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Biomedical subjects

I Kudo

Publications and source records attributed to I Kudo.

At least 109 records · Page 6Linked to original sources
PubMed1994

Characterization of cytosolic phospholipase A2 in rat mastocytoma RBL-2H3.

We previously reported that cultured mast cells expressed three discrete phospholipases A2 (PLA2S), one of which showed a remarkable preference for phospholipids bearing an arachidonoyl residue at the sn-2 position [M. Murakami, et al., J. Biochem., 111, 175 (1992)]. In the present study, we have purified and characterized this enzyme using rat mastocytoma RBL-2H3 as an enzyme source. The elution profiles of the arachidonoyl-preferential PLA2 activity of rat mastocytoma RBL-2H3 cells on several column chromatographies were indistinguishable from those of 85-kDa cytosolic PLA2 (cPLA2) characterized so far. The molecular mass of the partially purified PLA2 was estimated to be about 90 kDa by gel filtration and it hydrolyzed arachidonate-containing phospholipids preferentially in the presence of submicromolar Ca2+ concentrations. Furthermore, it was immunoprecipitated with an anti-rabbit cPLA2 antibody almost completely. From these observations, we have concluded that the arachidonoyl-preferential PLA2 in mast cells belongs to the "cPLA2" family.

Animals↗
PubMed1993

Participation in cellular prostaglandin synthesis of type-II phospholipase A2 secreted and anchored on cell-surface heparan sulfate proteoglycan.

Rat-liver-derived BRL-3A cells, which express both type-II phospholipase A2 (PLA2) and cytosolic PLA2 (cPLA2), generated prostaglandin E2 (PGE2) in the presence of fetal calf serum. When the cells were treated with tumor necrosis factor (TNF), PGE2 generation was greatly stimulated. The production of PGE2 observed in both cases was suppressed by a type-II PLA2-specific inhibitor, thielocin A1. Appreciable amounts of type-II PLA2 were released into the medium from the TNF-stimulated cells when heparin was added extracellularly. The release of type-II PLA2 from TNF-stimulated cells was also found in the presence of heparan sulfate or dextran sulfate, whereas other glycosaminoglycans showed no effects under the same conditions. These findings suggest that type-II PLA2 expressed in BRL-3A cells mostly associates with the cell surface by binding to cellular heparan sulfate proteoglycan. Removal of cell-surface-associated type-II PLA2, by either extracellular addition of heparin or by prior treatment of the BRL-3A cells with heparitinases, resulted in marked reduction of PGE2 synthesis in the cells. Exposure of BRL-3A cells to thrombin also induced the apparent secretion of type-II PLA2, and thrombin-stimulated PGE2 generation was suppressed by heparin effectively. Type-II PLA2 secreted and attached to heparan sulfate on the cell surface may therefore play an essential role in PGE2 synthesis by BRL-3A cells.

Animals↗
PubMed1993

Characteristics of lysophospholipase activity expressed by cytosolic phospholipase A2.

Evidence has accumulated to suggest that a wide variety of mammalian cells and tissues express a cytosolic phospholipase A2 with arachidonoyl preference (cPLA2). Purified rabbit platelet-derived cPLA2, as well as the human recombinant enzyme originally identified in the monocytic leukemic cell line U937, exhibit significant lysophospholipase activity. Several series of experiments indicated that a single protein mediated both activities. Treatment of the purified enzyme with p-bromophenacylbromide or an anti-(rabbit platelet cPLA2) monoclonal antibody, RHY-5, suppressed the activity of phospholipase A2 without any appreciable effect on lysophospholipase activity, suggesting that the domain(s) required for phospholipase A2 activity may be located separately from that for lysophospholipase activity. Lysophospholipase activity was appreciably detected above the critical micellar concentration of the substrate. Lysophosphatidylcholine was also hydrolyzed efficiently when it was incorporated into liposomes made of dialkylphosphatidylcholine. The hydrolysis of lysophospholipid was dependent on the fatty acid bound at the sn1 position; the relative rates of hydrolysis of 1-oleoyllysophosphatidylcholine, 1-palmitoyllysophosphatidylcholine, and 1-stearoyllysophosphatidylcholine were 23, 8, and 1, respectively. A similar order of reactivity was observed with lysophospholipid incorporated into dialkylphosphatidylcholine liposomes. cPLA2 may function not only as an arachidonate liberation enzyme but also as an enzyme responsible for degradation of certain molecular species of lysophospholipids formed in membranes.

Animals↗
PubMed1993

Triggering of degranulation in mast cells by exogenous type II phospholipase A2.

We have previously shown the possibility that endogenous type II phospholipase A2 (PLA2) might participate in degranulation in mast cells (MC) (Murakami, M., et al. 1992. Eur. J. Biochem. 209:257). Now we have examined whether or not exogenously added type II PLA2 triggers MC degranulation. When rat peritoneal connective tissue MC (CTMC) were exposed to purified rat type II PLA2 at concentrations of more than 10 micrograms/ml, significant release of histamine was observed, whereas PGD2 was not generated under the same conditions. Mouse peritoneal CTMC as well as bone marrow-derived immature MC also responded to PLA2. Preincubation of CTMC with tyrosine kinase inhibitors, genistein, and herbimycin A, but not with pertussis toxin, resulted in abolition of the sensitivity to PLA2. The ability of type II PLA2 to induce histamine release was inhibited by an antibody or chemicals, both of which blocked the catalytic activity of type II PLA2. Heparin or an antibody recognizing the heparin-binding domain of type II PLA2 also suppressed the MC-degranulating activity, probably due to inhibition of binding of PLA2 to the cells. The interaction between heparan sulfate on cell surface and the heparin-binding domain of type II PLA2 may be important for the induction of exocytosis. The catalytic domain of the enzyme is also crucially important for the degranulation induction. Furthermore, we found that nerve growth factor, one of the potent regulators of MC function, significantly potentiated type II PLA2-induced histamine release from rat CTMC. These results suggest the possible role of extracellular type II PLA2 in activation of CTMC primed with nerve growth factor at inflamed sites.

Animals↗
PubMed1993

Possible role of mammalian secretory group II phospholipase A2 in T-lymphocyte activation: implication in propagation of inflammatory reaction.

Both 2-lysophosphatidylcholine and cis-unsaturated fatty acids were previously shown to intensify agonist-induced cellular responses by enhancing the diacylglycerol-dependent activation of protein kinase C. Consistent with these observations, extracellular, secretory group II phospholipase A2, when added directly to human resting T lymphocytes, greatly potentiates their activation that was induced by a membrane-permeant diacylglycerol and ionomycin, as determined by the expression of the alpha subunit of the interleukin 2 receptor and thymidine incorporation into DNA. Diacylglycerol and ionomycin were essential for this cellular response, and phospholipase A2 alone showed no effect. The amount of 2-lysophosphatidylcholine produced in these cells by the exogenous phospholipase A2 was greatly increased in the presence of diacylglycerol and ionomycin, suggesting that the membrane phospholipids became susceptible to the phospholipase A2 when protein kinase C was activated. The results suggest that phospholipase A2 secreted into inflammatory sites plays a role in the propagation of cellular responses. Protein kinase C may function in the hydrolysis of membrane phospholipids by the exogenous phospholipase A2, and the products of this phospholipid hydrolysis enhance agonist-induced protein kinase C activation, thereby intensifying cellular responses.

Cell Membrane Permeability↗
PubMed1993

Molecular nature of phospholipases A2 involved in prostaglandin I2 synthesis in human umbilical vein endothelial cells. Possible participation of cytosolic and extracellular type II phospholipases A2.

A lysate of unstimulated human umbilical vein endothelial cells (HUVEC) exhibited phospholipase A2 (PLA2) activity, which hydrolyzed phospholipids bearing arachidonate more preferentially than those bearing linoleate at the sn-2 position. An anti-rabbit cytosolic PLA2 monoclonal antibody absorbed the activity, whereas an anti-human type II PLA2 monoclonal antibody did not. HUVEC treated with thrombin generated prostaglandin I2 (PGI2), and the PLA2 activity of the thrombin-stimulated cells was absorbed almost completely by the anti-cytosolic PLA2 antibody. HUVEC treated with tumor necrosis factor (TNF) also generated PGI2. PGI2 generation by TNF-treated cells was suppressed partially by extracellular addition of the anti-type II PLA2 antibody. PLA2 activity in a lysate of TNF-stimulated cells was increased about 2-3-fold, and about half of the increased activity was suppressed by the anti-type II PLA2 antibody. Addition of heparin together with TNF resulted in release of type II PLA2 in the medium. Thus, both cytosolic and type II PLA2s may be involved in agonist-stimulated PGI2 synthesis in HUVEC. Furthermore, exogenously added type II PLA2 was bound to the cell surface and synergistically enhanced PGI2 generation in TNF-stimulated HUVEC. This binding was blocked by either heparin or a monoclonal antibody recognizing the heparin-binding domain of type II PLA2. Taken together, type II PLA2 generated endogenously as well as added exogenously may be captured on the HUVEC surface via heparan sulfate proteoglycan and may contribute to cellular arachidonate metabolism.

Antibodies, Monoclonal↗
PubMed1993

De novo synthesis of phospholipase A2 and prostacyclin production by proliferating rat smooth muscle cells.

We investigated the role of phospholipase A2 (PLA2) in cell cycle-dependent alterations of endogenous prostacyclin (PGI2) synthesis in aortic smooth muscle cells in culture (VSMC) from Wistar Kyoto rats. Randomly cycling VSMC generated more PGI2 than the stationary cells. Cell cycle analysis showed that PGI2 production capacity was increased from the G0/G1 through the early DNA synthetic (S) phases. Enzyme analysis revealed that, although there were different mechanisms underlying this increase in the PGI2 production during the G0/G1, the peak at 4 hours coincided with a sharp increase in PLA2 activity. This increase in PLA2 activity was preceded by an increased expression of functional PLA2 messenger RNA, and protein synthesis inhibition prevented most of the increase in PGI2 production at 4 hours. These data indicate that endogenous PGI2 generation is mainly increased during the G0/G1 period and that this event is secondary to de novo synthesis of PLA2 and probably, at least in part, to cyclooxygenase induction. This mechanism provides a negative feedback regulating VSMC proliferation.

Animals↗
PubMed1993

Preferential hydrolysis of phosphatidylethanolamine in rat ischemic heart homogenates during in vitro incubation.

Phospholipid-hydrolyzing activities were examined in rat hearts with ischemia induced by occlusion of the left main coronary artery. When homogenates of ischemic heart were incubated in vitro at 37 degrees C, a significant amount of phosphatidylethanolamine (PE) was degraded, whereas the contents of other phospholipids did not change significantly. During the incubation, a stoichiometrical amount of lysoPE was concomitantly formed. The lysoPE formed had mainly saturated fatty acids and its composition resembled that of fatty acids detected at the sn-1 position in the glycerol backbone of heart PE. No appreciable PE degradation was observed in homogenates prepared from nonischemic rat heart. No difference in phospholipase activities was found between ischemic and nonischemic heart homogenates when exogenous radioactive phospholipids were used as substrates. Rabbit anti-rat 14-kDa type II phospholipase A2 antibody suppressed the degradation of PE observed in ischemic heart homogenates. These findings indicate that the type II phospholipase A2 activity may be involved in the breakdown of endogenous PE in ischemic heart homogenates.

Animals↗
PubMed1993

Dynamics and participation of type II phospholipase A2 in rat zymosan-induced pleurisy.

Intrapleural injection of zymosan into rats induces acute inflammation characterized by plasma leakage and cellular influx. The level of type II phospholipase A2 (PLA2) increased in the pleural fluid as well as in exudating leukocytes after the injection of zymosan. Rather low PLA2 activity was found in cell lysates, though treatment of such lysates at low pH increased the PLA2 activity drastically. The appearance of "acid-extracted" PLA2 activity in leukocytes preceded that of the extracellular enzyme activity, suggesting that pleural leukocytes might be one of the origins of the extracellular enzyme. Treatment of exudating pleural leukocytes with purified rat type II PLA2 elicited the production of prostaglandin E2, but not platelet-activating factor appreciably. These findings indicate that type II PLA2 might play a role in the progression of inflammation through the production of eicosanoids in the present inflammation model.

Animals↗
PubMed1992

Release of 14-kDa group-II phospholipase A2 from activated mast cells and its possible involvement in the regulation of the degranulation process.

Group II phospholipase A2 was detected in appreciable amounts in rat peritoneal mast cells. The effect of several inhibitors specific to 14-kDa group-II phospholipase A2, including two proteinaceous inhibitors and a product of microorganisms with a low molecular mass, on mast-cell activation was examined. When rat peritoneal mast cells were sensitized with IgE and then challenged with antigen, the specific phospholipase-A2 inhibitors suppressed histamine release in a concentration-dependent manner. By contrast, these inhibitors showed no effect on prostaglandin generation under the same conditions. Histamine release from rat peritoneal mast cells subjected to non-immunochemical stimuli, such as concanavalin A, the Ca2+ ionophore A23187, compound 48/80 and substance P was also suppressed. When rat peritoneal mast cells were treated with 14-kDa-group-II-phospholipase-A2-specific inhibitors, washed and stimulated, histamine release was not affected appreciably. Similar suppressive effects of the inhibitors on histamine release were observed with mouse cultured bone-marrow-derived mast cells. When bone-marrow-derived mast cells were activated, they secreted both a soluble and an ecto-enzyme form of 14-kDa group-II phospholipase A2, although appearance of the enzyme associated with the external surface of cells was observed transiently. An appreciable amount of membrane phospholipids was degraded during activation of mast cells, which was decreased by treatment with 14-kDa-group-II-phospholipase-A2 inhibitor. These observations suggest that degranulation and eicosanoid generation in mast cells are regulated independently by discrete phospholipases A2 and that the 14-kDa group-II phospholipase A2 released from mast cells during activation may play an essential role in the progression of the degranulation process.

Animals↗
PubMed1992

Change in phospholipid composition of mouse bone marrow-derived mast cells during cultivation with fibroblasts.

A mouse bone marrow-derived mast cell (BMMC) clone, designated as MC-MKM, was established. MC-MKM cells released histamine and generated lipid chemical mediators such as prostaglandin D2 or platelet-activating factor upon stimulation with IgE and antigen. It is known that BMMCs acquired the characteristics of connective tissue mast cells (CTMCs) during coculture with fibroblasts. In the present study, we found that cellular phospholipid composition changed drastically when MC-MKM cells were cultured for 2 weeks in the presence of mouse 3T3 fibroblasts. In MC-MKM cells, phosphatidylcholine was the predominant phospholipid class, followed by phosphatidylethanolamine. During coculture with fibroblasts for 2 weeks, total phospholipid content in MC-MKM cells increased 2.5-fold. Among major phospholipid classes, a percentage of phosphatidylethanolamine increased most dramatically in accordance with a decrease in that of phosphatidylcholine, while no appreciable change in composition of other phospholipids was observed. The phospholipid composition of cocultured MC-MKM cells was closer to that of rat peritoneal CTMCs than that of the starting MC-MKM cells. The present findings that BMMCs converted their phenotype into CTMC-like cells by the interaction with fibroblasts in terms of phospholipid composition are not contradictory to the previous observations that BMMCs changed into CTMC-like cells.

3T3 Cells↗
PubMed1992

Characteristics and possible functions of mast cell phospholipases A2.

Phospholipase A2 activity in lysates of mast cells and their related cells [mouse bone marrow-derived IL-3 dependent mast cells (BMMC), rat connective tissue mast cells (CTMC), and rat mastocytoma RBL-2H3 cells] was measured using phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylcholine (PC) as exogenous substrates. Both BMMC and RBL cells showed rather high phospholipase A2 activity, whereas CTMC showed only weak activity. These cells contained at least three types of phospholipase A2. Type 1 enzyme showed no appreciable affinity to heparin, and preferentially hydrolyzed either PC or PE, both of which have an arachidonic acid at the sn-2 position. The activity was absorbed by monoclonal antibody against rabbit platelet cytosolic 85-kDa phospholipase A2. Type 2 enzyme had an affinity to heparin, and was completely inhibited by anti-rat platelet 14-kDa secretory phospholipase A2. This enzyme could be expressed as an "ecto-type" enzyme on the cell surface and might be secreted from cells when mast cells are activated. Type 3 enzyme also had an affinity to heparin, but was separated from type 2 enzyme on reverse-phase HPLC. This enzyme did not interact with anti-14-kDa secretory enzyme antibody. Purified type 3 enzyme (30-kDa) specifically hydrolyzed PS. p-Bromophenacylbromide inhibited all types of phospholipase A2, whereas mepacrine inhibited type 2 and type 3 enzymes, but not type 1 enzyme. Type 2 enzyme was also inhibited by the specific antibody, complement degradation product, and a small-molecular-weight inhibitor. Histamine release was inhibited by all these inhibitors, whereas PGD2 production was inhibited only by p-bromophenacylbromide. Possible roles for these phospholipases A2 in mast cell function are proposed.

Animals↗
PubMed1992

Immunochemical detection of arachidonoyl-preferential phospholipase A2.

Monoclonal antibodies were raised against rabbit platelet cytosolic arachidonoyl-preferential phospholipase A2. The antibodies precipitated the arachidonoyl-preferential phospholipase A2 activity in the soluble fraction of a rabbit platelet lysate in combination with an immobilized anti-mouse immunoglobulin antibody, and reacted predominantly with a protein exhibiting a molecular weight of approximately 88,000 on immunoblotting analysis. All three antibodies established so far reacted with human platelet arachidonoyl-preferential phospholipase A2 as effectively as the rabbit platelet enzyme. One of them reacted with the rat platelet arachidonoyl-preferential enzyme, whereas none of them reacted with rabbit platelet secretory 14-kDa group II phospholipase A2. The existence of an immunologically related phospholipase A2 was further shown in rabbit granulocytes, brain, lung, and liver, rat and mouse mast cells, and human monocytoma U937 cells. Thus, an arachidonoyl-preferential phospholipase A2 with similar structural properties appeared to be expressed in a variety of cells and tissues.

Animals↗
PubMed1992

Detection of three distinct phospholipases A2 in cultured mast cells.

Phospholipase A2 activity in lysates of mast cells such as rat mastocytoma RBL-2H3 cells and mouse bone marrow-derived IL-3-dependent mast cells (BMMC) was measured using phosphatidylcholine (PC), phosphatidylethanolamine (PE), or phosphatidylserine (PS) as a substrate. Both types of cells exhibited phospholipase A2 activity with a similar pH profile; the optimum pH observed with PS as a substrate was 5.5-7.4, whereas that with PE or PC was 8.0-9.0. PE and PC bearing an arachidonate at the sn-2 position were cleaved more efficiently by PE, PC-hydrolyzing phospholipase A2 than phospholipids with a linoleate. A monoclonal antibody raised against rabbit platelet 85-kDa cytosolic phospholipase A2 absorbed the PE, PC-hydrolyzing activity. PS-hydrolyzing activity was purified from RBL-2H3 cells and BMMC by sequential heparin-Sepharose, butyl-Toyo-pearl, and reverse-phase HPLC. On reverse-phase HPLC, the PS-hydrolyzing activity of RBL cells was separated into two peaks, A and B. The peak B activity was inhibited by the anti-rat 14-kDa group II phospholipase A2 antibody, while the peak A activity was not. The partially purified peak A activity hydrolyzed PS about 10-fold more efficiently than PE at optimum pH of 5.5-7.4. No appreciable hydrolysis was observed with PC or phosphatidylinositol (PI). Thus, mast cells may express at least three distinct phospholipases A2; 14-kDa group II phospholipase A2, 85-kDa cytosolic arachidonate preferential phospholipase A2, and a novel phospholipase A2 that shows high substrate specificity for PS.

Animals↗
PubMed1992

Histopathological reactions of calcium phosphate cement.

Calcium phosphate cement (CPC) consisting of Ca4 (PO4)2O and CaHPO4 (2H2O) was recently developed. This study evaluated in vivo aspects of CPC and CPC mixtures compared to those of commercial hydroxyapatite (HP) and several endodontic materials: Grossman's cement (GC), calcium hydroxide-iodine paste (CHP) and gutta-percha plate (GP). Biocompatibility of subcutaneous implants in Donryu rats was evaluated after one month. Results showed very slight inflammatory reactions from CPC, CPC mixtures and HP. The materials were surrounded by thin fibrous connective tissues with a small number of lymphocytes and plasma cells. Severe inflammatory reactions were provoked by GC. Granulation tissues induced by CHP resembled those of pseudoxanthomatous granuloma. The GP material was encapsulated by relatively thick fibrous connective tissues with little inflammatory reactions.

Animals↗