Effect of hesperetin, a citrus flavonoid, on the liver triacylglycerol content and phosphatidate phosphohydrolase activity in orotic acid-fed rats.

The effect of dietary hesperetin on the hepatic lipid content and the enzyme activities involved in triacylglycerol (TG) synthesis in rats fed diets with or without 1% orotic acid (OA) was studied. Hepatic TG content was raised by approximately 5-fold after administration of OA for 10 days. The OA-feeding significantly increased the activity of hepatic microsomal phosphatidate phosphohydrolase (PAP), which is the rate-limiting enzyme for TG synthesis. Hepatic glucose-6-phosphate dehydrogenase (G6PDH) and malic enzyme activities were also increased. An addition of 1% hesperetin to the OA-supplemented diet resulted in the decrease of the hepatic TG content by 44% and of microsomal PAP activity. Dietary hesperetin alone neither affected liver TG content nor PAP activity significantly. OA-feeding caused an increased liver cholesterol level, whereas simultaneous addition of hesperetin and OA reduced its content to the control level. A slight reduction of hepatic cholesterol by hesperetin was also observed in the OA-free dietary group. The present study demonstrated that dietary hesperetin can reduce the hepatic TG accumulation induced by OA, and this was associated with the reduced activity of TG synthetic enzyme, PAP.

Troglitazone treatment increases plasma vascular endothelial growth factor in diabetic patients and its mRNA in 3T3-L1 adipocytes.

Troglitazone is one of the thiazolidinediones, a new class of oral antidiabetic compounds that are ligands of peroxisome proliferator-activated receptor-gamma. This study on vascular endothelial growth factor (VEGF), also known as vascular permeability factor, was prompted by our clinical observation that the characteristics of troglitazone-induced edema were very similar to those caused by vascular hyperpermeability. When Japanese diabetic patients were screened for plasma VEGF, we found levels to be significantly (P < 0.001) increased in troglitazone-treated subjects (120.1 +/- 135.0 pg/ml, n = 30) compared with those treated with diet alone (29.2 +/- 36.1 pg/ml, n = 10), sulfonylurea (25.8 +/- 22.2 pg/ml, n = 10), or insulin (24.6 +/- 19.0 pg/ml, n = 10). Involvement of troglitazone in increased VEGF levels was further supported by the plasma VEGF levels in five patients before treatment (20.2 +/- 7.0 pg/ml), after 3 months of troglitazone treatment (83.6 +/- 65.9 pg/ml), and 3 months after discontinuation (28.0 +/- 11.6 pg/ml). We further demonstrated that troglitazone, as well as rosiglitazone, at the plasma concentrations observed in patients, increased VEGF mRNA levels in 3T3-L1 adipocytes. VEGF is an angiogenic and mitogenic factor and is currently considered the most likely cause of neovascularization and hyperpermeability in diabetic proliferative retinopathy. Although increased VEGF may be beneficial for subjects with macroangiopathy and troglitazone is currently not available for clinical use, vascular complications, especially diabetic retinopathy, must be followed with great caution in subjects treated with thiazolidinediones.

Associations of medical status and physical fitness with periodontal disease.

To determine the possible associations of medical status and physical fitness with periodontal disease, a cross-sectional study was conducted. The subjects were 517 males and 113 females aged 23 to 83 years who participated in a multiphasic health test at the Aichi Prefectural Center of Health Care, Japan, from 1992 to 1997. Their periodontal status was assessed by means of the CPITN scoring system. To assess the strength of associations between the examined factors and the score, odds ratios were computed using ordinal logistic models. Conventional risk factors such as old age, smoking habits, and higher fasting plasma glucose and simplified debris index increased the risk of periodontal disease. Hypertension, hematuria, leucocytosis or thrombocytosis, positive C-reactive protein and higher serum alkaline phosphatase were positively associated with the score, whereas higher serum high-density lipoprotein cholesterol was related to a lower risk. Poor physical fitness affecting aerobic capacity, foot balance and reaction was associated with a higher CPITN score. These associations were independent of the conventional risk factors. Although these new potential risk factors should be further investigated for their causal relationship, our findings suggested a close relationship of oral health to medical status and physical fitness.

Favorable life-style modification and attenuation of cardiovascular risk factors.

In order to develop an effective counseling system for prevention of cardiovascular diseases, the association of a favorably changed life-style with improved risk factors was examined. Participants were 7,321 office workers aged 30-69 years from in and around Nagoya city. The age-adjusted odds ratio (OR) and its 95% confidence interval (CI) were calculated to assess the likelihood of risk factor improvement by favorable life-style modifications during a 3-year period. Those who began to eat breakfast and increased their vegetable intake normalized their previously abnormal diastolic blood pressure with more than twice the likelihood (adjusted OR [95% CI] 2.89 [1.29-6.46] and 2.60 [1.18-5.75], respectively). 'Began to eat breakfast' was also significantly associated with normalized total cholesterol (TC) (1.84, [1.05-3.21]). 'Stopped eating till full' significantly normalized the body mass index (2.03; [1.25-3.28]), uric acid (1.65; [1.07-2.52]) and TC (1.43; [1.04-1.97]). Those who started regular exercise significantly normalized their high-density lipoprotein-cholesterol (HDL-C) abnormality with 1.69-times the likelihood (1.69; [1.24-2.29]) and those who began to walk briskly also improved their TC abnormality (1.85; [1.19-2.89]). HDL-C was normalized with 2.55-times the likelihood in those who quit smoking (2.55; [1.68-3.86]). Because favorable life-style modifications can attenuate abnormal cardiovascular risk factors, then proper advice on specific risk factors should be routinely given at each health check-up in order to prevent the onset of cardiovascular diseases in subsequent years.

A study on how a 6-month aerobic exercise program can modify coronary risk factors depending on their severity in middle-aged sedentary women.

It is well known that physical exercise can reduce coronary risk factors. But how an aerobic exercise modifies coronary risk factors in relation to severity and physical fitness is still controversial.Fifty-four middle-aged women (mean age, 55 years) completed a 6-month on-site and home-based anaerobic threshold-level exercise program. The changes in coronary risk factor profiles were observed during the pre-intervention and intervention periods. Before the intervention (during control period), most coronary risk factors showed a rather unfavorable trend. After the program, their mean body weight decreased from 56.7 to 55.7 kg (p>0.05) and the proportion of body fat from 30.9 to 27.9% (p>0.05) without any reduction in lean body mass. Systolic blood pressure (SBP) decreased from 129.0 to 125.0 mm Hg (p>0.05) and diastolic blood pressure from 79.5 to 76.6 mm Hg (p>0.05). Fasting plasma glucose (FPG) declined from 109.6 to 103.4 mg/dl (p>0.05). Changes in SBP and FPG were most remarkable in their respective worst tertile. Serum lipids improved only modestly. Maximum oxygen uptake increased from 23.6 to 26.1 ml/kg/min (p>0.01). However, no significant correlations were found between changes in coronary risk factors and those in physical fitness. We conclude that the 6-month aerobic exercise program would modify women's coronary risk factors depending on their initial values, probably independently of the changes in physical fitness.

Relation of electrocardiographic left ventricular hypertrophy with and without T-wave changes to systemic blood pressure, body mass, and serum lipids and blood glucose levels in Japanese men.

Left ventricular (LV) hypertrophy, especially combined with an abnormal ST-T, is considered 1 of many coronary risk factors. Seven hundred forty-nine Japanese men were selected according to their electrocardiographic findings, i.e., normal electrocardiogram, LV hypertrophy without an abnormal ST-T segment, LV hypertrophy with a flat T wave, and LV hypertrophy with a negative T wave. Coronary risk factors were compared among these 4 age-matched groups. Groups with LV hypertrophy with negative or flat T waves had larger body mass index (24.9 vs 22.9 kg/m2), higher mean systemic blood pressure (111 vs 95 mm Hg), larger LV mass (265 vs 157 g), higher blood glucose (110 vs 100 mg/dl), higher serum triglyceride (148 vs 122 mg/dl), higher total cholesterol (206 vs 198 mg/dl), and lower high-density lipoprotein cholesterol (47 vs 54 mg/dl) than the normal group or the group with LV hypertrophy without T-wave change. Among these risk factors, blood pressure and glucose remained higher even after the adjustment by body mass index or by body mass index and blood pressure. Electrocardiographic LV hypertrophy with a changed T wave signified higher risk of coronary artery disease in Japanese men.

Stage-dependent changes in membrane currents in rats with monocrotaline-induced right ventricular hypertrophy.

Sequential changes in action potential configuration, 4-amino-pyridine-sensitive transient outward current (Ito), and L-type calcium current (ICa) in association with hypertrophy were investigated in ventricular myocytes from rats with monocrotaline (MCT)-induced pulmonary hypertension. The tissue weight ratio of right ventricle (RV) to left ventricle plus septum 14 and 28 days after a subcutaneous injection of MCT increased by 29.7 and 77.2%, respectively. Action potential duration (APD) of RV cells from MCT rats increased progressively, prolonged by 73.2 and 92.2% on days 14 and 28, respectively. The current density of Ito in RV cells from MCT rats on day 14 (32.5 +/- 4.5 pA/pF, n = 13) was significantly larger than in controls (26.8 +/- 4.5 pA/pF, n = 8; P < 0.05). On day 28, however, Ito density in MCT rats (15.3 +/- 4.6 pA/pF, n = 9) was significantly less than in controls (27.3 +/- 4.2 pA/pF, n = 10; P < 0.05). There were no differences in the voltage dependence of steady-state activation and inactivation of Ito between MCT and control rats. ICa density in MCT rats on day 14 (15.7 +/- 2.6 pA/pF, n = 10) was significantly larger than in controls (10.0 +/- 2.3 pA/pF, n = 10; P < 0.05), but there was no significant difference in Ito density between MCT rats (8.3 +/- 3.7 pA/pF, n = 10) and controls (11.6 +/- 3.0 pA/pF, n = 10) on day 28. These findings suggest that hypertrophy of mammalian hearts may cause stage-dependent changes in Ito and ICa density of ventricular myocytes. The APD prolongation in the early stage of hypertrophy may be caused mainly by an increase in ICa density, whereas the APD prolongation in the late stage may be ascribed to a reduction in Ito density.

Oxygen uptake and its relation to physical activity and other coronary risk factors in asymptomatic middle-aged Japanese.

PURPOSE: Low physical activity is considered to be an important risk factor for atherosclerotic coronary artery disease. However, few data are reported on the Japanese general population. The authors have studied whether oxygen uptake in physical fitness evaluation is a quantitative index for physical activity and whether or not it has a relation to coronary risk factors. METHODS: Five hundred thirteen asymptomatic Japanese (40-64 years of age, 282 males and 231 females) were tested on a cycle ergometer for measurement of peak oxygen uptake (peak VO2) and oxygen uptake at anaerobic threshold (VO2AT). Physical activity was estimated by pedometer score. Data for oxygen uptake were adjusted by age or by age and body mass index (BMI), then its relationship to the following risk factors was investigated: physical activity, BMI, blood pressure, total cholesterol, HDL and LDL cholesterol, fasting blood glucose, and triglycerides. RESULTS: Subjects in the highest peak VO2 quartile walked significantly more than those in the lowest quartile in both males and females. Those in the highest quartile showed lower BMI, lower blood pressure, lower triglyceride, and higher HDL cholesterol. The same relationship was observed for VO2AT. CONCLUSIONS: Higher fitness level determined by peak VO2 or VO2AT is related to higher physical activity and lower coronary risk factors in the asymptomatic middle-aged Japanese. These data provide support for exercise prescription in the primary prevention of coronary heart disease in Japan.

Modulation of pacemaker activity of sinoatrial node cells by electrical load imposed by an atrial cell model.

To investigate the electrotonic modulation of sinoatrial (SA) node pacemaker activity by atrial muscle, single or multiple (2-7) SA node cells isolated from rabbit hearts were connected to a membrane model [resistance-capacitance (R-C) circuit] of an atrial cell through an external circuit that mimics the gap junctional conductance (Gc) between cells. When Gc was 0 nS (uncoupled conditions), all the preparations generated regular and stable spontaneous action potentials with a mean cycle length (SCL) of 263 +/- 45 ms (+/- SD, n = 35). Step increases of Gc were associated with a progressive prolongation of SCL. At sufficiently high values of Gc, the spontaneous activity became irregular and finally stopped. We defined the threshold Gc causing an appreciable SCL irregularity as the minimum Gc at which the ratio of SD to mean of SCL was > 0.3. The threshold Gc for a single SA node cell was calculated to be 0.58 nS. In the presence of acetylcholine (ACh; 0.05-0.2 microM), the coupling-induced inhibition of spontaneous activity was greatly increased, and the threshold Gc for a single SA node cell was decreased in a concentration-dependent manner. These findings show that the pacemaker activity of SA node cells is easily inhibited when the cells are coupled to a passive atrial cell model and the inhibition is amplified by ACh. Computer simulation using a modified Oxsoft HEART model indicates that the passive atrial cell model acts as a current sink, imposing a substantial outward current on the SA node cell, and ACh amplifies the effect by activating an additional outward current.

Electrophysiological effects of Ro 22-9194, a new antiarrhythmic agent, on guinea-pig ventricular cells.

1. Cardiac effects of Ro 22-9194 were examined in papillary muscles and single ventricular myocytes isolated from guinea-pigs and compared with those of moricizine. 2. In papillary muscles, both Ro 22-9194 (> or = 10 microM) and moricizine (> or = 1 microM) caused a significant dose-dependent decrease in the maximum upstroke velocity (Vmax) and a shortening of the action potential duration. 3. In the presence of either drug, trains of stimuli at rates > or = 0.2 Hz led to an exponential decline in Vmax. This use-dependent block was enhanced at higher stimulation frequencies. A time constant (tau R) for Vmax recovery from the use-dependent block was 9.3 s for Ro 22-9194 and 26.4 s for moricizine. 4. The curves relating membrane potential and Vmax in single myocytes were shifted by Ro 22-9194 (30 microM) or by moricizine (3 microM) in a hyperpolarizing direction by 8.4 mV and 8.0 mV respectively. 5. In myocytes treated with Ro 22-9194 (30 microM), a 10 ms conditioning clamp to 0 mV caused a significant decrease in Vmax of the subsequent test action potential; further prolongation of the clamp pulse duration resulted in a modest enhancement of the Vmax inhibition. In the presence of moricizine (3 microM), a similar conditioning clamp > 200 ms caused a significant Vmax reduction; the longer the clamp pulse duration, the greater the Vmax reduction. 6. Ro 22-9194 > or = 30 microM caused a slight decrease of calcium inward current (ICa) of myocytes without affecting the delayed rectifier potassium current (IK). 7. These findings suggest that the primary electrophysiological effect of Ro 22-9194 as an antiarrhythmicagent is, like moricizine, a use- and voltage-dependent inhibition of sodium channels. From the onset and offset kinetics of the use-dependent block, Ro 22-9194 belongs to the intermediate kinetic Class I drugs, while moricizine is a slow kinetic drug. From the state-dependence of sodium channel block, Ro 22-9194 may belong to activated channel blockers, while moricizine belongs to inactivated channel blockers.

Computer simulation of the electrotonic modulation of pacemaker activity in the sinoatrial node by atrial muscle.

Electrotonic interaction between the sinoatrial (SA) node and surrounding atrial muscle was investigated in a computer simulation using a modified Oxsoft HEART model (Oxsoft, Oxford, UK). When an SA node cell model was coupled to a passive atrial membrane model (RC circuit) with various coupling conductances (Gc), there was a Gc-dependent prolongation of spontaneous cycle length (SCL). At a sufficiently high value of Gc, the spontaneous activity was finally stopped. A nonlinear relationship between Gc and SCL was obtained, similar to that observed in experiments on rabbit SA node cells. When the muscarinic potassium current (iK,ACh) was activated in the SA node cell model, the coupling-induced inhibition of pacemaker activity was potentiated. Although coupling current and iK,ACh were additive, their effects on SCL were more than additive because of the nonlinear dependence of SCL on net current. A decrease in the input resistance of the atrial membrane model to stimulate the activation of iK,ACh in atrial muscle was also shown to potentiate the coupling-induced inhibition of SA node spontaneous activity.

Inhomogeneity of cellular activation time and Vmax in normal myocardial tissue under electrical field stimulation.

To clarify the mechanism underlying the ectopic excitation after countershock, the cellular activation processes of cardiac tissue with a low-potential-gradient electric field (LPEF) were investigated in experiments using guinea pig papillary muscles and in computer simulation. Action potential upstrokes in papillary muscles during longitudinal propagation (LP) or transverse propagation (TP) were different from those of nonpropagating ones in single ventricular cells in terms of lower maximum upstroke velocity (Vmax) (LP, 231 V/s; TP, 309 V/s) and the presence of a linear ascending segment in the phase-plane plot. High Vmax (409 V/s) close to the single cell (512 V/s) was obtained in the muscle at the collision of LP (LC). Field stimulation of the muscles with LPEF < 5 V/cm caused inhomogeneous excitation suggesting multiple wave fronts, which collide with each other, and a wide spatial dispersion of Vmax (132-388 V/s). Phase-plane plots of action potential with lower Vmax were similar to LP or TP, whereas those with higher Vmax were similar to LC. In the two-dimensional discrete sheet composed of 51 x 51 elements of modified Beeler-Reuter model, the inhomogeneous excitation induced by LPEF is mimicked by setting a random variation of stimulus onset in each element. LPEF may induce inhomogeneous excitation with multiple wave fronts through a complex electrotonic interaction. This would provide a basis for the genesis of ectopic focal excitation.

[Molecular and biophysical aspects of cardiac Na channel].

Voltage-dependent ion channels mediate action potential and conduction in cardiac cells. In the past ten years, their single channel properties and protein components have been identified, isolated and restored to functional form in the purified state. Na channel has a primary alpha subunit, which is composed of four homologous domains, each of which has six transmembrane segments. Site-directed mutation has probed the function of specific regions or even of single amino acids. The ion-conducting pore, molecular voltmeter, ion filter and regulatory system of sodium channel have been characterized by coordination of biophysical, biochemical and molecular genetic methods.

Contribution of potassium accumulation in narrow extracellular spaces to the genesis of nicorandil-induced large inward tail current in guinea-pig ventricular cells.

The mechanism of nicorandil-induced large inward tail current (Itail) in single guinea-pig ventricular cells was investigated using the whole-cell patch-clamp technique. In the presence of 0.5-1.0 mM nicorandil, an activator of adenosine 5'-triphosphate (ATP)-sensitive K+ current (IKATP), a depolarization pulse causing a large outward current was followed by a large inward Itail on the repolarization step to the holding potential at -85 mV. The larger the outward current, the greater the Itail. The amplitude of Itail increased as a single exponential function (tau = 74.9 ms) as the duration of preceding depolarization was prolonged. Both the outward current and Itail were inhibited nearly completely after application of glibenclamide (1 microM), a specific blocker of IKATP. Substitution of K+ with Cs+ in both the external and internal solutions resulted in a virtual elimination of Itail. Itail was well preserved under the condition where Ca2+ entry during the preceding depolarization was largely inhibited or where external Na+ was replaced by Li+. A transient positive shift of reversal potential for the net current was observed at the peak of Itail). At 30 mM external K+ concentration, Itail was almost eliminated. From these findings, its is concluded that the Itail is a K+ current associated with an alteration of the K+ equilibrium potential (EK) following a substantial K+ efflux. This EK change is most likely explained by an accumulation of K+ in transverse tubules (T-tubules) since Itail was not induced in atrial cells in which T-tubules are poorly developed.

Long-term study of recurrent vasospastic angina using coronary angiograms during ergonovine provocation tests.

Chronologic changes of coronary spasm were examined by repeated ergonovine provocation tests during angiography. A total of 322 patients who had variant angina without severe atherosclerosis demonstrated a positive response to the first test. Ninety of these patients had recurrent variant anginal symptoms after an angina-free period of 38 +/- 12 months (mean +/- SD). Of these 90 patients, 76 (84%) had symptoms or electrocardiographic (ECG) findings similar to those of the first test. The initial 9 of these 76 patients underwent a second provocation test and showed coronary responses analogous to those on the first test. Of the 90 patients, 14 (16%) had different symptoms or ECG findings from those elicited at the first episode. All 14 patients again had a positive response to a second ergonovine test and the following angiographic changes were observed in the three major vessels between the two tests. Of the 21 vessels that had spasm on the first test, eight vessels (19%) did not have spasm on the second test. Of the 21 vessels that did not demonstrate spasm on the first test, 10 (24%) demonstrated spasm on the second test. In the present study it is concluded that the majority of patients with recurrent angina seemed to have consistency in the location of coronary spasm, while in some patients the fluctuation of coronary spasm was confirmed by two ergonovine provocation tests.

Voltage clamp of the cardiac sodium current at 37 degrees C in physiologic solutions.

The cardiac sodium current was studied in guinea pig ventricular myocytes using the cell-attached patch voltage clamp at 37 degrees C in the presence of 145 mM external sodium concentration. When using large patch pipettes (access resistance, 1-2 M omega), the capacity current transient duration was typically 70 microseconds for voltage clamp steps up to 150 mV. At 37 degrees C the maximum inward sodium current peaked in approximately 200 microseconds after the onset of a clamp step and at this strong depolarization, less than 10% of the sodium current developed during the capacity transient. The sodium current developed smoothly and the descending limb of the current-voltage relationship usually spanned a range of 40 mV. Moreover, currents reduced by inactivation of sodium channels could be scaled to superimpose on the maximum current. Current tails elicited by deactivation followed a monoexponential time course that was very similar for currents of different sizes. Data obtained over a range of temperatures (15 degrees-35 degrees C) showed that the steady-state inactivation and conductance-voltage curves were shifted to more negative voltages at lower temperatures. These results demonstrate the feasibility of investigating the sodium current of mammalian cardiac cells at 37 degrees C in normal physiological solutions.