SD0006 promotes nucleus pulposus cell proliferation via the p38MAPK/HDAC4 pathway.

OBJECTIVE: p38 mitogen-activated protein kinase (p38MAPK) is a critical negative regulator for nucleus pulposus (NP) cell metabolism contributing to intervertebral disc degeneration (IDD). Histone deacetylase 4 (HDAC4) has the ability to mediate cell proliferation and is affected by p38MAPK. It is unclear whether the p38 MAPK inhibitor SD0006 (SD) can regulate IDD. Our study aims to explore the effect of SD in the progression of IDD, as well as its potential mechanism. PATIENTS AND METHODS: NP cells isolating from mild degenerated NP tissues were cultured, and IL-1ß or Asiatic acid (AA) was used to activate p38MAPK and accelerate the NP cell degradation. Then, SD was used to reject the p38MAPK activation. After that, the levels of phosphorylated p38MAPK (p-p38), HDAC4, proliferating cell nuclear antigen (PCNA), inflammatory factor, proliferative cell rate, and cell cycle were determined by Western blot, RT-PCR, flow cytometry, and immunofluorescence, respectively. RESULTS: The results showed that the activation of p38MAPK by IL-1ß and AA decreased the HDAC4 expression, affected the collagen-Ⅱ expression, upregulated the TNF-α, IL-6, MMP-3, and ADAMTS mRNA levels, and prevent the NP cell proliferation by mediating cell cycles. However, the application of SD alleviated the negative effect of p-p38 by upregulating HDAC4, anti-inflammation, and promoting cell proliferation, while the blocking of HDAC4 expression partly abolished the effect of SD. CONCLUSIONS: SD can prevent NP cell degeneration by promoting cell proliferation and suppressing inflammation via p38MAPK/HDAC4 pathway.

[Quantitative measurement of citric acid in urine using tandem liquid chromatography mass spectrometry].

Objective: To find a suitable method for the determination of citric acid in the urine of patients with stones, in order to provide a new method and basis for the prevention and treatment of stone. Methods: Liquid chromatography tandem mass spectrometry was used to analyze the citric acid in urine directly. And the accuracy, stability, repeatability and other indicators of the results were detected. Results: The results showed a good linear relationship with the concentration of citric acid in urine. y=50.31x+ 0.002 6 (R(2)=0.994 21). The results were stable, reproducible [intra-day (Coefficient of Variance) CV ≈1% and inter-day CV<10%], and the accuracy of which was comparable with that of the enzyme method (n=20, R=0.97). Conclusion: Using the method of this study to detect the content of citric acid in urine has the advantages of simple operation, good repeatability, accurate results, and low price. So it is worth to be popularized and applied in clinical practice.

[Quantitative measurement of oxalic acid in urine by liquid chromatography combined with tandem mass spectrometry].

Objective: To find a suitable method for the determination of oxalic acid in the urine of patients with stones, in order to provide a new method and basis for the prevention and treatment of stone. Methods: Liquid chromatography combined with tandem mass spectrometry was used to analyze oxalic acid in urine directly.The accuracy, stability, repeatability and other indicators of the results were tested. Results: The results showed a good linear relationship with the concentration of oxalic acid in urine. y=58.524x-15.246 (R(2)=0.979 02). The results were stable, reproducible (the intra-day and inter-day coefficient of variation was less than 10% and 15%, respectively), and the accuracy was comparable with that of the enzyme method (N=20, R=0.93). Conclusion: Using the method of this study to detect the content of oxalic acid in urine has the advantages of simple operation, good repeatability, accurate results, and low price. It is worth to be popularized and applied in clinical practice.

[Effect of HIF-2α on regulating CDCP1 to promote hepatocellular carcinoma metastasis].

Objective: To investigate the effect of hypoxia inducible factor 2α (HIF-2α) on regulating CUB domain-containing protein 1 (CDCP1) and its role in hepatocellular carcinoma metastasis. Methods: HIF-2α-knocked down and HIF-2α-stably overexpressing cells (MHCC97H) were prepared by small interfering RNA (siRNA) and lentivirus transfection, respectively. The expression of CDCP1 protein and mRNA in the above cells was detected by western blot and real-time PCR. The effect of HIF-2α on cell invasion ability was determined by Transwell assay. Furthermore, immunohistochemical staining was performed to detect the expression of CDCP1 in human HCC tissue samples. Results: Both HIF-2α and CDCP1 were induced under hypoxic conditions. The activation of CDCP1 under hypoxic conditions was dependent on the expression of HIF-2α.When HIF-2α was overexpressed, the mRNA level of CDCP1 was greatly upregulated (5.92±0.28, P<0.05). When HIF-2α was knocked down by siRNA for 48 h and 72 h, the expression of CDCP1 was significantly downregulated (48 h: 0.25±0.04; 72 h: 0.18±0.02, all P<0.05). Moreover, analysis of human HCC samples showed that CDCP1 expression was correlated with tumor-free survival (P<0.05). Conclusions: The results of this study indicate that the expression of CDCP1 is regulated by HIF-2α and is correlated with the progression of HCC. Inhibition of HIF-2α/CDCP1 may play certain inhibitory role in the metastasis of HCC.

Effect of Xin Mai Jia on atherosclerosis in rats.

We investigated the therapeutic effect of Xin Mai Jia (XMJ) on atherosclerosis (AS) in rats. Rat models of AS were established by peritoneally injecting vitamin D, feeding a high-fat diet, and inducing balloon injuries in rats. The stomachs of the rats were irrigated continuously for 10 weeks with XMJ. Blood lipid- and hemorheology-related indices of blood samples were detected. Pathological changes in the right common carotid arterial tissues were also determined. The protein expression levels of endothelial nitric oxide synthase, angio-tensin-1, and endothelin-1 were determined by western blotting. XMJ reduced cholesterol, trigylecride, and low-density lipoprotein levels as well as blood viscosity, sedimentation, and hematocrit. Furthermore, XMJ alleviated vascular endothelial injury and reduced/eliminated atherosclerotic plaques. In contrast, XMJ significantly increased the endothelium-dependent relaxing response of the AS rat models. The western blotting results showed that XMJ upregulated endothelial nitric oxide synthase but downregulated angiotensin-1 and endothelin-1. XMJ prevented the development of AS by regulating blood lipid levels, hemorheology, and vascular function.

Bispectral index for monitoring anesthetic depth in patients with severe burns receiving target-controlled infusion of remifentanil and propofol.

This study evaluated the feasibility and effectiveness of using the bispectral index (BIS) to monitor anesthetic depth in patients with severe burns receiving intravenous target-controlled infusion (TCI) of remifentanil and propofol. We randomly assigned 80 patients undergoing elective escharectomy (<1 week) to BIS (A) and control (B) groups. All patients received remifentanil and propofol as intravenous TCI anesthesia. Clinical data were recorded at different time points. The time from drug withdrawal to eye opening upon the patient hearing his/her name called and upon reaching an Aldrete score of 9 points was also recorded. During anesthesia maintenance, the target concentrations of remifentanil and propofol in group A were significantly lower than that in group B (2.12 ± 0.35 vs 2.50 ± 0.21 ng/mL and 2.54 ± 0.22 vs 2.86 ± 0.31 µg/mL, respectively; P < 0.01). The time from drug withdrawal to eye opening upon the patient hearing his/her name called and reaching an Aldrete score of 9 points in group A was considerably shorter than that in group B (7.90 ± 0.58 vs 8.35 ± 0.66 min and 9.15 ± 0.69 vs 11.13 ± 0.96 min, respectively; P < 0.01). In both groups, mean arterial pressure and heart rate values at each time point after loss of consciousness were significantly lower than the baseline values (P < 0.05), with the exception of 2 min after intubation. The use of BIS to monitor anesthetic depth in patients with severe burns receiving TCI of remifentanil and propofol during the perioperative period reduces propofol consumption and shortens the consciousness recovery time in patients.

Opposing effect of p38 CCDPK and p44/42 CCDPK signaling on TNF-alpha-induced apoptosis in bovine aortic endothelial cells.

AIM: To investigate the pro-apoptotic role of tumor necrosis factor alpha (TNF-alpha) in cultured bovine aortic endothelial cells (BAEC) and its underlied apoptotic signaling pathways. METHODS: BAEC were cultured and passaged in Dulbecco's modified Eagle's medium (DMEM). Morphologic changes and quantification of apoptotic cells were determined under fluorescence microscope after TNF-alpha treated BAEC for 24 h with Hoechst 33258 staining. Cell viability was determined with MTT method. DNA fragmentation was visualized by agarose gel electrophoresis. The expression of phospho-p38 and phospho-p44/42 Ca2+-calmodulin dependent protein kinase (CCDPK, formerly called MAPK) was measured by Western blotting. RESULTS: TNF-alpha elicited typical apoptotic morphologic changes (chromatic condensation, nucleus fragmentation) and DNA fragmentation. At 1000-5000 kU/L, incubation with TNF-alpha for 24 h induced BAEC apoptosis and both of phospho-p38 and phospho-p44/42 CCDPK expression in a concentration-dependent manner. Interestingly, TNF-alpha-stimulated activation of p44/42 CCDPK was completely blocked, TNF-alpha-induced apoptosis was markedly increased by preincubation with U0126, a specific p44/42 CCDPK inhibitor. However, SB203580, a specific p38 CCDPK inhibitor, completely blocked TNF-alpha-stimulated activation of p38 CCDPK, and enhanced the expression of phospho-p44/42 CCDPK induced by TNF-alpha, substantially inhibited the pro-apoptotic effect of TNF-alpha. CONCLUSION: TNF-alpha simultaneously activates p38 CCDPK and p44/42 CCDPK, and these two CCDPK signaling pathways appeared to play opposing roles in TNF-alpha-induced apoptosis in BAEC.

[Genetic transformation of hairy roots in Trichosanthes kirilowii Maxim. by Ti and Ri plasmids].

OBJECTIVE: To establish a hairy root culture system by double transformation for Trichosanthes kirilowii. METHOD: 1. Crown galls were induced by direct infection of sterile seedlings of T. kirilonii with Agrobacterium tumefaciens C58, and then the hairy roots were obtained from the regenerated plants by infection with A. rhzogenes 15834; 2. Transformation of Ti and Ri plasmids was inspected by high-pressure-paper electrophoresis; 3. The protein contents in the tissues of T. kirilowii were inspected by spectrophotometer and SDS-PAGE. RESULT: A hairy root culture system has been established successfully by double transformation with Ti and Ri plasmids in T. kirilowii. CONCLUSION: Compared with the ordinary hairy roots, the double transformed hairy roots grow faster but retain similar protein contents.

High glucose enhances H2O2-induced apoptosis in bovine aortic endothelial cells.

AIM: To investigate the effect of high glucose on hydroperoxide (H2O2)-induced apoptosis in cultured bovine aortic endothelial cells (BAEC). METHODS: BAEC were cultured and passaged in normal glucose (5.5 mmol.L-1, NG) and high glucose (25 mmol.L-1, HG). Morphologic changes and quantification of apoptotic cells were determined under fluorescence microscope after H2O2-treated BAEC for 24 h with Hoechst 33258 staining. DNA fragmentation was visualized by agarose gel electrophoresis. The expression of phospho-p38 Ca(2+)-calmodulin dependent protein kinase (CCDPK, formerly called MAPK) was measured by Western blotting. RESULTS: H2O2 elicited typical apoptotic morphologic changes (chromatic condensation, nucleus fragmentation). At 100 -300 mumol.L-1, both NG- and HG-BAEC incubated with H2O2 for 24 h increased cell apoptosis and phospho-p38 CCDPK expression in a concentration-dependent manner. In HG-BAEC, H2O2 induced DNA fragmentation at a lower concentration than that in NG-BAEC, and the apoptotic cell count in HG-BAEC was also higher than that of NG-BAEC (P < 0.05). Similarly, the expression of phospho-p38 CCDPK induced by H2O2 was up-regulated in HG-BAEC (P < 0.05). CONCLUSION: High glucose enhances H2O2-induced apoptosis in BAEC, which is related to high expression of phospho-p38 CCDPK.

High glucose impairs endothelium-dependent relaxation in rabbit aorta.

AIM: To study the effects of high glucose on endothelium-dependent relaxation (EDR) and the action of L-arginine, superoxide dismutase (SOD), or glucose re-normalization in aorta. METHODS: Measurement of EDR of the isolated rabbit thoracic aortic rings. RESULTS: Elevated glucose (25 mmol.L-1) caused profound impairment of acetylcholine (ACh)-induced relaxation, EC50: 1.6 mumol.L-1 (95% CL: 7.9 nmol.L(-1)-6.3 mumol.L-1) vs normal glucose (5.5 mmol.L-1) EC50: 0.08 mumol.L-1 (95% CL: 0.02 mumol.L(-1)-0.3 mumol.L-1) (P < 0.01), which not reversed followed by a further 24 h incubation in normal glucose M199, EC50: 2.0 mumol.L-1 (95% CL: 0.2 pmol.L(-1)-12.5 mumol.L-1). However, aortic rings incubated with mannitol (19.5 mmol.L-1) relaxed to ACh normally. L-arginine 1 mmol.L-1 or SOD 150 U.L-1 restored ACh relaxation in elevated glucose to normal, EC50: 0.16 mumol.L-1 (95% CL: 0.04 mumol.L(-1)-0.8 mumol.L-1) and 0.16 mumol.L-1 (95% CL: 0.03-0.63 mumol.L-1). The relaxation in response to sodium nitroprusside was not different between rings exposed to normal or elevated glucose. CONCLUSION: Hyperglycemia impaired EDR, which was not reversible by glucose re-normalization, increased free radical production and altered L-arginine metabolism were involved in this endothelium dysfunction.

Antisense Ca(2+)-calmodulin dependent protein kinase oligonucleotide inhibits bFGF-induced proliferation of rat vascular smooth muscle cells.

AIM: To investigate the role of Ca(2+)-calmodulin dependent protein kinase (CCDPK) on basic fibroblast growth factor (bFGF)-induced vascular smooth muscle cell (VSMC) proliferation and the inhibitory effect of antisense CCDPK oligonucleotides (ODN). METHODS: Before being exposed to bFGF, cultured rat VSMC CCDPK activity was inhibited by pretreatment with either a phosphorothioate-protected 17-mer antisense CCDPK ODN-directed against the initiation of translation sites of the p42 and p44 CCDPK isoform or with CCDPK kinase inhibitor PD98059. All ODN were introduced into cells by liposomal transfection. DNA synthesis was measured by [3H]thymidine incorporation. P44- and p42-CCDPK protein expression and phosphorylation were measured by Western blot. RESULTS: PD98059 inhibited bFGF-induced phosphorylation of CCDPK and DNA synthesis. Antisense CCDPK ODN 0.2-0.8 mumol.L-1 reduced both p44- and p42-CCDPK expression and phosphorylation of CCDPK in a concentration-dependent manner and DNA synthesis induced by bFGF. Lipofectin alone or sense and random CCDPK ODN did not affect p44- and p42-CCDPK protein expression or bFGF-induced phosphorylation of CCDPK or DNA synthesis. CONCLUSION: bFGF-stimulated rat VSMC proliferation is mediated by CCDPK. The antisense CCDPK ODN can inhibit bFGF-induced VSMC proliferation through down-regulating p44- and p42-CCDPK level.

Prevention of intima hyperplasia by mitogen-activated protein kinase antisense oligodeoxynucleotide.

AIM: To investigate the preventive effect of Ca(2+)-calmodulin dependent kinase (CCDPK) (formerly: mitogen-activated protein kinase or MAPK) antisense phosphorothioate oligodeoxynucleotide (ODN) on vascular smooth muscle cell (VSMC) proliferation in vitro and on intima hyperplasia after injury in vivo. METHODS: Liposomal transfection was used to introduce phosphorothioate-protected 17-mer antisense CCDPK ODN directed against the initiation of translation sites of the p42 and p44 CCDPK isoforms into cultured rat VSMC to deplete CCDPK and DNA synthesis induced by endothelin-1 (ET) or platelet derived growth factor (PDGF). A 17-mer sense and a random sequence CCDPK ODN were used as controls. CCDPK protein p44 and p42 levels were measured by Western blot. DNA synthesis was measured by [3H]thymidine incorporation. In in vivo study, rat balloon angioplasty was performed by a 2F Fogarty catheter. The antisense CCDPK ODN 200 micrograms was administered to the adventitial surface of the injured carotid artery by pluronic gel 30% (w/v) solution. Two weeks after vascular injury, carotid arteries were removed and cross sections were made and stained with hematoxylin/eosin for patho-histological examination. Fluorecein isothiocynate (FITC)-labeled and phosphorothioate-protected ODN was used to detect the uptake of ODN in vitro and in vivo. RESULTS: CCDPK antisense ODN (0.4 mumol.L-1) reduced p42/p44 protein expression and inhibited VSMC [3H]thymidine incorporation stimulated by ET and PDGF. Antisense CCDPK ODN treatment at 2 wk after injury resulted in a significant inhibition of intima hyperplasia, compared with untreated vessels. CONCLUSION: The p42/p44-CCDPK antisense ODN inhibits in vitro stimulated rat VSMC proliferation and in vivo injured arterial intima hyperplasia.

High glucose inhibits expression of inducible and constitutive nitric oxide synthase in bovine aortic endothelial cells.

AIM: To investigate the effects of high glucose on the expression of nitric-oxide synthase (NOS) in cultured bovine aortic endothelial cells (BAEC). METHODS: BAEC were cultured and passaged in normal glucose (NG) 5.5 mmol.L-1, high glucose (HG) 25 mmol.L-1, or high osmolarity (glucose 5.5 mmol.L-1 + mannitol 19.5 mmol.L-1, Mann-BAEC), lipopolysaccharides (LPS)-induced nitric oxide (NO) production was assessed by Griess reaction. The expression of inducible NOS (iNOS) and constitutive NOS (ecNOS) was determined by Western blot. RESULTS: At a concentration range from 0.5 to 2 mg.L-1, LPS stimulated NO production in NG-BAEC in a concentration-dependent manner. NO production reached the peak level at LPS 1 mg.L-1. HG inhibited NO production, when compared with NG- and Mann-BAEC (nitrite mumol.L-1: HG-BAEC 43 +/- 8, vs NG-BAEC 71 +/- 11, Mann-BAEC 70 +/- 9, n = 4 experiments, P < 0.01). iNOS expression was decreased by 39.9% and 39.3%, and ecNOS by 28% and 24% respectively in HG-BAEC, when compared with NG- or Mann-BAEC. However, no marked difference was observed in the LPS-induced NO production and the expression of iNOS and ecNOS between NG- and Mann-BAEC. CONCLUSIONS: Inhibition of BAEC NO production by HG was mainly due to a decreased expression of NOS protein.

Platelet-derived growth factor stimulated vascular smooth muscle cell proliferation and its molecular mechanism.

AIM: To study the molecular mechanism of platelet-derived growth factor (PDGF)-BB-stimulated vascular smooth muscle cell (VSMC) proliferation. METHODS: DNA synthesis was measured by [3H]thymidine incorporation. Phosphorylation of the 42- and 44-kDa Ca(2+)-calmodulin dependent protein kinase (CCDPK) was measured by Western blotting method. The expression of c-myc specific mRNA was detected by in situ hybridization. RESULTS: PDGF-BB (2 micrograms.L-1) induced DNA synthesis and activated CCDPK in a concentration-dependent manner and a induced a marked c-myc mRNA expression. Egtazic acid (EGTA, 5 mmol.L-1), genistein (400 mumol.L-1) or PD 98059 (50 mumol.L-1) reduced PDGF-BB (2 micrograms.L-1)-induced CCDPK activities and inhibited VSMC [3H]thymidine incorporation (P < 0.05). PD 98059 (50 mumol.L-1) also inhibited PDGF-BB (2 micrograms.L-1)-induced c-myc mRNA expression. CONCLUSION: PDGF stimulated VSMC proliferation by activation of p44/p42 CCDPK, which is mediated by Ca2+ and protein tyrosine kinase (PTK), and up-regulation of c-myc mRNA expression.

Effects of p38 and p42/p44 CCDPK signaling on H2O2-induced apoptosis in bovine aortic endothelial cells.

AIM: To investigate the effects of p38 and p42/p44 Ca(2+)-calmodulin dependent protein kinases (CCDPK) signaling on hydroperoxide (H2O2)-induced apoptosis in cultured bovine aortic endothelial cells (BAEC). METHODS: Morphologic changes and quantification of apoptotic cells were determined under fluorescence microscope after a 24-h treatment of BAEC by H2O2. Cell viability was determined with MTT method. DNA fragmentation was visualized by agarose gel electrophoresis. The expression of phospho-p38 and phospho-p42/p44 CCDPK was measured by Western blotting. RESULTS: H2O2 elicited typical apoptotic morphologic changes (chromatic condensation, nucleus fragmentation) and DNA fragmentation. At 100-500 mumol.L-1, incubation of BAEC with H2O2 for 24 h also induced phospho-p38 and phospho-p42/p44 CCDPK expression in a concentration-dependent manner. Interestingly, H2O2-induced apoptosis was markedly increased by preincubation with U0126, a specific p42/p44 CCDPK inhibitor. However, SB203580, a specific p38 CCDPK inhibitor, enhanced the expression of phospho-p42/p44 CCDPK induced by H2O2, but had no effect on BAEC survival. CONCLUSION: p42/p44 CCDPK signaling appears to play protective roles in H2O2-induced apoptosis in BAEC, whereas p38 CCDPK is not the main signaling pathway mediating H2O2-induced cellular apoptosis.

High density lipoproteins enhanced antiaggregating activity of nitric oxide derived from bovine aortal endothelial cells.

In the present study, the effect of high density lipoproteins (HDLs) on the antiaggregating activity of nitric oxide (NO) derived from endothelial cells was investigated with the use of cultured bovine aortal endothelial cells (BAECs). The BAECs were placed in an aggregometer in contact with rabbit platelets after blocking cyclo-oxygenase with acetylsalicylic acid. Under this circumstance, the antiaggregating effect of endothelial cells was exclusively dependent on the release of NO, which was further confirmed by prevention of antiaggregating activity of BAECs with 1 mmol/L N(G)-Nitro-L-arginine. When this system was used, thrombin (0.1 U/ml) evoked 67.33+/-7.52% aggregation of rabbit platelets (2 10(8)/ml). This effect was inhibited by NO derived from endothelial cells (1 10(5) 1 10(6)/ml) in a cell number dependent manner. HDLs (1 mg/ml), added into the system immediately before BAECs, enhanced this antiaggregating effect of NO. However, incubating BAECs with HDLs for an hour and removing the HDLs by centrifugation did not have the same effect, unless HDLs were present during aggregation. No direct effect of HDLs on platelet aggregation was observed. The above findings suggest that HDLs can enhance the antiaggregating effects of BAECs mediated by direct interaction with NO.

Nitric oxide derived from endothelial cells inhibits Na+/H+ exchange in rabbit platelets activated by thrombin.

AIM: To study the effect of nitric oxide (NO) derived from endothelial cells on Na+/H+ exchange in rabbit platelets activated by thrombin. METHODS: Intracellular Ca2+ ([Ca2+]i) and intracellular pH (pHi) were measured by the dual-wavelength fluorophotometer with the fluorescent probes Fura-2 and 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF). Effects of NO on rabbit platelets were tested by cultured bovine endothelial cells (BAEC). RESULTS: BAEC (0.1-1 x 10(9).L-1) inhibited thrombin (100 U.L-1)-induced platelet aggregation in a concentration-dependent manner. This inhibiting effect was abolished by preincubating BAEC with NG-nitro-L-arginine 1 mmol.L-1. When the [Ca2+]i store was depleted with ionomycin in the presence of egtazic acid (EGTA), the increase in pHi induced by thrombin was inhibited. Refilling intracellular Ca2+ store partially reversed this effect. BAEC 2 x 10(8).L-1 inhibited thrombin (100 U.L-1)-induced elevation of pHi and mobilization of intracellular Ca2+ store (P < 0.01). No direct effect of endothelial cells on unstimulated rabbit platelets was observed. CONCLUSION: NO derived from endothelial cells inhibited thrombin-induced rabbit platelet activation by inhibiting thrombin-induced [Ca2+]i mobilization and then inhibiting the consequent Na+/H+ exchange in rabbit platelets.

Regulatory role of protein tyrosine phosphorylation in platelet activating factor-induced signal transduction in platelets.

AIM: To study the role of protein tyrosine phosphorylation (PTP) in platelet activating factor (PAF)-induced platelet signal transduction cascade. METHODS: Washed rabbit platelets were used to test the inhibitory effect of genistein (Gen) on platelet aggregation and serotonin secretion. Intracellular Ca2+ ([Ca2+]i) and pH (pHi) were measured by a dual wavelength fluorophotometer with Fura 2-AM and BCECF-AM. PTP was determined with a specific anti-phosphotyrosine monoclonal antibody by Western blotting. RESULTS: Pretreatment with Gen (100 and 200 mumol.L-1) inhibited PAF (20 nmol.L-1)-stimulated platelet serotonin release by 23.7% +/- 2.0% and 41% +/- 8%, respectively. Similar inhibitory effects of Gen were observed on PAF-evoked increase of [Ca2+]i and intracellular alkalization. PAF also elicited a pronounced increase in PTP of several bands with M(r) 70,000, 60,000, 50,000, 42,000/40,000, and 34,000, which were suppressed markedly by Gen 200 and 400 mumol.L-1. Pretreatment with staurosporine (Sta) 20 nmol.L-1, BAPTA 200 mumol.L-1, and egtazic acid 2 mmol.L-1 to inhibit PKC activation, [Ca2+]i elevation, and Ca2+ influx respectively, also showed an inhibitory effects on the formation of PTP. CONCLUSION: PTP is involved in multiple signal transduction pathways induced by PAF, on which PKC activation and calcium mobilization play a regulatory role.

Characteristics of apyrase (EC 3.6.1.5) on cultured bovine endocardial endothelial cells.

Apyrase activities in some tissues and cells, such as peripheral vascular endothelial cells, have been reported, but these in endocardium endothelial cells have not been reported. The present study was to characterise the properties of bovine endocardium endothelial cells (BEEC)-associated apyrase. Apyrase activity was assayed by inorganic phosphate release, which could be inhibited concentration-dependently by NaN3, an apyrase inhibitor. NaF (20 mmol/L), another inhibitor of apyrase, also markedly inhibited the activity. EDTA or EGTA (1 mmol/L) could also inhibit the activity completely. However, the inhibitor for Na+/K(+)-ATPase, ouabain (3 mmol/L) did not affect the enzyme activity. BEEC apyrase activity was dependent on divalent cations (Ca2+ or Mg2+) and pH value.

Inhibition of platelet aggregation by bovine endocardial apyrase.

AIM: To study the anti-aggregatory effect of bovine endocardial endothelial cell (EEC)-associated apyrase. METHODS: Cultured bovine EEC was used. Adenosine diphosphate (ADP) was analyzed by reversed phase HPLC, and rabbit platelet aggregation was measured turbimetrically. RESULTS: Incubation of EEC with ADP 500 mumol.L-1 resulted in a progressive decrease in ADP concentration, which was paralleled by the decrease in platelet aggregating potential of the unmetabolized ADP. In the presence of aspirin (Asp 1 mmol.L-1)-treated EEC 1 x 10(9) cells.L-1, the aggregation of Asp (1 mmol.L-1) and methylene blue (10 mumol.L-1)-treated platelets in response to thrombin 500 U.L-1 and platelet activating factor (PAF 1 nmol.L-1) was markedly inhibited and was reversible, which was very similar to that in apyrase-treated platelets. The supernatants of EEC had no effect on platelet aggregation. EEC inhibited ADP (5 mumol.L-1)-induced platelet aggregation, but failed to inhibit adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S, an unmetabolizable structural analog of ADP, 15 mumol.L-1)-induced platelet aggregation. CONCLUSION: ADP hydrolysis by EEC-associated apyrase is a major anti-thrombotic mechanism of bovine EEC.