Procalcitonin as a postmortem sepsis marker. A comparison of the validity of results obtained from blood serum, aqueous humour and cerebrospinal fluid.

In recent years, serum levels of the prohormone procalcitonin have been investigated in a number of studies in relation to postmortem sepsis diagnostics, as macroscopic and histomorphological findings are, as a rule, nonspecific. However, due to advanced haemolysis, it is often not possible to determine serum procalcitonin (PCT) levels in cases of sepsis-related death. Moreover, the impact of postmortem interval on PCT levels is largely unclarified. In view of this, the present pilot study investigated PCT levels in the serum, aqueous humour, and cerebrospinal fluid in a study population of 25 persons who died of sepsis and a control population of 25 deaths unrelated to sepsis. Using the Mann-Whitney U test, statistically significant differences in PCT levels were determined for all the analysed samples from the study and control populations. Logistic regression analysis was used to calculate cut-off values for sepsis diagnosis for all the sample types. Furthermore, the serum elimination rates published by Tsokos et al. (Int J Legal Med 114:237-243, 2001) were used to calculate the PCT levels at the time of death for the cases with a known postmortem interval. The results of our study demonstrate that, taking account of the postmortem elimination process, it is possible to infer the value at the time of death from the procalcitonin levels measured in all three sample types and to interpret this with the aid of a defined cut-off value. The findings need to be verified based on a larger study population.

Extracellular protein disulfide isomerase regulates feedback activation of platelet thrombin generation via modulation of coagulation factor binding.

BACKGROUND: Protein disulfide isomerase (PDI) controls platelet integrin function, tissue-factor (TF) activation, and concentrates at fibrin and thrombus formation sites of vascular injury. OBJECTIVE: To investigate the involvement of surface thiol isomerases and especially PDI, in thrombin-mediated thrombin amplification on human platelets. METHODS/RESULTS: Using a newly developed thrombin-dependent platelet thrombin generation assay, we observed that the feedback activation of thrombin generation on the platelet surface does not depend on TF, as anti-TF antibodies inhibiting TF-induced thrombin formation in platelet-depleted plasma had no effect compared with vehicle-treated controls. Feedback activation of thrombin generation in the presence of platelets was significantly diminished by membrane impermeant thiol blockers or by the thiol isomerase-inhibitors bacitracin and anti-PDI antibody RL90, respectively. Platelet thrombin formation depends on binding of coagulation factors to the platelet surface. Therefore, involvement of thiol isomerases in this binding was investigated. As shown by confocal microscopy and flow cytometry, thrombin-stimulated platelets exhibited increased surface-associated PDI as well as extracellular disulfide reductase activity compared with unstimulated platelets. Flow cytometric analysis revealed that membrane impermeant thiol blockers or PDI inhibitors, which had been added after platelet stimulation and after phosphatidylserine exposure to exclude their influence on primary platelet activation, significantly inhibited binding of all coagulation factors to thrombin-stimulated platelets. CONCLUSIONS: Thus, surface-associated PDI is an important regulator of coagulation factor ligation to thrombin-stimulated platelets and of subsequent feedback activation of platelet thrombin generation. Cell surface thiol isomerases might be therefore powerful targets to control hemostasis and thrombosis.

Impaired vitamin B12 metabolic status in healthcare workers occupationally exposed to nitrous oxide.

BACKGROUND: Previous studies demonstrated inactivation of vitamin B12 by nitrous oxide (N(2)O). The intraoperative exposure to N(2)O was shown to induce megaloblastic anaemia and myelopathy in subjects with subclinical vitamin B12 deficiency. In contrast, no data concerning the influence of occupational exposure to N(2)O on vitamin B12 metabolic status are available to date. In the present study, the vitamin B12 status in operating theatre personnel was assessed in relation to the extent of exposure. METHODS: Ninety-five operating theatre nurses with the history of exposure to N(2)O and 90 unexposed counterparts were examined. Vitamin B12 and folic acid were measured by immunoassay. Total homocysteine (tHcy), an indicator of impaired vitamin B12 metabolism, was determined by high performance liquid chromatography. N(2)O concentration was monitored by adsorption gas chromatography and mass spectrometry. RESULTS: No significant differences were found between both groups with respect to haematological parameters and folic acid. However, subjects exposed to N(2)O presented with lower vitamin B12 [372.8 (12.1) vs 436.8 (13.2) pmol litre(-1), P<0.001] and higher tHcy [11.2 (0.5) vs 8.9 (0.5) micromol litre(-1), P=0.006]. The changes in vitamin B12 status were aggravated in subjects exposed to N(2)O in concentrations substantially exceeding occupational exposure limit (180 mg m(-3)) [vitamin B12: 341.9 (17.7) vs 436.8 (13.2) pmol litre(-1), P=0.006; tHcy: 12.9 (0.7) vs 8.9 (0.5) micromol litre(-1), P=0.047]. CONCLUSIONS: Exposure to N(2)O in healthcare workers is associated with alterations of vitamin B12 metabolic status, the extent of which depends on the level of exposure.

Maternal serum free beta-hCG and PAPP-A in patients with habitual abortion-influence on first-trimester screening for chromosomal abnormalities.

OBJECTIVE: To explore if maternal serum free beta-hCG and pregnancy-associated plasma protein A (PAPP-A) levels in the first-trimester of pregnancy are altered in patients with habitual abortions and if there is an effect on first-trimester screening for Down syndrome. METHODS: A retrospective study was conducted on 913 normal singleton fetuses that underwent first-trimester combined screening for Down syndrome. Maternal serum PAPP-A and free beta-hCG were compared between patients with (n = 64) and without habitual abortions (n = 849). RESULTS: The medians +/- SD log(10) MoM of PAPP-A and free beta-hCG +/- SD in patients with and without habitual abortions were 0.063 +/- 0.28 versus - 0.014 +/- 0.27 and - 0.001 +/- 0.27 versus - 0.018 +/- 0.31, with a p value of 0.042 and 0.87, respectively. The screen positive rate setting the cut off at 1:350 looking at the background risk for trisomy 21 was 71.4% in women with and 81.2% in women without habitual abortion, after combined first-trimester screening it was 7.8% in women with and 10.1% in women without recurrent abortion. CONCLUSIONS: Patients with habitual abortions have slightly increased maternal serum PAPP-A levels in the first-trimester. This marginal difference seems not to effect risk calculation in combined first-trimester screening for trisomy 21.

Influence of maternal systemic lupus erythematosus on first-trimester combined screening for chromosomal abnormalities.

OBJECTIVE: To explore the effect of maternal systemic lupus erythematosus (SLE) on first-trimester screening markers for Down syndrome. METHODS: A retrospective study was conducted on 1150 normal singleton fetuses that underwent first-trimester combined screening for Down syndrome. Fetal delta nuchal translucency (NT), maternal serum PAPP-A and free beta-hCG were compared between pregnancies with SLE (n = 10) and without preexisting maternal disease (n = 1140). RESULTS: The medians +/- SD for delta NT, log(10) MoM of PAPP-A and free beta-hCG +/- SD in pregnancies with SLE and without maternal disease were - 0.18 +/- 0.29 versus - 0.18 +/- 0.33, 0.005 +/- 0.32 versus 0.02 +/- 0.26, and 0.22 +/- 0.19 versus - 0.014 +/- 0.28, with a p value of 0.7, 0.98 and 0.03, respectively. CONCLUSIONS: Patients with preexisting SLE have increased maternal serum-free beta-hCG levels in the first-trimester. But, because of the multimodal procedure of risk calculation there is no significant difference in the screen-positive rate after the combined first-trimester screening for trisomy 21.

Paracrine stimulation of vascular smooth muscle proliferation by diadenosine polyphosphates released from proximal tubule epithelial cells.

The purinergic receptor system plays an important role in the regulation of both vascular and tubular functions within the kidney; however, the release of purinergic agonists other than ATP by renal tissue is not known. In this investigation, we determine if kidney tissue is a source of diadenosine polyphosphates, which have high affinity for the P(2X) and P(2Y) receptors. Both diadenosine pentaphosphate and hexaphosphate were identified by matrix-assisted laser desorption ionization-mass spectrometry in extracts purified from both whole porcine kidney and from cloned cells of the LLC-PK1 cell line. Both polyphosphates in nanomolar concentrations were found to significantly stimulate the proliferation of vascular smooth muscle cells derived from rat thoracic aortas. The purinergic-receptor antagonist, suramin, did not significantly affect the growth-stimulatory properties of the polyphosphates. The growth stimulation of vascular smooth muscle cells by platelet-derived growth factor was potentiated by both diadenosine polyphosphates. We conclude that diadenosine polyphosphates are endogenous purinergic agonists of the kidney that have physiologic and pathophysiologic relevance. These epithelial cell metabolic products have vasoregulatory properties while linking the energy supply and tubular function.

Does vaginal bleeding influence first-trimester markers for Down syndrome?

OBJECTIVES: To examine the effect of early vaginal bleeding on first-trimester screening markers for Down syndrome. METHODS: A retrospective study was conducted on 1755 normal singleton fetuses that underwent first-trimester combined screening for Down syndrome on the basis of ultrasound and maternal serum markers. Fetal delta-nuchal translucency (NT), maternal serum pregnancy-associated plasma protein A (PAPP-A) and free beta-hCG were compared between pregnancies with (n = 252) and without (n = 1503) an episode of vaginal bleeding. Subgroup analysis for the intensity of bleeding (spotting n = 191; light n = 32; heavy n = 29) was performed. RESULTS: The median +/- SD (log(10)) for delta-NT, multiple of medians (MoM) PAPP-A and MoM free beta-hCG (corrected for maternal weight, smoking and ethnicity) was - 0.17 +/- 0.62, 1.10 +/- 0.28, 1.1 +/- 0.28 and - 0.15 +/- 0.51, 0.98 +/- 0.26, 0.94 +/- 0.3 in pregnancies with and without a history of early vaginal bleeding, which were not significantly different. Exclusion of patients with spotting from the vaginal bleeding group revealed significantly higher maternal serum free beta-hCG MoM values (median +/- SD (log(10))) compared to patients without bleeding, 1.29 +/- 0.27 vs 0.96 +/- 0.3(p = 0.011). Screen-positive (cut off of 1:350) rate after combined first-trimester screening was 28.1% in patients with light vaginal bleeding and 8.4% in patients without bleeding (p = 0.001). CONCLUSIONS: Light vaginal bleeding before first-trimester combined screening for Down syndrome leads to a higher screen-positive rate after combined first trimester screening, without a significant difference in serum levels of the screening markers.

Tangier disease: still more questions than answers.

High-density lipoproteins (HDLs) play a central role in transporting cholesterol from peripheral tissues to the liver for elimination from the body. Impairment of HDL-mediated cholesterol transport favors cholesterol deposition in the arterial wall and promotes development of arteriosclerosis. Tangier disease is a severe HDL deficiency syndrome characterized by the accumulation of cholesterol in tissue macrophages and prevalent atherosclerosis. A three-decade search for a culprit in Tangier disease led to the identification of mutations in a cell membrane protein called ABCA1, which mediates the secretion of excess cholesterol from cells into the HDL metabolic pathway. Because of its ability to deplete cells of cholesterol and to raise plasma HDL levels, ABCA1 has become a promising therapeutic target for preventing cardiovascular disease.

Suppression of endothelial cell apoptosis by high density lipoproteins (HDL) and HDL-associated lysosphingolipids.

Apoptotic cell death following injury of vascular endothelium is assumed to play an important role in the pathogenesis of atherosclerosis. In this report, we demonstrate that high density lipoproteins (HDL), a major anti-atherogenic lipoprotein fraction, protect endothelial cells against growth factor deprivation-induced apoptosis. HDL blocked the mitochondrial pathway of apoptosis by inhibiting dissipation of mitochondrial potential (Deltapsi(m)), generation of reactive oxygen species, and release of cytochrome c into the cytoplasm. As a consequence, HDL prevented activation of caspases 9 and 3 and apoptotic alterations of the plasma membrane such as increase of permeability and translocation of phosphatidylserine. Treatment of endothelial cells with HDL induced activation of the protein kinase Akt, an ubiquitous transducer of anti-apoptotic signals, and led to phosphorylation of BAD, a major Akt substrate. Suppression of Akt activity both by wortmannin and LY-294002 or by a dominant negative Akt mutant abolished the anti-apoptotic effect of HDL. Two bioactive lysosphingolipids present in HDL particles, sphingosylphosphorylcholine and lysosulfatide, fully mimicked the survival effect of HDL by blocking the mitochondrial pathway of apoptosis and potently activating Akt. In conclusion, the present study identifies HDL as a carrier of endogenous endothelial survival factors and suggests that inhibition of endothelial apoptosis by HDL-associated lysosphingolipids may represent an important and novel aspect of the anti-atherogenic activity of HDL.

High density lipoproteins induce cell cycle entry in vascular smooth muscle cells via mitogen activated protein kinase-dependent pathway.

In this study we found that HDL acts as a potent and specific mitogen in vascular smooth muscle cells (VSMC) by stimulating entry into S-phase and DNA synthesis in a time- and concentration-dependent manner, induction of cyclins D1, E, and A, as well as activation of cyclin D-dependent kinases as inferred from phosphorylation of the retinoblastoma protein (pRb). Moreover, HDL induced activation of the mitogen-activated protein kinase pathway including Raf-, MEK-1, and ERK1/2, as well as the expression of proto-oncogen c-fos, which is controlled by ERK1/2. PD98059, an inhibitor of MEK-1 blocked the mitogenic activity of HDL and cyclin D1 expression. HDL-induced VSMC proliferation, cell cycle progression, cyclin D1 expression, and activation of the Raf-1/MEK-1/ERK1/2 cascade were blocked by preincubation of cells with pertussis toxin indicating involvement of trimeric G-protein. By contrast, none of these responses was inhibited by the protein kinase C inhibitor, GF109203X. The mitogenic effects of native HDL were not mimicked by apo A-I, reconstituted HDL containing apo A-I, or cholesterol-containing liposomes. In conclusion, HDL possesses an intrinsic property to induce G-protein- and MAP-kinase-dependent proliferation and cell cycle progression in VSMC. The strong and specific mitogenic effect of HDL should be taken into account, when therapeutic strategies to elevate the plasma level of these lipoproteins are developed.

Effects of diets containing olive oil, sunflower oil, or rapeseed oil on the hemostatic system.

Various studies have already shown that the fatty acid composition of dietary fat has different effects on hemostasis and platelet function. However, knowledge on this topic is incomplete. In the present study, fifty-eight healthy students received either a 4-week rapeseed oil [high content of monounsaturated fatty acids (MUFA) and high n-3/n-6 PUFA ratio], an olive oil (high content of MUFA, low n-3/n-6 PUFA ratio) or a sunflower oil (low content of MUFA, low n-3/n-6 PUFA ratio) diet. In each group, effects on hemostatic parameters were compared with a wash-in diet rich in saturated fatty acids with respect to intermediate-time effects on the hemostatic system and platelet function. With the olive oil diet, a reduction of coagulation factors VIIc, XIIc, XIIa, and Xc was found, whereas sunflower oil led to lower values of coagulation factors XIIc, XIIa, and IXc. In all study groups levels of plasmin-alpha2-antiplasmin were lower in week 4 than at baseline. Lower fibrinogen binding on platelets was found after the sunflower oil diet, whereas expression of CD62 and spontaneous platelet aggregation were slightly higher after the olive oil diet. However, given the major differences in the fatty acid compositions of the diets, the differences between the groups with respect to hemostasis tended to be small. Therefore, the clinical significance of the present findings remains to be evaluated.

xIAP induces cell-cycle arrest and activates nuclear factor-kappaB : new survival pathways disabled by caspase-mediated cleavage during apoptosis of human endothelial cells.

Survival of human vascular endothelial cells depends on their ability to activate the transcription factor nuclear factor-kappaB (NF-kappaB), a regulator of antiapoptotic genes, such as the X chromosome-linked inhibitor of apoptosis protein (xIAP). In the present study, we demonstrated expression of xIAP in the endothelial lining of normal human arteries and veins and elevated levels in highly malignant human endothelial tumors. Using retroviral infection of human endothelial cells, we identified two novel survival mechanisms mediated by xIAP in endothelial cells. First, xIAP can activate the transcription factor NF-kappaB, a known survival factor for human endothelial cells. This positive feedback loop induced by xIAP is mediated via phosphorylation and sustained degradation of inhibitor (I) kappaBalpha. Second, xIAP can inhibit cell proliferation via downregulation of cyclins A and D1 and induction of the cyclin-dependent kinase inhibitors p21(Cip1/Waf1) and p27(Kip1). Cleavage of xIAP by caspases during endothelial cell apoptosis disables both of these biological functions of xIAP. Thus, caspase-mediated cleavage of xIAP interrupts a positive regulatory cytoprotective loop between NF-kappaB and xIAP and increases the vulnerability of the cell to apoptosis by releasing it from an xIAP-mediated quiescent state.

High density lipoproteins and arteriosclerosis. Role of cholesterol efflux and reverse cholesterol transport.

High density lipoprotein (HDL) cholesterol is an important risk factor for coronary heart disease, and HDL exerts various potentially antiatherogenic properties, including the mediation of reverse transport of cholesterol from cells of the arterial wall to the liver and steroidogenic organs. Enhancement of cholesterol efflux and of reverse cholesterol transport (RCT) is considered an important target for antiatherosclerotic drug therapy. Levels and composition of HDL subclasses in plasma are regulated by many factors, including apolipoproteins, lipolytic enzymes, lipid transfer proteins, receptors, and cellular transporters. In vitro experiments as well as genetic family and population studies and investigation of transgenic animal models have revealed that HDL cholesterol plasma levels do not necessarily reflect the efficacy and antiatherogenicity of RCT. Instead, the concentration of HDL subclasses, the mobilization of cellular lipids for efflux, and the kinetics of HDL metabolism are important determinants of RCT and the risk of atherosclerosis.

Acceleration of reverse cholesterol transport.

A low level of high-density lipoprotein (HDL) cholesterol is an important risk factor for coronary heart disease. Levels of HDL cholesterol and composition of HDL subclasses in plasma are regulated by many factors, including apolipoproteins, lipolytic enzymes, lipid transfer proteins, receptors, and cellular transporters. Reverse transport of cholesterol from cells of the arterial wall to the liver is an important mechanism by which HDL exerts its anti-atherogenic properties. Enhancement of reverse cholesterol transport is considered as a potential target for anti-atherosclerotic drug therapy. It is suggested, however, that the serum level of HDL cholesterol does not necessarily reflect the efficacy of reverse cholesterol transport.

Activation of phosphatidylinositol-specific phospholipase C by HDL-associated lysosphingolipid. Involvement in mitogenesis but not in cholesterol efflux.

Our earlier studies demonstrated that high-density lipoproteins (HDLs) stimulate multiple signaling pathways, including activation of phosphatidylcholine-specific phospholipases C and D (PC-PLs) and phosphatidylinositol-specific phospholipase C (PI-PLC). However, only activation of PC-PLs was linked to the HDL-induced cholesterol efflux. In the study presented here, the role of HDL-induced PI-PLC activation was studied. In human skin fibroblasts, HDL potently induced PI-PLC as inferred from enhanced phosphatidylinositol bisphosphate (PtdInsP(2)) turnover and Ca(2+) mobilization. The major protein component of HDL, apo A-I, did not induce PtdInsP(2) turnover or Ca(2+) mobilization in these cells. Both HDL and apo A-I promoted cellular cholesterol efflux, whereas only HDL induced fibroblast proliferation. Inhibition of PI-PLC with U73122 or blocking intracellular Ca(2+) elevation with Ni(2+) or EGTA markedly reduced the extent of HDL-induced cell proliferation but had no effect on cholesterol efflux. In fibroblasts from patients with Tangier disease which are characterized by defective cholesterol efflux, neither HDL-induced PtdInsP(2) breakdown and Ca(2+) mobilization nor cell proliferation was impaired. HDL-induced fibroblast proliferation, PtdInsP(2) turnover, and Ca(2+) mobilization were fully mimicked by the lipid fraction isolated from HDL. Analysis of this fraction with high-performance liquid chromatography (HPLC) and time-of-flight secondary ion mass spectroscopy (TOF-SIMS) revealed that the PI-PLC-inducing activity is identical with two bioactive lysosphingolipids, namely, lysosulfatide (LSF) and sphingosylphosphorylcholine (SPC). Like native HDL, LSF and SPC induced PtdInsP(2) turnover, Ca(2+) mobilization, and fibroblast proliferation. However, both compounds did not promote cholesterol efflux. In conclusion, two agonist activities are carried by HDL. Apo A-I stimulates phosphatidylcholine breakdown and thereby facilitates cholesterol efflux, whereas LSF and SPC trigger PI-PLC activation and thereby stimulate cell proliferation.

High-density lipoproteins inhibit fibrinogen binding on adenosine diphosphate-activated monocytes.

High levels of fibrinogen and low levels of high-density lipoprotein (HDL) cholesterol were reported to be risk factors for coronary heart disease. CD11b/CD18, a fibrinogen-binding protein, is expressed on the surface of monocytes, which play a crucial role in the formation of atherosclerotic lesions. In the present study, we investigate the effects of antibodies against CD11b and CD18, as well as HDL3 and low-density lipoprotein (LDL) cholesterol on fibrinogen binding on monocytes. We find that binding of fibrinogen on monocytes activated with adenosine diphosphate (ADP) was reduced to 66.0+/-8.3% (mean +/- SD) in the presence of anti-CD11b antibodies (12.5 microg/ml; P < or = 0.02) and to 54.5+/-4.9% in the presence of anti-CD18 antibodies (20 microg/ml; P < or = 0.01), respectively. Fibrinogen binding on Cytochalasin-B-activated monocytes was reduced to 79.8+/-6.0% in the presence of anti-CD18 (20 microg/ml; P < or = 0.05). Incubation of ADP-activated monocytes with HDL3 (0.5 g/l) led to a lowering of fibrinogen binding to 65.0+/-6.6% (P < or = 0.05). No effect of HDL3 on fibrinogen binding was seen on Cytochalasin-B-activated monocytes. A slight, non-significant stimulatory effect of LDL on fibrinogen binding on ADP-activated but not on Cytochalasin-B-activated monocytes was additionally observed. Neither incubation with HDL3 or with LDL had a significant influence on ADP-activated cellular binding of anti-CD11b or anti-CD18 antibodies. The inhibition of fibrinogen binding on monocytes in the presence of HDL3 is a major new finding of this study. Since inhibition of fibrinogen binding in the presence of HDL might impair both monocyte recruitment to the arterial wall and foam cells formation, our findings suggests a novel mechanism by which HDL may prevent development of arteriosclerosis.

Involvement of phospholipase D in store-operated calcium influx in vascular smooth muscle cells.

In non-excitable cells, sustained intracellular Ca2+ increase critically depends on influx of extracellular Ca2+. Such Ca2+ influx is thought to occur by a 'store-operated' mechanism, i.e. the signal for Ca2+ entry is believed to result from the initial release of Ca2+ from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Here we show that the depletion of cellular Ca2+ stores by thapsigargin or bradykinin is functionally linked to a phosphoinositide-specific phospholipase D (PLD) activity in cultured vascular smooth muscle cells (VSMC), and that phosphatidic acid formed via PLD enhances sustained calcium entry in this cell type. These results suggest a regulatory role for PLD in store-operated Ca2+ entry in VSMC.

D609-phosphatidylcholine-specific phospholipase C inhibitor attenuates thapsigargin-induced sodium influx in human lymphocytes.

Previously, we reported that the phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor tricyclodecan-9-yl xanthogenate (D609) potentiates thapsigargin-induced Ca(2+) influx in human lymphocytes. In the present study we examined the effect of D609 on the thapsigargin-induced Na(+) entry. We found that the early phase of the thapsigargin-induced increase in the intracellular Na(+) concentration (approx. 1-2 min after stimulation) was attenuated after preincubation of lymphocytes with D609. By contrast, thapsigargin-induced Na(+) influx was not affected in the presence butan-1-ol, which inhibits phosphatidylcholine-specific phospholipase D (PC-PLD). The thapsigargin-induced Na(+) influx could be mimicked by PC-PLC exogenously added to the lymphocyte suspension, whereas addition of PC-PLD had no effect. In addition, thapsigargin stimulated formation of the physiological PC-PLC products, diacylglycerol. Cell-permeable diacylglycerol analogue, dioctanoyl-glycerol (DOG), produced time- and concentration-dependent increase in the intracellular Na(+) concentration. Both thapsigargin- and DOG-induced Na(+) increases were not affected in the presence of Na(+)/H(+) antiport inhibitor, HOE609, or Na(+)/Ca(2+) antiport inhibitor, dimethylthiourea, as well as in the presence of Co(2+) and Ni(2+), which block store-operated Ca(2+) entry. By contrast, markedly reduced thapsigargin- and DOG-induced Na(+) influx were noted in the presence of flufenamic acid, which blocks the non-selective cation current (I(CRANC)). In conclusion, our results suggest that diacylglycerol released due to the PC-PLC activation contributes to the thapsigargin-induced Na(+) entry.

Phospholipase A(2) is involved in thapsigargin-induced sodium influx in human lymphocytes.

Previously, we reported that emptying of intracellular Ca(2+) pools with endoplasmatic Ca(2+)-ATP-ase inhibitor thapsigargin leads to the Na(+) influx in human lymphocytes (M. Tepel et al., 1994, J. Biol. Chem. 269, 26239-26242). In the present study we examined the mechanism underlying the thapsigargin-induced Na(+) entry. We found that the thapsigargin-induced increase in Na(+) concentration was effectively inhibited by three structurally unrelated phospholipase A(2) (PLA(2)) inhibitors, p-bromophenacyl bromide, 3-(4-octadecyl)-benzoylacrylic acid (OBAA), and bromoenol lactone (BEL). The thapsigargin-induced Na(+) influx could be mimicked by PLA(2) exogenously added to the lymphocyte suspension. In addition, thapsigargin stimulated formation of arachidonic acid (AA), the physiological PLA(2) product. AA induced Na(+) entry in a time- and concentration-dependent fashion. Both, thapsigargin-induced Na(+) influx and AA liberation were completely inhibited in the presence of tyrosine kinase inhibitor genistein but not in the absence of extracellular Ca(2+). Collectively, these data show that thapsigargin-induced Na(+) entry is associated with tyrosine kinase-dependent stimulation of PLA(2).