The effect of cholesterol on membrane potentials, proliferation and migration of cultivated endothelial cells]

The influence of cholesterol added as cholesterol containing liposomes [sphingomyelin/cholesterol (1: 1; mol/mol)] or as cholesterol suspensions on cultured bovine aortic endothelial cells was investigated. 10(-6) mol/l cholesterol enhanced membrane potential, 10(-5) mol/l caused depolarisation. The proliferation of the cells was dependent on the concentration influenced in opposite directions too. The proliferation was stimulated by 10(-6) mol/l cholesterol and inhibited by 10(-5) mol/l. The migratory rate of the cells was increased by 10(-6) mol/l and 10(-5) mol/l cholesterol. Our results suggest that exogenous cholesterol is integrated in membranes of the endothelial cells and causes in this way changes of membrane potential, proliferation rate and migratory activity.     

Tyrosine splitting aminopeptidases on cultivated anterior hypophyseal and vascular endothelial cells]

Activities of aminopeptidases for a tyrosine peptide hydrolysis were characterized with Tyrosyl-7-amino-4-methyl-coumarin as substrate on in vitro cultivated anterior pituitary cells, respectively, on aortic endothelial cells. Furthermore the corresponding activities were measured in different fractions of the cells. The activities of the enzymes in soluble fractions of the cell homogenates are comparable with aminopeptidases of cytosolic compartments of other tissue samples. On the other hand remarkable differences exist between Km- and IC50-values of the membrane preparations of both cell types. Furthermore, the substrate degradation on intact cells by provable membrane bound ectoenzymes is identically for both cell types and this degradation is insensitive for amastatin. Our results are discussed with special respect for the importance of the degradation of biological active peptides with N-terminal tyrosine by aminopeptidases on their physiological targets.     

Unproductive cleavage and the inactivation of protease-activated receptor-1 by trypsin in vascular endothelial cells

1 Using fura-2 fluorometry of [Ca(2+)](i) in response to thrombin, trypsin and protease-activated receptor activating peptides (PAR-APs), we determined whether trypsin cleaves protease-activated receptor 1 (PAR1) and activates it in the endothelial cells of the porcine aortic valves and human umbilical vein. 2 Once stimulated with thrombin, the subsequent application of trypsin induced a [Ca(2+)](i) elevation similar to that obtained without the preceding stimulation with thrombin in the valvular endothelial cells. However, the preceding stimulation with trypsin abolished the subsequent response to thrombin, but not to bradykinin or substance P. 3 The response to PAR1-AP (SFLLRNP) was significantly (P<0.05) reduced by the preceding stimulation with thrombin and PAR1-AP in the valvular endothelial cells, while, importantly, it remained unaffected by the preceding stimulation with either trypsin or PAR2-AP (SLIGRL). The response to PAR2-AP was reduced by the preceding stimulation with trypsin and PAP2-AP. PAR1-AP attenuated the subsequent responses not only to thrombin and PAR1-AP but also to trypsin and PAR2-AP, while PAR2-AP specifically attenuated the subsequent responses to trypsin and PAR2-AP. 4 In human umbilical vein endothelial cells, a higher affinity PAR1-AP (haPAR1-AP) (Ala-pF-Arg-Cha-HArg-Tyr-NH(2)) specifically attenuated the responses to thrombin but not trypsin. On the other hand, the response to haPAR1-AP was significantly (P<0.05) attenuated by the preceding stimulation with thrombin but not trypsin. 5 In conclusion, trypsin cleaved PAR1 but did not activate it in the endothelial cells. Moreover, the trypsin-cleaved PAR1 was no longer responsive to thrombin.     

西洋参茎叶皂甙单体P－F_（11）对培养大鼠心肌细胞动作电位的作用

从西洋参茎叶皂甙中分离提取的Ｐｓｅｕｄｏｇｉｎｓｅｎｏｓｉｄｅ－Ｆ１１（３、１０、３０ｍｇ·Ｌ－１，Ｐ－Ｆ１１）使培养的Ｗｉｓｔａｒ大鼠心肌细胞动作电位的幅值、动作电位时程、阈电位、最大舒张电位、０期最大除极速率和复极１０％、５０％水平的动作电位时程剂量依赖性增大。Ｐ－Ｆ１１（１０ｍｇ·Ｌ－１）的作用可被维拉帕米（２μｍｏｌ·Ｌ－１）所对抗，Ｐ－Ｆ１１（３０ｍｇ·Ｌ－１）与ＢａｙＫ８６４４（０．７５ｍｍｏｌ·Ｌ－１）比较对心肌细胞动作电位的电参数的作用相似。实验结果表明，Ｐ－Ｆ１１可能具有钙通道激活作用。     

西洋参茎叶皂甙单体P－F_（11）对培养大鼠心肌细胞动作电位的作用

从西洋参茎叶皂甙中分离提取的Ｐｓｅｕｄｏｇｉｎｓｅｎｏｓｉｄｅ－Ｆ１１（３、１０、３０ｍｇ·Ｌ－１，Ｐ－Ｆ１１）使培养的Ｗｉｓｔａｒ大鼠心肌细胞动作电位的幅值、动作电位时程、阈电位、最大舒张电位、０期最大除极速率和复极１０％、５０％水平的动作电位时程剂量依赖性增大。Ｐ－Ｆ１１（１０ｍｇ·Ｌ－１）的作用可被维拉帕米（２μｍｏｌ·Ｌ－１）所对抗，Ｐ－Ｆ１１（３０ｍｇ·Ｌ－１）与ＢａｙＫ８６４４（０．７５ｍｍｏｌ·Ｌ－１）比较对心肌细胞动作电位的电参数的作用相似。实验结果表明，Ｐ－Ｆ１１可能具有钙通道激活作用。     

Investigations of the influence of essential oils and their main components on the adenosine uptake by cultivated endothelial cells

The inhibition of adenosine uptake by endothelial cells which increases the local concentration of the neuromodulator adenosine and the release of adenine nucleotides may be involved in the pharmacological effects of essential oils via interaction between vascular endothelium and the nervi olfactorii of the nose mucosa.     

超声波对胰蛋白酶活力影响的机理研究

目的探索超声波对胰蛋白酶催化的影响效果和作用方式。方法研究胰蛋白酶经不同频率、不同功率、不同时间的超声波处理后酶活力的变化;用动力学参数变化和光谱学探索其影响催化的机理。结果经超声波处理后酶活力普遍升高。用15 kHz5、0 W超声波处理胰蛋白酶3 min,酶活力可提高45.4%。将粗酶用SephadexG75凝胶色谱,分部液在聚丙烯酰胺凝胶电泳检验显示出一条带,说明酶被纯化到电泳纯程度。对纯酶进行动力学分析,结果表明经超声波处理后Km值变小,Vmax值也降低,说明超声波处理使酶对底物的亲和力增大。超声波处理不会改变胰蛋白酶分子的构型,但会改变酶分子的构象。结论超声波对胰蛋白酶催化活性的提高可能是由于改变酶分子的构象、增强酶分子对底物的亲和力的结果。     

A novel action of palmitoyl-L-carnitine in human vascular endothelial cells

Palmitoyl-L-carnitine (palcar), which accumulates in ischemic heart, affects cellular functions of vascular endothelium in the ischemic area. The aim of this study was to examine the effects of palcar on intracellular Ca(2+) concentration ([Ca(2+)](i)) in vascular endothelial cells in comparison with those of sphingosine-1-phosphate (S1P) and to investigate the underlying mechanisms. Application of palcar at a concentration range between 0.3 and 3 micro M elevated [Ca(2+)](i) in huvecs, and its potency was about 30 times lower than that of S1P. When human umbilical vein endothelial cells (huvecs) were treated with 100 ng/ml pertussis toxin (PTX) for 15 h, they failed to respond to palcar or S1P, but did respond to 3 micro M histamine (His), suggesting that the response induced by palcar as well as S1P is mediated by a PTX-sensitive GTP binding protein, Gi. Although the sensitivity to palcar and S1P varied widely among huvecs from individuals, response to 3 micro M palcar in each huvec clearly paralleled that to 0.3 micro M S1P (r = 0.79, P<0.001). On the other hand, pre-treatment of huvecs with palcar abolished subsequent S1P-induced elevation of [Ca(2+)](i), but not the His-induced elevation. Our data indicate that palcar has a novel action on huvecs as a potential agonist of receptors for S1P. Effective inhibition of the response to S1P by palcar suggests that palcar affects functions regulated by S1P.     

Synthesis of somatostatin analogs resistant to the action of trypsin

The synthesis of a series of octapeptides based on the somatostatin analog cyclo(-Asn-Phe-Phe-d -Trp-Lys-Thr-Phe-Gaba-) containing the substitutions [Aap⁹], [d -Lys⁹], [l -Trp⁸, d -Lys⁹], [l -Orn⁹] and [d -aThr¹⁰] is reported. The analogs were designed and have been shown to inhibit proteolysis at the proposed (1) primary cleavage site between Lys⁹-Thr¹⁰ and thereby increase their stability to enzymic attack.     

Anti-NO action of carvedilol in cell-free system and in vascular endothelial cells

1. Carvedilol, an adrenoceptor blocker with antioxidant activity, was studied for its ability to interact with NO in a cell-free condition and in an endothelial cell line (ECV304). 2. In a cell-free system, carvedilol attenuated NO-dependent reduction of carboxy-2-phenyl-4,4, 5,5-tetramethyl-imidazoline-1-oxyl-3-oxide induced by a NO donor, 1-hydroxy-2-oxo-3-(aminopropyl)-3-isopropyl-1-triazene (NOC5), which was determined by electron paramagnetic resonance (EPR) spectrometry. The EPR study also showed that nitrosylhaemoglobin formation in rat red blood cells by the addition of NO-saturated solution was attenuated by prior incubation with 0.1 - 10 microM carvedilol. 3. NO-induced fluorescence in 4,5-diaminofluorescein-2 diacethyl (DAF-2DA)-loaded ECV304 cells was attenuated by carvedilol but not by labetalol. The IC(50) of carvedilol for NOC5 or sodium nitroprusside-induced fluorescence of DAF-2DA in ECV304 cells was 1. 0x10(-7) M, which was similar to the reported IC(50) of carvedilol for the antioxidant effect. 4. Cell toxicity induced by a NO donor determined by the number of viable cells after 24 h treatment with 2-2'(hydroxynitrosohydrazino)bis-ethanamine was significantly attenuated by pretreatment with 1 microM carvedilol. 5. Both free and cell-associated carvedilol quenched NO. Because NO mediates both physiological and pathophysiological processes, NO quenching by the drug may have diverse clinical implications depending upon specific functions of local NO in tissues where carvedilol is distributed.     

Potentiation of the action of bradykinin on smooth muscle by chymotrypsin, chymotrypsinogen and trypsin

Chymotrypsin, chymotrypsinogen and trypsin sensitized the guinea-pig isolated ileum and rat isolated uterus preparations to the action of bradykinin, whilst the responses to histamine, acetylcholine and 5-hydroxytryptamine were unaffected. Chymotrypsin caused a quick contraction of the guinea-pig ileum which was abolished by mepyramine and therefore probably mediated by histamine. Trypsin contracted the rat uterus as well as the guinea-pig ileum; the latter contraction was slow, resistant to mepyramine and gave rise to tachyphylaxis. It is suggested that isolated smooth muscle preparations should be treated with chymotrypsin for use in the estimation of minute amounts of bradykinin.     

Vasorelaxant effects of pramanicin, an anti-fungal agent: selective action on endothelial cells

A newly discovered antifungal agent, pramanicin, within the therapeutically effective concentration range (4-100 microM), inhibits the tone of phenylephrine (PE)-precontracted dog carotid arterial rings in a concentration-dependent manner and leads to gradual development of relaxation. However, pramanicin had no effect on rings precontracted with 100 mM KCl or on endothelium-denuded rings. Thus, inhibition by pramanicin of PE-induced contraction was endothelium-dependent. Preincubation of 100 microM pramanicin with carotid arterial rings for 30 min did not significantly affect the concentration-contraction response to PE, but almost completely inhibited the endothelium-dependent relaxation response to subsequent addition of 3 microM carbachol or 100 microM pramanicin. This irreversible inhibition of endothelium-dependent relaxation, which is independent of extracellular Ca2+, suggests possible endothelial cell damage by pramanicin. Pretreatment of the endothelium-intact vascular rings with L-N(G)-nitro-arginine (100 microM) inhibited the relaxation of PE-precontracted rings induced by 3 microM carbachol or 100 microM pramanicin, suggesting that the generation of nitric oxide (NO) in endothelial cells mediates the slow vascular relaxation induced by pramanicin. We conclude that pramanicin has little direct effect on the contractility of smooth muscle cells, but causes an initial slow endothelium-dependent, NO-mediated vascular relaxation. This is followed by a cytotoxic effect on vascular endothelial cells, eventually resulting in the loss of vasorelaxant function.     

The effect and mechanism of excessive iodine on the endothelial function of human umbilical vein endothelial cells

Iodine excess (IE) can cause thyroid dysfunction, thyroid diseases can adversely affect cardiovascular function. Accordingly, this study was to explore the direct and indirect effects of IE on endothelial function. Nthy-ori 3-1 and HUVECs cells were treated with potassium iodide (KI). CCK-8, LDH leakage, Elisa, RT-PCR and Western blotting were used to detect relevant indicators. Results showed that a certain level of KI can directly and indirectly reduce the viability of HUVECs and increase cytotoxicity. KI decreased the expression of ET-1 and VWF in HUVECs, inhibited the secretion of ET-1 in culture medium, and increased the expression of IL-6 and TNFα in HUVECs or Nthy-ori 3-1 cells alone. In the co-culture system, KI decreased the expression of ET-1 and THBD and increased the expression of TNFα and IL-6. Collectively, IE can directly and indirectly inhibit endothelial function of endothelial cells, which may be related to its induced inflammatory response.     

中药保护内皮祖细胞的有效成分及作用机制

近5年来中药界学人发现22种中药成分可有效保护内皮祖细胞（EPCs）。分别通过调节氧化应激反应、抑制EPCs凋亡、阻遏炎症因子的分泌、增强EPC动员、迁移、分化等生物学功能发挥对EPCs的保护作用。对此做系统论述及整理,并总结目前研究中存在的问题及以后的研究方向。