Mayumi Mori

Studies on sphingomyelinase of Bacillus cereus: Hydrolytic and hemolytic actions on erythrocyte membranes

Abstract Purified sphingomyelinase of Bacillus cereus was incubated with sheep erythrocytes at 37 °C, either in the absence or presence of divalent metal ions such as Mg 2+ and Ca 2+ . During incubation, the enzyme at first was adsorbed onto the surfaces of sheeps erythrocytes; then these cells became fragile to cold shock (hot-cold hemolysis). The enzyme further attacked sheep erythrocytes by hydrolyzing sphingomyelin, made these cells more temperature sensitive, and finally hemolyzed them during incubation at 37 °C. Mg 2+ stimulated both the hemolytic and hydrolytic activities of sphingomyelinase, while Ca 2+ was inhibitory. In the presence of both Mg 2+ and Ca 2+ , the processes of sphingomyelin hydrolysis and cell lysis were more accelerated than those in the presence of Mg 2+ alone. These divalent cations were essentially without effect on the adsorption of enzyme to erythrocytes. By scanning electron microscopy, it was revealed that sphingomyelinase of Bacillus cereus induced invagination of sheep erythrocytes in parallel with hydrolysis of sphingomyelin on the cell surfaces and finally transformed these cells into spherocytes.

Antidiuretic Effects of Oxotremorine Microinjected into the Hypothalamic Supraoptic and Paraventricular Nuclei in a Water-Loaded and Ethanol-Anesthetized Rat

The effect of oxotremorine, a muscarinic agonist, on urine outflow compared with the effects of other cholinergic agonists and inhibitory effects of cholinergic antagonists upon the cholinergic actions were studied by microinjecting drugs stereotaxically, unilaterally into the supraoptic (SON) or paraventricular nuclei (PVN) in the hypothalamus of the rat which was loaded with water and anesthetized with ethanol. Oxotremorine decreased the urine outflow in dose- and time-dependent manners when microinjected into these nuclei. The median effective doses (ED50) were approx. 0.3 and 0.2 nmol in SON an PVN, respectively, being much less than ED50 values for nicotine. The time course of the antidiuretic effects was relatively slow, with the minimal urine outflow at approx. 30 min and the duration of one or longer hours. The antidiuretic effects of oxotremorine in these nuclei as well as the effects of acetylcholine and nicotine were completely blocked by pretreatment with atropine. The pretreatment with hexamethonium inhibited partially the effects of nicotine, but was unable to inhibit the effects of oxotremorine and acetylcholine. The data suggest that the antidiuretic effects of cholinergic agonists in SON and PVN are predominantly mediated through a muscarinic type of acetylcholine receptor. A possible mechanism for the antidiuretic effects is discussed.

Mechanisms underlying the hydrogen peroxide-induced, endothelium-independent relaxation of the norepinephrine-contraction in guinea-pig aorta

The mechanisms underlying the hydrogen peroxide-induced relaxation of the norepinephrine-contraction were studied by measuring isometric force, myosin light chain (MLC(20)) phosphorylation and cyclic GMP in endothelium-denuded muscle from the guinea-pig aorta. Norepinephrine (5.2+/-1.3 microM) produced a phasic, followed by a tonic contraction. Hydrogen peroxide (10 and 100 microM), glyceryl trinitrate (30 and 300 nM) and 8-bromo cyclic GMP (30 and 100 microM) did not change the basal tone, but reduced the norepinephrine-induced contraction. Phosphorylation of MLC(20) (percentage of phosphorylated to total MLC(20)) was increased 1 min (5.9+/-1.0% vs. 35.9+/-4.9%) and, to a lesser extent, 20 min (3.7+/-1.7% vs. 13.9+/-1.6%) after the addition of norepinephrine. Hydrogen peroxide (100 microM) did not modify basal MLC(20) phosphorylation, but reduced the increase in MLC(20) phosphorylation induced by 1-min exposure to norepinephrine (20.9+/-4.1%). Its effect was abolished by catalase. When the tissue was incubated for 20 min with norepinephrine in the presence of hydrogen peroxide, norepinephrine-induced MLC(20) phosphorylation was not changed (13.6+/-1.5%), as compared to that in the absence of hydrogen peroxide. Hydrogen peroxide relaxed norepinephrine-stimulated aortas in a concentration-dependent fashion with EC(50) values of 5.9+/-0.2 microM. The relaxation was inhibited by soluble guanylate cyclase inhibitors and increased by an inhibitor of cyclic GMP-selective phosphodiesterase. In aorta precontracted with norepinephrine, hydrogen peroxide (100 microM) relaxed the tissue by 89+/-11% and almost doubled tissue concentrations of cyclic GMP, whereas sodium nitroprusside (1 microM) relaxed the tissue by 100% and increased cyclic GMP concentrations 30-fold. It is suggested that the inhibitory effects of hydrogen peroxide on the norepinephrine-induced phasic and sustained contractions are explained by a decrease in MLC(20) phosphorylation and by an alteration in MLC(20) phosphorylation-independent mechanisms, respectively. The effects of hydrogen peroxide were in part mediated by cyclic GMP.

Pharmacological characteristics of bombesin receptor mediating hypothermia in the central nervous system of rats.

Bombesin (BN) and structurally related peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB), injected into the lateral ventricle produce multiple effects such as hypothermia, anorexia and hormone release. In this study, the pharmacological characteristics of BN receptors mediating hypothermia in the central nervous system (CNS) were investigated using free-moving male Wistar rats. Intracerebroventricular injections of BN, GRP and NMB produced hypothermia in a dose-dependent manner. The BN (0.3 microg)-induced effect showed a short latency and a 4-h duration with a potency increased by more than 100 times compared to the NMB-induced effect. Pretreatment with [D-Tyr(6)]BN(6-13)methylester, a GRP receptor antagonist, inhibited the BN (0.3 microg)- and NMB (7 microg)-induced hypothermia. On the other hand, BIM23127, an NMB receptor antagonist, did not influence the hypothermia. Of the protein kinase C (PKC) inhibitors, chelerythrine, Go6983, staurosporine and GF109203X, the first two partially blocked the BN-induced hypothermia. A PKC activator, phorbol-12,13-dibutyrate, decreased the rectal temperature. Genistein (a tyrosine kinase inhibitor), Y-27632 (a Rho kinase inhibitor) and PD98059 (a MAPK inhibitor) tended to suppress the BN-induced hypothermia, however, these were not significant. The inhibitory effect of a mixture of the three inhibitors, chelerythrine, genistein and Y-27632, on the BN-induced hypothermia was of a similar degree to that of chelerythrine alone. The BN receptor mediating the hypothermia seem to be the GRP subtype, and the effect involves activation of PKC.

In vivo evidence that activation of tyrosine kinase is a trigger for lipopolysaccharide-induced fever in rats.

We measured the rectal temperature of free-moving, conscious rats after intracerebroventricular (i.c.v.) injections of lipopolysaccharide (LPS) and interleukin-1beta (IL-1beta) with or without various antagonists to investigate the mechanisms involved in LPS-induced fever. LPS (3 microg) elicited significant increases in rectal temperature, which lasted from 0.5 h to more than 8 h after administration. This febrile response was inhibited by pretreatment with L-nitro-arginine (LNA), indomethacin (IND), genistein (GEN), tyrphostin 46 and anti-rat IL-1beta antibody (anti-IL-1beta Ab), but was not inhibited by pretreatment with daidzein or chelerythrine (CHE) into the ventricle. LPS (0.3 microg) following orthovanadate (i.c.v.) produced fever, although the small amount of LPS (0.3 microg) or orthovanadate alone showed no effect on rectal temperature. I.c.v. injections of IL-1beta also induced fever of approximately 4-h duration. This effect was inhibited by pretreatment with IND and anti-IL-1beta Ab, but was not inhibited by pretreatment with LNA, GEN or CHE into the ventricle. These findings demonstrate that in the central nervous system, LPS increases IL-1beta production after activation of tyrosine kinase and NO synthase, and IL-1beta promotes prostaglandin production resulting in increased rectal temperature. Activation of tyrosine kinase in the central nervous system is probably a trigger for the febrile response induced by LPS.

Mechanisms underlying anorexia after microinjection of bombesin into the lateral cerebroventricle.

Intracerebroventricular (i.c.v.) injections of bombesin (BN) and gastrin-releasing peptide (GRP) dose-dependently decreased food intake in male Wistar rats fasted for 17 h. Neuromedin B (NMB) did not show any effect on food intake. After BN administration, locomotor activity did not significantly change, compared with a vehicle-injected group. The anorexia induced by BN (0.3 microg) was perfectly inhibited by pretreatment with a GRP-receptor antagonist, [D-Tyr(6)]BN(6-13) methyl ester (10 microg), an NO synthase inhibitor, L-nitro-arginine (30 microg), and a PKG inhibitor, H-9 (2 microg). The cGMP concentration in the hypothalamus increased 1 h after administration when compared with the vehicle-injected group. On the other hand, an NMB-receptor antagonist, BIM23127 (10 microg), and the protein kinase (PK) C inhibitors, chelerythrine (2 microg) and Go6983 (2 microg), inhibited only the late phase of the anorexia. A PKC activator, phorbol 12, 13-dibutyrate (3 microg), injected into the ventricle decreased food intake. These findings suggest that BN suppresses food intake mainly mediated through the GRP receptor and NO-cGMP-PKG pathway, and NMB receptor and PKC is partly involved in the late phase of the anorexia.

Involvement of protein kinase C and tyrosine kinase in lipopolysaccharide-induced anorexia.

Injections of lipopolysaccharide (LPS, 3 microg) into the lateral ventricle elicited anorexia with fever and also decreased body weight in rats. The LPS-induced anorexia was inhibited by intracerebroventicular (i.c.v.) injections of anti-interleukin (IL)-1beta antibody (Ab), chelerythrine, genistein and tyrphostin 46, but not by injections of indomethacin. Consecutive injections of orthovanadate and LPS (0.3 microg, a dose of LPS that did not show any effect on food intake, body weight or body temperature) reduced body weight, but did not induce anorexia. On the other hand, injections of IL-1beta (50 ng) did not influence food intake, although they decreased body weight and produced fever. The IL-1beta-induced decrease in body weight was inhibited by injections of genistein, but not by injections of chelerythrine or indomethacin. These findings suggest that the LPS-induced anorexia is independent of hyperthermia and involves IL-1beta generation, tyrosine kinase (TK) and protein kinase C (PKC). This is the first in vivo evidence that activation of TK and PKC induced by LPS is linked to anorexia.

Effects of fentanyl, injected into the hypothalamic supraoptic and paraventricular nuclei, in a water-loaded and ethanol-anesthetized rat

The analgesic fentanyl, having a predominantly mu-opioid agonist activity, when injected into the supraoptic or paraventricular nucleus of the hypothalamus in a water-loaded and ethanol-anesthetized rat, induced a potent antidiuretic effect in a time- and dose-dependent manner. The outflow of urine decreased to a minimal level of approximately 5% of the initial control, at 20-40 min and recovered to approximately 80% at 90 min after injection of fentanyl (30 nmol). The median effective dose (ED50) for the antidiuretic effect of fentanyl was approximately 13 nmol, when injected into the supraoptic or paraventricular nucleus, being nearly equipotent with morphine. The osmotic pressure of urine increased up to approximately 200% of control, at the minimal rate of outflow of urine when fentanyl (30 nmol) was injected into the supraoptic or paraventricular nucleus. Transient but significant decreases in mean blood pressure and in rate of respiration were observed when fentanyl (30 nmol) was injected into the supraoptic or paraventricular nucleus. The antidiuretic and the autonomic effects (transient decreases in mean blood pressure and rate of respiration) were inhibited by the previous injection of an opioid receptor antagonist, naloxone (300 or 600 nmol) into the nuclei. The results suggest that the effects of fentanyl were induced through opioid receptors in the nuclei.

Antidiuretic and thermogenic effects of intracerebroventricular prostaglandin H2 in ethanol-anaesthetized rats.

When PGH2 was administered intracerebroventricularly at doses of 5 and 15 nmoles in ethanol-anaesthetized rats, alcohol diuresis was inhibited and rectal temperature, blood pressure and heart rate were all significantly increased.

Canola and hydrogenated soybean oils accelerate ectopic bone formation induced by implantation of bone morphogenetic protein in mice

Highlights • BMP-induced bone formation was evaluated in mice fed a Soy, Can, or H2-Soy diet.• Ectopic bone formation was greater in the Can and H2-Soy groups.• Altered levels of VK2 and VK-dependent protein(s) might affect the bone formation.