Evidence for a role of oxygen-derived free radicals and protein kinase C in nitrate tolerance

The anti-ischemic effects of organic nitrates are rapidly attenuated due to the development of nitrate tolerance. The mechanisms underlying this phenomenon likely involve several independent factors. As a vasodilator, nitroglycerin activates compensatory neurohumoral mechanisms such as the renin-angiotensin system and increases catecholamine and vasopressin levels, all of which may attenuate its vasodilator potency. Tolerance may be also due to the inability of the vessel to dilate after prolonged treatment with the nitrate. More recent experimental studies have challenged traditional tolerance concepts by demonstrating that tolerance is not associated with sulfhydryl group depletion, reduced nitroglycerin biotransformation, or desensitization of the target enzyme guanylyl-cyclase. Experimental and clinical observations suggest that tolerance may be the consequence of intrinsic abnormalities of the vasculature, including enhanced endothelial production of oxygen-derived free radicals secondary to an activation of NAD(P)H-dependent oxidases and an activation of PKC. Superoxide degrades nitric oxide derived from nitroglycerin (NTG) while C activation causes enhanced sensitivity of the vasculature to circulating neurohormones such as catecholamines, angiotensin II, and serotonin, all of which may compromise the vasodilator potency of NTG. Interestingly, these vascular consequences of in vivo NTG treatment such as superoxide production and PKC activation can be mimicked in vitro by incubating cultured endothelial and smooth muscle cells with angiotensin II. Furthermore, nitrate tolerance and rebound following sudden cessation of prolonged NTG therapy can be prevented by concomitant treatment with high-dose angiotensin-converting enzyme inhibition, angiotensin type 1 receptor blockade, or antioxidants such as hydralazine. Thus one can conclude that neurohumoral counterregulatory mechanisms such as increased circulating levels of angiotensin II may be at least in part responsible for tolerance mechanisms at the cellular level.     

Involvement of free radicals in the cardioprotective effect of defibrotide

We have developed an estrogen bioassay using the Ishikawa human endometrial adenocarcinoma cell line grown in 96-well microtiter plates. Alkaline phosphatase enzyme activity (Alkp) in these cells was markedly stimulated by estrogen (E2), and this enzyme can be easily quantified in situ using a chromogenic substrate. Estradiol induces AlkP at levels as low as 10-8M. The induction of AlkP is specific for estrogens as for other steroids and other steroidal materials (Progesterone, Tamoxifen, clomiphene citrate, Ru 486, ICI) could not produce a similar effect. The stimulation of AlkP in Ishikawa cells is not only specific for estrogen, it is highly reproducible and sensitive and permits large numbers of samples to be assayed with ease. The non-radioactive nature of the assay makes it attractive to developing countries.     

Omeprazole attenuates oxygen-derived free radical production from human neutrophils

To look for patients with extreme urea rebound, we drew intradialytic samples one third of the way into dialysis during routine modeling for 3 months. The samples taken postdialysis were obtained after stopping the blood pump, without any slow flow period. Using the Smye equations, the intradialytic urea level was used to predict urea rebound, expressed as Kt/V-equilibrated minus Kt/V-single pool (deltaKt/V). Results were averaged for the 3-month period in 369 patients. Mean estimated deltaKt/V was -0.20 +/- 0.13, which was similar to but slightly higher than the predicted value (-0.6 x K/V + 0.03) of -0.19 +/- 0.04. In 27 patients, extreme rebound (mean deltaKt/V < -0.40) was found. Sixteen of these patients consented to further study, but only after access revision in four patients. In these patients, additional slow flow samples after 15 seconds and 2 minutes of slow flow, respectively, were drawn one third of the way into dialysis and postdialysis, and a sample was drawn 30 minutes after dialysis. On restudy, postdialysis rebound was still high with full flow samples deltaKt/V = -0.40 +/- 25, but was much lower (-0.18 +/- 0.07) and similar to predicted rebound (-0.19 +/- 0.05; P = NS) when based on 15-second slow flow samples. Eight of the 16 had marked (>15%) access recirculation by urea sampling, and deltaKt/V based on full flow post samples correlated with access recirculation (r = -0.91). The results suggest that the Smye method is valuable for identifying patients with aberrantly large postdialysis rebound values. When the postdialysis samples are drawn without an antecedent slow flow period, most patients with extreme rebound values turn out to have marked access recirculation.     

Respiratory failure in acute pancreatitis: the role of free fatty acids

Hypertriglyceridemia has been noted in patients with acute pancreatitis and respiratory failure. Utilizing an isolated, perfused, canine pulmonary lobe, the effect of triglyceride infusion on pulmonary function was evaluated. When heparin was used to anticoagulate the perfusion circuit, the addition of triglyceride to the autologous blood perfusate resulted in massive weight gain (226 gm), intrapulmonary shunting (36%), and a marked drop in pulmonary compliance (congruent to 50%). Heparin activates lipoprotein lipase, and therefore some triglyceride in the perfusate was lipolyzed with a resultant increase in serum free fatty acids (FFAs) to 253 mumole/dl. When anticoagulation of the perfusion circuit was accomplished by defibrinogenation with Arvin, the addition of triglyceride to the autologous blood perfusate caused minimal weight gain (28 gm), no intrapulmonary shunting, and only a slight decrease in pulmonary compliance (22%). Arvin has no effect on lipoprotein lipase, and the FFA level in the perfusate remained normal (less than 70 mumole/dl). Thus it appears that FFA release secondary to the action of pulmonary lipoprotein lipase on blood triglyceride is the important pathogenic step in the induction of respiratory failure in this model.     

Pre-eclampsia and oxygenated free radicals

Preeclampsia, clinically defined by arterial hypertension, oedema and proteinuria is a frequently occurred complication of pregnancy. This disease seems to be linked to oxidative stress within placenta. The local accumulation of lipid peroxides, resulting from free radicals production increase altered prostacyclin/thromboxane synthesis. Increased production of lipid peroxides, thromboxane and/or cytokines triggered vascular and organic dysfunctions observed in preeclampsia. Changes in lipoprotein metabolism, namely increase in plasma very low density lipoproteins concentrations (VLDL) and oxidized low density lipoproteins (LDL) concentrations could participate to endothelial dysfunctions observed during preeclampsia.     

Cerebral vasospasm and free radicals

The literature implicating free radical reactions in the genesis of cerebral vasospasm following aneurysmal subarachnoid hemorrhage is reviewed. While this condition has features of a prototypical free radical-mediated disease and a plausible theory can be outlined, data to support the theory are limited. An association of lipid peroxidation with vasospasm has been observed, but more sophisticated techniques for detection of free radicals and for detection of free radical damage to arterial wall proteins and nucleic acids have not been used. There are conflicting reports about efficacy of various antioxidant treatments for vasospasm. In these studies, concomitant experiments have usually not confirmed that the treatments have decreased free radicals or lipid peroxides in cerebrospinal fluid. Because smooth muscle contraction is involved in vasospasm, it would be interesting to investigate the actions of free radicals on smooth muscle cells using, for example, isometric tension recordings and patch clamp techniques. Studies of cardiac myocytes indicate that free radicals alter conductances through potassium and calcium channels and through the sodium-calcium exchanger and may result in elevations in intracellular calcium. Few studies have been performed on cerebral smooth muscle cells. In one study, exposure of cerebrovascular smooth muscle cells to free radicals resulted in increased outward currents, decreased membrane resistance, cell contraction, appearance of membrane blebs, and cell death. In summary, more investigations using better experimental techniques are required before free radicals and reactions induced by them can be said with certainty to be the primary cause of vasospasm.     

Interaction of free radicals with proteins

This review describe the generation of free radicals in the cells under the influence of exogenously and endogenously acting factors. The interactions of free radicals with proteins and amino acids and the consequences of these effects are also presented.     

Chronic pancreatitis, acute pancreatitis

MRCP has been recognized as a safe and noninvasive diagnostic method. In the present study we evaluated the usefulness of MRCP in diagnosis of chronic and acute pancreatitis. Two-dimensional fast asymmetric spin-echo (FASE) MRCP was performed in 40 patients with chronic pancreatitis and 13 with acute pancreatitis. In 29 patients (72.5%) with chronic pancreatitis and 9 (66.7%) with acute pancreatitis, main pancreatic duct (MPD) was visualized entirely. MRCP could demonstrate the characteristic findings of chronic pancreatitis such as dilatation and irregularity of MPD in most cases. In acute pancreatitis, MRCP indicated that MPD was normal in diameter, but irregular in configuration compared with that of the control group. MRCP may facilitate the diagnosis of chronic and acute pancreatitis.     

Oxidizing agents and free radicals in biomedicine

The reactions which involve oxidants and free radicals species have played an essential role at the beginning of aerobic life, and they are an intrinsic part in the regulation of the cellular processes. However, as a result of the derived toxic effects of these oxidative processes, antioxidants molecules appeared in the very early stages of the evolution and they are able to control the production of these reactants and their dangerous effects. Oxidants and antioxidants have a clear function in the cellular physiology. When this delicate balance change many biochemical and cellular reactions are altered, and that may cause different pathologic diseases. In addition, free radicals of oxygen are thought to play a growing role in the biological process of ageing (1). It is not unusual to find papers about these reactants in every topic of the biomedicine. The main oxidants and free radicals in the organisms are oxygen-related agents, which are globally called reactive oxygen intermediates (ROI). These unavoidable, useful and dangerous biological oxidants are well characterized, although the recent implications they received in some pathologies deserve, in our opinion, a general review.     

Involvement of free radicals and histamine in the potentiating action of cigarette smoke exposure on ethanol-induced gastric mucosal damage in rats

Cigarette smoking has been associated with peptic ulcer diseases. We studied the effects of cigarette smoke exposure on ethanol-induced gastric mucosal damage and its relationship with vascular integrity and the possible role of free radicals and histamine. Male Sprague-Dawley rats were exposed to cigarette smoke followed by ethanol administration (70% v/v). Smoke exposure alone dose-dependently reduced basal blood flow and increased xanthine oxidase (XO) activity but superoxide dismutase (SOD) activity remained unaffected in gastric mucosa. Cigarette smoking followed by ethanol administration significantly potentiated mucosal lesion formation along with augmentation of the mucosal blood flow, vascular permeability and myeloperoxidase (MPO) activity. The potentiating effect of smoking on ethanol-induced gastric mucosal lesion and MPO activity was abolished by pretreatment with allopurinol, terfenadine or ranitidine. Terfenadine and ranitidine also reduced the increased mucosal blood flow and vascular permeability induced by smoking and ethanol combined. These findings suggested that cigarette smoke adversely affected the defense mechanisms of the gastric mucosa by reducing the mucosal blood flow which in turn led to ischemia and increased XO activity. Activation of XO together with histamine H1 and H2 receptors stimulation could lead to neutrophil aggregation and vascular damage. However, the potentiating action of cigarette smoke on ethanol ulceration is unlikely through reduction of SOD activity in gastric mucosa.     

Presence of free radicals in pigment gallstone in vivo

OBJECTIVE: To clarify whether free radical, which may play a role in pigment gallstone formation, is present in pigment gallstones in vivo. MATERIALS AND METHODS: Free radical signal of gallstones from 18 patients was detected by electron paramagnetic resonance spectroscopy at 77K under anaerobic condition and in air (control). As soon as the anaerobic determination was finished, the fresh anaerobic sample was exposed to air and stored in a freezer at -20 degrees C. RESULTS: Free radical signal (g = 2.0038) was detected in fresh anaerobic samples containing more than 2% bilirubin compound, and the signal intensity correlated linearly with the content of calcium bilirubinate (r = +0.95, P < 0.0005). During the storage at -20 degrees C and exposure to air, the signal intensity of each anaerobic sample and its control increased gradually, eventually reaching the same stable level. Fe(III) signal intensity was enhanced synchronously and related linearly with free radical signal (r = +0.99, P < 0.0005). CONCLUSIONS: Free radical exists originally in pigment gallstones in vivo, and it may play an important role in pigment gallstone formation. The free radical signal carried by gallstones may be strengthened by the action of oxygen in air on bilirubin. The transition metal ions probably take part in the formation of bilirubin free radical.     

Role of polymorphonuclear leucocytes and oxygen-derived free radicals in the formation of gastric lesions induced by HCl/ethanol, and a possible mechanism of protection by anti-ulcer polysaccharide

This study examined the role of oxygen-derived free radicals in the pathogenesis of gastric mucosal lesions induced by HCl/ethanol. Superoxide dismutase, and catalase, and their combination reduced gastric lesion formation in mice. Gastric lesions were also reduced in mice treated with cyclophosphamide or anti-neutrophils, but not in mice treated with allopurinol or desulphated-carrageenan. Cobra venom factor did not reduce lesion formation. These results suggested that oxygen-free radicals may contribute to the formation of gastric mucosal lesions induced by HCl/ethanol, and that oxygen radicals were generated from neutrophils but not from xanthine oxidase. Anti-ulcer pectic polysaccharide, bupleuran 2IIc, which was recently isolated from the roots of Bupleurum falcatum L., showed potent inhibition of HCl/ethanol-induced gastric lesions in mice. Bupleuran 2IIc seemed to scavenge hydroxyl radical effectively. It was suggested that this anti-ulcer polysaccharide may provide protection to the gastric mucosa by scavenging oxygen-free radicals.     

The role of oxygen free radicals in acute renal failure complicating obstructive jaundice: an experimental study

Oxidant injury is considered to be an important mechanism in the pathophysiology of acute renal failure. It has been thought that decrease in extracellular and intracellular fluid and endotoxemia seen in obstructive jaundice may cause an increase in production of oxygen free radicals and impairment in antioxidant defense mechanism. This study is designed to investigate the possible role of oxidant injury in renal failure seen in jaundiced patients. In this study, 28 rats were divided into four groups: Control (C)(N = 7); Renal ischemia (RI)(N = 7); Obstructive jaundice+renal ischemia (OJ+RI)(N = 7); Obstructive jaundice (OJ)(N = 7). All groups were compared with each other according to renal failure findings and enzyme activities, such as Xanthine oxidase (XOD), Superoxide Dismutase (SOD) and Catalase in renal cortex and Glutathione Peroxidase (GSH-Px), in blood at 3rd day after ischemia and reperfusion. Renal failure findings monitored by blood urea and creatinine levels, seemed more evident in OJ+RI than RI group (p < 0.05). When compared with RI, in OJ+RI group, increase in XOD activity at 3rd day was statistically significant [0.259 +/- 0.01 U/g (tissue) and 0.362 +/- 0.03 U/g (tissue) respectively] (p < 0.05). SOD and GSH-Px activities of each ischemic group at 3rd day were decreased compared to non-ischemic groups. This fall was significant (p < 0.05). But there was no statistical difference between jaundiced and non-jaundiced groups. Alterations in catalase activities also had no statistical significance. These findings may suggest that the injury induced by oxygen free radicals at re-oxygenation of tissue after ischemia may also play a role in the pathogenesis of acute renal failure developed in obstructive jaundice.     

The role of oxygen free radicals in isoniazid-induced hepatotoxicity

This report describes the glycosaminoglycans, collagen and elastin--composition of leiomyosarcoma. Studies were performed on leiomyosarcoma removed during surgery. The material was taken from 7 patients. Rearrangement of extracellular matrix in leiomyosarcoma has been found. There was an increase of total collagen content in comparison to control uterus. In both tissues type I collagen was found to be the predominant one. Both tissues, normal and neoplastic contain all known glycosaminoglycans types. Among them heparan sulphate was found to be the most abundant. A slight decrease in the content of this glycosaminoglycan was observed in leiomyosarcoma. A possible role of these alterations in tumour biology is discussed.     

Therapy of acute pancreatitis

The mortality rate in acute pancreatitis (AP) is significantly lower in patients hospitalized directly at the intensive care unit than in patients admitted to hospital in 2 weeks after the assessment of diagnosis, prophylactic administration of low-molecular protease inhibitor reduces the occurrence of post ERCP pancreatitis a well a coincident complications. Despite rational considerations concerning the significance of pryphylactic administration of antibodies (ATB) in severe AP, there still not enough convincing data which could be recommended a standard therapy. One of the concepts of causal therapy of AP. Suggest that inhibition of exocrine pancreatic enzymatic secretion reduces autodigestion of the gland (setting the gland at rest). The reports on success of secretin-inhibiting substances a glucagon, calcitonin, atropine and somatostatin require confirmation in randomized or accurately defined comparable groups. The initial studies on the therapeutic significance of lexipanphate-antagonist of platelet activating factor (PAF) in acute pancreatitis is promising. A long-term lavage had reduced the mortality.     

Therapy of acute pancreatitis

Therapy in patients with acute pancreatitis (AP) is primarily conservative and follows the rules of generally accepted principles. A very important basis in the treatment of AP is the interdisciplinary approach to this disease which demands teamwork between clinicians, intensive care specialists and surgeons. Patients with a necrotising course should be hospitalised on the ICU and should receive maximum intensive care measures and antibiotics. Indications for surgical interventions in severe AP are infected pancreatic necrosis and non-response to intensive care therapy.