A single intravenous injection of KRN5500 (antibiotic spicamycin) produces long-term decreases in multiple sensory hypersensitivities in neuropathic pain.

UNLABELLED: Neuropathic pain is a significant clinical problem. Currently, there are no drugs that produce complete amelioration of this type of pain. We have previously shown that KRN5500, a derivative of the antibiotic spicamycin, produces a prolonged (7-day), and significant reduction in neuropathic pain, but not nociceptive pain. Herein, we provide further evidence for the efficacy of this drug in inhibiting pain after IV injection in a spared nerve injury model of neuropathic pain. A single IV dose of the drug produces an increase in pain thresholds to punctuate mechanical stimuli and to cold stimuli over a period of 7 days, whereas IV injection of the vehicle is without any effect. No change in pain threshold was observed in the contralateral foot. In addition, a significant antiallodynic effect to mechanical stimuli was observed at 1, 2, 4, and 6 wk. The drug may be a potential candidate for cancer-related neuropathic pain as well as a marker for discovery of effective analgesics for neuropathic pain. IMPLICATIONS: We examined the effect of a novel drug (KRN5500) on nerve damage pain. After the successful effects of this drug in a single human, we have shown that the drug infused as a single application at different doses in a rat model of nerve damage pain produces pain relief in this model for many weeks.

A water-soluble synthetic spicamycin derivative (San-Gly) decreases mechanical allodynia in a rodent model of neuropathic pain.

The goal of this study was to examine the effects of synthetic spicamycin derivative, SAN-Gly, on mechanical allodynia in a spared nerve injury animal model of neuropathic pain. Adult male rats underwent surgical ligation and cutting of the common peroneal and tibial nerves, which produced a mechanical allodynia within 2-4 days. One week after the surgery, SAN-Gly was administered via intravenous injection. Mechanical allodynia was measured using von-Frey hairs. Spicamycin produced a significant reduction in mechanical allodynia for up to 6 weeks. This study demonstrates that SAN-Gly may be of potential use in treating patients with neuropathic pain.

Systemic lipopolysaccharide and interleukin-1beta activate the interleukin 6: STAT intracellular signaling pathway in neurons of mouse trigeminal ganglion.

We have studied the activation of signal transducers and activators of transcription (STATs) in trigeminal ganglion after intraperitoneal injection of lipopolysaccharide (LPS) and Interleukin-1beta (IL-1beta). Using electrophoretic mobility shift assays we have shown that STAT1 and STAT3 are activated within 1 to 2 h. of injection of either LPS or IL-1beta. Eight hours after LPS injection the DNA binding activity of these complexes is still elevated while induction by IL-1beta returns to baseline levels within 4 h. By immunohistochemistry, using an antibody specific for the tyrosine phosphorylated, activated form of STAT-1, we show that this induction occurs in sensory neurons. IL-6 may be important in this cascade since induction of STATs by IL-1beta is blocked in Interleukin-6 knock out mice.

Cyclic AMP-dependent activation of the proenkephalin gene requires phosphorylation of CREB at serine-133 and a Src-related kinase.

The transcription factor CREB [cyclic AMP response element (CRE)-binding protein] is activated by several kinase pathways on phosphorylation of serine-133. Phosphorylation of CREB at serine-133 is required for the induction of target gene expression. The proenkephalin gene is a target of cyclic AMP-dependent agonists like forskolin, and its expression is driven by the enhancer element CRE-2. It has been shown that CREB binds CRE-2 in extracts from striatum and hypothalamus. However, these studies did not show a functional requirement for CREB serine-133 phosphorylation in CRE-2 function. We demonstrate that CREB binds CRE-2 in primary astrocyte cultures and that transcriptional activation of CRE-2 requires CREB phosphorylation at serine-133. In addition, it has recently been shown that, at least in some contexts, CREB phosphorylation is not sufficient to activate target gene expression and that another intracellular signal seems to be required. Therefore, we also sought to determine if another signaling event, in addition to CREB phosphorylation, might be involved in cyclic AMP-mediated induction of the proenkephalin gene. We have found that the inhibition of src-related nonreceptor tyrosine kinases blocks forskolin-induced proenkephalin gene expression without having any effect on serine-133-phosphorylated CREB levels and that constitutively activated src kinase can activate the proenkephalin promoter.

PhosphoCREB and CREM/ICER: positive and negative regulation of proenkephalin gene expression in the paraventricular nucleus of the hypothalamus.

In the hypothalamic paraventricular nucleus (PVN), the proenkephalin gene may be upregulated by lipopolysaccharide (LPS) and downregulated by the GABA-A agonist muscimol. Candidate transcription factors regulating the proenkephalin gene in opposite directions are cAMP-response-element-binding protein (CREB) (when phosphorylated, a positive regulator) and cAMP-responsive modulatory inducible cAMP early repressor (CREM/ICER) (a negative regulator). Our results demonstrate that CREM alpha,beta,gamma transcripts and ICER are induced in the PVN by LPS and remain elevated for periods of up to 12 h. PhosphoCREB is elevated after LPS administration, peaking at 8 h, but remaining elevated over control levels at 12 h. Phospho-CREB induction by LPS is also seen in primary hypothalamic cultures. Cotransfection of ICER with ENK-CAT12 into primary hypothalamic cultures produced a decrease in chloramphenicol acetyl transferase (CAT) levels following cAMP or LPS stimulation. PhosphoCREB is downregulated and CREM/ICER is upregulated in the PVN by muscimol, suggesting that the regulation of these transcription factors may underlie the inhibitory effect of muscimol on target genes in the PVN.

Influence of cocaine on the JAK-STAT pathway in the mesolimbic dopamine system.

Chronic exposure to cocaine produces characteristic biochemical adaptations within the rat ventral tegmental area (VTA), a brain region rich in dopaminergic neurons implicated in the reinforcing and locomotor-activating properties of cocaine. Some of these changes are mimicked by chronic ciliary neurotrophic factor (CNTF) infusions into the same brain area. We show in this study that chronic cocaine treatment regulates the signal transduction pathway used by CNTF specifically in the VTA. There is an increase in immunoreactivity of Janus kinase (JAK2), a CNTF-regulated protein tyrosine kinase, in the VTA after chronic but not acute cocaine administration. This increase is not seen in the nearby substantia nigra or several other brain regions studied. Furthermore, this increase in JAK2 is not seen after chronic administration of other psychotropic drugs and was not observed for JAK1. The increase in JAK2 levels is associated with an increased responsiveness of the system to acute CNTF infusion into the VTA, as measured by induction in this brain region of signal transducers and activators of transcription (STAT) DNA binding activity and of Fos-like proteins, two known functional endpoints of JAK activation. Double-labeling immunohistochemical studies show that JAK2 immunoreactivity in the VTA is enriched in dopaminergic and nondopaminergic cells, both of which exhibit increased JAK2 immunoreactivity after chronic cocaine treatment. These findings suggest a scheme whereby some of the effects of chronic cocaine on VTA dopaminergic neurons are mediated directly by regulation of the JAK-STAT pathway in these cells, as well as perhaps indirectly by regulation of this pathway in nondopaminergic cells.

6-Hydroxydopamine lesions of rat substantia nigra up-regulate dopamine-induced phosphorylation of the cAMP-response element-binding protein in striatal neurons.

Destruction of the substantia nigra produces striatal D1 dopamine receptor supersensitivity without increasing receptor number or affinity, thus implicating postreceptor mechanisms. The nature of these mechanisms is unknown. Increased striatal c-fos expression ipsilateral to a unilateral lesion of the substantia nigra in rats treated with appropriate dopamine agonists provides a cellular marker of D1 receptor supersensitivity. D1 receptors are positively linked to adenylate cyclase and therefore to cAMP-dependent protein kinase. Because expression of the c-fos gene in response to cAMP- and Ca2+/calmodulin-regulated protein kinases depends on phosphorylation of cAMP-response element-binding protein (CREB) at Ser-133, we examined CREB phosphorylation after dopaminergic stimulation in cultured striatal neurons and in the striatum of rats after unilateral 6-hydroxydopamine ablation of the substantia nigra. Using an antiserum specific for CREB phosphorylated at Ser-133, we found that dopamine increases CREB phosphorylation in cultured striatal neurons. This effect was blocked by a D1 antagonist. L-Dopa produced marked CREB phosphorylation in striatal neurons in rats ipsilateral, but not contralateral, to a 6-hydroxydopamine lesion. This response was blocked by a D1 antagonist, but not a D2 antagonist, and was reproduced by a D1 agonist, but not a D2 agonist. These findings are consistent with the hypothesis that D1 receptor supersensitivity is associated with upregulated activity of cAMP-dependent or Ca2+/calmodulin-dependent protein kinases, or both, following dopamine denervation of striatal neurons.

Activating transcription factor-3 stimulates 3',5'-cyclic adenosine monophosphate-dependent gene expression.

Activating transcription factor-3 (ATF-3) is one member of a large family of leucine zipper transcription factors which bind to promoters responsive to cAMP and phorbol ester at the related cAMP (CRE) and phorbol ester response elements. We report here that ATF-3 is coexpressed with the neuropeptide precursor proenkephalin in human neuroblastoma SK-N-MC cells. Cotransfection experiments indicate that activation of proenkephalin gene expression by ATF-3 is dependent upon both the catalytic subunit of the cAMP-dependent protein kinase and the CRE-2 element. The CRE-2 element is essential for second messenger-inducible expression and is known to bind AP-1-like transcription factors. ATF-3 expressed in bacteria or from rabbit reticulocyte lysates binds to the proenkephalin CRE-2 element as a homodimer and as a heterodimer with Jun-D, another activator of proenkephalin transcription. ATF-3 stimulates binding of Jun-D to the proenkephalin CRE-2 element and acts synergistically with Jun-D to induce proenkephalin gene expression. Sequential immunoprecipitations of ATF-3 from SK-N-MC cells expressing proenkephalin indicate that ATF-3 is complexed with Jun-D in vivo and that both proteins are highly phosphorylated. Together, our results suggest that ATF-3 may play an important role in the regulation of gene expression by cAMP-dependent intracellular signaling pathways.

The cAMP-response-element-binding protein interacts, but Fos protein does not interact, with the proenkephalin enhancer in rat striatum.

The proenkephalin gene is a well-studied model of transcription factor-target gene interaction in the nervous system and has been proposed as a regulatory target of the protein product of the immediate-early gene c-fos. This regulatory mechanism has been proposed, in part, because the cAMP response element 2 (CRE-2) site, the key DNA regulatory element within the proenkephalin second-messenger-inducible enhancer, avidly binds AP-1 proteins, including Fos, in vitro. However, we observe a dissociation in the time course of activation of c-fos and proenkephalin mRNA in rat striatum after administration of the dopamine D2 receptor antagonist haloperidol. This result prompted us to investigate the composition of protein complexes in striatal nuclear extracts that bind to the CRE-2 site. Even though our striatal nuclear extracts had substantial basal and haloperidol-inducible AP-1-binding activities that contained Fos, we could not detect Fos in complexes bound to the CRE-2 element. Instead, as determined by antibody supershift analysis, we detect CRE-binding protein (CREB)-like proteins binding to CRE-2 in both basal and haloperidol-stimulated conditions. Finally, we show that haloperidol induces CREB protein phosphorylation in striatum.

Regulation of opioid gene expression: a model to understand neural plasticity.

The recent finding that neurotransmitters and drugs that affect neurotransmission have important influences on gene expression suggests that drug-induced alterations in gene expression may underlie many long-term effects of addictive drugs, for example, dependence and drug-seeking behaviors. These long-term adaptive responses to opiate drugs have been particularly difficult to understand at a mechanistic level. Data presented here indicate that the gene encoding the opioid precursor proenkephalin is highly regulated by neural activity, second-messenger pathways, and PKA. These observations raise the possibility that drugs of abuse (e.g., opiates acting through opiate receptors) may act at the genetic level to modulate the expression of endogenous opiates and that these effects may underlie one component of the brain's long-term adaptive response to exogenous opiates. The transgenic animals described above can be used to investigate opiate drug-induced changes in proenkephalin gene expression, allowing rapid analysis of changes in proenkephalin gene expression in highly restricted populations of neurons in a fashion previously impossible. In addition, by analyzing the effects of specific enhancer mutations on tissue-specific and transsynaptic regulation of proenkephalin expression, transgenic models will permit mechanistic investigations within the intact nervous system that cannot otherwise be undertaken. Investigation of mechanisms underlying this process requires the analysis of intracellular signaling pathways, responsive DNA regulatory elements, and the transcription factors transducing synaptic signals into gene regulation. In the studies described herein, we demonstrate that AP-1 complexes consisting of different Jun proteins differentially regulate proenkephalin transcription at the CRE-2 element. c-Jun constitutively activates proenkephalin transcription, whereas JunD activates in a fashion completely dependent on the activation of second-messenger pathways and the cAMP-dependent PKA. JunB alone has no effect on proenkephalin gene expression, yet this molecule effectively blocks activation mediated by JunD and, hence, may act as a repressor. These data are consistent with a model (figure 4) in which preexisting JunD mediates the rapid cAMP-dependent activation of the proenkephalin enhancer, whereas IEGs such as JunB or c-Fos mediate the protein synthesis-dependent inactivation. Because c-Jun activates proenkephalin transcription constitutively, induction of c-Jun may lead to a further and prolonged activation of proenkephalin gene expression. Hence, the ratio of c-Jun to JunB induction may determine whether proenkephalin is repressed or further activated.

cAMP-dependent regulation of proenkephalin by JunD and JunB: positive and negative effects of AP-1 proteins.

We demonstrate that JunD, a component of the AP-1 transcription factor complex, activates transcription of the human proenkephalin gene in a fashion that is completely dependent upon the cAMP-dependent protein kinase, protein kinase A. Activation of proenkephalin transcription by JunD is dependent upon a previously characterized cAMP-, phorbol ester-, and Ca(2+)-inducible enhancer, and JunD is shown to bind the enhancer as a homodimer. Another component of the AP-1 transcription complex, JunB, is shown to inhibit activation mediated by JunD. As a homodimer JunB is unable to bind the enhancer; however in the presence of c-Fos, high-affinity binding is observed. Furthermore, JunD is shown to activate transcription of genes linked to both cAMP and phorbol ester response elements in a protein kinase A-dependent fashion, further blurring the distinction between these response elements. These results demonstrate that the transcriptional activity of an AP-1-related protein is regulated by the cAMP-dependent second-messenger pathway and suggest that JunD and other AP-1-related proteins may play an important role in the regulation of gene expression by cAMP-dependent intracellular signaling pathways.

The effect of depolarization on expression of the human proenkephalin gene is synergistic with cAMP and dependent upon a cAMP-inducible enhancer.

Membrane depolarization is a critical component of neural signaling; in recent years there also has been a great deal of evidence that membrane depolarization can regulate neural gene expression. Therefore, excitatory neurotransmission may be an important mechanism of neural plasticity. We have investigated the intracellular pathways and DNA regulatory elements through which membrane depolarization activates expression of the neural gene encoding human proenkephalin. In PC12 and C6-glioma cells, depolarization-induced expression of a transfected proenkephalin fusion gene was proportional to extracellular calcium concentration and was inhibited by verapamil. Activation of the gene by KCl-induced depolarization or the calcium ionophore A23187 was dependent upon and synergistic with cAMP in PC12 and C6-glioma cells, but neither depolarization nor treatment with A23187 affected cAMP levels. Trifluoperazine and W7 inhibited depolarization-induced gene expression but did not affect expression induced by the adenylyl cyclase activator forskolin. At the level of the DNA, depolarization-induced activation is conferred on the proenkephalin gene by a previously characterized cAMP-inducible enhancer. Multiple copies of a single component element of that enhancer, containing the CGTCA sequence motif characteristic of cAMP regulatory elements, can reconstitute the entire repertoire of responses to both cAMP and depolarization. These data suggest a model in which membrane depolarization activates gene expression through a calcium-dependent pathway, potentially involving calmodulin, and in which the transcriptional responses to both cAMP and calcium are transduced by the same DNA element.

Purification and characterization of FMRFamidelike immunoreactive substances from the lobster nervous system: isolation and sequence analysis of two closely related peptides.

In the preceding paper (Kobierski et al: J. Comp. Neurol. 266:1-15, '87) FMRFamidelike immunoreactivity (FLI) was localized to specific cells and processes in the nervous system of the lobster Homarus americanus. In an effort to establish a role for this material we have purified and characterized a variety of immunoreactive peptides that can be extracted from the secretory pericardial organs. By using gel-filtration chromatography and three different HPLC systems, it has been established that little or no authentic FMRFamide is present. Of the major immunoreactive components two peptides were purified in sufficient quantity for microsequence analysis and have been tentatively identified as the octapeptides Ser-Asp-Arg-Asn-Phe-Leu-Arg-Phe-amide (FLI 3) and Thr-Asn-Arg-Asn-Phe-Leu-Arg-Phe-amide (FLI 4). Both of these are novel neuropeptides with some sequence homology to the previously described FMRFamide family. The pericardial organs release FLI when depolarized with 100 mM K+ in the presence of calcium. Between 75 and 80% of this release is accounted for by FLI 3 and FLI 4. One of these peptides (FLI 4) has been synthesized and shown to cochromatograph with the endogenous immunoreactive material. Preliminary studies show that this peptide can act as a modulator of exoskeletal and cardiac neuromuscular junctions.

FMRFamidelike peptides of Homarus americanus: distribution, immunocytochemical mapping, and ultrastructural localization in terminal varicosities.

The distribution of FMRFamidelike peptides was studied in the nervous system of the lobster Homarus americanus by using immunocytochemical and radioimmunological techniques. By radioimmunoassay FMRFamidelike immunoreactivity (FLI) was found in low levels (ca. 1 pmol/mg protein) throughout the ventral nerve cord and in much higher amounts (60-100 pmol/mg protein) in the neurosecretory pericardial organs. Immunocytochemical studies showed FLI in approximately 300-350 cell bodies, and in distinct neuropil regions, neuronal fiber tracts, and varicose endings. Specificity of the immunostaining was tested by preabsorbing the antiserum with FMRFamide, with peptides having similar carboxyl termini to FMRFamide (Met-enkephalin-Arg-Phe, Phe-Met-Arg-Tyr-amide), with several amidated peptides (alpha-melanocyte-stimulating hormone, substance P, oxytocin), and with proctolin, a peptide found widely distributed in the lobster nervous system. Of these substances, only FMRFamide blocked the staining. In addition to the pericardial organs, significant levels of FLI were found in neurosecretory regions associated with thoracic second roots and in the connective tissue sheath that surrounds the ventral nerve cord. In all three regions, immunocytochemical studies showed the FLI to be localized to fine fibers and associated terminal varicosities lying close to the surface of the tissue, with no obvious target in their immediate vicinity. When examined at the ultrastructural level, the immunoreactive varicosities of the thoracic second roots and of the ventral nerve cord sheaths were found a few microns from the surface of the tissue and contained electron-dense granules. In the immunoreactive nerve cord sheath endings, in addition to the large, dense granules, small, clear vesicles were found. The appearance and location of these terminals suggest a neurohormonal role for FMRFamidelike peptides in lobsters. The observation that low levels of FLI are found in the hemolymph supports this suggestion. In addition, the localization of FLI to particular neuronal somata, fiber tracts, and neuropil regions suggests possible functional roles for these peptides in (1) integration of visual and olfactory information, (2) function of the anterior and posterior gut, and (3) the control of exoskeletal muscles.