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Published online before print August 7, 2007

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(Journal of Leukocyte Biology. 2007;82:1266-1277.)
© 2007 by Society for Leukocyte Biology

Store-operated calcium entry mediates intracellular alkalinization, ERK1/2, and Akt/PKB phosphorylation in bovine neutrophils

Laboratory of Molecular Pharmacology, Institute of Pharmacology, Faculty of Veterinary Science, Universidad Austral de Chile, Valdivia, Chile

¹ Correspondence: Laboratory of Molecular Pharmacology, Institute of Pharmacology, Faculty of Veterinary Science, Universidad Austral de Chile, P.O. Box 567, Valdivia, Chile. E-mail: rburgos1{at}uach.cl

Neutrophil’s responses to G protein-coupled chemoattractants are highly dependent on store-operated calcium (Ca²⁺) entry (SOCE). Platelet-activating factor (PAF), a primary chemoattractant, simultaneously increases cytosolic-free Ca²⁺, intracellular pH (pH_i), ERK1/2, and Akt/protein kinase B (PKB) phosphorylation. In this study, we looked at the efficacy of several putative SOCE inhibitors and whether SOCE mediates intracellular alkalinization, ERK1/2, and Akt/PKB phosphorylation in bovine neutrophils. We demonstrated that the absence of external Ca²⁺ and the presence of EGTA reduced the intracellular alkalinization and ERK1/2 phosphorylation induced by PAF, apparently via SOCE influx inhibition. Next, we tested the efficacy of several putative SOCE inhibitors such as 2-aminoethoxydiphenyl borate (2-APB), capsaicin, flufenamic acid, 1-{β-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride (SK&F 96365), and N-(4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP2) on Ca²⁺ entry induced by PAF or thapsigargin. 2-APB was the most potent SOCE inhibitor, followed by capsaicin and flufenamic acid. Conversely, SK&F 96365 reduced an intracellular calcium ([Ca²⁺]_i) peak but SOCE partially. BTP2 did not show an inhibitory effect on [Ca²⁺]_i following PAF stimuli. 2-APB strongly reduced the pH_i recovery, whereas the effect of flufenamic acid and SK&F 96365 was partial. Capsaicin and BTP2 did not affect the pH_i changes induced by PAF. Finally, we observed that 2-APB reduced the ERK1/2 and Akt phosphorylation completely, whereas the inhibition with flufenamic acid was partial. The results suggest that 2-APB is the most potent SOCE inhibitor and support a key role of SOCE in pH alkalinization and PI-3K–ERK1/2 pathway control. Finally, 2-APB could be an important tool to characterize Ca²⁺ signaling in neutrophils.

Key Words: calcium channel inhibitor • signal transduction • platelet-activating factor

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