Hiroshi Nagoshi

Direct evidence of nitric oxide production from bovine aortic endothelial cells using new fluorescence indicators : diaminofluoresceins

The measurement of nitric oxide (NO) is important for direct examination of the regulatory roles of NO in various biological systems. Diaminofluoresceins (DAFs), new fluorescence indicators for NO, were applied to detect the release of NO from bovine aortic endothelial cells (ECs). DAFs react with NO to yield the corresponding green‐fluorescent triazolofluoresceins, which provide the advantages of specificity, sensitivity and a simple protocol for the direct detection of NO. Using these DAFs, we could detect the generation of NO not only from inducible NO synthase expressed in macrophages, but also from constitutive NO synthase expressed in ECs.

Prostaglandin D2 Inhibits Inducible Nitric Oxide Synthase Expression in Rat Vascular Smooth Muscle Cells

Vascular smooth muscle cells (VSMCs) as well as macrophages have been shown to generate a substantial amount of NO in inflammatory vascular lesions. Prostaglandin (PG) D2 (PGD2) is produced by inflammatory cells, including mast cells and macrophages. We investigated whether PGD2 modulates NO metabolism in rat VSMCs. PGD2 at a concentration of 10(-7) mol/L or greater dose-dependently inhibited nitrite accumulation in the medium of cultured VSMCs stimulated with interleukin 1beta (IL-1beta). In a dose-response analysis of IL-1beta and nitrite accumulation, PGD2 was seen to decrease the maximal ability of VSMCs to generate NO, arguing against competition by PGD2 at cytokine receptors. Northern analysis showed that PGD2 suppresses induction of inducible NO synthase (iNOS) mRNA in IL-1beta-stimulated VSMCs, with consequent inhibition of iNOS protein expression in Western analysis. A thromboxane A2 (TXA2) analogue, U46619 (10(-5) mol/L), produced less inhibition of NO generation than did PGD2. Neither the PGI2 analog carbaprostacyclin nor PGE1 showed any inhibition. PGD2 dose-dependently inhibited NO generation despite the addition of the TXA2 antagonist SQ29548. PGJ2, delta12-PGJ2, and 15-deoxy-delta12,14-PGJ2, all metabolites of PGD2, were as potent as or slightly stronger than PGD2 in the inhibition of NO generation. These data suggest that PGD2 suppresses NO generation in VSMCs by inhibiting iNOS mRNA expression, most likely through the cascade of the PGJ2 series rather than through the TX receptor or cAMP upregulation. Such action makes it likely that PGD2 regulates NO metabolism in vascular lesions.

Stress-Induced Elevation of Ouabainlike Compound in Rat Plasma and Adrenal

Recent observations demonstrate the presence of neurosteroids and their rapid increase in response to acute stress. In view of a steroidal nature of ouabainlike compound, we tested the hypothesis that ouabainlike compound may participate in a homeostatic response to acute stress. Male Wistar rats were subjected to acute stress by swimming in water (22 degrees C) for 10 minutes. The levels of ouabainlike compound in plasma, hypothalamus, pituitary, and adrenal at 10, 40, and 70 minutes (n = 8 for each) after the end of swim stress were compared with nonstressed control levels (n = 10). Ouabainlike compound was measured by a radioimmunoassay for ouabain. Plasma levels of corticosterone and catecholamines were also measured. Plasma corticosterone concentrations increased rapidly at 10 minutes (P < .01) and then declined. A trend for a rise in plasma catecholamines was found at 10 minutes. Adrenal levels of ouabainlike compound concomitantly increased at 10 minutes (P < .01, control: 58.9 +/- 5.9 pmol ouabain equivalents per gram; 10 minutes: 92.5 +/- 4.8; 40 minutes: 47.3 +/- 9.6; 70 minutes: 45.1 +/- 6.3). In contrast, the response of plasma ouabainlike compound was slow and doubled at 40 minutes (P < .01, control: 115 +/- 12 pmol ouabain equivalents per liter; 10 minutes: 132 +/- 23; 40 minutes: 226 +/- 53; 70 minutes: 117 +/- 16). Ouabainlike compound levels in hypothalamus and pituitary remained unaltered. These findings suggest that ouabainlike compound may function as a stress hormone.

Elevation of Ouabainlike Compound Levels With Hypertonic Sodium Chloride Load in Rat Plasma and Tissues

A major biologically active endogenous digitalis-like factor in the mammalian body may be an isomer of ouabain (ouabainlike compound, OLC). However, the exact role of OLC in sodium homeostasis is still unclear, and acute isotonic volume expansion does not enhance the secretion of OLC. We tested the hypothesis that OLC may be more important in the response to acute hypertonic NaCl load rather than isotonic volume expansion. We injected intraperitoneally 2 mL of 20% NaCl solution into male Wistar rats (n=34) and measured OLC levels in plasma, hypothalamus, pituitary, and adrenal at baseline (n=10) and 1, 2, and 4 hours (n=8 for each). In response to hypertonic NaCl loading, plasma Na-K ratio was elevated at 2 and 4 hours (P<.01). OLC levels in pituitary increased (P<.01) at 1 hour. Thereafter, plasma OLC levels increased at 2 and 4 hours (P<.05; basal, 75+/-11 pmol/L [+/-SEM]; 1 hour, 55+/-11; 2 hours, 130+/-24; 4 hours, 156+/-20). Concomitantly, OLC levels in adrenal increased at 2 and 4 hours (P<.01; basal, 1.7+/-0.2 pmol/g; 1 hour, 4.5+/-0.9; 2 hours, 5.0+/-0.7; 4 hours, 6.8+/-2.2). A significant correlation was observed between OLC levels in plasma and adrenal (P<.05). Plasma Na-K ratio positively correlated with OLC levels in plasma (r=.51, P<.01) and adrenal (r=.48, P<.01). Similar injection of physiological saline solution or hypertonic sucrose solution in physiological saline did not increase OLC levels in plasma and tissues. These findings indicate the elevation of OLC levels in plasma, pituitary, and adrenal in response to acute hypertonic NaCl load in rats and suggest that OLC may be involved in the response to the hypernatremic state.

Angiotensin-Converting Enzyme Inhibition Delays Onset of Glucosuria With Regression of Renal Injuries in Genetic Rat Model of Non-Insulin-Dependent Diabetes Mellitus.

Background: The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is a new genetic model of non-insulin-dependent diabetes mellitus (NIDDM). We investigated whether angiotensin inhibition influences the onset of NIDDM and brings about a regression of renal injury in diabetes mellitus. Methods and Results: Six-week-old OLETF rats were treated with the angiotensin-con verting enzyme (ACE) inhibitors imidapril or enalapril for 16 weeks. Systolic blood pres sure is increased in an age-dependent manner in OLETF rats. In this study, the elevation in systolic blood pressure was dose-dependently reduced by ACE inhibitor treatment. In OLETF rats, plasma concentrations of insulin and glucose increased and the glucosuria occurred at the age of 22 weeks. Simultaneously, OLETF rats exhibited proteinuria and nodular lesions in glomeruli. The ACE inhibitor treatment almost completely reduced glu cosuria, and also decreased plasma concentrations of insulin and glucose in OLETF rats. ACE inhibitor treatment lessened the proteinuria and attenuated morphologically the severity of nodular lesions in OLETF rats. Moreover, increases in plasminogen activator inhibitor 1 (PAI-1) in OLETF rats were reduced by the ACE inhibitor treatment, and the improvement of glomerular lesions was related to decreases of PAI-1 and angiotensin II levels in plasma but not to improvement of glucose metabolism. Conclusions: ACE inhibitors delay onset of NIDDM with attenuation of kidney injury. The regression of kidney lesions is probably due to angiotensin reduction but not to glucose metabolism per se. ACE inhibitor drug therapy may be useful in preventing NIDDM and the subsequent renal injury in patients with NIDDM.

Role of Ouabain-like Compound in the Regulation of Transmembrane Sodium and Potassium Gradients in Rats

A major biologically active Na,K-ATPase inhibitor in the mammalian body may be ouabain-like compound. We investigated the potential roles of circulating ouabain-like compound in the regulation of Na+ and K+ homeostasis in terms of Na+ and K+ distribution between the cells and the extracellular fluid (internal balance). First, we developed a population of rats immunized against ouabain to block the action of ouabain-like compound. We measured plasma and intracellular Na+ and K+ concentrations in skeletal muscle and determined Na+ (extracellular-to-intracellular concentration ratio) and K+ (intracellular-to-extracellular concentration ratio) gradients in immune rats. We examined also the ability to respond to hypertonic NaCl load in immune rats. Consistent lower plasma K+ levels and steeper Na+ and K+ gradients were observed in immune rats. K+ handling in response to hypertonic NaCl load was altered, and lower plasma K+ level was maintained in immune rats. Second, we used PST-2238, a newly developed anti-ouabain agent, to block the action of ouabain-like compound and examined its effect on plasma Na+ and K+ concentrations. Chronic administration of PST-2238 significantly lowered plasma K+ levels in rats with subtotal nephrectomy. These findings collectively suggest that ouabain-like compound may determine at least in part the internal Na+ and K+ distribution and the transmembrane cation gradients in vivo in rats.

A New Adenosine Subtype-1 Receptor Antagonist, FK-838, Attenuates Salt-Induced Hypertension in Dahl Salt-Sensitive Rats

We investigated the effects of the adenosine type-1 receptor antagonist FK-838 on salt-induced hypertension in Dahl-Sea salt-sensitive (Dahl S) rats. Dahl S rats fed a high-salt (4% NaCl) diet for 4 weeks were treated with FK-838 or hydrochlorothiazide for 4 weeks and alterations in kidney function and morphologic changes were assessed. FK-838 attenuated the development of hypertension in Dahl S rats, and caused a decrease in aortic weight in a dose dependent fashion. The adenosine antagonist did not produce any detectable metabolic disturbance. The blood pressure reduction by FK-838 was associated with attenuation of glomerular and arterial injury in the kidney. The renal protective effect of FK-838 treatment was associated with a reduction of plasma renin activity and plasma aldosterone concentration. In contrast, the thiazide diuretic, which produced almost the same blood pressure reduction as FK-838, did not attenuate renal damage. These data indicate that adenosine A1 receptor antagonism reduces salt-induced hypertension and the consequent renal injuries.

MODULATION OF VASCULAR CALCIUM CHANNEL ACTIVITY IN RESPONSE TO ACUTE VOLUME EXPANSION IN RATS

The mechanisms of the increased resistance in hypertension are still unclear. Several studies have indicated that the potential-sensitive Ca2+ channels (PSC) are altered in arteries isolated from hypertensive patients or animals. An expansion of body fluid volume may trigger local autoregulatory responses or may induce the release of humoral factors, either of which could increase systemic vascular resistance and cause volume-dependent forms of hypertension. We tested the hypothesis that volume expansion per se may cause the alterations of PSC similar to those seen in hypertension. For this, we examined the alterations of PSC in aortas from volume-expanded rats with the use of dihydropyridine-type Ca2+ channel activator, BayK 8644, in parallel with the changes in endothelium-dependent relaxation. Volume expansion was produced by a rapid intravenous infusion of saline (10% of body weight) over 30 min in rats. At the end of infusion, rats were killed and aorta removed for in vitro measurement of isometric tension. Relaxation to acetylcholine (10(-7)-10(-5) mol/L, % relaxation to 10(-7) mmol/L norepinephrine contraction) was not significantly changed. In contrast, contractile response to BayK 8644 (10(-9)-10(-6) mol/L, % response to 50 mmol/L KCl) was significantly enhanced in rats with volume expansion (12 control rats: 11.6 +/- 4.9%; 18 volume-expanded rats: 40.9 +/- 10.4% at 10(-6) mol/L, p < 0.05). These findings suggest that acute volume expansion could induce a similar enhanced vascular Ca2+ channel activity to that seen in hypertension in rats.

Digitalis-like factors from human urine.

We isolated two candidates for endogenous digitalis-like factors from human urine based on the inhibition of [3H]ouabain binding to intact human erythrocytes. The more-polar ouabain-displacing compound-1 (ODC-1) closely resembled ouabain in biological, physicochemical, and chromatographic properties. Moreover, anti-ouabain IgG dose-dependently neutralized the action of ODC-1. The less-polar ODC-2 behaved identically to digoxin in three analytical high-performance liquid chromatography and thin layer chromatography systems. Fast atom bombardment mass spectrum and proton nuclear magnetic resonance spectrum supported the notion that ODC-2 may be indistinguishable from digoxin. The possibility that substances quite similar to cardenolides are synthesized in the mammalian body must be seriously considered.

Relation of 24-h ambulatory blood pressure with plasma potassium in essential hypertension.

The established associations between blood pressure (BP) and electrolytes are mostly based on either dietary intake or urinary excretion data. We measured office BP, ambulatory BP (ABP) using the automated oscillometric ABPM-630 device, and plasma electrolytes in 82 essential hypertensive patients to examine the relation between BP and plasma electrolytes. Significant negative correlations were observed between plasma potassium concentration and 24-h systolic BP (r = -0.336) and diastolic BP (r = -0.298) in our patients. Plasma potassium concentration inversely correlated also with both daytime and nighttime systolic and diastolic BPs. There was no relation between office BP and plasma potassium concentration. These findings indicate that in essential hypertensives plasma potassium concentration is inversely related to ABP including daytime and nighttime BPs and suggest that potassium may be a factor determining the whole day BP in essential hypertension.