro-vascular diseases did not differ from controls with respect to any of the three peptides. PC2 levels were also increased, whereas, those of prodynorphin and pro7B2/7B2 were not changed in AD. Dynorphin A levels correlated with the neuritic plaque density. These results along with the known non-opioid ability of dynorphin A to induce neurodegeneration suggest a role for this neuropeptide in AD neuropathology.

Study of molecular events in cells by fluorescence correlation spectroscopy.

To understand processes in a living cell, sophisticated and creative approaches are required that can be used for gathering quantitative information about large number of components interacting across temporal and spatial scales without major disruption of the integral network of processes. A physical method of analysis that can meet these requirements is fluorescence correlation spectroscopy (FCS), which is an ultrasensitive and non-invasive detection method capable of single-molecule and real-time resolution. Since its introduction about 3 decades ago, this until recently emerging technology has reached maturity. As commercially built equipment is now available, FCS is extensively applied for extracting biological information from living cells unattainable by other methods, and new biological concepts are formulated based on findings by FCS. In this review, we focus on examples in the field of molecular cellular biology. The versatility of the technique in this field is illustrated in studies of single-molecule dynamics and conformational flexibility of proteins, and the relevance of conformational flexibility for biological functions regarding the multispecificity of antibodies, modulation of activity of C5a receptors in clathrin-mediated endocytosis and multiplicity of functional responses mediated by the p53 tumor suppressor protein; quantitative characterization of physicochemical properties of the cellular interior; protein trafficking; and ligand-receptor interactions. FCS can also be used to study cell-to-cell communication, here exemplified by clustering of apoptotic cells via bystander killing by hydrogen peroxide.

Dynorphin A (1-17) induces apoptosis in striatal neurons in vitro through alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor-mediated cytochrome c release and caspase-3 activation.

Dynorphin A (1-17), an endogenous opioid neuropeptide, can have pathophysiological consequences at high concentrations through actions involving glutamate receptors. Despite evidence of excitotoxicity, the basic mechanisms underlying dynorphin-induced cell death have not been explored. To address this question, we examined the role of caspase-dependent apoptotic events in mediating dynorphin A (1-17) toxicity in embryonic mouse striatal neuron cultures. In addition, the role of opioid and/or glutamate receptors were assessed pharmacologically using dizocilpine maleate (MK(+)801), a non-equilibrium N-methyl-D-aspartate (NMDA) antagonist; 6-cyano-7-nitroquinoxaline-2,3-dione, a competitive alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate antagonist; or (-)-naloxone, a general opioid antagonist. The results show that dynorphin A (1-17) (>or=10 nM) caused concentration-dependent increases in caspase-3 activity that were accompanied by mitochondrial release of cytochrome c and the subsequent death of cultured mouse striatal neurons. Moreover, dynorphin A-induced neurotoxicity and caspase-3 activation were significantly attenuated by the cell permeable caspase inhibitor, caspase-3 inhibitor-II (z-DEVD-FMK), further suggesting an apoptotic cascade involving caspase-3. AMPA/kainate receptor blockade significantly attenuated dynorphin A-induced cytochrome c release and/or caspase-3 activity, while NMDA or opioid receptor blockade typically failed to prevent the apoptotic response. Last, dynorphin-induced caspase-3 activation was mimicked by the ampakine CX546 [1-(1,4-benzodioxan-6-ylcarbonyl)piperidine], which suggests that the activation of AMPA receptor subunits may be sufficient to mediate toxicity in striatal neurons. These findings provide novel evidence that dynorphin-induced striatal neurotoxicity is mediated by a caspase-dependent apoptotic mechanism that largely involves AMPA/kainate receptors.

Cytotoxic effects of dynorphins through nonopioid intracellular mechanisms.

Dynorphin A, a prodynorphin-derived peptide, is able to induce neurological dysfunction and neuronal death. To study dynorphin cytotoxicity in vitro, prodynorphin-derived peptides were added into the culture medium of nonneuronal and neuronal cells or delivered into these cells by lipofection or electroporation. Cells were unaffected by extracellular exposure when peptides were added to the medium. In contrast, the number of viable cells was significantly reduced when dynorphin A or "big dynorphin," consisting of dynorphins A and B, was transfected into cells. Big dynorphin was more potent than dynorphin A, whereas dynorphin B; dynorphin B-29; [Arg(11,13)]-dynorphin A(-13)-Gly-NH-(CH(2))(5)-NH(2), a selective kappa-opioid receptor agonist; and poly-l-lysine, a basic peptide more positively charged than big dynorphin, failed to affect cell viability. The opioid antagonist naloxone did not prevent big dynorphin cytotoxicity. Thus, the toxic effects were structure selective but not mediated through opioid receptors. When big dynorphin was delivered into cells by lipofection, it became localized predominantly in the cytoplasm and not in the nuclei. Big dynorphin appeared to induce toxicity through an apoptotic mechanism that may involve synergistic interactions with the p53 tumor-suppressor protein. It is proposed that big dynorphin induces cell death by virtue of its net positive charge and clusters of basic amino acids that mimic (and thereby perhaps interfere with) basic domains involved in protein-protein interactions. These effects may be relevant for a pathophysiological role of dynorphins in the brain and spinal cord and for control of death of tumor cells, which express prodynorphin at high levels.

Endogenous opioids and oligodendroglial function: possible autocrine/paracrine effects on cell survival and development.

Previous work has shown that oligodendrocytes (OLs) express both micro- and kappa-opioid receptors. In developing OLs, micro receptor activation increases OL proliferation, while the kappa-antagonist nor-binaltorphimine (NorBNI) affects OL differentiation. Because exogenous opioids were not present in our defined culture medium, we hypothesized that NorBNI blocked endogenous opioids produced by the OLs themselves. To test this, intact and partially processed proenkephalin and prodynorphin-derived peptides were assessed in OLs using immunocytochemistry or Western blot analysis, or both. Immature OLs possessed large amounts of intact and partially processed proenkephalin precursors, as well as posttranslational products of prodynorphin including dynorphin A (1-17). With maturation, however, intact or partially processed proenkephalin was expressed by only about 50% of OLs, while dynorphin A (1-17) was undetectable. To assess the function of OL-derived opioids, the effect of kappa-agonists/antagonists on OL differentiation and death was explored. kappa-Agonists alone had no effect. In contrast, NorBNI significantly increased OL death. Additive OL losses were evident when NorBNI was paired with toxic levels of glutamate, suggesting that kappa-receptor blockade alone is sufficient to induce OL death. Thus, the results indicate that OLs express proenkephalin and prodynorphin peptides in a developmentally regulated manner, and further suggest that opioids produced by OLs modulate OL maturation and survival through local (i.e., autocrine and/or paracrine) mechanisms.

p53 Latency. C-terminal domain prevents binding of p53 core to target but not to nonspecific DNA sequences.

The p53 transcription factor is either latent or activated through multi-site phosphorylation and acetylation of the negative regulatory region in its C-terminal domain (CTD). How CTD modifications activate p53 binding to target DNA sequences via its core domain is still unknown. It has been proposed that nonmodified CTD interacts either with the core domain or with DNA preventing binding of the core domain to DNA and that the fragments of the CTD regulatory region activate p53 by interfering with these interactions. We here characterized the sequence and target specificity of p53 activation by CTD fragments, interaction of activating peptides with p53 and target DNA, and interactions of "latent" p53 with DNA by a band shift assay and by fluorescence correlation spectroscopy. In addition to CTD fragments, several long basic peptides activated p53 and also transcription factor YY1. These peptides and CTD aggregated target DNA but apparently did not interact with p53. The potency to aggregate DNA correlated with the ability to activate p53, suggesting that p53 binds to target sequences upon interactions with tightly packed DNA in aggregates. Latent full-length p53 dissociated DNA aggregates via its core and CTD, and this effect was potentiated by GTP. Latent p53 also formed complexes via both its core and CTD with long nontarget DNA molecules. Such p53-DNA interactions may occur if latent p53 binding to DNA via CTD prevents the interaction of the core domain with target DNA sites but not with nonspecific DNA sequences.

Clustering of apoptotic cells via bystander killing by peroxides.

Clustering of apoptotic cells is a characteristic of many developing or renewing systems, suggesting that apoptotic cells kill bystanders. Bystander killing can be triggered experimentally by inducing apoptosis in single cells and may be based on the exchange of as yet unidentified chemical cell death signals between nearby cells without the need for cell-to-cell communication via gap junctions. Here we demonstrate that apoptotic cell clusters occurred spontaneously, after serum deprivation or p53 transfection in cell monolayers in vitro. Clustering was apparently induced through bystander killing by primary apoptotic cells. Catalase, a peroxide scavenger, suppressed bystander killing, suggesting that hydrogen peroxide generated by apoptotic cells is the death signal. Although p53 expression increased the number of apoptoses, clustering was found to be similar around apoptotic cells whether or not p53 was expressed, indicating that there is no specific p53 contribution to bystander killing. Bystander killing through peroxides emitted by apoptotic cells may propagate tissue injury in different pathological situations and be relevant in chemo-, gamma-ray, and gene therapy of cancer.

IGF-II enhances trichostatin A-induced TGFbeta1 and p21(Waf1,Cip1, sdi1) expression in Hep3B cells.

Cell growth and division are controlled through the actions of cyclin-dependent kinases (CDKs) and cyclin dependent kinase inhibitors (CKIs). Treatment of cell lines with Trichostatin A leads to induction of one of these CKIs, p21, and growth arrest. Induction of p21 can also occur through the actions of TGFbeta1. Latent TGFbeta1 can be activated by the M6P/IGF2R. In the present study we have examined the effect of TSA on members of the IGF axis, the CKIs p21 and p27, and also TGFbeta1 in Hep3B cells. The only member of the IGF axis to be affected by treatments was IGF2. Expression of another gene from the same chromosomal location, H19, was also affected. TGFbeta1 expression was greatly enhanced by TSA. In addition, both CKIs, p21 and p27, were upregulated by TSA. Effects of adding IGF-II or TGFbeta1 to TSA-treated cells on p21 induction were examined. The results show that the induction of p21 by TSA can be modulated by additions of IGF-II whereas addition of TGFbeta1 affects its own expression but not p21. In conclusion, the results indicate that the induction of p21 and cell growth arrest caused by Trichostatin A may involve multiple signaling pathways.

A novel neuron-specific DNA end-binding factor in the murine brain.

To characterize the distribution of transcription factor AP-1 and YY1 DNA-binding activities in the rat brain, the labeled target oligonucleotides were loaded on brain sections and after incubation and washing, the residual signal was registered by autoradiography. The binding was predominantly associated with neurons and was regionally specific with highest levels in the cerebellum, hippocampus, and piriform cortex. The identified binding factor was not, however, sequence-specific, but apparently recognized DNA ends and was activated by long double-stranded DNA. UV cross-linking identified the molecular mass of the factor to be about 80 kDa. The factor was not found in soluble brain extracts, suggesting its association with membranes or the nuclear matrix. Despite apparent similarities with Ku protein, which targets DNA-ends, the DNA end-binding activity was present in brains of Ku86- and Ku70-deficient mice. Since DNA end-binding factors are generally involved in DNA repair, the same function may be suggested for the novel factor identified in the present study.

Opioids modulate cell division in the germinal zone of the late embryonic neocortex.

Opioid effects on cell division in the embryonic cerebral cortex were examined using two experimental approaches: (i) the presence of opioid receptors in the embryonic day 16 mouse neocortex was tested using immunohistochemical techniques; (ii) the values of the indices of [3H]thymidine pulse labelled cells and mitotic indices were estimated in the ventricular zone of the embryonic day 16 mouse neocortex 2.5, 4.5 and 8.5 h after administration to pregnant females of selected opioid receptor agonists or the opioid antagonist naloxone. The immunohistochemical study demonstrated that distinct subpopulations of the ventricular zone cells express mu, delta or kappa opioid receptors. Acute exposure of mouse embryos to mu, delta and kappa opioid receptor agonists or naloxone differentially affects the indices of [3H] thymidine pulse labelled cells and mitotic indices indicating changes in the cell cycle composition. Treatment with the mu opioid receptor agonist D-Ala2-MePhe4, Gly-ol5-enkephalin (DAGO), or the partially selective kappa opioid receptor agonist bremazocine, increased the [3H]thymidine labelling and mitotic indices. In contrast, the delta receptor agonist (D-Ser8)-leucine enkephalin-Thr (DSLET) produced a decrease in the labelled cell indices and mitotic indices. Naloxone provided a biphasic effect: a decrease in the values of labelled cell indices 2.5 h after naloxone administration, followed by an increase in the values of the indices at 4.5 and 8.5 h. These results suggest that the endogenous embryonic/maternal opioid systems are involved in the regulation of cell division in the ventricular zone of the late embryonic cortex.

Autoantigen Ku in the brain. Developmentally regulated expression and subcellular localization.

A double-stranded DNA end-binding factor with high levels of expression in brain and testis of adult mice was identified as the Ku protein, earlier described as an autoantigen in connective tissue diseases and found to be essential for recombination of the immunoglobulin genes and DNA repair. High Ku levels were found in the cerebellum and pituitary gland, lower levels in the hippocampus, hypothalamus and white matter structures. Ku levels were much higher in embryonic rat brain than in the adult brain, suggesting a role of the Ku protein in brain development. In embryonic rat brain, Ku was associated with cell nuclei, but was predominantly located in the cytosol in the adult rat cerebellum and hippocampus. The abundant expression of Ku in the brain suggests the involvement of Ku autoantibodies in the pathogenesis of neuropsychiatric complications in connective tissue diseases.

Long-term morphine treatment increases Ku protein DNA end-binding activity.

Human neuroblastoma SH-SY5Y and small-cell lung carcinoma U1690 cells of neuroendocrine origin were exposed to morphine for 1 h, 3 h or 5 days. These treatments did not alter activities of AP-1, NF-kappa B and YY1 transcription factors in SH-SY5Y cells or NF-kappa B and YY1 in U1690 cells. Five-day morphine treatment, however, caused a twofold increase in the activity of a sequence-non-specific, spermidine-activated DNA-binding factor in U1690 cells. The morphine effect was prevented by the antagonist naloxone. The DNA-binding factor bound preferentially to double-stranded DNA ends. This fact and data on subunit composition, molecular masses of subunits, and supershift/inhibition by specific antibodies in a band shift assay, show the spermidine-activated factor to be identical with the Ku protein, the DNA-binding subunit of DNA-dependent protein kinase. The effect observed may be one of the mechanisms through which opioids influence gene regulation.

The Leu-enkephalin-encoding sequence DNA-binding factor (LEF) is the transcription factor YY1.

The Leu-enkephalin-encoding sequence DNA-binding factor (LEF) with high affinity for the Leu-enkephalin-encoding sequences in the prodynorphin and proenkephalin genes has earlier been identified. This factor is composed of three subunits of about 60, 70 (the major DNA-binding subunit), and 95 kDa, respectively. Estimated molecular mass, sequence specificity of DNA-binding, and supershift/inhibition with specific antibodies in a band shift assay showed that the DNA-binding subunit of LEF is identical to the multifunctional transcription factor YY1. However, an antibody against the C-terminus of YY1 distinguished the YY1 complexes with a Leu-enkephalin-encoding sequence and canonical YY1 binding site oligonucleotides, suggesting different protein conformations in complexes with these two DNA fragments.

The single-stranded DNA end binding site of p53 coincides with the C-terminal regulatory region.

p53 is a transcription factor that binds double-stranded (ds) DNA in a sequence-specific manner. In addition, p53 can bind the ends of single-stranded (ss) DNA. We previously demonstrated that ssDNA oligonucleotides interact with the C-terminal domain of p53 and stimulate binding to internal segments of long ssDNA by the p53 core domain. Here we show that the p53 C-terminal domain can recognize staggered ss ends of dsDNA. We have mapped the binding site for ssDNA ends to residues 361-382 in human p53 using a p53 deletion mutant (p53-delta 30) lacking the 30 C-terminal amino acid residues and a series of 22mer peptides. The binding site for DNA ends coincides with a region previously implicated in regulation of sequence-specific DNA binding by the core domain. The interaction of the C-terminal regulatory domain with the ends of ssDNA or with the protruding ends of dsDNA stimulates both sequence-specific and non-specific DNA binding via the core domain. Electron microscopy demonstrated the simultaneous binding of p53 to dsDNA and a ssDNA end. These results suggest a model in which interaction of the p53 C-terminal tail with DNA ends generated after DNA damage causes activation of sequence-specific p53 DNA binding in vivo and may thus provide a molecular link between DNA damage and p53-mediated growth arrest and apoptosis.

[Leu5]enkephalin-encoding sequences are targets for a specific DNA-binding factor.

A DNA-binding factor with high affinity and specificity for the [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes has been characterized. The factor has the highest affinity for the [Leu5]-enkephalin-encoding sequence in the dynorphin B-encoding region of the prodynorphin gene, has relatively high affinity for other [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes, but has no apparent affinity for similar DNA sequences coding for [Met5]-enkephalin in the prodynorphin or proopiomelanocortin genes. The factor has been named [Leu5]enkephalin-encoding sequence DNA-binding factor (LEF). LEF has a nuclear localization and is composed of three subunits of about 60, 70, and 95 kDa, respectively. The highest levels were observed in rat testis, cerebellum, and spleen and were generally higher in late embryonal compared to newborn or adult animals. LEF activity was also recorded in human clonal tumor cell lines. LEF inhibited the transcription of reporter genes in artificial gene constructs where a [Leu5]enkephalin-encoding DNA fragment had been inserted between the transcription initiation site and the coding region of the reporter genes. These observations suggest that the [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes also have regulatory functions realized through interaction with a specific DNA-binding factor.

p53 binds single-stranded DNA ends through the C-terminal domain and internal DNA segments via the middle domain.

We have previously reported that wild-type p53 can bind single-stranded (ss) DNA ends and catalyze renaturation of ss complementary DNA molecules. Here we demonstrate that p53 can also bind to internal segments of ss DNA molecules via a binding site (internal DNA site) distinct from the binding site for DNA ends (DNA end site). Using p53 deletion mutants, the internal DNA site was mapped to the central region (residues 99-307), while the DNA end site was mapped to the C-terminal domain (residues 320-393) of the p53 protein. The internal DNA site can be activated by the binding of ss DNA ends to the DNA end site. The C-terminal domain alone was sufficient to catalyze DNA renaturation, although the central domain was also involved in promotion of renaturation by the full-length protein. Our results suggest that the interaction of the C-terminal tail of p53 with ss DNA ends generated by DNA damage in vivo may lead to activation of non-specific ss DNA binding by the central domain of p53.

Prodynorphin gene expression relates to NF-kappa B factors.

The prodynorphin gene contains several kappa B motifs, suggesting that kappa B-specific DNA-binding factors may regulate its expression. Prodynorphin is known to be expressed in human tumor cell lines [Geiger et al., Regul. Peptides, 34 (1991) 181-188] and we report here that several DNA-binding factors of the NF-kappa B/c-Rel-family are present in the same cells. Three main kappa B-specific factors, presumably a p50 homodimer, NF-kappa B which is a p50/p65 heterodimer and a p65/c-Rel heterodimer were identified using an electromobility shift assay (EMSA), immunoabsorption and UV cross-linking experiments. Minor factors consisting of a novel kappa B-specific protein of about 125 kDa (p125) or being hetero-oligomeric, composed of p125 and either of three other subunits, namely p50, p65 and c-Rel, were also identified. The homo-oligomer of p125 may be identical to the kappa B-specific factor BETA, previously found only in brain [Korner et al., Neuron, 3 (1989) 563-572]. Comparison of prodynorphin mRNA levels with levels of the kappa B-specific DNA-binding factors revealed a negative correlation with the level of p50 homodimer, and a positive correlation with the ratio of the levels of p65/c-Rel to NF-kappa B. No association was found with proenkephalin mRNA levels which were significant in only one cell line. The p50 homodimer, but not p65/c-Rel and NF-kappa B, bound specifically to a DNA-motif within the dynorphin A-encoding gene sequence. This sequence is located in exon 4 and similar to the consensus kappa B-sequence. The dynorphin A-encoding sequence may represent an intragenic target for the p50 homodimer, which when bound to the sequence suppresses transcription.