Novel competitive inhibitor of NAD(P)H oxidase assembly attenuates vascular O(2)(-) and systolic blood pressure in mice.

We previously reported enhanced expression of the p67(phox) and gp91(phox) components of NAD(P)H oxidase in angiotensin (Ang) II-induced hypertension, suggesting de novo assembly in response to Ang II. To examine the direct involvement of NAD(P)H oxidases in Ang II-induced O(2)(-) production, we designed a chimeric peptide that inhibits p47(phox) association with gp91(phox) in NAD(P)H oxidase (gp91ds-tat). This was achieved by linking a 9-amino acid peptide (aa) derived from HIV-coat protein (tat) to a 9-aa sequence of gp91(phox) (known to interact with p47(phox)). As a control, we constructed a chimera containing tat and a scrambled gp91 sequence (scramb-tat). We found that gp91ds-tat decreased O(2)(-) levels in aortic rings treated with Ang II (10 pmol/L) but had no effect on either the O(2)(-)-generating enzyme xanthine oxidase or potassium superoxide-generated O(2)(-). We infused vehicle, Ang II (0.75 mg. kg(-1). d(-1)), Ang II+gp91ds-tat (10 mg. kg(-1). d(-1)), or Ang II+scramb-tat intraperitoneally in C57Bl/6 mice and measured systolic blood pressure (SBP) on days 0, 3, 5, and 7 of infusion. SBP increased by day 3 in mice given Ang II and Ang II+scramb-tat but was significantly lower with Ang II+gp91-tat. On day 7, SBP was still significantly inhibited in mice given Ang II+gp91ds-tat, whereas Ang II-induced O(2)(-) production was inhibited throughout the aorta as detected by dihydroethidium staining, consistent with the ability of this inhibitor to block the various vascular NAD(P)H oxidase isoforms. These data support the hypothesis that inhibition of the interaction of p47(phox) and gp91(phox) (or its homologues) can block O(2)(-) production and attenuate blood pressure elevation in mice.

Selective inactivation of NADPH oxidase 2 causes regression of vascularization and the size and stability of atherosclerotic plaques.

BACKGROUND: A variety of NADPH oxidase (Nox) isoforms including Noxs 1, 2, 4 and 5 catalyze the formation of reactive oxygen species (ROS) in the vascular wall. The Nox2 isoform complex has arguably received the greatest attention in the progression of atherogenesis in animal models. Thus, in the current study we postulated that specific Nox2 oxidase inhibition could reverse or attenuate atherosclerosis in mice fed a high-fat diet. METHODS: We evaluated the effect of isoform-selective Nox2 assembly inhibitor on the progression and vascularization of atheromatous plaques. Apolipoprotein E-deficient mice (ApoE-/-) were fed a high fat diet for two months and treated over 15 days with Nox2ds-tat or control sequence (scrambled); 10 mg/kg/day, i.p. Mice were sacrificed and superoxide production in arterial tissue was detected by cytochrome C reduction assay and dihydroethidium staining. Plaque development was evaluated and the angiogenic markers VEGF, HIF1-α and visfatin were quantified by real time qRT-PCR. MMP-9 protein release and gelatinolytic activity was determined as a marker for vascularization. RESULTS: Nox2ds-tat inhibited Nox-derived superoxide determined by cytochrome C in carotid arteries (2.3 ± 0.1 vs 1.7 ± 0.1 O2(•-) nmol/min*mg protein; P < 0.01) and caused a significant regression in atherosclerotic plaques in aorta (66 ± 6 µm(2) vs 37 ± 1 µm(2); scrmb vs. Nox2ds-tat; P < 0.001). Increased VEGF, HIF-1α, MMP-9 and visfatin expression in arterial tissue in response to high-fat diet were significantly attenuated by Nox2ds-tat which in turn impaired both MMP-9 protein expression and activity. CONCLUSION: Given these results, it is quite evident that selective Nox inhibitors can reverse vascular pathology arising with atherosclerosis.

A local paracrine and endocrine network involving TGFß, Cox-2, ROS, and estrogen receptor ß influences reactive stromal cell regulation of prostate cancer cell motility.

The tumor microenvironment plays a critical role in supporting cancer cells particularly as they disengage from limitations on their growth and motility imposed by surrounding nonreactive stromal cells. We show here that stromal-derived androgenic precursors are metabolized by DU145 human prostate cancer (PCa) cells to generate ligands for estrogen receptor-ß, which act to limit their motility through transcriptional regulation of E-cadherin. Although primary human PCa-associated fibroblasts and the human WPMY-1-reactive prostate stromal cell line maintain this inherent estrogen receptor (ER)ß-dependent motility inhibitor activity, they are subverted by TGF-ß1 pro-oxidant signals derived from cocultured DU145 PCa cells. Specifically, stromal-produced H(2)O(2), which requires Cox-2, acts as a second paracrine factor to inhibit ERß activity in adjacent DU145 cells. Chromatin immunoprecipitation analysis reveals that ERß recruitment to the E-cadherin promoter is inhibited when H(2)O(2) is present. Both neutralization of H(2)O(2) with catalase and prevention of its production by silencing Cox-2 expression in stromal cells restore the motility-suppression activity of stromal-derived ERß ligand precursors. These data suggest that reactive stromal cells may still have a capacity to limit cancer cell motility through a local endocrine network but must be protected from pro-oxidant signals triggered by cancer cell-derived TGF-ß1 to exhibit this cancer-suppressive function.

Polylysine induces a rapid Ca2+ release from sarcoplasmic reticulum vesicles by mediation of its binding to the foot protein.

The addition of polylysine to a heavy fraction of sarcoplasmic reticulum (SR) vesicles produces a rapid Ca2+ release with no appreciable lag period. The polylysine concentration for half-maximal activation (C1/2) is approximately 0.99 micrograms/ml, or 0.3 microM, the lowest C 1/2 for Ca2+ release-inducing reagents reported in the literature. The time course and the [Ca2+] dependence of polylysine-induced release are similar to those of caffeine-induced Ca2+ release. At higher concentrations of polylysine (e.g., 10 micrograms/ml), however, little or no Ca2+ release occurs. Upon photolysis of SR vesicles with the photocrosslinkable radiolabeled polylysine derivative, [3H]succinimidyl azido benzoate polylysine, 0.28 and 0.52-1.2 mol polylysine were bound to 1 mol of the 400-kDa foot protein at activating (3 micrograms/ml) and inhibitory (10 micrograms/ml) concentrations of polylysine, respectively. On the other hand, the amounts of polylysine bound to the other SR proteins (mol/mol) were negligible (e.g., less than or equal to 0.0127 mol polylysine/mol calsequestrin). This suggests that the binding of polylysine to the foot protein is responsible not only for the induction of release but also for inactivation. These results provide direct evidence that the receptor for the chemical trigger of Ca2+ release is localized within the foot protein. Ruthenium red, which inhibits polylysine-induced Ca2+ release, does not inhibit polylysine binding to the foot protein, suggesting that the polylysine binding domain of the foot protein is different from the channel domain.

Proteolytic fragments of phosphoinositide-specific phospholipase C-delta 1. Catalytic and membrane binding properties.

Active proteolytic fragments of phosphoinositide-specific phospholipase C-delta 1 (PLC-delta 1) were generated by trypsin digestion of the native protein. Brief proteolysis produced a 77-kDa fragment that contained the highly conserved X and Y regions but lacked the amino-terminal domain (amino acids 1-60). Prolonged digestion of PLC-delta 1 produced two fragments, one of 45 kDa that contained the entire X region and another of 32 kDa that consisted of the entire Y region and COOH-terminal domain. The 45- and 32-kDa fragments were isolated as an active heterodimeric complex. The 77-kDa fragment and the complex catalyzed calcium-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in detergent/phospholipid mixed micelles. When compared with the native enzyme, both the 77-kDa fragment and the complex exhibited a reduced capacity to processively hydrolyze PIP2; increasing the mole fraction of PIP2 in the mixed micelle surface greatly increased the rate of PIP2 hydrolysis catalyzed by the native enzyme but not the fragments. Both fragments also exhibited a reduced affinity for substrate; the native enzyme bound to bilayer vesicles consisting of phosphatidylcholine and PIP2 with high affinity (Ka approximately 10(6) M-1), whereas the fragments bound weakly (Ka < 10(4) M-1). These results demonstrate that the X, Y, and COOH-terminal regions form a calcium-dependent catalytic core that is resistant to proteolysis. The amino-terminal domain appears to be essential for high affinity binding to PIP2 but not catalysis. These observations are consistent with the idea that the amino-terminal domain forms part of a PIP2 binding site, which anchors PLC-delta 1 to the membrane surface during processive hydrolysis of its substrate.

D-myo-inositol 1,4,5-trisphosphate inhibits binding of phospholipase C-delta 1 to bilayer membranes.

The binding of phosphoinositide-specific phospholipase C-delta 1 (PLC-delta 1) to bilayer membranes composed of phosphatidylcholine (PC) and phosphatidylinositol 4,5-bisphosphate (PIP2) was measured in the presence or absence of inositol phosphates. Binding was inhibited by the natural D-isomer of myo-inositol 1,4,5-trisphosphate (D-InsP3), but not by the L-isomer. The concentration of D-InsP3 required to decrease binding by 50% was 5.4 +/- 0.5 microM. 1-(alpha-Glycerophosphoryl)-D-myo-inositol 4,5-bisphosphate and D-myo-inositol 2,4,5-trisphosphate were nearly as effective as D-Ins(1,4,5)P3. D-myo-inositol monophosphate with phosphate esterified at either positions 1 or 2 of the myo-inositol ring, had no significant effect on binding. D-myo-inositol 1,4-bisphosphate weakly inhibited the binding, whereas the 4,5-isomer was nearly as potent as D-InsP3. Neither ATP nor inorganic phosphate significantly affected binding. As expected, D-Ins(1,4,5)P3 but not L-Ins(1,4,5)P3 decreased the initial rate of PIP2 hydrolysis in bilayer vesicles. The concentration required to decrease hydrolysis by 50% was 12.4 +/- 0.5 microM. A catalytic fragment of PLC-delta 1 that lacks a domain necessary for high affinity PIP2 binding was prepared as previously described (Cifuentes, M. E., Honkanen, L., and Rebecchi, M. J. (1993) J. Biol. Chem. 268, 11586-11593). In contrast to the native enzyme, the rate of PIP2 hydrolysis, catalyzed by the fragment, was not affected by D-Ins(1,4,5)P3. These data suggest that high affinity binding of the enzyme to PIP2 and processive catalysis, involve specific recognition of the 4- and 5-position phosphates of the inositol ring. Our results are consistent with feedback inhibition by the polar head group product, D-Ins(1,4,5)P3, at a step that precedes catalysis, namely interfacial recognition.

Hormonal control of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression in rat hepatoma cells.

The hormonal control of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression was studied in the rat hepatoma cells, FTO-2B. In contrast to another hepatoma cell line (HTC), the enzyme in FTO-2B cells displays both kinase and bisphosphatase activities. As in rat liver, the mRNA in FTO-2B cells is 2.2-kilobases in length. However, the 5' region of the mRNA differs from the mRNA in the liver in that it contains sequences unique to 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA from skeletal muscle. These results suggest that the mRNA in FTO-2B cells may represent an additional alternative splicing product of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene. Exposure of FTO-2B cells to media containing either insulin (10(-7) M) or dexamethasone (10(-6) M) induced about a 10-fold increase in the level of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA within 6-10 h of hormone treatment. The concentrations of insulin or dexamethasone giving half-maximal stimulation were 10(-9) M and 2 x 10(-8) M, respectively, and dibutyryl cyclic AMP (5 x 10(-7) M) completely prevented the increase in enzyme mRNA induced by these hormones. Exposure of cells to glucose-free medium abolished the insulin-mediated enhancement in 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA, but not that induced by dexamethasone. No alteration in the degradation rate of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA was noted when cells were treated with insulin. Run-on transcription assays with isolated nuclei showed an increase in the relative transcription rate of the gene in cells treated with either insulin or dexamethasone. The time course of transcription activation preceded the increase in the level of the mRNA, indicating that the main mechanism for the induction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase expression by insulin and dexamethasone is mediated by stimulation of gene transcription.

Upregulation of p67(phox) and gp91(phox) in aortas from angiotensin II-infused mice.

Although NAD(P)H oxidase-derived superoxide (O(2)(-)) is increased during the development of angiotensin II (ANG II)-dependent hypertension, vascular regulation at the protein level has not been reported. We have shown that four major components of NAD(P)H oxidase are located primarily in the vascular adventitia as a primary source of vascular O(2)(-). Here we compare vascular levels of O(2)(-) and NAD(P)H oxidase in normotensive and ANG II-infused hypertensive mice and show that, after 7 days of ANG II infusion (750 microg. kg(-1). day(-1) ip) in C57B1/6 mice, systolic blood pressure was increased compared with that after sham infusion, concomitant with increased O(2)(-) in the thoracic aorta as measured using lucigenin (25 microM)-enhanced chemiluminescence. Both p67(phox) and gp91(phox) were detectable by Western blotting in aortic homogenates, and we observed increased protein levels of NAD(P)H oxidase subunits. These ANG II-induced increases were normalized by simultaneous treatment with the AT(1) receptor antagonist losartan. Moreover, the primary location of these subunits was the adventitia as detected immunohistochemically. Our results suggest that ANG II-induced increases in O(2)(-) are due to increased adventitial NAD(P)H oxidase activity, brought about by the heightened expression and interaction of its components.