Autophagy protects against redox-active trace metal-induced cell death in rabbit synovial fibroblasts through Toll-like receptor 4 activation.

Reactive oxygen species (ROS) are implicated to play a role in initiating rheumatoid arthritis (RA) pathogenesis. We have investigated the mechanism(s) by which essential redox-active trace metals (RATM) may induce cell proliferation and cell death in rabbit synovial fibroblasts. These fibroblast-like synovial (FLS) cells, which express Toll-like receptor 4 (TLR4), were used as a model system that plays a role in potentially initiating RA through oxidative stress. Potassium peroxychromate (PPC, [Cr5+]), ferrous chloride (FeCl2, [Fe2+]), and cuprous chloride (CuCl, [Cu+]) in the indicated valency states were used as exogenous pro-oxidants that can induce oxidative stress through TLR4 coupled activation that also causes HMGB1 release. We measured the proliferation index (PI) of FLS, and examined the effect of RATM oxidants on apoptosis and autophagy by fluorescence cell-sorting flow cytometry (FC). Cell cycle was analysed by FC and autophagy-related protein expression levels were measured by western blot. Our data showed that as RATM as prooxidants increased intracellular ROS (iROS) that can induce oxidative stress. Whereas iROS increased PI in FLS, these reactive species also protected cells against apoptosis by inducing autophagy. Our results indicate that ROS/TLR4-coupled activation may contribute to the pathogenesis of RA in FLS by induction of autophagy. The signalling pathway by which inflammation and its tissue destructive sequel may occur in RA underlies the need for developing therapeutic agents that can inhibit release of tissue-damaging high mobility group box 1 (HMGB1), cytokines, and possess both trace metal chelating capacity and oxidant scavenging properties in a directed combinatorial therapy for RA.

Role of Toll-like receptor 4/oxidant-coupled activation in regulating the biosynthesis of omega-3 polyunsaturated fatty acid derivative resolvin D1 in primary murine peritoneal macrophage.

We have previously shown that reactive oxygen species (ROS) as prooxidants can activate Toll-like receptor 4 (TLR4) with the potential to initiate, propagate and maintain "sterile" inflammation of innate immunity, which plays a mediatory role in a host of human disease states. We now present new evidence that ROS can also activate TLR4 to counter the inflammatory phenotype by increasing the production of resolvin D1 (RvD1), which is a specialized anti-inflammatory and pro-resolving lipid mediator. We used primary murine peritoneal macrophages (pM) derived from both TLR4-WT and TLR4-KO mice as a cellular model. We used potassium peroxychromate (PPC) as a direct in vitro source of exogenous ROS. PPC treatment increased intracellular ROS levels, which decreased intracellular total antioxidant capacity, thus suggesting an enhanced cellular oxidative stress. PPC and LPS-EK (a TLR4-specific agonist) increased pro-inflammatory TNFα production with noeffect on IL-10, an anti-inflammatory cytokine. Treatment with the prooxidant increased the expression of 12 lipoxygenase (12-LOX) and 5-lipoxygenase (5-LOX) only in pM derived from TLR4 WT but not in pM from TLR4-KO mice. 5-LOX and 12-LOX are the key enzymes in the RvD1 biosynthetic pathway. In addition, PPC increased the expression of RvD1 receptor, a member of G-protein-coupled receptor only in pM from TLR4-WT mice. Our data support the involvement of TLR4-mediated oxidant-induced pro-inflammatory phenotypes that are in opposition to the production of anti-inflammatory/pro-resolution phenotypes in macrophages. Now, we show that through TLR4 activation, exogenous oxidants can play a role both in producing proinflammatory phenotypes at the same time that it enhances resolution of inflammation to maintain a state of cellular homeostasis and prevent tissue damage/disease.

Redox-active trace metal-induced release of high mobility group box 1(HMGB1) and inflammatory cytokines in fibroblast-like synovial cells is Toll-like receptor 4 (TLR4) dependent.

Rheumatoid arthritis (RA) is a chronic autoimmune systemic inflammatory disease that is characterized by synovial inflammation and bone erosion. We have investigated the mechanism(s) by which essential trace metals may initiate and propagate inflammatory phenotypes in synovial fibroblasts. We used HIG-82, rabbit fibroblast-like synovial cells (FLS), as a model system for potentially initiating RA through oxidative stress. We used potassium peroxychromate (PPC, Cr+5), ferrous chloride (FeCl2, Fe+2), and cuprous chloride (CuCl, Cu+) trace metal agents as exogenous pro-oxidants. Intracellular ROS was quantified by fluorescence microscopy and confirmed by flow cytometry (FC). Protein expression levels were measured by western blot and FC, while ELISA was used to quantify the levels of cytokines. Trace metal agents in different valence states acted as exogenous pro-oxidants that generate reactive oxygen species (ROS), which signal through TLR4 stimulation. ROS/TLR4- coupled activation resulted in the release of HMGB1, TNF-α, IL-1ß, and IL-10 in conjunction with upregulation of myeloid-related protein (MRP8/14) inflammatory markers that may contribute to the RA pathophysiology. Our results indicate that oxidant-induced TLR4 activation can release HMGB1 in combination with other inflammatory cytokines to mediate pro-inflammatory actions that contribute to RA pathogenesis. The pathway by which inflammatory and tissue erosive changes may occur in this model system possibly underlies the need for functioning anti-HMGB1-releasing agents and antioxidants that possess both dual trace metal chelating and oxidant scavenging properties in a directed combinatorial therapy for RA.

Lipopolysaccharide (LPS)-mediated priming of toll-like receptor 4 enhances oxidant-induced prostaglandin E2 biosynthesis in primary murine macrophages.

Agonists and pseudo-agonists for toll-like receptor 4 (TLR4) are common in our environment. Thus, human exposure to these agents may result in "priming or sensitization" of TLR4. A body of evidence suggests that LPS-mediated sensitization of TLR4 can increase the magnitude of responses to exogenous agents in multiple tissues. We have previously shown that reactive oxygen and nitrogen species (RONS) stimulate TLR4. There is no evidence that LPS-primed TLR4 can influence the magnitude of responses to oxidants from either endogenous or exogenous sources. In the present study, we directly tested the hypothesis that LPS-primed TLR4 will sensitize primary murine peritoneal macrophages (pM) to oxidant-mediated prostaglandin E2 (PGE2) production. We used potassium peroxychromate (PPC) and potassium peroxynitrite (PPN) as direct in vitro sources of exogenous RONS. Our results showed that a direct treatment with PPC or PPN alone as sources of exogenous oxidants had a limited effect on PGE2 biosynthesis. In contrast, pM sensitized by prior incubation with LPS-EK, a TLR4-specific agonist, followed by oxidant stimulation exhibited increased transcriptional and translational expression of cyclooxygenase-2 (COX-2) with enhanced PGE2 biosynthesis/production only in pM derived from TLR4-WT mice but not in TLR4-KO mice. Thus, we have shown a critical role for LPS-primed TLR4 in oxidant-induced inflammatory phenotypes that have the potential to initiate, propagate and maintain many human diseases.

Exogenous oxidants activate nuclear factor kappa B through Toll-like receptor 4 stimulation to maintain inflammatory phenotype in macrophage.

Disturbances in redox equilibrium in tissue can lead to inflammatory state, which is a mediatory factor in many human diseases. The mechanism(s) by which exogenous oxidants may activate an inflammatory response is not fully understood. Emerging evidence suggests that oxidant-induced Toll-like receptor 4 (TLR4) activation plays a major role in "sterile" inflammation. In the present study, we used murine macrophage RAW-Blue cells, which are chromosomally integrated with secreted embryonic alkaline phosphatase (SEAP) inducible by NF-κB. We confirmed the expression of TLR4 mRNA and protein in RAW-Blue cells by RT-PCR and Western blot, respectively. We showed that oxidants increased intracellular reactive oxygen species production and lipid peroxidation, which resulted in decreased intracellular total antioxidant capacity. Consistent with the actions of TLR4-specific agonist LPS-EK, exogenous oxidants increased transcriptional activity of NF-κB p65 with subsequent release of NF-κB reporter gene SEAP. These effects were blocked by pretreatment with TLR4 neutralizing pAb and TLR4 signaling inhibitor CLI-095. In addition, oxidants decreased the expression of IκBα with enhanced phosphorylation at the Tyr42 residue. Finally, oxidants and LPS-EK increased TNFα production, but did not affect IL-10 production, which may cause imbalance between pro- and anti-inflammatory processes, which CLI-095 inhibited. For biological relevance, we confirmed that oxidants increased release of TNFα and IL-6 in primary macrophages derived from TLR4-WT and TLR4-KO mice. Our results support the involvement of TLR4 mediated oxidant-induced inflammatory phenotype through NF-κB activation in macrophages. Thus exogenous oxidants may play a role in activating inflammatory phenotypes that propagate and maintain chronic disease states.

Toll-like receptor 4 signaling: A common pathway for interactions between prooxidants and extracellular disulfide high mobility group box 1 (HMGB1) protein-coupled activation.

Necrotic cells passively release HMGB1, which can stimulate TLR4 in an autocrine fashion to potentially initiate "sterile" inflammation that maintains different disease states. We have shown that prooxidants can induce NF-κB activation through TLR4 stimulation. We examined whether prooxidants enhance HMGB1-induced TLR4 signaling through NF-κB activation. We used LPS-EK as a specific agonist for TLR4, and PPC and SIN-1 as in situ sources for ROS. As model systems, we used HEK-Blue cells (stably transfected with mouse TLR4), RAW-Blue™ cells (derived from murine RAW 264.7 macrophages) and primary murine macrophages from TLR4-KO mice. Both HEK-Blue and RAW-Blue 264.7 cells express optimized secreted embryonic alkaline phosphatase (SEAP) reporter under the control of a promoter inducible by NF-κB. We treated cells with HMGB1 alone and/or in conjunction with prooxidants and/or inhibitors using SEAP release as a measure of TLR4 stimulation. HMGB1 alone and/or in conjunction with prooxidants increased TNFα and IL-6 released from TLR4-WT, but not from TLR4-KO macrophages. Pro-oxidants increased HMGB1 release, which we quantified by ELISA. We used both fluorescence microscopy imaging and flow cytometry to quantify the expression of intracellular ROS. TLR4-neutralizing antibody decreased prooxidant-induced HMGB1 release. Prooxidants promoted HMGB1-induced NF-κB activation as determined by increased release of SEAP and TNF-α, and accumulation of iROS. HMGB1 (Box A), anti-HMGB1 and anti-TLR4-neutralizing pAbs inhibited HMGB1-induced NF-κB activation, but HMGB1 (Box A) and anti-HMGB1 pAb had no effect on prooxidant-induced SEAP release. The present results confirm that prooxidants enhance proinflammatory effects of HMGB1 by activating NF-κB through TLR4 signaling.

Activation of c-Src: a hub for exogenous pro-oxidant-mediated activation of Toll-like receptor 4 signaling.

To study the role of c-Src kinase in pro-oxidant-induced stimulation of Toll-like receptor 4 (TLR4), we used lipopolysaccharide from Escherichia coli K12 (LPS-EK) and monophosphoryl lipid A, as TLR4-specific agonists and positive controls, and SIN-1 and potassium peroxychromate as pro-oxidant sources. We used the HEK-Blue mTLR4 cell line, which is stably transfected with mouse TLR4 and expresses optimized SEAP reporter under the control of a promoter inducible by NF-κB transcription factor. The level of SEAP released due to TLR4 stimulation was a measure of NF-κB activation. Treatment with either the pro-oxidants or LPS-EK increased SEAP release and TNF-α production in these cells. These treatments also increased intracellular reactive oxygen species accumulation, with an enhanced production of nitric oxide and TBARS to confirm oxidant stress in these cells. Pretreatment with c-Src kinase inhibitors, PP2 and Ca-pY, which act by different mechanisms, decreased these parameters. Pretreatment with SSG, a c-Src activator, enhanced the effects promoted by LPS-EK and pro-oxidants and rescued cells from the PP2- and Ca-pY-induced effects. Curiously, pro-oxidants, but not TLR4 agonist, increased the ratio of TNF-α to IL-10 released, suggesting that pro-oxidants can initiate and maintain an imbalance of TNF-α production over IL-10. To different degrees, both pro-oxidants and TLR4 agonist increased formation of c-Src complexes with TLR4 and IκB-α as coimmunoprecipitates. Both pro-oxidants and TLR4 agonist increased c-Src phosphorylation of the Tyr42 residue in IκB-α, but the pro-oxidant-induced effect was more robust and much longer lasting. Taken together, these studies provide a mechanism whereby c-Src assumes a central role in pro-oxidant-induced NF-κB activation in TLR4 signaling. Pro-oxidant-induced activation of TLR4 through c-Src/NF-κB/IκB-α coupling provides a basis for a molecular dissection of the initiation and maintenance of sterile inflammation that may serve as a "pathophysiologic primer" for many diseases.

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-1ß 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.

Regulation of IL-1ß-induced cyclooxygenase-2 expression by interactions of Aß 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 (Aß) 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 Aß 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-1ß as well as cyclooxygenase-2 (COX-2) with associated prostaglandin E2 (PGE(2)), 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-1ß and nitric oxide (NO) as co-stimulators of Aß(1-40) in enhancing the expression of COX-2 and production of PGE(2) in the presence of recombinant human apolipoprotein E4 (apoE4). Neither Aß(1-40) nor its reverse sequence analog Aß(40-1) alone had a significant effect on COX-2 expression and PGE(2) production in the cells. In contrast, after co-incubation with apoE4, Aß(1-40) increased IL-1ß-induced COX-2 expression and PGE(2) production. Aß(12-28), which binds with high affinity to apoE4, blocked apoE4-mediated effects on Aß(1-40). Furthermore, (±)-S-Nitroso-N-acetylpenicillamine (SNAP), an agent that releases nitric oxide (NO) in situ, alone did not affect IL-1ß-induced COX-2 expression, but increased PGE(2) production only. Addition of Aß(1-40) preincubated with apoE4 to H4 cells in the presence of SNAP led to an additive IL-1ß-induced COX-2 expression and PGE(2) production. These observations indicate that increased PGE(2) resulted from increased nitrosative stress, which is enhanced by apoE4. Thus a molecular understanding of the interactions of apoE4 with Aß, NO and IL-1ß 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.

Regulation of the cyclooxygenase-2 system by interleukin-1beta through mitogen-activated protein kinase signaling pathways: a comparative study of human neuroglioma and neuroblastoma cells.

Glial activation and inflammation following brain injury may initiate and maintain the process of neurodegeneration. Both glia and neurons synthesize proinflammatory mediators such as interleukin 1 beta (IL-1beta), cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2 (COX-2), and prostaglandins. The molecular mechanisms by which IL-1beta regulates inflammatory genes such as cPLA2 and COX-2 in glial and neuronal cells are poorly understood. We have studied IL-1beta-mediated gene regulation in an established glial and neuronal human cell lines. We report that IL-1beta induced cPLA2 and COX-2 mRNA and protein expression and subsequent prostaglandin E2 (PGE2) release in a time-dependent manner in H4 neuroglioma cells. Both SB203580 and PD98059 [p38 and p42/44 mitogen-activated protein kinase (MAPKs) inhibitors, respectively] reduced IL-1beta-induced PGE2 production, while only SB203580 reduced both cPLA2 and COX-2 expression. Similarly, in SKNSH neuroblastoma cells, both SB203580 and PD98059 reduced IL-1beta-induced PGE2 release, with no detectable COX-2 and cPLA2 protein expression in these cells. Our results indicate that the signaling mechanisms of p38 and p42/44 MAPKs play a role in IL-1beta-mediated PGE2 release in both of these cell lines, with differences upstream at the level of cPLA(2)/COX-2 expression. IL-1beta-induced cPLA2 and COX-2 gene expression is modulated through the p38 MAPK pathway in both neuroglioma and neuroblastoma cells. Understanding the signaling mechanisms involved in IL-1beta-mediated inflammatory processes in both glia and neuronal cells may provide potential targets for therapeutic intervention for neurological disorders.

Modulation of peripheral inflammation in sensory ganglia by nuclear factor (kappa)B 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 Freund's 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.

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-1beta 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-1beta 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-1beta 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-1beta-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-1beta-induced SP synthesis and secretion, and p60c-src kinase activity in DRG neurons. Whereas, IL-1 receptor antagonist and cycloheximide inhibited IL-1beta-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-1beta 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-1beta-induced p60c-src activation.

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

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

Unilateral intraplantar injection of Freund's 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 radioreceptor-assay (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.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of lysergic acid diethylamide (LSD) and adjuvant-induced inflammation on desensitization to and metabolism of substance P in the mouse spinal cord.

We have previously shown that the caudally directed biting and scratching response to repeated intrathecal (i.t.) injections of substance P (SP) is decreased by the third injection of SP and that this apparent desensitization to SP is less pronounced in mice pretreated with Freund's adjuvant. This study was designed to study the mechanism of this desensitization to SP and to examine the effect of lysergic acid diethylamide tartrate (LSD) on desensitization. Our results indicate that while 25 micrograms of LSD/kg body weight i.p. in naive mice had no effect on the response to a single injection of SP, LSD decreased the development of desensitization to SP-induced behaviors. In contrast, identical injections of LSD in adjuvant-pretreated mice not only failed to prevent desensitization but enhanced the degree of apparent desensitization to SP. Tolerance developed to the effects of LSD on desensitization to SP-induced behaviors in both adjuvant- and saline-pretreated mice. When injected i.t. with SP, LSD failed to alter the degree of desensitization to SP-induced behaviors, suggesting that the effect of LSD is not produced at the spinal cord level. Separation and quantification of SP and its metabolites in the spinal cord using high performance liquid chromatography (HPLC) techniques indicated that either a single injection of LSD or pretreatment with Freund's adjuvant produced similar patterns of changes in the concentrations of SP-related peptides in mouse spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)