Recombinant human tumor necrosis factor alpha induces calcium oscillation and calcium-activated chloride current in human neutrophils. The role of calcium/calmodulin-dependent protein kinase

• Published in: The Journal of biological chemistry 1993-01, Vol.268 (3), p.2134-2140
• Main Authors: SCHUMANN, M. A, GARDNER, P, RAFFIN, T. A
• Format: Article
• Language: English
• Subjects: Analysis of the immune response. Humoral and cellular immunity; Biological and medical sciences; Calcium - metabolism; Calcium - pharmacology; Calcium-Calmodulin-Dependent Protein Kinases; Chloride Channels; Cytosol - metabolism; Egtazic Acid - analogs & derivatives; Egtazic Acid - pharmacology; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; Immunobiology; Ionomycin - pharmacology; Lymphokines, interleukins ( function, expression); Membrane Proteins - drug effects; Membrane Proteins - physiology; Neutrophils - drug effects; Neutrophils - physiology; Nitrobenzoates - pharmacology; Peptide Fragments - metabolism; Peptide Fragments - pharmacology; Peptides - pharmacology; Protein Kinase C - antagonists & inhibitors; Protein Kinase C - metabolism; Protein Kinase C - pharmacology; Protein Kinases - metabolism; Recombinant Proteins - pharmacology; Regulatory factors and their cellular receptors; Tumor Necrosis Factor-alpha - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)53972-7

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.