Centrally administered isoproterenol induces sympathetic outflow via brain prostaglandin E2-mediated mechanisms in rats.

Brain ß-adrenoceptor stimulation can induce elevations of plasma levels of noradrenaline. However, there have been no detailed studies related to signaling pathways downstream of ß-adrenoceptors responsible for central sympathetic outflow. In the present study, we pharmacologically examined the possibility that centrally administered isoproterenol can induce elevations of plasma noradrenaline levels in a brain prostaglandin-dependent manner. In addition, we also examined whether or not intracerebroventricular administration of isoproterenol could release endogenously synthesized prostaglandin (PG) E2 in the hypothalamic paraventricular nucleus (PVN) by using the brain microdialysis technique combined with liquid chromatography-ion trap tandem mass spectrometry (LC-ITMS(n)). Under urethane anesthesia, a femoral venous line was inserted for infusion of saline and a femoral arterial line was inserted for collecting blood samples. Next, animals were placed in a stereotaxic apparatus for application of test agents. Catecholamines in the plasma were extracted by alumina absorption and were assayed by high-performance liquid chromatography with electrochemical detection. Quantification of PGE2 in rat PVN microdialysates was performed by the LC-ITMS(n) method. We demonstrated that centrally administered isoproterenol-induced elevations of plasma noradrenaline could be mediated via activation of ß-adrenoceptors and the downstream phospholipase A2-cyclooxygenase pathway. Furthermore, PGE2 in the PVN and the PGE2 receptor EP3 subtype appear to play an important role in the process. Our results suggest that central isoproterenol-induced sympathetic outflow is mediated via brain PGE2 in a PGE2 receptor EP3 subtype-dependent manner.

Changes in hypothalamic neurotransmitter and prostanoid levels in response to NMDA, CRF, and GLP-1 stimulation.

Determination of neuroactive substances, such as neurotransmitters and prostanoids, in the extracellular space of the living brain is a very important technique in neuroscience. The hypothalamic paraventricular nucleus (PVN) is one of the most important autonomic control centers in the brain. Recently, we demonstrated that thromboxane (Tx) A2 in the PVN may play an important role in adrenomedullary outflow evoked by N-methyl-D-aspartate (NMDA), corticotrophin-releasing factor (CRF), and glucagon-like peptide-1 (GLP-1) stimulation using microdialysis sampling and liquid chromatography-ion trap tandem mass spectrometry (LC-ITMS(n)). In the present study, we investigated whether centrally administered NMDA, CRF, and GLP-1 can release five neurotransmitters, acetylcholine (ACh), histamine, glutamate (Glu), γ-aminobutyric acid (GABA), and serotonin (5-HT), along with six prostanoids, TxB2, prostaglandin (PG) E2, PGD2, 15-deoxy-∆(12,14) (15d)-PGJ2, 6-keto-PGF1α, and PGF2α in rat PVN microdialysates after optimization of LC-ITMS(n) conditions . All stimulations increased the levels of 5-HT, TxB2, PGE2, and PGF2α (except for 5-HT stimulated with GLP-1). Only NMDA increased the levels of ACh, Glu, and GABA. Treatment with dizocilpine maleate (MK-801), a noncompetitive NMDA receptor antagonist, attenuated the NMDA-induced increase in the levels of neuroactive substances except for Glu. This is the first study to use LC-ITMS(n) to analyze neurotransmitters and prostanoids in the same microdialysates from rat PVN.

Copper-dependent inhibition and oxidative inactivation with affinity cleavage of yeast glutathione reductase.

Effects of copper on the activity and oxidative inactivation of yeast glutathione reductase were analyzed. Glutathione reductase from yeast was inhibited by cupric ion and more potently by cuprous ion. Copper ion inhibited the enzyme noncompetitively with respect to the substrate GSSG and NADPH. The Ki values of the enzyme for Cu(2+) and Cu(+) ion were determined to be 1 and 0.35 µM, respectively. Copper-dependent inactivation of glutathione reductase was also analyzed. Hydrogen peroxide and copper/ascorbate also caused an inactivation with the cleavage of peptide bond of the enzyme. The inactivation/fragmentation of the enzyme was prevented by addition of catalase, suggesting that hydroxyl radical produced through the cuprous ion-dependent reduction of oxygen is responsible for the inactivation/fragmentation of the enzyme. SDS-PAGE and TOF-MS analysis confirmed eight fragments, which were further determined to result from the cleavage of the Met17-Ser18, Asn20-Thr21, Glu251-Gly252, Ser420-Pro421, Pro421-Thr422 bonds of the enzyme by amino-terminal sequencing analysis. Based on the kinetic analysis and no protective effect of the substrates, GSSG and NADPH on the copper-mediated inactivation/fragmentation of the enzyme, copper binds to the sites apart from the substrate-sites, causing the peptide cleavage by hydroxyl radical. Copper-dependent oxidative inactivation/fragmentation of glutathione reductase can explain the prooxidant properties of copper under the in vivo conditions.

Mass spectrometric determination of prostanoids in rat hypothalamic paraventricular nucleus microdialysates.

The hypothalamic paraventricular nucleus (PVN) is one of the most important autonomic control centers in the brain. Several kinds of prostanoids, such as prostaglandin (PG) E2, are considered to act in the PVN as mediators of autonomic responses. In the present study, we used liquid chromatography ion trap tandem mass spectrometry (LC-ITMS(n)) to simultaneously quantify four prostanoids, thromboxane (Tx) B2, PGE2, PGD2 and 15-deoxy-∆(12,14) (15d)-PGJ2 in PVN microdialysates from urethane-anesthetized rats. The quantification limits were estimated to be 0.05ng/mL for TxB2, 0.025ng/mL for PGE2, 0.1ng/mL for PGD2, and 0.5ng/mL for 15d-PGJ2. The RSD% obtained from all prostanoids was <15%, indicating an acceptable level of reproducibility. LC-ITMS(n) analysis of rat PVN microdialysates revealed that TxA2 may play an important role in adrenomedullary outflow evoked by centrally administered N-methyl-d-aspartate, corticotrophin-releasing factor and glucagon-like peptide-1. This is the first study to use LC-ITMS(n) to analyze prostanoid levels in rat PVN microdialysates. This LC-ITMS(n) method will be useful for investigating the potential involvement of prostanoids in brain function.

Inhibition of angiotensin I converting enzyme by subtilisin NAT (nattokinase) in natto, a Japanese traditional fermented food.

Angiotensin I converting enzyme (ACE) was inhibited by the culture medium of Bacillus subtilis subsp. natto, which ferments boiled soy beans to natto, a Japanese traditional food. Subtilisin NAT (nattokinase) produced by B. subtilis also inhibited ACE, and the inhibition was markedly stimulated by heat treatment of subtilisin at 120 °C for 15 min. Inhibition of ACE by subtilisin was of a mixed type: the decrease in V(max) and the increase in K(m) value. SDS-polyacrylamide gel electrophoresis showed that heat treatment of subtilisin caused inactivation with fragmentation of the enzyme protein into small peptides. The inhibitory action of subtilisin was not due to an enzymatic action of protease, but may be ascribed to the potent ACE-inhibitory peptides such as LY and FY, amino acid sequences in subtilisin. HPLC-MS analysis of heat-inactivated subtilisin confirmed that LY and FY were liberated by fragmentation of the enzyme. Inhibition of ACE by subtilisin and its degradation peptides such as LY and FY may participate in the suppression of blood pressure by ingestion of natto.

Iron-dependent oxidative inactivation with affinity cleavage of pyruvate kinase.

Treatment of rabbit muscle pyruvate kinase with iron/ascorbate caused an inactivation with the cleavage of peptide bond. The inactivation or fragmentation of the enzyme was prevented by addition of Mg2+, catalase, and mannitol, but ADP and PEP the substrates did not show any effect. Protective effect of catalase and mannitol suggests that hydroxyl radical produced through the ferrous ion-dependent reduction of oxygen is responsible for the inactivation/fragmentation of the enzyme. SDS-PAGE and TOF-MS analysis confirmed five pairs of fragments, which were determined to result from the cleavage of the Lys114-Gly115, Glu117-Ile118, Asp177-Gly178, Gly207-Val208, and Phe243-Ile244 bonds of the enzyme by amino-terminal sequencing analysis. Protection of the enzyme by Mg2+ implies the identical binding sites of Fe2+ and Mg2+, but the cleavage sites were discriminated from the cofactor Mg2+-binding sites. Considering amino acid residues interacting with metal ions and tertiary structure, Fe2+ ion may bind to Asp177 neighboring to Gly207 and Glu117 neighboring to Lys114 and Phe243, causing the peptide cleavage by hydroxyl radical. Iron-dependent oxidative inactivation/fragmentation of pyruvate kinase can explain the decreased glycolytic flux under aerobic conditions. Intracellular free Mg2+ concentrations are responsible for the control of cellular respiration and glycolysis.

Oxidative inactivation of reduced NADP-generating enzymes in E. coli: iron-dependent inactivation with affinity cleavage of NADP-isocitrate dehydrogenase.

Treatment of E. coli extract with iron/ascorbate preferentially inactivated NADP-isocitrate dehydrogenase without affecting glucose-6-phosphate dehydrogenase. NADP-Isocitrate dehydrogenase required divalent metals such as Mg(2+), Mn(2+ )or Fe(2+) ion. Iron/ascorbate-dependent inactivation of the enzyme was accompanied with the protein fragmentation as judged by SDS-PAGE. Catalase protecting the enzyme from the inactivation suggests that hydroxyl radical is responsible for the inactivation with fragmentation. TOF-MS analysis showed that molecular masses of the enzyme fragments were 36 and 12, and 33 and 14 kDa as minor components. Based on the amino acid sequence analyses of the fragments, cleavage sites of the enzyme were identified as Asp307-Tyr308 and Ala282-Asp283, which are presumed to be the metal-binding sites. Ferrous ion bound to the metal-binding sites of the E. coli NADP-isocitrate dehydrogenase may generate superoxide radical that forms hydrogen peroxide and further hydroxyl radical, causing inactivation with peptide cleavage of the enzyme. Oxidative inactivation of NADP-isocitrate dehydrogenase without affecting glucose 6-phosphate dehydrogenase shows only a little influence on the antioxidant activity supplying NADPH for glutathione regeneration, but may facilitate flux through the glyoxylate bypass as the biosynthetic pathway with the inhibition of the citric acid cycle under aerobic growth conditions of E. coli.