Hitoshi Okada

Phosphatidylinositol metabolism in hypertrophic rat heart.

The accumulation of inositol 1,4,5-trisphosphate (IP3) after hormonal stimulation has a physiological role, possibly by alteration of Ca2+ levels in cardiac myocyte. However, this accumulation has not been studied under pathophysiological conditions. In this report, we examine phosphatidylinositol metabolism during cellular response to norepinephrine in pressure-overloaded hypertrophic rat heart. After stimulation with norepinephrine, the accumulations of IP3 and diacylglyceride significantly increased in isolated myocytes from stroke-prone spontaneously hypertensive rat (SHRSP) heart, indicating phosphatidylinositol-specific phospholipase C activity increased in SHRSP heart cells. Protein kinase C activity was also enhanced in SHRSP, with a marked increase in particulate activity. We determined the intracellular calcium concentration and found it to be higher in SHRSP than in Wistar-Kyoto (WKY) rats at 30-40 weeks of age. Ca2+ influx was also elevated in SHRSP stimulated by norepinephrine. In SHRSP heart, cytosolic Ca2+ concentration may rise quickly in response to some stimuli, such as alpha 1-adrenergic stimulation, which is shown to be one of the pathways that increases cytosolic Ca2+ levels in hypertrophied rat heart. These data suggest that a part of the phosphatidylinositol-turnover pathway, such as the phosphatidylinositol 4,5-bisphosphate-IP3-Ca2+ pathway or the diacylglyceride-protein kinase C pathway, may play an important role in the development of hypertrophy in SHRSP heart.

Increased Calcium Release from Sarcoplasmic Reticulum Stimulated by Inositol Trisphosphate in Spontaneously Hypertensive Rat Heart Cells

It is known that inositol (1, 4, 5)-trisphosphate (IP3) stimulates Ca2+ release from sarcoplasmic reticulum (SR) in several tissues, but in cardiac myocytes this phenomenon has not been confirmed. The purpose of the present study was to confirm the effect of (1, 4, 5)-IP3 on Ca2+ release from SR in cardiac myocytes. The effect of IP3 on Ca2+ release from SR in hypertrophic cardiac cells was also determined.We examined the effects of IP3 on Ca2+ release from cardiac myocyte SR by the bigital-image method in a single cell. We also determined the effect of IP3 on calcium release from isolated SR. SR was prepared from spontaneous hypertensive rat hearts and Wistar kyoto rat hearts. The SR was prelabeled with45Ca2+, and then incubated with the indicated concentrations of IP3 for 1 min at 37°C. In cardiac myocytes treated with saponin, Ca2+ release stimulated by 10 μM (1, 4, 5)-IP3 was detected by fura-2. In45Ca2+ prelabeled SR, the maximal Ca2+ release was achieved at 10 μM IP3 incubated for 1 min. The release of Ca2+ was higher in Sr of SHR than in the SR of WKY. IP3 stimulates Ca2+ release from cardiac SR, and this release is greater in SHR than in WKY. However, it is uncertain whether this phenomenon plays a role in cardiac hypertrophy.

Alteration of extracellular matrix in dilated cardiomyopathic hamster heart.

The purpose of this study was to characterize the collagen in hereditary dilated cardiomyopathic hamster hearts, and to examine the participation of the collagen in the occurrence and progression of cardiomyopathy.BIO 53.58 hamsters (5, 10, 20 weeks old) were used as the model of dilated cardiomyopathy. Flb hamsters were used as controls. The collagen content was almost constant at any age in the Flb hamsters, but increased with age in BIO 53.58 hamsters. Type III collagen increased significantly in BIO 53.58 hamsters at 10 weeks. The acetic acid solubility of collagen decreased in BIO 53.58 hamsters as the fibrosis progressed, but was unchanged in controls. Reducible crosslinks showed a tendency to decrease progressively in BIO 53.58 hamsters. There were no differences between Flb and BIO 53.58 hamsters at 5 weeks, but its expression in BIO 53.58 hamsters at 10 and 20 weeks of age increased compared to Flb controls.These findings indicate that in the early phase of cardiomyopathy the extracellular matrix of the myocardium is rich in type III collagen. In the later phase, the matrix resembles that of hard tissues, whose collagen is mainly of type I collagen and is insoluble. These data suggest that the increased collagen synthesis may impair the cardiac function in the development of cardiomyopathy.

Leukaemia inhibitory factor and oncostatin M modulate expression of urokinase plasminogen activator and fibrinogen

BackgroundPathogenetic effects of active immune cell products on the coagulation and fibrinolytic system proteins in liver and endothelial cells - primary sites of synthesis of these proteins - have not been elucidated. MethodsWe incubated highly differentiated human hepatoma cells (Hep G2) and human umbilical vein endothelial cells (HUVECs) for 24 h with recombinant human leukaemia inhibitory factor (LIF) and oncostatin M (OSM) - cytokines that are implicated in acute phase reactions during inflammation and which bind to the same cell surface receptor unit (glycoprotein gp130). UF was. also given to mice in vivo. Contents of coagulation and fibrinolytic system proteins in cell culture supematants and in mouse liver were determined. ResultsLIF increased the accumulation of urokinase plasminogen activator (u-PA) in the Hep G2 cell culture supematants determined by enzyme-linked immunosorbent assay (ELISA) (0.21 ± 0.03 (SE) μg/ml at baseline; 0.40 ± 0.05 μg/ml at 100 μ/ml, P< 0.05; 0.57 ± 0.06 ng/ml at 500 U/ml, P< 0.01; n = 9) without altering total protein content. OSM elicited a similar effect (0.25 ± 0.04 ng/ml at baseline, 0.62 ± 0.19 ng/ml at 1 ng/ml; P<0.05, n = 6). A monoclonal antibody against gp130 abrogated the response to both agents (n = 9). Rasminogen activator inhibitor type-1 (PAI-1) (assayed by ELISA, n = 9), the PAI-1 binding protein, vitronectin (immunoprecipitation, n = 3) and tissue factor (ELISA, n = 3) were not affected by LIF, but fibrinogen production increased up to twofold with LIF (500 U/ml; Western blot, n = 3). In HUVECs, synthesis of tissue type plasminogen activator or PAI-1 was not altered by UF or OSM (ELISA, n = 9). In vivo, intraperitoneal recombinant murine LIF (2 μg) increased liver concentrations of u-PA by 30% and fibrinogen by 220% in mice. ConclusionsLIF and OSM produced by immune cells may modify fibrinolysis and coagulation by altering expression of u-PA and fibrinogen, thereby contributing to coagulopathy.

Angiotensin II Potentiates Collagen Synthesis in the Hypertrophied Heart

In cardiac hypertrophy,collagen accumulation in the myocardial interstitium is associated with hypertrophy of cardiomyocytes [1]. Collagen remodeling causes impaired ventricular compliance,abnormal electrical conduction and impaired oxygenation of the cardio-myocytes [2].At present,factors that regulate cardiac collagen metabolism are not elucidated.Eghbali et al [3] found that cardiac fibroblasts are mainly responsible for the synthesis of fibrillar type I and III co11agen.Recent studies have demonstrated that angiotensin II (A II),in addition to its inotropic and chronotropic actions,may exert effects by acting as a growth factor in the car¬diovascular system and that there may be local autocrine or paracrine renin-angiotensin system in several tissues [4].There are no data concerning the effects of A II on cardiac nonmyocytes.In this study we examined the effects of A II on collagen synthesis by cardiac fibroblasts in normotensive and hypertensive rats.

Biochemical properties potentially rendering human brain microvasculature vulnerable to proteolytic injury associated with the use of fibrinolytic drugs.

BACKGROUNDnDeterminants of predisposition to intracranial bleeding in response to the administration of thrombolytic drugs have not yet been well characterized.nnnOBJECTIVEnTo delineate factors involved, by characterizing susceptibility of human cerebral microvascular endothelium (HCME) to injury associated with inflammatory cytokines, levels of which are typically elevated in blood in patients who have suffered a myocardial infarction or stroke and been treated with thrombolytic drugs.nnnMETHODSnElaboration of fibrinolytic system proteins by HCME exposed either to interleukin-1 beta or to tumor necrosis factor-alpha (TNF) in serum-free medium for 24 h was characterized. Cell-conditioned medium was assayed for tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), and plasminogen activator inhibitor type 1--(PAI-1) by enzyme-linked immunosorbent assay. To determine whether the induction of u-PA was mediated by oxygen-centered radicals, the following were added to media: superoxide dismutase (a scavenger of O2-.), catalase (a scavenger of O2-. and H2O2) and dimethylthiourea (a scavenger of OH.).nnnRESULTSnInterleukin-1 beta had no effect upon elaboration of fibrinolytic system proteins by HCME. By contrast, TNF selectively increased elaboration of u-PA. Accumulation of t-PA and PAI-1 remained unchanged. Accumulation of u-PA was inhibited by cycloheximide, implying that there was a requirement for protein synthesis. Dimethylthiourea abolished the increase elaboration of u-PA induced by TNF completely, catalase did so partially, and SOD did not do so at all.nnnCONCLUSIONnThe propensity of HCME to elaborate u-PA rather than PAI-1 appears to render cerebral microvasculature particularly vulnerable to proteolytic attack in settings in which inflammatory cytokines are elaborated locally or in which their concentrations in blood are elevated.