Tomohiro Osanai

Study on the Relationship Between Plasma Nitrite and Nitrate Level and Salt Sensitivity in Human Hypertension Modulation of Nitric Oxide Synthesis by Salt Intake

BACKGROUND High salt intake suppresses the effect of nitric oxide (NO) in the peripheral resistance vessels in animal models. We tested the hypothesis that the modulation of endogenous NO is related to salt sensitivity in human hypertension. METHODS AND RESULTS Inpatients with essential hypertension (n=24) were maintained on a normal-salt diet (12 g/d NaCl) for 3 days, a low-salt diet (2 g), a high-salt diet (20 to 23 g), and a low-salt diet for 7 days. Normotensive subjects (n=16) were maintained on the first 2 salt diets. The hypertensive patients whose average 24-hour blood pressure was increased by >5% by salt loading were assigned to group 1 (n=8) and the others to group 2 (n=16). Nitrate plus nitrite (NO(x)) was measured by the Griess method, and asymmetrical dimethylarginine (ADMA) by high-performance liquid chromatography. The plasma NO(x) level during the normal-salt diet was lower in group 1 than in group 2 and the normotensive group. After salt loading, the plasma NO(x) level was decreased and reversed after the second salt restriction. Plasma ADMA level was increased after salt loading and decreased after salt restriction. The change in plasma NO(x) level was correlated inversely with those in blood pressure (r=-0.59, P=0.0007) and plasma ADMA level (r=-0.64, P=0.003) after salt loading and restriction. CONCLUSIONS Modulation of NO synthesis by salt intake may be involved in a mechanism for salt sensitivity in human hypertension, presumably via the change in ADMA.

Effect of Shear Stress on Asymmetric Dimethylarginine Release From Vascular Endothelial Cells

Abstract—We demonstrated recently that plasma concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase, are increased by high salt intake concomitantly with a decrease in plasma levels of NO in human hypertension. We investigated the effect of shear stress on ADMA release in 2 types of cells: transformed human umbilical vein endothelial cells (HUVECs; cell line ECV-304) and HUVECs. Exposure of ECV-304 cells and HUVECs to shear stress with the use of a cone-plate viscometer enhanced gene expression of protein arginine methyltransferase (PRMT-1), ADMA synthase. In HUVECs, the ratio of PRMT-1 to glyceraldehyde 3-phosphate dehydrogenase mRNA was increased by 2-fold by a shear stress of ≥15 dyne/cm2. A dominant-negative mutant of I&kgr;B kinase &agr; and troglitazone at 8 &mgr;mol/L, an activator of peroxisome proliferator–activated receptor &ggr;, abolished the shear stress–induced increase in PRMT-1 gene expression in parallel with the blockade of nuclear factor (NF)-&kgr;B translocation into the nucleus. The activity of dimethylarginine dimethylaminohydrolase, the degradation enzyme of ADMA, was unchanged after shear stress ≤15 dyne/cm2 and was enhanced by 1.48±0.06-fold (P <0.05) by shear stress at 25 dyne/cm2. The release of ADMA was increased by 1.64±0.10-fold (P <0.05) by shear stress at 15 dyne/cm2 but was not affected by shear stress at 25 dyne/cm2. These results indicate that shear stress enhances gene expression of PRMT-1 and ADMA release via activation of the NF-&kgr;B pathway. Shear stress at higher magnitudes facilitates the degradation of ADMA, thus returning ADMA release levels to baseline.

Effect of allopurinol pretreatment on free radical generation after primary coronary angioplasty for acute myocardial infarction

Allopurinol, an inhibitor of xanthine oxidase, was shown to improve the regional ventricular function after coronary artery occlusion and reperfusion in animal models. The effects of oral administration of allopurinol on a transient increase in free radical generation after primary percutaneous transluminal coronary angioplasty (PTCA) in patients with acute myocardial infarction (AMI) and on their clinical outcomes were examined. Thirty-eight AMI patients undergoing primary PTCA were randomly assigned to control (group 1, n = 20) and allopurinol treatment groups (group 2, n = 18). Allopurinol (400 mg) was administered orally just after the admission (approximately 60 min before reperfusion). Free radical production was assessed by successive measurement of urinary excretion of 8-epi-prostaglandin F2&agr; (PGF2&agr;) after PTCA. Urinary 8-epi-PGF2&agr; excretion was increased by twofold at 60–90 min after PTCA compared with the baseline value in group 1. This increase was completely inhibited in group 2. Plasma allopurinol concentration was 1,146 ± 55 ng/ml in group 2 when reperfusion was achieved. Slow flow in the recanalized coronary artery after PTCA occurred less frequently in group 2 than in group 1. Cardiac index determined just after reperfusion and left ventricular ejection fraction at 6 months after PTCA were both significantly greater in group 2 than in group 1 although pulmonary capillary wedge pressure was similar in the two groups. In conclusion, allopurinol pretreatment is effective in inhibiting generation of oxygen-derived radicals during reperfusion therapy and the recovery of left ventricular function in humans.

Characteristics of Intracerebral Hemorrhage During Rivaroxaban Treatment Comparison With Those During Warfarin

Background and Purpose— Neuroradiological characteristics and functional outcomes of patients with intracerebral hemorrhage (ICH) during novel oral anticoagulant treatment were not well defined. We examined these in comparison with those during warfarin treatment. Methods— The consecutive 585 patients with ICH admitted from April 2011 through October 2013 were retrospectively studied. Of all, 5 patients (1%) had ICH during rivaroxaban treatment, 56 (10%) during warfarin, and the other 524 (89%) during no anticoagulants. We focused on ICH during rivaroxaban and warfarin treatments and compared the clinical characteristics, neuroradiological findings, and functional outcomes. Results— Patients in the rivaroxaban group were all at high risk for major bleeding with hypertension, abnormal renal/liver function, stroke, bleeding history or predisposition, labile international normalized ratio, elderly, drugs/alcohol concomitantly (HAS-BLED) score of 3 and higher rate of past history of ICH. Moreover, multiple cerebral microbleeds (≥4) were detected more frequently in rivaroxaban group than in warfarin (80% versus 29%; P=0.04). Hematoma volume in rivaroxaban group was markedly smaller than that in warfarin (median: 4 versus 11 mL; P=0.03). No patient in the rivaroxaban group had expansion of hematoma and surgical treatment. Rivaroxaban group showed lower modified Rankin Scale at discharge relative to warfarin, and the difference between modified Rankin Scale before admission and at discharge was smaller in rivaroxaban than in warfarin (median: 1 versus 3; P=0.047). No patient in the rivaroxaban group died during hospitalization, whereas 10 (18%) warfarin patients died. Conclusions— Rivaroxaban-associated ICH occurs in patients at high risk for major bleeding. However, they had a relatively small hematoma, no expansion of hematoma, and favorable functional and vital outcomes compared with warfarin-associated ICH.

High plasma level of asymmetric dimethylarginine in patients with acutely exacerbated congestive heart failure: role in reduction of plasma nitric oxide level.

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is elevated in congestive heart failure (CHF) concomitantly with the higher levels of nitric oxide (NO) and cytokines. We investigated the association among ADMA, NO, and cytokines in human CHF. Blood was collected from 25 patients with acutely exacerbated chronic CHF (acute CHF, mean age 61 ± 3 years), 23 patients with chronic compensated CHF (chronic CHF, mean age 62 ± 2 years), and 26 control subjects (mean age 51 ± 1 years). ADMA was measured by high-performance liquid chromatography. Tumor necrosis factor-Α (TNF-Α) was measured by enzyme-linked immunosorbent assay. Nitrate plus nitrite (NOx) was measured by the Griess method. The plasma levels of ADMA and TNF-Α were higher in patients with acute CHF than in those with chronic CHF and control subjects (both P ≪ 0.05). The plasma level of NOx was higher in patients with chronic CHF than in those with acute CHF and control subjects (both P ≪ 0.01). The plasma level of TNF-Α was positively correlated with that of ADMA in combination with patients with acute and chronic CHF (r = 0.31, P ≪ 0.01). The plasma level of ADMA was, furthermore, negatively correlated with that of NOx (r = −0.29, P ≪ 0.05). These findings indicate that ADMA is related to exacerbation of chronic CHF by suppression of the compensatory higher level of plasma NO.

Intracellular Signaling for Vasoconstrictor Coupling Factor 6. Novel Function of β-Subunit of ATP Synthase as Receptor

Coupling factor 6 (CF6), a component of adenosine triphosphate (ATP) synthase, is circulating and functions as an endogenous vasoconstrictor by inhibiting cytosolic phospholipase A2. We showed a high plasma level of CF6 in human hypertension. The present study focused on the identification and characterization of a receptor for CF6 and its post-receptor signaling pathway. Incubation of human umbilical vein endothelial cells (HUVECs) with an excess of free CF6 reduced by 50% the immunoreactivity for the antibody to &bgr;-subunit of ATP synthase at the cell surface, but unaffected that for the &agr;-subunit antibody. A significant displacement of radioligand was observed at 3×10−9 through 10−7 M unlabeled CF6, and the Kd was 7.6 nM. Adenosine diphosphate (ADP) at 10−7 M and &bgr;-subunit antibody suppressed the binding of 125I-CF6 by 81.3±9.7% and 32.0±2.0%, respectively, whereas the &agr;-subunit antibody unaffected it. The hydrolysis activity of ATP to ADP was increased by 1.6-fold by CF6 at 10−7 M, and efrapeptin at 10−5 M, an inhibitor of ATP synthase, blocked it. CF6 at 10−7 M decreased intracellular pH in 2′,7′-bis(carboxyethyl-5 (6))-carboxyfluorescein-loaded HUVEC. Amyloride at 10−4 M augmented the pH decrease in response to CF6, whereas efrapeptin at 10−5 M blocked it. Arachidonic acid release was suppressed by CF6, and it was reversed by efrapeptin at 10−5 M or &bgr;-subunit antibody or ADP at 10−7 M. The &bgr;-subunit antibody suppressed coupling factor 6–induced increase in blood pressure. These indicate that membrane-bound ATP synthase functions as a receptor for CF6 and may have a previously unsuspected role in the genesis of hypertension by modulating the concentration of intracellular hydrogen.

Vasoconstrictor effect of aldosterone via angiotensin II type 1 (AT1) receptor: possible role of AT1 receptor dimerization.

AIMS We recently demonstrated that aldosterone induces a non-genomic vasoconstrictor effect on rat coronary arterioles and that this effect was blocked by angiotensin II type 1 receptor (AT1) blockers. Intracellular transglutaminase enhances AT1 signalling by cross-linking AT1 homodimers. The purpose of this study was to confirm the AT1-dependency of the vasoconstrictor effect of aldosterone using AT1a knockout (AT1aKO) mice and to investigate the role of intracellular transglutaminase and AT1 dimerization in this effect. METHODS AND RESULTS The mesenteric arterioles (60-160 microm) were isolated from C57BL/6J (wild-type, WT) and AT1aKO mice, and the internal diameter was measured by video microscopy. Aldosterone (10(-13) to 10(-6) M), but not hydrocortisone, produced a dose-dependent vasoconstriction in WT mice; the maximal diameter change was -8.6 +/- 0.3% from the baseline (P < 0.001). This vasoconstrictor effect was unaffected by the mineralocorticoid receptor antagonist spironolactone or eplerenone, the AT2 antagonist PD123319, the glucocorticoid receptor antagonist RU486, or endothelium denudation. Aldosterones vasoconstrictor effect was negligible in AT1aKO mice. The AT1 blockers valsartan or candesartan suppressed aldosterone-induced vasoconstriction in WT mice. The transglutaminase inhibitors cystamine and monodansyl cadaverine also suppressed the vasoconstrictor effect of aldosterone, without affecting the vasoconstrictor effect of angiotensin II in WT mice. AT1 dimer protein levels were increased in WT mesenteric arterioles treated with 10(-7) M aldosterone, and the transglutaminase inhibitor and AT1 blocker blocked this aldosterone-induced formation of AT1 dimer. Treatment with 10(-7) M aldosterone for 10 min increased the transglutaminase activity by 2.5 +/- 0.2-fold in cultured vascular smooth muscle cells and by 1.2 +/- 0.1-fold in the mesenteric arterioles. These increases were abolished by transglutaminase inhibitors. CONCLUSION Aldosterone produces a non-genomic, endothelium-independent vasoconstrictor effect by enhancing intracellular transglutaminase activity and presumably inducing AT1 dimer formation in mesenteric arterioles.

Nitric oxide-dependent vasodilator mechanism is not impaired by hypertension but is diminished with aging in the rat aorta.

This study was designed to elucidate the effects of hypertension and aging on nitric oxide (NO)-mediated relaxation response to acetylcholine in the rat aorta. NO-mediated relaxation response was assessed as the relaxation response to acetylcholine after treatment with cyclooxygenase inhibitor in KCl-precontracted aortic rings. The endothelium-dependent relaxation responses to acetylcholine were lower in aortic rings isolated from spontaneously hypertensive rats (SHRs) at ages 16-20 and 55-60 weeks compared with those seen in age-matched Wistar-Kyoto (WKY) rats. Aging induced a reduction of the relaxation response to acetylcholine in aortic rings from WKY rats but not from SHRs. Pretreatment with indomethacin enhanced the relaxation response to acetylcholine in only SHRs at ages 16-20 and 55-60 weeks, thereby cancelling the difference in the relaxation response between WKY rats and SHRs. Simultaneous administration of indomethacin and NG-nitro-L-arginine methyl ester abolished the relaxation response to acetylcholine in both strains. Thus NO-mediated relaxation response to acetylcholine was similar between WKY rats and SHRs at ages 16-20 and 55-60 weeks, respectively, and was attenuated with aging to the same degree in both strains. In conclusion, NO-mediated relaxation response to acetylcholine in the aorta is attenuated with aging but not impaired by hypertension.

A Novel Inhibitory Effect on Prostacyclin Synthesis of Coupling Factor 6 Extracted from the Heart of Spontaneously Hypertensive Rats

The possible presence of an unknown prostacyclin synthesis inhibitory substance has been reported in some strains of rats. We purified the inhibitory substance from the heart of spontaneously hypertensive rats by collecting active fractions after gel-filtration column chromatography and two steps of reverse-phase high performance liquid chromatography. The amino acid composition and automated gas-phase sequencing of the full-length substance and fragments cleaved by AspN indicated that the prostacyclin-inhibitory peptide was identical to coupling factor 6. Recombinant rat coupling factor 6, which was synthesized using a cleavable fusion protein strategy, attenuated base-line and bradykinin (10−6 m)-induced prostacyclin synthesis and [3H]arachidonic acid (AA) release in human umbilical vein endothelial cells in a dose-dependent manner (10−9–10−7 m). Exogenous AA- and prostaglandin H2-induced prostacyclin synthesis were unchanged even after treatment with 10−7 mrecombinant coupling factor 6. Base-line and bradykinin-induced [3H]AA release were suppressed by arachidonyltrifluoromethyl ketone, a relatively specific inhibitor of cytosolic phospholipase A2 at 40 μm, and simultaneous administration of coupling factor 6 showed no further effect. Neither oleyloxyethyl phosphorylcholine at 1 μmnor bromoenol lactone at 1 μm affected AA release. Preincubation (1 min) with 10−7 m recombinant coupling factor 6 had no influence on adenosine diphosphate- and collagen-induced platelet aggregations. We conclude that coupling factor 6 possesses a novel function of prostacyclin synthesis inhibition in endothelial cells via suppression of Ca2+-dependent cytosolic phospholipase A2, although it is unclear whether coupling factor 6 functions in normal conditions or only in pathophysiological states.

Effect of vasoconstrictor coupling factor 6 on gene expression profile in human vascular endothelial cells: enhanced release of asymmetric dimethylarginine.

Background Coupling factor 6 (CF6), a component of ATP synthase, inhibits phospholipase A2 and induces vasoconstriction. However, because arachidonic acid acts in the widespread fields of vascular biology, CF6 might exert profound effects in addition to vasoconstriction. We investigated the effect of CF6 on the gene expression profile in human umbilical vein endothelial cells. Methods and results The increased gene expression after 24-h exposure to CF6 at 10−7 mol/l, assessed by cDNA microarray (n = 3), included neuregulin-1 (1.84 ± 0.07 fold compared with control, P < 0.05) and relaxin-1 (1.74 ± 0.20, P < 0.05), both relating to congestive heart failure, urokinase type plasminogen activator receptor (1.77 ± 0.24, P = 0.06) and estrogen receptor β (1.74 ± 0.36, P = 0.08), both relating to vascular inflammation and cell infiltration, and protein arginine methyltransferase (PRMT-1; 1.73 ± 0.20, P < 0.05). Out of these genes, the enzyme relating to the synthesis (PRMT-1) of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), was further examined concomitantly with the degradation enzyme, dimethylarginine dimethylaminohydrolase 2 (DDAH-2). The ratio of PRMT-1 to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, measured by real-time quantitative reverse transcription-polymerase chain reaction, was increased by 9 ± 2% (n = 10, P < 0.01) at 48 h after CF6 at 10−7 mol/l, whereas the ratio of DDAH-2 to GAPDH was decreased by 12 ± 2% (n = 8, P < 0.01). DDAH-2 protein and activity were decreased by 28 ± 5% (n = 5, P < 0.01) and 19 ± 2% (n = 6, P < 0.01) by CF6, respectively. ADMA release was enhanced by 20 ± 8% and NOS activity was decreased by 13 ± 1% (both n = 8, P < 0.05) by CF6. Conclusions CF6 changes the gene expression profile to be proatherogenic and functions as a novel stimulator for ADMA release by enhancing its synthesis and suppressing its degradation.