Ethylene is differentially regulated during sugar beet germination and affects early root growth in a dose-dependent manner.

MAIN CONCLUSION: By integrating molecular, biochemical, and physiological data, ethylene biosynthesis in sugar beet was shown to be differentially regulated, affecting root elongation in a concentration-dependent manner. There is a close relation between ethylene production and seedling growth of sugar beet (Beta vulgaris L.), yet the exact function of ethylene during this early developmental stage is still unclear. While ethylene is mostly considered to be a root growth inhibitor, we found that external 1-aminocyclopropane-1-carboxylic acid (ACC) regulates root growth in sugar beet in a concentration-dependent manner: low concentrations stimulate root growth while high concentrations inhibit root growth. These results reveal that ethylene action during root elongation is strongly concentration dependent. Furthermore our detailed study of ethylene biosynthesis kinetics revealed a very strict gene regulation pattern of ACC synthase (ACS) and ACC oxidase (ACO), in which ACS is the rate liming step during sugar beet seedling development.

Why is folate effective in preventing neural tube closure defects?

Neural tube defects (NTDs) originate from a failure of the embryonic neural tube to close. The pathogenesis of NTDs is largely unknown. Fortunately, adequate maternal folate application is known to reduce the risk of human NTDs. However, why folate reduces NTDs is largely unknown. The main cause for NTDs is the disturbance of the cell growth in the neuroepithelium. Of course, rapid cell growth needs enough synthesis of nuclei acids. Interestingly, folate is used as a source for the synthesis of nucleic acids. Furthermore, glycine cleavage system (GCS) is essential for the synthesis of nucleic acids from folate, and very strongly expressed in neuroepithelial cells, suggesting that these highly proliferating cells need enough synthesis of nuclei acids and high amounts of folate. Taken together, I speculate the following hypothesis; (1) The closure of the neural tube requires rapid growth of neuroepithelial cells. (2) High rates of nuclei acids synthesis are needed for the rapid growth. (3) GCS, which is requisite in nucleic acid synthesis from folate, is expressed very strongly and functions robustly in neuroepithelial cells. (4) Pregnant women require 5-10-fold higher amounts of folate compared to non-pregnant women. (5) So, folate-deficient situations are easy to occur in neuroepithelial cells, resulting in NTDs. (6) Thus, folate is effective to prevent NTDs.

Use of fed-batch cultivation for achieving high cell densities for the pilot-scale production of a recombinant protein (phenylalanine dehydrogenase) in Escherichia coli.

A fed-batch process for the high cell density cultivation of E. coli TG1 and the production of the recombinant protein phenylalanine dehydrogenase (PheDH) was developed. A model based on Monod kinetics with overflow metabolism and incorporating acetate utilization kinetics was used to generate simulations that describe cell growth, acetate production and reconsumption, and glucose consumption during fed-batch cultivation. Using these simulations a predetermined feeding profile was elaborated that would maintain carbon-limited growth at a growth rate below the critical growth rate for acetate formation (mu < mu(crit)). Two starvation periods are incorporated into the feed profile in order to induce acetate utilization. Cell concentrations of 53 g dry cell weight (DCW)/L were obtained with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the total cell protein. The yield of PheDH was 129 U/mL with a specific activity of 1.2 U/mg DCW and a maximum product formation rate of 0.41 U/mg DCW x h. The concentration of aectate was maintained below growth inhibitory levels until 3 h before the end of the fermentation when the concentration reached a maximum of 10.7 g/L due to IPTG induction of the recombinant protein.

Inhibition of interleukin-1-alpha-induced nitric oxide synthase in vascular smooth muscle and full reversal of interleukin-1-alpha-induced hypotension by N omega-amino-L-arginine.

BACKGROUND: Interleukin-1-alpha (IL-1) is a cytokine with potentially therapeutic immunoproliferative and tumoricidal activities. Preliminary clinical studies suggest that use of IL-1 may be restricted by dose-limiting hypotension. PURPOSE: The purpose of this study was to investigate the role of nitric oxide (NO.) as a possible mediator of this hypotension. METHODS: Cytokine-treated rat aortic smooth muscle cells were assayed for nitrite production, a stable breakdown product of nitric oxide. Nitric oxide synthase from smooth muscle cells was partially characterized in cytosol preparations using a novel Fe(2+)-myoglobin method to test for nitric oxide production. To determine the role of NO. on the immunorestorative and antineoplastic activity of IL-1, N omega-amino-L-arginine (NAA) or N omega-monomethyl-L-arginine (NMA), inhibitors of nitric oxide synthase, were added to either cultures of IL-1-dependent T cells or A375 melanoma cells exposed to IL-1. To investigate the effects of NAA in vivo, pentobarbital anesthetized dogs, which were made hypotensive by administration of IL-1, received a single intravenous bolus dose of NAA. The effects of NAA were then reversed by the administration of L-arginine. RESULTS: Our results show that cultured IL-1-activated rat aortic smooth muscle cells synthesize nitric oxide, a potent vasodilator. Induction of nitric oxide synthase is augmented by interferon-gamma and blocked by IL-1 receptor antagonist and by inhibitors of RNA or protein synthesis. Nitric oxide synthesis by IL-1-activated smooth muscle cells is inhibited by NAA, NMA, and N omega-nitro-L-arginine (NNA) with ED50 (i.e., effective dose for 50% inhibition) values of 20, 60, and 1000 microM, respectively; this rank order of inhibition is characteristic of an agonist-unregulated, inducible isoform of nitric oxide synthase. In smooth muscle cells, inhibition of NO. synthesis by NAA is reversed by excess L-arginine. Consistent with the induction of unregulated NO. synthesis in vascular smooth muscle in vivo, administration of IL-1 (50 micrograms/kg) to dogs caused a 33.5% decrease in systemic vascular resistance and a 28% decrease in blood pressure within 3 hours. Subsequent administration of NAA (20 mg/kg) rapidly and completely reversed the hypotension and increased systemic vascular resistance; these effects of NAA were reversed by L-arginine. Neither the immunoproliferative nor the tumoricidal activity of IL-1 was diminished by NAA. CONCLUSIONS: Our results indicate that (a) vascular smooth muscle is a likely source as well as a target of IL-1-induced NO. synthesis, causing vasodilatation and hypotension, (b) nitric oxide synthase inhibitors can fully reverse this hypotension, and (c) the therapeutically useful properties of IL-1 are not diminished by nitric oxide synthase inhibitors. IMPLICATIONS: Administration of inhibitors of nitric oxide synthase can reverse the pathological cardiovascular effects of IL-1 at concentrations that do not interfere with the potentially useful immunoproliferative or tumoricidal effects of this cytokine. In the context of the current clinical trials of IL-1, this finding would represent a very significant advantage.

Distinct patterns of nitric oxide production in hepatic macrophages and endothelial cells following acute exposure of rats to endotoxin.

Hepatic macrophages and endothelial cells play an important role in the clearance of endotoxin from the portal circulation. These cells are activated by endotoxin to release reactive mediators including superoxide anion, hydrogen peroxide, and nitric oxide, which have been implicated in hepatic inflammation and tissue injury. In the present studies we analyzed mechanisms regulating the production of nitric oxide by hepatic macrophages and endothelial cells following in vivo exposure to endotoxin. Rats were injected intravenously with Escherichia coli lipopolysaccharide (LPS, 5 mg/kg). Cells were isolated from the animals 48 h later by in situ perfusion of the liver with collagenase and pronase followed by differential centrifugation and centrifugal elutriation. We found that macrophages and endothelial cells from both untreated and endotoxin-treated rats readily synthesized nitric oxide following in vitro stimulation with interferon-gamma (IFN-gamma) and LPS alone and in combination. This response was dependent on l-arginine and was blocked by two nitric oxide synthase inhibitors, NG-monomethyl-l-arginine and l-canavanine. Macrophages produced more nitric oxide in response to LPS or LPS plus IFN-gamma than endothelial cells. In addition, nitric oxide production by both cell types in response to LPS plus IFN-gamma was increased after treatment of rats with endotoxin. Macrophages appeared to be more sensitive than endothelial cells to the in vivo effects of this inflammatory stimulus. Northern and Western blot analysis demonstrated that nitric oxide production by macrophages and endothelial cells in response to LPS plus IFN-gamma was due to increased expression of an inducible form of nitric oxide synthase (iNOS) mRNA and protein. Using fluorescence image analysis, iNOS protein was found to be localized in the cytoplasm of the cells. Treatment of rats with endotoxin was associated with increased expression of iNOS protein in the macrophages. The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also stimulated nitric oxide production by macrophages and endothelial cells from endotoxin-treated rats, although not as effectively as LPS and IFN-gamma. Macrophages were more responsive than endothelial cells to TPA. Furthermore, depletion of the cells of glutathione using buthionine sulfoximine had no major effect on nitric oxide production by macrophages but resulted in small but significant inhibition in endothelial cells. This suggests that this sulfhydryl-containing tripeptide does not regulate intracellular levels of reactive nitrogen intermediates in activated macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)

Stimulation of human nitric oxide synthase by tetrahydrobiopterin and selective binding of the cofactor.

To check the stimulatory potency of the tetrahydro forms of the two major pteridines occurring in human tissues, neopterin and biopterin, NO synthase was purified 6000-fold from human cerebellum. Tetrahydrobiopterin stimulated the activity up to 4.5-fold in a concentration dependent manner with a maximum above 1 microM, whereas tetrahydroneopterin was completely inactive in concentrations up to 100 microM. Tetrahydrobiopterin, but not neopterin derivatives, were copurified with the NO synthase activity. Our results demonstrate that human cerebellum contains a tetrahydrobiopterin dependent NO synthase activity.

Characterization of nitric oxide synthases in non-adrenergic non-cholinergic nerve containing tissue from the rat anococcygeus muscle.

Tissue homogenates prepared from rat anococcygeus muscle converted L-arginine to L-citrulline indicating the presence of nitric oxide (NO) synthase. NO synthase activity was also found in crude and partially-purified soluble and particulate fractions prepared from the homogenates. Both soluble and particulate NO synthase were dependent on NADPH, 5,6,7,8-tetrahydrobiopterin and calcium, and inhibited by NG-nitro-L-arginine. Tissue homogenates or crude cytosolic and membrane fractions from rat vas deferens, which does not contain NO releasing non-adrenergic non-cholinergic neurones, had no NO synthase activity.

Relaxation of rat thoracic aorta induced by the Ca(2+)-ATPase inhibitor, cyclopiazonic acid, possibly through nitric oxide formation.

1 The effect of the Ca(2+)-ATPase inhibitor, cyclopiazonic acid (CPA), was studied on rat thoracic aortic ring preparations. 2 At concentrations above 0.3 microM, CPA induced relaxation in the arteries precontracted with phenylephrine. Removal of the endothelium abolished CPA-induced relaxation. 3 The nitric oxide (NO) synthase inhibitor NG-nitro L-arginine (3-300 microM), the free radical scavenger haemoglobin (0.1-3 microM), the soluble guanylate cyclase inhibitor, LY83583 (0.1-10 microM), each inhibited the endothelium-dependent relaxation to CPA. The potassium channel blocker, glibenclamide (10 microM) and cyclo-oxygenase inhibitor, indomethacin (100 microM for 60 min and then washed out) did not alter the action of CPA. 4 The calmodulin inhibitors calmidazolium (3-10 microM) and W-7 (100 microM) also abolished CPA-induced relaxation. 5 CPA (10 microM) increased guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels in arteries with an intact endothelium, without affecting adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. 6 The inhibitors of NO synthesis and actions, the calmodulin inhibitor and removal of the endothelium abolished the CPA-stimulated increase in the levels of cyclic GMP. 7 In Ca(2+)-free solution, CPA failed to induce relaxation or to stimulate cyclic GMP production. Relaxation to nitroprusside was not affected under these conditions. 8 These results suggest that CPA can stimulate NO synthesis, possibly by inhibiting a Ca(2+)-ATPase, which replenishes Ca2+ in the intracellular storage sites in endothelial cells. Depletion of the Ca2+ store in the endothelium may then trigger influx of extracellular Ca2+, contributing to an increase in free Ca2+ in the endothelial cells, which activates NO synthase and NO formation.

Glibenclamide-induced inhibition of the expression of inducible nitric oxide synthase in cultured macrophages and in the anaesthetized rat.

1. We have investigated whether glibenclamide, an inhibitor of ATP-sensitive potassium channels, influences the induction of the calcium-independent isoform of nitric oxide synthase (iNOS) in cultured J774.2 macrophages activated by bacterial endotoxin (E.coli lipopolysaccharide; LPS), as well as in the lung and aorta of rats with endotoxic shock. 2. Pretreatment of J774.2 macrophages with glibenclamide (10(-7) to 10(-5) M for 30 min) dose-dependently inhibited the accumulation of nitrite caused by LPS (1 microgram ml-1). In contrast, pretreatment of macrophages with tetraethylammonium (10(-4) to 10(-2) M for 30 min), a non-selective inhibitor of potassium channels, did not affect the rise in nitrite caused by LPS. At the highest concentration (10(-5) M) used, cromakalim, an opener of ATP-sensitive potassium channels, caused a small, but significant inhibition of nitrite formation in macrophages activated with LPS, while lower concentrations (10(-7) to 3 x 10(-6) M) were without effect. 3. The inhibition by glibenclamide (3 microM) of the increase in nitrite induced by LPS in J774.2 macrophages was weaker when glibenclamide was given several hours after LPS, indicating that glibenclamide inhibits the induction, but not the activity, of iNOS. In contrast, the degree of inhibition of nitrite formation caused by the nitric oxide synthase (NOS) inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) was similar when this agent was given up to 10 h after LPS. 4.In anaesthetized rats, LPS caused a fall in mean arterial blood pressure (MAP) from 120 +/-(time 0)to 98 +/- mmHg at 180 min (P<0.05, n = 6). Treatment of LPS-rats with glibenclamide (1 mg kg-1, i.v.at 60 min after LPS) caused a rapid and sustained rise in MAP (e.g. MAP at 180 min after LPS:122 +/-4 mmHg; n =6, P <0.05 when compared to LPS-rats). The maximum of the rise in MAP produced by glibenclamide (1 mg kg-1 , i.v.) was similar when the drug was given either at 60 or 180 min after LPS. However, the duration of the pressor response was significantly longer when glibenclamide was given at 60 min, rather than at 180 min after LPS.5. LPS-treatment caused a significant reduction of the pressor responses elicited by noradrenaline (NA,1 microg kg-1, i.v.) from 35 +/- 2 to 19 +/- 1 mmHg at 60 min and 20 +/- 2 mmHg at 180 min (P<0.05).Treatment of LPS-rats with glibenclamide (1 mg kg-1, i.v. at 60 min) caused a significant restoration of the pressor responses elicited by NA from 19 +/- 1 mmHg at 60 min (prior to glibenclamide injection) to 29 +/- 3 mmHg at 180 min (P<0.05).6. Endotoxaemia for 180 min resulted in a significant increase in a calcium-independent NOS activity(which was taken to represent iNOS activity) in the lung from 0.17 +/- 0.1 (control, n =4) to 6.21 +/- 0.48 pmol mg-1 min-1 (n =6, P<0.05). Injection of glibenclamide (1 mg kg-1, i.v.) at 60 min after LPS attenuated the increase in iNOS activity caused by endotoxaemia in the lung by 43 +/- 7%(n = 6, P <0.05). In contrast, injection of glibenclamide at 180 min after LPS did not result in a significant inhibition of iNOS activity (n = 6, P <0.05. 7. Thoracic aortae obtained from rats at 180 min after LPS showed a significant reduction in the contractions elicited by noradrenaline (NA, 10-9 to 10-6 M). Treatment of LPS-rats with glibenclamide(1 mg kg-1, i.v. at 60 min after LPS) significantly alleviated this LPS-induced hyporeactivity to NA ex vivo. In contrast, when aortic rings from LPS-rats were incubated in vitro with glibenclamide (10 microM for 20 min), glibenclamide did not reverse the vascular hyporeactivity to NA. However, L-NAME (300 microM for 20 min) significantly enhanced the contractile response to NA in aortic rings obtained from LPS-rats(P<0.05, n=6).8. No significant amounts of tumour necrosis factor-alpha (TNF alpha) were detectable in the plasma before the injection of LPS. Endotoxaemia for 90 min resulted in a significant rise in plasma TNFalpha levels(0.05 +/- 0.05 ng ml-1 at time 0, 3.78 +/- 0.24 ng ml-1 at 90 min, n = 6, P < 0.05). Treatment of LPS-rats with glibenclamide (1 mg kg-1, i.v. at 15 min prior to LPS, n = 5) did not significantly reduce the rise in plasma TNF alpha levels caused by endotoxin.9. Thus, glibenclamide inhibits the induction, but not the activity, of iNOS in vitro and in vivo. This inhibition of iNOS induction may contribute to the beneficial haemodynamic effects of glibenclamide in endotoxic shock.

Co-induction of nitric oxide synthase and cyclo-oxygenase: interactions between nitric oxide and prostanoids.

1. Lipopolysaccharide (LPS) co-induces nitric oxide synthase (iNOS) and cyclo-oxygenase (COX-2) in J774.2 macrophages. Here we have used LPS-activated J774.2 macrophages to investigate the effects of exogenous or endogenous nitric oxide (NO) on COX-2 in both intact and broken cell preparations. NOS activity was assessed by measuring the accumulation of nitrite using the Griess reaction. COX-2 activity was assessed by measuring the formation of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) by radioimmunoassay. Western blot analysis was used to determine the expression of COX-2 protein. We have also investigated whether endogenous NO regulates the activity and/or expression of COX in vivo by measuring NOS and COX activity in the lung and kidney, as well as release of prostanoids from the perfused lung of normal and LPS-treated rats. 2. Incubation of cultured murine macrophages (J774.2 cells) with LPS (1 microgram ml-1) for 24 h caused a time-dependent accumulation of nitrite and 6-keto-PGF1 alpha in the cell culture medium which was first significant after 6 h. The formation of both 6-keto-PGF1 alpha and nitrite elicited by LPS was inhibited by cycloheximide (1 microM) or dexamethasone (1 microM). Western blot analysis showed that J774.2 macrophages contained COX-2 protein after LPS administration, whereas untreated cells contained no COX-2. 3. The accumulation of 6-keto-PGF1 alpha in the medium of LPS-activated J774.2 macrophages was concentration-dependently inhibited by chronic (24 h) exposure to sodium nitroprusside (SNP; 1-1000 microM). Sodium nitroprusside (1-1000 microM) also acutely (30 min) inhibited COX-2 activity in broken cell preparations of LPS-activated (12 h) J774.2 macrophages, in a similar concentration dependent manner. Addition of adrenaline (5 mM) and glutathione (0.1 mM) increased the activity of COX-2 in broken cell preparations. In the presence of these co-factors, SNP inhibited prostanoid production only at the highest concentration used (1 mM). When J774.2 cells were incubated in the presence of LPS (1 microg ml-1) and NG-monomethyl-L-arginine (L-NMMA: 1 mM) for 12 h, SNP at the highest concentration used (1 mM) acutely (30 min) inhibited the activity of COX-2 in cell homogenates with co-factors. However, when J774.2 macrophages were incubated for 24 or 12 h with LPS (1 microg ml-1)and L-NMMA (1 mM), the addition of SNP (0.001-1I000 microM) increased in a concentration-dependent manner the accumulation of 6-keto-PGF1a in intact cells (measured at 24 h) and COX-2 activity in cell homogenates in the presence of co-factors (determined at 12 h). SNP (1 mM; together with LPS for 12 h)decreased the amount of COX-2 protein induced by LPS in J774.2 macrophages.4. Indomethacin (30 1AM) abolished the formation of 6-keto-PGFa by LPS-activated macrophages, but had no effect on the release of nitrite. Conversely, L-NMMA, at the highest concentrations used (1 and 10 mM), increased the release of 6-keto-PGFIa an effect which was reversed by excess L-arginine (3 mM)but not by D-arginine. Similarly, the decrease in nitrite formation caused by L-NMMA was partially reversed by L-arginine (3 mM), but not by D-arginine. L-NMMA (10 mM; together with LPS for 12 h)increased the amount of COX-2 protein induced by LPS in J774.2 macrophages.5. In separate experiments, J774.2 macrophages were activated with LPS (1 microg ml-1), and L-NMMA(10 mM) was added for various times (0.5-24 h) before the collection of mediun at 24 h. L-NMMAenhanced the release of 6-keto-PGFI,, in a time-dependent manner, with the maximal enhancement seen when the NOS inhibitor was incubated with the cells for 24 h. 6. In experiments on male Wistar rats, we investigated the effect of L-NMMA on the release of prostanoids (6-keto-PGF1a prostaglandin E2, thromboxane B2) elicited by arachidonic acid (AA,30nmol) from ex vivo perfused kidneys and lungs. The release from the organs from normal and LPS-treated rats was unaffected by L-NMMA intraperitoneally (30 mg kg-1) for 6 h together with LPS(5 mg kg-1) or LPS vehicle. Similarly, acute (5 min) in vitro exposure to L-NMMA (1 mM) of the perfused organs from control and LPS-treated animals did not change the release of prostanoids elicited by AA (30 nmol).7. These results show that LPS causes the induction of iNOS and COX-2 in J774.2 macrophages. The co-release of NO and PGI2 induced by LPS is dependent on protein synthesis and occurs after a lag-time of 6-12 h. The formation of COX metabolites has no effect on NOS activity whereas NO inhibits both COX-2 activity and induction. These results demonstrate that NOS and COX can be co-induced in vitro and that under these conditions large amounts of NO inhibit the degree of COX expression and activity.In the absence of endogenous NO, lesser amounts of exogenous NO increase the activity of COX-2. In those situations in vivo when the level of NO induction is relatively low, NO does not regulate the increased activity of COX.

Aminoguanidine attenuates the delayed circulatory failure and improves survival in rodent models of endotoxic shock.

1. We have investigated the effects of aminoguanidine, a relatively selective inhibitor of the cytokine-inducible isoform of nitric oxide synthase (iNOS), on the delayed circulatory failure, vascular hyporeactivity to vasoconstrictor agents, and iNOS activity in a rat model of circulatory shock induced by bacterial endotoxin (E. coli lipopolysaccharide; LPS). In addition, we have evaluated the effect of aminoguanidine on the 24 h survival rate in a murine model of endotoxaemia. 2. Male Wistar rats were anaesthetized and instrumented for the measurement of mean arterial blood pressure (MAP) and heart rate (HR). Injection of LPS (10 mg kg-1, i.v.) resulted in a fall in MAP from 115 +/- 4 mmHg (time 0, control) to 79 +/- 9 mmHg at 180 min (P < 0.05, n = 10). The pressor effect of noradrenaline (NA, 1 microgram kg-1, i.v.) was also significantly reduced at 60, 120 and 180 min after LPS injection. In contrast, animals pretreated with aminoguanidine (15 mg kg-1, i.v., 20 min prior to LPS injection) maintained a significantly higher MAP (at 180 min, 102 +/- 3 mmHg, n = 10, P < 0.05) when compared to rats given only LPS (LPS-rats). Cumulative administration of aminoguanidine (15 mg kg-1 and 45 mg kg-1) given 180 min after LPS caused a dose-related increase in MAP and reversed the hypotension. Aminoguanidine also significantly alleviated the reduction of the pressor response to NA: indeed, at 180 min, the pressor response returned to normal in aminoguanidine pretreated LPS-rats. 3. Thoracic aortae obtained from rats at 180 min after LPS showed a significant reduction in the contractile responses elicited by NA (10-9- 10-6 M). Pretreatment with aminoguanidine (15 mg kg- 1, i.v.,at 20 min prior to LPS) significantly prevented this LPS-induced hyporeactivity to NA ex vivo.4. Endotoxaemia for 180 min resulted in a significant increase in iNOS activity in the lung from 0.6 +/- 0.2 pmol mg-1 min-1 (control, n = 4) to 4.8 +/- 0.3 pmol mg-1 min-1 (P<0.05, n = 6). In LPS-rats treated with aminoguanidine, iNOS activity in the lung was attenuated by 44+/- 5% (n = 6, P <0.05).Moreover, when added in vitro to lung homogenates obtained from LPS-rats, aminoguanidine and N omega-nitro-L-arginine methyl ester (L-NAME; 10-8 to 10-3 M) caused a concentration-dependent inhibition of iNOS activity (n = 3-6, IC50: 30 +/- 12 and 11 +/- 6pEM, respectively P>0.05). In contrast,aminoguanidine was a less potent inhibitor than L-NAME of the constitutive nitric oxide synthase in rat brain homogenates (n = 3-6, IC50 is 140 +/- 10 and 0.6 +/- 0.1 I1M, respectively, P<0.05). In addition, the inhibitory effect of aminoguanidine on iNOS activity showed a slower onset than that of L-NAME(maximal inhibition at 90 min and 30 min, respectively).5. Treatment of conscious Swiss albino (T/O) mice with a high dose of endotoxin (60 mg kg-1, i.p.)resulted in a survival rate of only 8% at 24 h (n = 12). However, therapeutic application of aminoguanidine (15 mg kg-1, i.p. at 2 h and 6 h after LPS) increased the 24 h survival rate to 75%(n = 8), whereas L-NAME (3 mg kg-1, i.p. at 2 h and 6 h after LPS) did not affect the survival rate(11%, n=9).6 Thus, aminoguanidine inhibits iNOS activity and attenuates the delayed circulatory failure caused by endotoxic shock in the rat and improves survival in a murine model of endotoxaemia. Aminoguanidine,or novel, more potent selective inhibitors of iNOS may be useful in the therapy of septic shock.

Comparison of effects of chronic and acute administration of NG-nitro-L-arginine methyl ester to the rat on inhibition of nitric oxide-mediated responses.

1. Vascular responses to acetylcholine and sodium nitroprusside in vivo and in vitro, in the isolated perfused kidney and in rings of rat thoracic aorta, were measured in rats treated chronically with NG-nitro-L-arginine methyl ester (L-NAME; approx, 70 mg kg-1) and compared to responses in age-matched control animals, and age-matched animals after the acute administration of L-NAME (3-100 mumol kg-1). Parallel experiments examined alterations in responsiveness in rings of trachea and anococcygeus muscles taken from the same animals. 2. Chronic oral administration of L-NAME elevated the blood pressure in anaesthetized animals from 114 +/- 5 mmHg to 153 +/- 11 mmHg (n = 5). The hypotensive responses to both acetylcholine (1 nmol kg-1) and sodium nitroprusside (10 nmol kg-1) were enhanced by chronic L-NAME treatment (n = 5-7) whereas acute L-NAME administration enhanced only the response to sodium nitroprusside (n = 5). 3. After chronic treatment with L-NAME, the basal perfusion pressure in the isolated perfused kidney was elevated. However, vasodilator responses to either acetylcholine (1 nmol) or sodium nitroprusside (3 nmol) were unaltered (n = 5-7). The vasodilatation induced by acetylcholine was inhibited in a concentration-dependent manner by the administration of acute L-NAME (0.1 - 100 microM; n = 5), such that significant inhibition was seen at 10 microM L-NAME. The response to sodium nitroprusside was unaffected by L-NAME. 4. The relaxations of isolated rings of rat thoracic aorta induced by acetylcholine were inhibited in tissues prepared from rats treated chronically with L-NAME (n = 5-7). Acute administration of L-NAME (0.1-100 microM) concentration-dependently inhibited the relaxations induced by acetylcholine in this preparation, with significant inhibition occurring at 1 microM L-NAME (n = 5). Responses to sodium nitroprusside were unaffected by either chronic or acute exposure to L-NAME (n = 5-7).5. Relaxations of precontracted anococcygeus muscles induced by electrical field stimulation, or contractions of rings of trachea induced by carbachol or endothelin-1, were unaffected by chronic oral administration of L-NAME (n = 4-6). Acute addition of L-NAME (0.1-100 microM) to the organ baths inhibited in a concentration-dependent manner the relaxations of anococcygeus muscles taken from control animals, with a significant effect being seen at a concentration of 10 micro.M (n = 4-6).6. Our cardiovascular data are consistent with chronic oral administration of L-NAME inhibiting the production of nitric oxide (NO) within the vasculature, although the pattern of inhibition is not uniform between different tissues. Despite the inhibition of endothelial NO production, chronic L-NAME does not alter the vasodepressor activity of acetylcholine in vivo or in the isolated perfused kidney. This maybe explained by an enhanced responsiveness of guanylyl cyclase pathways, the increased release of vasodilators other than nitric oxide or a decreased importance of nitric oxide in resistance vessels compared with conductance vessels. The resistance of peripheral neuronal NO responses to chronic treatment with L-NAME indicates that selective inhibition of different isoforms of NOS may be achieved in vivo.

Effect of lipopolysaccharide on nitric oxide synthase activity in rat proximal tubules.

Renal proximal tubules isolated from the rat possess nitric oxide synthase (NOS) activity that is calcium/calmodulin dependent and stereoselectively inhibited by NG-monomethyl-arginine (NMMA). In the absence of added Ca2+ and calmodulin, activity was reduced 84 +/- 13% compared with the activity in the presence of 2 mM Ca2+ and 25 micrograms/mL calmodulin. Inhibition by EGTA (10 mM) was 95 +/- 5% and by calmidazolium (R24571, 250 microM) was 99 +/- 1%. Inhibition by L-NMMA (100 microM) was 78 +/- 13% and by D-NMMA (100 microM) was 7 +/- 7%. The majority of NOS activity was found in the soluble fraction. NOS activity in isolated proximal tubules was also examined 6 hr after a single i.v. injection of lipopolysaccharide. Activity was increased significantly (P < 0.05) in the soluble fraction by 2-fold [from 0.320 +/- 0.052 to 0.648 +/- 0.046 (nmol/mg protein/30 min)] and in the particulate fraction by 3-fold [from 0.081 +/- 0.030 to 0.256 +/- 0.034 (nmol/mg protein/30 min)]. All activities were inhibited by EGTA. These data demonstrate that proximal tubules express a calcium/calmodulin-dependent NOS activity that is increased in vivo by lipopolysaccharide.

Inhibition of a type B monoamine oxidase inhibitor, (E)-2-(4-fluorophenethyl)-3-fluoroallylamine (MDL-72974A), on semicarbazide-sensitive amine oxidases isolated from vascular tissues and sera of different species.

(E)-2-(4-Fluorophenethyl)-3-fluoroallylamine hydrochloride (MDL-72974A) has been discovered recently to be a very potent and highly selective type B monoamine oxidase inhibitor. We have found that this inhibitor is also capable of inhibiting semicarbazide-sensitive amine oxidases (SSAOs) obtained from vascular tissues and sera of different species. The inhibition of SSAO by MDL-72974A was irreversible and time dependent. It was competitive without preincubation of the enzyme with the inhibitor and demonstrated a mixed-type of inhibition when the enzyme was preincubated with the inhibitor. The IC50 values were estimated to be 2 x 10(-9) M, 5 x 10(-9) M, 8 x 10(-8) M and 2 x 10(-8) M for SSAO from dog aorta, rat aorta, bovine aorta and human umbilical artery, respectively. SSAO obtained from bovine serum was relatively insensitive to MDL-72974A (IC50 = 3 x 10(-7) M. Following intraperitoneal administration of MDL-72974A, rat brain MAO-B was inhibited with the ED50 value being about 0.2 mg/kg. Rat aorta SSAO was also inhibited and to a similar extent by the same dose. MDL-72974A is the most potent SSAO inhibitor that has been described thus far.

Inhibitory effect of arcaine on nitric oxide synthase in the rat brain.

Nitric oxide is synthesized by nitric oxide synthase (NOS) from L-Arg, which contains a guanidino group. Arcaine is the diguanidino compound and a derivative of Arg. This study was conducted to investigate the effects of arcaine on rat brain NOS activity, using nitrite and nitrate as indicators. Arcaine inhibited NOS activity in a linear mixed manner (K1 = 18.68 microM). Almost all previously reported NOS inhibitors were synthesized by substituting the guanidino nitrogen of Arg, but the guanidino nitrogens of arcaine were not substituted. Arcaine was also reported to be a competitive antagonist of the polyamine site on the N-methyl-D-aspartic acid (NMDA) receptor. Arcaine appears to be an excellent drug to investigate not only the chemical nature of NOS but also the functional and structural relationship between NOS and NMDA receptors.

Nitric oxide synthase inhibited by audouine in the rat brain.

Nitric oxide synthase (NOS) synthesizes nitric oxide (NO) from L-arginine (Arg) which has a guanidino group in its molecule. Audouine, a derivative of Arg, is the diguanidino compound. In this study, the effects of audouine on rat brain NOS activity were investigated by measuring nitrite and nitrate formation. Audouine inhibited NOS activity in a competitive (Ki = 2.10 microM) and partially uncompetitive (Ki = 49.7 microM) manner. Audouine is not substituted at the guanidino nitrogen, in contrast to most previously reported NOS inhibitors which were synthesized by substituting the guanidino nitrogen of Arg. Audouine is a novel inhibitor of NOS and should be useful for investigating the chemical nature of NOS and the roles of NO in the central nervous system.

Differential induction of nitric oxide synthase in hepatocytes during endotoxemia and the acute-phase response.

OBJECTIVE: Nitric oxide (NO) is a potent biologic mediator produced by hepatocytes following exposure to cytokines and lipopolysaccharide (LPS). These cytokines are also known to regulate induction of the hepatic acute-phase response. The objective of this study was to determine whether inducible nitric oxide synthase (iNOS), the enzyme that produces NO, is expressed as part of the hepatic acute-phase response. DESIGN: The gene expression for inducible NOS (iNOS) as well as alpha 1-acid glycoprotein (AGP), an established acute-phase reactant, was measured by Northern blot analysis in rat hepatocytes in vivo during endotoxemia (LPS injection) and during the acute-phase response produced by hindlimb turpentine injection. Hepatocyte iNOS messenger RNA (mRNA) levels were correlated with iNOS activity and circulating plasma nitrite and nitrate levels. In vitro, iNOS and AGP mRNA levels were determined in cultured hepatocytes stimulated with interleukin 6 (IL-6), interleukin 1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), or dexamethasone. RESULTS: The AGP mRNA levels were increased in vivo following both LPS and turpentine injection, while iNOS expression was induced only by LPS injection. Hepatocyte iNOS activity and plasma nitrite and nitrate levels also increased after LPS treatment. In vitro, the cytokine combination IL-6, IL-1 beta, and TNF-alpha induced hepatocyte iNOS expression but had minimal effects on AGP in the absence of dexamethasone. Addition of dexamethasone alone markedly increased AGP mRNA levels, with further increases seen with TNF-alpha or IL-1 beta addition. In contrast, dexamethasone decreased iNOS expression. CONCLUSION: The results show that hepatocyte iNOS expression is not part of the acute-phase response induced by remote inflammation and indicates that iNOS is differentially regulated from the acute-phase reactant, AGP.

Differential accumulation of transcripts for ACC synthase and ACC oxidase homologs in etiolated mung bean hypocotyls in response to various stimuli.

Ethylene can be produced by a variety of developmental and environmental factors such as ripening, the plant hormone auxin, and mechanical wounding via a biosynthetic pathway including AdoMet synthase, ACC synthase, and ACC oxidase steps. ACC synthase and ACC oxidase are known to be encoded by multigene families, and are believed to be differentially expressed in response to various stimuli. In mung bean, ACC synthase is encoded by 7 genes, ACS1, ACS2 ACS3, ACS4, ACS5, ACS6, and ACS7, and ACC oxidase by 2 genes, ACO1 and ACO2. In this study, was have investigated differential accumulation of transcripts for ACC synthase and ACC oxidase homologs in etiolated mung bean hypocotyls under various conditions by the semiquantitative RT-PCR method. Primers which can specifically bind and amplify each cDNAs of ACS1, ACS2, ACS3, ACS4, ACS5, ACS6, ACS7, and ACO1, and ACO2 were designed and used to monitor the responses to various stimuli. Transcripts of ACO1 and ACO2 were accumulated constitutively in the hypocotyl segments even without andy treatment. After cold treatment on intact plant, transcripts of ACS5, ACS6, and ACS7 were accumulated in the hypocotyl segments. We also found the excision of hypocotyl segments and incubation in a buffer solution, a typical way of chemical treatments to hypocotyl segments, lowered the level of ACO2 transcripts with little change of the level of ACO1 transcripts. In response to incubation with IAA (0.1 mM) of excised hypocotyl segments, transcripts of ACS1, ACS6, and ACS7 were accumulated and the level of ACO2 transcripts was increased. Transcripts of ACS1, ACS2, ACS3, ACS5, ACS6 and ACS7 were induced by incubation with OGA (50 micrograms/ml), while the transcripts of ACS4 were accumulated and the level of ACO2 transcripts was increased by incubation with 1 mM LiCl. Our results strongly suggest that all seven ACC synthase genes and two ACC oxidase genes must be active and each gene is differentially regulated by a different subset of the inducing factors.

Effects of nitric oxide (NO) synthesis inhibition on the development of supersensitivity to stereotypy and locomotion stimulating effects of methamphetamine.

The present study examined the effects of nitric oxide (NO) synthase inhibitor, N omega-nitro-L-arginine methyl ester (LNAME; 30 and 60 mg/kg, i.p.) on the development of supersensitivity to stereotypy as well as locomotion stimulating effects of methamphetamine (MA) (3.22 and 0.805 mg free base/kg, s.c., respectively). Rats treated with MA for 10 days showed enhancement in MA-induced stereotypy and locomotor activity. Rats pretreated with LNAME prior to MA also showed enhancement in the two types of behavior, also they showed significantly reduced stereotypy scores compared to those treated with MA alone. The results suggest that NO synthesis is not critically involved in the development of behavioral supersensitivity to stereotypy stimulating as well as locomotion stimulating effect of MA. However, No synthesis may have a modulatory role in behavioral sensitization in stereotypy.

Excitotoxic action of NMDA agonists on nigrostriatal dopaminergic neurons: modulation by inhibition of nitric oxide synthesis.

Focal infusions of N-methyl-D-aspartate (NMDA) or an endogenous NMDA agonist, quinolinic acid (QUIN), into the substantia nigra pars compacta (SNc) of adult Sprague-Dawley rats resulted in a dose-dependent depletion of ipsilateral striatal tyrosine hydroxylase (TH) activity, a biochemical marker for dopaminergic neurons. To assess the intermediary role of nitric oxide in the neurotoxicity elicited by these toxins, their action was tested in animals treated with N omega-nitro-L-arginine methyl ester (L-NAME). Systemic injections (2 injections; 8 h apart) of L-NAME (100, 150 and 250 mg/kg) produced a dose-related inhibition of cerebellar nitric oxide synthase (NOS) activity. The time-course of cerebellar NOS inhibition following L-NAME (250 mg/kg) was rapid in onset and lasted for at least 24 h following the second injection. An L-NAME treatment regimen of 250 mg/kg, with the second injection given 24 h prior to assessment of NOS activity, produced an 87 and 91% inhibition of cerebellar and nigral NOS activity, respectively. Intranigral infusion of 40 and 60 nmol QUIN reduced ipsilateral striatal TH activity by 62 and 75%, respectively. However, 40 and 60 nmol QUIN infusions into animals pretreated with L-NAME (250 mg/kg) reduced striatal TH activity by 83 and 96%, respectively. Intranigral infusion of 15 and 30 nmol NMDA produced a 48 and 77% decrease in striatal TH activity, respectively, whereas the same doses of NMDA given to animals pretreated with L-NAME (250 mg/kg) resulted in a 59 and 88% decrease in TH activity.(ABSTRACT TRUNCATED AT 250 WORDS)