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Notes: KATP Channel and Na/K ATPase. Introduction: Functional interaction between KATP channel and Na/K ATPase was studied in single guinea pig ventricular myocytes because both membrane molecules are known to he involved in ischemic episodes. Methods and Results: KATP channel currents were recorded at 36°C by using whole cell, cell attached, inside-out, and open cell-attached modes of patch clamp techniques on enzymatically isolated ventricular myocytes. In the whole cell mode, ouabain (1 μM) reversibly inhibited the KATP currents induced by metabolic stress (ATP-free pipette solution and 1 mM NaCN), but not those activated by cromakalim (100 μM), a KATP channel opener. In the cell-attached mode, ouabain concentration dependently inhibited KATP, channel opening induced by metabolic suppression (5.5 μM 2-deoxyglucose and 1 mM CN). Half-inhibition concentration for ouabain was 21.0 ± 5.5 nM and the Hill coefficient was 0.8 ± 0.1 (n = 26). However, ouabain did not have an effect on the channel activity induced by cromakalim (100 μM). In the inside-out mode, ouabain applied to the internal side of membrane did not affect the channel. In the open cell-attached mode made by preincubation with streptolysin-0 (0.08 U/mL), the KATP channels were not activated by the metabolic inhibitors but were by reducing extracellular ATP concentrations, because subsarcolenimal ATP concentration could he controlled through tiny membrane holes. The channels thus activated were not suppressed by ouabain. Conclusion: The inhibition of Na/K ATPase by ouahain appeared to block the KATP channels by accumulating subsarcolemmal ATP caused by a decrease of the transition from ATP to ADP. In the presence of ischemic episodes, the administration of digitalis compounds may affect the opening of KATP channels, which is primarily protective against the development of irreversible myocardial damage.

Notes: 1. It is reported that α1-receptors and adenosine A1-receptors are involved in the ischaemic preconditioning (PC) effect on infarct size (IS). However, it is still unclear to what extent α1-receptors and adenosine A1-receptors contribute to the mechanism of PC. Therefore, we investigated the extent of the contribution of α1-receptors and adenosine A1 receptors to the PC effect on IS and examined the relationship between these receptors and protein kinase C.2. Infarct size was measured in rabbits subjected to 30 min ischaemia and 48 h reperfusion. Tyramine (Tyr) was intravenously administered before 30 min ischaemia in the absence or presence of bunazosin (BN, α1-receptor blocker) and staurosporine (ST), a protein kinase C inhibitor, respectively. R(-)N6-(2-phenylisapropyl)-adenosine (PIA), a selective adenosine A1 agonist, was intravenously administered before 30 min ischaemia in the absence or presence of 8-p-sulphophenyltheophylline (8SPT), an adenosine blocker and ST, respectively. In the PC groups, BN, BN + PIA, 8SPT, 8SPT + Tyr or placebo saline was injected before or during PC.3. Both Tyr and PIA reduced the IS, which was blocked by BN and 8SPT, respectively. The IS-reducing effect of Tyr or PIA was blocked by ST. The IS-reducing effect of PC was completely blocked by BN and 8SPT, respectively. The blocking effect of BN on the IS-reducing effect of PC was abolished by adding PIA during PC ischaemia. The blocking effect of 8SPT on the IS-reducing effect of PC was abolished by adding Tyr before PC ischaemia.4. These data suggest that an α1-receptor dependent pathway exists and an adenosine A1-receptor dependent pathway, stimulation of both of which activates protein kinase C, then reduces the IS. However, exclusive stimulation of a single α1-receptor dependent pathway or a single adenosine A1-receptor dependent pathway alone is not sufficient but the summation of these pathways is required to achieve a PC effect on IS in rabbits.

Notes: 1. We investigated the role of vascular smooth muscle α-adrenoceptor subtypes in the vasoconstrictor response of femoral arteries and veins to dopamine and whether the vasoconstriction is modified by endothelium-dependent relaxation mediated via the activation of α2-adrenoceptors in ring preparations of femoral arteries and veins from mongrel dogs.2. Dopamine contracted both arteries and veins in a dose-dependent manner and this contraction was inhibited by pre-treatment with phentolamine or prazosin. Pretreatment with yohimbine shifted the dose-response curve for dopamine to the right in femoral veins, but not in arteries.3. Phenylephrine contracted femoral arteries and veins in a dose-dependent manner and this contraction was inhibited by pretreatment with prazosin.4. Clonidine produced a bell-shaped dose-response curve in femoral veins and this curve was shifted upwards by pretreatment with NG-nitro-l-arginine (l-NNA). In contrast, femoral arteries were not affected by clonidine. NG-Nitro-l -arginine potentiated contractile responses to dopamine in both veins and arteries. This potentiation was inhibited by yohimbine or by the removal of the endothelium in both arteries and veins.5. These results suggest that dopamine contracts femoral arteries via stimulation of α1-adrenoceptors and contracts femoral veins via stimulation of both α1- and α2-adrenoceptors and that these contractions are attenuated by the vasodilator action of dopamine via α2-adrenoceptor-mediated release of endothelium-derived relaxing factor.

Notes: 1. We investigated the effects of losartan and captopril on noradrenaline (NA) release and vascular reactivity to NA in the pithed rat.2. The pressor responses to sympathetic nerve stimulation (SNS) before and after i.v. administration of captopril (1 mg/kg), losartan (1 and 10 mg/kg), sodium nitroprusside (SNP; 5 μg/kg per min), losartan (1 mg/kg) + captopril (1 mg/kg), captopril (1 mg/kg) + losartan (1 mg/kg) or the bradykinin B2 receptor antagonist HOE 140 (1 mg/kg) + captopril (1 mg/kg) were measured. Plasma NA concentrations were measured during 60 s SNS before and after losartan (1 mg/kg), captopril (1 mg/kg), SNP (5 μg/kg per min) or HOE 140 (1 mg/kg) + captopril (1 mg/kg). Pressor responses to exogenous NA were measured before and after administration of losartan (1 mg/kg), captopril (1 mg/kg), HOE 140 (1 mg/kg) + captopril (1 mg/kg) or the nitric oxide synthase (NO) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg) + captopril (1 mg/kg).3. Captopril, losartan and SNP decreased frequency-response curves to a similar extent. The captopril-induced decrease in pressor responses to SNS was restored by pretreatment with HOE 140. Adding captopril to losartan decreased the curve more than did adding losartan to captopril. Both losartan, captopril and HOE 140 + captopril significantly decreased the plasma NA concentration after SNS (34.1±5.0, 27.4±2.6 and 41.4+8.1%, respectively). Sodium nitroprusside did not change the plasma NA concentration after SNS (3.8±28.2%). The dose-response curves to i.v. NA were not affected by losartan, but were significantly decreased by captopril. However, responses to NA that were reduced by captopril were restored to control values by pretreatment with HOE 140 or L-NAME.4. We suggest that both losartan and captopril decrease pressor responses to SNS by inhibiting NA release from sympathetic nerve endings; however, captopril also decreases ‘vascular reactivity’ to NA, which is mediated by nitric oxide produced by activation of the bradykinin B2 receptors.

Notes: 1. In the present study, we attempted to clarify whether the antidiabetic drug miglitol, an α-glucosidase inhibitor, has a protective effect against anginal ischaemia. We had reported previously that miglitol reduces myocardial infarct size through inhibition of glycogenolysis during ischaemia in rabbits. However, the effect of miglitol on anginal ischaemia remains unknown.2. In open-chest beagle dogs with a severely stenosed left anterior descending coronary artery, an epicardial electrode was attached to the surface of the risk area of the left ventricle and a microdialysis probe was implanted into the myocardium to measure ST segment changes and interstitial lactate accumulation. The first episode of anginal ischaemia was induced by atrial pacing and phenylephrine infusion (50–100 µg/min) for 10 min. The second episode of anginal ischaemia was induced 210 min after the first episode. Miglitol (10 mg/kg, i.v.) was administered to the miglitol group (n = 10) 30 min before the second episode of anginal ischaemia, whereas saline was administered to the control group (n = 10). Regional myocardial blood flow was measured using coloured microspheres.3. There was no significant difference in regional myocardial blood flow in the risk and non-risk areas between the first and second episodes of anginal ischaemia and between the miglitol and control groups. During the first and second episodes of anginal ischaemia, the ST segment was decreased to a similar extent in the control group. Although ST segment depression during the first episode of anginal ischaemia was similar in both groups, ST segment depression during the second episode of anginal ischaemia was significantly attenuated in the miglitol-treated group compared with the control group (1.3 ± 0.4 vs 2.2 ± 0.4 mV, respectively). Miglitol significantly attenuated myocardial interstitial lactate accumulation in the risk area.4. In conclusion, in the present study miglitol improved ST segment depression and attenuated the accumulation of myocardial interstitial lactate during anginal ischaemia without altering regional myocardial blood flow. Miglitol has an anti-anginal ischaemia effect via a mechanism that is independent of regional myocardial blood flow.

Notes: Aims:  We investigated the relationship between levels of plasma soluble Fas (sFas) and stages of diabetic nephropathy, with special reference to apoptosis and clinical features of diabetic nephropathy in 168 patients with diabetic nephropathy.Results:  There was a positive correlation between plasma sFas and creatinine levels, between sFas levels and urinary protein levels, and between sFas levels and urinary albumin. There was a negative correlation between plasma sFas levels and creatinine clearance. Plasma sFas levels in the early stage (stages 1, 2, 3A) and advanced stage (stages 3B and 4) were 2.6 ± 0.1 and 5.4 ± 0.5 ng/mL, respectively. Plasma sFas level of the advanced stage was significantly higher than that of the early stage. The number of proliferating cell nuclear antigen (PCNA) positive cells was significantly lower in the advanced stage than in the early stage. The number of in situ nick-end labelling (TUNEL) positive cells was also significantly lower in the advanced stage than in the early stage, suggesting the suppression of apoptosis.Conclusion:  These data suggest that apoptosis is involved in the advancement of diabetic nephropathy, and that plasma sFas level might be a predicting factor for prognosis.

Notes: 1. We investigated the acute effects of adrenaline infusion on plasma lipid levels in vehicle- and adriamycin-treated rabbits. Lipids were measured before and 30 and 60 min after the commencement of continuous intravenous administration of adrenaline (0.06 |xg/kg per min) or saline in pentobarbital-anaesthetized rabbits.2. Adrenaline infusion significantly increased plasma free fatty acid (P 〈 0.05) and noradrenaline (NA) levels (P 〈 0.05) in vehicle-treated control rabbits, but not in adriamycin-treated rabbits. However, adrenaline had no effect on plasma total cholesterol, free cholesterol, high-density lipoprotein-cholesterol, triglyceride or phospholipid levels.3. Pretreatment with propranolol almost completely inhibited increased plasma free fatty acid and NA levels associated with adrenaline infusion, suggesting that adrenaline increases plasma free fatty acid and NA levels via the stimulation of p-adreno-ceptors in vehicle-treated rabbits.4. It is suggested that both the production of plasma free fatty acids and the release of NA via the activation of β-adrenoceptors is reduced in rabbits with adriamycin-induced cardiomyopathy. This may be related to the down-regulation of β-adrenoceptors caused by elevated plasma NA levels induced by cardiac failure.

Notes: 1. In the present study, we investigated the effect of 1-(3- tert-butyl-2-hydroxy-5-methoxyphenyl)-3-(3-pyridylmethyl) urea hydrocloride (T-0970), a novel water-soluble low-molecular weight free radical scavenger, on the generation of hydroxyl radicals in vivo and on myocardial infarct size in an in vivo model of myocardial infarction in rabbits.2. T-0970 scavenged hydroxyl radicals generated in the myocardium during reperfusion, as assessed by using a microdialysis technique and HPLC in an in vivo model with 30 min coronary occlusion and 30 min reperfusion in rabbits.3. Another group of rabbits was subjected to 30 min coronary occlusion and 48 h reperfusion. The control group (n = 10) was infused with saline for 190 min from 10 min before occlusion to 180 min after reperfusion. The treatment group (T-0970 group; n = 10) was injected with a bolus 2.5 mg/kg T-0970 and then infused with T-0970 for 190 min from 10 min before reperfusion to 180 min after reperfusion at a rate of 100 μg/kg per min. The T-0970 + CHE group (n = 5) was given chelerythrine (CHE; a selective inhibitor of protein kinase C (PKC); 5 mg/kg, i.v.) 10 min before the administration of T-0970. The T-0970 + 5-HD group (n = 5) was given 5-hydroxydecanoate (5-HD; an inhibitor of mitochondrial KATP channels; 5 mg/kg, i.v.) 10 min before the administration of T-0970. The CHE and 5-HD groups were given CHE (5 mg/kg, i.v.) and 5-HD (5 mg/kg, i.v.) 20 min before reperfusion, respectively. After 48 h reperfusion, infarct size was measured histologically and expressed as a percentage of the area at risk (AAR). In another series of experiments, the control (n = 5) and T-0970 (n = 5) groups were killed 4 h after reperfusion following 30 min coronary occlusion and DNA fragmentation in myocytes was assessed using in situ dUTP nick end-labelling (TUNEL) at the light microscopic level.4. Infarct size, as a percentage of AAR, in the T-0970 group was significantly reduced compared with the control group (21±4 vs 41±4%, respectively; P 〈 0.05). This reduction of infarct size by T-0970 was abolished by pretreatment with CHE and 5-HD. Neither CHE nor 5-HD alone had any effect on infarct size. The percentage of infarcted myocytes with DNA fragmentation by TUNEL in the T-0970 group was significantly reduced compared with the number in the control group (4.0±1.5 vs 10.7±1.9%, respectively; P 〈 0.05).5. T-0970, a free radical scavenger, improved reperfusion injury. This effect seemed to be mediated by activation of PKC, the opening of mitochondrial KATP channels and inhibition of DNA fragmentation.

Notes: 1. We investigated the effects of 1-(3-tert-butyl-2-hydroxy- 5-methoxyphenyl)-3-(3-pyridylmethyl)urea hydrochloride (T-0162), a novel low-molecular weight free radical scavenger, on the generation of superoxide anions and hydroxyl radicals in vitro and in vivo and on myocardial infarct (MI) size in an in vivo model of MI in rabbits.2. It was found that T-0162 scavenged both superoxide anions and hydroxyl radicals in a concentration-dependent manner in vitro.3. In an in vivo rabbit model with 30 min coronary occlusion and 30 min reperfusion, T-0162 scavenged hydroxyl radicals generated in the myocardium during reperfusion.4. Anaesthetized open-chest Japanese white male rabbits were subjected to 30 min coronary occlusion and 48 h reperfusion. The control group (n = 10) was infused with 10% lecithin solution for 220 min from 10 min before occlusion to 180 min after reperfusion. The pretreatment group (n = 10) was infused with T-0162 dissolved in 10% lecithin solution for 220 min from 10 min before occlusion to 180 min after reperfusion at a rate of 400 μg/kg per min. The post-treatment group (n = 10) was injected with an i.v. bolus of 10 mg/kg T-0162 and was then infused with 400 μg/kg per min T-0162 for 190 min from 10 min before reperfusion to 180 min after reperfusion. After 48 h reperfusion, infarct size was measured histologically and expressed as a percentage of area at risk (AAR).5. There was no significant difference in haemodynamic parameters among the three groups throughout the experimental period. The per cent infarct size of the AAR in the T-0162 groups (24.8±4.3 and 30.5±3.9% for pre- and post- treatment groups, respectively) was significantly reduced compared with control (44.7±4.1%; P 〈 0.05). There was no significant difference in the AAR among the three groups.6. In conclusion, T-0162 reduces MI size through the inhibition of reperfusion injury.

Notes: Summary To define the reliability of the tracing method of fine fibrosis at low magnification (×10), the percentage area of fine fibrosis was compared between the traced pictures at magnifications of × 10 and × 250, using an image analyzer (Olympus VIP-21). A total of 25 tissue areas, each approximately 4 × 7 mm, from the inner and middle thirds of the left ventricular free wall and ventricular septum were selected from ten hearts with hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) and were stained with Masson trichrome. The percentage areas of fine fibrosis traced at a magnification of ×10 correlated well with those traced at a magnification of × 250 (Y=1.08X−1.1,r=0.95,P〈0.01). It is concluded that the overall percentage area of fibrosis is the same at magnifications of × 10 and × 250, despite the fact that at × 10 one is unable to detect individual fibers which can be detected at × 250. The tracing method of fine cardiac fibrosis at a magnification of ×10 is reliable. Therefore, fine fibrosis in large tissue sections of hearts with cardiomyopathy, such as entire transverse sections of the left ventricular wall, can be quantitatively analyzed by this method.