IntroductionRecent studies have shown that histones, the chief protein component of chromatin, are released into the extracellular space during sepsis, trauma, and ischemia-reperfusion injury, and act as major mediators of the death of an organism. This study was designed to elucidate the cellular and molecular basis of histone-induced lethality and to assess the protective effects of recombinant thrombomodulin (rTM). rTM has been approved for the treatment of disseminated intravascular coagulation (DIC) in Japan, and is currently undergoing a phase III clinical trial in the United States.MethodsHistone H3 levels in plasma of healthy volunteers and patients with sepsis and DIC were measured using enzyme-linked immunosorbent assay. Male C57BL/6 mice were injected intravenously with purified histones, and pathological examinations were performed. The protective effects of rTM against histone toxicity were analyzed both in vitro and in mice.ResultsHistone H3 was not detectable in plasma of healthy volunteers, but significant levels were observed in patients with sepsis and DIC. These levels were higher in non-survivors than in survivors. Extracellular histones triggered platelet aggregation, leading to thrombotic occlusion of pulmonary capillaries and subsequent right-sided heart failure in mice. These mice displayed symptoms of DIC, including thrombocytopenia, prolonged prothrombin time, decreased fibrinogen, fibrin deposition in capillaries, and bleeding. Platelet depletion protected mice from histone-induced death in the first 30 minutes, suggesting that vessel occlusion by platelet-rich thrombi might be responsible for death during the early phase. Furthermore, rTM bound to extracellular histones, suppressed histone-induced platelet aggregation, thrombotic occlusion of pulmonary capillaries, and dilatation of the right ventricle, and rescued mice from lethal thromboembolism.ConclusionsExtracellular histones cause massive thromboembolism associated with consumptive coagulopathy, which is diagnostically indistinguishable from DIC. rTM binds to histones and neutralizes the prothrombotic action of histones. This may contribute to the effectiveness of rTM against DIC.

Orexin neurons contribute to cardiovascular, respiratory and analgesic components of the fight-or-flight response against stressors. Here, we examined whether the same is true for stress-induced hyperthermia. We used prepro-orexin knockout mice (ORX-KO) and orexin neuron-ablated mice (ORX-AB) in which the latter lack not only orexin, but also other putative neurotransmitter/modulators contained in the orexin neurons. In response to repetitive insertion of a temperature probe into their rectum (handling stress), ORX-KO mice showed a normal temperature change as compared to that of wild-type littermates (WT) while ORX-AB showed an attenuated response. Stress-induced expression of uncoupling protein-1, a key molecule in non-shivering thermogenesis in the brown adipose tissue (BAT), was also blunted in ORX-AB but not in ORX-KO. When the BAT was directly activated by a β3 adrenergic agonist, there was no difference in the resultant BAT temperature among the groups, indicating that BAT per se was normal in ORX-AB. In WT and ORX-KO, handling stress activated orexin neurons (as revealed by increased expression of c-Fos) and the resultant hyperthermia was largely blunted by pre-treatment with a β3 antagonist. This observation further supports the notion that attenuated stress-induced hyperthermia in ORX-AB mice was caused by a loss of orexin neurons and abnormal BAT regulation. This study pointed out, for the first time, the possible importance of co-existent neurotransmitter/modulators in the orexin neurons for stress-induced hyperthermia and the importance of integrity of the orexin neurons for full expression of multiple facets of the fight-or-flight response. Abbreviations BAT, brown adipose tissue; DMH, dorsomedial hypothalamus; ORX-AB, orexin neuron-ablated mice; ORX-KO, orexin knockout mice; PBS, phosphate-buffered saline; REM, rapid eye movement; UCP, uncoupling protein; WT AB , wild-type littermate of the orexin neuron-ablated mice; WT KO , wild-type littermate of the orexin knockout mice.

SUMMARY1. To clarify the nature of the noradrenaline (NA)-induced contraction, the effects of NA on inositol phospholipid metabolism and the actions of inositol 1,4,5-trisphosphate (InsP3) on skinned muscle of the rabbit mesenteric artery were investigated.2. NA, in concentrations over 1 nm, reduced the amount of phosphatidylinositol 4,5-bisphosphate (PI-P2) and increased the amount of phosphatidic acid (PA). The maximum reduction in the amount of PI-P2 and the maximum increase in the amount of PA were observed in the presence of 1 /LM-NA. With prolonged application of NA, the PI-P2 was gradually restored to near the control level, but with repeated applications of NA separated by rinses with Krebs solution, there was a consistent reduction of PI-P2.3. The NA-induced PI-P2 breakdown was inhibited by the ax-adrenoceptor blocking agent, prazosin.4. After incubation of the tissue with radioactive inositol-containing solution, NA transiently increased the amount of radioactive InsP3 which was followed by increases in the amount of inositol 1,4-bisphosphate (InsP2) and inositol monophosphate (InsP).5. After accumulation of Ca by saponin-treated muscle cells of the dog mesenteric artery dispersed by collagenase, InsP3 released Ca stored in cells but InsP2 did not. A23187 (5/M) but not InsP3 (up to 10 tM), depleted Ca accumulated in the presence of ATP.6. In saponin-treated skinned muscle tissues, InsP3 in concentrations over 0 3 ,UM, produced contraction following accumulation of Ca into the store site. InsP3 released Ca from the same source as caffeine. The release of Ca by InsP3 appeared in a concentration-dependent manner and this release also depended on the amount of Ca stored in cells (the median effective dose (ED50) was 3 0 /tM in 0-1 tM-Ca and 1-0/tM in 0-3 /tM-Ca).7. We concluded that NA activates ax-adrenoceptors, thus hydrolysing P1-P2 and synthesizing InsP3. This product can release Ca stored in cells as estimated from the contraction in skinned muscle tissues, and also reduces the residual amount of Ca T. HASHIMOTO AND OTHERS stored in skinned dispersed muscle cells. Contraction evoked by NA through pharmacomechanical coupling can be explained as a function of InsP3.

SUMMARY1. The effects were investigated of inorganic phosphate (Pi) on the Ca2+-dependent and Ca2+-independent contractions evoked in chemically skinned smooth muscles of the rabbit mesenteric artery.2. The relation between the concentration of Ca2+ and tension showed a sigmoidal curve in the range ofpCa 7-5 5. Pi (over 1 mM) inhibited the Ca2+-induced contraction, shifted the pCa-tension curve to the right and increased the Hill number from 2 to 3. Calmodulin did not change the Hill number and attenuated the inhibitory action of Pi as estimated from the shift of the curve, but this agent did not modify the increased Hill number in the presence of Pi.3. P1 consistently inhibited the Ca2+-independent contractions provoked by application of trypsin-treated myosin light chain kinase, of MgATP following adenosine-5'-o-(3-thiotriphosphate) (ATP S) and Ca2+, and of a solution containing high Mg2+. These inhibitory actions of Pi were inversely proportional to the amplitude of the contraction. When Pi was applied simultaneously with ATP S and Ca2+, there was no change in the amplitude of Ca2+-independent contractions provoked by the application of MgATP.4. The amplitude of the rigor contraction evoked by ATP-free solution was less than 70 ofthat of the 10 /uM-Ca2+-induced contraction. When ATP was removed from the solution during the Ca2+ contraction, the rigor contraction was also generated.Pi did not inhibit either type of contraction.5. With a decrease in the concentration of Ca2+ from 10/M to below 1 nm, the tissue relaxed at a slower rate than the rate of rise of the Ca2+-induced contraction. The slow relaxation was not modified by a change in the concentration of EGTA or addition of 1 /tM-calmodulin.6. Pi reduced, and high Mg2+ prolonged the time required for the relaxation. This action of Pi was not prevented in the presence of calmodulin or of high Mg2+.

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