Sustained adrenergic signaling leads to increased metastasis in ovarian cancer via increased PGE2 synthesis.

Adrenergic stimulation adversely affects tumor growth and metastasis, but the underlying mechanisms are not well understood. Here, we uncovered a novel mechanism by which catecholamines induce inflammation by increasing prostaglandin E2 (PGE2) levels in ovarian cancer cells. Metabolic changes in tumors isolated from patients with depression and mice subjected to restraint stress showed elevated PGE2 levels. Increased metabolites, PTGS2 and PTGES protein levels were found in Skov3-ip1 and HeyA8 cells treated with norepinephrine (NE), and these changes were shown to be mediated by ADRB2 receptor signaling. Silencing PTGS2 resulted in significantly decreased migration and invasion in ovarian cancer cells in the presence of NE and decreased tumor burden and metastasis in restraint stress orthotopic models. In human ovarian cancer samples, concurrent increased ADRB2, PTGS2 and PTGES expression was associated with reduced overall and progression-free patient survival. In conclusion, increased adrenergic stimulation results in increased PGE2 synthesis via ADRB2-Nf-kB-PTGS2 axis, which drives tumor growth and metastasis.

Effect of plant density and mixing ratio on crop yield in sweet corn/mungbean intercropping.

In order to evaluate the ear and forage yield of sweet corn (Zea mays L. var. Saccarata) in pure stand and intercropped with mung bean (Vigna radiata L.), a field experiment was conducted at Varamin region on summer 2006. Experiment was carried out in a split plot design based on randomized complete blocks with 4 replications. Plant density with 3 levels [Low (D1), Mean (D2) and High (D3) respecting 6, 8 and 10 m(-2) for sweet corn, cultivar S.C.403 and 10, 20 and 30 m(-2) for mung bean cultivar, Partow] was arranged in main plots and 5 mixing ratios [(P1) = 0/100, (P2) = 25/75, (P3) = 50/50, (P4) = 75/25, (P5) = 100/0% for sweet corn/mung bean, respectively] were arranged in subplots. Quantitative attributes such as plant height, sucker numbers, LER, dry matter distribution in different plant organs were measured in sweet corn economical maturity. Furthermore the yield of cannable ear corn and yield components of sweet corn and mung bean were investigated. Results showed that plant density has not any significant effect on evaluated traits, while the effect of mixing ratio was significant (p < 0.01). Therefore, the mixing ratio of 75/25 (sweet corn/mung bean) could be introduced as the superior mixing ratio; because of it's maximum rate of total sweet corn's biomass, forage yield, yield and yield components of ear corn in intercropping. Regarding to profitability indices of intercropping, the mixing ratio 75/25 (sweet corn/mung bean) in low density (D1P2) which showed the LER = 1.03 and 1.09 for total crop yield before ear harvesting and total forage yield after ear harvest respectively, was better than corn or mung bean monoculture.

Heme oxygenase isoform-specific expression and distribution in the rat kidney.

BACKGROUND: The heme oxygenase (HO) genes, HO-1 and HO-2, are the limiting steps in heme degradation and in the regulation of renal heme-dependent enzymes. Previously we reported that selective overexpression of renal HO-1 resulted in a decrease of microsomal heme and the cytochrome P450-dependent arachidonic acid metabolite, 20 HETE, a vasoconstrictor. The present study was undertaken to explore the relative expression and contribution of each of the HO isoforms to HO activity in the rat kidney. METHODS AND RESULTS. Renal HO activity increased above control levels after an injection of the inducers of HO activity, heme or SnCl2. Stannous Mesoporphyrin (SnMP), a nonselective inhibitor of HO, when used alone or in combination with heme or SnCl2, decreased HO activity. Heme alone and combined with SnCl2 decreased the levels of heme content by 13 and 35%, respectively. Western blot analysis showed that both SnCl2 and heme readily induced HO-1 protein, whereas HO-2 was constitutively expressed. Immunohistochemistry showed the distribution of the HO-1 isoform primarily in proximal convoluted tubules. Western blot analysis exhibited relatively higher levels of HO-1 in isolated proximal tubules and relatively higher HO-2 levels in the thick ascending limbs of the loop of Henle and preglomerular arterioles. In vivo administration of HO-1 and HO-2 antisense oligodeoxynucleotides further confirmed that HO-2, but not HO-1, contributed to the basal HO activity; however, following induction of HO with heme, antisense to HO-1, but not to HO-2, inhibited the induced levels of HO activity. CONCLUSION: These results suggest that HO-2 is constitutively expressed in the rat kidney mainly within tubular and arteriolar structures, and its activity may modulate physiological function under basal conditions. On the other hand, the basal levels of expression of HO-1 in the rat kidney are relatively low, and its contribution to HO activity and the regulation of hemoproteins such as cytochrome P450 become apparent only under pathophysiological conditions causing HO induction.

CYP4A1 antisense oligonucleotide reduces mesenteric vascular reactivity and blood pressure in SHR.

The cytochrome P-450 4A (CYP4A)-derived arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) affects renal tubular and vascular functions and has been implicated in the control of arterial pressure. We examined the effect of antisense oligonucleotide (ODN) to CYP4A1, the low K(m) arachidonic acid omega-hydroxylating isoform, on vascular 20-HETE synthesis, vascular reactivity, and blood pressure in the spontaneously hypertensive rat (SHR). Administration of CYP4A1 antisense ODN decreased mean arterial blood pressure from 137 +/- 3 to 121 +/- 4 mmHg (P < 0.05) after 5 days of treatment, whereas treatment with scrambled antisense ODN had no effect. Treatment with CYP4A1 antisense ODN reduced the level of CYP4A-immunoreactive proteins along with 20-HETE synthesis in mesenteric arterial vessels. Mesenteric arteries from rats treated with antisense ODN exhibited decreased sensitivity to the constrictor action of phenylephrine (EC(50) 0.69 +/- 0.17 vs. 1.77 +/- 0.40 microM). Likewise, mesenteric arterioles from antisense ODN-treated rats revealed attenuation of myogenic constrictor responses to increases of transmural pressure. The decreased vascular reactivity and myogenic responses were reversible with the addition of 20-HETE. These data suggest that CYP4A1-derived 20-HETE facilitates myogenic constrictor responses in the mesenteric microcirculation and contributes to pressor mechanisms in SHR.

Adenoviral vector-mediated transfer of human heme oxygenase in rats decreases renal heme-dependent arachidonic acid epoxygenase activity.

Intravenous administration of an adenovirus human heme oxygenase (HO)-1 gene construct to rats resulted in functional expression of human HO-1 in brain, heart, lung, liver, and kidney. Because accurate assessment of human HO-1 mRNA in various tissues by Northern analysis is not sufficiently sensitive, we developed a method for quantifying human HO-1 mRNA copies with quantitative reverse transcription- polymerase chain reaction techniques; this allowed us to use the same primers for both the sample and internal standard. Administration of the adenovirus human HO-1 gene resulted in the detection of human HO-1 mRNA in various tissues with the highest levels seen in the kidney followed, in order, by lung > liver > brain > heart. Human HO-1 was detectable for up to 4 weeks in all tissues studied. Administration of adenovirus human HO-1 resulted in maximal increase of HO activity after 1 to 2 weeks in rats. The increase in HO activity due to gene transfer also was associated with a parallel decrease (approximately 25%) in cytochrome P-450 (CYP) content and in CYP-dependent arachidonic acid metabolism. In addition, we investigated the possibility that the human HO-1 gene altered the expression of the endogenous rat enzyme after administration of cobalt chloride s.c. Cobalt chloride administration resulted in increased HO activity in all tissues examined in rats transduced with the human HO-1 gene to the same degree as in nontransduced rats. The metal was a more potent inducer of renal HO activity than was the adenoviral-mediated human HO-1 vector. The increase in HO activity after adenoviral-mediated human HO-1 transfer was associated with a decrease in microsomal heme-CYP and CYP activity. The increase in HO-1 activity after adenovirus-mediated human HO-1 gene transfer may prove useful as a means of selectively increasing enzyme activity in a specific organ and regulating homeostasis by modulation of vasoactive molecules such as carbon monoxide and bilirubin and, in addition, providing a means of delivering the human HO-1 gene for experimental purposes.

Contribution of cytochrome P-450 4A1 and 4A2 to vascular 20-hydroxyeicosatetraenoic acid synthesis in rat kidneys.

20-Hydroxyeicosatetraenoic acids (20-HETE), a biologically active cytochrome P-450 (CYP) metabolite of arachidonic acid in the rat kidney, can be catalyzed by CYP4A isoforms including CYP4A1, CYP4A2, and CYP4A3. To determine the contribution of CYP4A isoforms to renal 20-HETE synthesis, specific antisense oligonucleotides (ODNs) were developed, and their specificity was examined in vitro in Sf9 cells expressing CYP4A isoforms and in vivo in Sprague-Dawley rats. Administration of CYP4A2 antisense ODNs (167 nmol. kg body wt-1. day-1 iv for 5 days) decreased vascular 20-HETE synthesis by 48% with no effect on tubular synthesis, whereas administration of CYP4A1 antisense ODNs inhibited vascular and tubular 20-HETE synthesis by 52 and 40%, respectively. RT-PCR of microdissected renal microvessel RNA indicated the presence of CYP4A1, CYP4A2, and CYP4A3 mRNAs, and a CYP4A1-immunoreactive protein was detected by Western analysis of microvessel homogenates. Blood pressure measurements revealed a reduction of 17 +/- 6 and 16 +/- 4 mmHg in groups receiving CYP4A1 and CYP4A2 antisense ODNs, respectively. These studies implicate CYP4A1 as a major 20-HETE synthesizing activity in the rat kidney and further document the feasibility of using antisense ODNs to specifically inhibit 20-HETE synthesis and thereby investigate its role in the regulation of renal function and blood pressure.

Cytochrome P450-derived arachidonic acid metabolism in the rat kidney: characterization of selective inhibitors.

We characterized the inhibitory activity of several acetylenic and olefinic compounds on cytochrome P450 (CYP)-derived arachidonic acid omega-hydroxylation and epoxidation using rat renal cortical microsomes and recombinant CYP proteins. Among the acetylenic compounds, 6-(2-propargyloxyphenyl)hexanoic acid (PPOH) and N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide were found to be potent and selective inhibitors of microsomal epoxidation with IC50 values of 9 and 13 microM, respectively. On the other hand, 17-octadecynoic acid inhibited both omega-hydroxylation and epoxidation of arachidonic acid with IC50 values of 7 and 5 microM, respectively. The olefinic compounds N-methylsulfonyl-12, 12-dibromododec-11-enamide (DDMS) and 12, 12-dibromododec-11-enoic acid (DBDD) exhibited a high degree of selectivity inhibiting microsomal omega-hydroxylation with an IC50 value of 2 microM, whereas the IC50 values for epoxidation were 60 and 51 microM for DDMS and DBDD, respectively. Studies using recombinant rat CYP4A isoforms showed that PPOH caused a concentration-dependent inhibition of omega-hydroxylation and 11, 12-epoxidation by CYP4A3 or CYP4A2 but had no effect on CYP4A1-catalyzed omega-hydroxylase activity. On the other hand, DDMS inhibited both CYP4A1- and CYP4A3- or CYP4A2-catalyzed arachidonic acid oxidations. Inhibition of microsomal activity by PPOH, but not DDMS, was time- and NADPH-dependent, a result characteristic of a mechanism-based irreversible inhibitor. These studies provide information useful for evaluating the role of the CYP-derived arachidonic acid metabolites in the regulation of renal function and blood pressure.