Investigation into the relationship between calyculin A-mediated potentiation of NADPH oxidase activity and inhibition of store-operated uptake of calcium by human neutrophils

• Published in: Biochemical pharmacology 2004-11, Vol.68 (9), p.1721-1728
• Main Authors: Oommen, Joyce, Steel, Helen C., Theron, Annette J., Anderson, Ronald
• Format: Article
• Language: English
• Subjects: Adult; Biological and medical sciences; Biological Transport - drug effects; Calcium - metabolism; Calyculin A; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Fura-2 - chemistry; Humans; Immunobiology; In Vitro Techniques; Manganese - chemistry; Membrane potential; Membrane Potentials - drug effects; Myeloid cells: ontogeny, maturation, markers, receptors; NADPH oxidase; NADPH Oxidases - metabolism; Neutrophils - drug effects; Neutrophils - enzymology; Oxazoles - pharmacology; Oxygen Consumption; Polynuclears; Spectrometry, Fluorescence; Store-operated calcium influx; Superoxides - metabolism
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/j.bcp.2004.07.004

The primary objective of the current study was to investigate possible relationships between calyculin A (CA)-mediated potentiation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and inhibition of store-operated uptake of Ca 2+ by chemoattractant-activated human neutrophils. Treatment of neutrophils with 100 nM CA, but not at lower concentrations (12.5–50 nM), prior to the addition of the N-formylated chemotactic tripeptide, N-formyl- l-methionyl- l-leucyl- l-phenylalanine (FMLP) (1 μM), both potentiated and prolonged the activity of NADPH oxidase which was accompanied by exaggerated membrane depolarisation, delayed and attenuated membrane repolarisation, and inhibition of store-operated Ca 2+ influx. Inclusion of diphenylene iodonium chloride (DPI, 10 μM), an inhibitor of NADPH oxidase, antagonised the effects of CA on NADPH oxidase activity and the membrane repolarisation responses of FMLP-activated neutrophils, but failed to restore store-operated influx of Ca 2+. Similarly, CA also inhibited store-operated influx of Ca 2+ into FMLP-activated neutrophils from a patient with chronic granulomatous disease, a primary immunodeficiency disorder characterised by the absence of a functional NADPH oxidase. CA also inhibited the store-operated influx of Ca 2+ into control neutrophils treated with 1 μM thapsigargin, a selective inhibitor of the endomembrane Ca 2+-ATPase, which does not activate NADPH oxidase. Taken together, these observations demonstrate that augmentation of NADPH oxidase activity is not primarily involved in CA-mediated inhibition of the store-operated influx of Ca 2+ into activated human neutrophils.