The neuroprotective action of 3,3'-diindolylmethane against ischemia involves an inhibition of apoptosis and autophagy that depends on HDAC and AhR/CYP1A1 but not ERα/CYP19A1 signaling.

There are no studies examining the effects of 3,3'-diindolylmethane (DIM) in neuronal cells subjected to ischemia. Little is also known about the roles of apoptosis and autophagy as well as AhR and ERα signaling and HDACs in DIM action. We demonstrated for the first time the strong neuroprotective capacity of DIM in mouse primary hippocampal cell cultures exposed to ischemia at early and later stages of neuronal development. The protective effects of DIM were mediated via inhibition of ischemia-induced apoptosis and autophagy that was accompanied by a decrease in AhR/CYP1A1 signaling and an increase in HDAC activity. DIM decreased the levels of pro-apoptotic factors, i.e., Fas, Caspase-3, and p38 mitogen-activated protein kinase (MAPK). DIM also reduced the protein levels of autophagy-related Beclin-1 (BECN1) and microtubule-associated proteins 1A/1B light chain (LC3), partially reversed the ischemia-induced decrease in Nucleoporin 62 (NUP62) and inhibited autophagosome formation. In addition, DIM completely reversed the ischemia-induced decrease in histone deacetylase (HDAC) activity in hippocampal neurons. Although DIM inhibited AhR/CYP1A1 signaling, it did not influence the protein expression levels of ERα and ERα-regulated CYP19A1 which are known to be controlled by AhR. This study demonstrated for the first time, that the neuroprotective action of 3,3'-diindolylmethane against ischemia involves an inhibition of apoptosis and autophagy and depends on AhR/CYP1A1 signaling and HDAC activity, thus creating the possibility of developing new therapeutic strategies that target neuronal degeneration at specific molecular levels.

Influence of behavioral traits in the inter-individual variability of nociceptive, emotional and cognitive manifestations of neuropathic pain.

Neuropathic pain is a complex disorder associated with emotional and cognitive deficits that may impair nociceptive manifestations. There is high inter-individual variability in the manifestations of human neuropathic pain, which largely depends on personality traits. We aim to identify the influence of different behavioral traits in the inter-individual vulnerability to neuropathic pain manifestations using behavioral, electrophysiological and genetic approaches. We first selected mice with extreme social and emotional traits and look for correlation with the spontaneous neuronal activity in the central amygdala. Neuropathic pain was induced to these mice to evaluate the influence of behavioral traits on nociceptive manifestations and gene expression profiles in the amygdala. Our results show an association of the spontaneous central amygdala neuronal activity with the sociability behavior. We demonstrate that low sociable, high anxious and low depressive phenotypes develop enhanced nociceptive hypersensitivity after nerve injury. However, greater emotional alterations and cognitive impairment are observed in high sociable, anxious-like and depressive-like mice, indicating that nociceptive, emotional and cognitive manifestations of neuropathic pain do not correlate with each other. Gene analyses identify high Pdyn and Il6 levels in the amygdala as indicative of enhanced nociceptive hypersensitivity and reveal an association between high Gadd45 expression and attenuated emotional and cognitive manifestations of neuropathic pain.

Regulation of the extracellular signal-regulated kinases following acute and chronic opioid treatment.

The adaptations in extracellular signal-regulated kinase (ERK) pathway activity result in alterations in transcription of several genes that can be essential for development of both opioid tolerance and dependence. In this study, we investigated the effect of acute and prolonged opioid treatment on ERK pathway activity in SH-SY5Y cells. Acute administration of morphine and DAMGO stimulated ERK activity and this stimulation required activation of Ca(2+)/calmodulindependent kinase II (CaMKII) and protein kinase C (PKC). In contrast, prolonged morphine treatment decreased the level of phosphorylated ERK. The pr ecipitation of withdrawal further decreased the ERK1/2 activity. The principal finding of these studies is demonstration that the activation of CaMKII and PKC is required for ERK stimulation following acute opioid treatment while in a chronic morphine treatment and withdrawal, the up-regulation of PKC and CaMKII pathways seems to be engaged in the ERK inhibition. These results provide evidence that both opioid administration and opioid withdrawal, affecting similar intracellular pathways, can exert different effects on ERK activity.

Morphine-induced changes in the activity of proopiomelanocortin and prodynorphin systems in zymosan-induced peritonitis in mice.

We have shown that supplementation of proinflammatory agent with a high dose of morphine not only abolishes inflammation-related pain symptoms but also inhibits influx of leukocytes to the inflamed peritoneal cavity. Present investigations focused on effects of morphine on proopiomelanocortin and prodynorphin systems during zymosan-induced peritonitis. Males of SWISS mice were ip injected with zymosan (Z, 40 mg/kg) or zymosan with morphine (ZM, 20 mg/kg). At time 0 (controls) and 4 and 24h after stimulation, peritoneal leukocytes (PTLs) were counted, PTL levels of opioid peptides (beta-endorphin and dynorphin) measured by radioimmunoassays, while mRNAs coding their respective precursors (POMC and PDYN) and receptors (MOR and KOR) determined by QRT-PCR. Influx of inflammatory PTLs, mainly PMNs, was significantly delayed by morphine co-injection. Total levels of beta-endorphin and dynorphin corresponded with PTL numbers, while levels per cell were similar in all groups except of beta-endorphin, decreased in ZM at 4h. Levels of both peptides in peritoneal fluid were increased in Z and ZM groups at 4h, while at 24h only in case of beta-endorphin in Z group. POMC was increased only in ZM group at 4h of peritonitis, while PDYN in both Z and ZM groups at the same time. MOR mRNA was increased 24h after injection in Z and ZM groups, while KOR mRNA was similar in all groups except of decrease in Z at 24h. In conclusion, endogenous opioids and their receptors are involved in zymosan-induced peritonitis and affected in various ways by morphine co-injection.

Effects of morphine on gene expression in the rat amygdala.

Influence of morphine self-administration on gene expression in the rat amygdala was studied using rat genome DNA arrays U34A from Affymetrix. Animals were trained to self-administer morphine, each having two 'yoked' control animals, receiving passive injections of either morphine or saline. After 40 sessions of self-administration, amygdalae were removed, total RNA was isolated and used to prepare probes for Genechip arrays. The treatment was found to significantly change abundance of 29 transcripts. Analysis by means of reverse transcription real-time PCR showed significant changes in abundance of five transcripts: gamma protein kinase C (PKC), upstream binding factor 2 (UBF2), lysozyme, noggin and heat shock protein 70 (hsp70). After 30 days of forced abstinence from morphine self-administration, abundance of hsp70 and lysozyme returned to basal levels. Changes in abundance of UBF2 persisted, and abundance of three additional genes, namely nuclear factor I/A, gamma1 subunit of GABAA receptor and the neuronal calcium sensor 1, changed. Additionally, acute as well as chronic intraperitoneal morphine administration changed the abundance of PKC gamma, gamma1 subunit of GABAA and hsp70 genes.

Acute delta-opioid receptor activation induces CREB phosphorylation in NG108-15 cells.

A growing body of evidence supports an important role of the transcription factor cAMP responsive element binding protein (CREB) in mediating opioid-induced changes in the cAMP pathway. Regulation of CREB and subsequent changes in gene expression may underlie some long-term cellular adaptations associated with the administration of opioid drugs. The effect of morphine on the level of the transcription factor CREB, as well as CREB phosphorylation, was investigated in NG108-15 cells. Morphine and the delta-opioid receptor agonist [D-Pen(2,5)]enkephalin (DPDPE) produced a dose-dependent increase in CREB phosphorylation. The effect was reversed by naloxone and naltrindole, respectively. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), the protein kinase inhibitor staurosporine, as well as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), an inhibitor of protein kinase C and cAMP-dependent protein kinase, but not N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-8), an inhibitor of cAMP- and cGMP-dependent protein kinase, blocked the opioid-induced CREB phosphorylation. The obtained results suggest that in the cells studied opioids affect, via the delta-opioid receptor, stimulatory intracellular mediator systems involving Ca(2+)/calmodulin and the protein kinase C pathway.

Effect of opioids on Ca2+/cAMP responsive element binding protein.

Ca2+/cAMP response element binding protein (CREB) is an important factor linking the opioid-regulated secondary messenger systems to alterations in gene expression. Opioids regulate CREB level, its phosphorylation and binding to its corresponding response element in the promoters of several genes implicated in drug addiction. CREB mediates the action of opioids on the expression of several genes in brain regions responsible for drug-seeking behavior and manifestation of signs of dependence. Moreover, alterations in CREB level can effect the rewarding properties of morphine and regulate the self-administration of cocaine. At the cellular level CREB acts as convergence point for different cellular pathways. Opioids affect two different intracellular mediator systems: inhibitory--connected with cAMP, and stimulatory--involving calcium and the PKC pathway. Both can affect CREB but in different phases of opiate action. The presence of this biphasic mechanism can explain the phenomenon of the induction of some CRE-controlled genes after both acute and chronic morphine administration. Cellular studies also highlight the relevance of other ATF/CREB family members which can affect Ca2+/cAMP response element (CRE) controlled transcription as well as other transcription factors which make the opioid induction longer lasting.

The effect of antisense oligodeoxynucleotides on nitric oxide secretion from macrophage-like cells.

Nitric oxide (NO) plays an important role in cellular signaling and host defense, and it also contributes to the deleterious effects of immune response. Until recently, the lack of specific inhibitors of various forms of nitric oxide synthase (NOS) hampered a stringent evaluation of the role played by inducible NOS (iNOS) in cell damage. The present study investigated the use of antisense oligodeoxynucleotides (AS-ODNs) to selectively inhibit the expression of iNOS. AS-ODNs (1-10 microM) inhibited, in a time-dependent and dose-dependent manner, iNOS activity in RAW 264.7 murine macrophages. Maximal inhibitory effect was >90%, and control ODNs had little or no effect on NO production. Treatment with AS-ODNs decreased iNOS protein and mRNA level in studied cell, and control ODNs again were ineffective. The decreased levels of the target mRNA in AS-ODN-treated samples suggest that the AS-ODNs used act as substrates for ribonuclease (RNase) H. Lipofection enhanced the effect of AS-ODNs on iNOS activity. However, this potentiation appears to be different from the antisense effect, in which the AS-ODNs studied were involved. Liposaccharide/interferon-gamma (LPS/IFN-gamma) induced iNOS, and increased NO production impaired the viability of macrophages. Treatment of RAW 264.7 cells with 10 microM AS-ODNs prevented the NO-induced lethal cell damage.