Thiomicrorhabdus immobilis sp. nov., a mesophilic sulfur-oxidizing bacterium isolated from sediment of a brackish lake in northern Japan.

A novel sulfur-oxidizing bacterium, strain Am19T, was isolated from sediment of a brackish lake. Strain Am19T grew chemolithoautotrophically on inorganic sulfur compounds, and heterotrophic growth was not observed. Cells were rod-shaped with length of 1.1-3.0 µm and diameter of 0.5-0.8 µm. Growth was observed at 5-37 °C with an optimum growth temperature of 30 °C. The pH range for growth was 5.6-8.5 with an optimum pH of 6.6-7.0. Major fatty acids were summed feature 3 (C16: 1ω7c and/or C16: 1ω6c), summed feature 8 (C18: 1ω7c and/or C18: 1ω6c) and C16: 0. The sole respiratory quinone was ubiquinone-8. The complete genome of strain Am19T is composed of a circular chromosome with length of 2.5 Mbp and G + C content of 42.7 mol%. Phylogenetic analysis based on genomic data indicated that strain Am19T belongs to the genus Thiomicrorhabdus but is distinct from any existing species. Analysis of the 16S rRNA gene supported creation of a new species to accommodate strain Am19T. On the basis of genomic and phenotypic characteristics, strain Am19T (= NBRC 114602 T = BCRC 81336 T) is proposed as the type strain of a new species, with name of Thiomicrorhabdus immobilis sp. nov.

Sulfurimicrobium lacus gen. nov., sp. nov., a sulfur oxidizer isolated from lake water, and review of the family Sulfuricellaceae to show that it is not a later synonym of Gallionellaceae.

A facultatively anaerobic sulfur-oxidizing bacterium, strain skT11T, was isolated from anoxic lake water of a stratified freshwater lake. As electron donor for chemolithoautotrophic growth, strain skT11T oxidized thiosulfate, tetrathionate, and elemental sulfur under nitrate-reducing conditions. Oxygen-dependent growth was observed under microoxic conditions, but not under fully oxygenated conditions. Growth was observed at a temperature range of 5-37 °C, with optimum growth at 28 °C. Strain skT11T grew at a pH range of 5.1-7.5, with optimum growth at pH 6.5-6.9. Heterotrophic growth was not observed. Major components in the cellular fatty acid profile were C16:1 and C16:0. The complete genome of strain skT11T consisted of a circular chromosome with a size of 3.8 Mbp and G + C content of 60.2 mol%. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that the strain skT11T is related to sulfur-oxidizing bacteria of the genera Sulfuricella, Sulfurirhabdus, and Sulfuriferula, with sequence identities of 95.4% or lower. The analysis also indicated that these three genera should be excluded from the family Gallionellaceae, as members of another family. On the basis of its genomic and phenotypic properties, strain skT11T (= DSM 110711 T = NBRC 114323 T) is proposed as the type strain of a new species in a new genus, Sulfurimicrobium lacus gen. nov., sp. nov. In addition, emended descriptions of the families Gallionellaceae and Sulfuricellaceae are proposed to declare that Sulfuricellaceae is not a later synonym of Gallionellaceae.

Questiomycins, Algicidal Compounds Produced by the Marine Bacterium Alteromonas sp. D and Their Production Cue.

Questiomycin A (1) along with three new compounds, questiomycins C-E (2-4), were isolated from culture of Alteromonas sp. D, an algicidal marine bacterium, guided by algal lethality assay using the raphidophyte, Chattonella antiqua, one of the causative organisms of harmful algal bloom. The structures of 1-4 were assigned on the basis of their spectrometric and spectroscopic data. Compounds 1 to 4 exhibited algicidal activity against C. antiqua with LC50 values ranging from 0.18 to 6.37 M. Co-cultivation experiment revealed that 1 was produced only when the microalgae and the bacterium are in close contact, suggesting that some interactions between them trigger the biosynthesis of questiomycins. These results suggested that the algicidal bacteria such as Alteromonas sp. D can control microalgae chemically in marine ecosystem.

Therapeutic effects of the allosteric protein tyrosine phosphatase 1B inhibitor KY-226 on experimental diabetes and obesity via enhancements in insulin and leptin signaling in mice.

The anti-diabetic and anti-obesity effects of the allosteric protein tyrosine phosphatase 1B (PTP1B) inhibitor 4-(biphenyl-4-ylmethylsulfanylmethyl)-N-(hexane-1-sulfonyl)benzoylamide (KY-226) were pharmacologically evaluated. KY-226 inhibited human PTP1B activity (IC50 = 0.28 µM), but did not exhibit peroxisome proliferator-activated receptor γ (PPARγ) agonist activity. In rodent preadipocytes (3T3-L1), KY-226 up to 10 µM had no effects on adipocyte differentiation, whereas pioglitazone, a PPARγ agonist, markedly promoted it. In human hepatoma-derived cells (HepG2), KY-226 (0.3-10 µM) increased the phosphorylated insulin receptor (pIR) produced by insulin. In db/db mice, the oral administration of KY-226 (10 and 30 mg/kg/day, 4 weeks) significantly reduced plasma glucose and triglyceride levels as well as hemoglobin A1c values without increasing body weight gain, while pioglitazone exerted similar effects with increases in body weight gain. KY-226 attenuated plasma glucose elevations in the oral glucose tolerance test. KY-226 also increased pIR and phosphorylated Akt in the liver and femoral muscle. In high-fat diet-induced obese mice, the oral administration of KY-226 (30 and 60 mg/kg/day, 4 weeks) decreased body weight gain, food consumption, and fat volume gain with increases in phosphorylated STAT3 in the hypothalamus. In conclusion, KY-226 exerted anti-diabetic and anti-obesity effects by enhancing insulin and leptin signaling, respectively.

Novel (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acids: peroxisome proliferator-activated receptor γ selective agonists with protein-tyrosine phosphatase 1B inhibition.

A novel series of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives were synthesized and (S)-2-[(2E,4E)-hexadienoyl]-7-(2-{5-methyl-2-[(1E)-5-methylhexen-1-yl]oxazol-4-yl}ethoxy)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (14i) was identified as a potent human peroxisome proliferator-activated receptor γ (PPARγ) selective agonist (EC(50)=0.03 µM) and human protein-tyrosine phosphatase 1B (PTP-1B) inhibitor (IC(50)=1.18 µM). C(max) after oral administration of 14i at 10mg/kg was 2.2 µg/ml (4.5 µM) in male SD rats. Repeated administration of 14i and rosiglitazone for 14 days dose-dependently decreased plasma glucose levels, ED(50)=4.3 and 23 mg/kg/day, respectively, in male KK-A(y) mice. In female SD rats, repeated administration of 14i at 12.5-100mg/kg/day for 28 days had no effect on the hematocrit value (Ht) and red blood cell count (RBC), while rosiglitazone significantly decreased them from 25mg/kg/day. In conclusion, 14i showed about a fivefold stronger hypoglycemic effect and fourfold or more weaker hemodilution effect than rosiglitazone, indicating that 14i is 20-fold or more safer than rosiglitazone. Compound 14i is a promising candidate for an efficacious and safe anti-diabetic drug targeting PPARγ and PTP-1B.

Novel acyl-CoA: cholesterol acyltransferase inhibitor: indoline-based sulfamide derivatives with low lipophilicity and protein binding ratio.

To find a novel acyl-CoA: cholesterol acyltransferase inhibitor, a series of sulfamide derivatives were synthesized and evaluated. Compound 1d, in which carboxymethyl moiety at the 5-position of Pactimibe was replaced by a sulfamoylamino group, showed 150-fold more potent anti-foam cell formation activity (IC(50): 0.02 microM), 1.6-fold higher log D(7.0) (4.63), and a slightly lower protein binding ratio (93.2%) than Pactimibe. Compound 1i, in which the octyl chain at the 1-position in 1d was replaced by an ethoxyethyl, showed markedly low log D(7.0) (1.73) and maintained 3-fold higher anti-foam cell formation activity (IC(50): 1.0 microM), than Pactimibe. The plasma protein binding ratio (PBR) of 1i was much lower than that of Pactimibe (62.5% vs. 98.1%), and its partition ratio to the rabbit atherosclerotic aorta after oral administration was higher than that of Pactimibe. Compound 1i at 10 microM markedly inhibited cholesterol esterification in atherosclerotic rabbit aortas even when incubated with serum, while Pactimibe had little effect probably due to its high PBR. In conclusion, compound 1i is expected to more efficiently inhibit the progression of atherosclerosis than Pactimibe.

Novel tetrahydroisoquinoline derivatives with inhibitory activities against acyl-CoA: cholesterol acyltransferase and lipid peroxidation.

To find a novel acyl-CoA: cholesterol acyltransferase (ACAT) inhibitor with anti-lipid peroxidative activity, a series of tetrahydroisoquinoline derivatives were synthesized and evaluated. A compound with a N-(4-hydroxy-2,3,5-trimethylphenyl)carbamoyl moiety at the 3-position and an octanoyl moiety at the 2-position (7) was demonstrated to show anti-foam cell formation activity stronger than and anti-lipid peroxidative activity comparable to those of Pactimibe, while it was hardly absorbed orally. To increase its bioavailability, the acyl chain at the 2-position was shortened and various polar or basic moieties were introduced at the 7-position of 7. Among the synthesized derivatives, (S)-7-dimethylamino-N-(4-hydroxy-2,3,5-trimethylphenyl)-2-isobutyryl-1,2,3,4-tetrahydroisoquinoline-3-carboxamide hydrochloride (21) showed about 16-fold stronger anti-foam cell formation activity, 3-fold stronger hepatic ACAT inhibitory activity, similar anti-low density lipoprotein (LDL) oxidative activity and 2-fold more potent protective activity against macrophage cell death by oxidative stress in comparison with Pactimibe. Compound 21 was efficiently absorbed after oral administration at 10 mg/kg in rats and dogs and its C(max) values were higher than its IC(50) values for in vitro activities. In conclusion, a tetrahydroisoquinoline structure is a useful scaffold for designing a phenolic anti-oxidative ACAT inhibitor, and compound 21 is expected to effectively prevent atherosclerosis.

Lipopolysaccharide-induced impairment of nitric oxide-mediated vasorelaxation and protective effects of nitric oxide synthesis inhibitors in isolated rat mesenteric arteries.

Isolated rat mesenteric arteries were incubated with lipopolysaccharide (LPS) for 6 h and then mounted in an organ bath to investigate their responses to various relaxants. Exposure to LPS moderately reduced acetylcholine (ACh)-induced endothelium-dependent relaxation (EDR), and markedly reduced sodium nitroprusside (SNP)-induced endothelium-independent relaxation (EIR). It did not affect ACh-induced EDR under treatment with a nitric oxide synthase (NOS) inhibitor, which is mediated by an endothelium-derived hyperpolarizing factor (EDHF), and forskolin-induced EIR. N-(3-(Aminomethyl)benzyl)acetamidine (1400 W), an inducible nitric oxide synthase (iNOS) inhibitor, actinomycin D, an RNA polymerase inhibitor, cycloheximide, a protein synthesis inhibitor, and dexamethazone reduced the nitric oxide (NO) production and reversed the reduced ACh-induced EDR and SNP-induced EIR. In LPS-treated mesenteric artery, L-arginine-induced relaxation was not affected by removal of endothelium, indicating muscular inducible nitric oxide synthase (iNOS) induction. Pre-exposure to SNP (NO donor) also moderately reduced ACh-induced EDR and markedly reduced SNP-induced EIR with little effect on ACh-induced EDHF-mediated EDR. In conclusion, in vitro exposure to LPS desensitized vascular smooth muscle cells to endogenous and exogenous NO by overproduction of muscular iNOS-derived NO, and an iNOS inhibitor and iNOS induction inhibitors prevented the LPS-induced desensitization.

Novel indoline-based acyl-CoA: cholesterol acyltransferase inhibitor: Effects of introducing a methanesulfonamide group on physicochemical properties and biological activities.

A novel series of indoline-based acyl-CoA: cholesterol acyltransferase (ACAT) inhibitors with a methanesulfonamide group at the 5-position were synthesized and their lipophilicity and biological activities were evaluated. Hepatic ACAT inhibitory and anti-foam cell formation activity increased dependent on lipophilicity of derivatives with various alkyl chains at the 1-position. The logD(7.0)-biological activity curve of the derivatives with a methanesulfonamide group shifted leftward compared to that of Pactimibe derivatives with a carboxymethyl group, and derivatives with no substituent, suggesting that a methanesulfonamide group plays an important role in the interaction with ACAT protein. Among derivatives, N-(1-ethyl-5-methanesulfonylamino-4,6-dimethylindolin-7-yl)-2,2-dimethylpropanamide (1b) had about twofold lower logD(7.0) than Pactimibe, while it showed twofold higher hepatic ACAT inhibition than and the same anti-foam cell formation as Pactimibe, respectively. The C(max) of 1b (10mg/kg, po) was higher than that of Pactimibe in rats. The plasma protein binding ratio of 1b was lower than that of Pactimibe: 64.8% and 97.9%, respectively. Compound 1b showed greater inhibitory effects on hepatic cholesterol secretion in mice than Pactimibe. In conclusion, the introduction of a methanesulfonamide group is effective to design less lipophilic, more efficacious and more bioavailable indoline-based ACAT inhibitors than previous indoline-based inhibitors.

NK1 receptor-mediated endothelium-dependent relaxation and contraction with different sensitivity to post-receptor signaling in pulmonary arteries.

In rabbit intrapulmonary arteries, substance P (SP) has been reported to induce endothelium-dependent relaxation (EDR) and endothelium-dependent contraction (EDC) via tachykinin NK(1) receptors, and endothelium-independent contraction (EIC) via tachykinin NK(2) receptors. The present study pharmacologically examined whether these opposite responses (EDR and EDC) are mediated by the same NK(1) receptor. Five tachykinin agonists, including septide, a reportedly atypical NK(1) agonist, caused concentration-dependent EDR in the presence of NK2 antagonist (SR-48968) + TXA2 synthetase inhibitor (ozagrel), which blocked EIC and EDC, in pre-contracted arteries, and concentration-dependent EDC in the presence of NK2 antagonist (SR-48968) + nitric oxide synthase inhibitor (l-N(G)-nitro-arginine methyl ester), which blocked EIC and EDR, in non-contracted arteries. The EC(50) values of these agonists for EDR were smaller than those for EDC, indicating that the affinities of NK(1) agonists to NK(1) receptors are different between EDR and EDC. However, the rank order of their potency for EDR and EDC was the same: SP = septide > SP methyl ester (SPME) > neurokinin A > neurokinin B. [Ala(5), beta-Ala(8)]-alpha-neurokinin fragment 4-10 (NK2 agonist) and senktide (NK3 agonist) caused no responses. Two structurally different NK(1) antagonists, CP-99994 and SR-140333, shifted the concentration-EDC and -EDR curves of SPME, a selective NK(1) agonist, and septide rightward and suppressed their maximal responses in a similar concentration-dependent manner, indicating that the affinities of NK(1) antagonists to NK1 receptors are similar between EDR and EDC. U-73122, a phospholipase C inhibitor, and thapsigargin, 2,5-di-tert-butylhydroquinone, and ruthenium red, all intracellular Ca2+ release blockers, inhibited SP-induced EDR and EDC. Effective concentrations of ionomycin (Ca2+ ionophore) causing EDR were also lower than those causing EDC. Taken together, SP-induced EDR and EDC are mediated by activation of the same NK1 receptor followed by an increase in intracellular Ca2+, and sensitivity to Ca2+ may be higher in the EDR than EDC pathway.

Regional heterogeneity of substance P-induced endothelium-dependent contraction, relaxation, and -independent contraction in rabbit pulmonary arteries.

AIMS: The present study examined whether substance P (SP)-induced endothelium-dependent TXA(2)-mediated contraction (EDC), nitric oxide (NO)-mediated relaxation (EDR), and endothelium-independent contraction (EIC) are different between the rabbit proximal and distal intrapulmonary arteries. MAIN METHODS: The helically cut strips of isolated proximal and distal arteries were fixed vertically between hooks in organ bath, and changes in isometric tension were measured. KEY FINDINGS: SP-induced EDC was greater in the distal than proximal arteries, and EDR was greater in the proximal than distal arteries. However, under the complete blockade of NK(2) receptors and NO production, SP (10(-9)-3x10(-7) M)-induced EDC did not differ between proximal and distal arteries. Under the complete blockade of NK(2) receptors and TXA(2) production, SP (3x10(-10)-3x10(-8) M)-induced EDR was greater in the proximal than distal arteries. Neither contraction induced by U-46619, a TXA(2) agonist, nor relaxation by sodium nitroprusside, an NO donor, was different between both portions of the arteries. Both ionomycin (10(-8) M)- and l-arginine (1 mM)-induced EDRs were also significantly greater in the proximal than distal arteries. Under the blockade of NK(1) receptors and NO and TXA(2) production, SP (10(-7) M)-induced EIC was greater in the distal than proximal arteries. In summary, the capacity for NO production is higher in the proximal than distal arteries, resulting in SP-induced higher EDR and lower EDC in the proximal arteries. SIGNIFICANCE: These regional differences in responses to SP may play important roles in maintaining the homogenous distribution of blood flow in the lung.

Novel indoline-based acyl-CoA:cholesterol acyltransferase inhibitor with antiperoxidative activity: improvement of physicochemical properties and biological activities by introduction of carboxylic acid.

A series of novel indoline derivatives with an ionizable moiety were synthesized to find a bioavailable acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor with antiperoxidative activity. [7-(2,2-Dimethylpropanamido)-4,6-dimethyl-1-octylindolin-5-yl]acetic acid hemisulfate (2, pactimibe sulfate) with low lipophilicity and high water solubility showed good oral absorption and inhibitory activity against foam cell formation in THP-1 cells exposed to acetyl-LDL after differentiation (IC50: 0.3 microM) and an antiperoxidative effect in LDL of hypercholesterolemic rabbits (IC50: 1.0 microM). 2 inhibited macrophage, hepatic, and intestinal ACAT activity (IC50: 1.9, 0.7, and 0.7 microM, respectively). Maximal plasma concentration after oral administration of 2 at 10 mg/kg was 0.9 microg/mL in rats, 3.0 microg/mL in rabbits, and 11.2 microg/mL in dogs. Repeated administration of 2 lowered plasma LDL/VLDL cholesterol in hypercholesterolemic rabbits at 1 mg/kg/day, rats and dogs at 3 mg/kg/day, and in normocholesterolemic hamsters at 3 mg/kg/day. 2 is a promising candidate for antihyperlipidemic and antiatherosclerotic drugs.

Synthesis and biological evaluation of (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acids: a novel series of PPAR gamma agonists.

A novel series of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives were synthesized and biologically evaluated. (S)-2-Benzyl-7-[2-(5-methyl-2-phenyloxazol-4-yl)ethoxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (10, KY-021) was identified as a novel peroxisome proliferators-activated receptor (PPAR) gamma agonist, which showed potent activity in human PPAR gamma (EC50=11.8 nM). KY-021 reduced plasma glucose and triglyceride levels at 3 mg/kg/d for 7 d in male KK-Ay mice. KY-021 also decreased plasma triglyceride levels at 0.3-3 mg/kg/d for 6 d, and improved oral glucose tolerance at 1 and 3 mg/kg/d for 7 d in male Zucker fatty rats. Maximal plasma concentration of KY-021 after oral administration at 10 mg/kg was 6.6 microg/ml and 2.1 microg/ml in male ICR mice and male SD rats, respectively. Repeated oral administration of KY-021 at 30 mg/kg/d for 10 weeks had little toxicity in male SD rats. These results demonstrated that KY-021 has great potential as an efficacious and safe drug for diabetes.

Impairment of endothelium-dependent ACh-induced relaxation in aorta of diabetic db/db mice--possible dysfunction of receptor and/or receptor-G protein coupling.

Diabetes is a risk factor of ischemic heart disease, cerebral ischemia, and atherosclerosis, in which endothelial dysfunction plays a role in the pathogenesis. We examined vascular responses in the aorta of pre-diabetic db/db mice with normoglycemia, hyperlipidemia, and hyperinsulinemia (6 weeks old), and diabetic db/db mice with hyperglycemia, hyperlipidemia, and hyperinsulinemia (11 weeks old) in comparison with age-matched non-diabetic db/+ mice. Prostaglandin F2alpha (PGF2alpha)-induced contraction was significantly enhanced in the aorta of diabetic but not pre-diabetic db/db mice compared to age-matched non-diabetic db/+ mice. Acetylcholine (ACh), adenosine-5'-diphosphate (ADP), NaF, a G protein activator and A-23187, a Ca-ionophore, caused endothelium-dependent and nitric oxide (NO)-mediated relaxation, and sodium nitroprusside (SNP), an NO donor, caused endothelium-independent relaxation in the pre-contracted aorta of db/db mice. Maximal endothelium-dependent ACh-induced relaxation was reduced in diabetic but not pre-diabetic db/db mice compared to age-matched db/+ mice, while maximal SNP-induced relaxation was not different between diabetic and non-diabetic mice. ACh-induced relaxation in diabetic db/db mice was not affected by ozagrel, a thromboxane A2 (TXA2) synthetase inhibitor, or acetylsalicylic acid (aspirin), a cyclooxygenase inhibitor, suggesting no involvement of endogenous TXA2 or prostanoids in the reduction of relaxation. Maximal endothelium-dependent ADP-, A-23187-, and NaF-induced relaxation was not reduced in diabetic db/db mice. EC50 values for ACh- and SNP-induced relaxation were increased in diabetic but not pre-diabetic db/db mice, suggesting decreases in sensitivity to NO in diabetic mice. Two-week treatment with KV-5070, a PPARgamma agonist, lowered plasma glucose, triglyceride (TG), and insulin but not cholesterol, and reversed the reduced ACh-induced relaxation. In conclusion, ACh-induced endothelium-dependent relaxation is impaired in diabetic db/db mice, probably due to the dysfunction of ACh receptors and/or receptor-G protein coupling. Endothelial dysfunction was not genetic and was considered to be initiated primarily by hyperglycemia, and was improved by anti-diabetic treatment with a PPARgamma agonist.

Effects of an anti-oxidative ACAT inhibitor on apoptosis/necrosis and cholesterol accumulation under oxidative stress in THP-1 cell-derived foam cells.

THP-1 cell-derived foam cells were exposed to oxidative stress through combined treatment with acetylated LDL (acLDL) and copper ions (Cu2+). The foam cells showed caspase-dependent apoptotic changes on exposure to oxidative stress for 6 h, and necrotic changes with the leakage of LDH after 24 h. KY-455, an anti-oxidative ACAT inhibitor, and ascorbic acid (VC) but not YM-750, an ACAT inhibitor, prevented apoptotic and necrotic changes. These preventive effects of KY-455 and VC were accompanied by the inhibition of lipid peroxidation in culture medium containing acLDL and Cu2+, suggesting the involvement of oxidized acLDL in apoptosis and necrosis. Foam cells accumulated esterified cholesterol (EC) for 24 h in the presence of acLDL without Cu2+, which was suppressed by KY-455 and YM-750. Foam cells showed necrotic changes and died in the presence of acLDL and Cu2+. KY-455 but not YM-750 prevented cell death and reduced the amount of EC accumulated. The foam cells treated with VC further accumulated EC without necrotic changes for 24 h even in the presence of acLDL and Cu2+. YM-750 as well as KY-455 inhibited lipid accumulation when co-incubated with VC in foam cells exposed to oxidative stress. It is concluded that an anti-oxidative ACAT inhibitor or the combination of an antioxidant and an ACAT inhibitor protects foam cells from oxidative stress and effectively reduces cholesterol levels, which would be a promising approach in anti-atherosclerotic therapy.

Effect of combination of nitric oxide synthase and cyclooxygenase inhibitors on carrageenan-induced pleurisy in rats.

In the present study, we examined the effects of L-nitroarginine methylester (L-NAME), a non-selective nitric oxide synthase (NOS) inhibitor, indomethacin (IND), a non-selective COX inhibitor and a combination of these agents (L-NAME+IND) on carrageenan-induced pleurisy in rats. Exudate volume, albumin leakage, leukocyte influx, exudate and plasma nitrite/nitrate (NO(x)) levels and exudate PGE(2) levels increased markedly 6 h after an intrapleural injection of 2% carrageenan. First, the effects of L-NAME and IND alone were investigated. L-NAME non-significantly reduced exudate volume by 26% at 10 mg/kg (i.p.), and significantly by 45% at 30 mg/kg. IND dose-dependently decreased the exudate volume at 0.3-10 mg/kg (p.o.) and the effect reached the maximal level at 1 mg/kg (33%). Second, the effects of L-NAME (10 mg/kg, i.p.), IND (1 mg/kg, p.o.) and L-NAME+IND were examined. L-NAME and IND alone at the dose employed significantly reduced the exudate volume and albumin levels by 21-26%. L-NAME but not IND tended to reduce the increased exudate and plasma NO(x) by 18% and 19%, respectively. IND but not L-NAME significantly reduced leukocyte numbers and PGE(2) levels in the exudates by 25% and 77%, respectively. L-NAME+IND significantly reduced exudate volume, albumin leakage, leukocyte number, PGE(2) and NO(x) by 43%, 41%, 31%, 80% and 37%, respectively. The inhibitory effects of L-NAME+IND on exudate volume, albumin leakage and NO(x) levels were greater than those of L-NAME and IND alone. In conclusion, a non-selective NOS inhibitor and COX inhibitor showed anti-inflammatory effects at the early phase of carrageenan-induced pleurisy, and a combination of both inhibitors had a greater effect than each alone probably via the potentiation of NOS inhibition. The simultaneous inhibition of NOS and COX could be a useful approach in therapy for acute inflammation.

Synergistic effect of nitric oxide synthase and cyclooxygenase inhibitors on carrageenan-induced paw edema in rats.

The present study examined the effects of L-nitroarginine methylester (L-NAME, CAS 50903-99-6), a non-selective nitric oxide synthase (NOS) inhibitor, indometacin (IND, CAS 3305-29-1), a non-selective cyclooxygenase (COX) inhibitor, and a combination of these agents (L-NAME + IND) on carrageenan-induced paw edema for 4 h after the injection of carrageenan in rats. L-NAME at 10 and 30 mg/ kg but not 3 mg/kg (i.p.) decreased paw volume slightly but significantly only at 1 h after the carrageenan injection. IND reduced paw volume slightly at 1 and 3 mg/kg, and markedly at 10 mg/kg (p.o.). A combination of L-NAME and IND at a subthreshold dose (3 mg/kg, i.p. and 1 mg/kg, p.o., respectively) caused a marked reduction of paw edema, which was also confirmed by histopathological examinations. A combination of N-(3-(aminomethyl)benzyl)acetamidine (1400W, CAS 180001-34-7), a selective inhibitor of inducible NOS, and IND at 3 mg/kg, i.p., and 1 mg/kg, p.o., respectively, did not show synergistic anti-inflammatory effects. In conclusion, the combination of non-selective NOS and COX inhibitors had synergistic anti-inflammatory effects on carrageenan-induced paw edema at an early stage, suggesting negative crosstalk between the endogenous NOS-NO and COX-PG pathways in the early stages of acute inflammation.

Effects of selective iNOS inhibition on type II collagen-induced arthritis in mice.

Nitric oxide as well as prostaglandins has been reported to play an important role in inflammatory diseases including arthritis. In the present study, the effects of iNOS inhibition on development of disease were examined in type II collagen-induced arthritis (CIA) in male DBA/1J mice. From 4 weeks after the first immunization with bovine type II collagen, 1400W (10 mg/kg/day, p.o.), a selective iNOS inhibitor, indomethacin (1 mg/kg/day, p.o.), a cyclooxygenase (COX) inhibitor, or 1400W + indomethacin was administered for 8 weeks. Immunization with type II collagen evoked arthritic inflammation of paws and bone destruction accompanied by increases in urinary nitrite/nitrate (NOx) excretion, plasma NOx and PGE2 levels. Administration of 1400W reduced urinary NOx excretion and increased plasma PGE2 levels, while it had no effect on arthritic inflammation or bone destruction. Indomethacin slightly reduced the inflammatory signs and bone destruction with marked reduction of plasma PGE2. Combination of 1400W and indomethacin reduced urinary NOx and PGE2 levels, and showed greater amelioration of inflammatory signs and bone destruction than either alone. In conclusion, 1400W, a selective iNOS inhibitor, failed to prevent CIA probably due to its increasing effect on PGE2 production, but showed a synergistic ameliorative effect in combination with indomethacin.

Hypolipidemic and antioxidant activity of the novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor KY-455 in rabbits and hamsters.

The hypolipidemic and antioxidant effects of N-(4,6-dimethyl-1-pentylindolin-7-yl)-2,2-dimethylpropanamide (CAS 178469-71-1, KY-455), a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, were examined in hyperlipidemic rabbits and normolipidemic hamsters. KY-455 inhibited rabbit intestinal, hepatic, macrophage and adrenal ACAT with IC50 values of 0.4, 0.9, 2.9 and 4.1 micromol/l, respectively. KY-455 also inhibited rabbit plasma and LDL-peroxidation (IC50: 0.4 and 1.7 micromol/l, respectively). In rabbits fed a high-cholesterol diet, treatment with KY-455 (30 mg/kg/day) for 8 days markedly lowered serum esterified, free, low-density lipoprotein (LDL)-cholesterol, and hepatic esterified cholesterol levels. KY-455 tended to inhibit ex vivo hepatic ACAT activity 5 h after the final administration. KY-455 also inhibited ex vivo peroxidation of plasma lipids 1 and 5 h after the final administration in rabbits. In normolipidemic hamsters fed a regular diet, treatment with KY-455 (30 mg/kg, twice a day) for 4 days significantly reduced serum esterified, free and LDL-cholesterol, and hepatic esterified and free cholesterol levels. A single administration of KY-455 (30 mg/kg) significantly inhibited ex vivo hepatic ACAT activity in hamsters. In conclusion, KY-455 showed in vitro inhibitory effects on LDL-peroxidation and macrophage ACAT activity at similar concentrations, and in vivo hypolipidemic and ex vivo antioxidative effects at the same dose. Long-term administration of KY-455 is expected to prevent the progress of atherosclerosis by lowering plasma lipid levels, inhibiting both LDL-oxidation and accumulation of cholesterol in macrophages.

Role of intracellular Ca2+ in endothelium-dependent contraction and relaxation of rabbit intrapulmonary arteries.

We examined whether Ca(2+) mobilizers induce endothelium-dependent contraction and relaxation (EDC and EDR) in isolated rabbit intrapulmonary arteries. Ionomycin (10(-7) M) and A-23187 (10(-7) M), both Ca(2+) ionophores, and thapsigargin (10(-6) M), an endoplasmic reticulum Ca(2+)-ATPase inhibitor, caused a contraction in the non-contracted preparations, and a transient relaxation followed by a transient contraction and sustained relaxation in the precontracted preparations. Endothelium-removal abolished the contraction and transient relaxation (EDC and EDR) but not sustained relaxation (endothelium-independent relaxation, EIR). In the noncontracted preparations, ionomycin-induced EDC was significantly attenuated by quinacrine (10(-5) M), manoalide (10(-6) M), both phospholipase A(2) inhibitors, indomethacin (10(-5) M) and aspirin (10(-4) M), both COX inhibitors, and ozagrel (10(-5) M), a TXA(2) synthetase inhibitor. In the precontracted arteries, EDR was markedly reduced by L-NAME (10(-4) M), a NOS inhibitor, and methylene blue (10(-6) M), a guanylate cyclase inhibitor, and was enhanced by indomethacin, aspirin and ozagrel, probably due to inhibition of EDC. ZM230487, a 5-lipoxygenase inhibitor, had no effect on EDR. EIR was not affected by L-NAME, indomethacin or ZM230487. Arachidonic acid (10(-6) M) evoked EDC sensitive to indomethacin and ozagrel. L-Arginine (10(-3) M) caused EDR sensitive to L-NAME in the ionomycin-stimulated preparations. In conclusion, Ca(2+) mobilizers cause EDC and EDR via production of TXA(2) and NO, respectively.