Biomedical subjects

E Gilad

Publications and source records attributed to E Gilad.

At least 19 recordsLinked to original sources

PubMed2006

A mathematical model of plants as ecosystem engineers.

Understanding the structure and dynamics of plant communities in water-limited systems often calls for the identification of ecosystem engineers--key species that modify the landscape, redistribute resources and facilitate the growth of other species. Shrubs are excellent examples; they self-organize to form patterns of mesic patches which provide habitats for herbaceous species. In this paper we present a mathematical model for studying ecosystem engineering by woody plant species in drylands. The model captures various feedbacks between biomass and water including water uptake by plants' roots and increased water infiltration at vegetation patches. Both the uptake and the infiltration feedbacks act as mechanisms for vegetation pattern formation, but have opposite effects on the water resource; the former depletes the soil-water content under a vegetation patch, whereas the latter acts to increase it. Varying the relative strength of the two feedbacks we find a trade-off between the engineering capacity of a plant species and its resilience to disturbances. We further identify two basic soil-water distributions associated with engineering at the single patch level, hump-shaped and ring-shaped, and discuss the niches they form for herbaceous species. Finally, we study how pattern transitions at the landscape level feedback to the single patch level by affecting engineering strength.

Biomass↗

PubMed2006

Lipopolysaccharide hyporesponsiveness as a risk factor for intensive care unit hospitalization in infants with respiratory syncitial virus bronchiolitis.

Factors such as genetic heterogeneity in the immune response contribute to respiratory syncytial virus (RSV) bronchiolitis severity. Such heterogeneity may manifest by an aberrant proliferation of phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) in response to lipopolysaccharide (LPS). The proliferation of PBMC was analysed in 52 infants: 21 ambulatory infants with mild RSV bronchiolitis (group I), 26 hospitalized infants with RSV bronchiolitis on ward (group II) and five intensive care unit (ICU) hospitalized infants (group III). Proliferation was analysed in response to negative control, PHA (LPS) and LPS/PHA. The TLR4 mutations were genotyped using reverse-transcriptase-polymerase chain reaction. The optical density (OD) post-LPS/PHA of group II (1.27 +/- 0.63) was significantly higher than group II (0.65 +/- 0.38, P = 0.005) or group I (0.63 +/- 0.33, P = 0.003), suggesting hyporesponsiveness to the LPS attenuation effect. None of the ICU hospitalized infants demonstrated OD readings post-LPS/PHA under the 0.75 threshold as opposed to group I (67% under 0.75) and group II (69%) (P < 0.05). The responses to negative-control, LPS and PHA stimulation alone were similar across groups. The presence of TLR4 mutations (Asp299Gly and Thr399Ile) were associated with severe RSV bronchiolitis and were significantly over-represented in groups II and III. These findings suggest that impairments of PBMC function manifested by hyporesponsiveness to LPS as well as the presence of TLR4 mutations are associated with an increased risk for more severe RSV bronchiolitis in previously healthy infants. A certain threshold of LPS hyporesponsiveness may have a very high negative predictive value for ICU hospitalization, even better than the determination of known TLR4 mutations for this purpose.

Ambulatory Care↗

PubMed2005

Fulminant neurological deterioration in a neonate with Leigh syndrome due to a maternally transmitted missense mutation in the mitochondrial ND3 gene.

Leigh syndrome can result from both nuclear and mitochondrial DNA defects. Mutations in complex V genes of the respiratory chain were considered until recently as the most frequent cause for mitochondrial inherited Leigh syndrome, while gene defects in complex I were related to recessive Leigh syndrome. Recently few reports of mutations in the mitochondrial-encoded complex I subunit genes causing Leigh syndrome have been reported. We describe a 1-month-old baby who acutely deteriorated, with abrupt onset of brainstem dysfunction, due to basal ganglia lesions extending to the brainstem. A muscle biopsy demonstrated complex I deficiency. Subsequent analysis of the mitochondrial genome revealed a homoplastic T10191C mutation in the ND3 gene (in blood and muscle), resulting in a substitution of serine to proline. Hair root analysis revealed a 50% mutant load, reflecting heteroplasmy in early embryonic stages. The mutation was also detected in his mother (5%). Western blot analysis revealed a decrease of the 20 kDa subunit (likely ND6) and of the 30 kDa subunit (NDUFA9), which is probably due to instability attributed to the inability to form subcomplexes with ND3. This is the first description of infantile Leigh syndrome due to a maternally transmitted T10191C substitution in ND3 and not due to a de novo mutation. This mutation is age and tissue dependent and therefore may not be amenable to prenatal testing.

DNA, Mitochondrial↗

PubMed2004

Ecosystem engineers: from pattern formation to habitat creation.

Habitat and species richness in drylands are affected by the dynamics of a few key species, termed "ecosystem engineers." These species modulate the landscape and redistribute the water resources so as to allow the introduction of other species. A mathematical model is developed for a pair of ecosystem engineers commonly found in drylands: plants forming vegetation patterns and cyanobacteria forming soil crusts. The model highlights conditions for habitat creation and for high habitat richness, and suggests a novel mechanism for species loss events as a result of environmental changes.

Biophysics↗

PubMed2002

Reversible fulminant lactic acidosis and liver failure in an infant with hepatic cytochrome-c oxidase deficiency.

Cytochrome-c oxidase (COX) is the most common respiratory chain complex involved in liver failure, either as a single enzyme deficiency or as part of multiple enzyme deficiencies. We describe an infant who presented with fulminant lactic acidosis in the neonatal period. The lactic acidosis resolved spontaneously but liver and pancreatic insufficiency ensued. Isolated cytochrome-c oxidase deficiency was found in liver but not in muscle and fibroblasts. mtDNA rearrangements or depletion were ruled out. By the age of one year, liver and pancreatic functions have normalized completely and neurodevelopment is normal.

Acidosis, Lactic↗

PubMed2001

Save a child's heart: we can and we should.

BACKGROUND: Congenital heart disease (CHD) causes the death of thousands of children in developing countries. At the Wolfson Medical Center (WMC), a prototype program has been developed to address this issue. METHODS: Since 1996, indigent children have been referred to the program, with the cooperation of partners in developing countries. The project's aims are to (a) train their medical personnel at WMC, (b) travel to participating countries to teach, evaluate patients, operate, and promote the development of local centers, and (c) treat children with CHD, at WMC, who lack a local option for care either due to prohibitive costs or unavailability. The project's personnel are state employees who volunteer to treat additional patients within the framework of their salaries, and community volunteers. RESULTS: The program has seven partner sites in six countries, including two provinces in China (Hebei and Gansu), Ethiopia, Moldova, Nigeria, the Palestinian Authority, and Tanzania. Five physicians and 10 nurses have been trained from five participating countries. Over the past 4 years, 11 teaching trips have been made abroad, and operations have been performed at four partner sites. A total of 386 patients have been operated on-360 at WMC and 26 at other sites. There have been 17 (4.3%) acute deaths. Follow-up is 92% complete with 3 late deaths reported. CONCLUSIONS: Hospital-based regional centers can be created to promote the care of children with CHD in developing countries. Good results and follow-up care can be provided with appropriate planning.

Child↗

PubMed2000

Is nebulized aerosol treatment necessary in the pediatric emergency department?

BACKGROUND: Infants and small children admitted to the pediatric emergency department (PED) with acute wheezing episodes (AWE) are currently treated with nebulized wet aerosol (NWA). OBJECTIVE: To determine the efficacy of MDI with Nebuchamber (Astra AB; Lund, Sweden), a nonelectrostatic spacer device (NESD), as compared to NWA in the treatment of an unselected population of babies and small children with AWE. DESIGN: Randomized, double-blind, placebo-controlled trial. Forty-two children referred to the PED (median age +/- SD, 16 +/- 15 months) with AWE received either placebo MDI through a NESD (four puffs) and salbutamol 0.5 mL (2.5 mg) as a NWA (group I, n = 19), or salbutamol MDI and 0.5 mL of saline solution administered in the same manner as above (group II, n = 23). This treatment was repeated three times every 20 min. RESULTS: The respiratory rates (RRs) at baseline were as follows: group I, 45 +/- 11.2 breaths/min; and group II, 52.3 +/- 11.3 breaths/min (p = not significant [NS]). After the first, second, and third interventions, the percent fall from baseline of the RR were as follows: group I, 8.9, 13.1, and 17.9%, respectively; group II, 8. 6, 14.6, and 18.6%, respectively. There was no significant difference at any time in the results between the two groups. The clinical scores (CSs) at baseline were as follows: group I, 6.6 +/- 1.3; group II, 6.8 +/- 1.49 (p = NS). After the first, second, and third interventions, the percent fall from baseline of the CS were as follows: group I, 9.1, 17.9, and 23.2%, respectively; group II, 8. 6, 18.9, and 24.7%, respectively. These results, also, did not differ significantly at any time between the two groups. Hospitalization rate and side effects did not differ between the two groups. CONCLUSIONS: We conclude that even in the group of unselected very young children (mean age < 2 years) with AWE, the use of MDI with NESD is at least as effective as the use of NWA. As opposed to data from an adult population, no plateau was reached in the dose-response curve using the above doses over time.

Administration, Inhalation↗

PubMed1999

Endotracheal tolazoline: pharmacokinetics and pharmacodynamics in dogs.

Tolazoline is a potent vasodilator of both arteries and veins and has a powerful effect on the pulmonary vasculature, reducing hypoxic pulmonary vasoconstriction and lowering pulmonary artery pressure. Intravenous tolazoline lowers the mean pulmonary arterial pressure and resistance and increases the cardiac index when given to infants with persistent pulmonary hypertension of the newborn (PPHN). Endotracheally administered tolazoline decreases mean pulmonary arterial pressure and pulmonary vascular resistance, and improves oxygenation without the harmful decline in systemic arterial pressure. The purpose of our study was to examine the pharmacokinetic and pharmacodynamic characteristics of endotracheal tolazoline in order to determine the relationship between endotracheal tolazoline administration, plasma concentration and its effects on the cardiovascular and respiratory systems. Tolazoline was administered endotracheally to 7 newborn dogs, and its serum concentration and the haemodynamic parameters were monitored for 270 min post-delivery. Results are expressed as median and quartiles. It was found that 15 s after dosing, tolazoline plasma concentrations started to increase significantly above baseline levels, reaching a maximum of 2.64 (1.36; 13.16) microg/ml. The extent of tolazoline absorption was 305 (148;453) microg/ min/ml. The volume of distribution was 3.4 (1.6;7.4) 1/kg. The total body clearance was 12.1 (10.9;23.9) ml/min/kg and the elimination half-life was 225 (171;303) min. Endotracheal tolazoline produced an initial short-lived decrease in mean blood pressure in all the dogs, but thereafter the blood pressure increased gradually above baseline levels. Immediately following endotracheal tolazoline significant tachycardia developed, peaking at 90 min. Subsequently, the heart rate gradually decreased and stabilized at values above baseline for 200 min. A single endotracheal dose of tolazoline is effectively absorbed and produces measurable pharmacological effects. Determining the optimal endotracheal dose of tolazoline in the clinical setting requires additional evaluation.

Animals↗

PubMed1999

Melatonin receptors in PC3 human prostate tumor cells.

Melatonin, secreted nocturnally by the pineal gland, can bind to human benign prostate epithelial cells and attenuate their growth and viability. In the present study, melatonin binding and responses were explored in the human steroid-independent PC3 prostatic tumor cells. PC3 cells bound 125I-melatonin with low affinity (Kd ca. 0.9 nM) at high as well as low cell density. Melatonin enhanced cGMP and 3H-thymidine incorporation at low, but attenuated them at high cell density. In addition, melatonin inhibited cAMP at low, but augmented it at high cell density. These effects were associated with an increase in cell count at low- but not high-density cultures. Pertussis toxin treatment suppressed 125I-melatonin binding and ablated all the effects of melatonin on 3H-thymidine incorporation, cAMP, and cGMP at both cell densities. Cholera toxin treatment failed to block the effects of melatonin on 3H-thymidine incorporation, but prevented the modulation by melatonin of cAMP at low and cGMP at high cell density. The cGMP analog 8-Br-cGMP, inhibited melatonin's effects on 3H-thymidine incorporation at both cell densities. H89, a protein kinase A inhibitor, prevented melatonin's effects on 3H-thymidine incorporation at low but not high cell density. These results provide the first demonstration of direct interaction of melatonin with hormone-insensitive prostate tumor cells. The melatonin receptors in the PC3 cells are coupled to pertussis toxin-sensitive G proteins to induce cell density-dependent changes in cGMP, cAMP, and cell growth.

Binding Sites↗

PubMed1999

Salbutamol metered-dose inhaler with spacer for hyperkalemia: how fast? How safe?

OBJECTIVE: To determine the efficacy of inhaled salbutamol (rapidly delivered, using a metered-dose inhaler with a spacer device [MDI-S]) in lowering the serum potassium levels in patients with hyperkalemia. DESIGN: A randomized, double-blind, placebo-controlled trial. PATIENTS: Seventeen chronic renal failure patients referred to the Nephrology Unit between October 1, 1997 and March 31, 1998 for hemodialysis were randomized. INTERVENTION AND RESULTS: Group 1 received salbutamol followed by a placebo. Group 2 received a placebo followed by salbutamol. Each patient inhaled 1,200 microg salbutamol or a placebo through an MDI-S within 2 min. Blood samples were obtained repeatedly before inhalation and after 1, 3, 5, 10, and 60 min. The pulse rate and blood pressure were repeatedly measured. Insulin levels were examined in a subset of patients (n = 10) before, and 1 and 5 min following inhalation. Salbutamol's known side effects, palpitation, tachycardia tremor, and headache, were recorded. Potassium levels rose after 1 min following the completion of treatment and then decreased steadily thereafter. A rise of > or = 0.1 mEq/L was seen in 10 of 17 patients (59%) during the treatment period and there was no change (0%) seen during the placebo period (p < 0.0001). Within 3 min after inhalation of salbutamol, potassium levels declined as a function of time. Potassium levels in those patients taking the placebo did not change as a function of time (p < 0.001). The difference between the placebo and the salbutamol-treated periods reached significance after 5 min (p < 0.05). The serum glucose levels rose following inhalation of salbutamol, with a significant rise after 3 min. The heart rate rose significantly within the first 5 min following inhalation. Serum insulin levels remained unchanged 1 min after inhalation; however, after 5 min, a significant elevation was detected. CONCLUSION: Salbutamol inhalation of 1,200 microg, using an MDI-S, has a relatively rapid onset of action that induces a consistent reduction in serum potassium levels, starting 3 to 5 min following delivery. Unexpectedly, a paradoxical elevation was detected in serum potassium levels in the first minutes following inhalation. This effect, although minor (0.15 mEq/L above baseline), may cast some doubt on the role of salbutamol inhalation as the first treatment for excessive hyperkalemia.

Administration, Inhalation↗

PubMed1998

Melatonin receptors in benign prostate epithelial cells: evidence for the involvement of cholera and pertussis toxins-sensitive G proteins in their signal transduction pathways.

BACKGROUND: Melatonin, the hormone secreted nocturnally by the pineal gland, binds to epithelial cells from the human benign prostate, and can reduce their growth and viability. The possible involvement of GTP binding proteins cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in melatonin responses in these cells were investigated. METHODS: The effects of melatonin on cAMP and cGMP were assessed in prostate cells untreated or pretreated with pertussis toxin (PTX) or cholera toxin (CTX). RESULTS: Melatonin augmented cAMP but reduced cGMP in the epithelial cells (maximal responses at 10 nM). The increase in cAMP was attenuated by PTX, but not by CTX, whereas the decrease in cGMP was attenuated by CTX, but not by PTX. CTX, but not PTX, abolished the melatonin-mediated suppression of 3H-thymidine incorporation. In addition, melatonin facilitated the CTX- and PTX-mediated ADP ribosylation of 44- and 41-kilodalton proteins, respectively. The cGMP analogue 8-bromo-cGMP, negated the melatonin-mediated decrease in 3H-thymidine incorporation, whereas H89, a protein kinase A inhibitor, did not inhibit melatonin's effect. CONCLUSIONS: Melatonin receptors in the human benign prostate epithelial cells enhance cAMP and inhibit cGMP through PTX- and CTX-sensitive G proteins, respectively. The decrease in DNA synthesis may be secondary to the melatonin-mediated decrease in cGMP.

Adenosine Diphosphate Ribose↗

PubMed1998

Protective effects of 3-aminobenzamide, an inhibitor of poly (ADP-ribose) synthase in a carrageenan-induced model of local inflammation.

A cytotoxic cycle triggered by oxidant-induced DNA single strand breakage and subsequent activation of the nuclear enzyme poly (ADP-ribose) synthetase have been shown to contribute to the cellular injury during various forms of oxidant stress in vitro. The aim of the present study was to investigate the role of poly (ADP-ribose) synthetase in a model of acute local inflammation (carrageenan-induced pleurisy), where oxyradicals, nitric oxide and peroxynitrite are known to play a crucial role in the inflammatory process. The results show that the poly (ADP-ribose) synthetase inhibitor 3-aminobenzamide (given at 1-30 mg/kg) inhibits the inflammatory response (pleural exudate formation, mononuclear cell infiltration, histological injury). Moreover, 3-aminobenzamide reduces the formation of nitrotyrosine, an indicator of the formation of peroxynitrite, in the lung. The present results demonstrate that 3-aminobenzamide, presumably by inhibition of poly (ADP-ribose) synthetase, exerts potent anti-inflammatory effects. Part of the anti-inflammatory effects of 3-aminobenzamide may be related to a reduction of neutrophil recruitment into the inflammatory site.

Animals↗

PubMed1998

Melatonin inhibits expression of the inducible isoform of nitric oxide synthase in murine macrophages: role of inhibition of NFkappaB activation.

The role of melatonin as an immunomodulator is well established. Recent reports showed that melatonin exerts protective effects in septic and hemorrhagic shock and in inflammation. The expression of the inducible isoform of nitric oxide synthase (iNOS) makes an important contribution to the pathophysiology of shock and inflammation. We studied, in cultured murine macrophages, the role of melatonin in the regulation of the expression of iNOS and defined the mode of melatonin's action. Our results show that melatonin, at 1 microM-1 mM, decreased the production of nitrite/nitrate (the breakdown products of NO) as well as the production of 6-keto-prostaglandin F1alpha (the major stable breakdown product of prostacyclin) in macrophages stimulated with bacterial lipopolysaccharide (10 microg/ml). We observed that melatonin reduces iNOS steady-state mRNA levels and iNOS protein expression in the same concentration range (1 microM-1 mM). Melatonin, up to 10 mM, exerted only a slight direct inhibitory effect on iNOS activity. Using iNOS promoter-luciferase constructs, we found that melatonin inhibits iNOS promoter activation. Inhibition of iNOS expression was associated with inhibition of activation of the transcription factor nuclear factor kappa B (NFkappaB). We conclude that melatonin inhibits NO production in immunostimulated macrophages mainly by inhibiting the expression of iNOS. This is due to inhibition of iNOS transcription, in part through inhibition of NFkappaB activation. Inhibition of iNOS-derived NO production by melatonin may contribute to the anti-inflammatory effects of this pineal secretory product.

Animals↗

PubMed1998

Evidence for a local action of melatonin on the rat prostate.

PURPOSE: Melatonin, secreted by the pineal gland at night, inhibits pubertal development in rats and possibly humans. We have recently found functional specific binding sites for 125I-labeled melatonin (125I-melatonin) in human benign prostate tissue, localized in the microsomal fraction of the glandular epithelium. The aim of the present study was to set up an animal (rodent) model for the growth inhibitory effects of melatonin on the prostate. MATERIALS AND METHODS: Putative melatonin in the rat ventral prostate were explored by means of autoradiography and receptor binding assays and the ability of melatonin to inhibit stimulated prostate growth was tested in vivo. RESULTS: In vitro autoradiography and equilibrium binding experiments demonstrated specific binding sites for 125I-labeled melatonin (125I-melatonin) associated with the microsomal fraction of the rat ventral prostate cells (apparent dissociation constant 0.9 nM). 125I-melatonin binding was inhibited by 2-iodomelatonin > 6-hydroxy-melatonin > melatonin = N-(2,4 dinitrophenyl)-5-methoxytryptamine whereas similar concentrations of serotonin, 5-methoxytryptamine, and tryptamine were less potent. The guanine nucleotide analogs, guanosine 5'-0-[3-thiotriphosphate] and guanosine 5'-0-[2-thio-diphosphate], inhibited specific 125I-melatonin binding whereas 5'-guanylyl imido-diphosphate was less potent. Daily injections of testosterone to castrated rats induced regrowth of the prostate and increased the weight of the seminal vesicles. Administration of melatonin to the rats through drinking water prevented the testosterone-mediated regrowth of the prostate but had no effect on the seminal vesicles' weight. CONCLUSIONS: The results demonstrate putative melatonin receptors in the rat prostate and suggest a direct suppression by melatonin of testosterone-dependent prostate growth.

Animals↗

PubMed1997

Putative melatonin receptors in the blind mole rat harderian gland.

The blind mole rat (Spalax ehrenbergi) displays daily and seasonal rhythms. Melatonin, secreted nocturnally by the pineal gland, is also produced in the harderian gland and affects its morphology in rodents. We report here on the presence of putative melatonin receptors in the blind mole rat harderian gland, located in the microsome-enriched fraction of the cells. Equilibrium 125I-melatonin binding studies indicated high- and low-affinity melatonin binding sites in the female (apparent Kd 10 pM and 2.4 nM, respectively) and low-affinity sites in the male (apparent Kd 2.6 nM) mole rat. The binding sites were not significantly affected by season. Castration increased the density of high-affinity binding sites in males and low-affinity binding in females. 125I-melatonin binding to the gonadectomized mole rat preparation was inhibited by serotonin > 2-iodomelatonin > or = memelatonin > 5-methoxytryptamine. The guanine nucleotide analogs, guanosine 5'-O-[3-thio-triphosphate] and guanosine 5'-O-[2-thio-diphosphate], inhibited specific 125I-melatonin binding, whereas 5'-guanylyl imido-diphosphate was less potent. These results indicate for the first time the presence of GTP-sensitive melatonin binding sites in the blind mole rat harderian gland, and suggest that their expression is under control of sex steroids.

Animals↗

PubMed1997

Protection by inhibition of poly (ADP-ribose) synthetase against oxidant injury in cardiac myoblasts In vitro.

Peroxynitrite and hydroxyl radical are reactive oxidants produced during myocardial reperfusion injury. In various cell types, including macrophages and smooth muscle cells, peroxynitrite and hydrogen peroxide cause DNA single strand breakage, which triggers the activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS), resulting in cytotoxicity. Using 3-aminobenzamide and nicotinamide, inhibitors of PARS, we investigated the role of PARS in the pathogenesis of myocardial oxidant injury in H9c2 cardiac myoblasts in vitro. Peroxynitrite (100-1000 microM), hydrogen peroxide (0.3-10 microM) and the NO donor compounds S-nitroso-N-accetyl-DL-penicillamine (SNAP) and diethyltriamine NONOate all caused a dose-dependent reduction of the mitochondrial respiration of the cells, as measured by the mitochondrial-dependent conversion of MTT to formazan. Peroxynitrite and hydrogen peroxide, but not the NO donors caused activation of cellular PARS activity. The suppression of mitochondrial respiration by peroxynitrite and hydrogen peroxide, but not by the NO donors, was ameliorated by pharmacological inhibition of PARS. The protection by the PARS inhibitors diminished at extremely high concentrations of the oxidants. Hypoxia (1 h) followed by reoxygenation (1-24 h) also resulted in a significant activation of PARS, and caused a suppression of mitochondrial respiration, which was prevented by inhibition of PARS. Similar to the results obtained with the pharmacological inhibitors of PARS, a fibroblast cell line which derives from the PARS knockout mouse was protected against the suppression of mitochondrial respiration in response to peroxynitrite and reoxygenation, but not to NO donors, when compared to the result of cells derived from wild-type animals. Based on our data, we suggest that activation of PARS plays a role in the myocardial oxidant injury.

Animals↗

PubMed1997

Melatonin is a scavenger of peroxynitrite.

Peroxynitrite is a toxic oxidant formed from the reaction of superoxide and nitric oxide under conditions of inflammation and oxidant stress. Here we demonstrate that the pineal neurohormone melatonin inhibits peroxynitrite-mediated oxidant processes. Melatonin caused a dose-dependent inhibition of the oxidation of dihydrorhodamine 123 to rhodamine in vitro. Moreover, in cultured J774 macrophages, melatonin inhibited the development of DNA single strand breakage in response to peroxynitrite and reduced the suppression of mitochondrial respiration. Thus, melatonin appears to be a scavenger of peroxynitrite. This action may contribute to the antioxidant and antiinflammatory effects of melatonin in various pathophysiological conditions.

Animals↗

PubMed1997

The inhibitory effects of mercaptoalkylguanidines on cyclo-oxygenase activity.

1. It has been proposed that in inflammatory conditions, in which both the inducible isoforms of nitric oxide synthase (iNOS) and cyclo-oxygenase (COX-2) are induced, inhibition of NOS also results in inhibition of arachidonic acid metabolism. In the present study we have investigated whether mercaptoalkylguanidines, a novel class of selective iNOS inhibitors, may also influence the activity of cyclo-oxygenase (COX). Therefore, the effect of mercaptoethylguanidine (MEG) and related compounds on the activity of the constitutive (COX-1) and the inducible COX (COX-2) was investigated in cells and in purified enzymes. Aminoguanidine, NG-methyl-L-arginine (L-NMA) and NG-nitro-L-arginine methyl ester (L-NAME) were also studied for comparative purposes. 2. Western blot analysis demonstrated a significant COX-1 activity in unstimulated J774 macrophages and in unstimulated human umbilical vein endothelial cells (HUVEC). Immunostimulation of the J774 macrophages by endotoxin (lipopolysaccharide of E. coli, LPS 10 micrograms ml-1) and interferon gamma (IFN gamma, 100 u ml-1) for 6 h resulted in a significant induction of COX-2, and a down-regulation of COX-1. No COX-2 immunoreactivity was detected in unstimulated HUVEC or unstimulated J774 cells. Therefore, in subsequent studies, the effect of mercaptoalkylguanidines on COX-1 activity was studied in HUVEC stimulated with arachidonic acid for 6 h, and in J774 cells stimulated with arachidonic acid for 30 min. The effect of mercaptoalkylguanidines on COX-2 activity was studied in immunostimulated J774 macrophages, both on prostaglandin production by endogenous sources, and on prostaglandin production in response to exogenous arachidonic acid stimulation. In addition, the effect of mercaptoalkylguanidines on purified COX-1 and COX-2 activities was also studied. 3. In experiments designed to measure COX-1 activity in HUVEC, the cells were stimulated by arachidonic acid (15 microM) for 6 h. This treatment induced a significant production of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha, the stable metabolite of prostacyclin), while nitrite production was undetectable by the Griess reaction. MEG (1 microM to 3 mM) caused a dose-dependent inhibition of the accumulation of 6-keto-PGF1 alpha, with an IC50 of 20 microM. However, aminoguanidine, L-NAME or L-NMA (up to 3 mM) did not affect the production of 6-keto-PGF1 alpha in this experimental system. In experiments designed to measure COX-1 activity in J774.2 macrophages, the cells were stimulated by arachidonic acid (15 microM) for 30 min; this also induced a significant production of 6-keto-PGF1 alpha and MEG (1 microM to 3 mM), aminoguanidine (at 1 and 3 mM), but neither L-NAME nor L-NMA inhibited the production of prostaglandins. 4. In experiments designed to measure prostaglandin production by COX-2 with endogenous arachidonic acid, J774.2 cells were immunostimulated for 6 h in the absence or presence of various inhibitors. In experiments designed to measure prostaglandin production by COX-2 with exogenous arachidonic acid, J774.2 cells were immunostimulated for 6 h, followed by a replacement of the culture medium with fresh medium containing arachidonic acid and various inhibitors. Both of these treatments induced a significant production of 6-keto-PGF1 alpha. Nitrite production, an indicator of NOS activity, was moderately increased after immunostimulation. MEG (1 microM to 3 mM) caused a dose-dependent inhibition of the accumulation of COX metabolites. Similar inhibition of LPS-stimulated 6-keto PGF1 alpha production was shown by other mercaptoalkylguanidines (such as N-methyl-mercaptoethylguanidine, N,N'-dimethyl-mercaptoethylguanidine, S-methyl-mercaptoethylguanidine and guanidino-ethyldisulphide), with IC50 values ranging between 34-55 microM. However, aminoguanidine, L-NAME and L-NMA (up to 3 mM) did not affect the production of prostaglandins.5. In comparative experiments indomethacin, a non selective COX inhibitor, and NS-398, a selective COX-2 inhibitor, reduced (LPS) stimulated 6-keto-PGF1alpha production in J774 macrophages in a dose-dependent manner without affecting nitrite release. Indomethacin, but not NS-398, inhibited 6-keto-PGF1alpha production in the HUVECs. 6.The inhibitory effect of MEG was due to direct inhibition of the catalytic activity of COX as indicated in experiments with purified COX-1 and COX-2. MEG dose-dependently inhibited the purified COX-1 and COX-2 activity with IC50 values of 33microM and 36microM, respectively. Aminoguanidine (at the highest concentrations) inhibited the formation of COX-1 metabolites, without affecting COX-2 activity. High doses of L-NAME (3mM) decreased COX-1 activity only, while L-NMA (up to 3mM) had no effect on the activity of either enzyme. 7.These results suggest that MEG and related compounds are direct inhibitors of the constitutive and the inducible cyclo-oxygenases, in addition to their effects on the inducible NOS. The additional effect of mercaptoalkylguanidines on COX activity may contribute to the beneficial effects of these agents in inflammatory conditions where both iNOS and COX-2 are expressed.

6-Ketoprostaglandin F1 alpha↗