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Biomedical subjects

T Kambe

Publications and source records attributed to T Kambe.

At least 37 records · Page 2Linked to original sources
PubMed1999

Functional coupling between various phospholipase A2s and cyclooxygenases in immediate and delayed prostanoid biosynthetic pathways.

Several distinct phospholipase A2s (PLA2s) and two cyclooxygenases (COXs) were transfected, alone or in combination, into human embryonic kidney 293 cells, and their functional coupling during immediate and delayed prostaglandin (PG)-biosynthetic responses was reconstituted. Signaling PLA2s, i.e. cytosolic PLA2 (cPLA2) (type IV) and two secretory PLA2s (sPLA2), types IIA (sPLA2-IIA) and V (sPLA2-V), promoted arachidonic acid (AA) release from their respective transfectants after stimulation with calcium ionophore or, when bradykinin receptor was cotransfected, with bradykinin, which evoked the immediate response, and interleukin-1 plus serum, which induced the delayed response. Experiments on cells transfected with either COX alone revealed subtle differences between the PG-biosynthetic properties of the two isozymes in that COX-1 and COX-2 were favored over the other in the presence of high and low exogenous AA concentrations, respectively. Moreover, COX-2, but not COX-1, could turn on endogenous AA release, which was inhibited by a cPLA2 inhibitor. When PLA2 and COX were coexpressed, AA released by cPLA2, sPLA2-IIA and sPLA2-V was converted to PGE2 by both COX-1 and COX-2 during the immediate response and predominantly by COX-2 during the delayed response. Ca2+-independent PLA2 (iPLA2) (type VI), which plays a crucial role in phospholipid remodeling, failed to couple with COX-2 during the delayed response, whereas it was linked to ionophore-induced immediate PGE2 generation via COX-1 in marked preference to COX-2. Finally, coculture of PLA2 and COX transfectants revealed that extracellular sPLA2s-IIA and -V, but neither intracellular cPLA2 nor iPLA2, augmented PGE2 generation by neighboring COX-expressing cells, implying that the heparin-binding sPLA2s play a particular role as paracrine amplifiers of the PG-biosynthetic response signal from one cell to another.

Animals↗
PubMed1999

Local renin-angiotensin system and mitogen-activated protein kinase activation in rat aorta.

We previously reported that endogenous angiotensin II is released to cause mitogen-activated protein (MAP) kinase stimulation in the media portion of the vasculature. In this study, we examined whether a functional renin-angiotensin system is indeed present within the media of the vasculature. In rat aortic strips, endothelium removal produced an increase of MAP kinase activity. The MAP kinase activation was inhibited either by the renin inhibitor pepstatin A or by the angiotensin-converting enzyme inhibitor captopril. The degree of the inhibition of the MAP kinase activation by pepstatin A, captopril and the angiotensin receptor antagonist losartan was almost the same. Pepstatin A inhibited MAP kinase activation induced by renin but not by angiotensin I and angiotensin II. Captopril inhibited the MAP kinase activation induced by angiotensin I but not by angiotensin II. In nephrectomized rat aortic strips, endothelium removal also produced an increase in MAP kinase activity, but the MAP kinase activation was considerably small and minimally inhibited by losartan. Nephrectomy produced a marked decrease in plasma renin activity. These findings suggest that an apparently fully intact and functional renin-angiotensin system is present in the media of the rat vasculature and this system serves to increase MAP kinase activity. It appears that renin plays the determining role in the regulation of angiotensin generation also in the media and the major source of the renin is renin of kidney origin.

Angiotensin I↗
PubMed1999

Evidence for the involvement of platelet-derived growth factor in the angiotensin II-induced growth of rat vascular smooth muscle cells.

This study examines the possible involvement of endogenous platelet-derived growth factor (PDGF) in the angiotensin II-induced growth of rat aortic smooth muscle cells. In quiescent confluent cells, anti-PDGF-AB neutralizing antibody inhibited angiotensin II-induced DNA synthesis and protein synthesis. PDGF-AA, -AB, and -BB produced concentration-dependent increases in DNA synthesis and protein synthesis. Genistein did not inhibit PDGF-AB-induced [3H]thymidine incorporation and [3H]leucine incorporation. PDGF-AB stimulated mitogen-activated protein (MAP) kinases, and PDGF-induced MAP kinase activation was inhibited by genistein. Angiotensin II induced PDGF-A chain messenger RNA expression, and genistein inhibited angiotensin-induced PDGF gene expression. These findings suggest that endogenous PDGF is, at least in part, involved in angiotensin II-induced cell growth in rat vascular smooth muscle cells. It appears that genistein inhibits angiotensin II-induced DNA synthesis partly by inhibiting PDGF-A gene expression.

Angiotensin II↗
PubMed1998

Induction of cyclooxygenase-2 by secretory phospholipases A2 in nerve growth factor-stimulated rat serosal mast cells is facilitated by interaction with fibroblasts and mediated by a mechanism independent of their enzymatic functions.

Mast cells exhibit a biphasic (immediate and delayed) eicosanoid-biosynthetic response after stimulation with particular cytokines or Fc epsilonRI (high affinity receptor for IgE) cross-linking. Treatment of rat serosal connective tissue mast cells (CTMC) with nerve growth factor (NGF) induced only the delayed phase of PGD2 generation that depended on inducible cyclooxygenase-2 (COX-2), but not constitutive COX-1, even though the subcellular distributions of these isoforms were similar. Experiments using several phospholipase A2 (PLA2) isozyme-specific probes and inhibitors suggested that both constitutive cytosolic PLA2 and inducible type IIA secretory PLA2 (sPLA2) are involved in NGF-initiated, COX-2-dependent, delayed PGD2 generation in rat CTMC. A type IIA sPLA2 inhibitor, but neither cytosolic PLA2 nor COX inhibitors, reduced, while adding exogenous type IIA sPLA2 augmented, NGF-induced COX-2 expression and its attendant PGD2 generation, indicating that the sPLA2-mediated increase in delayed PGD2 generation was attributable mainly to enhanced COX-2 expression. Type IIA sPLA2 and its close relative type V sPLA2 associated with fibroblastic cell surfaces increased NGF-induced COX-2 expression more efficiently than the soluble enzymes, revealing a particular juxtacrine sPLA2 presentation route. Surprisingly, catalytically inactive type IIA sPLA2 mutants, which were incapable of promoting arachidonic acid release from cytokine-primed cells, retained the ability to enhance COX-2 expression in CTMC, indicating that the COX-2-inducing activities of sPLA2 are independent of their catalytic functions.

Animals↗
PubMed1998

The functions of five distinct mammalian phospholipase A2S in regulating arachidonic acid release. Type IIa and type V secretory phospholipase A2S are functionally redundant and act in concert with cytosolic phospholipase A2.

We examined the relative contributions of five distinct mammalian phospholipase A2 (PLA2) enzymes (cytosolic PLA2 (cPLA2; type IV), secretory PLA2s (sPLA2s; types IIA, V, and IIC), and Ca2+-independent PLA2 (iPLA2; type VI)) to arachidonic acid (AA) metabolism by overexpressing them in human embryonic kidney 293 fibroblasts and Chinese hamster ovary cells. Analyses using these transfectants revealed that cPLA2 was a prerequisite for both the calcium ionophore-stimulated immediate and the interleukin (IL)-1- and serum-induced delayed phases of AA release. Type IIA sPLA2 (sPLA2-IIA) mediated delayed AA release and, when expressed in larger amounts, also participated in immediate AA release. sPLA2-V, but not sPLA2-IIC, behaved in a manner similar to sPLA2-IIA. Both sPLA2s-IIA and -V, but not sPLA2-IIC, were heparin-binding PLA2s that exhibited significant affinity for cell-surface proteoglycans, and site-directed mutations in residues responsible for their membrane association or catalytic activity markedly reduced their ability to release AA from activated cells. Pharmacological studies using selective inhibitors as well as co-expression experiments supported the proposal that cPLA2 is crucial for these sPLA2s to act properly. The AA-releasing effects of these sPLA2s were independent of the expression of the M-type sPLA2 receptor. Both cPLA2, sPLA2s-IIA, and -V were able to supply AA to downstream cyclooxygenase-2 for IL-1-induced prostaglandin E2 biosynthesis. iPLA2 increased the spontaneous release of fatty acids, and this was further augmented by serum but not by IL-1. Finally, iPLA2-derived AA was not metabolized to prostaglandin E2. These observations provide evidence for the functional cross-talk or segregation of distinct PLA2s in mammalian cells in regulating AA metabolism and phospholipid turnover.

Animals↗
PubMed1998

Evidence that angiotensin II, endothelins and nitric oxide regulate mitogen-activated protein kinase activity in rat aorta.

We measured the activity of mitogen-activated protein (MAP) kinases, enzymes believed to be involved in the pathway for cell proliferation, in rat aortic strips with or without endothelium, and examined effects of angiotensin receptor antagonists, endothelin receptor antagonists and nitric oxide (NO)-related agents. Endothelium removal produced an activation of MAP kinase activity in the strips, whereas the enzyme activity was not affected in the adventitia. The MAP kinase activation was inhibited by either the angiotensin AT1 receptor antagonist losartan or the endothelin ETA receptor antagonist BQ 123. The combination of both antagonists caused an additive inhibition. The angiotensin AT2 receptor antagonist PD 123,319 and the endothelin ETB receptor antagonist BQ 788 did not affect the MAP kinase activation. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) caused an activation of MAP kinase in the endothelium-intact aorta and the MAP kinase activation was inhibited by losartan or BQ123. The NO releaser nitroprusside inhibited the MAP kinase activation induced by endothelium removal or angiotensin II. These results suggest that even in isolated arteries, NO of endothelial origin tonically exert MAP kinase-inhibiting effects and endogenous angiotensin II and endothelins in the media are tonically released to cause MAP kinase-stimulating effects in medial smooth muscle.

Angiotensin II↗
PubMed1998

Embryonal carcinoma P19 cells produce erythropoietin constitutively but express lactate dehydrogenase in an oxygen-dependent manner.

Embryonic stem cells and embryonal carcinoma P19 cells produce erythropoietin (Epo) in an oxygen-independent manner, although lactate dehydrogenase A (LDHA) is hypoxia-inducible. To explore this paradox, we studied the operation of cis-acting sequences from these genes in P19 and Hep3B cells. The Epo gene promoter and 3' enhancer from P19 cells conveyed hypoxia-inducible responses in Hep3B cells but not in P19 cells. Together with DNA sequencing and the normal transcription start site of P19 Epo gene, this excluded the possibility that the noninducibility of Epo gene in P19 cells was due to mutation in these sequences or unusual initiation of transcription. In contrast, reporter constructs containing LDHA enhancer and promoter were hypoxia inducible in P19 and Hep3B cells, and mutation of a hypoxia- inducible factor 1 (HIF-1) binding site abolished the hypoxic inducibility in both cells, indicating that HIF-1 activation operates normally in P19 cells. Neither forced expression of hepatocyte nuclear factor 4 in P19 cells nor deletion of its binding site from the Epo enhancer was effective in restoring Epo enhancer function. P19 cells may lack an unidentified regulator(s) required for interaction of the Epo enhancer with Epo and LDHA promoters.

Base Sequence↗
PubMed1998

Effects of genistein and staurosporine on angiotensin II-induced DNA synthesis, protein synthesis and mitogen-activated protein kinase activation in vascular smooth muscle cells.

This study examines the effects of protein kinase inhibitors and activator on angiotensin II-induced DNA synthesis and protein synthesis of rat aortic smooth muscle cells. In quiescent confluent cells, angiotensin II induced a concentration-dependent increase in thymidine incorporation and leucine incorporation. The tyrosine kinase inhibitor genistein caused an inhibition of the angiotensin II-induced DNA synthesis but not of the agent-induced protein synthesis. The protein kinase C inhibitors staurosporine and calphostin C caused an inhibition of the angiotensin II-induced protein synthesis but not of the agent-induced DNA synthesis. The protein kinase C activator phorbol 12-myristate 13-acetate stimulated protein synthesis. Angiotensin II stimulated mitogen-activated protein (MAP) kinases and the angiotensin II-induced MAP kinase activation was inhibited by genistein but not by staurosporine. These findings suggest that angiotensin II-induced DNA synthesis is at least partly mediated via protein-tyrosine phosphorylation and angiotensin II-induced protein synthesis is at least partly mediated by activation of protein kinase C. It seems likely that MAP kinase activation is involved in DNA synthesis but not in protein synthesis induced by angiotensin II.

Angiotensin II↗
PubMed1997

Detection of secretory phospholipase A2s related but not identical to type IIA isozyme in cultured mast cells.

We previously reported that BALB/cJ mouse-derived bone marrow-derived mast cells (BMMC) exhibited two sequential phases of prostaglandin D2 (PGD2) generation in response to Fc(epsilon) receptor I (Fc(epsilon)RI) crosslinking and cytokine stimulation, the late phase of which was suppressed by an antibody raised against type IIA secretory phospholipase A2 (sPLA2). Here we report that BMMC derived from C57BL/6J mice, which are genetically deficient in type IIA sPLA2, display both immediate and delayed PGD2 generation normally. Lysates of C57BL/6J-derived BMMC contained a Ca2+-dependent PLA2 that was absorbed to a column conjugated with anti-type IIA sPLA2 antibody and had a similar molecular mass of 14 kDa, as assessed by immunoblotting. Therefore we speculate that a sPLA2 similar to, but distinct from, type IIA sPLA2 would compensate for type IIA sPLA2 deficiency in C57BL/6J-derived BMMC. We found that the two type IIA-related sPLA2 family members, type V and type IIC sPLA2s, were expressed in BMMC as well as in rat mastocytoma RBL-2H3 cells.

Animals↗
PubMed1997

Angiotensin type 2 receptor dephosphorylates Bcl-2 by activating mitogen-activated protein kinase phosphatase-1 and induces apoptosis.

We examined the cellular and signaling mechanism of angiotensin II (Ang II) type 2 (AT2) receptor-induced apoptosis in PC12W (rat pheochromocytoma cell line) cells that express abundant AT2 receptor but not Ang II type 1 receptor. In these cells, nerve growth factor (NGF) inhibited the internucleosomal DNA fragmentation induced by serum depletion, whereas Ang II antagonized this NGF cell survival action and induced apoptosis. We studied the mechanism of NGF and AT2 receptor interaction on apoptosis by examining their effects on the survival factor Bcl-2. AT2 receptor activation did affect intracellular Bcl-2 protein levels. Bcl-2 phosphorylation was stimulated by NGF, whereas AT2 receptor activation blocked this NGF effect. Pretreatment with antisense oligonucleotide of mitogen-activated protein (MAP) kinase phosphatase-1 enhanced the effects of NGF on MAP kinase activation and Bcl-2 phosphorylation but attenuated the inhibitory effects of AT2 receptor on MAP kinase, Bcl-2 phosphorylation, and apoptosis. Taken together, these results suggest that MAP kinase plays a critical role in inhibiting apoptosis by phosphorylating Bcl-2. The AT2 receptor inhibits MAP kinase activation, resulting in the inactivation of Bcl-2 and the induction of apoptosis.

Animals↗
PubMed1994

Effects of leukotriene D4 on myocardial blood flow and high energy phosphate concentration in anesthetized dogs.

The effects of intracoronary administration of leukotriene D4 (LTD4) on myocardial blood flow (MBF) and myocardial energy metabolism in anesthetized open-chest dogs were examined, and compared with those of coronary ligation. Two series of experiments were conducted. In the first, LTD4 (0-3.0 micrograms/kg) was injected into the left anterior descending coronary artery (LAD) and MBF was measured. While no changes in MBF were observed after 0.5 microgram/kg of LTD4, a significant decrease in MBF in the LAD area was apparent after 1.0 micrograms/kg of LTD4, with a return to baseline values by within 10 min after the injection. With 3.0 micrograms/kg of LTD4, MBF remained decreased up to 15 min after the injection. In the second study, myocardial high energy phosphate concentrations in the LAD area were determined 5 min after LTD4 administration and compared to those after ligation. ATP levels in the 1.0-3.0 micrograms/kg LTD4 groups were significantly less than those in the ligation group, although there were no associated significant differences in MBF values in the LAD area. These results indicate that LTD4 brings about changes in myocardial energy metabolism which are not secondary to reduced blood flow.

Anesthesia↗
PubMed1993

[The quantitative assay of (1-->3)-beta-D-glucan in culture medium of Candida albicans using G-test].

It has been difficult to diagnose the deep-seated fungal infection. Limulus test which originally has been developed to detect endotoxin in blood is also activated by (1-->3)-beta-D-glucan, the cell wall component of the fungi. Factor G in limulus lysate is activated by (1-->3)-beta-D-glucan and not by endotoxin. The quantitative assay of (1-->3)-beta-D-glucan is possible by the G-test using factor G. (1-->3)-beta-D-glucan in RPMI culture medium of Candida albicans was periodically measured using G-test and the effect of antifungal drug or neutrophil to the changes of (1-->3)-beta-D-glucan in the culture medium was studied. Increase in the level of (1-->3)-beta-D-glucan was in parallel with the growth of Candida albicans. G-test may be applied to the clinical diagnosis of fungal infection.

Candida albicans↗
PubMed1992

Primary structure of the alanine carrier protein of thermophilic bacterium PS3.

Purified alanine carrier proteins were cleaved into peptides either chemically after solubilization in 1,1,1,3,3,3-hexafluoro-2-propanol or proteolytically with lysylendopeptidase. From the amino acid sequence analyses of these peptides, we synthesized a DNA probe and utilized it for successful cloning of a gene encoding the alanine carrier protein (acp gene). The 5'-flanking region was determined by an inverse polymerase chain reaction, and an open reading frame consisting of 1,335 nucleotides was found. The amino acid sequence deduced from the open reading frame consists of 445 amino acids, and all the partial amino acid sequences determined are included in the sequence. Although the calculated M(r) of 47,803 is significantly larger than the apparent M(r) of 42,500 as reported previously (Hirata, H., Kambe, T., and Kagawa, Y. (1984) J. Biol. Chem. 259, 10653-10656), an in vitro translation experiment revealed that the product of the acp gene migrates at a position coinciding with that of the purified alanine carrier. Hydropathy analysis suggests that the protein contains at least 8 hydrophobic segments presumably spanning membrane. A homology search on a database reveals relatively high scores of homology with either the Escherichia coli melibiose carrier or the human Na+/glucose symporter, particularly in the region from Leu246 to Glu286. Furthermore, the region also reveals low but significant similarities to other Na(+)-coupled symporters.

Amino Acid Sequence↗
PubMed1991

Ischemic myocardial mitochondrial function and ultrastructural change--influence of regional myocardial blood flow.

Myocardial blood flow (MBF), tissue ATP content, mitochondrial respiratory function and mitochondrial ultrastructure were examined in 62 adult mongrel dogs weighing 6-14 kg in which acute myocardial ischemia had been produced under anesthesia. The left anterior descending coronary artery was dissected free for ligation before the first diagonal branch. MBF was measured before coronary ligation and 60 min following ligation. Then, samples of myocardium were taken and subjected to tissue ATP content assay, mitochondrial respiratory function measurement respiratory control index (RCI) and rate of oxygen consumption in state III (QO2III); and electron microscopic examination. Mitochondrial morphologic injury was evaluated quantitatively according to Schaper's criteria. MBF was significantly correlated with tissue ATP content, mitochondrial respiratory function and mitochondrial ultrastructural change. When MBF was less than 20 ml/min/100g, tissue ATP content (1.86 +/- 1.21 mumol/g wet weight) and mitochondrial respiratory function (RCI 2.51 +/- 0.59) were significantly lower than in the non-ischemic area (ATP 4.52 +/- 1.11, RCI 3.82 +/- 0.37), and mitochondrial ultrastructural injury had deteriorated significantly at an MBF below 40 ml/min/100 g. In conclusion, our findings show that when MBF is reduced, mitochondrial ultrastructural changes precede the depression in mitochondrial oxidative phosphorylation.

Adenosine Triphosphate↗