Yasutaka Ohta

Remnants of chylomicron and very low density lipoprotein impair endothelium-dependent vasorelaxation

Remnants of chylomicron and very low density lipoprotein (VLDL) have been implicated as potentially atherogenic. Since endothelial dysfunction is an early event in atherosclerosis, we examined effects of the remnants on endothelium-dependent vasorelaxation. The remnant lipoproteins were isolated from postprandial plasma in hyperlipidemic subjects using the immunoaffinity gel mixture of anti apo A-1 and anti apo B-100 monoclonal antibodies and ultracentrifugation. Rabbit aortic strips suspended in the organ chambers were incubated for 2 h with the preparations of lipoproteins and lipids. After incubation, the strips were tested with vasodilators after precontraction with phenylephrine (1 microM). The remnant lipoproteins (750-1500 microg triglyceride/ml) but not VLDL fraction (up to 1500 microg triglyceride/ml) impaired vasorelaxation in responses to acetylcholine, substance P and A23187. Carbamylated or methylated remnant lipoproteins, chemically modified remnant lipoproteins, had comparable impairment of the vasorelaxation as unmodified remnant lipoproteins. Incubation with lipid extracts from the remnant lipoproteins also exerted an inhibitory effect on the vasorelaxation. Relaxation to sodium nitroprusside was fully preserved in all aortas exposed to the lipoprotein preparations. Thus, the remnant lipoproteins impair endothelium-dependent arterial relaxation at the concentrations observed in the plasma in patients with coronary artery disease (500-2000 microg triglyceride of remnant lipoprotein/ml). The impairment may be in apoprotein receptor-independent manner, and the lipids in the remnants seem to contribute to the inhibitory effect. The endothelial dysfunction caused by the remnant lipoproteins may play a role in the high prevalence of atherosclerotic coronary artery disease in postprandial hyperlipidemic patients.

Membrane Active Lipids in Remnant Lipoproteins Cause Impairment of Endothelium-Dependent Vasorelaxation

We have recently found that remnant lipoproteins (RLPs) and their lipid fractions impair endothelium-dependent vasorelaxation (EDR). This study was aimed at clarifying mechanisms responsible for RLP-induced endothelial dysfunction in isolated rabbit aortas. RLPs were isolated from plasma in hyperlipidemic subjects by use of the immunoaffinity gel mixture of anti-ApoA1 and anti-ApoB100 monoclonal antibodies and ultracentrifugation. Organ chamber experiments showed that EDR impairment was restored by addition of reduced glutathione (GSH) or N-acetylcysteine, antioxidants, into the incubation buffer containing isolated rabbit aortas and RLPs (0.75 mg of triglyceride/mL). Furthermore, the incubation of isolated human red blood cells (RBCs) with RLP and its lipids converted the normal shape of RBCs to echinocytes, but coincubation with antioxidants suppressed the RLP-induced RBC transformation, suggesting that they exerted oxidative damage on RBC surface membranes. Studies with HPLC and the postcolumn chemiluminescence method showed that RLPs contain a substantial amount of phosphatidylcholine hydroperoxides. Peroxidized phosphatidylcholine also impaired EDR and had echinocytogenic action, both of which were suppressed by N-acetylcysteine. RLPs isolated from the plasma of patients under treatment with alpha-tocopherol, an antioxidant, had a lower level of phosphatidylcholine hydroperoxides (15% of the amount in nontreated patients), which was associated with a lack of the inhibitory action on EDR and with lesser effect on RBC transformation. Oxidative damage caused by lipid components in RLPs, especially peroxidized phospholipids, deteriorates cell surface membrane and may be at least partly responsible for RLP-induced impairment of EDR.

Suppression of Atherosclerotic Changes in Cholesterol-Fed Rabbits Treated With an Oral Inhibitor of Neutral Endopeptidase 24.11 (EC 3.4.24.11)

Neutral endopeptidase 24.11 (NEP), widely distributed in the body, hydrolyzes and inactivates a number of endogenous vasoactive peptides, some of which could alter various functions of cells present in the arterial wall. Recently NEP has been found to exist in the vascular endothelium. The aim of this study was to assess the influence of chronic NEP inhibition by daily administration of UK79300 (candoxatril), an orally active NEP inhibitor (NEPI), on the development of atherosclerotic changes in high-cholesterol-fed rabbits. Male New Zealand White rabbits were fed for 8 weeks as follows: normal rabbit diet (Normal, n = 15), 1.5% cholesterol diet (Cholesterol, n = 15), or 1.5% cholesterol diet containing NEPI (20 mg.kg-1.d-1) (Cholesterol+NEPI, n = 15). At the end of the dietary period, NEPI treatment was found to suppress the surface area of the aorta covered by plaques (% surface area: Cholesterol, 59 +/- 6 versus Cholesterol+NEPI, 36 +/- 7, P < .01) and decreased contents of cholesterol and cholesterol esters in the aortas. NEPI also reduced plasma total cholesterol by 27% of Cholesterol rabbits (1781 +/- 130 mg/dL). The endothelial function, estimated by the endothelium-dependent relaxation of the isolated aortas in response to acetylcholine, was preserved in Cholesterol+NEPI rabbits compared with that in Cholesterol rabbits. NEP enzymatic activities in plasma and the particulate fraction of the homogenates from the aortas in Cholesterol rabbits were both increased, 3.1- and 3.9-fold, respectively, above those in Normal rabbits, but the activities in Cholesterol+NEPI rabbits were significantly lower than those in Cholesterol rabbits. UK73967, an active form of UK79300, or phosphoramidon partly reversed the atherosclerotic impairment of relaxation of the isolated thoracic aortic rings from Cholesterol rabbits in response to exogenous additions of C-type natriuretic peptide (CNP) and substance P, which are NEP substrates known to exist endogenously in the vascular endothelium. The results suggest that the increased NEP activity plays a significant role in atherogenesis, and NEPIs might be therapeutically useful in the prevention of atherosclerosis. Reduction of plasma cholesterol and suppression of degradations in the arteries of endogenously released CNP, substance P, or possibly other kinins known to have anti-atherosclerotic actions may at least partially contribute to the inhibitory effects of NEPIs on atherosclerotic changes.

Ca2+calmodulin-dependent protein kinase II immunoreactivity in the rat hippocampus after forebrain ischemia

The influence of transient forebrain ischemia on the temporal alteration of Ca2+/calmodulin-dependent kinase II (CaM kinase II) in the rat hippocampus was analysed by the immunohistochemical method using antigen-affinity purified polyclonal antibodies against CaM kinase II of rat brain. Six to twenty-four hours after ischemia, CA1 and CA3 pyramidal cells, and dentate granule cells lost CaM kinase II immunoreactivity in neuronal perikarya, although immunoreactivity in the dendritic fields was preserved. The recovery of immunoreactivity of the CA3 pyramidal cells and dentate granule cells was noted 3 days after recirculation. Seven days after ischemia, immunoreactivity in the CA1 subfield was greatly reduced. These results suggest that CaM kinase II molecules in the CA1 subfield are preferentially located on the CA1 pyramidal cells and that CaM kinase II plays a critical role in the reconstruction of neuronal cytoskeleton and neuronal networks damaged by ischemic insult.

Fetal Calf Serum: Eliciting Phosphorylation of Ca++/Calmodulin-Dependent Protein Kinase II in Cultured Rat Granulosa Cells

It is well known that a number of factors affect the proliferation, differentiation, and steroidogenesis of cultured rat granulosa cells. Two multifunctional protein kinases, such as cAMP-dependent protein kinase and protein kinase C, are thought to mediate intracellular signals in granulosa cells. Follicle stimulating hormone (FSH) induces granulosa cell differentiation and the steroidogenesis, which are mediated by the action of cAMP-dependent protein kinase (1–3). In contrast, phorbol ester inhibits FSH actions, such as cAMP formation, LH receptor expression, cholesterol side-chain cleavage enzyme synthesis, and progesterone production (4–7) via the activation of protein kinase C.