Yasuki Ishizaki

Cerebral microvessel endothelium is producing endothelin.

Endothelin, a potent vasoconstrictor peptide, was recently isolated from the supernatant of the cultured endothelia of the porcine aorta and is now supposed to be the most likely candidate for the endothelium-derived contractile factor (EDCF), which is responsible for the endothelium-dependent vasoconstriction by various stimuli. In this study, the production of endothelin by the endothelia of porcine cerebral microvessels was revealed by the Northern blot analysis with porcine endothelin cDNA probe and the enzyme-linked immunosorbent assay (ELISA) with anti-porcine endothelin antibody. Our results raise the possibility that the endothelia of cerebral microvessels regulate the local blood flow within the brain through the production of endothelin.

A caspase inhibitor blocks ischaemia-induced delayed neuronal death in the gerbil

Caspases play a critical role in the cell death machinery in various cell types. Here we investigated the involvement of caspases in the delayed neuronal death after transient global forebrain ischaemia in the gerbil. Intrahippocampal injection of benzyloxycarbonyl‐Asp‐CH2‐dichlorobenzene (zD), an irreversible inhibitor of caspases, saved hippocampal CA1 neurones from chromatin condensation and DNA fragmentation at post‐ischaemia day 4, and these neurones maintained normal morphology at day 8 post‐insult. Intrahippocampal injection of interleukin‐1β (IL‐1β) after ischaemic insults did not influence the neuroprotective effect of zD, suggesting that the neuroprotective effect does not depend on the inhibition of mature IL‐1β production. Animals that received zD‐injection showed significant improvement in step‐through and step‐down passive avoidance learning at post‐ischaemia days 4 and 5, suggesting that neural functions were preserved in these animals. At post‐ischaemia day 4, the cleavage of poly(ADP‐ribose)polymerase was observed, and this cleavage was almost completely suppressed in zD‐injected hippocampus, suggesting involvement of caspase‐3 and caspase‐3‐like caspase in the delayed neuronal death. Our findings indicate that caspases play important roles in the delayed neuronal death after transient global forebrain ischaemia in the gerbil, and suggest that ischaemia‐induced brain damage can be blocked by caspase inhibitors.

TRPV2 Enhances Axon Outgrowth through Its Activation by Membrane Stretch in Developing Sensory and Motor Neurons

Thermosensitive TRP (thermo TRP) channels are well recognized for their contributions to sensory transduction, responding to a wide variety of stimuli including temperature, nociceptive stimuli, touch, and osmolarity. However, the precise roles for the thermo TRP channels during development have not been determined. To explore the functional importance of thermo TRP channels during neural development, the temporal expression was determined in embryonic mice. Interestingly, TRPV2 expression was detected in spinal motor neurons in addition to the dorsal root ganglia from embryonic day 10.5 and was localized in axon shafts and growth cones, suggesting that the channel is important for axon outgrowth regulation. We revealed that endogenous TRPV2 was activated in a membrane stretch-dependent manner in developing neurons by knocking down the TRPV2 function with dominant-negative TRPV2 and TRPV2-specific shRNA and significantly promoted axon outgrowth. Thus, for the first time we revealed that TRPV2 is an important regulator for axon outgrowth through its activation by membrane stretch during development.

Synthesis and release of acetylcholine by cultured bovine arterial endothelial cells

Using cultured endothelial cells prepared from bovine carotid artery, and a specific radioimmunoassay for acetylcholine (ACh), the synthesis and release of ACh by vascular endothelial cells were investigated directly. ACh content in the culture medium after 24 h of incubation in the presence of isoflurophate, a nonspecific cholinesterase inhibitor, was about 16 times higher than that in endothelial cells, indicating that ACh synthesized inside the cells was released rapidly. The presence of ACh in the culture medium was further confirmed qualitatively using high-performance liquid chromatography with an electrocapture detection. These results represent the first direct evidence that endothelial cells can synthesize and release ACh, suggesting the possibility that ACh acts not only as a neurotransmitter but also as an autacoid under certain conditions.

Presence of nitric oxide synthase activity in the neurons of the rat embryonal cerebrum

Nitric oxide (NO), apparently identical to endothelium-derived relaxing factor (EDRF) in blood vessels, has been reported to be formed in adult brain tissue, and the possible physiological significance of NO in neuron functions has been suggested. The present study was undertaken to demonstrate the biochemical evidence of the presence of NO synthase activity in intact neurons. In this report, we will demonstrate first that NO synthase is also present in fetal brain where neurons can easily be isolated from and can be cultured in vitro and then we will present the evidence that the cultured neurons are responsible for NO production. We will also show that the distribution pattern of NO synthase in adult brain detected by our biochemical method is well consistent with the immunohistochemical data reported so far. NO synthase activity, which was measured as the formation of [3H]citrulline from [3H]arginine in the presence of Ca2+ and reduced nicotinamide adenine dinucleotide phosphate (NADPH), existed in the homogenate of adult rat cerebrum, olfactory bulb and cerebellum. The cerebellum showed the strongest NO synthase activity and the cerebrum showed the least but significant activity. It was also revealed that NO synthase activity also existed in the homogenate of cerebra of rat embryos, the activity, however, was about half of that in adult cerebrum. Furthermore, the homogenate of the cultured neurons prepared from the rat embryonal cerebra has also shown NO synthase activity. These results suggest that NO has some important roles in the development of the neuron system.

The role of cerebral microvessel endothelium in regulation of cerebral blood flow through production of endothelin-1.

We have previously reported the production of endothelin-1 (ET-1) by the cerebral microvessel endothelia and suggested an important role of ET-1 and microvessel endothelia in the regulation of local blood flow within the brain. In the present study, radioimmunoassay of ET-1 revealed that ET-1, produced by cultured cerebral microvessel endothelia grown on a filter, is released mainly to the basal side (corresponding to the basement membrane side), not to the apical side (corresponding to the vascular lumen side). This might indicate that ET-1 constricts arterioles locally at the same place where it is produced by endothelia. The present results also show that cultured cerebral microvessel endothelia produce less ET-1 under low oxygen pressure and that they produce more ET-1 under low carbon dioxide pressure. Taken together, our results may suggest that the negative feedback regulation of cerebral blood flow through oxygen and carbon dioxide pressure is mediated by ET-1 produced locally by cerebral microvessel endothelia.

Genetic background conversion ameliorates semi-lethality and permits behavioral analyses in cystathionine β-synthase-deficient mice, an animal model for hyperhomocysteinemia

Cystathionine beta-synthase-deficient mice (Cbs(-/-)) exhibit several pathophysiological features similar to hyperhomocysteinemic patients, including endothelial dysfunction and hepatic steatosis. Heterozygous mutants (Cbs(+/-)) on the C57BL/6J background are extensively analyzed in laboratories worldwide; however, detailed analyses of Cbs(-/-) have been hampered by the fact that they rarely survive past the weaning age probably due to severe hepatic dysfunction. We backcrossed the mutants with four inbred strains (C57BL/6J(Jcl), BALB/cA, C3H/HeJ and DBA/2J) for seven generations, and compared Cbs(-/-) phenotypes among the different genetic backgrounds. Although Cbs(-/-) on all backgrounds were hyperhomocysteinemic/hypermethioninemic and suffered from lipidosis/hepatic steatosis at 2 weeks of age, >30% of C3H/HeJ-Cbs(-/-) survived over 8 weeks whereas none of DBA/2J-Cbs(-/-) survived beyond 5 weeks. At 2 weeks, serum levels of total homocysteine and triglyceride were lowest in C3H/HeJ-Cbs(-/-). Adult C3H/HeJ-Cbs(-/-) survivors showed hyperhomocysteinemia but escaped hypermethioninemia, lipidosis and hepatic steatosis. They appeared normal in general behavioral tests but showed cerebellar malformation and impaired learning ability in the passive avoidance step-through test, and required sufficient dietary supplementation of cyst(e)ine for survival, demonstrating the essential roles of cystathionine beta-synthase in the central nervous system function and cysteine biosynthesis. Our C3H/HeJ-Cbs(-/-) mice could be useful tools for investigating clinical symptoms such as mental retardation and thromboembolism that are found in homocysteinemic patients.

A Novel Subtype of Astrocytes Expressing TRPV4 (Transient Receptor Potential Vanilloid 4) Regulates Neuronal Excitability via Release of Gliotransmitters

Background: The functional differences among astrocyte subtypes are uncharacterized. Results: Only a subset of astrocytes expresses TRPV4 and regulates neurons. Conclusion: TRPV4+ astrocytes release ATP and glutamate to regulate neurons. Significance: Astrocytes can be classified by TRPV4 expression and by function. Astrocytes play active roles in the regulation of synaptic transmission. Neuronal excitation can evoke Ca2+ transients in astrocytes, and these Ca2+ transients can modulate neuronal excitability. Although only a subset of astrocytes appears to communicate with neurons, the types of astrocytes that can regulate neuronal excitability are poorly characterized. We found that ∼30% of astrocytes in the brain express transient receptor potential vanilloid 4 (TRPV4), indicating that astrocytic subtypes can be classified on the basis of their expression patterns. When TRPV4+ astrocytes are activated by ligands such as arachidonic acid, the activation propagates to neighboring astrocytes through gap junctions and by ATP release from the TRPV4+ astrocytes. After activation, both TRPV4+ and TRPV4− astrocytes release glutamate, which acts as an excitatory gliotransmitter to increase synaptic transmission through type 1 metabotropic glutamate receptor (mGluR). Our results indicate that TRPV4+ astrocytes constitute a novel subtype of the population and are solely responsible for initiating excitatory gliotransmitter release to enhance synaptic transmission. We propose that TRPV4+ astrocytes form a core of excitatory glial assembly in the brain and function to efficiently increase neuronal excitation in response to endogenous TRPV4 ligands.

Methionine excess in diet induces acute lethal hepatitis in mice lacking cystathionine γ-lyase, an animal model of cystathioninuria.

Physiological roles of the transsulfuration pathway have been recognized by its contribution to the synthesis of cytoprotective cysteine metabolites, such as glutathione, taurine/hypotaurine, and hydrogen sulfide (H(2)S), whereas its roles in protecting against methionine toxicity remained to be clarified. This study aimed at revealing these roles by analyzing high-methionine diet-fed transsulfuration-defective cystathionine γ-lyase-deficient (Cth(-/-)) mice. Wild-type and Cth(-/-) mice were fed a standard diet (1 × Met: 0.44%) or a high-methionine diet (3 × Met or 6 × Met), and hepatic conditions were monitored by serum biochemistry and histology. Metabolome analysis was performed for methionine derivatives using capillary electrophoresis- or liquid chromatography-mass spectrometry and sulfur-detecting gas chromatography. The 6 × Met-fed Cth(-/-) (not 1 × Met-fed Cth(-/-) or 6 × Met-fed wild type) mice displayed acute hepatitis, which was characterized by markedly elevated levels of serum alanine/aspartate aminotransferases and serum/hepatic lipid peroxidation, inflammatory cell infiltration, and hepatocyte ballooning; thereafter, they died of gastrointestinal bleeding due to coagulation factor deficiency. After 1 week on 6 × Met, blood levels of ammonia/homocysteine and hepatic levels of methanethiol/3-methylthiopropionate (a methionine transamination product/methanethiol precursor) became significantly higher in Cth(-/-) mice than in wild-type mice. Although hepatic levels of methionine sulfoxide became higher in 6 × Met-fed wild-type mice and Cth(-/-) mice, those of glutathione, taurine/hypotaurine, and H(2)S became lower and serum levels of homocysteine became much higher in 6 × Met-fed Cth(-/-) mice than in wild-type mice. Thus, transsulfuration plays a critical role in the detoxification of excessive methionine by circumventing aberrant accumulation of its toxic transamination metabolites, including ammonia, methanethiol, and 3-methylthiopropionate, in addition to synthesizing cysteine-derived antioxidants to counteract accumulated pro-oxidants such as methionine sulfoxide and homocysteine.

Possible involvement of a chloride-bicarbonate exchanger in apoptosis of endothelial cells and cardiomyocytes.

We examined the role of ion movement in staurosporine‐induced apoptosis of vascular endothelial cells. Cultured vascular endothelial cells from bovine carotid arteries were used. Apoptosis was determined by propidium iodide assay. Treatment of the endothelial cells with staurosporine (10nmol/l‐1μmol/l) for 6h induced nuclear fragmentation in a dose‐dependent manner. Staurosporine (1μmol/l) elicited apoptosis in 70.5±1.5% of cells. Concomitant treatment of endothelial cells with 1mmol/l of 4,4‐diisothiocyanatostilbene‐2,2‐disulfonic acid (DIDS), a chloride—bicarbonate exchange blocker, completely inhibited staurosporine‐induced apoptosis. Other ion transporter inhibitors such as dimethyl amiloride and anthracene‐9 carboxylic acid were less effective inhibitors of staurosporine‐induced apoptosis of endothelial cells. DIDS prevented staurosporine‐induced apoptosis of endothelial cells as well as cardiomyocytes. Next, we determined whether chloride ions or bicarbonate are involved in apoptosis. Incubation with a chloride ion removal buffer did not inhibit staurosporine‐induced apoptosis of endothelial cells. However, endothelial cell apoptosis was completely suppressed by an inhibitor of caspase, benzyloxycarbonyl‐Asp‐CH2‐O(C)O‐dichlorobenzene (zD‐dcb, 50μmol/l). Staurosporine (1μmol/l) increased the intracellular pH of endothelial cells, and DIDS (1mmol/l), but not a caspase inhibitor, inhibited this increase in pH caused by staurosporine. Our findings suggest that endothelial cell apoptosis induced by staurosporine may be associated with the Cl−and bicarbonate (HCO−3) ions. Thus, Cl−efflux from cells or HCO−3 influx to cells (which increases pH) may play an important role in signal transduction leading events such as activation of caspase in staurosporine‐induced apoptosis.