Abstract-Epidemiological evidence suggests a role for sex-dependent mechanisms in the pathophysiology of hypertension. It has been shown that 5␣-dihydrotestosterone (DHT) administration (56 mg/kg of body weight per day IP for 14 days) increases blood pressure, cytochrome P450 4A expression, and 20-hydroxyeicosatetraenoic acid synthesis in rats.We examined whether increased vascular 20-hydroxyeicosatetraenoic acid synthesis underlies endothelial dysfunction and hypertension in DHT-treated male Sprague-Dawley rats by using HET0016, a selective cytochrome P450 4A inhibitor. Coadministration of HET0016 (10 mg/kg per day IP for 14 days) to DHT-treated rats markedly reduced DHT-induced interlobar arterial production of 20-hydroxyeicosatetraenoic acid (14.3Ϯ1.5 versus 1.5Ϯ0.5 ng/mg of protein per hour; PϽ0.05), superoxide anion (246Ϯ47 versus 31Ϯ8 cpm/g of protein), and the levels of gp91-phox, p47-phox, and 3-nitrosylated proteins. Moreover, the maximal relaxing response to acetylcholine in phenylephrinepreconstricted renal interlobar arteries from DHT-treated rats (42.8Ϯ4.8%) significantly (PϽ0.05) increased in the presence of HET0016 (81.5Ϯ10.8%). Importantly, the administration of HET0016 negated DHT-induced hypertension; systolic blood pressure was reduced from 146Ϯ2 mm Hg in DHT-treated rats to 130Ϯ1 mm Hg ( The synthesis of 20-HETE is catalyzed primarily by enzymes of the cytochrome P450 (CYP) 4A family. 6,7 CYP4A proteins are present in vascular tissues and show distinct distribution along the vascular tree. 8 Suppression and overexpression of CYP4A proteins in small arteries and arterioles decreases and increases, respectively, vascular reactivity and myogenic tone 7,9,10 ; these effects can be reversed by the addition of 20-HETE or inhibition of its synthesis.CYP4A and 20-HETE synthesis have been linked to hypertension in numerous experimental models. In the spontaneously hypertensive rat, depletion or inhibition of CYP4A activity lowers blood pressure (BP). 11,12 Inhibition of vascular 20-HETE synthesis by intravenous administration of CYP4A1 or CYP4A2 antisense oligonucleotides decreases BP in normotensive and hypertensive rats, 6,7 whereas transduction with adenoviruses expressing the CYP4A2 protein increases vascular CYP4A expression and 20-HETE levels and augments BP. 13 A role for androgens in promoting elevation of BP is well recognized 14 and, according to recent studies, such a role may rely on increased synthesis of vascular 20-HETE. Hence, mice deficient in cyp4a14 (the mouse homologue of CYP4A2) displayed androgen-sensitive hypertension, which was reversed by castration. 15 In these mice, cyp4a12 expression (the mouse homologue of CYP4A8) is elevated and so is renal microsomal 20-HETE synthesis. Similarly, androgeninduced hypertension in rats treated with 5␣-dihydrotestosterone (DHT) has been associated with increased CYP4A8 expression and renal vascular 20-HETE synthesis. 16 The mechanisms by which 20-HETE promotes hypertension are primarily linked to its ability to sensitize constrictor responsi...

Phosphofructokinases (PFK; EC 2.7.1.11) are tetrameric enzymes that have a key role in the regulation of glycolysis; as such, they are subject to allosteric activation and inhibition by various metabolites. Eukaryotic PFKs are about twice the size of prokaryotic enzymes and are regulated by a wider repertoire of effectors: for example, the subunit molecular weights of rabbit muscle (RM) PFK and Bacillus stearothermophilus (Bs) PFK are 82,000 and 36,000, respectively. Both enzymes are activated by ADP (or AMP), but RM-PFK is also activated by fructose bisphosphates (FBP) and inhibited by ATP and citrate. This, together with other evidence, has led to speculation that mammalian PFKs have evolved by duplication of a prokaryotic gene, although previous peptide analysis failed to reveal internal homology in RM-PFK. Here we demonstrate clear homology among the N- and C-halves of RM-PFK and Bs-PFK, thus establishing an evolutionary relationship by series gene duplication and divergence. Furthermore, detailed knowledge of the Bs-PFK structure provides the basis for inferences concerning the structural organization of RM-PFK and the evolution of new effector sites in the enzyme tetramer.

We used the patch-clamp technique to study the effect of arachidonic acid (AA) on epithelial Na channels (ENaC) in the rat cortical collecting duct (CCD). Application of 10 μM AA decreased the ENaC activity defined by NPo from 1.0 to 0.1. The dose–response curve of the AA effect on ENaC shows that 2 μM AA inhibited the ENaC activity by 50%. The effect of AA on ENaC is specific because neither 5,8,11,14-eicosatetraynoic acid (ETYA), a nonmetabolized analogue of AA, nor 11,14,17-eicosatrienoic acid mimicked the inhibitory effect of AA on ENaC. Moreover, inhibition of either cyclooxygenase (COX) with indomethacin or cytochrome P450 (CYP) ω-hydroxylation with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) failed to abolish the effect of AA on ENaC. In contrast, the inhibitory effect of AA on ENaC was absent in the presence of N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH), an agent that inhibits CYP-epoxygenase activity. The notion that the inhibitory effect of AA is mediated by CYP-epoxygenase–dependent metabolites is also supported by the observation that application of 200 nM 11,12-epoxyeicosatrienoic acid (EET) inhibited ENaC in the CCD. In contrast, addition of 5,6-, 8,9-, or 14,15-EET failed to decrease ENaC activity. Also, application of 11,12-EET can still reduce ENaC activity in the presence of MS-PPOH, suggesting that 11,12-EET is a mediator for the AA-induced inhibition of ENaC. Furthermore, gas chromatography mass spectrometry analysis detected the presence of 11,12-EET in the CCD and CYP2C23 is expressed in the principal cells of the CCD. We conclude that AA inhibits ENaC activity in the CCD and that the effect of AA is mediated by a CYP-epoxygenase–dependent metabolite, 11,12-EET.

The effects of a series of 102 bisphosphonates on the inhibition of growth of Entamoeba histolytica and Plasmodium falciparum in vitro have been determined, and selected compounds were further investigated for their in vivo activity. Forty-seven compounds tested were active (IC 50 < 200 µM) versus E. histolytica growth in vitro. The most active compounds (IC 50 ∼ 4-9 µM) were nitrogen-containing bisphosphonates with relatively large aromatic side chains. Simple n-alkyl-1-hydroxy-1,1-bisphosphonates, known inhibitors of the enzyme farnesylpyrophosphate (FPP) synthase, were also active, with optimal activity being found with C9-C10 side chains. However, numerous other nitrogen-containing bisphosphonates known to be potent FPP synthase inhibitors, such as risedronate or pamidronate, had little or no activity. Several pyridine-derived bisphosphonates were quite active (IC 50 ∼ 10-20 µM), and this activity was shown to correlate with the basicity of the aromatic group, with activity decreasing with increasing pK a values. The activities of all compounds were tested versus a human nasopharyngeal carcinoma (KB) cell line to enable an estimate of the therapeutic index (TI). Five bisphosphonates were selected and then screened for their ability to delay the development of amebic liver abscess formation in an E. histolytica infected hamster model. Two compounds were found to decrease liver abscess formation at 10 mg/kg ip with little or no effect on normal liver mass. With P. falciparum, 35 compounds had IC 50 values <200 µM in an in vitro assay. The most active compounds were also simple n-alkyl-1-hydroxy-1,1-bisphosphonates, having IC 50 values around 1 µM. Five compounds were again selected for in vivo investigation in a Plasmodium berghei ANKA BALB/c mouse suppressive test. The most active compound, a C9 n-alkyl side chain containing bisphosphonate, caused an 80% reduction in parasitemia with no overt toxicity. Taken together, these results show that bisphosphonates appear to be useful lead compounds for the development of novel antiamebic and antimalarial drugs.