Azaspiracid-4 inhibits Ca 2+ entry by stored operated channels in human T lymphocytes

Abstract

Azaspiracids (AZs) are a new group of phycotoxins discovered in the Ireland coast that includes the isolated analogues: AZ-1, AZ-2, AZ-3, AZ-4 and AZ-5 and the recently described AZ-6–11. Azaspiracid toxic episodes show gastrointestinal illness, but neurotoxic symptoms are also observed in mouse bioassay. Despite their great importance in human health, so far its mechanism of action is largely unknown. In this report, we present the first data about the effect of AZ-4 on cytosolic calcium concentration [Ca 2+] i in freshly human lymphocytes. Cytoslic Ca 2+ variations were determined by fluorescence digital imaging microscopy using Fura2 acetoxymethyl ester (Fura2-AM). AZ-4 did not modify cytosolic Ca 2+ in resting cells. However, the toxin dose-dependent inhibited the increase in cytosolic Ca 2+ levels induced by thapsigargin (Tg). AZ-4 decreased Ca 2+-influx induced by Tg but did not affect the Ca 2+-release from internal stores induced by this drug. The effects of AZ-4 on Ca 2+-influx induced by Tg were reversible and not regulated by adenosine 3′,5′-cyclic monophosphate (cAMP) pathway. When AZ-4 was added before, after or together with nickel, an unspecific blocker of Ca 2+ channels, the effects were indistinguishable and additive. AZ-4 also inhibited maitotoxin (MTX)-stimulated Ca 2+-influx by 5–10%. Thus, AZ-4 appeared to be a novel inhibitor of plasma membrane Ca 2+ channels, affecting at least to store operated channels, showing an effect clearly different from other azaspiracid analogues.