Effect of free radical scavengers on myocardial function and Na+, K+-ATPase activity in stunned rabbit myocardium

Objectives. The role of reactive oxygen species (ROS) in the mechanism of myocardial stunning was investigated. Material and methods. Isolated Langendorff-perfused rabbit hearts were subjected to 15?min normothermic ischemia followed by 10?min reperfusion with Krebs-Henseleit solution±mannitol or histidine. Results. In hearts reperfused without free radical scavenger the left ventricular developed pressure as well as its maximal positive and negative first derivatives (+dP/dt, ?dP/dt) was significantly depressed, whereas end diastolic pressure (LVEDP) increased when compared to preischemic values. Treatment with mannitol had little protective effects, whereas singlet oxygen scavenger histidine significantly improved the recovery of LVEDP and ?dP/dt. Sarcolemmal Na⁺, K⁺-ATPase activity (control, 400±41 nmol P_i.min^?1.mg^?1) was depressed in untreated stunned hearts (260±27 nmol P_i.min^?1.mg^?1), but was almost completely recovered in hearts pretreated with histidine (364±27 nmol P_i.min^?1.mg^?1). The inhibition of Na⁺, K⁺-ATPase was only slightly prevented by mannitol (302±29 nmol P_i.min^?1.mg^?1l). Conclusions. The results suggest that ROS-induced inhibition of Na⁺, K⁺-ATPase activity is involved in the mechanism of postischemic contractile dysfunction and support the view that singlet oxygen may be one of the major causes of oxidative injury during ischemia and reperfusion.     

Adrenergic regulation of (Na+, K+)-ATPase activity in proximal tubules of spontaneously hypertensive rats

Increased renal nerve activity and sodium retention have been implicated in the development of hypertension in genetically transmitted forms of this disease. The present studies were designed to investigate the relationship between renal nerve integrity and renal proximal tubule (Na+, K+)-ATPase activity in spontaneously hypertensive rats (SHR). (Na+, K+)-ATPase activity of basolateral membranes (BLMs) enriched from proximal tubules of five-week-old SHR was greater, 328.6 +/- 18.9 nmol Pi/mg protein.min, than in age-matched genetic controls rats (Wistar-Kyoto, WKY, rats), 262.3 +/- 34.6 nmol Pi/mg protein.min (P less than 0.02). There was no detectable difference in (Na+, K+)-ATPase activity of 13-week-old SHR and WKY rats. Prior renal denervation was associated with a reduction in proximal tubule basolateral membrane (BLM) (Na+, K+)-ATPase activity, 316.8 +/- 23.8 to 223.1 +/- 23.9 nmol Pi/mg protein/min (P less than 0.02), in five-week SHR. However, denervation had no effect on renal (Na+, K+)-ATPase activity in either WKY rats, nor did sham-denervation in SHR. In addition, exogenous norepinephrine, 1 microM, produced a more pronounced stimulation of (Na+, K+)-ATPase activity in basolateral membranes from SHR as opposed to WKY controls (40.2% vs. 28.7%). Therefore, renal nerve integrity and exogenous catecholamines have a greater stimulatory influence on proximal tubule (Na+, K+)-ATPase activity in the early stages (prior to 5 weeks) of the development of hypertension in SHR than in age-matched WKY rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of surgically induced ischemia/reperfusion injury by oxygen free radical scavengers

Recent experimental work implicates oxygen free radicals as mediators of ischemia/reperfusion injury. A simple cardioplegic solution was designed to scavenge superoxide anion and hydroxyl free radical with superoxide dismutase (10 micrograms/ml), mannitol (325 mOsm/L), and KCl 25 mEq/L (FRS). Hemodynamic and subcellular functions were studied in seven in situ canine models of hypothermic global ischemia receiving FRS, compared to a group (n = 7) receiving hyperosmolar, hyperkalemic saline (HSK) and to a standard model of topical hypothermia (TH, n = 5). Following 60 minutes of ischemia (10 degrees to 15 degrees C), hearts were reperfused and rewarmed. After 45 minutes of reperfusion, left ventricular peak systolic pressure (LVPSP), developed pressure (LVDP), dP/dt max, -dP/dt max, compliance, and elastic stiffness constant (K) were improved in the FRS group and not significantly different from control. Sarcoplasmic reticulum (SR) calcium transport in the FRS group was significantly improved (control = 1.077 +/- 0.022, TH = 0.754 +/- 0.018, HSK = 0.725 +/- 0.05, and FRS = 0.966 +/- 0.05 mumol/mg-min). Calcium adenosine triphosphatase (ATPase) activity did not differ significantly from control at pH 7.0. In this model of hypothermic global ischemia and reperfusion, free radical scavengers provide significant protection of mechanical and subcellular function. These findings support the hypothesis that oxygen free radicals are important mediators of myocardial ischemia and reperfusion injury.     

Nitric oxide reduces the molecular activity of Na+,K+-ATPase in opossum kidney cells.

BACKGROUND: Nitric oxide (NO) directly inhibits fluid and solute reabsorption in the proximal tubule. In the present study, we investigated the effect of NO on the Na+, K+-ATPase of opossum kidney (OK) cells, a proximal tubule cell line, and its mechanisms. METHODS: Na+,K+-ATPase activity in the membrane fraction of OK cells was measured as the ouabain-sensitive ATP hydrolytic activity. The enzyme unit number on intact cells was measured by ouabain-binding assay. RESULTS: Incubation with 0.5 mM sodium nitroprusside (SNP), a NO donor, for two hours inhibited the catalytic activity of the membrane-associated Na+,K+-ATPase in OK cells to 65.5 +/- 9.7% of control (N = 6, P < 0.05 vs. control). This effect of SNP was concentration- and time-dependent. The NO scavenger hemoglobin blunted, while another NO donor spermine NONOate (5 microM) mimicked this effect of SNP. At all concentrations and time points tested, SNP did not alter the molecular number of Na+,K+-ATPase on intact OK cells, indicating that NO inhibited the molecular activity of Na+,K+-ATPase. The soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one (ODQ), blunted the inhibitory effect of SNP on the Na+,K+-ATPase activity. An exogenous cGMP analog similarly inhibited the Na+,K+-ATPase activity. Neither lipid soluble antioxidants vitamin E/probucol or thiol group compound DL-dithiothreitol (DTT) altered the inhibitory effect of SNP on the Na+,K+-ATPase activity. CONCLUSIONS: NO inhibited the molecular activity of the Na+,K+-ATPase of the OK proximal tubule cell line probably via cGMP-dependent mechanisms.     

Enhancement of Na+,K(+)-ATPase and Ca(2+)-ATPase activities in multi-cycle ischemic preconditioning in rabbit hearts.

OBJECTIVE: Ischemic preconditioning (IP) has been shown to attenuate intracellular Na+ accumulation and Ca2+ overload during ischemia and reperfusion, both of which are closely related to the outcome of myocardial damage. We compared the effects of single- and four-cycle IP in Na+,K(+)-activated adenosine 5'-triphosphatase (Na+,K(+)-ATPase) and Ca(2+)-activated adenosine 5'-triphosphatase (Ca(2+)-ATPase) activities in in vivo rabbit hearts, correlating these differences to the quality of protection against subsequent ischemia. METHODS: The morphological outcome was evaluated in in vivo rabbit hearts subjected to 30 min of coronary occlusion and reperfusion for 180 min by assessing the ratio of infarct volume to risk zone volume. The effects of single- and four-cycle preconditioning ischemia were then examined. Another set of in vivo rabbit hearts was subjected to the measurement of ATPase activities at the conclusion of final preconditioning ischemia and at 60 min after reperfusion following 30 min of ischemia. RESULTS: The infarct volume was reduced by single-cycle IP to 38% of that in the control group. The four-cycle IP further reduced the infarct volume, which was 11% of that in the control group. Na+,K(+)-ATPase activity at 60 min after reperfusion in the four-cycle group was increased to 172% of that in the control group (10.8 micromol ADP/h/mg protein), whereas no difference was found in the single-cycle group. On the other hand, Ca(2+)-ATPase activity at the conclusion of IP was increased by single-cycle IP, the value being 255% of that in the control group (4.9 micromol ADP/h/mg protein). The four-cycle IP further increased the activity, and the value was 158% of that in the single-cycle group. CONCLUSIONS: Since increases in Na+,K(+)-ATPase and Ca(2+)-ATPase activities contribute to the decrease in intracellular Ca2+ concentration, the enhancement of these activities by four-cycle IP may be involved in the additional protection.     

右美托咪啶后处理对大鼠离体心脏缺血再灌注时线粒体损伤的影响

目的 探讨右美托咪啶后处理对大鼠离体心脏缺血再灌注时线粒体损伤的影响.方法 健康雌性Wistar大鼠,体重220～250 g,成功制备Langendorff离体灌注模型的40个心脏随机分为5组(n=8):缺血再灌注组(A组)、右美托咪啶10 nmol/L组(B组)、右美托咪啶100 nmol/L组(C组)、线粒体通透性转换孔开放剂苍术苷组(D组)及右美托咪啶联合苍术苷组(E组).离体心脏经K-H液平衡灌注20 min后,采用全心停灌40 min再灌注60 min的方法制备离体心脏缺血再灌注模型.于再灌注即刻B组、C组、D组和E组分别灌注含10 nmol/L右美托咪啶、100 nmol/L右美托咪啶、20μmol/L苍术苷、100 nmol/L右美托咪啶和20 μmol/L苍术苷的K-H液10 min.再灌注结束即刻取心尖组织,分离线粒体,测定SOD、Na+ -K+ -ATP酶、Ca2+-ATP酶活性和MDA和Ca2+含量.结果 与A组比较,B组和C组线粒体SOD、Na+ -K+ -ATP酶和Ca2+ -ATP酶活性升高,MDA和Ca2+含量降低(P＜0.05),D组和E组上述指标比较差异无统计学意义(P＞0.05);与C组比较,D组和E组线粒体SOD、Na+-K+-ATP酶和Ca2+ -ATP酶活性降低,MDA和Ca2+含量升高(P＜0.05),B组上述指标比较差异无统计学意义(P＞0.05).结论 右美托咪啶后处理可减轻大鼠离体心脏缺血再灌注时的线粒体损伤,其机制可能与抑制线粒体通透性转换孔开放有关.     

Jejunal dopamine and Na,K-ATPase activity in early chronic renal insufficiency

AIM: The uninephrectomised and three-quarter nephrectomised (3/4nx) rats present dopamine-sensitive enhanced natriuresis. This is accompanied in uninephrectomised rats by a reduced jejunal Na(+),K(+)-ATPase activity with recovered sensitivity to inhibition by dopamine. The present study examined the jejunal Na(+),K(+)-ATPase activity and the role of dopamine in 3/4nx animals. METHODS: Fourteen days after surgery, the L-amino acid decarboxylase activity (AADC) activity, the enzyme responsible for the synthesis of dopamine, and the Na(+),K(+)-ATPase activity, were determined in jejunal epithelial cells from 3/4nx and Sham rats. In addition, the effect of dopamine (1 micromol/L) on jejunal Na(+),K(+)-ATPase activity was evaluated in both groups. RESULTS: The 3/4nx rats presented a reduced AADC activity in jejunal epithelial cells (V(max) in nmol/mg prot/15 min, 142 +/- 6 vs 190 +/- 10, P < 0.05). In addition, the jejunal Na(+),K(+)-ATPase activity was increased in 3/4nx rats (Pi release in nmol/mg prot/min, 137 +/- 1 vs 122 +/- 2, P < 0.05). However, dopamine was unable to inhibit the Na(+),K(+)-ATPase activity in jejunal epithelial cells from both 3/4nx and Sham animals. CONCLUSIONS: In contrast to uninephrectomy, the jejunal Na(+),K(+)-ATPase activity is increased in 3/4nx rats and is not sensitive to inhibition by dopamine.     

Modulation of pulmonary NA+ pump gene expression during cold storage and reperfusion

BACKGROUND: Reperfusion injury with pulmonary edema continues to be a major complication after lung transplantation. Alveolar fluid homeostasis is regulated by Na+/K+-ATPase activity on the basolateral surface of alveolar epithelial cells. Intact Na+/K+-ATPase is essential to the resolution of pulmonary edema. We characterized the effects of cold ischemia and reperfusion on expression of Na+/K+-ATPase mRNA and protein. METHODS: Baseline values for Na+/K+-ATPase mRNA and protein were determined from freshly harvested lungs with no cold storage time or reperfusion (group I). Group II lungs were analyzed after cold storage times of 12 or 24 hr without subsequent reperfusion. Group III lungs were analyzed after cold storage times of 12 or 24 hr with subsequent reperfusion. Lungs were flushed with either Euro-Collins (EC) or University of Wisconsin (UW) solution in each group. All samples were quantified for Na+/K+-ATPase mRNA and Na+/K+-ATPase protein. Physiological parameters including oxygenation and compliance were also measured. RESULTS: There were no significant differences in the level of mRNA and protein for samples that were cold stored without reperfusion (group II). With reperfusion (group III) there was a significant increase in the level of the Na+/K+-ATPase mRNA after 12 hr of storage for both EC and UW. After 24 hr of storage and subsequent reperfusion, lungs flushed with EC had significantly decreased Na+/K+-ATPase protein and mRNA, although lungs preserved with UW maintained their increased levels of Na+/K+-ATPase protein and mRNA. CONCLUSIONS: Our data suggest that ischemia-reperfusion injury results in an initial up-regulation of Na+/K+-ATPase mRNA. With prolonged injury in lungs preserved with EC, the level of the mRNA decreased with a corresponding decrease in the Na+/K+-ATPase protein. The different response seen in EC versus UW may be explained by better preservation of pump function with UW than EC and correlates with improved physiological function in lungs preserved with UW solution.     

Hypoxic reperfusion limits functional impairment following cardioplegic arrest in isolated rat heart

BACKGROUND: Reperfusion injury decreases both systolic contractility and diastolic compliance. Several studies indicate that the sustained decrease in diastolic compliance is mainly due to reactive oxygen intermediates (ROI) generation and calcium overload. METHODS: Male Wistar isolated rat hearts were divided into 2 groups (n=10 each), perfused according to Langendorff technique and exposed to 45 min of ischemia. Hearts belonging to the first group were reperfused with Krebs-Henseleit solution at 600 mmHg pO2; a 150 mmHg pO2 perfusate was utilized in the second group during the first minute and switched to 600 mmHg pO2 thereafter. Modifications in diastolic compliance and systolic contractility were assessed by changes in left ventricular end-diastolic (LVEDP) and developed pressure (LVDP), and first derivative of the pressure curve (dP/dt). RESULTS: Increase in LVEDP values, with respect to pre-ischemic data, were detected at 1, 5, 10, 20 min following reperfusion at 600 mmHg pO2, and were respectively: +40.17+/-18.61, +57.5+/-28.8, +59.8+/-30.5 and +63.2+/-34.1 mmHg. At 150 mmHg pO2 they were: +15.69+/-13.13, +22.4+/-14.1, +26.2+/-13.7 and +28.9+/-15.8, with a significant difference within the first 20 min (p<0.05). At high pO2 levels, LVDP decreased of 53.0+/-27.35, 60.5+/-22.6, 59.8+/-23.3 and 50.7+/-25.0 mmHg, versus 42.7+/-25.7, 38.0+/-29.5, 39.2+/-30.9 and 38.7+/-32.7 mmHg at lower pO2 (p=NS). The correspondent values of the dP/dt were 37.8+/-27.7, 30.1+/-17.2, 32.2+/-13.6 and 35.4+/-14.0% of pre-ischemic values at high pO2, versus 43.3+/-27.09, 51.9+/-25.1, 50.1+/-24.6 and 53.1+/-29.9% at lower pO2. Statistical significance was lower for LVDP and dP/dt than LVEDP. CONCLUSIONS: Diastolic functional impairment was partially reduced within the first 20 min following low pO2 reperfusion, but without any significant improvement of contractility.     

Glomerular Na+-K+-ATPase activity in acute and chronic diabetes and with aldose reductase inhibition

Sorbitol accumulation, myo-inositol depletion, and reduced Na+-K+-ATPase activity have been identified in experimental diabetes in several tissues in which diabetic complications occur. However, a reduction in renal Na+-K+-ATPase activity has not been universally reported, prompting us to examine the influence of diabetes duration on the activity of this enzyme complex in isolated glomeruli. Additionally, we examined the ability of the aldose reductase inhibitor sorbinil to directly stimulate glomerular Na+-K+-ATPase activity in vitro, an area of interest in view of the central position that the ability of sorbinil to restore Na+-K+-ATPase activity in vivo occupies in the interpretive scheme that links associated changes in sorbitol, myo-inositol, and Na+-K+-ATPase to enhanced polyol-pathway activity. Glomerular Na+-K+-ATPase activity was significantly decreased in rats with acute (less than 18 days) streptozocin-induced diabetes but was significantly greater than control values in rats with more chronic (greater than 32 days) diabetes. In vitro addition of sorbinil (1 x 10(-7) M) directly stimulated Na+-K+-ATPase in control (0.627 +/- 0.090 vs. 0.843 +/- 0.098 mumol P1.mg-1.min-1) but not diabetic glomeruli. These results indicate that the time of examination after induction of diabetes critically influences glomerular Na+-K+-ATPase activity and suggest that sorbinil, at least in normal glomerular tissue, has a membrane-associated effect that may be independent of its aldose reductase-inhibiting property.     

Cortical Na+,K+-ATPase activity, abundance and distribution after in vivo renal ischemia without reperfusion in rats

The aim of our work was to study the changes in activity, abundance and distribution of sodium, potassium-adenosine triphosphatase (Na+,K+-ATPase) in membranes of cortical tubular cells in an in vivo model of ischemic injury without reperfusion. Na+,K+-ATPase, alkaline phosphatase (AP) activities and their distribution in membranes isolated from renal cortex using a Percoll gradient were studied after different ischemic periods. Na+,K+-ATPase alpha-subunit protein abundance was analysed by Western-blot. Plasma urea and cortical adenosine 5' triphosphate (ATP) were also measured. In cortical homogenates 5 min of ischemia promoted a diminution in ATP content. Na+,K+-ATPase activity diminished after 40 min and AP after 100 min of ischemia. Na+,K+-ATPase activity in the Percoll gradient fractions after 5 min peaked at a higher density and was significantly decreased after 40 min. AP activity was decreased in typically enriched apical membranes after both times of ischemia. At each time studied Na+,K+-ATPase abundance was increased in cortical homogenates and membranes. Our results showed opposite effects of ischemia on Na+,K+-ATPase activity and abundance. Increased levels of Na+,K+-ATPase protein were observed. The enzyme would be rapidly delivered to membrane domains and become inactivated as ischemia persists.     

Improvement of leucocytic Na+ K+ pump activity in uremic patients on low protein diet.

Leucocytic Na+ K+ pump activity was assessed in 20 patients with advanced renal failure. Na+ K(+)-ATPase activity was reduced when compared with the values obtained from normal subjects (101.8 +/- 48.6 versus 165.13 +/- 8.9 microM of Pi hr-1.g-1; P less than 0.001) and the mean 86Rb uptake by U 937 cells was depressed by 38% after the addition of patients' sera. Subsequently, patients were put on a diet providing 0.3 g protein/kg body weight daily and supplemented with ketoacids. After three months of dietary treatment Na+ K(+)-ATPase activity increased to 142 +/- 48.3 (P less than 0.01) and reached normal values at the sixth month (162.8 +/- 54.70 microM of Pi hr-1.g-1; P less than 0.001) whereas 86Rb uptake increased by 23 percent when compared to initial values. These data suggest that among the different mechanisms which have been advanced to explain the defects in the Na+ pump observed in uremic patients, circulating inhibitors deriving from alimentary protein intake may affect cation transport.     

Enhanced functional recovery with venting during cardioplegic arrest in chronically damaged hearts

Thirty dogs with experimental myocardial infarction underwent cardiopulmonary bypass, hypothermic asanguineous K+ cardioplegia (1 hour), and reperfusion (30 minutes). Ten hearts were vented throughout, 5 only during arrest, and 5 only during reperfusion; 10 were not vented. Left ventricular (LV) performance and compliance were assessed by isovolumic (LV balloon) indexes before bypass and after reperfusion. Vented hearts recovered 116 +/- 8.3% of prearrest developed LV systolic pressure (DLVSP) and 131 +/- 13.6% of prearrest rate of rise of LV pressure (dP/dt). Nonvented hearts allowed to develop pressure during arrest (11.6 +/- 1.6 mm Hg) and reperfusion (65 +/- 4 mm Hg) recovered 50 +/- 3.9% of prearrest DLVSP and 55 +/- 5% of prearrest dP/dt (p less than 0.05). Reduction in LV compliance was comparable in both groups. Mitochondrial architecture (electron microscopy) was preserved in vented hearts, but was modestly disrupted in nonvented hearts, thus suggesting slight metabolic impairment. Functional recovery was nearly complete in hearts vented only during reperfusion (DLVSP, 94 +/- 10.4%; dP/dt, 89 +/- 12.6%), but venting only during arrest led to functional depression (DLVSP, 50 +/- 6.6%; dP/dt, 51 +/- 8%; p = 0.01). We conclude that venting chronically infarcted hearts during cardiac operations affords better myocardial protection by avoiding the damage that occurs during nonvented reperfusion.     

Stem cells improve right ventricular functional recovery after acute pressure overload and ischemia reperfusion injury

BACKGROUND: In many clinical scenarios, a relatively untrained right ventricle may be subjected to acute elevations in pulmonary artery and right ventricular pressures. The right and left heart are distinctly different in this regard and there is currently no in vivo model to study right ventricular ischemia in the setting of acute pressure overload. In acute injury, cardiomyocytes produce tumor necrosis factor, which mediates a proinflammatory pathway, eventually leading to myocardial dysfunction. Stem cells have been shown to reduce the production of proinflammatory mediators by the ischemic myocardium and protect the myocardium. Pretreatment with stem cells has been shown to protect the left ventricle. The effect of acute pressure overload to the untrained right ventricle is still not well understood. Furthermore, it is unclear whether pretreatment with stem cells would protect the right ventricle when it is subjected to acute pressure overload and concomitant ischemia reperfusion injury. The purpose of this study was (1) to create a simple model of acute pressure overload for the study of concomitant right ventricular ischemia and reperfusion, and (2) to evaluate the effect of pretreatment with stem cells prior to ischemia reperfusion injury. MATERIALS AND METHODS: Isolated rat hearts were perfused with the modified Langendorff technique with the latex balloon in the right ventricle instead of the left, with a pressure-transduced balloon being used to create an acute elevation in right ventricular pressure before ischemia. In the first of a two-series experiment, there were two experimental groups (N = 8 per group): one with right ventricular balloon end-diastolic pressure (EDP) of 5 mmHg (physiological), and the other with an EDP of 40 mmHg (pathologic). In the second series, the hearts with the higher balloon pressure (EDP 40 mmHg) were divided into two experimental groups (N = 5 per group). The control group was not pretreated. One group was pretreated with human mesenchymal stem cells 5 min immediately prior to ischemia reperfusion injury. Right ventricular developed pressure (RVDP), contractility (+dP/dt), and compliance (-dP/dt) were continuously assessed. Additionally, mesenchymal stem cells (MSCs) in culture were stressed by hypoxia and activation was determined by measuring vascular endothelial growth factor-A (VEGF) and hepatocyte growth factor (HGF) production by enzyme-linked immunosorbent assay. RESULTS: Recovery of RVDP, +dP/dt, and -dP/dt was significantly higher (P < 0.001) in the group with lower EDP compared to the group with the higher EDP [RVDP: 79.53 +/- 6.34 versus 54.28 +/- 10.76%; +dP/dt: 76.54 +/- 8.79 versus 38.75 +/- 19.74%; -dP/dt: 72.29 +/- 7.02 versus 30.54 +/- 12.44%]. In the higher EDP groups, pretreatment with human mesenchymal stem cells significantly improved myocardial function recovery (P < 0.01) when compared to controls [RVDP: 75.76 +/- 7.97 versus 59.10 +/- 11.18%; +dP/dt: 71.78 +/- 10.36 versus 54.93 +/- 12.64%; -dP/dt: 77.38 +/- 11.09 versus 59.30 +/- 15.20%]. Further, hypoxic MSCs demonstrated significantly greater VEGF and HGF release than controls. CONCLUSION: This compounded injury model allowed the study of right ventricular dysfunction in the setting of acute pressure overload and ischemia. Additionally, we have also demonstrated that pretreatment with stem cells of an acutely pressure overloaded right ventricle prior to ischemia reperfusion injury improves functional recovery. This is the first report of a modified Langendorff technique to study right ventricular function in the setting of acute pressure overload and ischemia and the effect of pretreatment with stem cells.     

Characterization of a (Ca2+ + Mg2+)-ATPase system in the osteoblast plasma membrane

A high affinity, calmodulin-sensitive (Ca2 + Mg2+)-ATPase was demonstrated in the plasma membrane preparation of three different osteosarcoma cell lines previously demonstrated to respond to parathyroid hormone with an increase in cytosolic calcium and a decrease in pH. The maximal velocity of the enzyme activity in the membrane preparations ranged from 0.83 to 2.42 nmol Pi released per min per mg protein with half-saturation constants of 26 nM of free Ca. The enzyme activity was not affected by Na+, K+, ouabain and azide, and exhibited an absolute requirement for Mg2+ ions. These results suggest a possible role for a membrane Ca2 + Mg2+-ATPase in initiating and perpetuating the ionic control of osteoblastic function.     

酸处理对大鼠离体心脏缺血再灌注损伤的影响及PI3K/Akt信号通路在其中的作用

目的 评价酸处理对大鼠离体心脏缺血再灌注损伤的影响及磷脂酰肌醇-3激酶/蛋白质丝氨酸苏氨酸激酶(PI3K/Akt)信号通路在其中的作用.方法 清洁级SD大鼠70只,雌雄不拘,体重450-550 g,随机分为7组(n=10):缺血再灌注组(I/R组)、缺血后处理组(IPO组)、酸处理组(H+组)、缺血后处理+碱处理组(IPO+OH-组)、碱处理组(OH-组)、酸处理+渥曼青霉索组(H++wort组)和渥曼青霉素组(wort组).采用Langendorff装置行离体心脏灌注,采用全心停灌30 min、再灌注中性K-H液(pH值7.4)120 min的方法 制备大鼠离体心脏缺血再灌注模型.IPO组于再灌注即刻灌注中性K-H液15 s,停灌15 s,重复6次,行缺血后处理;H+组于再灌注即刻灌注经80%O2-20%CO2饱和的酸性K-H液(pH值6.9)3 min;IPO+OH-组于再灌注即刻灌注经100%O2饱和的碱性K-H液(pH值7.8)3min,并行缺血后处理;OH-组于再灌注即刻灌注碱性K-H液3 min;H++wort组于再灌注即刻灌注含100 nmol/L渥曼青霉素的酸性K-H液3 min;wort组于再灌注即刻灌注含100 nmol/L渥曼青霉素的中性K-H液3 min.于缺血前和再灌注30 min时记录左心室舒张末期压(LVEDP)和左心室压力最大上升和下降速率(±dp/dtmax);于再灌注30 min时测定冠状动脉流出液一氧化氮(NO)浓度;于再灌注120 min时,计算心肌梗塞区与缺血危险区的质量比,来表示心肌梗塞范围.结果 与I/R组比较,IPO组和H+组再灌注时LVEDP降低,±dp/dtmax升高,心肌梗塞范围减小,冠状动脉流出液NO浓度升高,OH-组、H++wort组心肌梗塞范围增加,wort组心肌梗塞范围增加,冠状动脉流出液NO浓度降低,IPO+OH-组LVEDP降低(P<0.05或0.01),±dp/dtmax、心肌梗塞范围和冠状动脉流出液NO浓度差异无统计学意义(P>0.05);IPO组与H+组上述指标比较差异均无统计学意义(P>0.05).结论 酸处理可减轻大鼠离体心脏缺血再灌注损伤.其机制与激活PI3K/Akt信号通路有关.     

K(ATP) channel opener protects neonatal rabbit heart better than St. Thomas' solution

OBJECTIVES: Myocardial protection with ATP-sensitive potassium channel (K(ATP) channel) openers is as effective as St. Thomas' cardioplegia (StTCP) in adult rabbit hearts. This study compares the effectiveness of the K(ATP) channel opener pinacidil to StTCP in protecting neonatal rabbit hearts exposed to global ischemia. METHODS: Seventeen neonatal rabbit hearts (7-9 days old) perfused with Krebs-Henseleit buffer (KHB) on a Langendorff apparatus underwent 90 min of normothermic ischemia. Six (ischemia control) received no pretreatment before or during ischemia. Six others (pinacidil) received a 3-min infusion of 50 microM pinacidil in KHB without StTCP at the onset of ischemia. Five others (StTCP) received a 3-min infusion of StTCP at the onset of ischemia. After 60 min of KHB reperfusion, recovery of left ventricular (LV) performance and coronary flow (CF) were measured and compared to preischemia. A paired t test was used for comparison between drug-treated and untreated groups. RESULTS: Pinacidil-treated hearts had significantly better recovery of left ventricular developed pressure (47 +/- 3.8 mmHg vs 32 +/- 2.5 mmHg, P < 0.05), contractility (+dP/dt(max); 885.4 +/- 74 mmHg vs 643.7 +/- 65 mmHg, P < 0.05), left ventricular end diastolic pressure (10.5 +/- 0.9 mmHg vs 17.4 +/- 1.2 mmHg P < 0.05), compliance (-dP/dt(max); 994.2 +/- 86 mmHg vs 673.6 +/- 69 mmHg, P < 0.05), and CF (5.9 +/- 0.4 ml/min vs 4.2 +/- 0.2 ml/min, P < 0.05) compared to ischemic control. StTCP only improved the recovery of -dP/dt(max) (877.4 +/- 73 mmHg/s vs 673.6 +/- 69 mmHg/s, P < 0.05) and CF (5.7 +/- 0.3 ml/min vs 4.2 +/- 0.2 ml/min, P < 0.05) compared to control. CONCLUSIONS: Pinacidil pretreatment provided superior recovery of systolic performance compared to St. Thomas' cardioplegia solution in neonatal hearts. Myocardial protection by pretreatment with the K(ATP) channel opener pinacidil may be a new strategy for myocardial protection during pediatric cardiac surgery.     

The protective effect of mannitol, vitamin E, and glucocorticoid in experimental cerebral ischemia--influence on lipid peroxidation, energy metabolism and brain edema

At present we apply the three-drug-combination therapy consisting of mannitol, vitamin E and glucocorticoid (betamethasone) in the treatment of cerebral infarction at acute stage with favorable results. However, much of the action mechanism of these drugs remains unelucidated. For the purpose to elucidate the mechanism by which they exert actions and moreover to evaluate the efficacy of this therapy, we conducted experiments using highly ischemic whole brain models of rats. In this model, chemiluminescence value, energy metabolism, water content and concentrations of Na+, K+ were determined with time. During ischemic period chemiluminescence level increased with time, and more remarkable increase was seen after recirculation of the blood flow. However, in the group with administration of the three drugs combination increase in chemiluminescence was inhibited remarkably. In the analysis of intensity of chemiluminescence by wavelengths, the peaks were observed at 480, 520 - 530, 570, 620 - 640 and 680 - 700 nm. These wavelengths were taken to suggest the release of energy (luminescence) associated with the transition of singlet oxygen to the grounded state during the breakdown of the lipid hydroperoxide. By addition of vitamin E or beta-carotene (quencher of singlet oxygen) on the brain homogenate in vitro, luminescence was remarkably inhibited over whole ranges of wavelength. Determination of adenine nucleotide and carbohydrate revealed that these three drugs combination promoted their recovery at the period of recirculation of the blood flow after ischemia. In particular, increase in lactate was inhibited from the period of ischemia with prevention of progression of lactic acidosis. Moreover, in the group with administration of the three drugs combination and the group with administration of 20% or isotonic mannitol the increase in the cortical water content following recirculation of the blood flow was inhibited. From these result it is considered that each of the three drugs shows not only inhibition of lipid peroxidation as radical scavengers but also protective effect on lowering of activities of ion channel (Na+, K+-ATPase, etc.) by the free radicals.     

Ca2+-Mg2+-ATPase activity in kidney basolateral membrane in non-insulin-dependent diabetic rats. Effect of insulin

The direct effect of insulin on the high-affinity Ca2+-Mg2+-ATPase was studied in kidney proximal tubular basolateral membranes (BLM) obtained from control and streptozocin-induced non-insulin-dependent diabetes mellitus (NIDDM) rats. Plasma glucose of the diabetic animals was only mildly elevated (217 +/- 9 vs. 138 +/- 3 mg/dl). Both high- and low-affinity calcium-dependent Ca2+-Mg2+-ATPase activities were identified in the BLM. Enzyme activity in BLM from diabetic rats was higher at all Ca2+ concentrations tested due to a higher maximum velocity of the enzyme from NIDDM rats. The high-affinity Ca2+-Mg2+-ATPase activity was inhibited by trifluoroperazine (TFP) in both membranes. No difference in calmodulin content was found in the membranes from the diabetic and control rats. Insulin (16-200 microU/ml) significantly increased the high-affinity Ca2+-Mg2+-ATPase activity (17-40%) in membranes from control animals but had no effect on the enzyme activity in the membranes from the NIDDM rats. The basal activity of the enzyme at 0.1 microM free Ca2+ was higher in the BLM from the NIDDM animals compared to controls (17.8 +/- 0.5 vs. 14.7 +/- 0.8 nM Pi X mg-1 X min-1; P less than .02). There was no effect of insulin on the Ca2+-independent ATPase activity of BLM preparations. These findings demonstrate a defect in the ability of insulin to regulate the high-affinity Ca2+-Mg2+-ATPase activity in BLM from diabetic rats. Such a defect in enzyme activity may play a role in the mechanism of impaired insulin action observed in these NIDDM rats.