Pore Formation Triggered by Legionella spp. Is an Nlrc4 Inflammasome-Dependent Host Cell Response That Precedes Pyroptosis

Legionella pneumophila, the etiological agent of Legionnaires disease, is known to trigger pore formation in bone marrow-derived macrophages (BMMs) by mechanisms dependent on the type IVB secretion system known as Dot/Icm. Here, we used several mutants of L. pneumophila in combination with knockout mice to assess the host and bacterial factors involved in pore formation in BMMs. We found that regardless of Dot/Icm activity, pore formation does not occur in BMMs deficient in caspase-1 and Nlrc4/Ipaf. Pore formation was temporally associated with interleukin-1 beta secretion and preceded host cell lysis and pyroptosis. Pore-forming ability was dependent on bacterial Dot/Icm but independent of several effector proteins, multiplication, and de novo protein synthesis. Flagellin, which is known to trigger the Nlrc4 inflammasome, was required for pore formation as flaA mutant bacteria failed to induce cell permeabilization. Accordingly, transfection of purified flagellin was sufficient to trigger pore formation independent of infection. By using 11 different Legionella species, we found robust pore formation in response to L. micdadei, L. bozemanii, L. gratiana, L. jordanis, and L. rubrilucens, and this trait correlated with flagellin expression by these species. Together, the results suggest that pore formation is neither L. pneumophila specific nor the result of membrane damage induced by Dot/Icm activity; instead, it is a highly coordinated host cell response dependent on host Nlrc4 and caspase-1 and on bacterial flagellin and type IV secretion system.

Effects of creatine supplementation on homocysteine levels and lipid peroxidation in rats

Hyperhomocysteinaemia is an independent risk factor for CVD. Recent data show a relationship between homocysteine (Hcy) and free radical formation. Since creatine synthesis is responsible for most of the methyl group transfers that result in Hcy formation, creatine supplementation might inhibit Hcy production and reduce free radical formation. The present study investigated the effects of creatine supplementation on Hcy levels and lipid peroxidation biomarkers. Thirty rats were divided into three groups: control group; diet with creatine group (DCr; 2% creatine in the diet for 28 d); creatine overload plus diet with creatine group (CrO + D; 5 g creatine/kg by oral administration for 5 d + 2 % in the diet for 23 d). Plasma Hcy was significantly lower (P<0.05) in DCr (7.5 (SD 1.2) mu mol/l) and CrO + D (7.2 (SD 1.7) mu mol/l) groups compared with the control group (12.4 (SD 2.2) mu mol/l). Both plasma thiobarbituric acid-reactive species (TBARS) (control, 10 (SD 3.4); DCr, 4.9 (So 0.7); CrO + D, 2.4 (SD 1) mu mol/l) and plasma total glutathione (control, 4.3 (SD 1.9); DCr, 2.5 (SD 0.8); CrO + D, 1.8 (SD 0.5) mu mol/l) were lower in the groups that received creatine (P<0.05). In addition, Hcy showed significant negative correlation (P<0.05) with plasma creatine (r - 0.61) and positive correlation with plasma TBARS (r 0.74). Plasma creatine was negatively correlated with plasma TBARS (r - 0.75) and total peroxide (r - 0.40). We conclude that creatine supplementation reduces plasma Hcy levels and lipid peroxidation biomarkers, suggesting a protective role against oxidative damage. Modulating Hcy fort-nation may, however, influence glutathione synthesis and thereby affect the redox state of the cells.

Assessment of Vascular Effects of Tamoxifen and Its Metabolites on the Rat Perfused Hindquarter Vascular Bed

Tamoxifen has been suggested to produce beneficial cardiovascular effects, although the mechanisms for these effects are not fully known. Moreover, although tamoxifen metabolites may exhibit 30-100 times higher potency than the parent drug, no previous study has compared the effects produced by tamoxifen and its metabolites on vascular function. Here, we assessed the vascular responses to acetylcholine and sodium nitroprusside on perfused hindquarter vascular bed of rats treated with tamoxifen or its main metabolites (N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen, and endoxifen) for 2 weeks. Plasma and whole-blood thiobarbituric acid reactive substances (TBARS) concentrations were determined using a fluorometric method. Plasma nitrite and NOx (nitrite + nitrate) concentrations were determined using an ozone-based chemiluminescence assay and Griess reaction, respectively. Treatment with tamoxifen reduced the responses to acetylcholine (pD(2) = 2.2 +/- 0.06 and 1.9 +/- 0.05 after vehicle and tamoxifen, respectively; P < 0.05), while its metabolites improved these responses (pD(2) = 2.5 +/- 0.04 after N-desmethyl-tamoxifen, 2.5 +/- 0.03 after 4-hydroxy-tamoxifen, and 2.6 +/- 0.08 after endoxifen; P < 0.01). Tamoxifen and its metabolites showed no effect on endothelial-independent responses to sodium nitroprusside (P > 0.05). While tamoxifen treatment resulted in significantly higher plasma and whole blood lipid peroxide levels (37% and 62%, respectively; both P < 0.05), its metabolites significantly decreased lipid peroxide levels (by approximately 50%; P < 0.05). While treatment with tamoxifen decreased the concentrations of markers of nitric oxide formation by approximately 50% (P < 0.05), tamoxifen metabolites had no effect on these parameters (P > 0.05). These results suggest that while tamoxifen produces detrimental effects, its metabolites produce counteracting beneficial effects on the vascular system and on nitric oxide/reactive oxygen species formation.

Mechanisms involved in the pressor response to noradrenaline microinjection into the supraoptic nucleus of unanesthetized rats

We report on the cardiovascular effects of noradrenaline (NA) microinjection into the hypothalamic supraoptic nucleus (SON) as well as the central and peripheral mechanisms involved in their mediation. Microinjections of NA 1, 3, 10, 30 or 45 nmol/100 nL into the SON caused dose-related pressor and bradycardiac response in unanesthetized rats. The response to NA 10 nmol was blocked by SON pretreatment with 15 nmol of the alpha(2)-adrenoceptor antagonist RX821002 and not affected by pretreatment with equimolar dose of the selective alpha(1)-adrenoceptor antagonist WB4101, suggesting that local alpha(2)adrenoceptors mediate these responses. Pretreatment of the SON with the nonselective beta-adrenoceptor antagonist propranolol 15 nmol did not affect the pressor response to NA microinjection of into the SON. Moreover, the microinjection of the 100 nmol of the selective alpha(1)-adrenoceptor agonist methoxamine (MET) into the SON did not cause cardiovascular response while the microinjection of the selective alpha(2)adrenoceptor agonists BHT920 (BHT, 100 nmol) or clonidine (CLO, 5 nmol) caused pressor and bradycardiac responses, similar to that observed after the microinjection of NA. The pressor response to NA was potentiated by intravenous pretreatment with the ganglion blocker pentolinium and was blocked by intravenous pretreatment with the V(1)-vasopressin receptor antagonist dTyr(CH2)5(Me)AVP, suggesting an involvement of circulating vasopressin in this response. In conclusion, our results suggest that pressor responses caused by microinjections of NA into the SON involve activation of local alpha(2)-adrenoceptor receptors and are mediated by vasopressin release into circulation. (c) 2008 Published by Elsevier B.V.

Stimulation-Produced Analgesia From the Occipital or Retrosplenial Cortex of Rats Involves Serotonergic and Opioid Mechanisms in the Anterior Pretectal Nucleus

The electrical stimulation of the occipital (OC) or retrosplenial (RSC) cortex produces antinociception in the rat tail-flick test. These cortices send inputs to the anterior pretectal nucleus (APtN) which is implicated in antinociception and nociception. At least muscarinic cholinergic, opioid, and serotonergic mechanisms in the APtN are involved in stimulation-produced antinociception (SPA) from the nucleus. In this study, the injection of 2% lidocaine (.25 mu L) or methysergide (40 and 80 ng/.25 mu L) into the APtN reduced the duration but did not change the intensity of SPA from the OC, whereas both duration and intensity of SPA from the RSC were significantly reduced in rats treated with lidocaine or naloxone (10 and 50 ng/.25 mu L), injected into the ANN. Naloxone or methysegide injected into the APtN was ineffective against SPA from the OC or RSC, respectively. Atropine (100 ng/.25 mu L) injected into the ANN was ineffective against SPA from either the OC or RSC. We conclude that the APtN acts as an intermediary for separate descending pain inhibitory pathways activated from the OC and RSC, utilizing at least serotonin and endogenous opioid as mediators in the nucleus. Perspective: Stimulation-induced antinociception from the retrosplenial or occipital cortex in the rat tail-flick test depends on the activation of separate descending pain inhibitory pathways that utilize the APtN as a relay station. (C) 2011 by the American Pain Society

Aldosterone and angiotensin II synergistically stimulate migration in vascular smooth muscle cells through c-Src-regulated redox-sensitive RhoA pathways

Objective - Synergistic interactions between aldosterone (Aldo) and angiotensin II (Ang II) have been implicated in vascular inflammation, fibrosis, and remodeling. Molecular mechanisms underlying this are unclear. We tested the hypothesis that c-Src activation, through receptor tyrosine kinase transactivation, is critically involved in synergistic interactions between Aldo and Ang II and that it is upstream of promigratory signaling pathways in vascular smooth muscle cells (VSMCs). Methods and Results - VSMCs from WKY rats were studied. At low concentrations (10(-10) mol/L) Aldo and Ang II alone did not influence c-Src activation, whereas in combination they rapidly increased phosphorylation (P<0.01), an effect blocked by eplerenone ( Aldo receptor antagonist) and irbesartan (AT1R blocker). This synergism was attenuated by AG1478 and AG1296 ( inhibitors of EGFR and PDGFR, respectively), but not by AG1024 (IGFR inhibitor). Aldo and Ang II costimulation induced c-Src-dependent activation of NAD(P)H oxidase and c-Src-independent activation of ERK1/2 (P<0.05), without effect on ERK5, p38MAPK, or JNK. Aldo/Ang II synergistically activated RhoA/Rho kinase and VSMC migration, effects blocked by PP2, apocynin, and fasudil, inhibitors of c-Src, NADPH oxidase, and Rho kinase, respectively. Conclusions - Aldo/Ang II synergistically activate c-Src, an immediate signaling response, through EGFR and PDGFR, but not IGFR transactivation. This is associated with activation of redox-regulated RhoA/Rho kinase, which controls VSMC migration. Although Aldo and Ang II interact to stimulate ERK1/2, such effects are c-Src-independent. These findings indicate differential signaling in Aldo-Ang II crosstalk and highlight the importance of c-Src in redox-sensitive RhoA, but not ERK1/2 signaling. Blockade of Aldo/Ang II may be therapeutically useful in vascular remodeling associated with abnormal VSMC migration.

Mechanisms underlying the biphasic effect of vitamin K-1 (phylloquinone) on arterial blood pressure

Phylloquinone (vitamin K-1, VK1) is widely used therapeutically and intravenous administration of this quinone can induce hypotension. We aimed to investigate the mechanisms underlying the effects induced by VK1 on arterial blood pressure. With this purpose a catheter was inserted into the abdominal aorta of male Wistar rats for blood pressure and heart rate recording. Bolus intravenous injection of VK1 (0.5-20 mg kg(-1)) produced a transient increase in blood pressure followed by a fall. Both the pressor and depressor response induced by VK1 were dose-dependent. On the other hand, intravenous injection of VK1 did not alter heart rate. The nitric oxide synthase (NOS) inhibitor N-G-nitro-L-arginine methyl ester (L-NAME, 10 and 20 mg kg(-1)) reduced both the increase and decrease in blood pressure induced by VK1 (5 mgkg(-1)). On the other hand, indometacin (10 mg kg(-1)), a non-selective cyclooxygenase inhibitor, did not alter the increase in mean arterial pressure (MAP) induced by VK1. However, VK1-induced fall in MAP was significantly attenuated by indometacin. We concluded that VK1 induces a dose-dependent effect on blood pressure that consists of an acute increase followed by a more sustained decrease in MAP. The hypotension induced by VK1 involves the activation of the nitric oxide (NO) pathway and the release of vasodilator prostanoid(s).

Crucial role of neutrophils in the development of mechanical inflammatory hypernociception

Neutrophil migration is responsible for tissue damage observed in inflammatory diseases. Neutrophils are also implicated in inflammatory nociception, but mechanisms of their participation have not been elucidated. In the present study, we addressed these mechanisms in the carrageenan-induced mechanical hypernociception, which was determined using a modification of the Randall-Sellito test in rats. Neutrophil accumulation into the plantar tissue was determined by the contents of myeloperoxidase activity, whereas cytokines and PGE(2) levels were measured by ELISA and radioimmunoassay, respectively. The pretreatment of rats with fucoidin (a leukocyte adhesion inhibitor) inhibited carrageenan-induced hypernociception in a dose- and time-dependent manner. Inhibition of hypernociception by fucoidin was associated with prevention of neutrophil recruitment, as it did not inhibit the hypernociception induced by the direct-acting hypernociceptive mediators, PGE(2) and dopamine, which cause hypernociception, independent of neutrophils. Fucoidin had no effect on carrageenan-induced TNF-alpha, IL-1 beta, and cytokine-induced neutrophil chemoattractant 1 (CINC-1)/CXCL1 production, suggesting that neutrophils were not the source of hypernociceptive cytokines. Conversely, hypernociception and neutrophil migration induced by TNF-alpha, IL-1 beta, and CINC-1/CXCL1 was inhibited by fucoidin, suggesting that neutrophils are involved in the production of direct-acting hypernociceptive mediators. Indeed, neutrophils stimulated in vitro with IL-1 beta produced PGE(2), and IL-1 beta-induced PGE(2) production in the rat paw was inhibited by the pretreatment with fucoidin. In conclusion, during the inflammatory process, the migrating neutrophils participate in the cascade of events leading to mechanical hypernociception, at least by mediating the release of direct-acting hypernociceptive mediators, such as PGE(2). Therefore, the blockade of neutrophil migration could be a target to development of new analgesic drugs.

Role of complement C5a in mechanical inflammatory hypernociceptio: potential use of C5a receptor antagonists to control inflammatory pain

particularly neutrophil chemoattraction. Herein, the role of C5a in the genesis of inflammatory hypernociception was investigated in rats and mice using the specific C5a receptor antagonist PMX53 (AcF-[OP(D-Cha)WR]). Experimental approach: Mechanical hypernociception was evaluated with a modification of the Randall-Selitto test in rats and electronic pressure meter paw test in mice. Cytokines were measured by ELISA and neutrophil migration was determined by myeloperoxidase activity. Key results: Local pretreatment of rats with PMX53 (60-180 mg per paw) inhibited zymosan-, carrageenan-, lipopolysaccharide (LPS)- and antigen-induced hypernociception. These effects were associated with C5a receptor blockade since PMX53 also inhibited the hypernociception induced by zymosan- activated serum and C5a but not by the direct-acting hypernociceptive mediators, prostaglandin E-2 and dopamine. Underlying the C5a hypernociceptive mechanisms, PMX53 did not alter the cytokine release induced by inflammatory stimuli. However, PMX53 inhibited cytokine-induced hypernociception. PMX53 also inhibited the recruitment of neutrophils induced by zymosan but not by carrageenan or LPS, indicating an involvement of neutrophils in the hypernociceptive effect of C5a. Furthermore, the C5a-induced hypernociception was reduced in neutrophil-depleted rats. Extending these findings in rats, blocking C5a receptors also reduced zymosan- induced joint hypernociception in mice. Conclusions and implications: These results suggest that C5a is an important inflammatory hypernociceptive mediator, acting by a mechanism independent of hypernociceptive cytokine release, but dependent on the presence of neutrophils. Therefore, we suggest that inhibiting the action of C5a has therapeutic potential in the control of inflammatory pain.

Aripiprazole, an atypical antipsychotic prevents the motor hyperactivity induced by psychotomimetics, and psyphostimulants in mice

Aripiprazole is an atypical antipsychotic that acts as a partial agonist at the dopamine D-2 receptor. It has been mainly investigated in dopamine-based models of schizophrenia, while its effects on glutamate-based paradigms have remained to be further characterized. Due to its unique mechanism of action, aripiprazole has also been considered as a replacement medication for psychostimulant abuse. Thus, in the present study we tested the hypothesis that aripiprazole would prevent the motor hyperactivity induced by psychostimulant and psychotomimetic drugs that act either by dopaminergic or glutamatergic mechanisms. Male Swiss mice received injections of aripiprazole (0.1-1 mg/kg) followed by drugs that enhance the dopamine-mediated neurotransmission, amphetamine (3 mg/kg) or cocaine (5 mg/kg), or by glutamate NMDA-receptor antagonists, ketamine (60 mg/kg) or MK-801 (0.4 mg/kg). Independent groups also received aripiprazole (0.1-1 mg/kg) or haloperidol (0.5 mg/kg) and were tested for catalepsy. All doses of aripiprazole were effective in preventing the motor stimulant effects of amphetamine and cocaine. Moreover, the higher dose also prevented the effects of ketamine and MK-801. The present study reports the effects of aripiprazole in dopaminergic and glutamatergic models predictive of antipsychotic activity, suggesting that both may be useful for screening novel partial agonists with antipsychotic activity. It also shows that aripiprazole may prevent the acute effects of psychostimulant drugs without significant motor impairment. C) 2007 Elsevier B.V. All rights reserved.

Peroxisome proliferator-activated receptor-gamma ligand, 15-deoxy-Delta A(12,14)-prostaglandin J(2), reduces neutrophil migration via a nitric oxide pathway

Ligands for peroxisome proliferator-activated receptor gamma (PPAR-gamma), such as 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) have been implicated as a new class of anti-inflammatory compounds with possible clinical applications. Based on this concept, this investigation was designed to determine the effect of 15d-PGJ(2)-mediated activation of PPAR-gamma ligand on neutrophil migration after an inflammatory stimulus and clarify the underlying molecular mechanisms using a mouse model of peritonitis. Our results demonstrated that 15d-PGJ(2) administration decreases leukocyte rolling and adhesion to the inflammated mesenteric tissues by a mechanism dependent on NO. Specifically, pharmacological inhibitors of NO synthase remarkably abrogated the 15d-PGJ(2)-mediated suppression of neutrophil migration to the inflammatory site. Moreover, inducible NOS(-/-) mice were not susceptible to 15d-PGJ(2)-mediated suppression of neutrophil migration to the inflammatory sites when compared with their wild type. In addition, 15d-PGJ(2)-mediated suppression of neutrophil migration appeared to be independent of the production of cytokines and chemokines, since their production were not significantly affected in the carrageenan-injected peritoneal cavities. Finally, up-regulation of carrageenan-triggered ICAM-I expression in the mesenteric microcirculation vessels was abrogated by pretreatment of wild-type mice with 15d-PGJ(2), whereas 15d-PGJ(2) inhibited F-actin rearrangement process in neutrophils. Taken together these findings demonstrated that 15d-PGJ(2) suppresses inflammation-initiated neutrophil migration in a mechanism dependent on NO production in mesenteric tissues.

Vascular biology of magnesium and its transporters in hypertension

Magnesium may influence blood pressure by modulating vascular tone and structure through its effects on myriad biochemical reactions that control vascular contraction/dilation, growth/apoptosis, differentiation and inflammation. Magnesium acts as a calcium channel antagonist, it stimulates production of vasodilator prostacyclins and nitric oxide and it alters vascular responses to vasoconstrictor agents. Mammalian cells regulate Mg(2+) concentration through special transport systems that have only recently been characterized. Magnesium efflux occurs via Na(2+)-dependent and Na(2+)-independent pathways. Mg(2+) influx is controlled by recently cloned transporters including Mrs2p, SLC41A1, SLC41A2, ACDP2, MagT1, TRPM6 and TRPM7. Alterations in some of these systems may contribute to hypomagnesemia and intracellular Mg(2+) deficiency in hypertension and other cardiovascular pathologies. In particular, increased Mg(2+) efflux through dysregulation of the vascular Na(+)/Mg(2+) exchanger and decreased Mg(2+) influx due to defective vascular and renal TRPM6/7 expression/activity may be important in altered vasomotor tone and consequently in blood pressure regulation. The present review discusses the role of Mg(2+) in vascular biology and implications in hypertension and focuses on the putative transport systems that control magnesium homeostasis in the vascular system. Much research is still needed to clarify the exact mechanisms of cardiovascular Mg(2+) regulation and the implications of aberrant cellular Mg(2+) transport and altered cation status in the pathogenesis of hypertension and other cardiovascular diseases.

Gastroprotective effect of heme-oxygenase 1/biliverdin/CO pathway in ethanol-induced gastric damage in mice

Our objective was to evaluate the role of heme-oxygenase 1 (HO-1)/biliverdin/CO pathway in gastric defense against ethanol-induced gastric damage in mice. Mice were pre-treated with saline, hemin (HO-1 inducer), biliverdin (HO-1 product), dimanganese decacarbonyl (DMDC, CO donor) or zinc protoporphyrin IX (ZnPP IX, HO-1 antagonist). Another group received soluble guanylate cyclase (sGC) inhibitor (ODQ) 30 min before hemin, biliverdin or DMDC. After 30 min, gastric damage was induced by ethanol. After one hour, rats were sacrificed. Gastric lesions were measured using a computer planimetry program, and gastric corpus pieces were assayed for malonylaldehyde (MDA), glutathione (GSH) or bilirubin. HO-1 expression was determined after saline or ethanol administration by polymerase chain reaction (PCR) or immunohistochemistry. Ethanol (25% or 50%) induced gastric damage, increased MDA levels and reduced GSH in the gastric tissue. Ethanol 50% increased HO-1 mRNA transcripts, HO-1 immunoreactivity, and bilirubin concentration in gastric mucosa. Pre-treatment with hemin reduced gastric damage and MDA formation and increased GSH concentration in the gastric mucosa. ZnPP IX amplified the ethanol-induced gastric lesion, increased MDA formation and decreased GSH concentration in gastric mucosa. Biliverdin and DMDC reduced gastric damage and MDA formation and increased GSH concentration in the gastric tissue. ODQ completely abolished the DMDC protective gastric effect However, effects of hemin or biliverdin did not change with ODQ treatment. Our results suggest that HO-1/biliverdin/CO pathway plays a protective role against ethanol-induced gastric damage through mechanisms that can be dependent (CO) or independent (biliverdin) of sGC activation. (C) 2010 Elsevier B.V. All rights reserved.