Vasodilator effects of sarafotoxins and endothelin-1 in spontaneously hypertensive rats and rat isolated perfused mesentery

Sarafotoxins (SRTa, SRTb and SRTc) and ET-1 produced a potent vasodilator effect in spontaneously hypertensive rats in vivo and in rat isolated perfused mesenteries in vitro. Among these peptides SRTc demonstrated the most potent vasodilator activity, and was three times more active than SRTa in both preparations. These peptides induced endothelium-dependent vasodilatation in vitro and pretreatment with methylene blue inhibited this effect, while exposure to the antagonists of other vasodilators did not. In contrast, [nitrophenylsulfenylated Trp21]SRTc, SRTc(1-18) and reduced and S-carboxymethylated SRTc caused no vasodilatation in either animal model; the vasodilator effect of acetylated SRTc was less potent than that of SRTc. These results suggest that (i) the vasodilatations of these peptides may be exerted through the release of endothelium derived relaxing factor; (ii) the C-terminal Trp21 and disulfide bonds are essential; and (iii) the N-terminal amino group plays an important role in vasodilator activity.     

The role of intracellular Ca2+ in the degranulation of skinned mast cells

The intracellular pH of rat peritoneal mast cells was slightly acidic and compound 48/80 induced a decrease in the cytoplasmic pH of these cells. By means of chemical skinning, it was revealed that perfusion with Ca2+ or inositol 1,4,5-trisphosphate (IP3) induced degranulation dose-dependently in mast cells at concentrations higher than 10 microM and 0.1 microM, respectively. Na+ was essential for the release of histamine from mast cells. An assay based on the binding of 45Ca to mast cell fragments revealed that the intracellular Ca store of the mast cell is located in the endoplasmic reticulum. IP3 liberated Ca from the endoplasmic reticulum.     

Antiallergic effects of astemizole on immediate type hypersensitivity reactions.

Astemizole (0.5-5 mg/kg, p.o.) dose-dependently inhibited heterologous and homologous PCA reactions in rats at ID50 values of 1.48 mg/kg and 2.37 mg/kg, respectively. The inhibitory effect of astemizole on heterologous PCA was most remarkable when this compound was given p.o. 2 h prior to antigen challenge. Astemizole (0.1-5 mg/kg, p.o.) dose-dependently inhibited experimentally-induced asthma in guinea pigs at an ID50 of 0.86 mg/kg. Ex vivo, astemizole (0.5-5 mg/kg, p.o.) inhibited antigen-induced histamine release from lung pieces of sensitized guinea pigs. In in vitro experiments, the drug dose-dependently inhibited antigen-induced histamine and SRS-A releases from guinea pig lung pieces at concentrations of 0.05-10 microM. Furthermore, astemizole (0.1-10 microM) inhibited the histamine release induced by compound 48/80 and antigen-antibody reaction from rat peritoneal mast cells, and at 0.1-500 nM inhibited both leukotriene C4- and platelet-activating factor (PAF)-induced contraction of isolated guinea pig trachea at submicromolar concentrations. Astemizole not only inhibited 45Ca uptake into rat mast cells but also prevented the Ca2+ release from the intracellular Ca store induced by compound 48/80, although this compound did not affect the histamine release from permeabilized mast cells induced by Ca2+. Our results suggest that one of the antiallergic mechanisms of astemizole may be an inhibition of signal transduction from the mast cell membrane to the intracellular systems.     

Identification of vimentin in rat peritoneal mast cells and its phosphorylation in association with histamine release.

Stimulation of rat peritoneal mast cells with histamine releasers, such as compound 48/80 and substance P, caused a similar pattern of protein phosphorylations: the molecular weights of the two major phosphorylated proteins were 45 kDa and 59 kDa. When rat mast cells permeabilized with beta-escin were exposed to Ca2+ at concentrations higher than 0.6 microM, phosphorylated proteins of identical molecular weight were also detected. By a radioimmunoprecipitation assay using anti-vimentin mouse monoclonal antibody, the 59 kDa protein was identified as vimentin, one of the intermediate cytoskeletal proteins. Moreover, it became apparent that the phosphoamino acid in phosphorylated vimentin was a serine residue. Sequential changes in vimentin phosphorylation were similar to that of histamine release elicited by histamine releasers: phosphorylation took place within 5 s of stimulation and reached a maximum within 10 s. When permeabilized mast cells were treated with calphostin C, a specific protein kinase C inhibitor, phosphorylation was markedly inhibited. Fluorescence images of mast cells stained with FITC-labelled anti-vimentin antibody showed filamentous structures surrounding the granules in the cytoplasm. However, after exposure to compound 48/80, the filamentous structures promptly disappeared and a dim fluorescence was observed homogeneously in the cell indicating that a rapid depolymerization of vimentin had taken place. From the present study, it became clear that when rat peritoneal mast cells were stimulated, vimentin was rapidly phosphorylated by protein kinase C and this phosphorylation process seems to be related to histamine release.     

Histamine-induced depolarization and the cyclic AMP--protein kinase A system in isolated guinea pig adipocytes.

The relationship between histamine (Hi)-induced depolarization and the cyclic AMP system in adipocytes was studied in guinea pigs, which seem to be more sensitive than rats to Hi. Hi caused a dose-dependent depolarization in guinea pig mesenterial and epididymal adipocytes with EC50 values of 1.69 x 10(-7) M and 1.19 x 10(-7) M, respectively. Guinea pig adipocytes were 280-750 times more sensitive than rat adipocytes to Hi. Isoproterenol, forskolin and 3-isobutyl-1-methylxanthine (IBMX) also caused a depolarization, and the slopes of the concentration response lines for these drugs were almost the same as that for Hi. Furthermore, pretreatment with these drugs resulted in a potentiation of Hi-induced depolarization at lower concentrations which are not effective when each drug is used alone. In addition, Hi-induced depolarization was inhibited by pretreatment with prostaglandin E1 (PGE1) and insulin dose-dependently. The content of cyclic AMP in adipocytes was increased by Hi (10(-7) M) in association with a decrease in membrane potential. KT5720, a protein kinase A inhibitor, which provides no significant effect even at a concentration of 10(-6) M, showed an antagonistic effect on Hi-induced depolarization.     

A critical role of sterols in embryonic patterning and meristem programming revealed by the fackel mutants of Arabidopsis thaliana

Here we report a novel Arabidopsis dwarf mutant, fackel-J79, whose adult morphology resembles that of brassinosteroid-deficient mutants but also displays distorted embryos, supernumerary cotyledons, multiple shoot meristems, and stunted roots. We cloned the FACKEL gene and found that it encodes a protein with sequence similarity to both the human sterol reductase family and yeast C-14 sterol reductase and is preferentially expressed in actively growing cells. Biochemical analysis indicates that the fk-J79 mutation results in deficient C-14 sterol reductase activity, abnormal sterol composition, and reduction of brassinosteroids (BRs). Unlike other BR-deficient mutants, the defect of hypocotyl elongation in fk-J79 cannot be corrected by exogenous BRs. The unique phenotypes and sterol composition in fk-J79 indicate crucial roles of sterol regulation and signaling in cell division and cell expansion in embryonic and post-embryonic development in plants.     

Role of the cytoskeleton in Ca2+ release from the intracellular Ca store of rat peritoneal mast cells

In order to study the role of the cytoskeleton in histamine release from mast cells, the effects of cytochalasin D, cholchicine and vinblastine on Ca²⁺ release from the intracellular Ca store induced by compound 48/80 were investigated by means of a video-intensified microscopy system. When the quin 2-loaded mast cells were stimulated by 0.35 g/ml of compound 48/80, a rapid increase in intracellular Ca²⁺ was observed. At concentrations higher than 10^–6 M, both colchicine and vinblastine pretreatments significantly inhibited the increase in intracellular Ca²⁺ concentrations caused by compound 48/80, although cytochalasin D had no effect. When permeabilized mast cells were exposed to potassium-antimonate solution, microtubules became attached to the endoplasmic reticulum, where many dots of Ca-antimonate were observed; in some areas, the microtubules interconnected the endoplasmic reticulum and granules in the mast cells. From the results of the present study, it was assumed that microtubules play some important role in the processes leading to Ca²⁺ release from the intracellular Ca store.     

Apoptosis-inducing protein derived from hepatocyte selectively induces apoptosis in lymphocytes

The liver is where lymphocytes undergo activation-induced cell death (AICD) at the resolution phase of an immune response, which is crucial for homeostasis of the immune system and prevention of autoimmunity. Exploring the machinery of AICD in the liver, we found that a primary culture supernatant of murine hepatocytes had an antiproliferative effect on antigen-stimulated T clone and T lymphoma cells. Biological study showed that the antiproliferation was due to induction of apoptosis in a caspase-dependent manner. The apoptosis-inducing potential was sensitive to trypsin, heat (> 70 degrees ) and acid (< pH 5) treatment but could not be neutralized by anti-tumour necrosis factor-alpha, anti-Fas ligand, or anti-transforming growth factor-beta antibodies. Biochemical study of the isolated and purified apoptosis-inducing component from the supernatant showed that it was a protein with a molecular mass of about 68,000-70,000. It induced apoptotic change in murine T and B cells, and to a lesser degree, in human lymphoid cells, but not in macrophages. Biochemical and biological characteristics distinguish this protein from others that have been reported to induce apoptosis of lymphocytes. The identification of an apoptosis-inducing protein derived from murine hepatocytes, which selectively induces apoptosis in lymphocytes, suggests one possible mechanism for immune suppression in the liver.     

Role of microfilaments in the exocytosis of rat peritoneal mast cells

When rat peritoneal mast cells were treated with the potent histamine releaser compound 48/80 in the presence of tetramethylrhodamine-labeled G-actin, the fluorescent G-actin particles were bound to the surface of extruded granules and to the cell surface. When rhodamine-phalloidin was incorporated into permeabilized rat mast cells in a Ca2+-free medium, rhodamine fluorescence was observed on the cell surface accompanied by serpentine ridges which appeared in the resting state. After perfusion with a cytosol-like solution containing Ca2+, rhodamine fluorescence appeared on the cell surface as a distinct network formation. In some cases, circular fluorescences which appeared to surround the extruded pores were observed in the cell membrane. These findings indicate the existence of actin filaments in the cell membrane and/or subplasmalemmal network. In whole-mount preparations, the granules were surrounded very densely with microfilaments of various widths. After exposure to compound 48/80, granules protruding through the cell membrane were wrapped in many filaments. The extruded granules located in the periphery of the cells were connected by many filamentous structures and disruptions in the middle of these connections were occasionally observed. In some cases, circular configurations of microfilaments were observed at the bottom of the extruded granules and in others dense gatherings of microfilaments were seen just beneath the granules, as if the latter were being pushed up and out of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

General Pathology Of Kawasaki Disease

Systemic pathological alterations were studied in thirty-seven autopsied patients with Kawasaki disease. Systemic vasculitis was the most characteristic pathological finding and was present in all the patients. In addition to the vasculitis, there was a high incidence of inflammatory lesions in various organs and tissues: in the heart, endocarditis, myocarditis, and pericarditis; in the digestive system, stomatitis, sialoduct-adenitis, catarrhal enteritis, hepatitis, cholangitis, pancreatitis, and pancreas ductitis; in the respiratory system, bronchitis and segmental interstitial pneumonia; in the urinary system, focal interstitial nephritis, cystitis, and prostatitis; in the nervous system, aseptic leptomeningitis, choriomeningitis, ganglionitis, and neuritis; in the hematopoietic system, lymphadenitis, splenitis, and thymitis. Dermatitis, panniculitis or myositis were also observed in some patients. Therefore, Kawasaki disease is a systemic inflammatory disease which mainly affects the cardiovascular system. These systemic inflammatory lesions are considered to correspond to the variegated clinical manifestaitions. The relationship between Kawasaki disease and infantile polyarteritis nodosa (IPN) were discussed, based on the clinicopathological characteristics.