Michio Yajima

Protective role of nitric oxide synthase against ischemia-reperfusion injury in guinea pig myocardial mitochondria.

In guinea-pig myocardial mitochondria preparation, lowering the Ca2+ concentration or pH level in the perfusate rapidly elevated the fura-2 Ca2+ signal ([Ca2+]m). Pretreatment with 10(-4) M L-Arg inhibited the rapid [Ca2+]m influx, whereas administration of 10(-4) M L-NAME did not, suggesting some association between nitric oxide (NO*) synthase (NOS) activation and Ca2+ kinetics in mitochondria. Immunoblotting analysis showed that endothelial (e)-NOS was present in mitochondria, but not inducible (i)-NOS or brain (b)-NOS. Electron microscopy observations revealed that the e-NOS antibody-reactive site in the mitochondria was the inner cristae. The production of reactive oxygen species and NO* in isolated mitochondria was detected by the spin trapping technique with electron paramagnetic resonance (EPR) spectrometry. Pretreatment with 10(-5) M S-nitroso-N-acetyl-DL-penicillamine (SNAP) and 10(-5) M 3-[2-Hydroxy-1-(1-methylethyl)-2-nitrosohydrazino]-1-propananin e (NOC 5), which spontaneously generate NO*, completely inhibited the [Ca2+]m uptake. In addition, N-morpholino sydnonimine hydrochloride (SIN-1) (10(-5) M), which simultaneously generates NO* as well as *O2- and peroxynitrite anion (ONOO-), inhibited the increase in [Ca2+]m. ONOO- (3 x 10(-4) M) itself also inhibited this increase. Pretreatment with the *O2(-)-scavenger manganese superoxide dismutase or catalase (200 units/ml) completely inhibited the increase in [Ca2+]m caused by lowering of either the Ca2+ concentration or the pH in the perfusate. These results suggested that the formation of reactive oxygen species promoted the [Ca2+]m influx. The agents that inhibited the [Ca2+]m influx improved contractility even in Langendorff preparations after ischemia. Based on these findings, we concluded that e-NOS exists in mitochondria and that NO* may play an important protective role in reperfusion cardiac injury after ischemia, by inhibiting the Ca2+ influx into mitochondria which are otherwise damaged by *O2-.

Protective effects of antioxidative serotonin derivatives isolated from safflower against postischemic myocardial dysfunction

N-(p-Coumaroyl)serotonin (C) and N-feruroylserotonin (F) with antioxidative activity are present in safflower oil. The protective effects of C and F were investigated in perfused guinea-pig Langendorff hearts subjected to ischemia and reperfusion. Changes in cellular levels of high phosphorous energy, NO and Ca2+ in the heart together with simultaneous recordings of left ventricular developed pressure (LVDP) were monitored by an nitric oxide (NO) electrode, fluorometry and 31P-NMR. The rate of recovery of LVDP from ischemia by reperfusion was 30.8% in the control, while in the presence of C or F a gradual increase to 63.2 or 61.0% was observed. Changes of transient NO signals (TNO) released from heart tissue in one contraction (LVDP) were observed to be upside-down with respect to transient fura-2-Ca2+ signals (TCa) and transient O2 signals detected with a pO2 electrode. At the final stage of ischemia, the intracellular concentration of Ca2+ ([Ca2+]i) and the release of NO increased with no twitching and remained at a high steady level. The addition of C increased the NO level at the end of ischemia compared with the control, but [Ca2+]i during ischemia decreased. On reperfusion, the increased diastolic level of TCa and TNO returned rapidly to the control level with the recovery of LVDP. By in vitro EPR, C and F were found to directly quench the activity of active radicals. Therefore, it is concluded that the antioxidant effects of two derivatives isolated from safflower play an important role in ischemia-reperfusion hearts in close relation with NO.

Distinct involvement of NF-κB and p38 mitogen-activated protein kinase pathways in serum deprivation-mediated stimulation of inducible nitric oxide synthase and its inhibition by 4-hydroxynonenal

Cytokine‐induced expression of inducible nitric oxide synthase (iNOS) and concomitant production of nitric oxide (NO) involve activation of mitogen‐activated protein (MAP) kinases and are in most cases mediated by the transcription factor NF‐κB. We investigated the role of p38 MAP kinase activation and IκB phosphorylation in iNOS expression in a novel iNOS‐inducing model in mouse macrophages. Deprivation of serum from the culture medium of RAW 264.7 cells up‐regulated iNOS and NO production, which were inhibited by 4‐hydroxy‐2‐nonenal (HNE), a component of oxidatively modified low‐density lipoprotein (oxLDL). Serum withdrawal induced phosphorylation of Akt, IκB, and p38 MAP kinase. Pretreatment with the potent PI3 kinase inhibitor wortmannin, the NF‐κB inhibitor PDTC or the specific p38 MAP kinase inhibitor SB203580 each partially attenuated the induction of iNOS and NO production, demonstrating that both p38 activation and IκB phosphorylation are required for iNOS expression. SB203580, however, did not prevent the phosphorylation of Akt and IκB, suggesting that the p38 MAP kinase signal contributes to iNOS gene expression through an IκB‐phosphorylation‐independent pathway. HNE, which markedly inhibited iNOS expression and NO production, prevented the serum withdrawal‐triggered IκB phosphorylation but not that of Akt or p38 MAP kinase. A high concentration of HNE stimulated dephosphorylation of IκB but promoted activation of p38 MAP kinase. Taken together, these results suggest that NF‐κB and p38 MAP kinase lie in separate signal pathways for serum deprivation‐stimulated iNOS expression and NO production. HNE selectively suppresses the former pathway, targeting a site downstream of Akt. J. Cell. Biochem. 83: 271–280, 2001.

A role of anti-verotoxin antibody immunoreactive peptide, Virp5, from rat spinal cord

An anti-verotoxin 2 (VT2) antibody immunoreactive 5-kDa polypeptide (Virp5), has been obtained through screening of the rat spinal cord cDNA library with the aid of anti-VT2 antibody. Virp5 was mainly expressed in the central nervous system, liver and kidney, and localized at glia-like cells and nerve fibers in the central nervous system, vascular endothelial cells and hepatic cells in the liver, as well as epithelial cells of distal tubules in the kidney. Intravenous administration of purified Virp5 elicited a dose-dependent increase in blood pressure. These results suggest that Virp5 commonly exists in the body, being partly playing a role in regulating the blood pressure.