Bovine Carotid Artery Research Articles

Arachidonic acid pretreatment enhances smooth muscle cell migration via increased Ca 2+ influx

It is well known that vascular smooth muscle cell (SMC) migration is an initial step in atheromatous plaque formation. In the present study, we investigated the effects of arachidonic acid (AA, 20:4 n-6) on bovine carotid artery SMC migration using the modified Boyden chamber technique. SMCs pretreated with 2.5 μg/ml of AA for 2 days, showed an enhanced migration response to fetal bovine serum. AA pretreatment (0.5-5.0 μg/ml) increased fetal bovine serum-induced SMC migration dose-dependently, and maximum stimulation was observed at a concentration of 2.5 μg/ml. However, AA pretreatment did not enhance fetal bovine serum-induced endothelial cell migration. Using lipid analysis, we found that AA was substantially incorporated into cellular phospholipids. When SMC migration was induced by platelet derived growth factor (PDGF)-BB, instead of serum, the stimulative effect of AA pretreatment was retained. SMCs pretreated with AA showed greater mobilization of intracellular Ca 2+ in response to PDGF-BB than SMCs without AA pretreatment (controls). Nifedipine, a Ca 2+ channel blocker, and glycoletherdiamine-tetraacetic acid (EGTA) had no effect on PDGF-induced migration of controls but both drugs reduced the enhanced PDGE-induced migration of AA-pretreated SMCs to the control level. Baicalein, an inhibitor of 12-lipoxygenase, reduced PDGF-BB-induced migration of both control and AA pretreated SMCs, however the AA-pretreated cells still showed enhanced migration compared to control cells. These findings suggest that AA accelerates SMC migration in the thickening of the intima during atheroma formation, via stimulation of extracellular Ca 2+ influx.

Thrombin-induced vasospasm: Cellular signaling mechanisms

Background: In the setting of arterial injury, thrombin contributes to the hemostatic process by activating the coagulation cascade and platelets. We hypothesized that thrombin also contributes to hemostasis by inducing vasospasm. The purpose of this investigation was to characterize the cellular signaling mechanisms that modulate thrombin-induced vascular smooth muscle contractions. Methods: Contractile responses of intact bovine carotid artery smooth muscles were determined in a muscle bath. Contractile responses were correlated with phosphorylation events as determined with whole cell phosphorylation and two-dimensional gel electrophoresis and with immunoblotting of glycerol-urea or two-dimensional gels. Results: Thrombin (1 to 1000 units/ml) induced sustained vascular smooth muscle contractions of similar magnitude as the potent contractile agonist, endothelin. Thrombin-induced contractions were associated with increases in the phosphorylation of the myosin light chains (MLC 20) and in the tyrosine phosphorylation of mitogen-activated protein kinase. Conclusions: These data suggest that thrombin is a potent physiologic contractile agonist that may modulate some forms of vasospasm. Thrombin-induced contractions are associated with the activation of two cellular signaling pathways, the myosin light chain kinase and the mitogen-activated protein kinase pathways. (Surgery 1998;123:46-50.)

Thrombin-induced vasospasm: Cellular signaling mechanisms

Background: In the setting of arterial injury, thrombin contributes to the hemostatic process by activating the coagulation cascade and platelets. We hypothesized that thrombin also contributes to hemostasis by inducing vasospasm. The purpose of this investigation was to characterize the cellular signaling mechanisms that modulate thrombin-induced vascular smooth muscle contractions. Methods: Contractile responses of intact bovine carotid artery smooth muscles were determined in a muscle bath. Contractile responses were correlated with phosphorylation events as determined with whole cell phosphorylation and two-dimensional gel electrophoresis and with immunoblotting of glycerol-urea or two-dimensional gels. Results: Thrombin (1 to 1000 units/ml) induced sustained vascular smooth muscle contractions of similar magnitude as the potent contractile agonist, endothelin. Thrombin-induced contractions were associated with increases in the phosphorylation of the myosin light chains (MLC20) and in the tyrosine phosphorylation of mitogen-activated protein kinase. Conclusions: These data suggest that thrombin is a potent physiologic contractile agonist that may modulate some forms of vasospasm. Thrombin-induced contractions are associated with the activation of two cellular signaling pathways, the myosin light chain kinase and the mitogen-activated protein kinase pathways. (Surgery 1998;123:46-50.)

Small heat shock proteins and vasospasm in human umbilical artery smooth muscle.

Human umbilical artery smooth muscle is uniquely refractory to cyclic nucleotide-dependent vasorelaxation. Small heat shock proteins (HSPs) have been implicated as contractile regulatory proteins. Thus, we hypothesized that alterations in the phosphorylation of small HSPs may contribute to human umbilical artery smooth muscle vasospasm. Physiologic contractile responses were determined in a muscle bath and compared with phosphorylation events determined with whole-cell phosphorylation and 2-dimensional gel electrophoresis. Precontraction of bovine carotid artery smooth muscle with serotonin followed by relaxation with forskolin was associated with increases in the phosphorylation of HSP27 and HSP20. Precontraction of umbilical artery with serotonin followed by forskolin treatment led to increases in the phosphorylation of HSP27. However, the umbilical artery smooth muscle did not relax, nor was there an increase in the phosphorylation of HSP20 with forskolin treatment. These data suggest that impaired cyclic nucleotide-dependent relaxation of umbilical artery smooth muscle is associated with a lack of phosphorylation of HSP20.

Experimental investigation of the distribution of residual strains in the artery wall.

Arterial wall stresses are thought to be a major determinant of vascular remodeling both during normal growth and throughout the development of occlusive vascular disease. A completely physiologic mechanical model of the arterial wall should account not only for its residual strains but also for its structural nonhomogeneity. It is known that each layer of the artery wall possesses different mechanical properties, but the distribution of residual strain among the different mechanical components, and thus the true zero stress state, remain unknown. In this study, two different sets of experiments were carried out in order to determine the distribution of residual strains in artery walls, and thus the true zero stress state. In the first, collagen and elastin were selectively eliminated by chemical methods and smooth muscle cells were destroyed by freezing. Dissolving elastin provoked a decrease in the opening angle, while dissolving collagen and destroying smooth muscle cells had no effect. In the second, different wall layers of bovine carotid arteries were removed from the exterior or luminal surfaces by lathing or drilling frozen specimens, and then allowing the frozen material to thaw before measuring residual strain. Lathing material away from the outer surface caused the opening angle of the remaining inner layers to increase. Drilling material from the inside caused the opening angle of the remaining outer layers to decrease. Mechanical nonhomogeneity, including the distribution of residual strains, should thus be considered as an important factor in determining the distribution of stress in the artery wall and the configuration of the true zero stress state.

Glutaraldehyde-tanned bovine carotid artery graft for infrainguinal vascular reconstruction: 5-year follow-up

To assess the long-term patency of a modified biological conduit, the glutaraldehyde-tanned bovine carotid artery, in above-knee infrainguinal arterial reconstruction. Prospective follow-up of a cohort of 58 above-knee femoropopliteal grafts in 55 patients. Graft patency was assessed at yearly intervals with doppler ankle pressure measurements. The median follow-up period has been 67 months. Nine grafts occluded within 30 days of surgery and a further 19 graft closures have been observed in the follow-up period. The overall cumulative primary graft patency at 1, 3 and 5 years was 70%, 61% and 56%, respectively. If the 30-day graft failures are excluded, the primary graft patency rises to 83%, 74% and 68% at 1, 3 and 5 years, respectively. Six limbs have been amputated, four above the knee and two below the knee. There were no graft aneurysms and no graft infections. Results indicate that the modified bovine carotid artery graft with an above-knee anastomosis does not seem to be inferior to PTFE, but is inferior to reversed vein. Modified biological conduits offer a reasonable alternative to synthetic grafts for infrainguinal arterial reconstruction and appear to maintain acceptable long-term mechanical stability.

Mitogen-activated protein kinase activation: an alternate signaling pathway for sustained vascular smooth muscle contraction.

The vascular smooth muscle determines the dynamic caliber of the blood vessel and hence is the final effector cell in modulating vasomotor tone. Although considerable information is available regarding the physiologic agonists that induce contraction, less is known about the cellular signaling events that lead to long-lasting contractions or vasospasm. We examined the hypothesis that activation of mitogen-activated protein (MAP) kinase may be associated with sustained smooth muscle contractions. Physiologic contractile responses were determined in intact bovine carotid artery smooth muscles in a muscle bath. Corresponding signaling events were determined with immunoblots using antiphosphotyrosine antibodies or immunoprecipitation of whole-cell phosphorylated strips of muscle. The tyrosine kinase inhibitor, genestein, significantly inhibited the magnitude of contractions induced by phorbol ester, endothelin, angiotensin, and serotonin. In addition, genestein inhibited the sustained phase of contractions induced by serotonin. Serotonin-induced vascular smooth muscle contractions were temporally associated with an increase in the phosphorylation of MAP kinase. These data suggest that the activation of MAP kinase is associated with sustained vascular smooth muscle contractions. Pharmacologic manipulation of MAP kinase activation may lead to new approaches to treat pathologic circumstances of increased vasomotor tone such as vasospasm.

Cellular mechanisms of cyclic nucleotide-induced vasorelaxation

Endothelial-derived vasorelaxants such as prostacyclin and nitric oxide (NO) induce vascular smooth muscle relaxation through activation of cyclic nucleotide-dependent cellular signalling pathways. However, the specific events that lead to dissociation of actin and myosin and relaxation are not known. The purpose of this investigation was to determine the late phase signaling events that modulate vascular smooth muscle relaxation. Fresh bovine carotid artery smooth muscle (BCASM) contractile responses were determined in a muscle bath under Ca(2+)-containing and Ca(2+)-free conditions. Physiologic responses were correlated with phosphorylation events using whole cell phosphorylation and two-dimensional gel electrophoresis. Cyclic nucleotide-dependent vasorelaxation can occur without detectable changes in intracellular Ca2+ concentrations. However, vascular smooth muscles that had been precontracted with the phosphatase inhibitor calyculin were refractory to relaxation. Vascular smooth muscle relaxation was associated with an increase in the phosphorylation of two 20 kDa proteins under Ca(2+)-containing and Ca(2+)-free conditions. These results suggest that Ca(2+)-independent mechanisms may also modulate vascular smooth muscle relaxation. Two possible late phase signaling mechanisms include phosphatase activation and an increase in the phosphorylation of two 20 kDa phosphoproteins.

Susceptibility of endothelial cells to bovine herpesvirus type 4 (BHV-4)

The sensitivity of two different types of cells to bovine herpesvirus type 4 (BHV-4) was compared by median tissue culture infectious dose (TCID 50) assays. The bovine arterial endothelial (BAE) cell culture derived from bovine carotid arteries was 100–1000 times more sensitive to two strains of BHV-4, Movar 33 63 and DN 599, than Madin Darby bovine kidney (MDBK) cell line commonly used for the propagation of these viruses. BAE cell cultures infected with BHV-4 displayed cytopathic effects (CPE) earlier and more prominently than the MDBK cells infected with the same viruses. BAE cells were also more sensitive than MDBK cells in conventional plaque assays in that the former developed well characterized and easily recognized plaques after infection with the viruses. BAE cells, which are proved to be exceptionally susceptible to BHV-4, can be used in the detection and quantitation of BHV-4.

Enhancement of membrane fluidity in cholesterol-poor endothelial cells pre-treated with simvastatin.

We examined the membrane fluidity of the cholesterol-poor bovine carotid artery endothelial cells (BAEC). Cholesterol-poor BAEC were obtained by treating the cells with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors under 10% low density lipoprotein (LDL)-deficient serum condition for 2 days. Simvastatin reduced the intracellular cholesterol content significantly at a concentration of 0.1 microgram/ml. The reduction in the cholesterol content was accompanied by the enhancement of the cell membrane fluidity which was measured by a photobleaching technique. Additional data suggested that the reduction in cholesterol content referred to the reduction in the proliferation of BAEC.

The Impact of Balloon Embolectomy on the Function and Morphology of the Endothelium

Purpose.Intravascular interventions such as balloon embolectomy lead to endothelial injury. The extent and functional consequences of this endothelial injury are not known.Methods.Segments of bovine carotid artery were equilibrated in a vasomotor perfusion apparatus (VPA). Smooth muscle function was determined by the contractile responses to 10−5Mnorepinephrine (NE). Endothelial function was evaluated by the extent of vasorelaxation to the subsequent addition of 10−5Macetylcholine (ACh). To determine the pattern of injury throughout the length of the vessel, strips of artery from along the vessel length were also evaluated in a muscle bath. Morphology was examined by scanning electron microscopy (SEM).Results.No significant differences were noted in the contractile response to NE after embolectomy. Balloon embolectomy significantly decreased the relaxation response to ACh from 97 ± 1% before embolectomy to 89 ± 1% after embolectomy (n= 5 vessels,P< 0.01, ANOVA). In the muscle bath, the vasorelaxation of strips precontracted with NE was variable throughout the length of the vessel suggesting that endothelial injury was diffuse. The relaxation response to ACh was inhibited byl-NMMA. SEM revealed patchy areas of endothelial denudation.Conclusion.Embolectomy produces no change in smooth muscle function and partial injury to the endothelium in intact vessels. However, there remains sufficient endothelial function after balloon embolectomy to produce NO-dependent vasorelaxation and modulate vasomotor tone.

Renal Artery Smooth Muscle Is Refractory to Contraction by Angiotensin II

The vasomotor responses of vascular smooth muscle from different vascular smooth muscle beds have not been well characterized. The purpose of this study was to compare the contractile responses of vascular smooth muscle from two vascular beds, the peripheral vascular bed (carotid artery) and the visceral vascular bed (renal artery) to vasoactive agonists. Fresh bovine carotid and renal artery smooth muscle contractile responses to serotonin, endothelin, angiotensin, and dopamine were determined in a muscle bath. Serotonin and dopamine cause rapidly developing sustained contractions in carotid and renal artery smooth muscle. The magnitude of the contractile response to serotonin is significantly greater in carotid artery and the magnitude of the response to dopamine is similar between carotid and renal artery. Endothelin induces a slowly developing sustained contraction of greater magnitude in renal artery. Angiotensin II causes transient contractile responses in carotid artery but renal artery smooth muscle is uniquely refractory to angiotensin stimulation. This lack of response to angiotensin II may be protective in the role of the kidney in regulating blood pressure through the renin-angiotensin system. Differences in receptor expression or affinity or in postreceptor cellular signaling events may account for these differential responses to endogenous vasoactive agonists.

The synergistic effect of elastase and hydrogen peroxide on vascular endothelial cell injury is due to the production of hydroxylradical in the endothelial cells.

Protease inhibitors such as aprotinin and urinastatin inhibited vascular endothelial cell injury induced by PMA-stimulated leukocytes, although their inhibitors did not suppress the production of active oxygen species released from leukocytes. On the other hand, in the presence of pancreas elastase (10 micrograms/ml), hydrogen peroxide (50 microM) caused severe injury of endothelial cells isolated from the bovine carotid artery (% specific 51Cr release, % SR = 42.9 +/- 3.3%), although the % SR elicited by elastase or hydrogen peroxide alone, respectively, was below 1%. Elastase and hydrogen peroxide acted synergistically on the injury of endothelial cells from the bovine carotid artery similarly to that in the endothelial cells isolated from the bovine coronary artery and human umbilical vein. Furthermore, elastase derived from both pancreas and leukocyte induced this synergistic action on endothelial cell injury. To clarify the mechanism of vascular endothelial cell injury induced by the combination of elastase and hydrogen peroxide, we examined the effects of various radical scavengers and protease inhibitors. Deferoxamine mesylate completely inhibited the endothelial cell injury, while protease inhibitors such as antitrypsin and macroglobulin had a protective effect. Pretreatment of endothelial cells with deferoxamine mesylate also protected against this cytotoxicity. These findings suggested that the synergistic effect of elastase and hydrogen peroxide on the endothelial cell injury is due to the production of hydroxylradical in the endothelium and that this synergistic action might be partially involved in the endothelial cell injury induced by activated leukocytes.

Characterization of Phospholipase A(2) Activation by Plasmin in Cultured Bovine Endothelial Cells.

Treatment of cultured bovine carotid artery endothelial cells with 0.1 &mgr;M human plasmin has been reported to induce a receptor-mediated short burst of arachidonate release, which is a pertussis toxin-sensitive and extracellular calcium-dependent reaction. Plasmin-induced calcium influx in cells was significantly inhibited by pretreatment with pertussis toxin, indicating that the former was coupled with a pertussis toxin-sensitive guanosine 5'-triphosphate (GTP)-binding protein. Plasmin significantly induced the formation of lysophosphatidylcholine but not lysophosphatidylethanolamine. A cellular phospholipase A(2) with an arachidonyl specificity at the sn-2 position of phosphatidylcholine, which required submicromolar calcium, was identified as a cytosolic phospholipase A(2) by immunoblot analysis. By a cell-free enzyme activity assay and immunoblot analysis, plasmin was found to induce a translocation of the cytosolic phospholipase A(2) from the cytosol to the membrane. Taken together, the results suggest that plasmin bound to its putative receptor and activated a GTP-binding protein coupled to calcium influx channel, followed by translocation and activation of cytosolic phospholipase A(2) in endothelial cells. Copyright 1996 S. Karger AG, Basel

Kinase activation and smooth muscle contraction in the presence and absence of calcium

The intracellular signalling mechanisms that modulate the sustained vascular smooth muscle contractions that occur with vasospasm are not well understood. The purpose of this investigation was to examine cell signalling mechanisms that account for sustained vascular smooth muscle contraction, independent of increases in intracellular Ca2+ concentrations ([Ca2+]i). Fresh bovine carotid artery smooth muscles contractile responses were examined in a muscle bath. [Ca2+]i was depleted by use of the extracellular Ca2+ chelator, ethylene glycol-bis(beta-aminoethylether) N,N,N',N'-tetraacetic acid and the intracellular chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid. In Ca(2+)-free conditions, depolarizing the membrane with high extracellular KCI failed to elicit a contraction. In addition, in Ca(2+)-free conditions the ([Ca2+]i) was less than 10 nmol/L as determined with the Ca(2+)-indicator, Fura 2. The protein kinase C (PKC) activator, phorbol 12, 13-dibutyrate (PDBu), induced slowly developing sustained contractions in bovine carotid artery smooth muscle, and the magnitude of the contractile response to PDBu (10 nmol/L to 10 mumol/L) was the same in the presence and absence of Ca2+. PDBu induced contractions in Ca(2+)-free conditions were not inhibited by the myosin light chain kinase inhibitor, ML-9 (50 mumol/L), but were inhibited by the PKC inhibitor, staurosporine (50 nmol/L). These data suggest that vascular smooth muscle contractions can occur under conditions where the [Ca2+]i is low and fixed and that these contractions may be mediated by PKC.

Conversion of xanthine dehydrogenase to xanthine oxidase in bovine carotid artery endothelial cells induced by activated neutrophils: involvement of adhesion molecules

We have demonstrated that the endothelial cell-derived superoxide anion is deeply involved in the endothelial cell injury induced by activated neutrophils (Fujita, H., Morita, 1. and Murota, S. (1994) Arch. Biochem. Biophys. 309, 62–69). To clarify the mechanism underlying the increase in the endothelial cell-derived superoxide anion induced by activated neutrophils, the conversion of xanthine dehydrogenase (XD) to xanthine oxidase (XO) in cultured endothelial cells isolated from bovine carotid arteries was investigated. Although the endothelial cells expressed both XD and XO activity, the XO activity of unstimulated cells comprised about 12% of the total (XD + XO) activity. When endothelial cells were exposed to neutrophils activated with phorbol 12-myristate 13-acetate (PMA), XO activity rapidly increased about 3-fold over the control. Whereas treatment of endothelial cells with PMA alone or unstimulated neutrophils alone did not increase the XO activity at all. The increase in XO activity in endothelial cells was also observed on the treatment of the cells with neutrophils activated with leukotriene B4 or thrombin. To determine whether or not proteases released from activated neutrophils are involved in the increased conversion of XD to XO in endothelial cells, the effects of the elastase specific inhibitor, ONO-5046, and protease inhibitors, such as aprotinin, gabexate mesylate and urinastatin, were examined. However, these protease inhibitors did not suppress the conversion of XD to XO induced by PMA-activated neutrophils. Moreover, the treatment of endothelial cells with purified human neutrophil elastase and H 20 2 also did not affect the conversion at all. In contrast, monoclonal antibodies against CD11a and CD18 significantly inhibited the increased conversion of XD to XO induced by PMA-activated neutrophils. Moreover, tyrosine kinase inhibitors such as staurosporine and herbimysine also inhibited the increased conversion of XD to XO induced by PMA-activated neutrophils. These results indicate that the adhesion of activated neutrophils to endothelial cells via CD11a/CD18-ICAM-1 is involved in the conversion of XD to XO in endothelial cells induced by activated neutrophils.

Endothelin-1 as an Autocrine Growth Factor for Endothelial Cells

To elucidate the role of endothelin-1 (ET-1) as an autocrine growth factor for vascular endothelial cells (ECs), we studied the effects of phosphoramidon, an inhibitor of ET-1-converting enzyme, on the production of ET-1-like immunoreactivity (LI), [125I]ET-1 binding activity, ETB receptor mRNA expression, phosphoinositide breakdown, and DNA synthesis in cultured bovine carotid artery ECs. Phosphoramidon dose-dependently decreased ET-1-LI production but reciprocally increased [125I]ET-1 binding capacity. ET-1 and ET-3 equipotently inhibited the binding of [125I]ET-1 only in the presence of phosphoramidon. Northern blot analysis revealed that ETB receptor mRNA expression was more evident in phosphoramidon-treated cells than in nontreated cells. In the presence of phosphoramidon, ET-1 and ET-3 equipotently stimulated inositol 1,4,5-trisphosphate formation and [3H]-thymidine incorporation into cultured ECs. Both phosphoramidon and anti-ET-1 antibody inhibited basal [3H]thymidine incorporation. These data suggest that endogenous ET-1 constitutively secreted by ECs is an autocrine growth factor via ETB receptors.

Endothelin-1 as an Autocrine Growth Factor for Endothelial Cells

Summary: To elucidate the role of endothelin-1 (ET-1) as an autocrine growth factor for vascular endothelial cells (ECs), we studied the effects of phosphoramidon, an inhibitor of ET-l-converting enzyme, on the production of ET-1-like immunoreactivity (LI), [125I]ET-1 binding activity, ETB receptor mRNA expression, phosphoinositide breakdown, and DNA synthesis in cultured bovine carotid artery ECs. Phosphoramidon dose-dependently decreased ET-1-LI production but reciprocally increased [125I]ET-1 binding capacity. ET-1 and ET-3 equipotently inhibited the binding of [125I]ET-1 only in the presence of phosphoramidon. Northern blot analysis revealed that ETB receptor mRNA expression was more evident in phosphoramidon-treated cells than in nontreated cells. In the presence of phosphoramidon, ET-1 and ET-3 equipotently stimulated inositol 1,4,5-trisphosphate formation and [3H]-thymidine incorporation into cultured ECs. Both phosphoramidon and anti-ET-1 antibody inhibited basal [3H]thymidine incorporation. These data suggest that endogenous ET-1 constitutively secreted by ECs is an autocrine growth factor via ETB receptors.

Impaired cyclic nucleotide-dependent vasorelaxation in human umbilical artery smooth muscle

Activation of either the adenylate cyclase pathway with forskolin or the guanylate cyclase pathway with sodium nitroprusside fails to induce active relaxation of serotonin-precontracted human umbilical artery smooth muscle (HUASM) but causes active relaxation of serotonin-precontracted bovine carotid artery smooth muscle (BCASM). This difference in response appears to be unique to HUASM in that all other vascular muscles exhibit relaxation in response to these substances. Forskolin and sodium nitroprusside stimulation leads to respective increases in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) concentrations in HUASM and BCASM. cAMP- and cGMP-dependent protein kinases are both present in HUASM and can be activated in homogenates of HUASM by the addition of exogenous cAMP and cGMP, respectively. When either forskolin or nitroprusside acts in BCASM, two low-molecular-weight proteins display an increase in the extent of phosphorylation. Neither protein shows such an increase when HUASM is treated with either agent. Thus the inability of HUASM to display active relaxation appears to be secondary to impaired activation of cyclic nucleotide-dependent protein kinases. The refractoriness to active relaxation may contribute to the vasospasm that occurs in the umbilical vasculature with parturition.

Diacylglycerol formation induced by human plasmin in cultured endothelial cells

Treatment of cultured bovine carotid artery endothelial cells with 10 −7M plasmin increased the cellular diacylglycerol which was determined by the formation of [ 3H]palmitate-labeled diacylglycerol and diacylglycerol mass. Upon the stimulation with plasmin, a gradual increase in diacylglycerol formation was observed within 20 min then slightly declined. The maximal effect during the 1-h time course study was 45 and 55% increases in [ 3H]palmitate-labeled diacylgycerol and diacylglycerol mass, respectively, at 20 min after plasmin treatment. Formation of phosphatidylethanol was also studied in [ 3H]palmitate-prelabeled cells in the presence of ethanol. Treatment with plasmin for 20 min induced a significant 45% increase in phosphatidylethanol formation. The present results indicate that the plasmin-induced diacylglycerol formation in endothelial cells was at least in part mediated through the phospholipase D activation.