Apolipoprotein-mediated cellular cholesterol/phospholipid efflux and plasma high density lipoprotein level in mice.

Helical apolipoprotein(apo)s generate pre-beta-high density lipoprotein (HDL) by removing cellular cholesterol and phospholipid upon the interaction with cells. To investigate its physiological relevance, we studied the effect of an in vitro inhibitor of this reaction, probucol, in mice on the cell-apo interaction and plasma HDL levels. Plasma HDL severely dropped in a few days with probucol-containing chow while low density protein decreased more mildly over a few weeks. The peritoneal macrophages were assayed for apoA-I binding, apoA-I-mediated release of cellular cholesterol and phospholipid and the reduction by apoA-I of the ACAT-available intracellular cholesterol pool. All of these parameters were strongly suppressed in the probucol-fed mice. In contrast, the mRNA levels of the potential regulatory proteins of the HDL level such as apoA-I, apoE, LCAT, PLTP, SRB1 and ABC1 did not change with probucol. The fractional clearance rate of plasma HDL-cholesteryl ester was uninfluenced by probucol, but that of the HDL-apoprotein was slightly increased. No measurable CETP activity was detected either in the control or probucol-fed mice plasma. The change in these functional parameters is consistent with that observed in the Tangier disease patients. We thus concluded that generation of HDL by apo-cell interaction is a major source of plasma HDL in mice.

Desensitization, surface expression, and glycosylation of a functional, epitope-tagged type I PACAP (PAC(1)) receptor.

To study desensitization and glycosylation of the type I pituitary adenylate cyclase-activating polypeptide (PACAP) receptor (PAC(1)R), a hemagglutinin (HA) epitope was inserted within the N-terminal extracellular domain, allowing immunological detection of PAC(1)R both in intact and permeabilized cells. PAC(1)R was tagged without loss of functions in ligand binding and ligand-stimulated cAMP production. In transiently transfected COS-7 cells, PAC(1)R was localized both in the plasma membrane and the cytoplasm around the nucleus. By immunoblot analysis, the immunoreactive bands with relative molecular masses ranging from 45 to 70 kDa were detected in the membrane fractions of PAC(1)R-expressing COS-7 cells. Digestion of the membranes with endoglycosidase F or treatment of the cells with tunicamycin decreased the size of the receptor to major bands of smaller size (approximately 45 and 48 kDa), suggesting that these two forms of PAC(1)R represent core proteins. Flow cytometric analysis indicated that the agonist promoted a disappearance of cell surface receptor. In accordance with this observation, preexposure of cells to PACAP38 induced a desensitization of PAC(1)R to the agonist response, although it did not cause a reduction in PAC(1)R mRNA or protein level and even slightly elevated them. These results suggest that agonist-induced desensitization of PAC(1)R involves the receptor sequestration.

Effect of probucol in lecithin-cholesterol acyltransferase-deficient mice: inhibition of 2 independent cellular cholesterol-releasing pathways in vivo.

Cellular cholesterol release takes place by at least 2 distinct mechanisms: the lecithin-cholesterol acyltransferase (LCAT)-driven net efflux by cholesterol diffusion and the generation of high density lipoprotein (HDL) with cellular cholesterol and phospholipid on the cell-apolipoprotein interaction. Therefore, LCAT deficiency impairs the former pathway, and the latter can be inhibited by probucol, which interferes with the apolipoprotein-cell interaction. Hence, probucol was given to the LCAT-deficient mice in the attempt to suppress both of these pathways. The mice were fed low (0.2%) and high (1.2%) cholesterol diets containing 0.5% probucol for 2 weeks. LCAT deficiency and probucol markedly decreased plasma HDL, and the effects were synergistic. Tissue cholesterol content was lower in the adrenal glands and ovaries in the LCAT-deficient mice and in the probucol-treated mice, suggesting that HDL is a main cholesterol provider for these organs. It was also moderately decreased in the spleen of the low cholesterol-fed female mice and in the thyroid gland of the low cholesterol-fed male mice. On the other hand, the esterified cholesterol content in the liver was substantially increased by the probucol treatment with a high cholesterol diet in the LCAT-deficient mice but not in the wild-type mice. Among the groups, there was no significant difference in the tissue cholesterol levels in other organs, such as the liver, spleen, thymus, brain, erythrocytes, thyroid gland, testis, and aorta, resulting from either LCAT deficiency or probucol. Thus, the apolipoprotein-mediated mechanism plays a significant role in the export of cellular cholesterol in the liver, indicating that the liver is a major site of the HDL assembly. Otherwise, tissue cholesterol homeostasis can largely be maintained in mice even when the assembly of new HDL is inhibited by probucol in the absence of LCAT. Nonspecific diffusion of cholesterol perhaps adequately maintains the homeostasis in the experimental condition.

Walking 10,000 steps/day or more reduces blood pressure and sympathetic nerve activity in mild essential hypertension.

We investigated the effects of walking 10,000 steps/day or more on blood pressure and cardiac autonomic nerve activity in mild essential hypertensive patients. All subjects were males aged 47.0+/-1.0 (mean+/-SEM) years old. The original cohort consisted of 730 people in a manufacturing industry who measured the number of steps they walked each day using a pedometer. Eighty-three of these subjects walked 10,000 steps/day or more for 12 weeks. Thirty-two of these were hypertensives with systolic blood pressure (SBP) greater than 140 mmHg and/or diastolic blood pressure (DBP) greater than 90 mmHg. Thirty of these hypertensive subjects (HT) were examined twice, once during the pre- and once during the post-study period, for body mass index (BMI), maximal oxygen intake (Vo2max), blood pressure, heart rate (HR), and autonomic nerve activity by power spectral analysis of SBP and HR variability. In the HT group, walking 13,510+/-837 steps/day for 12 weeks lowered blood pressure (from 149.3+/-2.7/98.5+/-1.4 to 139.1+/-2.9/90.1+/-1.9 mmHg; p<0.01, respectively). In both the 34 normotensive controls and 17 hypertensive sedentary controls, blood pressure did not change. Walking also significantly lowered low-frequency fluctuations in SBP as an index of sympathetic nerve activity, from 1.324+/-0.192 to 0.738+/-0.154 mmHg2/Hz (p<0.05). VO2max rose significantly from 26.1+/-2.4 to 29.5+/-2.5 ml/kg/min (p<0.05). There were no changes in parasympathetic nerve activity, baroreceptor reflex sensitivity, or BMI. Our results indicate that walking 10,000 steps/days or more, irrespective of exercise intensity or duration, is effective in lowering blood pressure, increasing exercise capacity, and reducing sympathetic nerve activity in hypertensive patients.

Plasma concentrations of adrenomedullin correlate with the extent of pulmonary hypertension in patients with mitral stenosis.

OBJECTIVE: To examine the pathophysiological significance of adrenomedullin in the pulmonary circulation by investigating the relation between plasma concentrations of adrenomedullin and central haemodynamics in patients with mitral stenosis. METHODS: Plasma concentrations of adrenomedullin in blood samples obtained from the femoral vein, pulmonary artery, left atrium, and aorta were measured by a newly developed specific radio-immunoassay in 23 consecutive patients with mitral stenosis (16 females and seven males, aged 53 (10) years (mean (SD)) who were undergoing percutaneous mitral commissurotomy. RESULTS: Patients with mitral stenosis had higher concentrations of adrenomedullin than age matched normal controls (3.9 (0.3) v 2.5 (0.3) pmol/l, p < 0.001). There was a reduction in adrenomedullin concentrations between the pulmonary artery and the left atrium (3.8 (0.2) v 3.2 (0.4) pmol/l, p < 0.001). The venous concentrations of adrenomedullin correlated with mean pulmonary artery pressure (r = 0.65, p < 0.001), total pulmonary vascular resistance (r = 0.83, p < 0.0001), and pulmonary vascular resistance (r = 0.65, p < 0.001). Plasma concentrations of adrenomedullin did not change immediately after percutaneous mitral commissurotomy; however, they decreased significantly one week later. CONCLUSIONS: Plasma concentrations of adrenomedullin are increased in patients with mitral stenosis. This may help to attenuate the increased pulmonary arterial resistance in secondary pulmonary hypertension due to mitral stenosis.

Pulmonary capillary hemangiomatosis: a unique feature of congestive vasculopathy associated with hypertrophic cardiomyopathy.

A 34-year-old man with an 18-year history of hypertrophic cardiomyopathy died of worsening right-sided heart failure. Central venous pressure was greatly increased to 25 cm H2O before death. Postmortem examination revealed features of severe congestive vasculopathy, including those of pulmonary capillary hemangiomatosis in the lungs. Marked proliferation of capillaries was seen chiefly in alveolar septa and extending into pulmonary veins and arteries, causing severe luminal occlusion with recanalization. Diffusely distributed intra-alveolar edema and hemorrhage with collections of hemosiderin-laden macrophages were also seen, which suggested that the pulmonary capillary hemangiomatosis was associated with longstanding chronic passive congestion of the lung. It is possible that severe pulmonary passive congestion may be one of the causes of development of idiopathic pulmonary capillary hemangiomatosis.

Adrenal tumor producing 11-deoxycorticosterone, 18-hydroxy-11-deoxycorticosterone and aldosterone.

A case of adrenal tumor producing 11-deoxycorticosterone, 18-hydroxy-11-deoxycorticosterone and aldosterone is reported. A 55-year-old woman had hypertension, hypokalemia, low plasma renin activity and an adrenal tumor. The plasma level of aldosterone was normal, and the levels of 11-deoxycorticosterone and 18-hydroxy-11-deoxycorticosterone were extremely high. After the tumor removal, the plasma level of aldosterone decreased and plasma levels of 11-deoxycorticosterone and 18-hydroxy-11-deoxycorticosterone were normalized. The tumor was benign adenoma and the production of steroid hormones was under control of adrenocorticotropic hormone. The enzyme activity of 21-hydroxylation in the tumor was elevated and that of 11 beta-hydroxylation was decreased compared with the adjacent tissue.

Optical control of the magnetic anisotropy of ferromagnetic bilayered manganites.

Optical manipulation of the magnetic anisotropy is demonstrated for bilayered manganites, La2-2xSr1+2xMn2O7, by means of femtosecond Kerr-rotation measurements. Upon the photoexcitation on the x=0.32 crystal, the magnetization exhibits the precessional motion for about 1 ns, revealing the directional change of the magnetocrystalline anisotropy from the c axis to the ab plane. This change of the anisotropy induces the nonthermal decrease of the c-axis magnetization component for about 1 ns.

Noninvasive assessment of left ventricular diastolic filling in coronary artery disease by Doppler dipyridamole-stress testing.

To evaluate left ventricular diastolic function during dipyridamole-provoked myocardial ischemia, transmitral flow was studied in 73 patients with coronary artery disease and 8 normal subjects using pulsed Doppler echocardiography. Coronary vasodilating agents like dipyridamole can provoke myocardial ischemia in patients with coronary artery disease. The peak flow velocity of left ventricular rapid filling (R), that of atrial contraction (A) and the ratio of A to R (A/R) in each cardiac cycle were measured. The rapid filling phase was divided into two subphases at the point of R. The integral of the two subphases and atrial contraction were computed and designated IR1, IR2 and IA. The time intervals of the two subphases of rapid filling were designated TR1 and TR2. Of the 73 patients with coronary artery disease, 41 patients developed ischemia (positive responder = PR) and 32 patients did not (negative responder = NR) after dipyridamole infusion. In PR, A/R increased (p less than 0.05), IR2 decreased (p less than 0.01) and TR2 shortened (p less than 0.01) significantly. In NR and normal subjects, these indices remained unchanged. We observed mitral regurgitation (MR) in 13 PR patients during acute myocardial ischemia. A/R increased in patients without MR but A/R remained unchanged in patients with MR. These results suggest that in acute myocardial ischemia, changes in Doppler indices (A/R, IR2 and TR2) reflect a left ventricular diastolic abnormality, and that the masking of the diastolic abnormality was ascribed to the presence of MR.

Hemodynamic effect of amrinone depends on pretreatment vascular resistance in patients with evolving congestive heart failure: correlation between vascular resistance and neurohormonal activity.

We investigated the hemodynamic effects of amrinone and assessed its effects on neurohormonal factors in 15 patients with evolving congestive heart failure with various origins. We serially determined the pulmonary and systemic vascular-resistance indices after amrinone infusion and examined the relation between changes in hemodynamic parameters and changes in concentrations of norepinephrine, atrial natriuretic peptide, angiotensin II, and endothelin-1 in the pulmonary capillary wedge region (PCWR) and in the peripheral veins. Amrinone significantly reduced pulmonary vascular-resistance index (PVRI; Wood x m2) in patients with high PVRI (> or =15) before the infusion, significantly reduced systemic vascular-resistance index (SVRI; Wood x m2) in patients with high SVRI (> or =50) before the infusion, and had little effect on vascular resistances in patients with low PVRI (<15) and low SVRI (<50). The reduction in PVRI was correlated with the reduction in the endothelin-1 level (r = 0.75) in the PCWR, and the reduction in SVRI with norepinephrine level (r = 0.70) in the peripheral veins. The angiotensin II level did not change throughout the study. These findings suggest that amrinone had selective hemodynamic effects on pulmonary and systemic circulations with neurohormonal effects, according to PVRI and SVRI before infusion.

[Transmitral flow velocity patterns as influenced by preload, afterload and heart rate alterations: pulsed Doppler echocardiographic study].

The influences of preload, afterload and heart rate alterations on the pattern of left ventricular filling were investigated using pulsed Doppler echocardiography (PDE) in humans. Transmitral flow at the level of the valvular tip was recorded during dextran infusion, lower body negative pressure, angiotensin II infusion, and atrial and atrioventricular sequential pacings. Peak velocity of rapid filling (R), peak velocity of atrial contraction (A), the ratio of peak velocities (A/R), flow velocity integrals of the rapid filling phase (IR) and atrial contraction (IA) were obtained. PDE and the measurement of hemodynamics during lower body negative pressure (0, -10 mmHg, -20 mmHg) and dextran infusion (100 ml, 200 ml) were studied in 22 patients with ischemic heart disease. R decreased significantly after lower body negative pressure, and increased significantly during dextran infusion. Before and during angiotensin II infusion, PDE and the measurement of hemodynamics were studied in 14 patients with ischemic heart disease. The patients were categorized into 2 subgroups according to left ventricular function. During afterload stress, the A/R and IA increased in patients with normal left ventricular function; whereas, the A/R decreased in patients with poor left ventricular function. PDE was recorded during right atrial and atrioventricular sequential pacings at the heart rates of 60 to 100 beats/min in 29 patients with ischemic heart disease. When the heart rates increased, R decreased during atrial and atrioventricular sequential pacings. The A increased after the occurrence of the summation between the rapid and atrial filling waves. The A/R gradually increased with incremental heart rate. These results indicate that changes in the preload alter the peak velocity of left ventricular filling pattern of transmitral flow. The effects of the increasing afterload depend on the basal left ventricular function, with an increase in the peak velocity of atrial contraction being observed in the presence of normal left ventricular function. Both the peak velocity of rapid filling and atrial contraction were related to the heart rate and atrioventricular conduction delay. In assessing left ventricular filling dynamics using PDE, the influence of the preload, afterload and heart rate must be considered.

Single-beat estimation of the slope of the end-systolic pressure-volume relation in the human left ventricle.

This study assessed a new method of estimating the slope (Ees) of the end-systolic pressure-volume relation (ESPVR) from a single beat of the human heart. Left ventricular pressure was recorded with a high-fidelity micromanometer in patients with heart disease during left ventriculography. Peak isovolumic pressure at the end-disastolic volume was estimated by a curve-fitting technique from an isovolumic left ventricular pressure curve. The ESPVR line was drawn from the estimated peak isovolumic pressure-volume point tangential to the left upper corner of the pressure-volume loop. The slope of this estimated ESPVR line from single-beat analysis was compared with the slope of the ESPVR line obtained from three pressure-volume loops in 16 patients given angiotensin II or nitroglycerin infusion. The estimated Ees was 5.0 +/- 2.2 mm Hg/ml/m2, and the conventional Ees was 4.9 +/- 2.7 mm Hg/ml/m2. The estimated Ees showed a positive correlation with the conventional Ees (r = 0.91, p less than 0.001, SEE = 1.2 mm Hg/ml/m2). In the other 13 patients, after dobutamine infusion (5 micrograms/kg/min i.v.) the estimated Ees increased significantly from 5.6 +/- 1.4 to 7.4 +/- 2.0 mm Hg/ml/m2 (p less than 0.01). Thus, the estimated Ees approximated the conventional Ees and was sensitive to a positive inotropic intervention. We conclude that this single-beat analysis method facilitates assessment of the beat-by-beat ESPVR of the human heart.

[Lesion-related factors associated with restenosis after percutaneous transluminal coronary angioplasty in the absence of patient-related factors].

Restenosis after percutaneous transluminal coronary angioplasty (PTCA) is one of the biggest problems in the treatment of coronary artery disease. Although many studies have been performed on lesion-related factors, they are influenced by patient-related factors such as smoking, hyperlipidemia, and the presence of acute coronary syndrome. In this study, lesion-related factors were assessed in the absence of other factors by univariate and multivariate analysis. One hundred and nine lesions were reviewed in 37 consecutive patients with both restenotic lesion(s) and non-restenotic one(s) confirmed by coronary arteriography performed 4.4 +/- 2.2 months after PTCA. Angiographic findings before and immediately after angioplasty were compared between restenotic and non-restenotic lesions. The overall lesion-restenosis rate was 42%. Univariate analysis revealed that calcified lesions (p < 0.05), multiple irregularities (p < 0.01) before angioplasty, residual percentage stenosis (p < 0.05), and angiographical intraluminal haziness (p < 0.05) were related to restenosis. Intimal dissection after PTCA was not associated with restenosis. Multivariate analysis with multiple logistic regression revealed that multiple irregularities (t = 2.8) was the most predictive of restenosis before PTCA and residual percent stenosis (t = 2.6) after the procedure. Coronary lesions with calcification or multiple irregularities indicate high risk of restenosis after PTCA. Optimal dilatation of the lesions without intraluminal haziness regardless of intimal dissection is important to prevent restenosis.

[Feasibility of the left ventricular volume measurement by acoustic quantification method: comparison with ultrafast computed tomography].

Acoustic quantification (AQ: the real-time automated boundary detection system) allows instantaneous measurement of cardiac chamber volumes. The feasibility of this method was evaluated by comparing the left ventricular (LV) volumes obtained with AQ to those derived from ultrafast computed tomography (UFCT), which enables accurate measurements of LV volumes even in the presence of LV asynergy, in 23 patients (8 with ischemic heart disease, 5 with cardiomyopathy, 3 with valvular heart disease). Both LV end-diastolic and end-systolic volumes obtained with the AQ method were in good agreement with those obtained with UFCT (y = 1.04 x - 16.9, r = 0.95; y = 0.87x + 15.7, r = 0.91; respectively). AQ was reliable even in the presence of LV asynergy. Interobserver variability for the AQ measurement was 10.2%. AQ provides a new, clinically useful method for real-time accurate estimation of the left ventricular volume.

[Influence of preload alteration on the transmitral flow velocity in patients with old myocardial infarction: effects of nitroglycerin and lower body negative pressure].

We studied the influence of preload alteration on the pattern of transmitral flow as obtained by pulsed Doppler echocardiography. In protocol I, 14 patients with old myocardial infarction (OMI) were observed by simultaneous measurements of transmitral flow by PDE and left ventricular end-diastolic dimension (LVDd) using M-mode echocardiography during the control period and after sublingual nitroglycerin. In protocol II, eight patients with OMI were similarly observed during stepwise lower body negative pressure (LBNP: 0, -10 and -20 mmHg). The rapid filling phase was categorized in two subphases at peak R as the first half and second half phases. The integrals of each half phase and the atrial systolic phases (IR1, IR2 and IA) were measured by planimetry. After the administration of nitroglycerin, LVDd decreased significantly; whereas the mean arterial pressure decreased and heart rate increased significantly. The peak R and IR1 decreased significantly, but the peak A, IR2 and IA remained unchanged. During LBNP, LVDd decreased stepwise; whereas there was no change in the mean arterial pressure or heart rate. The changes in the peak R and IR1 paralleled the preload reduction during LBNP. The peak A, IR2, and IA remained unchanged. In conclusion, the first and second half volumes during the LV rapid filling phase can be influenced by various factors. The patterns of the transmitral flow velocity, the maximal velocity and first half volume in the LV rapid filling phase may be influenced by preload alteration.

Involvement of p38 MAP kinase pathway in the synergistic activation of PACAP mRNA expression by NGF and PACAP in PC12h cells.

We have recently shown that in PC12 cells, PACAP and NGF synergistically increase PACAP gene transcription and mRNA level, and that the MAPK/ERK kinase inhibitor PD98059 blocks the PACAP mRNA expression induced by either PACAP or NGF, but not that induced by the combination, suggesting involvement of multiple signaling pathways. Here we show that the p38 MAPK inhibitor SB203580 almost completely inhibits the PACAP mRNA expression induced by PACAP alone or in combination with NGF. PACAP induces neurite outgrowth and potentiates NGF-induced neurite outgrowth in PC12h cells. Unlike the case for the PACAP mRNA expression, SB203580 did not affect, but PD98059 reduced, PACAP and NGF-induced neurite outgrowth. These results indicate that PACAP receptors are coupled to the p38 signaling pathway, and that p38 plays a key role in the regulation of PACAP gene expression, while ERK, but not p38, MAPK is involved in PACAP and NGF-induced neurite outgrowth.

Synergistic induction of pituitary adenylate cyclase-activating polypeptide (PACAP) gene expression by nerve growth factor and PACAP in PC12 cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) gene expression was analyzed in PC12 cells. PC12 cells transfected with a PACAP promoter-luciferase reporter construct were utilized to investigate the effects of PACAP, either alone or in combination with nerve growth factor (NGF), on PACAP transcriptional response. PACAP induced transcription from the PACAP promoter through PACAP type I receptor (PAC1 receptor). PACAP gene transcription was also induced by NGF. Simultaneous treatment with PACAP and NGF resulted in a synergistic transcriptional response that was more than three times the predicted response, based on a simple additive effect of both agents. This synergism in transcriptional response paralleled the PACAP mRNA levels, as determined by RT-PCR and northern blotting. The level of PACAP mRNA peaked 3 h after stimulation and gradually returned to basal levels by 48 h. PC12 cells are known to express predominantly the hop isoform of the PAC1 receptor, which positively couples to both adenylate cyclase and phospholipase C. To determine the role of the cyclic AMP and protein kinase C pathways in PACAP gene expression, the effects of forskolin and phorbol 12-myristate 13-acetate (PMA) were then examined. PMA did not alter PACAP mRNA levels but enhanced forskolin-induced PACAP mRNA expression. Down-regulation of protein kinase C blocked the ability of PACAP to stimulate PACAP mRNA expression. The mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) kinase 1/2 (MEK1/2) inhibitor PD98059 also blocked the PACAP mRNA expression induced by either PACAP or NGF but not that induced by a combination of PACAP and NGF. These results suggest that PACAP stimulates the PACAP gene expression in PC12 cells at least in part through activation of adenylate cyclase and protein kinase C signaling pathways and that the ERK1/2 cascade is involved in PACAP and NGF-induced PACAP gene expression, although redundant signaling pathways may also be involved. The present finding showing that PACAP in combination with NGF causes a synergistic increase in PACAP gene expression in PC12 cells supports the idea that PACAP acts as an autocrine regulatory factor.

Development of non-bacterial thrombotic endocarditis after percutaneous transvenous mitral commissurotomy for severely calcified mitral stenosis.

We encountered a case of mitral stenosis, complicated with non-bacterial thrombotic endocarditis, that developed after percutaneous transvenous mitral commissurotomy (PTMC). A 71-year-old female Japanese patient had severe congestive heart failure and underwent PTMC for critical and severely calcified mitral stenosis. Four weeks later, the echocardiogram demonstrated a highly echoic protrusion in the postero-medial commissure of the mitral valve. There was little evidence of inflammation at that time. She had been anticoagulated adequately since she was admitted. The patient underwent replacement of the mitral valve. She did not show any evidence of systemic embolization. Microscopic evaluation showed only organized thrombus but no evidence of inflammation in the mitral valve. Silent development of non-bacterial thrombotic endocarditis after PTMC should be recognized as a rare but potentially lethal complication of PTMC.