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In previous studies, benzo[a]pyrene-7,8-dione (BPQ), a polycyclic aromatic hydrocarbon (PAH) o-quinone, was found to be 200-fold more potent as a nuclease than (+/-)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene, a suspect human carcinogen. The mechanism of strand scission mediated by naphthalene-1,2-dione (NPQ) and BPQ was further characterized using either phiX174 DNA or poly(dG).poly(dC) as the target DNA. Strand scission was extensive, dependent on the concentration of o-quinone (0-10 microM), and required the presence of NADPH (1 mM) and CuCl2 (10 microM). The production of reactive species, i.e., superoxide anion radical, o-semiquinone anion (SQ) radical, hydrogen peroxide (H2O2), hydroxyl radical (OH.), and Cu(I), was measured in the incubation mixtures. The formation of SQ radicals was measured by EPR spectroscopy under anaerobic conditions in the presence of NADPH. A Cu(II)/Cu(I) redox cycle was found to be critical for DNA cleavage. No strand scission occurred in the absence of Cu(II) or when Cu(I) was substituted, yet Cu(I) was required for OH* production. Both DNA strand scisson and OH. formation were decreased to an equal extent, albeit not completely, by the inclusion of OH. scavengers (mannitol, soduim benzoate, and formic acid) or Cu(I) chelators (bathocuproine and neocuproine). In contrast, although the SQ radical signals of NPQ and BPQ were quenched by DNA, no strand scission was observed. When calf thymus DNA was treated with PAH o-quinones, malondialdehyde (MDA) was released by acid hydrolysis. The formation of MDA was inhibited by OH. scavengers suggesting that OH* cleaved the 2'-deoxyribose moiety in the DNA to produce base propenals. These studies indicate that for PAH o-quinones to act as nucleases, NADPH, Cu(II), Cu(I), H2O2, and OH*, were necessary and that the primary species responsible for DNA fragmentation was OH., generated by a Cu(I)-catalyzed Fenton reaction. The genotoxicity of PAH o-quinones may play a role in the carcinogenicity and mutagenicity of the parent hydrocarbons.

Dihydrodiol dehydrogenase (DD; EC 1.3.1.20) catalyzes the oxidation of polycyclic aromatic hydrocarbon (PAH) trans-dihydrodiols (proximate carcinogens) to catechols which rapidly autoxidize to yield o-quinones (Smithgall, T. E., Harvey, R. G., and Penning, T. M. (1988) J. Biol. Chem 263, 1814-1820). Although this pathway suppresses the formation of the PAH anti- and syn-diol epoxides (ultimate carcinogens), the process of autoxidation is anticipated to yield reactive oxygen species (ROS). We now show that the NADP+ dependent oxidation of (+/-)-trans-1,2-dihydroxy-1,2-dihydronaphthalene (Npdiol) and (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (Bpdiol) catalyzed by homogeneous DD is accompanied by the consumption of molecular oxygen and the production of H2O2. With both trans-dihydrodiol substrates, oxygen consumption was stoichiometric with H2O2 production consistent with the reaction: QH2 + O2 = H2O2 + Q, where QH2 is the catechol and Q is the o-quinone. Using Npdiol or Bpdiol as substrates, a burst of superoxide anion production is catalyzed by DD which can be detected as the rate of cyt c reduction that is inhibited by superoxide dismutase. Using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as spin-trapping agent, secondary spin adducts corresponding to DMPO-CH3 were formed during the enzymatic oxidation of Npdiol and Bpdiol. The formation of the CH3. radical arises from the OH. attack of DMSO, which was used as cosolvent. These spin adducts were attenuated by superoxide dismutase and catalase, implying that O2-. and H2O2 are obligatory for the formation of DMPO-CH3. It is proposed that O2-. is the radical that propagates autoxidation and that the resultant H2O2 undergoes Fenton chemistry to produce the OH. radical. Identical spin adducts were observed using a superoxide anion generating system (hypoxanthine/xanthine oxidase) and DMPO as spin-trapping agent in the presence of DMSO. The ability of DD to generate ROS during the oxidation of PAH trans-dihydrodiols (proximate carcinogens) may have important implications for tumor initiation and promotion.

Because of the short half-life of NO, previous studies implicating NO in central nervous system pathology during infection had to rely on the demonstration of elevated levels of NO synthase mRNA or enzyme expression or NO metabolites such as nitrate and nitrite in the infected brain. To more definitively investigate the potential causative role of NO in lesions of the central nervous system in animals infected with neurotropic viruses or suffering from experimental allergic encephalitis, we have determined directly the levels of NO present in the central nervous system of such animals. Using spin trapping of NO and electron paramagnetic resonance spectroscopy, we confirm here that copious amounts of NO (up to 30-fold more than control) are elaborated in the brains of rats infected with rabies virus or borna disease virus, as well as in the spinal cords of rats that had received myelin basic protein-specific T cells.

Carbon monoxide (CO) poisoning has been reported to temporarily inhibit B2 integrin adherence molecules on leukocytes in previous studies in a rat model. The aim of this study was to investigate the mechanism for this effect. Studies were conducted using blood obtained from rats after they were exposed to CO and also with blood cells exposed to CO in vitro. Initial investigations indicated that inhibition of neutrophil (polymorphonuclear leukocyte, PMN) B2 integrin function was linked to the platelets in blood, as the effect was resolved by decreasing platelet number before PMN adherence was tested. The platelet effect could also be shown by incubating either platelet-rich plasma or whole blood with CO in vitro. The effect of platelets was blocked by superoxide radicals and by NG-nitro-L-arginine methyl ester, an inhibitor of nitric oxide (NO) synthase. These observations suggested that CO caused platelets to release NO, an agent known to inhibit the function of B2 integrins. The concentration of NO measured in suspensions of platelets from rats poisoned with CO according to the established model (exposure to 1000 ppm CO for 40 min and 3000 ppm CO for 20 min) was 47 nmol/10(8) platelets, in contrast to only 0.3 nmol NO/10(8) platelets from control rats. Enhanced NO release occurred despite a 60% inhibition of NO synthase activity, assessed by measuring conversion of [14C]L-arginine to citrulline. Exposure to only 1000 ppm CO for 1 hr caused platelets to release 74 nmol NO/10(8) platelets, and no inhibition of NO synthase occurred. Enhanced NO release, and inhibition of PMN adherence, did not occur after platelets were exposed to light from a quartz lamp to photodissociate CO from heme proteins. The data suggest that the NO flux from platelets increased when CO became bound to heme-containing platelet proteins, which normally scavage intraplatelet NO and thus prevent diffusion beyond the platelet membrane.

Protective effects of an aged garlic extract on the cardiotoxicity of doxorubicin (DOX) was evaluated using the mouse. DOX (1.5 mg/kg body wt i.p.) was administered three times per week for 40 days. An aged garlic extract, WG-1 (a preserved stock solution; Wakunaga Pharmaceutical) was administered intraperitoneally six times weekly. DOX caused changes in the electrocardiogram. In the control mice, the width of the QRS complex was 20 +/- 2.8 milliseconds, the R-R interval was 130 +/- 2.8 milliseconds, and the P-Q interval was 30 +/- 1.4 milliseconds. In mice treated with DOX for 40 days, the width of the QRS complex was 50 +/- 10 milliseconds (p < 0.05), the R-R interval was 240 +/- 30 milliseconds (p < 0.05), and the P-Q interval was 45 +/- 1.0 milliseconds (p < 0.01). These values were significantly smaller in mice treated with WG-1 + DOX than in mice treated with DOX. The width of the QRS complex was 29.3 +/- 5.8 milliseconds (p < 0.05), the R-R interval was 145.8 +/- 17.9 milliseconds (p < 0.01), and the P-Q interval was 37.8 +/- 3.5 milliseconds (p < 0.05). The lipid peroxidation in the heart homogenates prepared from DOX-treated mice, as measured by thiobarbituric acid-reactive substance (TBARS, nmol malondialdehyde/100 mg protein) was 332.5 +/- 67.0, which was significantly larger than that in the control mice (186.6 +/- 42.2) (p < 0.05). WG-1 decreased the level of TBARS in DOX-treated mice significantly. In the mice treated with WG-1 + DOX, TBARS was 221.3 +/- 31.6, which was significantly smaller than that of DOX-treated mice (p < 0.05). Histological study demonstrated that the heart treated with DOX had vacuolization in muscle cells, disrupted myofibrils, and swollen mitochondria. Mice that received WG-1 + DOX had no significant pathological lesions in the heart.

The effects of a protein-restricted diet on renal recovery following renal ischemia were studied. The renal function was assessed by measuring the inulin clearance (CIN), the p-aminohippurate clearance (CPAH), and the percent fractional sodium excretion (%FENa) 24 h after 45 min renal ischemia. In rats fed with a regular diet (containing 19.6% protein), CIN was 10.0 +/- 2.2 microL/min/100 g body weight (BW), CPAH 0.08 +/- 0.02 mL/min/100 g BW, and %FENa 14.8 +/- 2.0, 24 h after renal ischemia. In contrast, feeding rats with a no-protein diet (0% protein) for 1 week prior to the ischemic insult significantly improved renal recovery (CIN 48.0 +/- 9.3 microL/min/100 g BW, CPAH 0.16 +/- 0.04 mL/min/100 g BW, and %FENa 2.43 +/- 0.58). Feeding rats with a no-protein diet for 3 weeks prior to ischemic insult further improved the renal recovery (CIN 113 +/- 30 microL/min/100 g BW, CPAH 0.47 +/- 0.17 mL/min/100 g BW, and %FENa 1.55 +/- 0.29). When rats fed with a regular diet were exposed to 45 min of ischemia, the survival rate on day 7 was 16.7%. In rats fed with the no-protein diet for 1 week and for 3 weeks, the 7-day survival rate was 100% in each case. The survival rate of rats fed for 3 days instead of 7 days with the no-protein diet was 87.5%. When a no-protein feeding was shortened to 1 day, no beneficial effects were observed and survival rate was 14.3%. (ABSTRACT TRUNCATED AT 250 WORDS)

Effects of prostaglandin E (PGE) on acute pancreatitis have been controversial. This study shows the effects of PGE1 oligomer, MR-356, on trypsin-taurocholate-induced acute pancreatitis in rats. Divided intraperitoneal doses of 0.6 mg/rat were administered, which increased 24 h survival rates when the oligomer was given both at 1 h before and after (group A) and immediately and 3 h after (group B) induction of pancreatitis. In group A MR-356 significantly improved the survival rates at 18 h (94 vs 61%, P < 0.05) and 24 h (68 vs 33%, P < 0.05) when compared with controls. MR-356 improved the survival rates dose-dependently up to 0.6 mg/rat when given by the same protocol of group A. In group B MR-356 also improved the survival rate (72 vs 39%, P < 0.05) only at 24 h, while other parameters failed to improve. The present results suggest that the PGE1 oligomer may play a beneficial role in bile-induced pancreatitis, probably through its proposed effects of stabilization of lysosomal membranes, maintenance of microcirculation and inhibition of protease in the pancreas.

Two ester-type prostaglandin oligomeric compounds were synthesized, one from prostaglandin E1 (termed MR-356) and the other from prostaglandin B2 (termed OC-5186). Using in vivo [31P]MRS, the protective effects of these oligomers on forebrain ischemia (15 min) were evaluated in a rat model. Forebrain ischemia caused a decrease in intracellular high energy phosphates and intracellular pH (pHi) in the control and compounds-treated groups, but changes of these values in the OC-5186-treated group were significantly smaller than those in the control group. Moreover, the cerebral energy metabolism of the OC-5186-treated group returned to the preischemia level more rapidly than in the control group after forebrain ischemia. MR-356 had some effects, but the differences were not significant.

Previous studies have shown that brain tissue hypoxia results in increased N-methyl-D-aspartate (NMDA) receptor activation and receptor-mediated increase in intracellular calcium which may activate Ca++-dependent nitric oxide synthase (NOS). The present study tested the hypothesis that tissue hypoxia will induce generation of nitric oxide (NO) free radicals in cerebral cortex of newborn guinea pigs. Nitric oxide free radical generation was assayed by electron spin resonance (ESR) spectroscopy. Ten newborn guinea pigs were assigned to either normoxic (FiO2 = 21%, n = 5) or hypoxic (FiO2 = 7%, n = 5) groups. Prior to exposure, animals were injected subcutaneously with the spin trapping agents diethyldithiocarbamate (DETC, 400 mg/kg), FeSO4.7H2O (40 mg/kg) and sodium citrate (200mg/kg). Pretreated animals were exposed to either 21% or 7% oxygen for 60 min. Cortical tissue was obtained, homogenized and the spin adducts extracted. The difference of spectra between 2.047 and 2.027 gauss represents production of NO free radical. In hypoxic animals, there was a difference (16.75+/-1.70 mm/g dry brain tissue) between the spectra of NO spin adducts identifying a significant increase in NO free radical production. In the normoxic animals, however, there was no difference between the two spectra. We conclude that hypoxia results in Ca2+-dependent NOS mediated increase in NO free radical production in the cerebral cortex of newborn guinea pigs. Since NO free radicals produce peroxynitrite in presence of superoxide radicals that are abundant in the hypoxic tissue, we speculate that hypoxia-induced generation of NO free radical will lead to nitration of a number of cerebral proteins including the NMDA receptor, a potential mechanism of hypoxia-induced modification of the NMDA receptor resulting in neuronal injury.

Doxorubicin, a potent anticancer drug, is effective against a wide range of human neoplasms. However, the clinical uses of doxorubicin have been limited due to its serious cardiotoxic effects, which are likely the result of generation of free radicals and lipid peroxidation. S-Allylcysteine (SAC), an organosulfur compound purified from garlic, has been reported to have antioxidant and radical scavenging effects. Thus, we examined the effect of SAC on doxorubicin toxicity in mice. Severe doxorubicin toxicity was induced in mice by a single intraperitoneal injection (15 mg/kg body weight). SAC (30 mg/kg) was injected intraperitoneally daily for 5 days, starting two days prior to the administration of doxorubicin. Body weight was measured every alternate day. A measurement of serum creatine phosphokinase (CPK) and a histopathological analysis of the heart and liver was performed 6 days after the administration of doxorubicin. Death of any of the animals was recorded during the observation period. Doxorubicin injection induced a mortality rate of 58%, with SAC treatment reducing the doxorubicin-induced mortality rate to 30%. The severe body weight loss caused by doxorubicin (13%) was also significantly attenuated by SAC treatment (9%). Although an elevation of the level of serum CPK was observed following doxorubicin injection (5472 +/- 570 i.u./L), treatment with SAC significantly reduced the level of CPK (1923 +/- 635 i.u./L). Histological analysis demonstrated that heart and liver damage was significantly less severe in SAC treated mice than in mice receiving only doxorubicin. These results suggest that SAC research may ultimately lead to a resolution of the adverse effects of doxorubicin treatment in cancer chemotherapy.

Inhalation of the pulmonary irritant ozone is associated with an accumulation of macrophages in the lung. These cells, along with type II epithelial cells, are activated to release increased quantities of hydrogen peroxide and nitric oxide, two reactive mediators that have been implicated in tissue injury. In the present studies we determined whether pretreatment of rats with bacterially derived endotoxin, which modulates oxidant levels in tissues, could abrogate the effects of ozone on lung injury and nitric oxide production. Acute exposure of rats to ozone (2 parts per million, 3 h) resulted in nitric oxide production in the lung as measured by electron paramagnetic resonance spin trapping. This was correlated with expression of inducible nitric oxide synthase (iNOS) mRNA in the lung as determined by in situ hybridization. Particularly high levels of iNOS were evident in alveolar macrophages and type II cells. Alveolar macrophages isolated from ozone-treated rats also expressed increased iNOS mRNA and protein as measured by Northern and Western blotting, respectively, and produced more nitric oxide compared with cells from air-exposed animals. Treatment of rats with endotoxin (5 mg/kg, intravenously), 30 min prior to ozone, was found to abrogate ozone-induced increases in iNOS mRNA and protein expression, as well as nitric oxide production by alveolar macrophages. This was associated with a reduction in ozone-induced tissue injury as determined by levels of lung lavage fluid protein. Ozone inhalation also resulted in a reduction in intracellular glutathione in alveolar macrophages, an effect that was blocked by endotoxin administration. Taken together, these data provide evidence that the protective effects of endotoxin against ozone-induced injury are mediated, at least in part, by alterations in levels of lung oxidants and antioxidants.

Nitric oxide is a short-lived free radical and physiological mediator which has the potential to cause cytotoxicity. Studies were conducted to investigate whether nitric oxide, and the potent oxidant peroxynitrite, were generated in brain during experimental carbon monoxide (CO) poisoning in the rat. Nitric oxide production was documented by electron paramagnetic resonance spectroscopy, and found to be increased by ninefold immediately after CO poisoning. Evidence that peroxynitrite was generated was sought by looking for nitrotyrosine in the brains of CO-poisoned rats. Nitrotyrosine was found deposited in vascular walls, and also diffusely throughout the parenchyma in inummocytochemical studies. The affinity and specificity of an anti-nitrotyrosine antibody was investigated and a solid phase immunoradiochemical assay was developed to quantity nitrotyrosine in brain homogenates. A 10-fold increase in nitrotyrosine was found in the brains of CO-poisoned rats. Platelets were involved with production of nitrotyrosine in the early phase of exposure to CO. However, nitrotyrosine formation and leukocyte sequestration were not decreased in thrombocytopenic rats poisoned with CO according to the standard model. When rats were pre-treated with the nitric oxide synthase inhibitor, L-nitroarginine methyl ester, formation of both nitric oxide and nitrotyrosine in response to CO poisoning were abolished, as well as leukocyte sequestration in the microvasculature, endothelial xanthine dehydrogenase conversion to xanthine oxidase, and brain lipid peroxidation. We conclude that perivascular reactions mediated by peroxynitrite are important in the cascade of events which lead to brain oxidative stress in CO poisoning.

In the present studies, we analyzed the effects of acute endotoxemia on hepatocyte nitric oxide production and functional activity. Treatment of rats with 5 mg/kg of lipopolysaccharide (LPS), which induces acute endotoxemia, caused an increase in nitric oxide production in the liver, as measured by electron paramagnetic spin trapping, which was evident within 6 hours. This was associated with expression of inducible nitric oxide synthase (iNOS) messenger (m) RNA in hepatocytes and in sinusoidal cells throughout the liver lobule. Acute endotoxemia also caused alterations in hepatic structure, including hypertrophy, vacuolization, and chromosomal emargination, however these changes were not apparent for 24 to 48 hours. Hepatocytes isolated from endotoxemic rats released increased amounts of nitric oxide, measured by nitrite production, in response to interferon gamma (gamma-IFN) alone or in combination with LPS, tumor necrosis factor alpha, macrophage-colony stimulating factor, granulocyte/macrophage-colony stimulating factor, or hepatocyte growth factor. These results show that hepatocytes are sensitized by acute endotoxemia to respond to inflammatory mediators and growth factors. Increased nitrite production by hepatocytes was due to increased expression of iNOS mRNA and protein and was correlated with the time following induction of acute endotoxemia. Thus, cells isolated 48 hours after induction of acute endotoxemia released significantly more nitrite than cells recovered after 6 hours, a response that was not due to alterations in hepatocyte viability. Hepatocytes isolated from endotoxemic rats also exhibited a marked increase in proliferative capacity when compared with cells from control rats. Nitric oxide production by hepatocytes in vitro was associated with inhibition of cell growth and protein synthesis, which was reversed by the nitric oxide synthase inhibitor, NG-monomethyl-l-arginine (L-NMMA). Agarose gel electrophoresis showed extensive cytoplasmic DNA fragmentation in hepatocytes treated with LPS and gamma-IFN, a characteristic of apoptosis, which was also reversed by L-NMMA. These results, together with our findings that treatment of rats with an inhibitor of nitric oxide synthase partially reversed the structural alterations in the liver associated with acute endotoxemia suggest that nitric oxide may contribute to the pathophysiologic response to this bacterially derived toxin.

Nitric oxide is a short-lived cytotoxic mediator that has been implicated in the pathogenesis of endotoxin-induced tissue injury and septic shock. In the present studies we determined whether this mediator is produced in the lung during acute endotoxemia. We found that intravenous injection of rats with bacterially derived lipopolysaccharide (LPS), a condition that induces acute endotoxemia, caused a time-dependent increase in inducible nitric oxide synthase (iNOS) mRNA expression in the lung, which reached a maximum after 24 h. This was correlated with nitric oxide production in the lung as measured by electron paramagnetic spin trapping, which was detectable within 6 h. Alveolar macrophages (AMs) and interstitial macrophages (IMs) isolated from rats 6–12 h after induction of acute endotoxemia were also found to exhibit increased nitric oxide production in response to in vitro stimulation with interferon-γ (IFN-γ) and LPS measured by nitrite accumulation in the culture medium. The effects of acute endotoxemia on nitric oxide production by these cells were, however, transient and returned to control levels by 24 h in AMs and 36 h in IMs. Interestingly, although nitrite accumulation in the culture medium of IMs isolated 48 h after induction of acute endotoxemia and stimulated with low concentrations of IFN-γ and LPS was reduced, when compared with cells from control animals, these cells, as well as AMs, continued to express high levels of iNOS protein and mRNA. This was correlated with increased peroxynitrite production by the cells. Peroxynitrite has been shown to act as a nitrating agent and can generate nitrotyrosine residues in proteins. Using a specific antibody and immunohistochemistry, we found evidence of nitrotyrosine residues in sections of lungs 48 h after treatment of rats with endotoxin. These data suggest that nitric oxide produced by IMs and AMs can react with superoxide anion to form peroxynitrite. Taken together, the present studies demonstrate that AMs and IMs are activated following acute endotoxemia to produce reactive nitrogen intermediates and that both cell types contribute to inflammatory responses in the lung. J. Leukoc. Biol. 56: 759–768; 1994.

We investigated the effects of a non-protein diet on renal recovery in rats following 45-minute renal ischemia and mercuric chloride (3 mg/kg BW:S.C.) poisoning. The rats were fed a non-protein diet for one week before the renal insults. Renal functions were measured 24 hours after renal ischemia or 6 hours after mercuric chloride administration. In the ischemia investigation, the glomerular filtration rate (GFR), the renal plasma flow rate (RPFR) and the percent fractional sodium excretion (%FENa) of rats fed a regular diet (19.6% protein) were 25 +/- 7 microliters/min/g KW, 0.19 +/- 0.1 ml/min/g KW and 14.8 +/- 2.0, respectively. These values in the rats fed a non-protein diet showed better recovery, returning to a GFR of 114 +/- 32 microliters/min/g KW, an RPFR of 0.37 +/- 0.1 ml/min/g KW, and a %FENa of 2.43 +/- 0.6, respectively (p0.05). Furthermore, the seven-day survival rate was improved from 17% in the regular diet group to 100% in the non-protein diet group. In the mercuric chloride investigation, the renal functions in rats on a regular diet were shown by a GFR of 461 +/- 51 microliters/min/g KW, an RPFR of 1.91 +/- 0.2 ml/min/g KW, and a %FENa of 2.22 +/- 0.5. One-week feeding with a non-protein diet ameliorated the decrease in renal function, resulting in a GFR of 604 +/- 84 microliters/min/g KW, an RPFR of 2.15 +/- 0.5 ml/min/g KW, and a %FENa of 2.20 +/- 0.6. Consequently, there was a distinct beneficial effect on the survival of these rats.(ABSTRACT TRUNCATED AT 250 WORDS)

A monomeric derivative and several oligomeric derivatives were synthesized from prostaglandin B2. Their lipid solubility was studied by measuring their octanol-water partition coefficients. With EPR spectroscopy, the oligomeric derivatives were shown to have g = 2 signal, indicating these compounds have intrinsic free radicals. Measuring the rate of adenochrome formation, it was shown that these derivatives could scavenge superoxide anions. Using a spin-trapping technique employing DMPO, we found that these oligomers could also scavenge hydroxyl radicals. The calcium chelating activity of these compounds were also studied. In an in vitro rat model, these compounds inhibited lipid peroxidation as measured by the production of thiobarbituric acid reacting substances. Other prostaglandin oligomeric derivatives synthesized from PGE1 were also studied, and their properties were compared with these new compounds. Results suggest that both the water solubility and the chelating activity for calcium ions may not be related to their protective effects in ischemic or traumatic injury.

Forebrain ischemia was produced in the rat by bilateral occlusion of the common carotid arteries combined with hemorrhagic hypotension (30 mmHg). The whole cerebral cortex was homogenized in the presence of the spin trap agent N-tert-butyl-alpha-phenyl-nitrone, followed by a Folch extract. Spin-adducts were detected using electron spin resonance spectroscopy. The lipid peroxidation was estimated from both the amount of thiobarbituric acid reactive substance and the formation of conjugated diene. After 10 or 20 min of ischemia, reperfusion was initiated which induced an abrupt burst of free radical formation. The formation peaked at 5 min, and the peak value increased with the ischemia time. The degree of lipid peroxidation, which was measured after 20 min of reperfusion, also increased with the ischemia time. The results suggest that the lipid peroxidation may be the direct consequence of the action of free radicals formed during ischemia and reperfusion periods.

We have studied the question of the possible role of sarcoplasmic reticulum (SR) in the interaction of volatile anesthetics (such as halothane, enflurane and isoflurane) with muscle. We used two cardiac muscle models, i.e., isolated rat myocytes and Langendorff perfused rat hearts. We compared the results with those for skeletal muscle SR from rabbits, rats and pigs susceptible to malignant hyperthermia (MH). In both skeletal and cardiac muscle SR, volatile anesthetics enhanced the calcium release from the SR. In cardiac muscle, these agents are known to decrease contractility (negative inotropism). We found that caffeine, a well-known agent which releases calcium from the SR, also had a negative inotropic effect in cardiac muscle, raising the possibility of an unexpected link between the potentiation of calcium release and mechanism underlying the observed negative inotropism. Current understanding of anesthetic mechanisms does not include this possibility. We further found that both volatile anesthetics and caffeine decrease the content of calcium in the SR, suggesting that the increase of calcium permeability results in the decrease of calcium ions in the SR which are available for excitation-contraction (E-C) coupling. In MH-susceptible skeletal muscle, a similar increase in calcium permeability does not cause a decrease of contractility, but rather may contribute to a fatal syndrome of temperature increase provoked by abnormal contracture. This difference may be because in skeletal myoplasm calcium ions recycle internally, while in the cardiac muscle cell they are in dynamic equilibrium with extracellular calcium ions.

A combined impounder-surface K+ electrode was developed to measure change in K+ ion concentration of the cerebrospinal fluid (CSF) across the dura and arachnoid maters. To determine whether K+ permeability of the rat dura and arachnoid maters is due to an intrinsic permeability, a study was conducted using an atraumatic laminectomy model. Dorsal laminectomy was performed at T7-8, T12, and L1. Artificial CSF containing 4.2 mM, 24.2 mM, or 54.2 mM of K+ was administered by anterograde subdural infusion into the subarachnoid space from the proximal laminectomy site (T7-8), with effluent overflow at the distal laminectomy site (L1). K+ concentration on the dorsal aspect of the central laminectomy site (T12) was measured. It was found that changes in K+ concentrations of the infused solution were detected by the epidural surface electrode. This suggests that the intact rat spinal cord dura and arachnoid maters may be permeable to K+ in this laminectomy model. This study supports the use of the combined impounder-K+ electrode for measuring changes in K+ ion concentration of the CSF that can result from spinal cord trauma and ischemia.

Effect of Hypoxia on Nitric Oxide Free Radical Generation in the Cerebral Cortex of Newborn Guinea Pigs

Previous studies have shown that hypoxia increases the generation of oxygen free radicals in the brain of the term guinea pig fetus. The present study tests the hypothesis that free radical production during hypoxia changes with each stage of brain development. Pregnant guinea pigs at 40 days (preterm, n=12) and 60 days (term n=14) gestation were assigned to normoxic or hypoxic groups and exposed to 21% or 7% oxygen, respectively, for 60 minutes. Fetal cerebral cortical tissue was homogenized in N2 bubbled ice-cold 100 mMα -phenyl-N-tert-butylnitrone (PBN) to trap oxygen free radicals. Trapped PBN spin adducts were extracted in toluene and free radical generation was measured by electron spin resonance spectroscopy (ESR) performed at-20°C. Peak height of the spin adduct signal was used as an index of the intensity of free radical formation and expressed as units/g dry tissue. Fetal brain hypoxia was documented biochemically by decreased levels of ATP and phosphocreatine. In the preterm group, cortical tissue from hypoxic fetuses showed an increase in spin adducts (normoxic:41.4±3.5 units/g tissue vs. hypoxic:55.1±6.4 units/g tissue, 33% increase, p

We investigated the relations of brain edema, ion shifts, motor performance, and memory impairment using a focal ischemia model in rats. Cortical infarction was produced by ligation of the middle cerebral artery and the ipsilateral common carotid artery combined with temporary occlusion of the contralateral common carotid artery for 1 hour. Water content and sodium, potassium, and calcium concentrations were measured until Day 14 after the ischemic insult. Significant edema formation was observed; it peaked on Day 3 (p less than 0.001) and then declined. The tissue sodium concentration changed in a manner similar to that of water content, but the tissue potassium concentration changed in an opposite fashion. Massive accumulation of calcium was detected as early as Day 1 after ischemia (almost four times the normal level). The increased calcium concentration was sustained even up to Day 14. Motor performance examinations performed on Day 3, including inclined plane, balance beam, and prehensile tests, demonstrated significantly reduced (p less than 0.001) motor ability that did recover even by Day 7. Passive avoidance learning was carried out on Day 2, followed by a memory retention test on Day 3. Significant memory dysfunction was observed in ischemic compared with sham-operated rats (p less than 0.001). A high correlation coefficient (r = 0.91, p less than 0.01, n = 13) was obtained between water content and calcium concentration on Day 3. Both the total motor score and the degree of disturbance of the passive avoidance reaction also correlated well with water content.

The antisickling effect of cetiedil, alpha-cyclohexyl-3-thiopheneacetic acid 2-(hexahydro-1H-azepin-1-yl)-ethyl ester, has been investigated. The concentration of cetiedil required for 50% inhibition of erythrocyte sickling was about 140 micro M. An antisickling effect was achieved regardless of whether cetiedil was added before or after deoxygenation. The minimal gelling concentration of deoxy Hb S was increased by less than 10% in the presence of 218 micro M cetiedil. The oxygen equilibrium curves of Hb S were not significantly affected by cetiedil. Erythrocytes treated with high concentrations of cetiedil were swollen and became spheroidal. The antisickling effects of cetiedil might be due to an effect on erythrocyte membranes.

Release of Ca2+ from a heavy fraction of rabbit skeletal muscle sarcoplasmic reticulum was triggered by several different methods: (a) increasing extravesicular [Ca2+] [( CaO2+] from about 0.1 microM to 10 microM), (b), adding caffeine, (c) adding quercetin, and (d) substituting a solution containing equimolar choline+ for K+-containing solution (depolarization-induced Ca2+ release). The maximal rate of Ca2+ release triggered by caffeine or quercetin in the presence of 12.5 microM [CaO2+] (21-25 nmol of Ca2+/mg/s) is similar to that of the depolarization-induced Ca2+ release (19 nmol of Ca2+/mg/s), as determined by stopped flow spectrometry of changes in [CaO2+] with arsenazo III. The release is transient and all of the released Ca2+ is reaccumulated. The rates of Ca2+ release triggered by caffeine, quercetin, or membrane depolarization sharply decrease at high [CaO2+], suggesting a negative feedback effect of the released Ca2+. Inhibition of the release pathway allows the sarcoplasmic reticulum to reaccumulate Ca2+. The rate of Ca2+ release triggered by caffeine or quercetin, but not that triggered by membrane depolarization, is also reduced upon decreasing [CaO2+] to the submicromolar range. Passive efflux of intravesicular Ca2+ in solutions containing lower [CaO2+] in the absence of Mg.ATP is attenuated at about the same time (congruent to 1 min) regardless of the amounts of Ca2+ released, indicating that the opened Ca2+ channels close spontaneously. These results suggest that kinetically identical channels are responsible for Ca2+ release independent of the methods of triggering and this in vitro release is consistent with the physiological mechanism both in terms of the rapidity and the reversibility of Ca2+ release.

The Chelex resin method was found to be suitable for studying drug effects on Ca2+ binding of proteins. In comparison to conventional dialysis techniques, the Chelex method has the following advantages: Ca2+-EGTA buffer is not necessary, free Ca2+ concentration as low as 10(-9) M can be determined directly, and the reaction is complete in 30 min, thus creating fewer problems with protein denaturation at elevated temperatures. Methods to cope with problems inherent to this assay, such as the excluded volume effect of the resin and protein adsorption by the resin are described. The validity of the method was confirmed by the measurements of Ca2+ binding of troponin in the presence and absence of Mg2+. Using this method, it was demonstrated that ethanol concentration as high as 25% does not influence the Ca2+ binding of troponin.

The time course of intracellular depolymerization of hemoglobin S aggregates is directly observed by using microscope laser light-scattering spectroscopy in single sickle erythrocytes upon slow reoxygenation. From the correlation functions of the light intensity scattered from a single cell, we determine the average diffusion coefficient as well as the fraction of hemoglobin aggregates that have intracellular mobility. The oxygen saturation of the hemoglobin of the same cell is measured by single-cell absorption spectrophotometry. Combining the results obtained with these techniques and information on the cellular morphology, we propose a model for the depolymerization process of hemoglobin inside single erythrocytes as they transform from the fully sickled to the normal biconcave shape upon reoxygenation.

Several anti-psychotic drugs were found to have an in vitro antisickling effect. A linear relationship was found between the antisickling activity and the inhibition of calmodulin stimulated phosphodiesterase activity. These membrane-interacting compounds produce a "cup-shaped" red blood cell at the concentration where they demonstrate an antisickling effect. The volume of a "cup-shaped" red cell (with chlorpromazine) does not change significantly from that of an untreated control red cell. The anti-sickling mechanism of these membrane-interacting compounds is not known at this point.

Dichloroisoproterenol (DCI) and propranolol were found to inhibit sickling in vitro when they were added to red-cell suspensions prior to deoxygenation. The effectiveness was maximal between [Formula: see text]'s of 30 and 40 mmHg (1 mmHg = 133.322 Pa). When cells were sickled at a low oxygen tension [Formula: see text], and then DCI was added later, the drug decreased the degree of sickling while the suspension was maintained at the same oxygen tension. The antisickling effect of these drugs was not antagonized by isoproterenol, a β-adrenergic stimulator, by the addition of cAMP or increase of the intracellular calcium concentration. Other β-blockers, such as MJ1999 (sotalol) and timolol, did not show antisickling activity. It was also found that DCI, propranolol, and timolol had some effect on the delay time of gelation of sickle-cell hemoglobin (Hb S), as well as on the oxygen affinity of sickle cells.

General anesthetic agents can be divided on the basis of whether or not they "sensitize" the heart to the arrhythmogenic actions of catecholamines. There appear to be two separate means by which the catecholamines cause ventricular arrhythmias during anesthesia: one is related to a reduction in supraventricular driving rate caused both directly by the anesthetic and reflexly in response to the pressor effect of catecholamine injection; the other action (favoring ventricular fibrillation) is caused by direct anesthetic depression of the intraventricular conducting system. Of the anesthetics used today, halothane has the most pronounced cardiac sensitizing action. The same anesthetics can also be divided according to whether or not they stimulate sympathetic nervous system activity. Those that do (e.g., diethyl ether) abolish the barostatic reflexes. Those that do not (e.g. halothane) reset the barostatic reflexes to favor a reduced level of arterial pressure; they also tend to cause a reduction in sympathetic nervous activity efferent to the heart. Parasympathetic nervous actions are relatively insignificant. All general anesthetics cause myocardial depression at all concentrations that are clinically useful. The mechanism appears to involve a reduction in the Ca2+ available to the contractile elements. Exactly how anesthetics do this is unclear, but these drugs appear to decrease both the Ca2+ influx across the plasma membrane and the Ca2+ content of the sarcoplasmic reticulum.

We have developed two rheological methods for use in the study of the effect of anti-sickling drugs on in vitro sickling: (a) An automated instrument which measures the relationship between the oxygen partial pressure and blood viscosity, and (b) an instrument to measure the relationship between filtrability (through a 3 mu-pore Nuclepore filter) and the oxygen partial pressure. With these instruments, we found that both viscosity and filtration pressure of a sickle-cell suspension increased even at oxygen saturation values above 90%, levels at which morphological changes of sickle cells are not yet pronounced. The effect of anti-sickling drugs was demonstrated by a decrease of the filtration pressure at low oxygen saturation.

The effects of chronic ethanol ingestion on the rat kidney were studied. Rats were fed a liquid diet containing ethanol for 5 weeks to induce chronic alcoholism. Renal ischemia was introduced by clamping the renal artery and vein either for 10 or 20 min. The glomerular filtration rate (GFR) and the renal blood flow (RBF) were determined by using I125-iothalamate and I131-iodohippurate. In the absence of renal ischemia, there were no significant differences in the renal function between nonalcoholic rats (n = 5) and alcoholic rats (n = 5): 380 +/- 30 vs. 403 +/- 27 microliters/min/100 g body weight (BW) in GFR, and 3.1 +/- 0.1 vs. 3.1 +/- 0.2 ml/min/100 g BW in RBF. The recovery of GFR measured 2 h following 10-min renal ischemia in both groups was not significantly different; the values returned to 340 +/- 40 microliters/min/100 g BW (nonalcoholic rats) and 246 +/- 22 microliters/min/100 g BW (alcoholic rats), respectively. The changes of RBF following 10 min ischemia were also similar in both groups. However, the effects of alcoholism on the renal function became apparent when animals were subjected to more prolonged renal ischemia. In nonalcoholic rats (n = 5), GFR and RBF measured 2 h following 20 min renal ischemia were 245 +/- 51 microliters/min/100 g BW and 2.5 +/- 0.4 ml/min/100 g BW, whereas in alcoholic rats (n = 5) the GFR and RBF were significantly decreased to 93 +/- 15 microliters/min/100 g BW and 1.1 +/- 0.2 ml/min/100 g BW, respectively (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Release of Ca2+ from a heavy fraction of rabbit skeletal muscle sarcoplasmic reticulum was triggered by several different methods: (a) increasing extravesicular [Ca2+] [( CaO2+] from about 0.1 microM to 10 microM), (b), adding caffeine, (c) adding quercetin, and (d) substituting a solution containing equimolar choline+ for K+-containing solution (depolarization-induced Ca2+ release). The maximal rate of Ca2+ release triggered by caffeine or quercetin in the presence of 12.5 microM [CaO2+] (21-25 nmol of Ca2+/mg/s) is similar to that of the depolarization-induced Ca2+ release (19 nmol of Ca2+/mg/s), as determined by stopped flow spectrometry of changes in [CaO2+] with arsenazo III. The release is transient and all of the released Ca2+ is reaccumulated. The rates of Ca2+ release triggered by caffeine, quercetin, or membrane depolarization sharply decrease at high [CaO2+], suggesting a negative feedback effect of the released Ca2+. Inhibition of the release pathway allows the sarcoplasmic reticulum to reaccumulate Ca2+. The rate of Ca2+ release triggered by caffeine or quercetin, but not that triggered by membrane depolarization, is also reduced upon decreasing [CaO2+] to the submicromolar range. Passive efflux of intravesicular Ca2+ in solutions containing lower [CaO2+] in the absence of Mg.ATP is attenuated at about the same time (congruent to 1 min) regardless of the amounts of Ca2+ released, indicating that the opened Ca2+ channels close spontaneously. These results suggest that kinetically identical channels are responsible for Ca2+ release independent of the methods of triggering and this in vitro release is consistent with the physiological mechanism both in terms of the rapidity and the reversibility of Ca2+ release.

A method was developed to study sickling in vitro under physiological conditions using a small amount of blood (0.1 mL). The diluted blood suspension (2.1 mL) was placed in a flask and flushed with a gas mixture containing 5% CO2. In deoxygenation experiments, samples were withdrawn anaerobically into a microslide (optical path 0.1 mm) and red cell morphology was studied directly under a light microscope after both ends of the microslide were sealed. The blood suspension with a hematocrit value of 1% can be deoxygenated in less than 10 min, but it takes 30 min for the sickling of cells to reach a plateau. The degree of sickling increases with increasing osmolality of the medium, or with a decrease of the pH. With a citrate–phosphate–dextrose–adenine solution, sickle blood may be stored for this type of study for about 10 days at 4 °C. The blood may be stored for about 5 days with a noncitrate preservative. This method was found useful in examining the antisickling activity of various drugs.

Two prostaglandin oligomeric compounds, an acid-form compound and an ester-form compound, were synthesized from alprostadil (prostaglandin E1). They were found to provide significant protection to the rat spinal cord against contusion injury. After laminectomy at the T-11 segment of the spinal cord, a weight drop (10 g x 5 cm) caused a "dynamic" injury. The degree of recovery was estimated by several neurologic deficit indices; the Tarlov score, inclined plane test and hot plate test. In the control group (no drug), animals were still paralyzed 4 weeks after injury (Tarlov score 1 to 2). By administering these prostaglandin oligomers, either pre-injury (30 min before injury; one dose of 6 mg/kg i.p.) or post-injury (3 doses, each of 6 mg/kg i.p. at 30 min, 6 h, and 12 h after injury), the Tarlov scores recovered to 3.5 to 4.5 by 4 weeks, and animals were able to either support body weight or to walk with a slight deficit. Although both acid- and ester-forms of the compound demonstrated efficacy, the ester-form provided greater protection to the spinal cord. Other neurologic deficit indices also supported these observations.