Toshikazu Takabatake

Redox regulation of MAPK pathways and cardiac hypertrophy in adult rat cardiac myocyte

OBJECTIVES We analyzed the regulatory function of reactive oxygen species (ROS) on the hypertrophic signaling in adult rat cardiac myocytes: BACKGROUND The ROS regulate mitogenic signal transduction in various cell types. In neonatal rat cardiac myocyte, antioxidants have been shown to inhibit cardiac hypertrophy, and ROS are suggested to modulate the hypertrophic signaling. However, the conclusion may not reflect the situation of mature heart, because of the different natures between neonatal and adult cardiac myocytes. METHODS Cultured adult rat cardiac myocytes were stimulated with endothelin-1 (ET-1) or phenylephrine (PE), and intracellular ROS levels, the activities of mitogen-activated protein kinases (MAPKs; ERK, p38, and JNK), and 3H-phenylalanine incorporation were examined. We also examined the effects of antioxidant pretreatment of myocytes on MAPK activities and cardiac hypertrophy to analyze the modulatory function of redox state on MAPK-mediated hypertrophic signaling. RESULTS The ROS levels in ET-1- or PE-stimulated myocytes were maximally increased at 5 min after stimulation. The origin of ROS appears to be from NADH/NADPH oxidase, because the increase in ROS was suppressed by pretreatment of myocytes with NADH/NADPH oxidase inhibitor diphenyleneiodonium. Extracellular signal-regulated kinase (ERK) activity was increased by the stimulation of ET-1 or PE. In contrast, p38 and c-Jun-N-terminal protein kinase (JNK) activities did not change after these stimulations. Antioxidant treatment of myocytes suppressed the increase in ROS and blocked ERK activation and the subsequent cardiac hypertrophy induced by these stimuli. CONCLUSIONS These data demonstrate that ROS mediate signal transduction of cardiac hypertrophy induced by ET-1 or PE in adult rat cardiac myocytes.

ANTI-APOPTOTIC EFFECT OF ATORVASTATIN, A 3-HYDROXY-3-METHYLGLUTARYL COENZYME A REDUCTASE INHIBITOR, ON CARDIAC MYOCYTES THROUGH PROTEIN KINASE C ACTIVATION

1. Pleiotropic effects of statins, which are independent of lipid lowering, have been reported. In the present study, we examined the effect of a statin on apoptosis of adult rat cultured cardiac myocytes. We used the protein kinase C (PKC) inhibitors staurosporine (1 µmol/L), chelerythrine (10 µmol/L) and rottlerin (5 µmol/L) to induce myocyte apoptosis. The effect of atorvastatin (10−7 g/mL), a statin, on myocyte apoptosis induced by these PKC inhibitors was examined.

AN ANGIOTENSIN-CONVERTING ENZYME INHIBITOR PROTECTS AGAINST DOXORUBICIN-INDUCED IMPAIRMENT OF CALCIUM HANDLING IN NEONATAL RAT CARDIAC MYOCYTES

1. The effects of doxorubicin (DOX) on intracellular calcium transients were examined in neonatal rat cultured cardiac myocytes, as were the cardioprotective effects of an angiotensin‐converting enzyme (ACE) inhibitor on DOX‐induced impairment of calcium handling.

Effects of ACE inhibition and beta-blockade on collagen remodelling in the heart of Bio 14.6 hamsters.

1 The effects of angiotensin converting enzyme (ACE) inhibition and beta‐blockade on collagen in the heart and on plasma catecholamines and tissue angiotensin (Ang) I and II were examined in Bio 14.6 Syrian hamsters. Male hamsters (76–79 days old) were given low‐dose enalapril (3 mg/kg per day), high‐dose enalapril (30 mg/kg per day), atenolol (50 mg/kg per day) or vehicle for 65 days. Age and sex matched healthy F1b hamsters were used as controls. Collagen concentration was determined by measuring hydroxyproline content and the relative proportion of type I, III, and V collagens was obtained by non‐interrupted sodium dodecyl polyacrylamide gel electro‐phoresis (SDS‐PAGE). Per cent collagen area (PCA) was measured by pixel counting in myocardial tissue by a personal computer. 2 Although heartweight (HW) and bodyweight (BW) in F1b controls were significantly higher compared with drugtreated groups and vehicles, the HW/BW ratio in cardiomyopathic Bio 14.6 hamsters tended to be high compared with F1b controls and was decreased by each drug treatment. 3 Collagen concentration, total collagen content and PCA in the heart of Bio 14.6 hamsters were significantly higher than F1b controls. Collagen concentration and total collagen content were significantly decreased in all drug‐treated groups compared with vehicles. 4 The proportion of type I collagen tended to decrease while that of type III collagen tended to increase in all drugtreated groups compared with vehicles. Type V collagen in vehicle‐treated group was significantly higher than in F1b controls, while it tended to decrease in all drug‐treated groups compared with vehicles. 5 Plasma concentrations of catecholamines (adrenaline and noradrenaline) were decreased significantly by atenolol and high‐dose enalapril, but not by low‐dose enalapril. Tissue Angl remained unaltered in any of the drug‐treated hamsters. Tissue Angll was decreased by the high‐dose enalapril and beta‐blockade, and tended to be decreased by low‐dose enalapril treatment. 6 These results reveal that enalapril and atenolol produced similar beneficial effects on collagen remodelling in Bio 14.6 hamsters by decreasing the total amount of collagen, and also by changing collagen phenotypes through the inhibition of the renin‐angiotensin system. Both drugs also improved myocardial morphological integrity.

Effects of vasopeptidase inhibition on renal function and tubuloglomerular feedback in spontaneously hypertensive rats.

Vasopeptidase inhibitors are a novel class of antihypertensive agents that concomitantly inhibit angiotensin converting enzyme and neutral endopeptidase. Our purpose was to investigate the effects of omapatrilat, a vasopeptidase inhibitor, on renal function and tubuloglomerular feedback (TGF) response in anesthetized 9–10-week-old spontaneously hypertensive rats (SHR). Intravenous injection of omapatrilat at 10 μmol/kg decreased systemic blood pressure and renal vascular resistance. Renal plasma flow was unchanged, whereas glomerular filtration rate (GFR) and filtration fraction (FF) were reduced. Increased urinary sodium excretion of tubular origin was observed. These parameters remained unaltered with vehicle treatment. Micropuncture study revealed that the maximal reduction of early proximal flow rate (EPFR) induced by orthograde perfusion of Henles loop with artificial tubular fluid (ATF) was significantly reduced by omapatrilat treatment (28.5 ±3.1% vs. 72.0±2.8% of control) and was not significantly changed in the vehicle-treated group (vehicle 70.8±1.7% vs. control 71.0 ±2.1%). EPFR at zero perfusion was comparable between omapatrilat and vehicle treatment (29.7±2.2 vs. 31.3 ±2.1 nl/min, respectively). Luminal perfusion of 10-4 mol/l 7-nitroindazole in ATF abrogated the blunting of TGF response by omapatrilat but elicited no change in the vehicle-treated group. The suppression of the TGF mechanism and the reduction in FF suggest that omapatrilat respectively dilates the afferent and efferent arterioles. Under such conditions, reduction of GFR may indicate a fall in intraglomerular pressure. The restoration of nitric oxide signaling in the juxtaglomerular apparatus of SHR seems to participate in the inhibition of TGF by omapatrilat. These findings suggest that omapatrilat may provide a novel approach to the treatment of systemic and glomerular hypertension.

Renal Effects Of Efonidipine Hydrochloride, A New Calcium Antagonist, In Spontaneously Hypertensive Rats With Glomerular Injury

1. To obtain some insight into the renoprotective mechanism of the new calcium antagonist efonidipine hydrochloride, we evaluated the acute effects of efonidipine on proteinuria, glomerular haemodynamics and the tubuloglomerular feedback (TGF) mechanism in anaesthetized 24–25‐week‐old spontaneously hypertensive rats (SHR) with glomerular injury.

CONTRASTING EFFECTS OF ISOPROTERENOL AND PHOSPHODIESTERASE I11 INHIBITOR ON INTRACELLULAR CALCIUM TRANSIENTS IN CARDIAC MYOCYTES FROM FAILING HEARTS

1. Effects of a newly developed phosphodiesterase (PDE) I11 inhibitor, E‐1020, on intracellular calcium transients were compared with those of isoproterenol (ISO) in isolated single myocytes from failing hearts secondary to pulmonary hypertension induced by monocrotaline injection. Myocytes were isolated by enzyme digestion using a Langendorff apparatus. Changes in intracellular calcium concentrations ([Ca2+]i,) were recorded using a fura‐2 fluorescence microscopic technique. Cyclic AMP contents of the hearts were measured by radio‐immunoassay.

Sympathetic Regulation of the Renal Functions in Rats Reciprocally Congenic for Chromosome 1 Blood Pressure Quantitative Trait Locus

The role of the chromosome 1 blood pressure quantitative trait locus (QTL) on the sympathorenal interaction was studied using congenic strains. The two reciprocal congenic strains, WKYpch1.0 and SHRSPwch1.0, were respectively constructed by introgressing the stroke-prone spontaneously hypertensive rat (SHRSP)−derived fragment for the QTL into a Wistar-Kyoto rat (WKY) and vice versa. The role of the sympathetic nervous system in the kidney was evaluated by comparing the renal functions between denervated and sham-operated kidneys under anesthesia. The denervation was performed by stripping the adventitia off and applying 10% phenol to the blood vessels at the left renal hilus. Polyfructosan was continuously injected intravenously to determine the renal plasma flow and the glomerular filtration rate. A reciprocal and significant alteration in the renal norepinephrine (NE) content was observed between WKY and WKYpch1.0 and between SHRSP and SHRSPwch1.0. Concomitantly, the renal vascular resistance differed significantly between the congenic and the background parental strains. By contrast, no significant difference was observed in the fractional excretion of sodium, an index of the tubular function. While the denervation elicited a significant decrease of the renal NE content in all of the four strains studied, the significant effects of the denervation on the renal functions were observed only in SHRSP and WKYpch1.0, both of which harbored the SHRSP-derived QTL fragment. These results indicated that the chromosome 1 blood pressure QTL modulated the renal functions through the sympathetic nerve activity in the kidney.

Underrecognition of the Severity of Asthma and Undertreatment of Asthma in a Rural Area of Japan

Background and Aim. Revised guidelines were released in Japan in 2003 for the assessment, treatment, and management of adult asthmatics, and similar guidelines for child asthmatics were released in 2002. We reassessed the severity and possible undertreatment of asthma according to these guidelines in stable asthmatics. Methods. We reviewed medical records of 861 well-controlled asthmatic patients who, in April through June 2004 were cared for by 47 pulmonologists at 29 medical centers and 13 asthma clinics in a rural community in the San-in area of Japan. The physician obtained completed medical records about their symptoms and current treatment of the subjects, 726 adult and 135 children (aged 6 years or older) who were in stable condition and had had no exacerbations in the previous 3 months. The severity of asthma and current treatment for each patient were assessed according to the newly revised Japanese guidelines for the assessment, treatment, and management of adult and child asthmatics. Results. In adult and child asthmatics, the percentage of predicted forced expiratory volume at 1 second (FEV1.0) was smaller and has a narrower distribution range than the percentage of predicted peak expiratory flow (PEF). When the severity of asthma was classified according to symptoms alone, 50% and 35% of those classified as mildly asthmatics patients with adults and children, respectively, had moderate to severe airflow limitation. Inhaled corticosteroids were prescribed to 90.6% of adult and 14.9% of child patients. When we compared the treatments that patients were actually receiving against the optimal treatments indexed according to a combined symptoms-FEV1.0 classification, we found that 49% of adult asthmatics were overtreated, 21% were properly treated, and 30% were undertreated. Among children, the respective percentages were 35%, 25%, and 40%. Conclusion. In well-controlled adult and child asthmatics, the severity of asthma is poorly judged when symptoms alone are considered. We suggest that the severity of asthma should be assessed through a combination of symptoms and the measurement of FEV1.0 during office visits. We also suggest that the proper dose of inhaled steroid needed to maintain stable conditions should be judged according to this combined symptoms-FEV1.0 classification.

Enhanced sympathetic control of renal function in rats congenic for the hypertension-related region on chromosome 1.

1. Recent studies suggest that a quantitative trait locus (QTL) for blood pressure (BP) on rat chromosome 1 is associated with exaggerated sympathetic nervous activity. The aim of the present study was to examine whether this QTL can affect BP by altering sympathetic control of renal function.