Colony stimulating factors regulate nitric oxide and prostaglandin E2 production in rat cartilage chondrocytes

Colony stimulating factors (CSFs) are now widely used in cancer treatment and immunological disease therapy. Both granulocyte CSF (G-CSF) and granulocyte-macrophage CSF (GM-CSF) are used to increase neutrophil counts in Felty syndrome. In the present study, the effects of macrophage CSF (M-CSF), G-CSF, GM-CSF and interleukin-3 (IL-3) (10 ng/ml) on the production of nitric oxide and prostaglandin E2 (PGE2) by cartilage explants were examined over 24 and 48 h. The effects of these CSFs were also measured in combination with IL-1 beta (10 ng/ml). M-CSF, GM-CSF and IL-3 had no effect on nitrite production. However, both IL-1 beta and G-CSF caused a significant increase (p < 0.05) in nitrite levels at 48 h. NG-L-arginine-methyl-ester was used to inhibit nitrite production induced by G-CSF and this implicated nitric oxide synthase activity. When G-CSF and IL-1 beta were used in a combined treatment, nitrite levels were significantly increased (p < 0.05) at both 24 and 48 h. Both IL-3 alone and in combination with IL-1 beta caused elevated PGE2 production in this model. PGE2 levels were also significantly increased by stimulation with GM-CSF and IL-3 combined with IL-1 beta. These findings demonstrate that GM-CSF, G-CSF and IL-3 may induce changes in the production of inflammatory mediators such nitric oxide and PGE2 in cartilage chondrocytes. Hence, CSFs may play a vital role in influencing cartilage metabolism in rheumatoid and osteoarthritis.     

Up-regulation of hepatic prostaglandin E receptors in vivo induced by prostaglandin synthesis inhibitors

Up-regulation in vivo of liver plasma membrane receptors for prostaglandin E (PGE) was studied in Sprague-Dawley rats using the prostaglandin synthesis inhibitors, acetylsalicylic acid (ASA) and indomethacin (Indo). Following 4 days of treatment with ASA, the concentration of receptors and the affinity for binding were both significantly increased (Ro - +37%, KA = +62%). Following 4 days of treatment with Indo, the number of receptors was increased but the binding-site affinity was decreased (Ro = +40%, KA = -71%). Animals were then examined after treatment with either ASA or Indo for 1 day, a time when there was no significant decrease in PGE. After 1 day of treatment, the opposite changes in binding-site affinity were again observed, but there were no changes in the number of receptors with either drug, suggesting that the changes in affinity resulted from non-prostaglandin-related effects of the drugs. To ascertain the physiologic consequences of up-regulation, adenylate cyclase activity was measured in control and up-regulated membranes. There were no significant changes in basal or in PGE-stimulated adenylate cyclase activity. These data demonstrate that decreased endogenous PGE causes up-regulation of PGE receptors, but that this is not accompanied by increased adenylate cyclase activity. These data may indicate that PGE-stimulated adenylate cyclase operates maximally under normal receptor concentrations and that therefore its activity cannot be increased by regulatory changes in receptor density.     

Additive effect of nitric oxide and prostaglandin-E2 synthesis inhibitors in endotoxin-induced uveitis in the rabbit

The involvement of nitric oxide (NO) and prostaglandin E₂ (PGE₂) was investigated in a model of intraocular inflammation induced by intravitreal injection of endotoxin (lipopolysaccharide, LPS, 10 ng) in rabbits. The severity of uveitis, the myeloperoxidase (MPO) activity in iris-ciliary body, and the protein concentration in aqueous humor were determined. Nitric oxide synthase (NOS) and cyclooxygenase (COX) activities were assessed respectively by nitrite and PGE₂ levels in aqueous humor. Treatment with inhibitors of NOS (N^G-nitro-L-arginine methyl ester, L-NAME, 50 mg/kg i.p.) or COX (diclofenac, 30 g, topically), alone or in combination, were compared to a salinetreated group. Diclofenac or L-NAME alone reduced or delayed the intensity of uveitis, and partially decreased the protein concentration in aqueous humor; diclofenac, but not L-NAME, partially reduced the polymorphonuclear leukocyte infiltration in the iris ciliary body as indicated by the MPO activity. Treatment with both inhibitors in combination diminished the clinical uveitis, the disruption of the blood-aqueous barrier and the MPO activity in the iris-ciliary body. We conclude that NO and PGE₂ have additive effects in endotoxin-induced uveitis in rabbits, and that the inhibition of both pathways would improve the therapeutical management of uveitis.accepted by M. J. Parnham     

A synthetic hydroxypropenone inhibits nitric oxide,prostaglandin E2, and proinflammatory cytokine synthesis

HMP [3-(2-hydroxyphenyl)-1-(5-methyl-furan-2-y-l) propenone] was evaluated for its ability to inhibit the synthesis of major proinflammatory mediators and cytokines in interferon-γ (IFN-γ)- and lipopolysaccharide (LPS)-induced RAW 264.7 cells and phorbol myristate acetate (PMA)-differentiated/LPS-induced U937 cells. HMP suppressed the production of nitric oxide (NO) with significant inhibitory effects at doses as low as 0.78?μM (P?<?0.05). Prostaglandin E2 (PGE2) secretion was also inhibited at doses of 12.5?μM and above (P?<?0.01). The secretion of both TNF-α and IL-6 were only inhibited at the highest dose used (25?μM; P?<?0.001). IL-1β secretion was also inhibited from 12.5?μM onwards (P?<?0.01). This inhibition was demonstrated to be caused by down-regulation of inducible enzymes, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), without direct effect upon iNOS or COX-2 enzyme activity. HMP only inhibited iNOS (P?<?0.001) and IL-1β (P?<?0.05) gene expression at the highest tested concentration. HMP did not affect the secretion of chemokines IL-8 and monocyte chemotactic protein-1 (MCP-1) and the anti-inflammatory cytokine IL-10. The most striking effect of HMP was its NO inhibitory activity and therefore we conclude that HMP is a selective inhibitor of iNOS.     

Mechanisms underlying dedepression of synaptic NMDA receptors in the hippocampus

N-Methyl-D-aspartate receptor (NMDAR)-mediated synaptic responses in hippocampal CA1 pyramidal cells are depressed during NMDAR-dependent long-term depression (LTD) due to mechanisms, in part, distinct from those underlying LTD of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated synaptic responses. The mechanisms underlying dedepression of synaptic NMDARs, however, are not known. We find that dedepression of NMDAR-mediated synaptic responses in the CA1 region of the rat hippocampus is input specific and does not require synaptic stimulation to be maintained. The induction of dedepression does not require activation of metabotropic glutamate receptors, L-type Ca(2+) channels, or release of Ca(2+) from intracellular stores. It does, however, rely on activation of NMDARs. In contrast to the dedepression of AMPAR-mediated synaptic responses, dedepression of NMDAR-mediated synaptic responses does not depend on activation of calcium/calmodulin-dependent protein kinase II, protein kinase C, cAMP-dependent protein kinase, or Src kinases. However, dedepression of synaptic NMDARs is significantly impaired by inhibitors of mitogen-activated protein kinase signaling. Specifically, inhibitors of extracellular signal-regulated kinase 1/2 prevented normal dedepression of synaptic NMDARs by a mechanism that did not require protein synthesis. These results provide further evidence that synaptic NMDARs can be bidirectionally modified by activity but by mechanisms distinct from those responsible for the activity-dependent, bidirectional modulation of synaptic AMPARs.     

Glycine enhances extracellular 45Ca2+ response to NMDA application investigated with microdialysis of rabbit hippocampus in vivo.

This study evaluates the role of glycine in in vivo modulation of the activity of excitatory amino acid receptors sensitive to N-methyl-D-aspartate (NMDA) in the rabbit hippocampus. Changes of extracellular calcium concentration were studied by the microdialysis technique combined with 45Ca-utilizing clearance method. The steady state level of amino acids in the dialysate was analyzed with HPLC. Pharmacologically active substances were applied directly to the hippocampus via the dialysis medium. Glycine concentration in the extracellular fluid of the hippocampus was in the range of 10(-5) M. Application of NMDA induced a drop of calcium concentration in the extracellular compartment of the hippocampus. This effect was inhibited by 7-Cl-kynurenic acid, an antagonist of glycine binding site on the NMDA receptors. Application of glycine and D-serine to the dialysis medium did not interfere with the basal level of extracellular calcium in the hippocampus, but resulted in an enhancement of the NMDA-induced decrease of calcium concentration. These results indicate that in the hippocampus the NMDA receptors are under constant positive modulation by endogenous glycine. Since glycine binding sites on the NMDA receptors are not saturated by an endogenous ligand, glycine may play a role in regulation of the NMDA receptor activity in the hippocampus in vivo.     

Effect of erythropoietin on nitric oxide production in the rat hippocampus using in vivo brain microdialysis

In order to investigate the role of erythropoietin (EPO) in the hippocampus, we studied the effect of EPO on nitric oxide (NO) production in the rat hippocampus using brain microdialysis. The dialysis probe was stereotaxically inserted into the rat hippocampus 24 h before the dialysis experiment. The perfusion fluid (Krebs-HEPES buffer, pH 7.4) was collected at 15-min intervals under freely moving conditions and NO metabolites (NOx) in the perfusate were immediately measured using a NOx-analyzing high performance liquid chromatography (HPLC)system. Following the collection of four fractions, 1 microl of EPO (10(-10) M, 10(-8) M and 10(-6) M) or vehicle (saline) was gently injected into the hippocampal tissue. The perfusion fluid was collected for 3 h after the injection. The NOx levels were unchanged by the injection of vehicle alone. After the injection of EPO, NOx levels gradually increased. The EPO-induced increase in NOx levels was significant at 10(-6) M EPO. The EPO-induced increases in NOx levels were eliminated in the presence of anti-EPO antibody. The increase in NOx levels induced by EPO was blunted by nicardipine, a Ca2+ channel blocker, but not by MK-801, an antagonist of N-methyl-D-aspartate (NMDA) receptors. These findings, taken together, suggest that EPO increased NO production in the rat hippocampus by activating voltage-gated Ca2+ channels, but not through NMDA receptors.     

AMPA and NMDA receptors regulate responses of neurons in the rat's inferior colliculus

The contribution of N-methyl-D-aspartate (NMDA) and AMPA receptors to auditory responses in the rat's inferior colliculus was examined by recording single-unit activity before, during, and after local iontophoretic application of receptor-specific antagonists. Tone bursts and sinusoidal amplitude modulated sounds were presented to one ear, and recordings were made from the contralateral central nucleus of inferior colliculus (ICC). The receptor specific antagonists, (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) for NMDA receptors and 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX) for AMPA receptors, were released at the recording site through a multi-barreled pipette. For most neurons, either CPP or NBQX alone resulted in a reversible reduction in the number of action potentials evoked by tonal stimulation. For neurons with an onset response pattern, NBQX either completely eliminated or greatly reduced the number of action potentials. CPP also reduced the number of action potentials but had a less pronounced effect than NBQX. For neurons with a sustained firing pattern, NBQX reduced the total number of action potentials, but had a preferential effect on the early part (first 10-20 ms) of the response. CPP also resulted in a reduction in the total number of action potentials, but had a more pronounced effect on the later part (>20 ms) of the response. These results indicate that both AMPA and NMDA receptors contribute to sound evoked excitatory responses in the ICC. They have a selective influence on early and late components of tone-evoked responses. Both receptor types are involved in generating excitatory responses across a wide range of sound pressure levels as indicated by rate level functions obtained before and during drug application. In addition, both CPP and NBQX reduced responses to sinusoidal amplitude modulated sounds. The synchrony of firing to the modulation envelope as measured by vector strength at different rates of modulation was not greatly affected by either CPP or NBQX in spite of the decrease in firing rate.     

G-protein-coupled receptors act via protein kinase C and Src to regulate NMDA receptors

The N-methyl-D-aspartate (NMDA) receptor contributes to synaptic plasticity in the central nervous system and is both serine-threonine and tyrosine phosphorylated. In CA1 pyramidal neurons of the hippocampus, activators of protein kinase C (PKC) as well as the G-protein-coupled receptor ligands muscarine and lysophosphatidic acid enhanced NMDA-evoked currents. Unexpectedly, this effect was blocked by inhibitors of tyrosine kinases, including a Src required sequence and an antibody selective for Src itself. In neurons from mice lacking c-Src, PKC-dependent upregulation was absent. Thus, G-protein-coupled receptors can regulate NMDA receptor function indirectly through a PKC-dependent activation of the non-receptor tyrosine kinase (Src) signaling cascade.     

Protein kinase CK2 modulates synaptic plasticity by modification of synaptic NMDA receptors in the hippocampus

Synaptic plasticity is the foundation of learning and memory. The protein kinase CK2 phosphorylates many proteins related to synaptic plasticity, but whether it is directly involved in it has not been clarified. Here, we examined the role of CK2 in synaptic plasticity in hippocampal slices using the CK2 selective inhibitors 5,6-dichloro-1-β- d -ribofuranosylbenzimidazole (DRB) and 4,5,6,7-tetrabromobenzotriazole (TBB). These significantly inhibited N -methyl- d -aspartate (NMDA) receptor-dependent long-term potentiation (LTP). DRB also inhibited NMDA receptor-mediated synaptic transmission, while leaving NMDA receptor-independent LTP unaffected. NMDA receptors thus appear to be the primary targets of CK2. Although both long-term depression (LTD) and LTP are induced by the influx of Ca²⁺ through NMDA receptors, surprisingly, LTD was not affected by CK2 inhibitors. We postulated that the LTP-selective modulation by CK2 is due to selective modulation of NMDA receptors, and tested two hypotheses concerning the modulation of NMDA receptors: (i) CK2 selectively modulates NR2A subunits possibly related to LTP, but not NR2B subunits possibly related to LTD; and (ii) CK2 selectively affects synaptic but not extrasynaptic NMDA receptors whose activation is sufficient to induce LTD. DRB decreased NMDA receptor-mediated synaptic transmission in the presence of selective NR2A subunit antagonist. The former hypothesis thus appears unlikely to be correct. However, DRB decreased synaptic NMDA receptor responses in cultured hippocampal neurons without affecting extrasynaptic NMDA receptor current. These findings support the latter hypothesis, that CK2 selectively affects LTP by selective modification of synaptic NMDA receptors in a receptor-location-specific manner.     

Expression of nitric oxide synthase in the developing rat hippocampus

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry was used to study the development of neurons synthesizing nitric oxide (NO) in the postnatal rat hippocampus. We show that NADPH-d reactive somata and processes are present from the day of birth until adulthood in the Ammon's horn. The dentate gyrus, however, has a more delayed period of nitric oxide synthase (NOS) expression with the staining appearing only by the end of the first postnatal week. Our results suggest that the time course of NOS expression correlates with the developmental sequence of events described in the hippocampus and that NO could be involved in the development of connections in this structure.     

Distinct roles of prostaglandin D2 receptors in chronic skin inflammation

Prostaglandin D2 (PGD2) is a prostanoid implicated in allergic inflammation. However, the roles of PGD2 in immune and allergic responses remain controversial. PGD2 exerts its effect through the CRTH2 and DP receptors. To elucidate functional differences of PGD2 and its receptors in chronic skin inflammation, chronic contact hypersensitivity (chronic CHS) and IgE-mediated chronic allergic skin inflammation (IgE-CAI) were induced in mice deficient in the CRTH2 and/or DP genes. DP (-/-) mice and CRTH2 (-/-)/DP (-/-) mice showed exacerbated chronic CHS, and conversely, CRTH2 (-/-) mice exhibited diminished skin responses. Skin responses correlated with local levels of IL-13, CCL11, and CCL22. These phenotypic changes in chronic CHS of mutant mice were similar to those in acute CHS despite the differences in the cytokine milieus; chronic CHS and acute CHS were mediated by Th2 and Th1/Th17 immunity, respectively. However, in IgE-CAI, DP (-/-) mice showed comparable skin responses to wild-type mice. Alleviation of IgE-CAI was observed in CRTH2 (-/-) mice, and as a consequence, CRTH2 (-/-)/DP (-/-) mice exhibited diminished IgE-CAI compared with wild-type mice. IgE-CAI in mutant mice correlated with local IL-4 and CCL22 production. Consistent with these results, a CRTH2-specific antagonist exerted inhibitory effects in both chronic CHS and IgE-CAI. The present study demonstrates that functional roles of PGD2 and its receptors appear to depend on the nature of the inflammation. Nevertheless, tools targeted against PGD2-CRTH2 signals could offer therapeutic potential for both types of chronic skin inflammation.     

In the hippocampus in vivo,nitric oxide does not appear to function as an endogenous antiepileptic agent

Using a reverberatory epilepiform discharge of hippocampal-parahippocampal circuits termed maximal dentate activation, this study investigated whether the local release of nitric oxide within these circuits functions as an antiepileptic agent. Two nitric oxide synthase inhibitors (l-nitro-arginine methyl ester and 7-nitro-indazole) and a guanylate cyclase inhibitor (methylene blue) were tested, and none had a significant effect on the time to onset or duration of maximal dentate activation. A membrane-permeable analogue of cyclic guanosine monophosphate (cGMP), 8-bromo-cGMP, caused an increase in the time to onset and a decrease in the duration of maximal dentate activation. The number of neurons expressing NADPH diaphorase activity (a marker for nitric oxide synthase) was also examined after repeated elicitation of maximal dentate activation. After 18 seizures there was a significant, but transient, decrease in the number of hilar/subgranular neurons that were NADPH diaphorase-positive. The decrease was only seen at 1 h after the last seizure. There was no induction of NADPH diaphorase activity. These results are not consistent with the hypothesis that, in hippocampal-parahippocampal circuits in vivo, nitric oxide is released in response to neuronal activity and then acts to terminate the neuronal activity.     

Synergistic interactions of dopamine D1 and glutamate NMDA receptors in rat hippocampus and prefrontal cortex: Involvement of ERK1/2 signaling

Interactions between dopamine and glutamate receptors are essential for the prefrontal cortical (PFC) and hippocampal cognitive functions. In order to understand the molecular basis of dopamine/glutamate interactions in rat PFC and hippocampus, we investigated (a) the effect of in vitro dopamine D1 receptor stimulation on glutamate N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits' phosphorylation and (b) the signal transduction pathway underlying these interactions, by examining the involvement of D1–extracellular regulated kinase 1/2 (ERK1/2) and D1/protein kinase A (PKA)/dopamine- and cyclic AMP-regulated phosphoprotein-32 (DARPP-32) signaling pathways. Furthermore, we compared the D1/NMDA/AMPA receptor interactions seen in PFC and hippocampus with those appearing in striatum, in which the D1 receptors' density is the highest within the mammalian brain. Our results showed that stimulation of D1 receptor by the specific agonist SKF38393 (10 μM) in PFC and hippocampal slices significantly increased the phosphorylation state of NR1ser897 and NR2Bser1303 subunits of NMDA receptor and of the GLUR1 (ser831 and ser845) subunit of AMPA receptor, as well as of ERK1/2, but not of DARPP-32. Interestingly, co-stimulation of D1 and NMDA receptors with an ineffective dose of SKF38393 (2 μM) and NMDA (5 μM) respectively, elevated further the phosphorylation level of NMDA and AMPA receptor subunits, as well as of ERK1/2, but not of DARPP-32. The D1- and D1/NMDA-induced phosphorylations were totally inhibited by SL327 (specific ERK1/2 inhibitor). Conversely, in striatal slices our data confirm that the D1-mediated phosphorylation of NMDA and AMPA receptor subunits relies on D1/PKA/DARPP-32 signaling. In conclusion, in PFC and hippocampus: (a) a strong synergistic interaction of D1 and NMDA receptors exists, which results in a significant ERK1/2 pathway activation, (b) the D1 and the D1/NMDA receptor-induced phosphorylation of NMDA and AMPA receptor subunits seems to rely on ERK1/2 signaling and could to some extent underlie the enhancement of NMDA and AMPA receptor currents mediated by D1 receptor activation.     

快速老化小鼠海马突触体内NMDA受体的表达变化

目的:观察NMDA受体在SAMP8小鼠海马突触体内的表达变化。方法:首先应用生物化学的方法分离海马突触蛋白,并对其进行鉴定。其次,Western Blot检测NMDA受体的主要亚基NR1、NR2A和NR2B在SAMP8小鼠海马突触体内的表达变化。结果:PSD-95和synaptophysin特异性抗体检测显示突触蛋白的分离是成功的。SAMP8小鼠海马内NR1、NR2A和NR2B在突触的表达均显著低于SAMR1小鼠。进一步分析NR1、NR2A和NR2B蛋白在突触的表达量占总表达量的比值,SAMP8小鼠同样显著低于SAMR1小鼠,而SAMR1和CD-1小鼠间没有显著性差异。结论:SAMP8小鼠海马突触体内NR1、NR2A和NR2B的蛋白表达水平均显著性降低,推测NMDA受体在突触表达水平的降低可能是导致受体功能失调,激发突触功能损伤信号途径的原因之一,进而导致SAMP8小鼠学习记忆功能的下降。     

NR2C and NR2D subunits of NMDA receptors in frog and turtle retina

Glutamate NMDA (N-methyl-d-aspartate) receptors are widely distributed in the central nervous system where they are involved in cognitive processes, motor control and many other functions. They are also well studied in the retina, which may be regarded as a biological model of the nervous system. However, little is known about NR2C and NR2D subunits of NMDA receptors, which have some specific features as compared to other subunits. Consequently the aim of the present study was to investigate their distribution in frog (Rana ridibunda) and turtle (Emys orbicularis) retinas which possess mixed and cone types of retina respectively. The experiments were performed using an indirect immunofluorescence method. Four antibodies directed to NR2C and NR2D subunits of NMDA receptor, as well as three antibodies directed to different splice variants of NR1 subunit, which is known to be obligatory for proper functioning of the receptor, were applied. All antibodies caused well expressed labeling in frog and turtle retinas. The NR2C and NR2D subunits were localized in glial Müller cells, while the NR1 subunit had both neuronal and glial localization. Our results show that glial NMDA receptors differ from neuronal ones in their subunit composition. The functional significance of the NMDA receptors and their NR2C and NR2D subunits, in particular for the neuron-glia interactions, is discussed.     

Differential effects of aging on NMDA receptors in the intermediate versus the dorsal hippocampus

There are discrepancies in the literature about the effects of aging on NMDA receptor expression in the hippocampus. The present study was designed to determine whether there are regional differences in how NMDA receptors are affected by aging. Brains from male C57BL/6 mice from three different age groups (3, 10, and 30 months of age) were sectioned coronally through the dorsal hippocampus and horizontally through the intermediate hippocampus. Sections were processed and analyzed for [3H]glutamate binding to NMDA receptors using receptor autoradiography and for mRNA for the zeta1 (NR1), epsilon1 (NR2A), and epsilon2 (NR2B) subunits of the NMDA receptor using in situ hybridization. There were more significant effects of aging within the intermediate hippocampus in NMDA-displaceable [3H]glutamate binding and epsilon2 mRNA densities than were seen in the dorsal hippocampus. There was no significant effect of age on densities of either the zeta1 or epsilon1 subunit mRNA. These results suggest that the aging process affects NMDA receptors more in the intermediate hippocampus than the dorsal.     

Inducible nitric oxide synthase mediates prostaglandin h2 synthase nitration and suppresses eicosanoid production

Nitric oxide (NO) modulates the biological levels of arachidonate-derived cell signaling molecules by either enhancing or suppressing the activity of prostaglandin H(2) isoforms (PGHS-1 and PGHS-2). Whether NO activates or suppresses PGHS activity is determined by alternative protein modifications mediated by NO and NO-derived species. Here, we show that inducible NO synthase (iNOS) and PGHS-1 co-localize in atherosclerotic lesions of ApoE(-/-) mouse aortae. Immunoblotting and immunohistochemistry revealed Tyr nitration in PGHS-1 in aortic lesions but markedly less in adjacent nonlesion tissue. PGHS-2 was also found in lesions, but 3-nitrotyrosine incorporation was not detected. 3-Nitrotyrosine formation in proteins is considered a hallmark reaction of peroxynitrite, which can form via NO-superoxide reactions in an inflammatory setting. That iNOS-derived NO is essential for 3-nitrotyrosine modification of PGHS-1 was confirmed by the absence of 3-nitrotyrosine in lesions from ApoE(-/-)iNOS(-/-) mice. Mass spectrometric studies specifically identified the active site residue Tyr385 as a 3-nitrotyrosine modification site in purified PGHS-1 exposed to peroxynitrite. PGHS-mediated eicosanoid (PGE(2)) synthesis was more than fivefold accelerated in cultured iNOS(-/-) versus iNOS-expressing mouse aortic smooth muscle cells, suggesting that iNOS-derived NO markedly suppresses PGHS activity in vascular cells. These results further suggest a regulatory role of iNOS in eicosanoid biosynthesis in human atherosclerotic lesions.     

The involvement of nitric oxide in corpus luteum regression in the rat: feedback mechanism between prostaglandin F(2alpha) and nitric oxide.

In the corpus luteum (CL), prostaglandin F(2alpha) (PGF(2alpha)) is a physiological agent with luteolytic actions. Nitric oxide (NO) is a messenger molecule capable of modulating diverse pathophysiological processes. The aim of the present study was to investigate the role of ovarian NO in PGE (a luteotrophic prostanoid) and PGF(2alpha) (a luteolytic prostanoid) production and in progesterone synthesis during CL regression in the rat. To obtain a longer functional CL, we used a pseudopregnant (PSP) rat model. By means of intrabursa ovarian sac treatment of two competitive nitric oxide synthase (NOS) inhibitors, N(G)-monomethyl-L-arginine (L-NMMA, 1 mg/kg) and N(W)-nitro-L-arginine methyl ester (L-NAME; 3 mg/kg), and sodium nitroprusside (SNP, 0.05 mg/kg) as a NO generator, we found that NO, produced by the ovarian tissue during the last 2 days of CL development (days 8 and 9), increased PGF(2alpha) production in the ovary and diminished serum progesterone concentrations leading to CL involution. We also proposed a positive feedback mechanism between PGF(2alpha) and NO, to ensure luteal regression. Thus, we injected intraperitoneally a luteolytic dose (3 microg/kg) of a synthetic PGF(2alpha) during the mid and late phase of CL development. Ovarian NOS activity was evaluated. The results confirmed our hypothesis; we did not see any effect in the mid-stage of CL development, but increased ovarian NOS activity was found in PGF(2alpha)-injected late pseudopregnant rats.