Hormonal regulation of sodium-dependent phosphate transport in opossum kidney cells

There are multiple regulators of renal proximal tubule sodium-dependent phosphate (Na(+)-Pi) transport, including 1,25-dihydroxyvitamin D (1,25-Vit. D), parathyroid hormone (PTH), insulin-like growth factor 1 (IGF-1), and arachidonic acid (AA) and/or its metabolites. The purpose of our studies was to determine whether the effect of these factors on Pi transport is synergistic or antagonistic. The control solution or the substances were added independently or coincidentally to opossum kidney (OK) cells before incubation for 4 h. 1,25-Vit. D (10(-8) M) had no significant effect on Pi transport ( upward arrow6.8%; p = 0.8). PTH (10(-7) M) significantly inhibited Pi transport by 39.6% (p < 0.0001). IGF-1 (10(-8) M) stimulated Pi transport by 19.6% (p < 0.0001). The AA metabolite 20-HETE (10(-7) M) had no significant impact on Pi transport ( downward arrow6.4; p = 0.3). The combined effect of 1,25-Vit. D and PTH was no different from PTH alone (p = 0.2). Likewise, addition of either 1,25-Vit. D or 20-HETE to IGF-1 failed to affect the magnitude of the increase on Pi transport induced by IGF-1 alone (p = 0.4, p = 0.6, respectively). The combination of 20-HETE and PTH was not different from that observed with PTH alone (p = 0.9). We conclude that in OK cells, PTH inhibits whereas IGF-1 stimulates Pi transport into OK cells. The effects of each of these hormones are independent and unaffected by either 1,25-Vit. D or 20-HETE.     

Effects of cytochrome P-450 metabolites of arachidonic acid on the epithelial sodium channel (ENaC)

Sodium reabsorption via the epithelial Na(+) channel (ENaC) in the aldosterone-sensitive distal nephron plays a central role in the regulation of body fluid volume. Previous studies have indicated that arachidonic acid (AA) and its metabolite 11,12-EET but not other regioisomers of EETs inhibit ENaC activity in the collecting duct. The goal of this study was to investigate the endogenous metabolism of AA in cultured mpkCCD(c14) principal cells and the effects of these metabolites on ENaC activity. Liquid chromatography/mass spectrometry analysis of the mpkCCD(c14) cells indicated that these cells produce prostaglandins, 8,9-EET, 11,12-EET, 14,15-EET, 5-HETE, 12/8-HETE, and 15-HETE, but not 20-HETE. Single-channel patch-clamp experiments revealed that 8,9-EET, 14,15-EET, and 11,12-EET all decrease ENaC activity. Neither 5-, 12-, nor 15-HETE had any effect on ENaC activity. Diclofenac and ibuprofen, inhibitors of cyclooxygenase, decreased transepithelial Na(+) transport in the mpkCCD(c14) cells. Inhibition of cytochrome P-450 (CYP450) with MS-PPOH activated ENaC-mediated sodium transport when cells were pretreated with AA and diclofenac. Coexpression of CYP2C8, but not CYP4A10, with ENaC in Chinese hamster ovary cells significantly decreased ENaC activity in whole-cell experiments, whereas 11,12-EET mimicked this effect. Thus both endogenously formed EETs and their exogenous application decrease ENaC activity. Downregulation of ENaC activity by overexpression of CYP2C8 was PKA dependent and was prevented by myristoylated PKI treatment. Biotinylation experiments and single-channel analysis revealed that long-term treatment with 11,12-EET and overexpression of CYP2C8 decreased the number of channels in the membrane. In contrast, the acute inhibitory effects are mediated by a decrease in the open probability of the ENaC. We conclude that 11,12-EET, 8,9-EET, and 14,15-EET are endogenously formed eicosanoids that modulate ENaC activity in the collecting duct.     

Quantitation of the Na(+)-Pi cotransporter in renal cortical brush border membranes. [14C]phosphonoformic acid as a useful probe to determine the density and its change in response to parathyroid hormone.

To determine the density of Na(+)-Pi symporters in brush border membranes (BBM) from rat renal cortex, [14C] phosphonoformic acid [( 14C] PFA), a competitive inhibitor of Na(+)-Pi cotransport, was employed as a probe. The [14C]PFA binding was measured in BBM vesicles (BBMV) under equilibrated conditions (extra-vesicular Na+, K+, and H+ = intravesicular Na+, K+, and H+) to avoid modulatory effects of these solutes. BBMV were preincubated in media without or with addition of molar excess of Pi (greater than 20 times) to determine the Pi-protectable PFA-binding sites, and then [14C] PFA binding was determined. Only the [14C]PFA binding in the presence of Na+ displaceable by an excess of Pi was saturated and was independent of intravesicular volume of BBMV. This value denoted as "Pi-protectable Na(+)-[14C]PFA binding," was analyzed by Scatchard plot showing BmaxPFA = 375 +/- 129 pmol of PFA/mg protein, KDPFA = 158 +/- 18 microM; the Hill coefficient was congruent to 1. For Na(+)-dependent binding of [3H]phlorizin, in the same BBMV, Bmax = 310 +/- 37 pmol/mg protein and KD V 2.2 +/- 0.5 microM. BBMV prepared from cortex of thyroparathyroidectomized rats infused with phosphaturic doses of parathyroid hormone (PTH) were compared with vehicle-infused controls. Administration of PTH resulted in decrease of BmaxPFA (-38%) and of Na(+)-gradient-dependent uptake of 32Pi (-35%), but KDPFA was not changed. Neither BmaxPhl and KDPhl for Na(+)-phlorizin binding, nor the Na(+)-gradient-dependent uptake of [3H]D-glucose differed between PTH-treated and control rats. We conclude: (a) measurement of Pi-protectable Na(+)-[14C]PFA binding determines numbers and affinity of Na(+)-Pi symporters in renal BBMV; (b) the affinity of PFA for Na(+)-Pi symporter is similar to apparent affinity for Pi (KmPi), as determined from measurements of Na(+)-gradient-dependent 32Pi uptake by BBMV; (c) both Na(+)-Pi symporter and [Na+]D-glucose symporters are present within renal BBM in a similar range of density; (d) PTH decreases the number of Na(+)-Pi cotransporters in BBMV commensurate with the parallel decrease of Na(+)-gradient-dependent Pi transport, whereas the affinity of Na(+)-Pi symporters for Pi is not changed. These observations support the hypothesis that PTH decreases capacity for Na(+)-dependent Pi reabsorption by internalization of Na(+)-Pi symporters in BBM of renal proximal tubules.     

The Na+/Pi-cotransporter of OK cells: reaction and tentative identification with N-acetylimidazole

Using an established renal epithelial cell line (OK cells) the effect of the amino-acid side-chain modifying reagent N-acetylimidazole (NAI) upon the sodium-dependent transport of phosphate (Pi) was investigated. After an incubation with 10 mM NAI for 20 min, cellular Na+/Pi uptake was inhibited by 70%. The presence of 5 mM Pi protected this transport function from being affected by NAI by 80 to 100%. Since the presence of sulfate was unable to protect the Na+/Pi transport inactivation by NAI and since the presence of Pi did not affect NAI inhibition of other transport systems, it is suggested that NAI interacts with the Pi transporter directly. The protective effect of Pi was used as a criterion to identify Pi-protectable [3H]NAI labelling of OK cell plasma membrane proteins. Pi protection was observed in four molecular mass regions: 31, 53, 104 and 176 kDa. Since the incorporation of [3H]NAI into these proteins was also affected by parathyroid hormone at 10(-10) M, it is concluded that the identified proteins represent possible candidates for the renal Na+/Pi cotransporter.     

Epoxyeicosatrienoic acids activate Na+/H+ exchange and are mitogenic in cultured rat glomerular mesangial cells

The present study examined responses of cultured rat glomerular mesangial cells to exogenous exposure of epoxyeicosatrienoic acids (EET's), products of cytochrome P450 epoxygenase. One day after administration of 8,9- or 14,15-EET, cultured rat mesangial cells demonstrated significant increases in [3H]thymidine incorporation (10(-7) M 14,15-EET: 120 +/- 7% of control; n = 6; P less than 0.025; 10(-6) M 14,15-EET: 145 +/- 10%; n = 20; P less than 0.0005; 10(-6) M 8,9-EET: 167 +/- 31%; n = 9; P less than 0.05), which was not affected by addition of the cyclooxygenase inhibitor indomethacin. In addition to stimulation of [3H]thymidine incorporation, the epoxides stimulated mesangial cell proliferation. 14,15-EET administration induced intracellular alkalinization of 0.2-0.3 pH units, which was prevented by extracellular Na+ removal and blunted by amiloride (0.5 mM). Following intracellular acidification with NH4Cl addition and removal, greater than 85% of 3 mM 22Na uptake into mesangial cells was inhibited by 1 mM amiloride, indicating Na+/H+ exchange. Under these conditions, 14,15-EET stimulated Na+/H+ exchange by 42% and 8,9-EET stimulated Na+/H+ exchange by 59%. Neither protein kinase C depletion nor addition of the protein kinase C inhibitor, staurosporine, affected this stimulation. In [3H]myo-inositol loaded mesangial cells, no significant stimulation of phosphoinositide hydrolysis was detected in response to administration of 14,15-EET. Twenty-four hours after addition of [14C]14,15-EET, greater than 90% was preferentially esterified to cellular lipids, with predominant incorporation into phosphatidylinositol, phosphatidylethanolamine, and diacylglycerol. Thus, these results demonstrate epoxyeicosatrienoic acids stimulate Na+/H+ exchange and mitogenesis in mesangial cells. These effects do not appear to be mediated via phospholipase C activation. In addition, 14,15-EET was selectively incorporated into cellular lipids known to mediate signal transduction. These observations extend the potential biologic roles of c-P450 arachidonate metabolites to include stimulation of cell proliferation and suggest a role for these compounds in vascular and renal injury.     

Normal molecular size of the Na(+)-phosphate cotransporter and normal Na(+)-dependent binding of phosphonoformic acid in renal brush border membranes of X-linked Hyp mice

X-linked Hyp mice have a specific defect in Na(+)-dependent phosphate (Pi) transport at the renal brush border membrane (BBM). In the present study we examined the effect of the Hyp mutation on the molecular size of the Pi transporting unit and on Na(+)-dependent 14C-phosphonoformic (PFA) binding in renal BBM vesicles. By radiation inactivation analysis, we demonstrated that the molecular size of the Na(+)-Pi cotransporter is similar in normal (242 +/- 16 kDa) and Hyp mice (227 +/- 39 kDa). Moreover, while BBM Na(+)-dependent Pi transport is significantly reduced in Hyp mice (249 +/- 54 vs 465 +/- 82 pmol/mg protein/6s), genotype differences in (1) Na(+)-dependent PFA binding (1020 +/- 115 vs 1009 +/- 97 pmol/mg protein/30 min), (2) Pi-displaceable Na(+)-dependent PFA binding (605 +/- 82 vs 624 +/- 65 pmol/mg protein/6s), and (3) phosphate uptake at Na(+)-equilibrium (67 +/- 10 vs 54 +/- 7 pmol/mg protein/6s) are not apparent. The present data demonstrate that the molecular size of the renal BBM Na(+)-Pi cotransporter is normal in Hyp mice and suggest that the number of Na(+)-Pi cotransporters may not be reduced in the mutant strain.     

Expression of the rat renal PiT-2 phosphate transporter.

BACKGROUND: NaPi-2a is the main sodium-dependent Pi (Na+-Pi) transporter in the apical membrane of the renal proximal tubule. Another group of Pi transporters, Glvr-1 (PiT-1) and Ram-1 (PiT-2), was identified. The PiT-2 cRNA induces Na+-dependent Pi uptake into Xenopus laevis oocytes. Prior studies have revealed the presence of the Pit-2 transporter in the kidney. OBJECTIVES: Further characterization of the PiT-2 transporter in the kidney and assessment of its developmental regulation. METHODS: Using primers specific for the PiT-2 mRNA and an antibody specific for the PiT-2 protein, we assessed the expression and developmental regulation of the renal PiT-2 mRNA and protein. RESULTS: RT-PCR analysis revealed that a 182 bp product was evident in the total kidney (TK), cortex (C), and medulla (M). Northern blots demonstrated a PiT-2 mRNA of approximately 4 kb (expected size) in the TK, C, and M. PiT-2 mRNA expression was similar in all kidney regions. RT-PCR and Northern blot analysis revealed that the PiT-2 cDNA was highly abundant in OK and MDCK culture cells. RT-PCR and Northern blot analysis revealed expected products at all ages studied. Densitometry demonstrated similar levels of expression of PiT-2 mRNA in the kidneys of older versus younger animals, and persistent expression in elderly rats. The PiT-2 protein was present in the TK, C, and M, and in OK and MDCK cells. PiT-2 protein abundance was similar at all ages studied. CONCLUSIONS: These studies further characterize the renal PiT-2 transporter and show that its expression is stable throughout development and ageing.     

Role of arachidonic acid lipoxygenase metabolites in acetylcholine-induced relaxations of mouse arteries

Arachidonic acid (AA) metabolites function as EDHFs in arteries of many species. They mediate cyclooxygenase (COX)- and nitric oxide (NO)-independent relaxations to acetylcholine (ACh). However, the role of AA metabolites as relaxing factors in mouse arteries remains incompletely defined. ACh caused concentration-dependent relaxations of the mouse thoracic and abdominal aorta and carotid, femoral, and mesentery arteries (maximal relaxation: 57 ± 4%, 72 ± 4%, 82 ± 3%, 80 ± 3%, and 85 ± 3%, respectively). The NO synthase inhibitor nitro-L-arginine (L-NA; 30 μM) blocked relaxations in the thoracic aorta, and L-NA plus the COX inhibitor indomethacin (10 μM) inhibited relaxations in the abdominal aorta and carotid, femoral, and mesenteric arteries (maximal relaxation: 31 ± 10%, 33 ± 5%, 41 ± 8%, and 73 ± 3%, respectively). In mesenteric arteries, NO- and COX-independent relaxations to ACh were inhibited by the lipoxygenase (LO) inhibitors nordihydroguaiaretic acid (NDGA; 10 μM) and BW-755C (200 μM), the K(+) channel inhibitor apamin (1 μM), and 60 mM KCl and eliminated by endothelium removal. They were not altered by the cytochrome P-450 inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (20 μM) or the epoxyeicosatrienoic acid antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 μM). AA relaxations were attenuated by NDGA or apamin and eliminated by 60 mM KCl. Reverse-phase HPLC analysis revealed arterial [(14)C]AA metabolites that comigrated with prostaglandins, trihydroxyeicosatrienoic acids (THETAs), hydroxyepoxyeicosatrienoic acids (HEETAs), and hydroxyeicosatetraenoic acids (HETEs). Epoxyeicosatrienoic acids were not observed. Mass spectrometry confirmed the identity of 6-keto-PGF(1α), PGE(2), 12-HETE, 15-HETE, HEETAs, 11,12,15-THETA, and 11,14,15-THETA. AA metabolism was blocked by NDGA and endothelium removal. 11(R),12(S),15(S)-THETA relaxations (maximal relaxation: 73 ± 3%) were endothelium independent and blocked by 60 mM KCl. Western immunoblot analysis and RT-PCR of the aorta and mesenteric arteries demonstrated protein and mRNA expression of leukocyte-type 12/15-LO. Thus, in mouse resistance arteries, 12/15-LO AA metabolites mediate endothelium-dependent relaxations to ACh and AA.     

Noncyclooxygenase metabolites of arachidonic acid amplify the vasopressin-induced Ca2+ signal in glomerular mesangial cells by releasing Ca2+ from intracellular stores

Noncyclooxygenase metabolites of arachidonic acid may be potent modulators of the mitogenic response of renal mesangial cells to the mitogenic vasoactive peptide arginine vasopressin (AVP). Since Ca2+ is a critical second messenger in the response of mesangial cells to AVP, and Ca2+ has been implicated in the regulation of growth, we determined whether noncyclooxygenase metabolites altered the phospholipase C-Ca2+ signalling cascade which is activated by AVP. Pretreatment of mesangial cells for 10 min with lipoxygenase and cytochrome P450 monooxygenase inhibitors, nordihydroguaiaretic acid (NDGA, 10(-5) M) or SKF-525A (2.5 x 10(-5) M), but not the cyclooxygenase inhibitor indomethacin (2 x 10(-5) M), reduced the magnitude of the AVP (10(-8) and 10(-7) M)-induced increase in cytosolic free Ca2+ concentration ([Ca2+]i) without affecting inositol trisphosphate production. With 10(-8) M AVP, [Ca2+]i increased to 250 +/- 47 nM in NDGA-treated cells versus 401 +/- 59 nM in control cells (p less than 0.01). [Ca2+]i, measured 2 min after exposure to AVP, was also lower with NDGA (152 +/- 21 nM) when compared with AVP alone (220 +/- 22 nM, p less than 0.01). 14,15-epoxyeicosatrienoic acid (EET) (10(-8) M), which had no effect on inositol trisphosphate production, completely reversed the NDGA-induced inhibition of the [Ca2+]i transient, whereas 5-hydroperoxyeicosatetraenoic acid (HPETE) (5 x 10(-7) M) did not. Pretreatment with higher concentrations of 14,15-EET (10(-7)-10(-6) M) markedly potentiated the AVP-induced increase in [Ca2+]i. NDGA-induced inhibition of the AVP-generated [Ca2+]i transient was also observed when cells were incubated in low Ca2+ media ([Ca2+] less than 5 x 10(-8) M), suggesting that NDGA pretreatment impaired intracellular release of Ca2+. Since NDGA had no direct effect on inositol 1,4,5-trisphosphate-induced Ca2+ release, we postulated that NDGA blocked production of a metabolite that releases Ca2+ from intracellular stores. 14,15-EET and 15-HPETE, but not 15-hydroxyeicosatetraenoic acid (each at 3 x 10(-7) M), raised [Ca2+]i when added directly to cells in low Ca2+ media. In permeabilized cells 14,15-EET and 15-HPETE (10(-7) M) potently released Ca2+ from intracellular stores. In summary, noncyclooxygenase metabolites of arachidonic acid, and in particular P450 metabolites, are potent endogenous amplifiers of the AVP-induced [Ca2+]i signal by mechanisms not directly involving phospholipase C activation. This effect is mediated, at least in part, by enhanced release of Ca2+ from intracellular storage sites by an inositol 1,4,5-trisphosphate-independent mechanism.     

Determination of cytochrome P450 metabolites of arachidonic acid in coronary venous plasma during ischemia and reperfusion in dogs

Arachidonic acid (AA) can be metabolized by cytochrome P450 enzymes to many biologically active compounds including 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), their corresponding dihydroxyeicosatrienoic acids (DHETs), as well as 19- and 20-hydroxyeicosatetraenoic acids (HETEs). These eicosanoids are potent regulators of vascular tone. However, their role in the ischemic myocardium has not been well investigated. In this study, we used a gas chromatographic-mass spectrometric technique to analyze total EETs, DHETs, and 20-HETE released into coronary venous plasma during coronary artery occlusion and reperfusion in anesthetized dogs. Pentafluorobenzyl esters (PFB-esters) of EETs and PFB-esters/trimethylsilyl ethers (TMS-ethers) of DHETs and 20-HETE were detected in the negative ion chemical ionization (NICI) using methane as a reagent gas. Under the conditions used, all four regioisomers of EET eluted from the capillary gas chromatographic column at similar retention times while four regioisomers of DHETs and 20-HETE eluted separately. The detection limits in plasma samples are 5 pg for total EETs, 40 pg for DHET, and 15 pg for 20-HETE. 14,15-DHET is the major regioisomer detected in the plasma samples while other regioisomers of DHETs are probably present at too low a concentration for detection. During the first 5 to 15 min of coronary occlusion, a slight decrease in the concentration of EETs, 14,15-DHET, and 20-HETE from the control values was observed in coronary venous plasma. At 60 min of occlusion, their concentrations significantly increased and remained elevated during 5 to 60 min of reperfusion. The concentrations decreased at 120 min of reperfusion. The NICI GC-MS was successfully used as a sensitive technique to determine cP450 metabolites of AA in plasma during prolonged occlusion-reperfusion periods. Furthermore, the results indicate that these metabolites may play a role in mediating ischemic-reperfusion injury.     

Efflux of inorganic phosphate from rat hepatocytes: lack of effect of insulin

Efflux of Pi from rat hepatocytes suspended in phosphate free-medium was studied by chemical assay of released Pi and by monitoring the loss in radioactivity of cells pre-labelled with [32P]-Pi. The release follows first-order kinetics fairly closely with a rate constant of 7 x 10(-3) min-1 approximately. Insulin at a concentration of 10(-8) M had no effect on the rate of Pi release and it is concluded therefore, that the insulin-stimulated accumulation of Pi described in the literature is the result of hormone action on Pi uptake by liver rather than on its release.     

Effect of soluble epoxide hydrolase inhibition on epoxyeicosatrienoic acid metabolism in human blood vessels

We investigated the effects of soluble epoxide hydrolase (sEH) inhibition on epoxyeicosatrienoic acid (EET) metabolism in intact human blood vessels, including the human saphenous vein (HSV), coronary artery (HCA), and aorta (HA). When HSV segments were perfused with 2 micromol/l 14,15-[3H]EET for 4 h, >60% of radioactivity in the perfusion medium was converted to 14,15-dihydroxyeicosatrienoic acid (DHET). Similar results were obtained with endothelium-denuded vessels. 14,15-DHET was released from both the luminal and adventitial surfaces of the HSV. When HSVs were incubated with 14,15-[3H]EET under static (no flow) conditions, formation of 14,15-DHET was detected within 15 min and was inhibited by the selective sEH inhibitors N,N'-dicyclohexyl urea and N-cyclohexyl-N'-dodecanoic acid urea (CUDA). Similarly, CUDA inhibited the conversion of 11,12-[3H]EET to 11,12-DHET by the HSV. sEH inhibition enhanced the uptake of 14,15-[3H]EET and facilitated the formation of 10,11-epoxy-16:2, a beta-oxidation product. The HCA and HA converted 14,15-[3H]EET to DHET, and this also was inhibited by CUDA. These findings in intact human blood vessels indicate that conversion to DHET is the predominant pathway for 11,12- and 14,15-EET metabolism and that sEH inhibition can modulate EET metabolism in vascular tissue.     

Inhibition of renal Na(+)-Pi cotransporter by mercuric chloride: role of sulfhydryl groups.

We studied the role of sulfhydryl groups in Na(+)-Pi cotransport across the renal brush border membrane (BBM), using HgCl2, an agent which penetrates membranes freely. HgCl2 inhibited the initial Na(+)-dependent 32Pi transport in a dose-dependent manner (IC50 = 54 microM). Na(+)-independent transport was not affected. The inhibitory effect persisted under Na+ equilibrium-exchange conditions. Additionally, HgCl2 had no effect on the diffusional uptake of 22Na up to 1 min incubation. Exposure to HgCl2 had no effect on vesicle integrity as determined by osmotic shrinking experiments. BBM vesicle (BBMV) volume, determined by D-glucose equilibrium uptake, was not affected at low HgCl2 concentrations, but decreased at higher concentrations (greater than 100 microM). Vesicle volumes, determined by flow cytometry, were not changed after exposure to HgCl2. Kinetic studies showed a reduction in the apparent Vmax for Pi transport from 1.40 +/- 0.13 to 0.75 +/- 0.19 nmoles/mg protein/5 sec, without a significant change in the apparent Km. In protection studies, dithiothreitol (DTT) completely protected against inhibition, but Pi, phosphonoformic acid (PFA), and Na+ gave no protection. The data suggest that sulfhydryl groups are essential for the function of Na(+)-Pi cotransporter of renal BBM.     

Incorporation of arachidonic and linoleic acid hydroperoxides into cultured human umbilical vein endothelial cells

The current study assessed the differential incorporation of 12-hydroperoxyeicosatetraenoic acid (12-HPETE), arachidonic acid (AA), 12-hydroxyeicosatetraenoic acid (12-HETE) and the linoleic acid (LA) oxidation products, 13-hydroxyoctadecadienoic acid (13-HODE) and 13-hydroperoxyoctadecadienoic acid (13-HPODE), into human umbilical vein endothelial cells (HUVEC). Approximately 80-90% of AA (10(-8)-10(-5)M) and 80% of LA (10(-8)-10(-5)M) were incorporated into HUVEC within 12h, while less than 50% of the hydroxy metabolites (12-HETE, 12-HPETE, 13-HODE, 13-HPODE) were incorporated into HUVEC over 48h. Further, treatment of HUVEC with either 12-HPETE or 13-HPODE (concentrations of 10(-5)M) had no effect on cell number at a 48h time point when compared with control. These results demonstrate that exogeneous hydroxy metabolites are incorporated into HUVEC to a lesser degree than were endogenous fatty acids. Further, we speculate that 12-HPETE and 13-HPODE are rapidly metabolized to substances without significant cytotoxic effects.     

Atrial natriuretic factor inhibits phosphate uptake in opossum kidney cells: as a model of renal proximal tubules

The cultured renal cell, an opossum kidney (OK) cell line, which contains several features characteristic of proximal tubular cells, was utilized to examine the direct effects of atrial natriuretic factor (ANF) and cyclic GMP (cGMP) on phosphate uptake. ANF at 2 x 10(-7) M significantly inhibited phosphate uptake by 10.1% of control (P less than 0.01). Incubation of the cells with ANF (10(-8) to 10(-6) M) resulted in an increment of intracellular cGMP in a dose dependent fashion. Exogenous addition of 8-bromo-cGMP (10(-4) M) also significantly inhibited phosphate uptake by 14.6%. These results suggest that ANF directly inhibits phosphate transport in renal proximal tubular cells, probably through stimulation of cGMP production.     

Liquid chromatographic-electrospray ionization-mass spectrometric analysis of cytochrome P450 metabolites of arachidonic acid.

Arachidonic acid (AA) can be metabolized by cytochrome P450 (CYP) enzymes to many biologically active compounds including 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), their corresponding dihydroxyeicosatrienoic acids (DHETs), and 20-hydroxyeicosatetraenoic acid (20-HETE). These eicosanoids are potent regulators of vascular tone. We developed a liquid chromatography-electrospray ionization-mass spectrometry method to simultaneously determine 5,6-, 8,9-, 11,12-, and 14,15-EETs; 5,6-, 8,9-, 11,12-, and 14,15-DHETs; and 20-HETE. [2H8]EETs, [2H8]DHETs, and [2H2]20-HETE were used as internal standards. These compounds are readily separated on a C18 reverse-phase column using water:acetonitrile with 0.005% acetic acid as a mobile phase. The internal standards, [2H8]EETs, [2H8]DHETs, and [2H2]20-HETE, eluted slightly faster than the natural eicosanoids. The samples were ionized by electrospray with fragmentor voltage of 120 V and detected in a negative mode. The negative ion detection gave a lower background than the positive ion detection for these compounds. These eicosanoids exhibited high abundance of the ions corresponding to [M - 1]-. The m/z = 319, 337, and 319 ions were used for quantitation of EETs, DHETs, and 20-HETE, respectively. The detection limits using selected ion monitoring of these compounds are about 1 pg per injection. The position of functional groups and water content of mobile phase had a significant effect on the sensitivity of detection. Water content of 40% was found to give maximal sensitivity. The method was used to determine EETs, DHETs, and 20-HETE in bovine coronary artery endothelial cells, dog plasma, rat astrocytes, and rat kidney microsome samples.     

Soluble epoxide hydrolase contamination of specific catalase preparations inhibits epoxyeicosatrienoic acid vasodilation of rat renal arterioles

Cytochrome P-450 metabolites of arachidonic acid, the epoxyeicosatrienoic acids (EETs) and hydrogen peroxide (H(2)O(2)), are important signaling molecules in the kidney. In renal arteries, EETs cause vasodilation whereas H(2)O(2) causes vasoconstriction. To determine the physiological contribution of H(2)O(2), catalase is used to inactivate H(2)O(2). However, the consequence of catalase action on EET vascular activity has not been determined. In rat renal afferent arterioles, 14,15-EET caused concentration-related dilations that were inhibited by Sigma bovine liver (SBL) catalase (1,000 U/ml) but not Calbiochem bovine liver (CBL) catalase (1,000 U/ml). SBL catalase inhibition was reversed by the soluble epoxide hydrolase (sEH) inhibitor tAUCB (1 μM). In 14,15-EET incubations, SBL catalase caused a concentration-related increase in a polar metabolite. Using mass spectrometry, the metabolite was identified as 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), the inactive sEH metabolite. 14,15-EET hydrolysis was not altered by the catalase inhibitor 3-amino-1,2,4-triazole (3-ATZ; 10-50 mM), but was abolished by the sEH inhibitor BIRD-0826 (1-10 μM). SBL catalase EET hydrolysis showed a regioisomer preference with greatest hydrolysis of 14,15-EET followed by 11,12-, 8,9- and 5,6-EET (V(max) = 0.54 ± 0.07, 0.23 ± 0.06, 0.18 ± 0.01 and 0.08 ± 0.02 ng DHET·U catalase(-1)·min(-1), respectively). Of five different catalase preparations assayed, EET hydrolysis was observed with two Sigma liver catalases. These preparations had low specific catalase activity and positive sEH expression. Mass spectrometric analysis of the SBL catalase identified peptide fragments matching bovine sEH. Collectively, these data indicate that catalase does not affect EET-mediated dilation of renal arterioles. However, some commercial catalase preparations are contaminated with sEH, and these contaminated preparations diminish the biological activity of H(2)O(2) and EETs.     

Purification and characterization of phosphoenolpyruvate carboxylase from Brassica napus (rapeseed) suspension cell cultures: implications for phosphoenolpyruvate carboxylase regulation during phosphate starvation, and the integration of glycolysis with nitrogen assimilation.

Phosphoenolpyruvate carboxylase (PEPC) specific activity increased by 250% following 8 to 10 days of Pi starvation of Brassica napus suspension cells. Densitometric scanning of PEPC immunoblots revealed a close correlation between PEPC activity and the amount of the antigenic 104-kDa PEPC subunit. To further assess the influence of Pi deprivation on PEPC, the enzyme was purified from Pi-sufficient (+Pi) and Pi-starved (-Pi) cells to electrophoretic homogeneity and final specific activities of 37-40 micromol phosphoenolpyruvate utilized per min per mg protein. Gel filtration, SDS/PAGE, and CNBr peptide mapping indicated that the +Pi and -Pi PEPCs are both homotetramers composed of an identical 104-kDa subunit. Respective pH-activity profiles, phosphoenolpyruvate saturation kinetics, and sensitivity to L-malate inhibition were also indistinguishable. Kinetic studies and phosphatase treatments revealed that PEPC of the +Pi and -Pi cells exists mainly in its dephosphorylated (L-malate sensitive) form. Thus, up-regulation of PEPC activity in -Pi cells appears to be solely due to the accumulation of the same PEPC isoform being expressed in +Pi cells. PEPC activity was modulated by several metabolites involved in carbon and nitrogen metabolism. At pH 7.3, marked activation by glucose 6-phosphate and inhibition by L-malate, L-aspartate, L-glutamate, DL-isocitrate, rutin and quercetin was observed. The following paper provides a model for the coordinate regulation of B. napus PEPC and cytosolic pyruvate kinase by allosteric effectors. L-Aspartate and L-glutamate appear to play a crucial role in the control of the phosphoenolpyruvate branchpoint in B. napus, particularly with respect to the integration of carbohydrate partitioning with the generation of carbon skeletons required during nitrogen assimilation.     

Structural identification of cytochrome P450-dependent arachidonate metabolites formed by rabbit medullary thick ascending limb cells.

The medullary thick ascending limb of Henle's loop (mTALH) contributes importantly to the regulation of extracellular fluid volume and composition and metabolizes arachidonic acid (AA) chiefly by a cytochrome P450 monooxygenase pathway. Rabbit mTALH cells, when incubated with radiolabeled [14C]AA, form products that segregate into two peaks designated P1 and P2 based on their reverse-phase high pressure liquid chromatography retention times. We have now definitively identified their chemical structures. mTALH cells, isolated from the rabbit outer medulla, were homogenized and incubated with [14C]AA in the presence of NADPH. The AA metabolites in P1 and P2 were identified by gas chromatographic-mass spectrometric methods, including fast atom bombardment, negative ion electron capture, and electron ionization. All mass spectrometric data, the lack of UV chromophores, and comparisons with authentic standards were consistent with P1 containing two principal components: 19-hydroxy-5,8,11,14 eicosatetraenoic acid (19-HETE) and 20 - hydroxy - 5,8,11,14 - eicosatetraenoic acid (20-HETE), P2 contained primarily 1,20-eicosa-5,8,11,14-tetraenedioic acid (20-COOH-AA). The biological properties of P1 and P2 were compared with those of the authentic standards of 19- and 20-HETE and 20-COOH-AA. P1 dose dependently relaxed precontracted mesenteric arterial rings, as did authentic (19S)- and (19R)-HETE, whereas 20-HETE relaxed at lower and contracted at higher concentrations. As P1 contained a mixture of 19- and 20-HETE, each of these AA metabolites presumably contributed to the vascular relaxation produced by P1. Neither P2 nor 20-COOH-AA exhibited vasoactivity, but each demonstrated a similar potency in inhibiting rabbit medullary Na(+)-K(+)-ATPase activity. As previously reported, P2 was a more potent inhibitor of Na(+)-K(+)-ATPase than P1. The lesser inhibitory activity of P1 presumably reflects the presence of similar amounts of 19-HETE, the least active metabolite, and 20-HETE, which resembles 20-COOH-AA in its capacity to inhibit Na(+)-K(+)-ATPase. Thus, the biological activity of the less polar peak, P1, can be accounted for by 19- and 20-HETE, and that of P2, by 20-COOH-AA.     

Characterization and identification of cytochrome P450 metabolites of arachidonic acid released by human peritoneal macrophages obtained from the pouch of Douglas

Cytochrome P450 metabolism of arachidonic acid (AA) was investigated in human peritoneal macrophages which play a central role in chronic pelvic diseases in women (for example in endometriosis). The formation of eicosanoids other than prostaglandins (PGs) by these cells is still unknown. In non-activated macrophages obtained from women in the reproductive age, the main [(3)H]-AA metabolites coeluted with epoxyeicosatrienoic acids, dihydroxyeicosatrienoic acids (DHETs) and hydroxyeicosatetraenoic acids (HETEs) in reverse-phase HPLC. After zymosan activation a shift to PGs pathway was observed. Treatment with low doses of 2,3,7,8-tetrachlorodibenzo- p -dioxin increased the formation of a metabolite coeluting with 5,6-DHET. By gas chromatography/mass spectrometry 5,6-DHET (after beta-naphthoflavone induction), and 14,15-DHET as well as 11,12-DHET (after AA stimulation) were identified as major epoxygenase metabolites, respectively. The enantioselective formation of 12(S)-HETE was demonstrated by chiral-phase HPLC. Our findings demonstrate that non-activated peritoneal macrophages produce substantial amounts of bioactive cytochrome P450 metabolites of AA.