Biomedical subjects

Masahiro Oike

Publications and source records attributed to Masahiro Oike.

17 recordsLinked to original sources

PubMed2006

Pivotal role of integrin alpha5beta1 in hypotonic stress-induced responses of human endothelium.

We have previously reported that both hypotonic stress (HTS) and lysophosphatidic acid (LPA) induce ATP release and a transient reorganization of actin through sequential activation of RhoA/Rho-kinase and focal adhesion kinase F-actin (FAK)/paxillin in human umbilical cord vein endothelial cells (HUVECs). LPA is known to induce the activation of RhoA via its specific receptors, but the mechanisms by which HTS initiates these intracellular signals are not known. The present study aimed to identify the molecule(s) that are unique to the sensing and/or transducing the mechanical stress. Reverse transcriptase-polymerase chain reaction revealed the expression of several integrin subunits in HUVECs. Anti-integrin alpha5beta1 antibody (Ab), but not anti-integrin alpha2, alpha6, alpha v, or beta4 antibodies, inhibited HTS-induced RhoA translocation, tyrosine phosphorylation of FAK and paxillin, ATP release, and actin reorganization. However, the LPA-induced ATP release and actin reorganization were not inhibited by any of these anti-integrin antibodies, indicating that integrin alpha5beta1 plays a pivotal role in the HTS-induced but not in the LPA-induced responses. It is therefore reasonable to assume that this particular subtype of integrin is involved in the initiation of the responses induced by mechanical stimuli in HUVECs.

Actins↗

PubMed2005

Excess l-arginine restores endothelium-dependent relaxation impaired by monocrotaline pyrrole.

The pyrrolizidine alkaloid plant toxin monocrotaline pyrrole (MCTP) causes pulmonary hypertension in experimental animals. The present study aimed to examine the effects of MCTP on the endothelium-dependent relaxation. We constructed an in vitro disease model of pulmonary hypertension by overlaying MCTP-treated bovine pulmonary artery endothelial cells (CPAEs) onto pulmonary artery smooth muscle cell-embedded collagen gel lattice. Acetylcholine (Ach) induced a relaxation of the control CPAEs-overlaid gels that were pre-contracted with noradrenaline, and the relaxation was inhibited by L-NAME, an inhibitor of NO synthase (NOS). In contrast, when MCTP-treated CPAEs were overlaid, the pre-contracted gels did not show a relaxation in response to Ach in the presence of 0.5 mM l-arginine. Expression of endothelial NOS protein, Ach-induced Ca2+ transients and cellular uptake of l-[3H]arginine were significantly smaller in MCTP-treated CPAEs than in control cells, indicating that these changes were responsible for the impaired NO production in MCTP-treated CPAEs. Since cellular uptake of l-[3H]arginine linearly increased according to its extracellular concentration, we hypothesized that the excess concentration of extracellular l-arginine might restore NO production in MCTP-treated CPAEs. As expected, in the presence of 10 mM l-arginine, Ach showed a relaxation of the MCTP-treated CPAEs-overlaid gels. These results indicate that the impaired NO production in damaged endothelial cells can be reversed by supplying excess l-arginine.

Animals↗

PubMed2005

Role of PRIP-1, a novel Ins(1,4,5)P3 binding protein, in Ins(1,4,5)P3-mediated Ca2+ signaling.

PRIP-1 was isolated as a novel inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] binding protein with a domain organization similar to phospholipase C-delta1 (PLC-delta1) but lacking the enzymatic activity. Further studies revealed that the pleckstrin homology (PH) domain of PRIP-1 is the region responsible for binding Ins(1,4,5)P3. In this study we aimed to clarify the role of PRIP-1 at the physiological concentration in Ins(1,4,5)P3-mediated Ca2+ signaling, as we had previously used COS-1 cells overexpressing PRIP-1 (Takeuchi et al., 2000, Biochem J 349:357-368). For this purpose we employed PRIP-1 knock out (PRIP-1-/-) mice generated previously (Kanematsu et al., 2002, EMBO J 21:1004-1011). The increase in free Ca2+ concentration in response to purinergic receptor stimulation was lower in primary cultured cortical neurons prepared from PRIP-1-/- mice than in those from wild type mice. The relative amounts of [3H]Ins(1,4,5)P3 measured in neurons labeled with [3H]inositol was also lower in cells from PRIP-1-/- mice. In contrast, PLC activities in brain cortex samples from PRIP-1-/- mice were not different from those in the wild type mice, indicating that the hydrolysis of Ins(1,4,5)P3 is enhanced in cells from PRIP-1-/- mice. In vitro analyses revealed that type1 inositol polyphosphate 5-phosphatase physically interacted with a PH domain of PRIP-1 (PRIP-1PH) and its enzyme activity was inhibited by PRIP-1PH. However, physical interaction with these two proteins did not appear to be the reason for the inhibition of enzyme activity, indicating that binding of Ins(1,4,5)P3 to the PH domain prevented its hydrolyzation. Together, these results indicate that PRIP-1 plays an important role in regulating the Ins(1,4,5)P3-mediated Ca2+ signaling by modulating type1 inositol polyphosphate 5-phosphatase activity through binding to Ins(1,4,5)P3.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Sequential activation of RhoA and FAK/paxillin leads to ATP release and actin reorganization in human endothelium.

We have investigated the cellular mechanisms of mechanical stress-induced immediate responses in human umbilical vein endothelial cells (HUVECs). Hypotonic stress (HTS) induced ATP release, which evoked a Ca(2+) transient, followed by actin reorganization within a few minutes, in HUVECs. Disruption of the actin cytoskeleton did not suppress HTS-induced ATP release, and inhibition of the ATP-mediated Ca(2+) response did not affect actin reorganization, thereby indicating that these two responses are not interrelated. ATP release and actin reorganization were also induced by lysophosphatidic acid (LPA). HTS and LPA induced membrane translocation of RhoA, which occurs when RhoA is activated, and tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin. Tyrosine kinase inhibitors (herbimycin A or tyrphostin 46) inhibited both HTS- and LPA-induced ATP release and actin reorganization, but did not affect RhoA activation. In contrast, Rho-kinase inhibitor (Y27632) inhibited all of the HTS- and LPA-induced responses. These results indicate that the activation of the RhoA/Rho-kinase pathway followed by tyrosine phosphorylation of FAK and paxillin leads to ATP release and actin reorganization in HUVECs. Furthermore, the fact that HTS and LPA evoke exactly the same intracellular signals and responses suggests that even these immediate mechanosensitive responses are in fact not mechanical stress-specific.

Actins↗

PubMed2004

[ATP release pathways in vascular endothelial cells].

Vascular endothelial cells regulate vascular tonus, growth, and angiogenesis in response to mechanical stresses. ATP release is one of well-known mechanosensitive responses in endothelial cells. Released ATP induces Ca(2+) responses and nitric oxide production in neighboring cells in an auto/paracrine manner. Mechanosensitive and agonist-induced ATP releases are also observed in other cell types, but the cellular mechanisms and pathways of ATP release are largely unknown. Reported candidates for ATP release pathways are ABC proteins including P-glycoprotein and CFTR, exocytosis of ATP-containing vesicles, and ATP-permeable anion channels. In vascular endothelium, vesicular exocytosis, volume-regulated anion channels (VRAC), and connexin hemichannels have been reported as candidates for ATP release pathways. We found that VRAC inhibitors suppressed hypotonic stress-induced ATP release in bovine aortic endothelial cells. Furthermore, extracellular ATP suppressed VRAC current in a voltage dependent manner, which could be fitted to the permeation-blocker model with a Kd(0) of 1 mM and delta value of 0.41. However, it should be noted that VRAC is probably not the only pathway for ATP release in the endothelium, because basal ATP release was not inhibited by VRAC inhibitors. Further investigations are definitely warranted to clarify the details and therapeutic significance of mechanosensitive ATP release in the endothelium.

Adenosine Triphosphate↗

PubMed2003

Constitutive nitric oxide production in bovine aortic and brain microvascular endothelial cells: a comparative study.

Vascular endothelium constitutively generates nitric oxide (NO) in large vessels and induces a relaxation of smooth muscle cells. However, little is known about the production of NO in microvessels, where smooth muscle layers are thin or absent. In this study, we have compared the constitutive production of NO in bovine brain microvascular endothelial cells (BBECs) with that in bovine aortic endothelial cells (BAECs). ATP, acetylcholine (ACh) and A23187 induced Ca(2+) transients both in BBECs and BAECs. In contrast, although ATP and A23187 evoked a similar degree of [Ca(2+)](i) increase in both types of cell, they failed to induce NO production in BBECs, as measured with an NO-sensitive fluorescent dye DAF-2, whereas in BAECs there was an increase in DAF-2 fluorescence. Hypotonic stress induced ATP release and subsequent NO production in BAECs, but not in BBECs. We have developed an in vitro model vessel system that consists of aortic smooth muscle cells embedded in a collagen gel lattice and overlaid with endothelial cells. Precontracted gels showed relaxation in response to ACh, when BAECs were overlaid. However, ACh-induced relaxation was not observed in BBEC-overlaid gels. Expression of eNOS protein as well as cellular uptake of l-[(3)H]arginine were significantly lower in BBECs than in BAECs. These results indicate that Ca(2+)-dependent NO production is at an undetectable level in BBEC, for which at least two factors, i.e. low levels of eNOS expression and l-arginine uptake, are responsible.

Adenosine Triphosphate↗

PubMed2003

Theophylline and cAMP inhibit lysophosphatidic acid-induced hyperresponsiveness of bovine tracheal smooth muscle cells.

We have established an in vitro model of airway hyperresponsiveness, using a bovine tracheal smooth muscle cell (BTSMC)-embedded collagen gel lattice. When the gel was pretreated with lysophosphatidic acid (LPA), which activates the small G protein RhoA, ATP- and high K+ solution-induced gel contraction was significantly augmented. This was not due to the modulation of Ca2+ mobilizing properties, since ATP- and high K+-induced Ca2+ transients were not significantly different between control and LPA-treated BTSMC. Y-27632, an inhibitor of Rho-kinase, suppressed the LPA-induced augmentation of gel contraction, whereas it did not inhibit the contraction of control gels. Theophylline (> or = 1 microM) reversed the LPA-induced augmentation of gel contraction, whereas it inhibited control gel contraction only with a very high concentration (100 microM). We confirmed that theophylline increased the intracellular concentration of cAMP ([cAMP]i) in BTSMC. Elevation of [cAMP]i with dibutyryl cAMP or forskolin also reversed the LPA-induced augmentation of gel contraction. Furthermore, theophylline, as well as dibutyryl cAMP and forskolin, suppressed the LPA-induced membrane translocation of RhoA, indicating that they prevented airway hyperresponsiveness by inhibiting RhoA. We conclude from these results that theophylline inhibits LPA-induced, RhoA/Rho-kinase-mediated hyperresponsiveness of tracheal smooth muscle cells due to the accumulation of cAMP.

Actins↗

PubMed2003

Involvement of proteinase-activated receptor-2 in mast cell tryptase-induced barrier dysfunction in bovine aortic endothelial cells.

We report here a direct modulation by mast cell tryptase of endothelial barrier function through activation of proteinase-activated receptor-2 (PAR-2). In cultured bovine aortic endothelial cells (BAECs), tryptase, trypsin and PAR-2 activating peptide impaired the barrier function as determined by the permeability of protein-conjugated Evans blue. The tryptase-induced barrier dysfunction was completely blocked by U73122, and partially reversed by xestospongin C, calphostin C or Y27632. The intracellular Ca(2+) was elevated by tryptase. It was notable that ioxaglate, a contrast material that degranulates mast cells, markedly increased the permeability when applied to BAECs in combination with mast cells, an action that was blocked by nafamostat, a potent tryptase inhibitor. Immunofluorescence analysis showed that actin stress fibre formation and disruption of VE-cadherin were observed after exposure to tryptase or ioxaglate in combination with mast cells. Therefore, it is suggested that mast cell tryptase impairs endothelial barrier function through activation of endothelial PAR-2 in a manner dependent on the phospholipase C activity.

Actins↗

PubMed2003

DOCK2 is essential for antigen-induced translocation of TCR and lipid rafts, but not PKC-theta and LFA-1, in T cells.

DOCK2 is a mammalian homolog of Caenorhabditis elegans CED-5 and Drosophila melanogaster Myoblast City which are known to regulate actin cytoskeleton. DOCK2 is critical for lymphocyte migration, yet the role of DOCK2 in TCR signaling remains unclear. We show here that DOCK2 is essential for TCR-mediated Rac activation and immunological synapse formation. In DOCK2-deficient T cells, antigen-induced translocation of TCR and lipid rafts, but not PKC-theta and LFA-1, to the APC interface was severely impaired, resulting in a significant reduction of antigen-specific T cell proliferation. In addition, we found that the efficacy of both positive and negative selection was reduced in DOCK2-deficient mice. These results suggest that DOCK2 regulates T cell responsiveness through remodeling of actin cytoskeleton via Rac activation.

Animals↗

PubMed2003

[Regulatory role of mechanical stress response in cellular function: development of new drugs and tissue engineering].

The investigation of mechanotransduction in the cardiovascular system is essentially important for elucidating the cellular and molecular mechanisms involved in not only the maintenance of hemodynamic homeostasis but also etiology of cardiovascular diseases including arteriosclerosis. The present review summarizes the latest research performed by six academic groups, and presented at the 75th Annual Meeting of the Japanese Pharmacological Society. Technology of cellular biomechanics is also required for research and clinical application of a vascular hybrid tissue responding to pulsatile stress. 1) Vascular tissue engineering: Design of pulsatile stress-responsive scaffold and in vivo vascular wall reconstruction (T. Matsuda); 2) Cellular mechanisms of mechanosensitive calcium transients in vascular endothelium (M. Oike et al.); 3) Cross-talk of stimulation with fluid flow and lysophosphatidic acid in vascular endothelial cells (K. Momose et al.); 4) Mechanotransduction of vascular smooth muscles: Rate-dependent stretch-induced protein phosphorylations and contractile activation (K. Obara et al.); 5) Lipid mediators in vascular myogenic tone (I. Laher et al.); and 6) Caldiomyocyte regulates its mechanical output in response to mechanical load (S. Sugiura et al.).

Animals↗

PubMed2003

Calcium-dependent injury of human microvascular endothelial cells induced by a variety of iodinated radiographic contrast media.

RATIONALE AND OBJECTIVES: The aim of the present study was to determine the possible mechanisms underlying the endothelial cell damage induced by iodinated radiographic contrast materials (RCM). METHODS: The cultured human skin microvascular endothelial cells (HMVECs) were exposed to various contrast media, and the cell viability was measured by mitochondrial enzyme activity. Nuclear damage was assessed by Hoechst 33342 staining and a fluorescent single-cell gel electrophoresis. The effects of contrast materials on the cellular ATP content and intracellular free Ca2+ concentration were subsequently examined. RESULTS: Although the iodinated RCM tested all caused the cell injury in HMVECs, ionic RCM including amidotrizoate and ioxaglate were more potent in producing the cell damage than nonionic RCM. It is unlikely that the contrast material-induced cell damage is associated with hyperosmolality, since hyperosmolar solution of mannitol or NaCl had no marked influence on the endothelial cell viability. Nuclear damage was noted in cells exposed to amidotrizoate. Amidotrizoate lowered cellular ATP content while elevating the intracellular free Ca2+ concentration. It was notable that the RCM-induced endothelial cell damage was reversed by the chelation of intracellular Ca2+ with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid but not by the removal of extracellular Ca2+. CONCLUSIONS: Both ionic and nonionic contrast materials caused nuclear damage of endothelial cells. The decrease in tissue ATP content and elevation of intracellular Ca2+ are likely to contribute to the contrast materials-induced endothelial cell damage.

Adenosine Triphosphate↗

PubMed2003

[Analysis of airway hyperresponsiveness using in vitro contraction model].

We have established an in vitro model of airway hyperresponsiveness, using bovine tracheal smooth muscle cells (BTSMC)-embedded collagen gel lattice. When the gel was pretreated with lysophosphatidic acid (LPA), which activates small G-protein RhoA, ATP- and high K+ solution-induced gel contraction was significantly augmented. This was not due to the modulation of Ca(2+)-mobilizing properties, since ATP- and high K(+)-induced Ca2+ transients were not different significantly between control and LPA-treated BTSMC. Y-27632, an inhibitor of Rho-kinase, suppressed the LPA-induced augmentation of gel contraction, whereas it did not inhibit the contraction of control gels. Theophylline (> or = 1 microM) reversed the LPA-induced augmentation of gel contraction, whereas it inhibited control gel contraction only with a very high concentration (100 microM) Theophylline suppressed the LPA-induced membrane translocation of RhoA, indicating that it prevented airway hyperresponsiveness by inhibiting RhoA. We conclude from these results that theophylline inhibits LPA-induced, RhoA/Rho-kinase-mediated hyperresponsiveness of tracheal smooth muscle cells.

Animals↗

PubMed2002

Functional implications of Ca2+ mobilizing properties for nitric oxide production in aortic endothelium.

We have investigated the relationship between Ca2+ mobilization and the cellular production of nitric oxide (NO) by using fura-2 and diaminofluorescein-2 (DAF-2), an NO-sensitive dye, in bovine aortic endothelial cells (BAEC). High concentrations of ATP (100 microM) or thapsigargin (1 micro M) depleted intracellular Ca2+ store sites with a single Ca2+ transient, and induced an increase in DAF-2 fluorescence even in Ca2+-free solution, thereby indicating that store depletion leads to NO production. The same level of increase in DAF-2 fluorescence was elicited by low concentrations of ATP (1 micro M), which induced Ca2+ oscillations but did not deplete store sites, only in the presence of extracellular Ca2+. Furthermore, inhibition of ATP (1 micro M)-induced Ca2+ entry with La3+ suppressed DAF-2 fluorescence. ATP (0.3 micro M), applied in Ca2+-free, Mn2+-containing solution induced Mn2+ entry-coupled fura-2 quenching, repeating shortly after each oscillation peak. These results indicate that NO is produced preferentially by entered Ca2+, and that Ca2+ oscillations, which are induced by low levels of stimulation, play a significant role in NO production by strongly modulating Ca2+ entry.

Adenosine Triphosphate↗

PubMed2002

Tumor cell apoptosis by irradiation-induced nitric oxide production in vascular endothelium.

We examined the effects of X-ray irradiation on endothelial nitric oxide (NO) production in bovine aortic endothelial cells (BAECs). Single irradiation of up to 60 Gy did not affect the expression of endothelial NO synthase (eNOS) mRNA, as assessed by reverse transcription-PCR, in BAECs. The basal level of intracellular Ca(2+) concentration and Ca(2+) mobilization induced by thapsigargin and ATP were also not affected by single irradiation. However, eNOS-mediated, Ca(2+)-dependent constitutional NO production could not be examined directly because irradiated BAECs showed continuous NO production, as measured by diaminofluorescein-2 fluorescence, without agonist stimulation. Expression of inducible NO synthase (iNOS) mRNA and protein was markedly increased by 2 Gy of irradiation, thereby indicating that the basal and continuous NO production in irradiated BAECs was due to the expression of iNOS. Hepatoma HepG2 cells cocultured with irradiated BAECs showed apoptosis in the presence of L-arginine, and the apoptosis was prevented by L-NAME (N(omega)-nitro-L-arginine methyl ester). These results indicate that single irradiation does not affect Ca(2+) mobilization and eNOS expression but induces the expression of iNOS in BAECs, and the latter property would be beneficial to induce apoptosis in the adjacent tumor cells.

Animals↗

PubMed2002

Propofol prevents endothelial dysfunction induced by glucose overload.

Surgical operations often induce acute hyperglycemia, which is known to affect endothelial functions. In this study, we examined the effects of propofol, a commonly used general anaesthetic, on bovine aortic endothelial cell (BAEC) dysfunction induced by glucose overload. 2 D-glucose overload (23 mM) induced an accumulation of superoxide anion (O2-), assessed by MCLA chemiluminescence, to a similar extent as that generated by 233 microU ml(-1) xanthine oxidase (XO) and 100 micro M xanthine. Propofol inhibited this accumulation with an IC50 of 0.21 micro M, whereas much higher concentrations of propofol were required to scavenge O2- generated by 250 microU ml(-1) XO and 100 microM xanthine (IC50: 13.5 micro M). 3 D-glucose overload attenuated ATP-induced NO production which was detected using diaminofluorescence-2 (DAF-2). The inhibition was reversed by propofol with an EC50 of 0.60 microM. In contrast, inhibitions caused by xanthine/XO were not altered by propofol (1 microM). 4 D-glucose overload suppressed ATP-induced Ca2+ oscillations and capacitative Ca2+ entry (CCE), which were both restored by superoxide dismutase, indicating that O2- was responsible. Propofol restored these attenuated Ca2+ oscillations and CCE with EC50 of 0.31 and 1.0 microM, respectively. 5 D-glucose overload (23 mM) increased the intracellular glucose concentration 4 fold, compared with cells exposed to 5.75 mM glucose, and 1 micro M propofol reduced this increase to 2.8 fold. 6 We conclude from these results that anaesthetic concentrations of propofol prevent the impairment of Ca2+-dependent NO production in BAEC induced by glucose overload. This effect is mainly due to the reduction of O2- accumulation, and involves, at least in part, the inhibition of cellular glucose uptake.

Animals↗

PubMed2002

Volume-regulated anion channels serve as an auto/paracrine nucleotide release pathway in aortic endothelial cells.

Mechanical stress induces auto/paracrine ATP release from various cell types, but the mechanisms underlying this release are not well understood. Here we show that the release of ATP induced by hypotonic stress (HTS) in bovine aortic endothelial cells (BAECs) occurs through volume-regulated anion channels (VRAC). Various VRAC inhibitors, such as glibenclamide, verapamil, tamoxifen, and fluoxetine, suppressed the HTS-induced release of ATP, as well as the concomitant Ca(2+) oscillations and NO production. They did not, however, affect Ca(2+) oscillations and NO production induced by exogenously applied ATP. Extracellular ATP inhibited VRAC currents in a voltage-dependent manner: block was absent at negative potentials and was manifest at positive potentials, but decreased at highly depolarized potentials. This phenomenon could be described with a "permeating blocker model," in which ATP binds with an affinity of 1.0 +/- 0.5 mM at 0 mV to a site at an electrical distance of 0.41 inside the channel. Bound ATP occludes the channel at moderate positive potentials, but permeates into the cytosol at more depolarized potentials. The triphosphate nucleotides UTP, GTP, and CTP, and the adenine nucleotide ADP, exerted a similar voltage-dependent inhibition of VRAC currents at submillimolar concentrations, which could also be described with this model. However, inhibition by ADP was less voltage sensitive, whereas adenosine did not affect VRAC currents, suggesting that the negative charges of the nucleotides are essential for their inhibitory action. The observation that high concentrations of extracellular ADP enhanced the outward component of the VRAC current in low Cl(-) hypotonic solution and shifted its reversal potential to negative potentials provides more direct evidence for the nucleotide permeability of VRAC. We conclude from these observations that VRAC is a nucleotide-permeable channel, which may serve as a pathway for HTS-induced ATP release in BAEC.

Adenosine Diphosphate↗

PubMed2002

Superoxide anion impairs contractility in cultured aortic smooth muscle cells.

We examined the effects of superoxide anion (O) generated by xanthine plus xanthine oxidase (X/XO) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) and muscle contractility in cultured bovine aortic smooth muscle cells (BASMC). Cells were grown on collagen-coated dish for the measurement of [Ca(2+)](i). Pretreatment with X/XO inhibited ATP-induced Ca(2+) transient and Ca(2+) release-activated Ca(2+) entry (CRAC) after thapsigargin-induced store depletion, both of which were reversed by superoxide dismutase (SOD). In contrast, Ca(2+) transients induced by high-K(+) solution and Ca(2+) ionophore A-23187 were not affected by X/XO. BASMC-embedded collagen gel lattice, which was pretreated with xanthine alone, showed contraction in response to ATP, thapsigargin, high-K(+) solution, and A-23187. Pretreatment of the gel with X/XO impaired gel contraction not only by ATP and thapsigargin, but also by high-K(+) solution and A-23187. The X/XO-treated gel showed normal contraction; however, when SOD was present during the pretreatment period. These results indicate that O(2)(-) attenuates smooth muscle contraction by impairing CRAC, ATP-induced Ca(2+) transient, and Ca(2+) sensitivity in BASMC.

Adenosine Triphosphate↗