Shinsuke Katoh

Hyperoxia induces the neuronal differentiated phenotype of PC12 cells via a sustained activity of mitogen-activated protein kinase induced by Bcl-2.

We previously reported that rat pheochromocytoma PC12 cells express the neuronal differentiated phenotype under hyperoxia through the production of reactive oxygen species (ROS). In the present study, we found that in this phenotype, Bcl-2, an apoptosis inhibitor, affects mitogen-activated protein (MAP)-kinase activity, which is known as a key enzyme of the signal-transduction cascade for differentiation. When PC12 cells were cultured under hyperoxia, a rapid increase in MAP-kinase activity, including that of both p42 and p44, was observed. Although the activity level then decreased quickly, activity higher than the control level was observed for 48 h. PD98059, an inhibitor of MAP kinase, suppressed the hyperoxia-induced neurite extensions, suggesting the involvement of MAP-kinase activity in the mechanism of differentiation induced by ROS. An elevation of Bcl-2 expression was observed after culturing PC12 cells for 24 h under hyperoxia. This Bcl-2 elevation was not affected by treatment with PD98059, suggesting that it did not directly induce neurite extension under hyperoxia. However, the blockade of the Bcl-2 elevation by an antisense oligonucleotide inhibited the sustained MAP-kinase activity and neurite extensions under hyperoxia. Further, in PC12 cells highly expressing Bcl-2, the sustained MAP-kinase activity and neurite extensions under hyperoxia were enhanced. These results suggested that MAP kinase is activated through the production of ROS, and the subsequent elevation of Bcl-2 expression sustains the MAP-kinase activity, resulting in the induction of the neuronal-differentiation phenotype of PC12 cells under hyperoxia.

The rescuing effect of nerve growth factor is the result of up-regulation of bcl-2 in hyperoxia-induced apoptosis of a subclone of pheochromocytoma cells, PC12h.

The rat pheochromocytoma cell line PC12 is useful for studying neuronal cell differentiation since this cell line differentiates into neuron-like cells in response to nerve growth factor (NGF). We demonstrated that PC12h cells, a subclone of PC12 cells, died under hyperoxia (50% O2). This cell death did not occur in the presence of antioxidant reagents. In the dead cells, DNA fragmentation and chromatin condensation were observed, suggesting that hyperoxia-induced apoptosis via reactive oxygen species (ROS). NGF effectively suppressed this hyperoxia-induced apoptosis. Accordingly, the amounts of bcl-2, a proto-oncogene product, increased in the cells rescued from apoptosis by NGF. Furthermore, bcl-2 antisense oligonucleotide canceled this rescuing effect of NGF. The present findings indicate that NGF rescues PC12h cells from hyperoxia-induced apoptosis via up-regulation of bcl-2.

Endothelin-1 stimulates cardiomyocyte injury during mitochondrial dysfunction in culture

To understand the pathophysiological role of endothelin-1 in the failing heart, we constructed a cellular mitochondrial impairment model and demonstrated the effect of endothelin-1. Primary cultured cardiomyocytes from neonatal rats were pretreated with rotenone, a mitochondrial complex I inhibitor, and the cytotoxic effect of endothelin-1 on the cardiomyocytes was demonstrated. Rotenone gradually decreased the pH of the culture medium with incubation time and caused slight cell injury. Endothelin-1 markedly enhanced the effect of rotenone that decreased the pH of the medium and enhanced cellular injury. The enhancement of the decrease in pH and cell injury induced by endothelin-1 was counteracted by the endothelin ET(A) receptor antagonist BQ123 or by maintaining the pH of the medium by the addition of 50 mM HEPES. Endothelin-1 markedly increased the uptake of 2-deoxyglucose and lactic acid production when the cardiomyocytes were pretreated with rotenone. These findings suggest that the stimulation of glucose uptake and anaerobic glycolysis followed by the increase in lactic acid accumulation in cardiomyocytes under the condition of mitochondrial impairment may be involved, at least in part, in the cellular injury by endothelin-1. Moreover, these findings suggest the possibility that the effect of endothelin-1 on myocardium is reversed by the condition of the mitochondria, and endogenous endothelin-1 may deteriorate cardiac failure with mitochondrial dysfunction. This may contribute to clarify the beneficial effect of endothelin receptor blockade in improving heart failures.

Norepinephrine modulates the zonally different hepatocyte proliferation through the regulation of transglutaminase activity

A neurotransmitter, norepinephrine (NE), amplifies the mitogenic effect of epidermal growth factor (EGF) in the liver by acting on the alpha(1)-adrenergic receptor coupled with G protein, Galpha(h). However, the molecular mechanism is not well understood. Galpha(h) is known as a transglutaminase 2 (TG2), a cross-linking enzyme implicated in hepatocyte proliferation. We investigated the effect of NE on EGF-induced cell proliferation and TG2 activity using hepatocytes isolated in periportal and perivenous regions of the liver, which differ in proliferative capacity. Periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) were isolated by the digitonin-collagenase perfusion technique. EGF or NE receptor binding was analyzed by Scatchard analysis. Changes in NE-induced DNA synthesis, EGF receptor (EGFR) dimerization and phosphorylation, and TG2 activity were measured. NE enhanced EGF-induced DNA synthesis, EGF-induced EGFR dimerization, and its phosphorylation in PVH but not in PPH. [(3)H]NE binding studies indicated that PVH was found to have a greater affinity and number of receptors than PPH. Furthermore, NE treatment decreased TG2 activity and increased phospholipase C activity in PVH although TG2 level showed no change. These results suggest that NE-induced amplification of EGF-induced DNA synthesis especially in PVH is caused by upregulation of EGFR activation through the switching of function from TG2 to Galpha(h).

Studies on 67Ga uptake by mouse granuloma tissues.

In connection with the uptake of 67Ga into the inflammatory tissues, such as granuloma tissues produced with turpentine oil, the influence of Fe3+ on the uptake of 67Ga into mouse granuloma and normal tissues and on the uptake of 125I-labeled transferrin and 59Fe were investigated. Fe3+ decreased the uptake of 67Ga into the liver and spleen, but had no influence on the uptake of 67Ga into the granuloma tissues. The uptake patterns of 125I-labeled transferrin and 59Fe in the granuloma tissues were not consistent with that of 67Ga at all. These results show that the uptake of 67Ga into the granuloma tissues occurs in a free, transferrin-unbound form, but into the liver and spleen in a transferrin-bound form.

67Ga in transferrin-unbound form is taken up by inflamed liver of mouse treated with CCl4

In order to investigate whether or not transferrin is involved in the uptake of67Ga by inflamed liver (acute inflammatory tissues) the uptake of67Ga by the liver of mice treated with carbon tetrachloride (CCl4) was studied. The serum GPT value reached its maximum on the 1st day after the CCl4 treatment. The uptake of67Ga by the liver also reached its maximum on the 1 st day after the CCl4 treatment and the amount uptaken into inflamed liver was about 6 times that uptaken into normal liver. On the other hand, the uptake of125I-transferrin into inflamed liver on the 1st day after CCl4 treatment was only about 1.6 times that into normal liver. Moreover, cold Fe3+ decreased the uptake of67Ga by normal liver but increased the uptake of67Ga by inflamed liver. These results show that transferrin plays an important role in the uptake of67Ga by normal liver but not by inflamed liver, i.e.67Ga in the transferrin-unbound form is preferentially taken up by inflamed liver.

Application of67Ga for the estimation of reticulocyte production

In order to estimate the production of reticulocytes, which have a larger number of transferrin receptors than erythrocytes, we used67Ga which is exclusively bound to transferrin in the blood. The pattern of uptake of67Ga by reticulocytes was quite similar to the time course of transglutaminase activity which might be involved in receptor-mediated endocytosis. The preinjection of Fe3+ decreased the uptake of67Ga by reticulocytes. These results suggested that67Ga in a transferrin-bound form was taken up by reticulocytes via receptor-mediated endocytosis. It was showed that the application of67Ga is very easy and useful for the estimation of reticulocyte production.

111In (III) uptake by inflammatory and normal tissues

Tissue distributions of111In (III) in the rats bearing granuloma, inflammatory tissue induced by turpentine oil, were compared with those of67Ga. The results showed that indium-111 resembles67Ga in the manner of uptake by inflammatory and normal soft tissues. The effect of cold-InCl3 on111In (III) uptake showed that transferrin is not involved in the uptake of111In (III) into inflammatory tissues but is involved in the uptake into liver and spleen.