Hakuo Takahashi

Cardiac-specific overexpression of angiotensin II AT2 receptor causes attenuated response to AT1 receptor-mediated pressor and chronotropic effects.

Angiotensin (Ang) II has two major receptor isoforms, AT1 and AT2. Currently, AT1 antagonists are undergoing clinical trials in patients with cardiovascular diseases. Treatment with AT1 antagonists causes elevation of plasma Ang II which selectively binds to AT2 and exerts as yet undefined effects. Cardiac AT2 level is low in adult hearts, whereas its distribution ratio is increased during cardiac remodeling and its action is enhanced by application of AT1 antagonists. Although in AT2 knock-out mice sensitivity to the pressor action of Ang II was increased, underlying mechanisms remain undefined. Here, we report the unexpected finding that cardiac-specific overexpression of the AT2 gene using alpha-myosin heavy chain promoter resulted in decreased sensitivity to AT1-mediated pressor and chronotropic actions. AT2 protein undetectable in the hearts of wild-type mice was overexpressed in atria and ventricles of the AT2 transgenic (TG) mice and the proportions of AT2 relative to AT1 were 41% in atria and 45% in ventricles. No obvious morphological change was observed in the myocardium and there was no significant difference in cardiac development or heart to body weight ratio between wild-type and TG mice. Infusion of Ang II to AT2 TG mice caused a significantly attenuated increase in blood pressure response and the change was completely blocked by pretreatment with AT2 antagonist. This decreased sensitivity to Ang II-induced pressor action was mainly due to the AT2-mediated strong negative chronotropic effect and exerted by circulating Ang II in a physiological range that did not stimulate catecholamine release. Isolated hearts of AT2 transgenic mice perfused using a Langendorff apparatus also showed decreased chronotropic responses to Ang II with no effects on left ventricular dp/dt max values, and Ang II-induced activity of mitogen-activated protein kinase was inhibited in left ventricles in the transgenic mice. Although transient outward K+ current recorded in cardiomyocytes from AT2 TG mice was not influenced by AT2 activation, this study suggested that overexpression of AT2 decreases the sensitivity of pacemaker cells to Ang II. Our results demonstrate that stimulation of cardia AT2 exerts a novel antipressor action by inhibiting AT1-mediated chronotropic effects, and that application of AT1 antagonists to patients with cardiovascular diseases has beneficial pharmacotherapeutic effects of stimulating cardiac AT2.

Estimation of Sulphoconjugated Catecholamine Concentrations in Plasma by High-Performance Liquid Chromatography

Free and sulphoconjugated catecholamine (CA) concentrations were measured in plasma using a fully automated and sensitive analyser equipped with a three-column system of high-performance liquid chromatography (HPLC). Although the free dopamine (DA) concentration has been below the detection limit of HPLC analysers thus far available, this new CA analyser can measure as little as 0·03 nmol/L. For the estimation of sulphoconjugated CA, we performed enzymatic deconjugation with arylsulphatase prior to HPLC analysis. The difference between free and total concentrations represents that of sulphoconjugated CA. The intraassay coefficient variation was less than 2·2% for free noradrenaline (NA) and adrenaline (A), 12·62% for free DA, and less than 4·3% for total A, and DA. These assays of free and sulphoconjugated CA are simple and can be performed routinely by a suitably equipped laboratory.

Identification of endogenous ouabain in culture supernatant of PC12 cells

Objective Ouabain-like factor (OLF), assayed as ouabain-like immunoreactivity (OLI), is thought to represent an endogenous digitalis-like factor. We found increased plasma OLI during the surgical removal of a pheochromocytoma. The elution volume of the OLI extracted from plasma and the pheochromocytoma tissue was the same as that for authentic ouabain, using reverse phase high-performance liquid chromatography. The present study was performed to characterize OLF from the culture supernatant of a rat pheochromocytoma cell line, PC12 cells. Design OLI from culture supernatant and chromatographic fractions were assayed by a sensitive enzyme-linked immunosorbent assay for ouabain. PC12 cells, subcultured in RPMI 1640 with 10% horse serum and 5% fetal bovine serum, were washed, and then cultured in Iscoves modified Dulbeccos medium (Life Technologies, Rockville, Maryland, USA) with 0.4% bovine serum albumin (without serum). Progesterone was added to augment the production or secretion of OLI. The conditioned medium was acidified to dissociate the binding protein, and OLI was purified by five steps of octadecylsilane (ODS) column chromatography. The structural identity of this OLI was determined by liquid chromatography and mass spectrometry (LC/MS). Results OLI in the culture medium increased after addition of progesterone in a dose-dependent manner. The concentration in the culture medium was approximately double of that in homogenized PC12 cells. After five rounds of ODS column chromatography, approximately 100 ng of OLI was purified from 2 l of culture supernatant, without fetal calf serum, in the presence of progesterone. The molecular size of purified OLI was found to be identical to authentic ouabain, based on analysis by LC/MS. Conclusion Mammalian cells originating from a rat pheochromocytoma cell line were found to produce and/or secrete OLF by the addition of progesterone.

Expression of Functional Tissue Factor on Small Vesicles of Lipopolysaccharide- Stimulated Human Vascular Endothelial Cells

We examined tissue factor expression on lipopolysaccharide-stimulated endothelial cells and their small vesicles by using specific antibodies and flow cytometry. Tissue factor functional activity was also assessed by activation of factor X. Endothelial cells were stimulated with 10 microg/ml of lipopolysaccharide in M-199/bovine serum albumin. Flow cytometry showed that expression of tissue factor on endothelial cells reached a maximum at 6 hours after stimulation, whereas that on small vesicles reached a maximum after 12 hours. Factor X activation mediated by factor VIIa and tissue factor was observed over a similar time course and was inhibited by the addition of antitissue factor antibody. Immunoelectron microscopy suggested that small vesicles with expression of some tissue factor were produced from the surface of endothelial cells. Our findings thus showed that tissue factor on endothelial cells produced by lipopolysaccharide stimulation was partly released to small vesicles. This may cause disseminated intravascular coagulation and related coagulation disorders.

Enhanced Production of Nitric Oxide May Be Involved in Acute Hypotension During Maintenance Hemodialysis

To investigate the possible involvement of endogenous nitric oxide (NO) in acute hypotension during maintenance hemodialysis, we measured the plasma concentration of the nitrate anion NO3-, a stable metabolite of NO, in 19 patients undergoing hemodialysis. We analyzed heart rate variability to estimate the relationship between autonomic nervous activity and NO production, low-frequency/high-frequency components (L/H) as a parameter of cardiac sympathetic activity, and high-frequency power as a parameter of cardiac vagal activity. Six patients developed severe hypotension (a change in mean blood pressure during dialysis > or = 20 mm Hg), four patients developed mild hypotension (a change in mean blood pressure < or = 19 mm Hg and > or = 1 mm Hg), and nine patients did not develop hypotension. The plasma levels of NO3- before dialysis were markedly elevated in the severely hypotensive group compared with the patients who showed no hypotension (566+/-122 micromol/L v 133+/-38 micromol/L; P < 0.01), and this difference disappeared midhemodialysis and after hemodialysis. The plasma concentration of NO3- before dialysis was significantly associated with both the change in mean blood pressure during dialysis (r= -0.735; P = 0.003) and the mean blood pressure after dialysis (r = -0.675; P = 0.0015). The L/H ratio was inhibited before or after dialysis in the severely hypotensive group compared with the nonhypotensive group, and hypotension during dialysis was correlated with the inhibited L/H ratio before (r = 0.784; P = 0.001) or after (r = 0.822; P = 0.001) dialysis. Plasma NO3- concentrations were correlated with the L/H ratio before (r = -0.553; P = .014) or after (r = -0.546; P = 0.015) dialysis. These results suggest that inhibited sympathetic activity is one of the causes of acute hypotension during dialysis, and the enhanced production of NO is involved in this inhibition of the sympathetic activity in patients having a hypotensive episode during dialysis. The plasma concentration of NO3- before dialysis may be a predictor of the risk of hypotension during dialysis in patients with end-stage renal disease.

Distribution of the endogenous digitalis-like substance (EDLS)-containing neurons labeled by digoxin antibody in hypothalamus and three circumventricular organs of dog and macaque

Endogenous digitalis-like substance (EDLS) is a newly discovered humoral agent which causes sodium-diuresis. EDLS is well known to have inhibitory activity to Na+,K(+)-ATPase and cross-immunoreactivity to digoxin antibody; however, its precise chemical structure has not yet been determined. We had previously developed a histochemical technique for EDLS, i.e., digoxin-immunohistochemistry, and demonstrated that EDLS was produced in the hypothalamic neurons. In the present study, the distribution of EDLS-containing neurons in the hypothalamus of dog and macaque was investigated using this technique, because anti-EDLS antibody cannot be obtained yet. In both species, EDLS neuronal somata were mainly localized in the paraventricular nucleus and the supraoptic nucleus and its accessory nuclei. A number of somata were also scattered in the other hypothalamic areas. The processes of these neurons ran from the area where the somata were located, through the lateral and basal area of the hypothalamus, to the infundibulum. These nerve fibers with varicosities were associated with the primary capillaries of hypophysial portal veins. A few immunopositive nerve fibers were also seen in the pituitary posterior lobe of both species. Intensive immunoreactivities were observed in the subfornical organ and organum vasculosum laminae terminalis. There were no differences between dog and macaque.

Benzamil blockade of brain Na+ channels averts Na+-induced hypertension in rats

To determine the possible involvement of brain amiloride-sensitive Na+channels in Na+-induced hypertension, we investigated the effects of benzamil hydrochloride, a specific blocker of these Na+channels, on the acute pressor mechanisms of intracerebroventricular infusion of hypertonic NaCl and the continuous pressor mechanisms of Na+-induced chronic hypertension, such as deoxycorticosterone acetate-salt hypertensive or stroke-prone spontaneous hypertensive rats, and of non-Na+-induced hypertension, such as renovascular hypertensive rats. Intracerebroventricular preinjection with benzamil (1 or 10 nmol/kg) abolished the increase in mean arterial pressure, heart rate, abdominal sympathetic discharge, and plasma vasopressin concentration induced by an acute increase in cerebrospinal Na+ concentrations at intracerebroventricular infusion of 1.5 M hypertonic NaCl. Continuous intracerebroventricular infusion of benzamil (1 or 10 nmol ⋅ kg-1 ⋅ day-1) for 7 days attenuated Na+-induced chronic hypertension in both deoxycorticosterone acetate-salt and stroke-prone spontaneous hypertensive rats, accompanied by reduction of urinary excretion of vasopressin and norepinephrine but not in renovascular hypertensive rats. Intravenous infusion of benzamil (10 nmol ⋅ kg-1 ⋅ day-1) for 7 days affected neither arterial pressure nor urinary excretion of vasopressin and norepinephrine in either model of hypertension. Benzamil-blockable brain amiloride-sensitive Na+ channels are expected to function as one of the Na+receptors in the brain and to be involved in the pressor mechanism of Na+-induced hypertension.To determine the possible involvement of brain amiloride-sensitive Na+ channels in Na(+)-induced hypertension, we investigated the effects of benzamil hydrochloride, a specific blocker of these Na+ channels, on the acute pressor mechanisms of intracerebroventricular infusion of hypertonic NaCl and the continuous pressor mechanisms of Na(+)-induced chronic hypertension, such as deoxycorticosterone acetate-salt hypertensive or stroke-prone spontaneous hypertensive rats, and of non-Na(+)-induced hypertension, such as renovascular hypertensive rats. Intracerebroventricular preinjection with benzamil (1 or 10 nmol/kg) abolished the increase in mean arterial pressure, heart rate, abdominal sympathetic discharge, and plasma vasopressin concentration induced by an acute increase in cerebrospinal Na+ concentrations at intracerebroventricular infusion of 1.5 M hypertonic NaCl. Continuous intracerebroventricular infusion of benzamil (1 or 10 nmol.kg-1.day-1) for 7 days attenuated Na(+)-induced chronic hypertension in both deoxycorticosterone acetate-salt and stroke-prone spontaneous hypertensive rats, accompanied by reduction of urinary excretion of vasopressin and norepinephrine but not in renovascular hypertensive rats. Intravenous infusion of benzamil (10 nmol.kg-1.day-1) for 7 days affected neither arterial pressure nor urinary excretion of vasopressin and norepinephrine in either model of hypertension. Benzamil-blockable brain amiloride-sensitive Na+ channels are expected to function as one of the Na+ receptors in the brain and to be involved in the pressor mechanism of Na(+)-induced hypertension.

Stimulation of nitric oxide release from rat spinal cord by prostaglandin E2.

1 We recently demonstrated that intrathecal administration of prostaglandin E2 (PGE2) and PGF2α induced allodynia through a pathway that includes the glutamate receptor and nitric oxide (NO)‐generating systems from pharmacological studies. In order to clarify the involvement of NO in prostaglandin‐induced allodynia, we measured NO released from rat spinal cord slices by a chemiluminescence method. 2 PGE2 stimulated NO release from both dorsal and ventral regions all along the spinal cord. PGE2 stimulated the release within 10 min and increased it in a time‐dependent manner. 3 The PGE2‐induced NO release was observed at 100 nM–10 μM. PGF2α stimulated the release at concentrations higher than 1 μM, but PGD2 (up to 10 μM) did not enhance it. 4 17‐Phenyl‐ω‐trinor PGE2 (EP1>EP3) and sulprostone (EP1

Endogenous nitric oxide production is augmented as the severity advances in patients with liver cirrhosis.

1 Since endothelium‐derived nitric oxide (NO) is a potent vasodilator and degraded into nitrous ions, we measured the serum nitrate ion (NO3−) and the amount of urinary excretions of NO3− as an index for endogenous NO to ascertain whether NO formation is augmented in patients with chronic liver diseases. 2 Using inpatients suffering from chronic liver diseases, serum levels and urinary excretions of NO3− were measured by using high‐performance liquid chromatography with an anion exchange column. 3 Among the four patient groups of normal controls, and those with chronic liver diseases such as chronic active hepatitis, compensated cirrhosis, and decompensated cirrhosis the serum level of NO3− showed the highest level in a patient group with decompensated cirrhosis. The amount of urinary excretion of NO3− was significantly increased in both groups of patients with liver cirrhosis compared with the control group and patients with chronic active hepatitis. Patients with chronic active hepatitis did not show any difference between the normal control group. The amount of urinary excretion of NO3− correlated significantly and negatively with the level of serum albumin (P<0.05) and counts of platelets (P< 0.01) in patients with compensated cirrhosis. 4 These findings suggest that the production of endogenous NO is augmented in patients with liver cirrhosis, particularly in a decompensated subgroup. Increases in the production of endogenous NO correspond to the progress of liver cirrhosis, but not in patients with chronic hepatitis.

Eicosapentaenoic Acid Stimulates Nitric Oxide Production And Decreases Cardiac Noradrenaline In Diabetic Rats

1. The aim of the present study was to investigate whether long‐term oral administration of eicosapentaenoic acid increases nitric oxide (NO) production and affects cardiac sympathetic activity in rats with diabetes mellitus.