Hemin treatment abrogates monocrotaline-induced pulmonary hypertension.

Treatment of rats with monocrotaline (MCT), a pyrrolizidine alkaloid plant toxin, is known to cause pulmonary hypertension (PH), and it has been used as a useful experimental model of PH. Recent findings suggested that pulmonary inflammation may play a significant role in the pathogenesis of MCT-induced PH. We also demonstrated that, following MCT administration to rats, there was a significant and sustained increase in the pulmonary expression of heme oxygenase-1 (HO-1), which is known to be induced by various oxidative stresses, including inflammation and free heme, and is thought to be essential in the protection against oxidative tissue injuries. In this study, we administered hemin (ferriprotoporphyrin chloride, 30 micromol/kg b.w., subcutaneously), a potent inducer of HO-1, every 3 days to rats following subcutaneous administration of MCT (60 mg/kg) and examined its effect on MCT-induced PH and pulmonary inflammation. MCT administration caused pulmonary arterial wall thickening with marked elevation of right ventricular pressure, in association with prominent pulmonary inflammation as revealed by the increase in gene expression of tumor necrosis factor-alpha and the number of infiltrated neutrophils in the lung. In contrast, hemin treatment of MCT-administered animals, which led to a further increase in pulmonary HO-1 mRNA expression, significantly ameliorated MCT-induced PH as well as tissue inflammation. These findings suggest that hemin treatment ameliorates MCT-induced PH possibly mediated through induction of pulmonary HO-1 which leads to the attenuation of pulmonary inflammation.

Site- and cell-type- specific induction of intestinal inducible nitric oxide synthase in a rat model of endotoxemia.

The intestine is one of the major organs that are involved in sepsis. The inducible isoform of nitric oxide synthase (iNOS) is known to play a critical role in the pathogenesis of septic tissue injury by generating excess amount of nitric oxide (NO) in response to cytokines and endotoxin. In this study, we examined changes in gene expression of iNOS in various regions of the intestine as well as the distribution of iNOS protein in the intestinal cells in a rat model of endotoxemia produced by intraperitoneal injection of lipopolysaccharide (LPS; 10 mg/kg). While iNOS mRNA was undetectable in the intestine of untreated control animals, it underwent marked induction following LPS treatment. Induction of iNOS mRNA in the ileum was marked and biphasic, while it was also marked but monophasic in the jejunum. The induction of iNOS mRNA was maximal in the ileum. The administration of interleukin-6 (IL-6) upregulated intestinal iNOS gene expression specifically in the ileum. Consistent with enhanced iNOS gene expression, iNOS protein was markedly expressed in the ileum after LPS treatment, exclusively in the mucosal epithelium both at crypt and villus cells, although more prominently in the former. These findings suggested that intestinal iNOS expression was upregulated both at transcriptional and protein levels not only in a site-specific, but also in a cell type-specific manner in a rat model of endotoxemia, possibly through increasing serum IL-6 levels. Differential regulation of iNOS expression along the longitudinal and crypt-villus axes of the gut might be a determinant of the pattern of sepsis-induced intestinal damage.

Increased cytotoxicity of carbon tetrachloride in a human hepatoma cell line overexpressing cytochrome P450 2E1.

Cytotoxic free radicals generated during the metabolism of carbon tetrachloride by cytochrome P450 2E1 (CYP2E1) are thought to cause hepatotoxicity. Here, the cytotoxic effects of carbon tetrachloride in a liver cell line expressing CYP2E1 (HLE/2E1) are compared with those in the mother cell line (HLE). The effects of carbon tetrachloride on the gene expression of HSP70, a potential marker of oxidative stress, were also examined. The viability of HLE/2E1 cells after exposure to carbon tetrachloride was significantly decreased compared with that of HLE cells. Northern blot analysis revealed that the HSP70 mRNA level was significantly increased after carbon tetrachloride treatment in both cell lines, while the magnitude of its increase was much greater in HLE/2E1 cells than in HLE cells. These results suggest that the oxidative stress induced by CYP2E1 plays an important role in the increase in cytotoxicity of carbon tetrachloride in CYP2E1-overexpressing cells.

Increased heme oxygenase-1 gene expression in the livers of patients with portal hypertension due to severe hepatic cirrhosis.

Surgical bleeding associated with splanchnic hyperaemia due to portal hypertension complicates the anaesthetic management of hepatic transplantation. Although the mechanism(s) of portal hypertension are not fully understood, carbon monoxide, a product of the heme oxygenase (HO) reaction, is thought to be one of the endogenous vasodilators in the liver. In this study, the expression of mRNA encoding inducible HO isozyme (HO-1) in the livers of patients with portal hypertension undergoing hepatic transplantation was determined in comparison with those without portal hypertension. HO-1 mRNA levels were significantly greater in the portal hypertension group than in the group without portal hypertension. In contrast with HO-1, the gene expression of non-specific delta-amino-levulinate synthase (ALAS-N), which is down-regulated by heme in the liver, was the same in both groups. These results suggest that HO-1 is up-regulated through heme-independent stimuli according to the development of portal hypertension, and that induced HO-1 plays a pathophysiological role in portal hypertension through carbon monoxide production.

Induction of renal metallothionein in rats with ischemic renal failure.

Metallothionein (MT) is induced by various types of oxidative stress. However, whether or not MT is induced in renal ischemia/reperfusion injury, in which oxidative stress is believed to play a major role, remains unknown. The present study investigated MT expression in the kidneys of rats with ischmic acute renal failure (IARF). Rats were subjected to 60 min of bilateral renal ischemia followed by reperfusion. Renal MT mRNA expression was then analyzed by Northern blotting. MT expression in ischemic kidney was also localized by in situ hybridization and immunohistochemistry. Renal MT mRNA expression, which was barely detectable in the sham-operated control kidney, increased significantly at 3 h afer reperfsion, continued to increase to a maximal level at 24 h that was maintained for 48 h. The level of MT mRNA expression returned to that of the control by day 4. A morphological study revealed that MT was expressed exclusively in the renal tubular epithelial cells, which are the targets of ischemia/reperfusion injury, and that MT predominated in the outer medulla in the IARF rat kidney at transcriptional and translational levels. These results suggest that MT induced in the IARF rat kidney plays an important role in protecting renal cells against oxidative stress induced by ischemia/reperfusion.

Plasma laudanosine levels in patients given atracurium during liver transplantation.

Atracurium, a nondepolarizing muscle relaxant, does not depend on the liver for clearance, but its principal metabolite, laudanosine, is eliminated primarily by the liver and is potentially neurotoxic. We measured atracurium and laudanosine levels in 15 adult patients during the three stages of liver transplantation to assess the effect of major impairment of liver function. Atracurium was given in a bolus dose of 0.5 mg/kg followed by continuous infusion at a rate adjusted to maintain 95-99% of total neuromuscular block, as judged by train of four response to facial nerve stimulation. Atracurium levels remained constant at 1.4-1.8 micrograms/mL during the 180-min preanhepatic and 75-min anhepatic stages but decreased to a mean of 1.0 microgram/mL by the end of the 180-min postanhepatic stage. In contrast, laudanosine levels increased during each stage, being 0.40 +/- 0.18, 0.50 +/- 0.22, and 0.43 +/- 0.16 micrograms/mL after the preanhepatic, anhepatic, and postanhepatic stages, respectively. The highest individual value recorded was 1.02 microgram/mL. We conclude that, despite increases in laudanosine levels during each stage of liver transplantation in patients receiving atracurium, those levels are only about one-tenth of the maximum values previously reported in humans.

[The effect of pressure support ventilation on breathing patterns and the work of breathing].

We assessed breathing patterns during pressure support ventilation (PSV) and its relationship with the work of breathing in 10 postoperative patients. With increasing levels of pressure support, minute ventilation and tidal volume increased with a decrease in respiratory frequency. Increased minute ventilation was achieved by increased mean inspiratory flow. Duty cycle, however, decreased with PSV. This decrease might allow the diaphragm a longer rest period between contractions, which might decrease the risk of diaphragmatic fatigue. Furthermore, PSV reduced the inspiratory work added by a ventilator to near zero. Oxygen consumption was also decreased with PSV. We conclude that PSV improved the breathing patterns and minimized the work of breathing spontaneously via a ventilator.

Induction of metallothionein synthesis in cultured cells by substances released from endotoxin-activated macrophages.

The involvement of macrophages in the induction of metallothionein (MT) synthesis by bacterial endotoxin was studied in vitro. Rat peritoneal macrophages were incubated with endotoxin. The incubation medium from endotoxin-activated macrophages accelerated MT synthesis by human hepatic Chang cells. However, the incubation medium from non-activated macrophages did not. Endotoxin added to the culture medium of Chang cells was ineffective in inducing MT synthesis. The contents of zinc, copper and cadmium, which are primary inducers of MT, in the incubation medium of macrophages in the presence of endotoxin were not different from those in the absence of endotoxin. These results suggest that MT synthesis is induced by endotoxin-treated macrophages.

Metallothionein and zinc metabolism in endotoxin shock rats.

Zinc metabolism in endotoxicosis was investigated in rats. The zinc concentration in the serum decreased, while zinc contents increased in the lung, kidney and liver. Marked increase in zinc level was observed in the liver. In the liver cells, zinc concentrations of mitochondria and cytosol increased, but not in microsome. Metallothioneins (MTs), which probably participate in zinc metabolism, were induced by endotoxin administration. Although the same level of MT-2 as that of MT-1 was induced by zinc, the level of MT-2 was 3 to 4 fold higher than that of MT-1 by administrations of endotoxin or glucocorticoid hormone. Since no metallothionein was induced directly by addition of endotoxin to the media of cultured cells, the endotoxin was found not to induce MTs directly. The effect of zinc on superoxide generation of polymorphonuclear leukocytes was also studied. Zinc was found to inhibit superoxide generation dose-dependently.