Effects of Azaspiracids 2 and 3 on intracellular cAMP, [Ca2+], and pH.

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. Toxic episodes of AZs show gastrointestinal illness as in diarrhetic shellfish poisoning, but neurotoxic symptoms are also observed in a 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 of AZ-2 and AZ-3 effects on intracellular cyclic adenosine monophosphate (cAMP), intracellular calcium ([Ca 2 + ] i ), and cytosolic pH levels (pH;) in freshly human lymphocytes. The variations of cAMP, calcium, and pH were determined by fluorescence digital imaging microscopy using recombinant fluorescein- and rhodamine-labeled protein kinase A, Fura2-AM, and 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester, respectively. Our experiments show that both analogues, AZ-2 and AZ-3, clearly increase cytosolic cAMP levels of human lymphocytes. In calcium studies, we found that only if cells are initially in a calcium-free medium, AZ-2 increases the intracellular calcium concentration with two components: Ca 2 + release from internal stores and Ca 2 + influx from extracellular medium. AZ-2 sensitive Ca 2 + stores seem to be different from the thapsigargin sensitive one. AZ-2-induced Ca 2 - influx is mediated through Ni 2 + and SKF96365 blockable channels, and it is additive with Tg-induced Ca 2 + influx. Surprisingly, AZ-3 does not empty intracellular stores but also increases cytosolic calcium levels. This AZ-3-induced Ca 2 + influx is mediated through Ni 2 + blockable channels, and it is not additive with Tg-induced Ca 2 + influx. In addition, AZ-3 slightly alkalinizes cytosol. In accordance with cAMP studies, we found that adenylyl cyclase (AC) modulation inhibits AZ-2- and AZ-3-evoked Ca 2 + increase and AZ-3-induced pH i rise. Thus, both analogues seem to involve an AC pathway, although its effects on [Ca 2 + ] i and pH; are quite different.