Schumann M A, Gardner P, Raffin T A
Division of Pulmonary and Critical Care Medicine, Stanford University School of Medicine, California 94305-5236.
J Biol Chem. 1993 Jan 25;268(3):2134-40.
The role of calcium in the action of tumor necrosis factor (TNF) on human neutrophils is not clear. With fluorescent cytometry, using the visible wavelength calcium probe, fluo-3, and patch clamping, we investigated whether TNF induces cytosolic free Ca2+ changes and Ca(2+)-activated Cl- current, respectively. Bath application of 1000 units/ml recombinant human TNF alpha (rhTNF alpha) induced a rise in cytosolic free Ca2+ in 75% of fluo-3-loaded cells, 25% of which displayed irregular patterns of oscillation. Addition of rhTNF alpha activated Cl- current in 80% of tested cells; the activated current was blocked by 10 microM 5-nitro-2-3-phenylpropylamino)benzoic acid, a Cl- channel blocker. The current was similarly activated by 1 microM ionomycin, a Ca2+ ionophore. To study the mechanism by which rhTNF alpha induced Ca(2+)-activated Cl- current, we examined the involvement of calcium/calmodulin-dependent protein kinase (CaM kinase). With intracellular application of the Ca2+ chelator 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (5 mM), the calmodulin antagonist (2 microM), CaM kinase II-(290-309), or the inhibitory peptide (10 microM), CaM kinase II-(273-302), the current was no longer activated by rhTNF alpha. The intracellular application of the control peptide (10 microM), CaM kinase II-(284-302), or the protein kinase C (PKC) inhibitory, PKC-(19-36), or control, [Glu27]PKC-(19-36), peptide (5 microM) did not block the rhTNF alpha-induced Cl- current. These results show that Ca2+ changes are associated with the effects of rhTNF alpha and that CaM kinase plays a role in the mechanism underlying rhTNF alpha-induced activation of Ca(2+)-activated Cl- current in human neutrophils.
钙在肿瘤坏死因子（TNF）作用于人类中性粒细胞过程中的作用尚不清楚。我们使用可见波长钙探针fluo-3通过荧光细胞术以及膜片钳技术，分别研究了TNF是否诱导胞质游离Ca2+变化和Ca(2+)-激活的Cl-电流。浴槽中加入1000单位/毫升重组人TNFα（rhTNFα）可使75%的加载fluo-3的细胞胞质游离Ca2+升高，其中25%表现出不规则的振荡模式。加入rhTNFα可使80%的受试细胞激活Cl-电流；激活的电流被10微摩尔5-硝基-2-（3-苯丙氨基）苯甲酸（一种Cl-通道阻滞剂）阻断。该电流同样可被1微摩尔离子霉素（一种Ca2+离子载体）激活。为研究rhTNFα诱导Ca(2+)-激活的Cl-电流的机制，我们检测了钙/钙调蛋白依赖性蛋白激酶（CaM激酶）的参与情况。通过细胞内应用Ca2+螯合剂1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸（5毫摩尔）、钙调蛋白拮抗剂（2微摩尔）、CaM激酶II-（290-309）或抑制性肽（10微摩尔）、CaM激酶II-（273-302），rhTNFα不再激活电流。细胞内应用对照肽（10微摩尔）、CaM激酶II-（284-302）或蛋白激酶C（PKC）抑制剂PKC-（19-36）或对照肽[Glu27]PKC-（19-36）（5微摩尔）并不阻断rhTNFα诱导的Cl-电流。这些结果表明，Ca2+变化与rhTNFα的作用相关，且CaM激酶在rhTNFα诱导人类中性粒细胞Ca(2+)-激活的Cl-电流的潜在机制中发挥作用。
