20-Hydroxyeicosatetraenoic acid is excreted as a glucuronide conjugate in human urine.

20-hydroxyeicosatetraenoic acid, a major renal P-450 metabolite of arachidonic acid, has been quantified in human urine using capillary gas chromatography/electron capture negative ion chemical ionization mass spectrometry. The urinary excretion of 20-hydroxyeicosatetraenoic acid was in the low pg/ml range. However, treatment of urine with beta-glucuronidase resulted in a 13- to 28-fold increase in its concentration. This suggests 20-hydroxyeicosatetraenoic acid differs from other eicosanoids in that it is excreted primarily as a glucuronide conjugate.     

Nicorandil is a potent dilator of endothelin-induced vascular contraction

Nicorandil, an antianginal drug, is known to open K+ channel and to increase cGMP production. The effects of nicorandil on vascular contraction induced by endothelin (ET), a potent newly discovered vasoconstrictor peptide, were investigated using helical strips from rat thoracic aorta. ET at a concentration of 5 x 10(-9) M induced strong and persistent contraction in the presence of extracellular Ca2+ and similar persistent but smaller contraction in the absence of extracellular Ca2+. Nicorandil at concentrations greater than 10(-7) M, strongly and dose-dependently inhibited ET-induced contraction in the presence of extracellular Ca2+. Nicorandil also suppressed ET-induced contraction in the presence of 10(-4) M methylene blue, an inhibitor of cGMP production, in the presence of extracellular Ca2+ but not in the absence of extracellular Ca2+. ET-induced contraction was also inhibited to lesser extents by the Ca2+ channel blockers nicardipine and verapamil. Nicorandil also strongly suppressed ET-induced increase in cytosolic free Ca2+ concentration in cultured vascular smooth muscle cells. These results suggest that nicorandil is a potent dilator of ET-induced vasoconstriction.     

20-Hydroxyeicosatetraenoic acid (20-HETE) is a novel activator of transient receptor potential vanilloid 1 (TRPV1) channel

TRPV1 is a member of the transient receptor potential ion channel family and is gated by capsaicin, the pungent component of chili pepper. It is expressed predominantly in small diameter peripheral nerve fibers and is activated by noxious temperatures >42 °C. 20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P-450 4A/4F-derived metabolite of the membrane phospholipid arachidonic acid. It is a powerful vasoconstrictor and has structural similarities with other TRPV1 agonists, e.g. the hydroperoxyeicosatetraenoic acid 12-HPETE, and we hypothesized that it may be an endogenous ligand for TRPV1 in sensory neurons innervating the vasculature. Here, we demonstrate that 20-HETE both activates and sensitizes mouse and human TRPV1, in a kinase-dependent manner, involving the residue Ser(502) in heterologously expressed hTRPV1, at physiologically relevant concentrations.     

19-Hydroxyeicosatetraenoic acid analogs: Antagonism of 20-hydroxyeicosatetraenoic acid-induced vascular sensitization and hypertension

19-Hydroxyeicosatetraenoic acid (19-HETE, 1), a metabolically and chemically labile cytochrome P450 eicosanoid, has diverse biological activities including antagonism of the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE, 2). A SAR study was conducted to develop robust analogs of 1 with improved in vitro and in vivo efficacy. Analogs were screened in vitro for inhibition of 20-HETE-induced sensitization of rat renal preglomerular microvessels toward phenylephrine and demonstrated to normalize the blood pressure of male Cyp4a14(-/-) mice that display androgen-driven, 20-HETE-dependent hypertension.     

20-Hydroxyeicosatetraenoic acid synthesis is increased in human neutrophils and platelets by angiotensin II and endothelin-1

The cytochrome P-450 arachidonic acid metabolite 20-HETE is central to the regulation of vascular tone, renal function, and blood pressure and is synthesized in the rat kidney in response to angiotensin II (ANG II) and endothelin-1 (ET-1). There are very few studies examining the cellular synthesis of 20-HETE in humans. We aimed to measure human neutrophil and platelet 20-HETE levels under basal conditions and after ANG II, ET-1, and calcium ionophore (CaI). 20-HETE was measured in human platelets and neutrophils after saline (control), CaI (2.5 μg/ml), and ANG II or ET-1 (10 nmol/l-1 μmol/l) incubations. The effect of cells, which were preincubated with the ω-hydroxylase inhibitor N-hydroxy-N'-(4-butyl-2-methylphenyl) (HET0016, 10 nM), ANG II types 1 or 2 (AT(1) or AT(2)) receptor inhibition with irbesartan (1 μmol/l) or PD-123319 (1 μmol/l), or endothelin receptor subtypes A or B (ET(A) or ET(B)) receptor inhibition with BQ-123 or BQ-778 (100 nmol/l), was studied. Neutrophil and platelet content and release of 20-HETE was significantly increased by CaI and blocked by the ω-hydroxylase inhibitor HET0016. ANG II and ET-1 significantly increased neutrophil and platelet content and release of 20-HETE. ANG II increased 20-HETE via the AT(2) receptor. ET-1 increased 20-HETE through the ET(B) receptor in platelets and both the ET(A) and ET(B) receptors in neutrophils. These studies show that human platelets and neutrophils synthesize 20-HETE in response to ANG II and ET-1. 20-HETE synthesis in both cell types was predominantly mediated via the AT(2) and ET(B) receptors. Stimulation via these receptor pathways has generally been thought to be cardioprotective and requires further studies in clinical situations associated with low-grade inflammation or where ANG II and ET-1 are elevated to clarify the role of 20-HETE.     

20-Hydroxyeicosatetraenoic acid (20-HETE) stimulates migration of vascular smooth muscle cells.

AIM: We tested the hypothesis that 20-HETE production contributes to platelet derived growth factor (PDGF)-BB stimulated migration of VSMC in a cell culture model. METHODS: Studies were performed with A10 cells which are a rat vascular smooth muscle derived cell line. Migration was determined using a Boyden chamber chemotactic assay. RESULTS: Pre-treatment of cells with two doses of 20-HETE (100 and 500 nM) significantly increased PDGF-BB stimulated VSMC migration by 34-58% of control; whereas, prior incubation of cells with inhibitors of 20-HETE production, 17-ODYA (1-25 M) or HET0016 (100 nM), significantly decreased PDGF-BB stimulated migration by 40-90%. 20-HETE mediated increase in PDGF-BB migration was completely prevented by the 20-HETE antagonist, WIT-002. In order to determine what second messenger pathways are involved in the 20-HETE mediated stimulation of VSM migration, experiments were performed with specific inhibitors of tyrosine kinase (tyrphostin 25, 10 microM), mitogen-activated extracellular signal-regulated kinase (MEK, PD98059, 20 microM and U0126, 10 microM), protein kinase C (Myr-PKC, 50 microM), and phosphoinositide 3-kinases (PI3Ks) (wortmannin, 50 nM). Blockade of MEK and PI3K all abolished the increase in 20-HETE mediated migration. CONCLUSION: 20-HETE stimulates PDGF-mediated VSM migration acting through pathways that involve MEK and PI3K.     

12-Hydroxyeicosatetraenoic acid is metabolized by beta-oxidation in mouse peritoneal macrophages. Identification of products and proposed pathway

The products derived from the metabolism of 12-hydroxyeicosatetraenoic acid (12-HETE) by mouse peritoneal macrophages were characterized by high performance liquid chromatography (HPLC) and GC-mass spectrometry. HPLC analysis demonstrated two predominant polar products and several minor ones. The proportion and amounts of these products were dependent on the concentration of 12-HETE, the number of macrophages incubated with the monohydroxy fatty acid, and the time of incubation. The products identified by GC-mass spectrometry suggested that 12-HETE had undergone beta-oxidation. The intermediates identified were: 3,12-dihydroxy-5,8,10,14, 20:4; 10-hydroxy-3,6,8,12, 18:4; 3,10-dihydroxy-6,8,12, 18:3; 8-hydroxy-4,6,10, 16:3; 6-hydroxy-4,8, 14:2; and 4-hydroxy, 12:1. The major products, as identified by HPLC and GC-mass spectrometry, were 8-hydroxy-4,6,10, 16:3 and 4-hydroxy, 12:1. A minor product, 10-hydroxy-6,8,12, 18:3 was postulated to arise from either the isomerization and reduction of 10-hydroxy-3,6,8,12, 18:4 or from chain elongation of 8-hydroxy-4,6,10, 16:3. Inhibiting cyclooxygenase and lipoxygenase activities by ibuprofen and nordihydroguaiaretic acid, respectively, did not inhibit the formation of these products. 82% to 98% of 12-HETE was converted and released into the medium as products of beta-oxidation. The remainder was taken up into cellular lipids. beta-Oxidation of 12-HETE was decreased by only 12 and 21% after inhibiting mitochondrial fatty acid oxidation by 89 and 93% by 5 and 100 microM concentrations of the mitochondrial fatty acid oxidation inhibitor, methyl palmoxirate, respectively. It is thus postulated that the beta-oxidation of 12-HETE by mouse peritoneal macrophages occurs in peroxisomes.     

Chebulagic acid is a potent alpha-glucosidase inhibitor

Chebulagic acid, isolated form Terminalia chebula Retz, proved to be a reversible and non-competitive inhibitor of maltase with a K(i) value of 6.6 muM. The inhibitory influence of chebulagic acid on the maltase-glucoamylase complex was more potent than on the sucrase-isomaltase complex. The magnitude of alpha-glucosidase inhibition by chebulagic acid was greatly affected by its origin. These results show a use for chebulagic acid in managing type-2 diabetes.     

Depside as potent inhibitor of prostaglandin biosynthesis: a new active site model for fatty acid cyclooxygenase

Forty depsides and depsidones, the esters of phenolcarboxylic acids, were examined for their inhibitory effect against prostaglandin biosynthesis with rabbit renal microsomes. 4-0-Methylcryptochlorophaeic acid was the most active inhibitor so far tested and its IC50 value was 0.34 muM. Kinetic investigation has shown that this depside acts competitively with respect to arachidonic acid as most of the non - steroidal antiinflammatory drugs. X-Ray analysis has revealed that 4-0-methylcryptochlorophaeic acid maintains its rigid conformation by forming a strong hydrogen bond between the hydroxyl and methoxyl groups. Comparison of CPK models between 4-0-methylcryptochlorophaeic acid and non-steroidal antiinflammatory drugs revealed that the carboxyl group and the two rings of these drugs are almost superimposable to those of the depside. This finding led us to propose a new active site model based on the three dimentional structure of the depside.     

Aurintricarboxylic acid is a potent inhibitor of phosphofructokinase.

Aurintricarboxylic acid (ATA) was found to be a very potent inhibitor of purified rabbit liver phosphofructokinase (PFK), giving 50% inhibition at 0.2 microM. The inhibition was in a manner consistent with interaction at the citrate-inhibitory site of the enzyme. The data suggest that inhibition of PFK by ATA was not due to denaturation of the enzyme or the irreversible binding of inhibitor, since the inhibition could be reversed by addition of allosteric activators of PFK, i.e. fructose 2,6-bisphosphate or AMP. Two other tricarboxylic acids, agaric acid and (-)-hydroxycitrate, were found to inhibit PFK. ATA at much higher concentrations (500 microM) was shown to inhibit fatty acid synthesis from endogenous glycogen in rat hepatocytes; however, protein synthesis was not altered.     

Ribonucleic acid is a component of the oligomeric, transformed mouse AtT-20 cell glucocorticoid receptor

The glucocorticoid receptor from mouse AtT-20 pituitary tumor cells exists in three forms. The largest form is an untransformed (non-DNA-binding), oligomeric species (9.1 S, 8.3 nm, Mr 319 000). Two transformed (DNA-binding) forms can be generated. One is an oligomeric protein (5.2 S, 6-8.3 nm, Mr 132 000-182 000), while the other is the monomeric, hormone-binding subunit (3.8 S, 6 nm, Mr 96 000). The composition of the oligomeric, transformed receptor and its relationship to the monomeric protein were examined. The 3.8S monomer can be isolated from DEAE-cellulose (0.12 M step elution) in a form that continues to sediment at about 3.8 S on molybdate-containing sucrose gradients and at about 4.2 S on molybdate-free gradients. Addition of a non-hormone-binding component isolated from the same DEAE-cellulose column (0.5 M KCl step) can apparently interact with the 3.8-4.2 S monomer, increasing its sedimentation coefficient to 5.2 S (on molybdate-containing gradients) or 6.6 S (on low-salt, molybdate-free gradients). This factor is a macromolecule (nondialyzable) and is heat-stable (100 degrees C, 20 min). A dose-dependent shift to the higher sedimentation coefficient is observed when increasing quantities of the 0.5 M step material are added to the receptor monomer. This activity is abolished when the 0.5 M step material is treated with ribonuclease A. Further, when RNA is purified from the 0.5 M step by phenol/chloroform extraction, its ability to increase the S value of the monomer is retained. Ribonuclease treatment of the untransformed, 9.1S, oligomeric complex does not cause a significant decrease in sedimentation rate, while the same treatment of the 5.2S, oligomeric, transformed receptor (obtained after Sephadex G-25 transformation) causes a decrease in sedimentation rate to about 3.8 S. The addition of bovine liver mRNA and rRNA does not cause a shift in sedimentation rate of the receptor monomer to a discrete, higher sedimenting receptor form. However, the addition of total rabbit liver tRNA or three distinct tRNA species causes a shift in sedimentation to a similar, but not identical, form as that with the 0.5 M step material. We propose that the 5.2S, oligomeric transformed glucocorticoid receptor is composed of one monomeric hormone-binding, protein subunit (Mr 96 000) and a low molecular weight RNA (Mr 36 000). This interaction may be important for the role of the receptor in regulating gene expression.     

17S,20S-Methanofusidic acid,a new potent semi-synthetic fusidane antibiotic

A novel fusidic acid type antibiotic having the side chain linked to the tetracyclic ring system via a spiro-cyclopropane system is described. 17S,20S-Methanofusidic acid is obtained by an efficient synthetic route including cyclopropanation of the Δ17(20) bond with attack solely from the least hindered -face. The spiro-cyclopropane system orients the side chain into a bioactive conformational space. The new 17S,20S-methanofusidic acid exerts antibacterial activity against several Gram-positive species with potency essentially equal to natural fusidic acid.     

O6C-20-nor-salvinorin A is a stable and potent KOR agonist

Salvinorin A (SalA) is a potent and selective agonist of the kappa-opioid receptor (KOR), but its instability has frustrated medicinal chemistry efforts. Treatment of SalA with weak bases like DBU leads to C8 epimerization with loss of receptor affinity and signaling potency. Here we show that replacement of C20 with H and replacement of O6 with CH₂ stabilizes the SalA scaffold relative to its C8 epimer, so much so that epimerization is completely supressed. This new compound, O6C-20-nor-SalA, retains high potency for agonism of KOR.     

The psoriasis drug monomethylfumarate is a potent nicotinic acid receptor agonist

Nicotinic acid has been used for several decades to treat dyslipidemia. In mice, the lipid-lowing effect of nicotinic acid is mediated by the Gi coupled receptor PUMA-G. In humans, high (GPR109A) and low (GPR109B) affinity nicotinic acid receptors have been characterized. Here we identify monomethylfumarate as a GPR109A agonist. Monomethylfumarate is the active metabolite of the psoriasis drug Fumaderm. We show that monomethylfumarate activates GPR109A in a calcium based aequorin assay, cAMP assay and demonstrate competitive binding with nicotinic acid. We show that GPR109A is highly expressed in neutrophils and epidermal keratinocytes, and that its expression is increased in human psoriatic lesions. Our findings provide evidence that GPR109A is a target for the drug Fumaderm and suggest that niacin should be investigated to treat psoriasis in addition to its role in treating lipid disorders.     

Quinolinic acid is a potent lipid peroxidant in rat brain homogenates

In this study, we describe the lipoperoxidative effect of quinolinic acid (QUIN) in vitro. The formation of thiobarbituric acid reactive products (TBA-RP), an index of lipid peroxidation, was measured in rat brain homogenates after incubation at 37°C for 30 min in the presence of QUIN and some structurally and metabolically related compounds such as Kynurenine, Kynurenic acid, Glutamate, Aspartate and Kainate. Concentrations of QUIN in the range of 20 to 80 M increased lipid peroxidation in a concentration-dependent manner from about 15% to about 50%. Kynurenic acid, a compound metabollically related to QUIN that can block its neurotoxic actions in vivo, also inhibited completely the QUIN-induced TBA-RP formation in our system. Lipid fluorescent material, another index of lipid peroxidation was also found increased by 49% after incubation with 40 M QUIN. It is concluded that lipid peroxidation may be a damaging process involved in the neurotoxicity of QUIN.     

Phenyl-n-butylborinic acid is a potent transition state analog inhibitor of lipolytic enzymes

The cholesterol esterase and lipoprotein lipase catalyzed hydrolyses of the water-soluble substrate p-nitrophenyl butyrate are competitively inhibited by butaneboronic acid and phenylboronic acid. Phenyl-n-butylborinic acid has been synthesized and characterized as an ultrapotent transition state analog inhibitor: Ki = 2.9 +/- 0.6 nM and 1.7 +/- 0.3 microM for the cholesterol esterase and lipoprotein lipase reactions, respectively. These results are interpreted in terms of transition state structure and stabilization.     

Phosphatidylinositol-3,5-Bisphosphate is a potent and selective inhibitor of acid sphingomyelinase

Acid sphingomyelinase (A-SMase, EC 3.1.4.12) catalyzes the lysosomal degradation of sphingomyelin to phosphorylcholine and ceramide. Inherited deficiencies of acid sphingomyelinase activity result in various clinical forms of Niemann-Pick disease, which are characterised by massive lysosomal accumulation of sphingomyelin. Sphingomyelin hydrolysis by both, acid sphingomyelinase and membrane-associated neutral sphingomyelinase, plays also an important role in cellular signaling systems regulating proliferation, apoptosis and differentiation. Here, we present a potent and selective novel inhibitor of A-SMase, L-alpha-phosphatidyl-D-myo-inositol-3,5-bisphosphate (PtdIns3,5P2), a naturally occurring substance detected in mammalian, plant and yeast cells. The inhibition constant Ki for the new A-SMase inhibitor PtdIns3,5P2 is 0.53 microM as determined in a micellar assay system with radiolabeled sphingomyelin as substrate and recombinant human A-SMase purified from insect cells. Even at concentrations of up to 50 microM, PtdIns3,5P2 neither decreased plasma membrane-associated, magnesium-dependent neutral sphingomyelinase activity, nor was it an inhibitor of the lysosomal hydrolases beta-hexosaminidase A and acid ceramidase. Other phosphoinositides tested had no or a much weaker effect on acid sphingomyelinase. Different inositol-bisphosphates were studied to elucidate structure-activity relationships for A-SMase inhibition. Our investigations provide an insight into the structural features required for selective, efficient inhibition of acid sphingomyelinase and may also be used as starting point for the development of new potent A-SMase inhibitors optimised for diverse applications.     

Transforming growth factor is a potent stimulator of testicular ornithine decarboxylase in immature mouse

Administration of transforming growth factor, type alpha (TGF-alpha), to eight-day old mice resulted in 22 fold increase of testicular ornithine decarboxylase activity. Two to seven fold increases of enzyme activities by TGF-alpha were also observed in intestine, kidney, spleen, liver, and heart. The maximal enzymatic responses were reached 2-4 h after TGF-alpha administration in vivo. The induction of tissue ornithine decarboxylase activity was accompanied by an increase in new protein synthesis. These effects were found to be comparable to those in littermates administered with mouse epidermal growth factor but were significantly more pronounced than with bovine growth hormone. Daily administration of TGF-alpha to newborn mice also produced a 1.8 fold increase of testicular weight after 14 days. The present studies therefore show that TGF-alpha is an epidermal growth factor-like mitogen and is likely to be important for the testicular development of immature animal.