Orisa J. Igwe

Hyperalgesia induced by peripheral inflammation is mediated by protein kinase C βII isozyme in the rat spinal cord

We have addressed the molecular mechanism(s) of hyperalgesia, which depends on increased excitability of dorsal horn neurons and on sensitization of primary afferent nociceptors, during peripheral inflammation. Following unilateral adjuvant-induced inflammation in the rat hind paw, time-course changes in behavioral hyperalgesia and functional activities of Ca2+/phospholipid-dependent protein kinase C isozymes were examined. Inflammation was characterized by increase in paw diameter, and behavioral hyperalgesia was quantified as paw withdrawal latency from a radiant heat source. Behavioral hyperalgesia on the injected paw was significantly increased. This was accompanied by a significant increase in total functional membrane-associated protein kinase C activity, whereas total cytosolic protein kinase C activity was unchanged on the sides of the lumbar spinal cord both contralateral and ipsilateral to the inflammation. Importantly, on the side of lumbar cord ipsilateral to the inflamed paw, the activity of membrane-associated protein kinase CbetaII was increased following the same time-course as the paw withdrawal latency decrease, suggesting an increased translocation of protein kinase Cbetall to the membrane related to behavioral hyperalgesia. A defined mixture of purified gangliosides, which inhibits intracellular protein kinase C translocation and activation, decreased inflammation-induced paw withdrawal latency, and specifically decreased the activity of membrane-associated protein kinase Cbetall on the side of the spinal cord ipsilateral to the inflammation. Quantitative immunohistochemical analyses demonstrated intensified protein kinase CbetaII-like immunoreactivity on the side of the spinal cord ipsilateral to the inflammation. Time-course for increases in the activity of membrane-associated protein kinase CbetaII, and in intensity of protein kinase CbetaII-immunoreactivity, paralleled inflammation-mediated changes in paw withdrawal latency and paw diameter. Our findings indicate an apparent involvement of protein kinase CbetaII isozyme specifically in the molecular mechanism(s) of thermal hyperalgesia.

Regulation of the second-messenger systems in the rat spinal cord during prolonged peripheral inflammation.

&NA; Unilateral intraplantar injection of Freunds complete adjuvant (FCA) into 1 hind paw of rats was used as a model of peripheral inflammation and persistent pain in order to examine time course effects of a continuous barrage of nociceptive input on the second‐messenger transducing systems in the spinal cord. cAMP, cGMP and inositol 1,4,5‐trisphosphate (insP3) were extracted from the lumbosacral cord at days 1, 7, 14, 21 and 42 following FCA injection and quantified by either radioreceptorassay (RRA) or radioimmunoassay (RIA). The lumbosacral contents of cAMP and cGMP when quantified in whole lumbosacral cord segment were not significantly changed by FCA treatment at all time points. InsP3 accumulation was significantly increased on days 14, 21 and 42 following FCA injection relative to sham‐treated time‐matched controls. However, cGMP and insP3 contents were significantly increased in the left longitudinal half of the lumbar enlargement ipsilateral to the injected paw on day 21 following FCA treatment, but not in the sham‐treated time‐matched controls. With [3H]insP3 as a ligand, Scatchard (Rosenthal) analyses of the concentration‐dependent saturation curves showed that the densities (Bmax) of insP3 receptors (insP3R) were significantly increased throughout the time course of adjuvant‐induced peripheral inflammation. The binding affinities (KD) for insP3R were significantly decreased on days 7, 14 and 21 following FCA injection corresponding to the times of most stable and peak inflammation. InsP3R from the cerebelli of the same rats as used in the lumbosacral insP3R characterization was used as a positive control in this study and did not show any change in both Bmax and KD as a result of FCA treatment, thus demonstrating that the changes in lumbosacral insP3R characteristics might be specific to the nociceptive sensory pathway such as the spinal cord. Thus it appears that sustained afferent nociceptive input induced by FCA injection increased the accumulation of cGMP, insP3 and insP3R density in the spinal cord through increased neuronal activities of functional receptors coupled to major classes of chemical mediators of nociception including neuropeptides and excitatory aminoacids. Changes in insP3 accumulation in the lumbosacral cord following FCA injection were significantly correlated with changes in insP3R density. Changes in the ratios of lumbosacral insP3 contents and insP3R density were also significantly correlated with changes in body weight and hind paw size induced by FCA injection. Thus, a relationship is established between modulation in the spinal cord insP3/insP3R system, an intracellular calcium‐mobilizing transduction system, and modifiable systemic variables induced by peripheral inflammation.

Inositol 1,4,5-trisphosphate arm of the phosphatidylinositide signal transduction pathway in the rat cerebellum during aging

To determine whether the intracellular calcium-mobilizing second messenger, inositol 1,4,5-trisphosphate (InsP3) and its receptor (InsP3R) display age-dependent coordinate regulation, InsP3 content and [3H]InsP3-binding characteristics were investigated in cerebellar particulate membranes prepared from male Fischer 344 rats at 3, 12 and 25 months of age. Cerebellar InsP3 content was significantly increased in 25-month-old rats compared with 3-month-old animals. Cerebellar InsP3R densities were significantly reduced at 12 and 25 months of age but InsP3-binding affinity was significantly decreased only in the 25-month-old animals. The present data strongly suggest that modulation of the phosphoinositide second messenger system may contribute to impaired neuronal responsiveness associated with the aging process in the cerebellum.

Interleukin 1-induced cyclooxygenase and nitric oxide synthase gene expression in the rat dorsal root ganglia is modulated by antioxidants.

Interleukin 1beta induced both nitric oxide synthase 2 (NOS-2) and cyclooxygenase 2 (COX-2) gene expression in dorsal root ganglion explant culture with increased NOS-2 and COX-2 activities, and corresponding increases in the production of nitric oxide and prostaglandin E(2). The proinflammatory cytokine also increased 8-isoprostaglandin F(2alpha) concentration, an index of oxidant stress-mediated production of lipid hydroperoxides/reactive oxygen species. The signaling mechanisms by which interleukin 1beta regulates NOS-2 and COX-2 genes remain obscure. Reactive oxygen species play an important role in inflammatory processes as mediators of injury, and potentially as intracellular signaling molecules in interleukin 1beta-mediated regulation of gene expression. The effects of antioxidants that act by different mechanisms on interleukin 1beta-mediated NOS-2 and COX-2 gene expression were studied in rat dorsal root ganglion explants. The oxidant scavenger pyrrolidine dithiocarbamate abolished interleukin 1beta-induced NOS-2 mRNA accumulation and decreased nitric oxide production in a concentration-dependent manner, thus indicating that this antioxidant decreased either the transcription of NOS-2 gene or the stability of NOS-2 mRNA. In contrast, pyrrolidine dithiocarbamate significantly inhibited COX-2 gene expression at the posttranscriptional level, since pyrrolidine dithiocarbamate did not affect interleukin 1beta-induced COX-2 mRNA transcripts but inhibited COX-2 protein expression and prostaglandin E(2) production. Rotenone, another antioxidant that attenuates reactive oxygen species production by inhibiting the mitochondrial electron transport system, failed to inhibit interleukin 1beta-induced NOS-2 and COX-2 mRNA-encoding transcripts. However, rotenone inhibited NOS-2 and COX-2 proteins and associated nitric oxide and prostaglandin E(2) production, respectively, suggesting a posttranscriptional target for interleukin 1beta-mediated regulation of NOS-2 and COX-2 gene expression. Furthermore, both pyrrolidine dithiocarbamate and rotenone also decreased interleukin 1beta-induced 8-isoprostaglandin F(2alpha) production. These results indicate that not only transcriptional regulation, but also posttranscriptional events are involved in a redox-sensitive regulation of interleukin 1beta-induced NOS-2 and COX-2 gene expression in the dorsal root ganglia. Overall, interleukin 1beta-induced oxidant stress appears to regulate NOS-2 and COX-2 gene expression primarily at the level of protein translation. By implicating reactive oxygen species production in interleukin-1beta receptor-activated molecular signaling in the dorsal root ganglia, our data suggest a possible novel target for intervention in cytokine-mediated inflammatory processes.

c-Src kinase activation regulates preprotachykinin gene expression and substance P secretion in rat sensory ganglia

Increased synthesis of substance P (SP) in the dorsal root ganglia (DRG) and enhanced axonal transport to and secretion from the primary afferent sensory neurons might enhance pain signalling in the spinal dorsal horn by modifying pronociceptive pathways. IL‐1β increases SP synthesis by enhancing the expression of preprotachykinin (PPT) mRNA encoding for SP and other tachykinins in the DRG. Stimulation of IL‐1 receptor by IL‐1β may induce the phosphorylation of tyrosine residues in many effector proteins through the activation of p60c‐src kinase. The hypothesis that the synthesis of SP in and secretion from the primary sensory ganglia are regulated by the activation of p60c‐src kinase induced by IL‐1β was tested. Pretreatment of DRG neurons in culture with herbimycin A, genistein or PP2, three structurally different nonreceptor tyrosine kinase inhibitors that act by different mechanisms, decreased the kinase activity of p60c‐src induced by the activation of IL‐1 receptor. PP3, a negative control for the Src family of tyrosine kinase inhibitor PP2 had no effect. Herbimycin A and genistein also decreased IL‐1β‐induced expression of PPT mRNA‐encoding transcripts and the levels of SP‐li synthesized in the cells and secreted into the culture medium in a concentration‐dependent manner. SB 203580 [a p38 mitogen‐activated protein kinase (p38 MAPK) inhibitor] and PD 98059 (a p44/42 MAPK kinase inhibitor) were ineffective in modulating IL‐1β‐induced SP synthesis and secretion, and p60c‐src kinase activity in DRG neurons. Whereas, IL‐1 receptor antagonist and cycloheximide inhibited IL‐1β‐evoked secretion of SP‐like immunoreactivity (SP‐li), actinomycin D decreased it significantly but did not entirely abolish it. These findings show that phosphorylation of specific protein tyrosine residue(s) following IL‐1 receptor activation might play a key role in IL‐1β signalling to modulate PPT gene expression and SP secretion in sensory neurons. In view of the role of SP as an immunomodulator, these studies provide a new insight into neural‐immune intercommunication in pain regulation in the sensory ganglia through the IL‐1β‐induced p60c‐src activation.

Prostaglandin E2 promotes proliferation of skeletal muscle myoblasts via EP4 receptor activation

We recently demonstrated that conditioned media (CM) from osteocytes enhances myogenic differentiation of myoblasts, suggesting that signaling from bone may be important for skeletal muscle myogenesis. The effect of CM was closely mimicked by prostaglandin E2 (PGE2), a bioactive lipid mediator in various physiological or pathological conditions. PGE2 is secreted at high levels by osteocytes and such secretion is further enhanced under loading conditions. Although four types of receptors, EP1 to EP4, mediate PGE2 signaling, it is unknown whether these receptors play a role in myogenesis. Therefore, in this study, the expression of EPs in mouse primary myoblasts was characterized, followed by examination of their roles in myoblast proliferation by treating myoblasts with PGE2 or specific agonists. All four PGE2 receptor mRNAs were detectable by quantitative real-time PCR (qPCR), but only PGE2 and EP4 agonist CAY 10598 significantly enhance myoblast proliferation. EP1/EP3 agonist 17-phenyl trinor PGE2 (17-PT PGE2) and EP2 agonist butaprost did not have any significant effects. Moreover, treatment with EP4 antagonist L161,982 dose-dependently inhibited myoblast proliferation. These results were confirmed by cell cycle analysis and the gene expression of cell cycle regulators. Concomitant with the inhibition of myoblast proliferation, treatment with L161,982 significantly increased intracellular reactive oxygen species (ROS) levels. Cotreatment with antioxidant N-acetyl cysteine (NAC) or sodium ascorbate (SA) successfully reversed the inhibition of myoblast proliferation and ROS overproduction caused by L161,982. Therefore, PGE2 signaling via the EP4 receptor regulates myogenesis by promoting myoblast proliferation and blocking this receptor results in increased ROS production in myoblasts.

Toll-Like Receptor 4–Mediated Nuclear Factor Kappa B Activation Is Essential for Sensing Exogenous Oxidants to Propagate and Maintain Oxidative/Nitrosative Cellular Stress

The mechanism(s) by which cells can sense exogenous oxidants that may contribute to intracellular oxidative/nitrosative stress is not clear. The objective of this study was to determine how cells might respond to exogenous oxidants to potentially initiate, propagate and/or maintain inflammation associated with many human diseases through NF-κB activation. First, we used HEK-Blue cells that are stably transfected with mouse toll-like receptor 4 (mTLR4) or mouse TLR2. These cells also express optimized secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of a promoter inducible by NF-κB transcription factor. These cells were challenged with their respective receptor-specific ligands, different pro-oxidants and/or inhibitors that act at different levels of the receptor signaling pathways. A neutralizing antibody directed against TLR4 inhibited responses to both TLR4-specific agonist and a prooxidant, which confirmed that both agents act through TLR4. We used the level of SEAP released into the culture media due to NF-κB activation as a measure of TLR4 or TLR2 stimulation. Pro-oxidants evoked increased release of SEAP from HEK-Blue mTLR4 cells at a much lower concentration compared with release from the HEK-Blue mTLR2 cells. Specific TLR4 signaling pathway inhibitors and oxidant scavengers (anti-oxidants) significantly attenuated oxidant-induced SEAP release by TLR4 stimulation. Furthermore, a novel pro-oxidant that decays to produce the same reactants as activated phagocytes induced inflammatory pain responses in the mouse orofacial region with increased TLR4 expression, and IL-1β and TNFα tissue levels. EUK-134, a synthetic serum-stable scavenger of oxidative species decreased these effects. Our data provide in vitro and related in vivo evidence that exogenous oxidants can induce and maintain inflammation by acting mainly through a TLR4-dependent pathway, with implications in many chronic human ailments.

Modulation of peripheral inflammation in sensory ganglia by nuclear factor κB decoy oligodeoxynucleotide: involvement of SRC kinase pathway

Nuclear factor kappa B (NF(kappa)B) transcription factor plays a key role in the expression of many genes involved in the inflammatory process. We used the Freunds Complete Adjuvant (FCA)-induced model of peripheral inflammation to investigate the anti-inflammatory effects of double stranded oligodeoxynucleotides (ODN) with consensus NF(kappa)B sequence as transcription factor decoys to inhibit NF(kappa)kappaB activation in the dorsal root ganglia (DRG). Local administration of the wild-type-, but not mutant-ODN decoy, dose-dependently inhibited edema formation and paw withdrawal latency as a measure of hyperalgesic response induced by FCA in rat paw. Biochemical assays performed in ipsilateral L4/L5 dorsal root ganglia obtained following FCA/wild-type ODN treatment showed: (1) an inhibition of the activity of c-Src kinase, a member of the non-receptor tyrosine kinase super family, (2) a decreased level of p65 NF(kappa)B subunit, and (3) an inhibition of cyclooxygenase-2 (COX-2) protein expression, a major pro-inflammatory enzyme transcriptionally controlled by NF(kappa)B. The present results indicate that the wild-type ODN decoy may act as a competitor for NF(kappa)B binding to its cognate recognition sequence as well as a modulator of c-Src activity in the DRG. The NF(kappa)B/c-Src interaction may represent a novel pathway for further exploring the molecular mechanism of inflammatory pain.

Regulation of β-amyloid precursor protein and inositol 1,4,5-trisphosphate receptor gene expression during differentiation of a human neuronal cell line

Abstract Retinoic acid-induced differentiation of SH-SY5Y human neuroblastoma cells results in the development of extensive neurite processes as well as changes in cell body morphology toward a neuronal phenotype. The authors have examined concurrent regulation of β-amyloid precursor protein (APP) and inositol 1,4,5-trisphosphate receptor (insP 3 R) gene expression in SY5Y cells during neuronal differentiation. Of the multiple APP mRNA transcripts expressed in this cell line, retinoic acid treatment significantly increased the expression of APP 695 transcript while the level of total APP remained unchanged. In the same time course, neuronal differentiation decreased the expression of insP 3 R at both the mRNA and protein levels. These findings demonstrate an inverse relationship between APP and insP 3 R gene expression during neuronal differentiation of SH-SY5Y cells and suggest a possible change in intracellular calcium homeostasis.

Regulation of IL-1β-Induced Cyclooxygenase-2 Expression by Interactions of Aβ Peptide, Apolipoprotein E and Nitric Oxide in Human Neuroglioma

Alzheimer disease (AD) is characterized by chronic neuroinflammation, which may lead to dysfunction in neuronal circuits. Although reactive microglia are found in association with accumulation of beta amyloid (Aβ) plaques in the AD brain, their contribution to neuronal cell loss remains speculative. A major genetic risk factor for sporadic AD is inheritance of the apolipoprotein (apo) E4 allele, which has been shown to contribute significantly to neurodegeneration in AD. Many studies have documented the ability of Aβ fibrils in vitro to induce microglia to undergo phenotypic activation, which results in the secretion and/or expression of a plethora of free radicals and pro-inflammatory mediators. These mediators, such as reactive nitrogen/oxygen species and IL-1β as well as cyclooxygenase-2 (COX-2) with associated prostaglandin E2 (PGE2), are believed to be neurotoxic and to contribute to the underlying cause of AD. We have used the human H4 neuroglioma cells as a model astroglial system to examine the interactions between IL-1β and nitric oxide (NO) as co-stimulators of Aβ1–40 in enhancing the expression of COX-2 and production of PGE2 in the presence of recombinant human apolipoprotein E4 (apoE4). Neither Aβ1–40 nor its reverse sequence analog Aβ40–1 alone had a significant effect on COX-2 expression and PGE2 production in the cells. In contrast, after co-incubation with apoE4, Aβ1–40 increased IL-1β-induced COX-2 expression and PGE2 production. Aβ12–28, which binds with high affinity to apoE4, blocked apoE4-mediated effects on Aβ1–40. Furthermore, (±)-S-Nitroso-N-acetylpenicillamine (SNAP), an agent that releases nitric oxide (NO) in situ, alone did not affect IL-1β-induced COX-2 expression, but increased PGE2 production only. Addition of Aβ1–40 preincubated with apoE4 to H4 cells in the presence of SNAP led to an additive IL-1β-induced COX-2 expression and PGE2 production. These observations indicate that increased PGE2 resulted from increased nitrosative stress, which is enhanced by apoE4. Thus a molecular understanding of the interactions of apoE4 with Aβ, NO and IL-1β on the regulation of the COX-2/prostaglandin pathway may open new avenues in understanding the mechanism of development of neurodegenerative disease such as AD.