Periodontitis increases vascular cyclooxygenase-2: potential effect on vascular tone.

BACKGROUND AND OBJECTIVE: It has been demonstrated that periodontitis induces a systemic inflammation, which may impair endothelial function. Cyclooxygenase-2 (COX-2) is an important enzyme in the inflammatory process and is responsible for prostacyclin production. We hypothesised that in periodontitis, an increase in vascular COX-2 expression may occur, which in turn may have a role in vascular homeostasis. Thus, we evaluated the vascular effects of COX-2 inhibition in an experimental rat model of periodontitis. MATERIAL AND METHODS: Experimental periodontitis was induced in rats by placing a cotton ligature around the cervix of both sides of the mandibular first molars and maxillary second molars. Sham-operated rats had the ligature removed immediately after the procedure. Mesenteric vessels were obtained for the study of COX-2 expression, and blood samples were collected for nitric oxide quantification. In another set of experiments, animals received etoricoxib (10 mg/kg/d, v.o.) or vehicle, and alveolar bone loss and cardiovascular parameters were evaluated. RESULTS: We observed an increase in COX-2 expression in mesenteric vessels harvested from animals with periodontitis, which was accompanied by a reduction in nitric oxide content. Etoricoxib treatment impaired the endothelium-dependent reduction in blood pressure in rats with periodontitis. CONCLUSION: Periodontitis increases vascular COX-2 expression, which is important in the maintenance of vascular homeostasis in this model. Despite the limitations of an animal study, these findings may have important implications regarding the safety of using selective COX-2 inhibitors in patients with periodontitis.

Apoptotic mimicry: phosphatidylserine liposomes reduce inflammation through activation of peroxisome proliferator-activated receptors (PPARs) in vivo.

BACKGROUND AND PURPOSE: Recently, there has been much attention paid to understanding the molecular mechanisms underlying apoptosis and the functional consequences of apoptotic body clearance by phagocytes. In an attempt to investigate this latter aspect, the present study evaluated the anti-inflammatory effects of in vivo administration of phosphatidylserine (PS) liposomes, a well-characterised membrane component expressed during apoptosis. The participation of peroxisome proliferator-activated receptors (PPARs) in PS-mediated effects was also investigated. EXPERIMENTAL APPROACH: The anti-inflammatory effect of PS liposomes on the delayed phase of carrageenan mouse paw oedema was studied. PS liposomes were injected at different doses and times, after carrageenan. Hind paws were collected for evaluation of interleukin-1beta (IL-1beta) levels, myeloperoxidase (MPO) and N-acetyl-glucosaminidase (NAG) activities and Evans blue dye leakage. Participation of PPAR pathways was explored by using PPAR antagonists (BADGE and GW9662). KEY RESULTS: Administration of PS, but not phosphatidylcholine (PC), liposomes (20-200 mg kg(-1), i.p., 8 h after carrageenan) reduced the paw oedema in a dose-dependent manner. PS liposomes were effective even when administered 24 and 48 h after carrageenan, a time at which indomethacin (1 mg kg(-1), i.p.) had no significant effects. Carrageenan-induced Evans blue leakage and IL-1beta production was decreased in PS-treated paws. The PPAR antagonists (BADGE and GW9662) partially prevented the anti-inflammatory effects of PS administration. CONCLUSIONS AND IMPLICATIONS: PS liposomes have anti-inflammatory effects in vivo that are at least partly dependent on PPAR activation. Therapeutic strategies mimicking apoptosis may be useful for the treatment of inflammatory disorders.

Kallikrein-kinin system activation by Lonomia obliqua caterpillar bristles: involvement in edema and hypotension responses to envenomation.

Lonomia obliqua envenomation induces an intense burning sensation at the site of contact and severe hemorrhage followed by edema and hypotension, and after few days death can occur usually due to acute renal failure. In order to understand more about the envenomation syndrome, the present study investigates the role played by kallikrein-kinin system (KKS) in edematogenic and hypotensive responses to the envenomation by L. obliqua. The incubation of L. obliqua caterpillar bristles extract (LOCBE) with plasma results in kallikrein activation, measured by cromogenic assay using the kallikrein synthetic substrate S-2302 (H-D-Pro-Phe-Arg-pNA). It was also showed that LOCBE was able to release kinins from low-molecular weight kininogen (LMWK). Moreover, it was demonstrated that previous administration of a kallikrein inhibitor (aprotinin) or bradykinin B2 receptor antagonist (HOE-140) significantly reduces the edema and hypotension in response to LOCBE, using mouse paw edema bioassay and mean arterial blood pressure analysis, respectively. The results demonstrate a direct involvement of the KKS in the edema formation and in the fall of arterial pressure that occur in the L. obliqua envenomation syndrome.

Depolymerization of macrophage microfilaments prevents induction and inhibits activity of nitric oxide synthase.

We have investigated the relationship between peritoneal murine macrophage cytoskeleton and nitric oxide (NO) synthase (NOS). Activation of the cells with lipopolysaccharide plus interferon-gamma (LI) induced iNOS, detected by nitrite or by labeled L-citrulline production and by a specific antibody against macrophage iNOS. Addition of cytochalasin B (a microfilament-depolymerizing agent) caused a dose-dependent inhibition in NO production by macrophages, whereas colchicine (a microtubule depolymerizing agent) inhibited it only by 20% and not dose-dependently. Addition of cytochalasin B together with LI abolished nitrite and L-citrulline accumulation as well as the amount of iNOS antigen in activated macrophage. Moreover, addition of cytochalasin B 6 or 12 h after stimulus, also decreased the nitrite and L-citrulline production by macrophages although iNOS antigen content by Western blot was the same in the presence or in the absence of cytochalasin B added 12 h after activation. Since cytochalasin B failed to inhibit iNOS activity directly, its inhibitory effects on NO production by macrophages is likely to be indirect, through microfilament network in central regions of cells, but not in filaments seen at pseudopodia or edging processes. Our findings demonstrate that disruption of microfilaments but not of microtubules prevents the iNOS induction process and inhibits its enzymatic activity in activated macrophages.

Leishmania-induced tyrosine phosphorylation in the host macrophage and its implication to infection.

Tyrosine phosphorylation is an important mechanism of cell regulation and has been recently implicated in defense strategies against a variety of pathogens. We have investigated the involvement of protein tyrosine kinase activity in the Leishmania attachment, invasion and survival within macrophages, as well as promastigote ability to trigger tyrosine phosphorylation, which could contribute to leishmanicidal activity. Treatment of murine macrophage monolayers with genistein, herbimycin A, tyrphostin 25 or staurosporine prior to infection decreased parasite invasion in a dose-dependent manner. Contrary, addition of sodium orthovanadate, a protein tyrosine phosphatase inhibitor, phosphotyrosine and p-nitrophenyl phosphate to the interaction medium, significantly increased parasite binding and internalization, whereas phosphoserine and phosphothreonine had no effect. The phosphatase activity of intact promastigotes was greater than that of macrophages. Western blot analysis revealed tyrosine-phosphorylated bands from 198 to 28 kDa following macrophage challenge with promastigotes. Uninfected macrophages displayed no detectable tyrosine phosphorylated proteins, possibly indicating an inducible process, while in parasites it was constitutive, as seen by the presence of 42, 40 and 35 kDa phosphoproteins on the Leishmania lysates. Immunofluorescence and immunogold detection of phosphotyrosine residues in some promastigote-macrophage attachment areas, but not in the vicinity of ingested parasites, suggest that Leishmania-induced tyrosine phosphorylation is an early, local and short-lived event. Genistein treatment of Leishmania-infected cells significantly enhanced the parasite burden. This antagonist also diminished nitric oxide production in resting and interferon gamma/lipopolysaccharide-activated infected macrophages, which may account for the increased parasite survival. We propose that protein tyrosine kinase-linked pathways regulate the Leishmania promastigote invasion and the macrophage microbicidal activity.

Nitric oxide inhibits irreversibly P815 cell proliferation: involvement of potassium channels.

Nitric oxide (NO) has been shown to inhibit both normal and cancer cell proliferation. Potassium channels are involved in cell proliferation and, as NO activates these channels, we investigated the effect of NO on the proliferation of murine mastocytoma cell lines and the putative involvement of potassium channels. NO (in the form of NO donors) caused dose-dependent inhibition of cell proliferation in the P815 cell line inducing growth arrest in the mitosis phase. Incubation with NO donor for 4 or 24 h had a similar inhibitory effect on cell proliferation, indicating that this effect is irreversible. The inhibitory effect of NO was completely prevented by the blockade of voltage- and calcium-dependent potassium channels, but not by blockade of ATP-dependent channels. NO inhibition of cell proliferation was unaffected by guanylate cyclase and by cytoskeleton disruptors. Therefore, NO inhibits cell proliferation irreversibly via a potassium channel-dependent but guanylate cyclase-independent pathway in murine mastocytoma cells.

A perfusion system for the long term study of macrophage activation.

1. A closed system was developed for perfusing J774 macrophages in columns. The cells were perfused for up to 100 h, at which time they were still viable. 2. Stimulation with increasing concentrations (0.01-10 micrograms ml-1) of bacterial lipopolysaccharide (LPS) caused the cells to produce increasing amounts of nitrite in the perfusion medium. This production was time-dependent, reaching a plateau by 48-50 h. 3. The nitrite accumulation caused by 0.1 microgram ml-1 of LPS was augmented by priming the cells for 2 h with increasing amounts of interferon-gamma. The nitrite accumulation also reached a plateau under these conditions. 4. N-iminoethyl-L-ornithine (L-NIO, 30 microM) completely inhibited the accumulation of nitrite whereas dexamethasone (0.3 microM) caused 60-70% inhibition. 5. Perfusion of the cells without L-arginine prevented the nitrite accumulation. Replacement of this amino acid after 20 or 50 h of perfusion led to a rapid generation of nitrite, the levels of which continued to increase for the duration of the experiment. 6. Thus, the perfusion system can be used to study the kinetics of the activation of the NO synthase and most likely other parameters in J774 cells and probably other cells in culture. An observation already of interest is that the 'disappearance' of the NO synthase after its activation can be prevented or reduced by removal of L-arginine from the medium.

Feedback inhibition of nitric oxide synthase activity by nitric oxide.

1. A murine macrophage cell line, J774, expressed nitric oxide (NO) synthase activity in response to interferon-gamma (IFN-gamma, 10 u ml-1) plus lipopolysaccharide (LPS, 10 ng ml-1). The enzyme activity was first detectable 6 h after incubation, peaked at 12 h and became undetectable after 48 h. 2. The decline in the NO synthase activity was not due to inhibition by stable substances secreted by the cells into the culture supernatant. 3. The decline in the NO synthase activity was significantly slowed down in cells cultured in a low L-arginine medium or with added haemoglobin, suggesting that NO may be involved in a feedback inhibitory mechanism. 4. The addition of NO generators, S-nitroso-acetyl-penicillamine (SNAP) or S-nitroso-glutathione (GSNO) markedly inhibited the NO synthase activity in a dose-dependent manner. The effect of NO on the enzyme was not due to the inhibition of de novo protein synthesis. 5. SNAP directly inhibited the inducible NO synthase extracted from activated J774 cells, as well as the constitutive NO synthase extracted from the rat brain. 6. The enzyme activity of J774 cells was not restored after the removal of SNAP by gel filtration, suggesting that NO inhibits NO synthase irreversibly.

Sequential induction of nitric oxide synthase by Corynebacterium parvum in different organs of the mouse.

1. The ability of Corynebacterium parvum (C. parvum) to induce nitric oxide (NO) synthase in the macrophage, spleen, liver, aorta, heart and brain, and to elevate plasma NO2-/NO3- in the mouse was investigated. In addition, the relationship between NO synthase activity and blood pressure was studied. 2. C. parvum (100 mg kg-1, i.p.) induced a time-dependent expression of a Ca(2+)-independent NO synthase in the macrophage, spleen, liver, aorta and heart. The time course of induction of the NO synthase varied such that the maximum enzyme activity was at day 8 in the macrophage and liver, day 12 in the spleen and heart and day 16 in the aorta. 3. There was no significant induction of a Ca(2+)-independent NO synthase in the brain, nor was there any change in the Ca(2+)-dependent enzyme in this organ, during the study period. 4. C. parvum produced a gradual decrease in blood pressure, with a maximum fall at day 16 (from 108 +/- 1 mmHg to 79 +/- 3 mmHg), which recovered gradually by day 28. 5. Plasma NO2-/NO3- was significantly elevated between days 8 and 24, with a maximum increase at day 12. 6. These results show that C. parvum induces a Ca(2+)-independent NO synthase in a number of tissues and that this induction occurs initially in macrophages and the liver. This suggests that induction of the NO synthase in the other tissues is secondary and probably the result of activation of macrophages and some cells of the liver. 7. Furthermore, the decrease in blood pressure induced by C. parvum is associated with the induction of NO synthase in the vasculature, whereas the increased concentration of plasma NO2-/NO3- seems to result from the generation of NO by a number of tissues.

Killing of lymphoblastic leukemia cells by nitric oxide and taxol: involvement of NF-kappaB activity.

Nitric oxide (NO) and taxol are cytotoxic towards leukemia and tumor cells and interfere with the transcription factor NF-kappaB activity. NO and taxol inhibited NF-kappaB activity and were cytotoxic to human and murine leukemia cells, but at a different magnitude (30% cell killing and 80% inhibition of NF-kappaB). Sub-effective concentrations of SNAP and taxol synergized in killing L-1210 cells but either alone or in combination completely inhibited NF-kappaB. Pyrrolidine dithiocarbamate (PDTC) was cytotoxic on its own and inhibited NF-kappaB activity. It potentiated NO and taxol killing but again there was no direct relationship between inhibition of NF-kappaB and cell killing. Neither NO nor taxol cytotoxicity was related to the cytoskeleton. Our results show that NO, taxol and PDTC induced apoptosis and NF-kappaB inhibition in leukemic cells but their cytotoxicity either alone or in combination, does not seem to be dependent on the inhibition of NF-KB activity.

Nitric oxide involvement in the anxiogenic-like effect of substance P.

This study investigates whether nitric oxide (NO) is involved in the anxiogenic profile of action of substance P (SP) in mice in the elevated plus-maze (EPM). Adult Swiss mice were injected with NOS inhibitors such as L-NOARG (20 nmol/kg) i.p., L-NAME (3 nmol per site), 7-NI (0.25 nmol per site) i.c.v. or vehicle (NaCl 0.9% i.p. or PBS i.c.v.). About 30 min (i.p. pretreatment) or 5 min later (i.c.v. pretreatment), the animals received i.c.v. injections of SP (10 pmol) or phosphate buffered saline (PBS) (2 microl). Afterwards, they were observed in the EPM. SP per se reduced the time spent on open arms, an anxiogenic-like effect. This effect was reverted by different NOS inhibitors and the NO donor. NOS inhibitors had no influence on the EPM parameters but the NO-releasing compound SNAP, as well as its parent thiol NAP, increased the animals' locomotor activity. 8-Br-cGMP (20 nmol), a permeable cGMP analog, promoted an anxiogenic-like effect per se and enhanced the SP effect on the EPM. Altogether, these results suggest a putative NO role in the mediation of the anxiogenic-like effect of SP.

Pharmacological properties of a new kinin-potentiating peptide generated from human serum proteins.

A kinin-potentiating peptide (KPP) generated from human plasma proteins on trypsin incubation was partially purified by ultrafiltration and ion-exchange chromatography and was characterized through some of its pharmacological properties. KPP itself was devoid of any action but it potentiated the guinea-pig ileum contractions elicited by several kinins, including an analog resistant to angiotensin-converting enzyme (ACE). In contrast, contractions induced by angiotensin II, histamine, acetylcholine, barium chloride and substance P were not potentiated. Not only did KPP have high specificity towards kinins, but its action started immediately and induced kinin potentiation in a dose-dependent and reversible manner. Furthermore KPP potentiated the bradykinin contracting effects on the rat uterus, a preparation with very poor ACE activity, and on guinea-pig ileum previously incubated with 1.10-phenanthroline, a metal chelator able to inhibit ACE and kininase I activities and with phosphoramidon, a specific inhibitor of neutral endopeptidase (NEP). The results suggest that the potentiating effect of KPP is due to a mechanism different from the inhibition of kinin metabolism by ACE, NEP and kininase I.

Comparative study on the mechanism of bradykinin potentiation induced by bradykinin-potentiating peptide 9a, enalaprilat and kinin-potentiating peptide.

The action of a kinin-potentiating peptide (KPP) obtained from tryptic digestion of human serum proteins was compared with that of bradykinin-potentiating peptide 9a (BPP9a; obtained from snake venom) and enalaprilat (a synthetic inhibitor of angiotensin-converting enzyme; ACE) as a means of understanding the mechanism of action of KPP on smooth muscle. KPP potentiated bradykinin-induced contractile effects in guinea-pig ileum and rat uterus, but not the bradykinin-induced relaxation of pre-contracted ileum, whereas BPP9a and enalaprilat potentiated both bradykinin effects. The receptor mediating both the contraction and the relaxation elicited by bradykinin in the ileum was found to be of the B2 type. KPP retained its potentiating effect in the presence of enalaprilat in the guinea-pig ileum and rat uterus, whereas the potentiation evoked by BPP9a was abolished. Enalaprilat inhibited the activity of purified ACE, whereas KPP was completely devoid of such an effect. The potentiating effect of KPP, but not that of BPP9a or enalaprilat, was blocked by compounds that inhibit phospholipase A2 and lipoxygenase activity but not by inhibitors of cyclo-oxygenase or phosphodiesterases. The results suggest that the potentiating effect of KPP (i) does not involve inhibition of ACE; (ii) is not due to an increased affinity of the receptor for bradykinin, and (iii) probably involves post-receptor events linked to phospholipase A2 and to the lipoxygenase pathway.

Involvement of soluble guanylate cyclase and calcium-activated potassium channels in the long-lasting hyporesponsiveness to phenylephrine induced by nitric oxide in rat aorta.

Excessive nitric oxide (NO) production by inducible NO synthase has been implicated in the hyporesponsiveness to vasoconstrictors present in septic shock. Here we show that a brief incubation (30 min) of rat aorta rings with NO donors renders the vessels hyporesponsive to phenylephrine for several hours. Contraction of rings without endothelium by phenylephrine (0.1 nM to 100 microM) was decreased by 50-60% after incubation (30 min) with sodium nitroprusside (3-300 microM) or S-nitroso-acetyl-D,L-penicillamine (SNAP; 70-200 microM). This decrease was characterized by reductions in maximal response and rightwards shifts of phenylephrine concentration/response curves, present even 130 min after NO donor removal. Soluble guanylate cyclase inhibitors methylene blue ( 10 microM) and 1H-(1,2,4)-oxadiazol-(4,3-a)quinoxalin-1-one (ODQ, 1 microM) or the potassium channel blockers TEA (tetraethylammonium; 10 mM) and charybdotoxin (100 nM) inhibited the hyporesponsiveness to phenylephrine induced by the NO donors. In contrast, 4-aminopyridine (1 mM) and glibenclamide (10 microM) had no effect. Our results show that incubation with NO donors reproduces the hyporesponsiveness to phenylephrine and that NO alone accounts for most, if not all, the refractoriness to vasoconstrictors present in septic shock. In addition, soluble guanylate cyclase activation and opening of potassium channels, more specifically the calcium-activated subtype, play a predominant role in this NO-induced hyporesponsiveness to phenylephrine in the rat aorta.

Nitric oxide production by BCG-infected pleural macrophages from C57Bl/6 and DBA-2 mice.

We studied the kinetics of in vivo nitrite production in the inflammatory reaction induced by M. bovis BCG into the pleural space. Pleural macrophages harvested from C57Bl/6 mice after acute BCG infection produced high levels of nitric oxide (NO). Enhanced production was obtained upon stimulation with LPS plus IFN-gamma. In sharp contrast, macrophages from DBA-2 mice produced low levels of NO, as nitrite, at the same time interval (24 h after BCG infection), being completely refractory to further stimulation. After the third day, NO production was similar in both strains. There was a close relationship between nitrite levels in the pleural exudate in vivo and those produced by harvested macrophages in vitro. In this in vivo system, the pattern of NO production by pleural macrophages one day after BCG infection was discrepant and unexpected in the response of C57Bl/6 and DBA-2 mice. However, this early response did not affect the late progressive NO production in both mice strains, that may be responsible to the late control of the mycobacteria growth.

Role of nitric oxide and superoxide in Giardia lamblia killing.

Giardia lamblia trophozoites were incubated for 2 h with activated murine macrophages, nitric oxide (NO) donors or a superoxide anion generator (20 mU/ml xanthine oxidase plus 1 mM xanthine). Activated macrophages were cytotoxic to Giardia trophozoites (approximately 60% dead trophozoites). The effect was inhibited (> 90%) by an NO synthase inhibitor (200 microM) and unaffected by superoxide dismutase (SOD, 300 U/ml). Giardia trophozoites were killed by the NO donors, S-nitroso-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) in a dose-dependent manner (LD50 300 and 50 microM, respectively). A dual NO-superoxide anion donor, 3-morpholino-sydnonimine hydrochloride (SIN-1), did not have a killing effect in concentrations up to 1 mM. However, when SOD (300 U/ml) was added simultaneously with SIN-1 to Giardia, a significant trophozoite-killing effect was observed (approximately 35% dead trophozoites at 1 mM). The mixtures of SNAP or SNP with superoxide anion, which yields peroxynitrite, abolished the trophozoite killing induced by NO donors. Authentic peroxynitrite only killed trophozoites at very high concentrations (3 mM). These results indicate that NO accounts for Giardia trophozoites killing and this effect is not mediated by peroxynitrite.

In vitro reduction of human blood kininogen by catecholamines.

Plasma kininogen levels were significantly reduced in normal human blood, but not in cell-free human plasma, following 10 min in vitro exposure to, in order of decreasing effectiveness, 6 microM adrenaline, noradrenaline or isopropyl-noradrenaline. Phenoxybenzamine (0.1 mM), an alpha-receptor blocking drug, and 0.5 mM aspirin, an inhibitor of prostaglandin (PG) synthesis, inhibited the action of adrenaline, whereas 0.1 mM propranolol, a beta-receptor blocker, and 0.5 mM indomethacin, another inhibitor of the formation of PG, failed to do so. The results suggest that catecholamines are able to activate cell-mediated activation of the kallikrein system in human blood and that this process can be inhibited by aspirin.

Polymer films with surfaces unmodified and modified by non-thermal plasma as new substrates for cell adhesion.

The surface properties of biomaterials, such as wettability, polar group distribution, and topography, play important roles in the behavior of cell adhesion and proliferation. Gaseous plasma discharges are among the most common means to modify the surface of a polymer without affecting its properties. Herein, we describe the surface modification of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) films using atmospheric pressure plasma processing through exposure to a dielectric barrier discharge (DBD). After treatment the film surface showed significant changes from hydrophobic to hydrophilic as the water contact angle decreasing from 95° to 37°. All plasma-treated films developed more hydrophilic surfaces compared to untreated films, although the reasons for the change in the surface properties of PS and PMMA differed, that is, the PS showed chemical changes and in the case of PMMA they were topographical. Excellent adhesion and cell proliferation were observed in all films. In vitro studies employing flow cytometry showed that the proliferation of L929 cells was higher in the film formed by a 1:1 mixture of PS/PMMA, which is consistent with the results of a previous study. These findings suggest better adhesion of L929 onto the 1:1 PS/PMMA modified film, indicating that this system is a new candidate biomaterial for tissue engineering.

Heme modulates smooth muscle cell proliferation and migration via NADPH oxidase: a counter-regulatory role for heme oxygenase system.

Accumulation of vascular smooth muscle cells (VSMC) in response to inflammatory stimuli is a key event in atherogenesis, which commonly occurs in sinuous vessels with turbulent blood flow what leads to hemolysis and consequent free heme accumulation, a known pro-oxidant and pro-inflammatory molecule. In this work, we investigated the effects of free heme on VSMC, and the molecular mechanisms underlying this process. Free heme induces a concentration-dependent migration and proliferation of VSMC which depends on the production of reactive oxygen species (ROS) derived from NADPH oxidase (NADPHox) activity. Additionally, heme activates redox-sensitive proliferation-related signaling routes, such as mitogen activated protein kinase (MAPK) and NF-κB, and induces heme oxygenase-1 (HO-1) expression. NADPHox-dependent proliferative effect of heme seems to be endogenously modulated by HO since the pretreatment of VSMC with HO inhibitors potentiates heme-induced proliferation and, in parallel, increases ROS production. These effects were no longer observed in the presence of heme metabolites, carbon monoxide and biliverdin. The data indicate that VSMC proliferation induced by heme is endogenously modulated by a critical counter-regulatory crosstalk between NADPHox and HO systems.

Early potassium channel blockade improves sepsis-induced organ damage and cardiovascular dysfunction.

BACKGROUND AND PURPOSE: There is increasing evidence that potassium channels are involved in the cardiovascular dysfunction of sepsis. This evidence was obtained after the systemic inflammation, cardiovascular dysfunction and organ damage had developed. Here we have studied the consequences of early interference with potassium channels on development of sepsis. EXPERIMENTAL APPROACH: Sepsis was induced by caecal ligation and puncture (CLP) or sham surgery in Wistar rats. Four hours after surgery, animals received tetraethylammonium (TEA; a non-selective potassium channel blocker) or glibenclamide (a selective ATP-sensitive potassium channel blocker). Twenty-four hours after surgery, inflammatory, biochemical, haemodynamic parameters and survival were evaluated. KEY RESULTS: Sepsis significantly increased plasma NO(x) levels, expression of inducible nitric oxide synthase (NOS-2) protein in lung and thigh skeletal muscle, lung myeloperoxidase, urea, creatinine and lactate levels, TNF-α and IL-1ß, hypotension and hyporesponsiveness to phenylephrine and hyperglycemia followed by hypoglycemia. TEA injected 4 h after surgery attenuated the increased NOS-2 expression, reduced plasma NO(x) , lung myeloperoxidase activity, levels of TNF-α and IL-1ß, urea, creatinine and lactate levels, prevented development of hypotension and hyporesponsiveness to phenylephrine, the alterations in plasma glucose and reduced late mortality by 50%. Glibenclamide did not improve any of the measured parameters and increased mortality rate, probably due to worsening the hypoglycemic phase of sepsis. CONCLUSIONS AND IMPLICATIONS: Early blockade of TEA-sensitive (but not the ATP-sensitive subtype) potassium channels reduced organ damage and mortality in experimental sepsis. This beneficial effect seems to be, at least in part, due to reduction in NOS-2 expression.