The response of red cell hexose monophosphate shunt after sulfhydryl inhibition

In this investigation, we studied the importance of cellular glutathione (GSH) in the hexose monophosphate shunt (HMPS) activity of unstimulated human erythrocytes and the mechanism by which pyruvate stimulates the HMPS. The rate of HMPS activity was measured by the production of radioactive CO2 from 14C-1-glucose or 14C-1-ribose using a vibrating reed electrometer and ionization chamber. HMPS activity was not significantly impaired by N-ethylmaleimide (NEM) in concentrations which bound all red cell GSH. Red cells incubated under carbon monoxide (CO), an experimental condition which eliminates peroxide production, still had HMPS activity which was 44% of the value under air. Pyruvate stimulation of the HMPS was unaffected by doses of NEM which bound all cellular GSH or by incubation under CO. These data indicated that pyruvate stimulation of the HMPS occurs by pathways which do not involve peroxide formation, GSH, or oxygen. This study indicates that sulfhydryl blockade of GSH does not necessarily inhibit HMPS activity and that HMPS activity in red cells may respond to reactions not linked directly to glutathione reduction.     

Factors affecting unstimulated flux through the hexose monophosphate shunt during incubations of human red blood cells

Factors affecting the flux of glucose through the hexose monophosphate shunt in unstimulated human red blood cells were studied in vitro. Reduction of oxyhemoglobin or free O₂ each accounted for about one-third of the total flux of reducing equivalents through the shunt. Approximately one third of total flux remained after removal of oxyheme activity and free O₂. Both deoxyhemoglobin and methemoglobin stimulated flux in the absence of free O₂ suggesting that the small amount of deoxyheme and metheme (1%), in equilibrium with the large pool of oxyheme (99%), may contribute to the total oxidizing effect of the heme group. The flux of reducing equivalents through the hexose monophosphate shunt in unstimulated red cells primarily involved oxidation and reduction of oxyhemoglobin or free O₂. In low phosphate buffer (1.2 mM), glutathione served as the source of reducing equivalents for the remaining “electron sinks” (after removal of oxyheme activity and free O₂) during the 1st hr of incubation so that glutathione stimulated flux through the hexose monophosphate shunt; during the 2nd hr of incubation, glutathione acted as a reservoir of reducing equivalents maintaining NADPH and inhibiting flux through the hexose monophosphate shunt. When red cells were incubated in high phosphate buffer (17.4 mM), glutathione behaved as an inhibitor of flux in the 1st hr of incubation in red cells lacking oxyheme activity and free O₂. The $\text{H}{}_2\text{PO}{}_4{}^{\mathrm{?}}\text{HPO}{}_4{}^{\mathrm{2?}}$ anion couple appears to alter the pattern of NADPH oxidation in red cells lacking oxyheme activity and free O₂. Flux was inhibited by incubation of red cells in a medium containing lactate (4 mM). Inhibition of flux by lactate was not dependent on heme, free O₂ or glutathione but all these factors had complex influences on lactate-mediated inhibition. The inhibitory effect of lactate on flux is complementary to the well-characterized stimulatory effect of pyruvate. The lactate/pyruvate couple may act by directly filling or creating electron sinks, by interacting with the $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ couple through lactic dehydrogenase or through transhydrogenation between the $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ and $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ couples.     

Catecholamine induced cardiac hypertrophy

Cardiac hypertrophy was induced in adult female Wistar rats by daily subcutaneous injections of isoproterenol (0.3 mg/kg body weight). Heart weight increased 39% after eight days of treatment. Left ventricular pressure development (positive dP/dt) in hearts four days after hypertrophy induction was significantly increased, while negative dP/dt remained unchanged. RNA polymerase activity in isolated myocyte and nonmyocyte nuclei was stimulated 29 and 23%, respectively 24 h after a single isoproterenol injection. In the myocyte fraction, RNA polymerase activation progressively increased up to four days of treatment and then returned to control values after eight days. In the nonmyocyte nuclear subset, RNA polymerase activity showed no further stimulation and gradually returned to control values after eight days of treatment. Chromatin template function was substantially stimulated in the early stage (one to four days) of hypertrophy in both myocyte and nonmyocyte fractions. Titration of chromatin against a fixed amount of RNA polymerase (5 micrograms) in the presence of rifampicin and heparin showed that less chromatin from hypertrophied hearts was required to saturate the enzyme. These results indicate that both myocyte and nonmyocte chromatin from hypertrophied hearts can support greater enzyme binding than normal chromatin. The alkaline sucrose density centrifugation profile of DNA in myocyte and nonmyocyte chromatin from day 4 hypertrophied hearts was less fragmented. These observations suggest that during the early phase of isoproterenol-induced cardiac hypertrophy, enhanced RNA polymerase activity and chromatin template function play a coordinated role in RNA synthesis. The increased template activity could be due to alterations in chromatin composition which was indicated by the change in their enzyme binding capacity and DNA fragmentation profile.     

Induction of plasminogen activator secretion in macrophages by electrochemical stimulation of the hexose monophosphate shunt with methylene blue.

Resident peritoneal macrophages were obtained from untreated mice and were cultured in medium 199 with or without 5% acid-treated fetal bovine serum. Three hours after harvesting, redox compounds--i.e., methylene blue, methyl viologen, or nitro blue tetrazolium--were added to the cultures of adherent cells. After 1 hr, the cells were washed and culturing was continued in the absence of redox compounds. The effects of the redox compounds were tested by assaying for hexose monophosphate (HMP) shunt activity and for plasminogen activator secretion, and the results were compared with the effects induced by phagocytic stimuli. Methylene blue caused a concentration-dependent stimulation of the HMP shunt, whereas methyl viologen and nitro blue tetrazolium were ineffective. Shunt stimulation by methylene blue was followed, after a lag of 2-4 days, by plasminogen activator secretion. The rate of secretion was dependent on the methylene blue concentration used. Methyl viologen and nitro blue tetrazolium were again ineffective, whereas phagocytosis of zymosan or sheep erythrocytes, which stimulates the HMP shunt, induced plasminogen activator secretion at rates similar to those induced by methylene blue. These results add further evidence to our hypothesis that the HMP shunt-dependent metabolic burst is involved in macrophage activation. Because methylene blue mimics the action of zymosan it appears that shunt stimulation by itself initiates the activation process independently of phagocytosis.     

Inhibition of hexose monophosphate shunt in young erythrocytes by pyrimidine nucleotides in hereditary pyrimidine 5' nucleotidase deficiency

Recent reports have suggested that haemolytic anaemia in pyrimidine 5' nucleotidase (P5'N) deficiency might be due to impaired erythrocyte hexose monophosphate shunt (HMS). To investigate the relationship between pyrimidine accumulation, HMS impairment and shortened red-cell survival, we tested glucose 6-phosphate dehydrogenase (G-6PD), HMS, P5'N activities and the UV spectrum in whole red cells and in red cells of different age from 2 P5'N-deficient patients with different degrees of haemolytic anaemia. In whole red cells we found a reduction of both G-6PD and stimulated HMS activity in the presence of a variable amount of pyrimidine nucleotides (37.79 and 17.88 mumol/gHb respectively). A drastic inhibition of stimulated HMS activity was already present in the lightest red-cell fractions from patient 1, who presented a more severe haemolytic anaemia. The variable degree of pyrimidines found among red cell fractions, with a minor accumulation in the older red cells, supports the hypothesis that pyrimidine accumulation and HMS impairment occur in the younger erythrocytes of P5'N-deficient patients.     

Stimulation of the hexose monophosphate shunt independent of hydrogen peroxide and superoxide production in rabbit alveolar macrophages during phagocytosis.

Phagocytosis and oxidative metabolism of human polymorphonuclear leukocytes (PMN) and rabbit alveolar macrophages (AM) were studied. Human PMN ingested a mean of 12 polyvinyl toluene latex particles (2 micrometer in diameter) per cell. There was stimulation of O2- and H2O2 production, light emission, and activation of the hexose monophosphate shunt during phagocytosis by human PMN. Rabbit AM ingested 51 latex particles (2 micrometer in diameter) per cell. There was no stimulation of the production of O2- and H2O2 or light emission associated with phagocytosis by rabbit AM, while the hexose monophosphate shunt was activated. Similar metabolic changes were obtained in both cell types when opsonized zymosan was used as phagocytic particles. 1-14C-glucoseoxidation was stimulated by H2O2 and methylene blue in both resting human PMN and rabbit AM. It is concluded that activation of the hexose monophosphate shunt in rabbit AM during phagocytosis is independent of O2- and H2O2 production.     

Glucose flux through the hexose monophosphate shunt and NADP(H) levels during in vitro ageing of human skin fibroblasts

In cultured human skin fibroblasts the glucose flux through the hexose monophosphate shunt (HMS) amounts to 4% of the glucose flux through the glycolytic pathway. Upon in vitro ageing the rate of glucose utilization through the HMS is decreased more than 50%. This decrease in HMS was not caused by a limiting enzymatic capacity since glucose utilization through the HMS could be raised at least 30-fold in both 'young' and 'aged' fibroblasts upon stimulation with phenazine methosulphate. This effect of in vitro ageing upon glucose metabolism was also not due to differences in proliferation rate between 'young' and 'aged' human fibroblasts, since there was no difference in glucose utilization between proliferating and growth-inhibited (confluently cultured) fibroblasts. The NADPH/NADP ratio was found to be decreased by 12% in 'aged' cells.     

Significance of myocardial alpha- and beta-adrenoceptors in catecholamine-induced cardiac hypertrophy

The role of alpha- and beta-adrenoceptors in the development of catecholamine-induced cardiac hypertrophy in vivo was investigated. Rats received a constant intravenous infusion of norepinephrine or sodium chloride (control) for 3 days. The norepinephrine infusion was combined with the alpha-blocker prazosin, the beta-blocker metoprolol, or both blockers. For modulation of the work load of the heart, the calcium channel blocker verapamil was added to the norepinephrine infusion. A further group of animals was treated with the alpha-adrenergic stimulator norfenephrine, which also was combined with prazosin or verapamil. Norepinephrine induced significant increases in mean aortic pressure, left ventricular dP/dtmax, heart rate, and total peripheral resistance. The left ventricular weight/body weight ratio was significantly elevated and was accompanied by an increase in the RNA concentration and the RNA/DNA ratio. Prazosin as well as metoprolol partially antagonized the increase in left ventricular weight and RNA concentration, whereas simultaneous prazosin and metoprolol treatment prevented the norepinephrine-induced alterations. Although combination of norepinephrine with verapamil resulted in considerable reduction of all functional parameters, the development of cardiac hypertrophy and the elevated RNA/DNA ratio were not significantly influenced. Stimulation of alpha-receptors with norfenephrine elicited an increase in total peripheral resistance and in left ventricular weight, which was abolished by prazosin. Verapamil did not affect the norfenephrine-induced cardiac hypertrophy, although it normalized essentially all functional parameters. Thus, the rapid development of cardiac hypertrophy in the norepinephrine model seems to be directly mediated by stimulation of myocardial alpha- and beta-adrenoceptors rather than by hemodynamic changes.     

Skeletal muscle abnormalities in rats with experimentally induced heart hypertrophy and failure

Background: In congestive heart failure (CHF), function and metabolism of skeletal muscles are abnormal. Aim: To evaluate whether the reduced oxidative capacity of skeletal muscles in CHF is due to impaired O₂ utilisation. Methods: CHF was induced in rats by injecting 50 mg/Kg monocrotaline. Several animals received the same dose of monocrotaline but only compensated right ventricular hypertrophy and no sign of congestion resulted. Two age- and diet-matched groups of control animals were also studied. In soleus and extensor digitorum longus (EDL) muscles, we studied skeletal muscle blood flow, oxidative capacity and respiratory function of skinned muscle fibres. Results: In CHF, we observed a decrease of muscle blood flow (statistically significant in the soleus, p < 0.05 vs. controls). In compensated rats, a similar trend in blood flow was observed. In both soleus and EDL, a significant reduction of high energy phosphate and a shift of the redox potential towards accumulation of reducing equivalents were observed. The reduction of energy charge was not correlated to the decrease of blood flow. In skinned myofibres, the ratio of O₂ utilised in the presence and in absence of ADP (an index of phoshorilating efficiency) was reduced from 8.9 ± 1.9 to 2.7 ± 0.2 (p < 0.001) and from 5.7 ± 1.0 to 2.0 ± 0.3 (p < 0.01) in soleus and EDL, respectively. Activity of the different complexes of respiratory chain was investigated by means of specific inhibitors, showing major abnormalities at the level of complex I. In fact, inhibition of VO₂ by rotenone was decreased from 83.5 ± 3.2 to 36.4 ± 9.6 % (p < 0.005) and from 81.8 ± 6.1 to 38.2 ± 7.4 % (p < 0.005) in soleus and EDL, respectively. Conclusions: In rats with CHF, abnormalities of oxidative phosphorylation of muscles occur and complex I of the respiratory chain seem to be primarily affected. The metabolic alterations of skeletal muscles in CHF may be explained, at least in part, by an impaired O₂ utilisation. Received: 22 February 2002, Returned for 1. revision: 14 March 2002, 1. Revision received: 5 June 2002, Returned for 2. revision: 21 June 2002, 2. Revision received: 23 August 2002, Accepted: 12 September 2002 Correspondence to: Dr. C. Ceconi     

Hemodynamic and pathological results from experimentally induced left ventricular hypertrophy in the rabbit.

Many studies of ventricular structure and function of the hypertrophied cardiac muscle have been performed using a model of right ventricular hypertrophy because a reliable experimental model for the chronically hypertrophied left ventricle was not available. Adult rabbits underwent thoractomy with placement of an ameroid band on the proximal aorta. The ameroid band closed to a maximal extent at 9 days. Systolic gradients were measured at 3 weeks. 37 animals (group I) were sacrificed at an average of 7 weeks, and 5 (group II) at 18 weeks. Specific cardiac chambers were weighed. The average left ventricular weight expressed as a percentage of body weight of control animals was 0.13 +/- 0.01 compared to group I 0.17 +/- 0.04 (P = 0.001) and group II 0.23 +/- 0.04. Hemodynamic data revealed significant aortic outflow obstruction with systolic gradients averaging at least 50 mm Hg. Left ventricular end-diastolic pressures were elevated in 13 of 29 animals compatible with left ventricular failure. These results indicate that this technique affords a reliable experimental model for inducing biventricular hypertrophy which can be evaluated hemodynamically.     

Elevated collagen content in volume overload induced cardiac hypertrophy

This investigation was designed to determine if chronic volume overload is associated with altered collagen content of five regions of the myocardium. Five adult cats were subjected to a 6-week period of chronic volume overload induced by atrial septotomy and five untreated animals served as controls. Significant (P < 0.05) right ventricular hypertrophy was present as indicated by the right ventricular body weight ratio. For control animals this ratio was 0.68 ± 0.04 g/kg; for volume overloaded animals it was 0.83 ± 0.05 g/kg.) The collagen content was assessed by measuring the hydroxyproline content of the dried cardiac muscle. Right ventricular endocardium hydroxyproline in volume overloaded animals was significantly elevated above that in control animals (in the latter it was 5.30 ± 0.36 μg/mg; in the former it was 6.33 ± 0.18 μg/mg) while the epicardial collagen content was unchanged. Similarly, the amount of collagen found in the left ventricle was significantly increased in the endocardium and normal in the epicardium. Septal collagen concentration was unaltered in volume overloaded animals. This study demonstrated that alterations in cardiac muscle collagen concentration are associated with volume overload and that these cellular changes are nonuniform.     

Urinary acidification in a patient with glucose-6-phosphate dehydrogenase deficiency. A reevaluation of the role of the hexose monophosphate shunt in renal acid secretion

The relationship between renal metabolism and urinary acidification is poorly understood. During the past decade evidence has accrued to suggest that the hexose monophosphate (HMP) shunt might serve in the process of urinary acidification by providing reducing equivalents for a redox-coupled membrane-bound proton pump that could transport protons into the tubular lumen. The major support for this hypothesis has come from the finding that HMP shunt activity increases with acute and chronic metabolic acidosis. In the present study, we examine the urinary acidification capacity of a young man with severe erythrocyte glucose-6-phosphate dehydrogenase (G-6-PD) deficiency and with unmeasurable G-6-PD activity in renal cortical tissue. We found that despite unmeasurable G-6-PD activity in renal tissue, the patient was capable of generating a maximally acid urine and increasing total acid secretion. Our findings suggest that the HMP shunt may not be necessary for the urinary acidification process.     

异丙肾上腺素致兔心肌肥厚模型的在体心脏电生理研究

目的探讨异丙肾上腺素(Iso)导致兔心肌肥厚在体心脏电生理指标的改变及其与室性心律失常(VAs)的关系。方法 24只兔随机分为Iso组(n=12)和对照组(n=12):Iso组给予异丙肾上腺素(0.3 mg.kg-1.d-1)耳缘静脉注射7天,而对照组注射生理盐水(1 ml.kg-1.d-1)7天。运用单相动作电位记录技术和程控刺激技术,观测在体心室部单相动作电位时程(MAPD)、心室有效不应期(VERP)、自发和诱发的室性心律失常。结果与对照组比较,ISO组MAPD30～MAPD90均延长,而且内外膜MAPD梯度增大,在300,400和500 ms固定频率刺激下VERP明显缩短,VAs发生率升高(P均<0.05)。结论心肌肥厚引起心室部MAPD延长和VERP缩短,VAs发生率增加。     

Nitric oxide reacts with intracellular glutathione and activates the hexose monophosphate shunt in human neutrophils: evidence for S-nitrosoglutathione as a bioactive intermediary.

We performed experiments to determine whether nitric oxide promoted the formation of intracellular S-nitrosothiol adducts in human neutrophils. At concentrations sufficient to inhibit chemoattractant-induced superoxide anion production, nitric oxide caused a depletion of measurable intracellular glutathione as determined by both the monobromobimane HPLC method and the glutathione reductase recycling assay. The depletion of glutathione could be shown to be due to the formation of intracellular S-nitrosoglutathione as indicated by the ability of sodium borohydride treatment of cytosol to result in the complete recovery of measurable glutathione. The formation of intracellular S-nitrosylated compounds was confirmed by the capacity of cytosol derived from nitric oxide-treated cells to ADP-ribosylate glyceraldehyde-3-phosphate dehydrogenase. Depletion of intracellular glutathione was accompanied by a rapid and concomitant activation of the hexose monophosphate shunt (HMPS) following exposure to nitric oxide. Kinetic studies demonstrated that nitric oxide-dependent activation of the HMPS was reversible and paralleled nitric oxide-induced glutathione depletion. Synthetic preparations of S-nitrosoglutathione shared with nitric oxide the capacity to inhibit superoxide anion production and activate the HMPS. These data suggest that nitric oxide may regulate cellular functions via the formation of intracellular S-nitrosothiol adducts and the activation of the HMPS.     

热休克蛋白27心肌特异性高表达诱导小鼠产生心肌肥厚

目的探讨不同表达量的热休克蛋白27（Hsp27）在小鼠心脏中的作用。方法构建在心肌中特异性表达Hsp27的不同表达量的转基因鼠（TG）,对照组为野生型鼠（WT）。采用蛋白印记测定Hsp27蛋白表达水平;分别测量心脏质量与体质量的比值和心脏质量与胫骨长度的比值（HW／BW和HW／TL）;采用实时定量荧光PCR（real time PCR）技术测定心肌肥厚标记物心房利钠肽（ANP）、脑利钠肽（BNP）的表达;采用超声心动图观察心功能及心脏结构。通过长期饲养,观察存活率。结果不同TG系鼠,Hsp27心肌特异性表达水平不同。高表达TG10鼠与WT鼠相比,其HW／BW和HW／TL比值显著增加（P〈0．01）,并且ANP和BNP的mRNA水平也明显增加（P〈0．01）。TG10鼠的心功能与WT鼠比较显著下降（P〈0．01）,射血分数（EF）降低了24．64％,短轴缩短率（VS）降低了29．37％。TG10鼠的生存率明显降低（P〈0．01）。结论高表达的Hsp27在TG鼠心肌肥厚发展中起着重要作用,提示Hsp27可以作为心肌病患者治疗的潜在靶点。