Abstract
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.
Highlights
From the SDiuision of Pulmonary and Critical Care Medicine and the §Cardiology Division, Departmeonf tMedicine, Stanford University School of Medicine, Stanford, California94305-5236
We studied the effect of recombinant human tumornecrosis factor a on the cytosolic free Ca2+level and on Ca2+activated C1- current in human neutrophils
We examined the recombinant human tumor necrosis factor a;PKC, protein kinaseC; effect of recombinant human tumornecrosis factor a (rhTNFa) on the cytosolic free Ca2+level in human
Summary
The pipette solution included (mM) NMDG-CI, 140; tagonist, CaM kinase I1 [290-309],were purchased from Sigma; EGTA, 0.5; MgCl,, 2; ATP ( M e salt), 2; and Hepes, 5 (pH 7.3 with ionomycin and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraa- NMDG-OH, 295mosmkg"). To alter the [Cl-] ratio(pipette control peptide, [GluZ7]PKC-(19-36),and Minimum Essential Me- compared to bath)w, e replaced portions of NMDG-CI (in thepipette) dium with Hanks' salts and L-glutamine from Gibco BRL; and the isosmotically with NMDG, or choline, and glutamic acid. PKC-(19-36), from Peninsula Laboratories, Inc. rhTNFa action and CaM kinase involvement, the [Cl-] ratio was 1:7 We purchased the fluorescent probe 2-(3,6-bis(acetyloxy)-2,7-di- (pipette compared to bath).Cs+ and Triscan alternatively be used as chloro-9H-xanthen-9-yl)benzoiaccid acetoxymethyl ester (fluo-3 major cations in the pipette and bath solutions, respectively, to give AM) and the surfactant Pluronic F-127 from Molecular Probes. Hanks' salts and L-glutamine weremixed carefully with the cell suspension, andthe cells attached within 10-15 min ina moist
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