Effects of Azaspiracids 2 and 3 on Intracellular cAMP,[Ca~(2+)],and pH

摘要

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_i)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_i are quite different.