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

• Published in: Chemical research in toxicology 2004-10, Vol.17 (10), p.1338-1349
• Main Authors: Román, Yolanda, Alfonso, Amparo, Vieytes, Mercedes R, Ofuji, Katsuya, Satake, Masayuki, Yasumoto, Takeshi, Botana, Luis M
• Format: Article
• Language: English
• Subjects: Calcium - metabolism; Calcium Channel Blockers - pharmacology; Calcium Channels - metabolism; Cations, Divalent; Cells, Cultured; Cytosol - chemistry; Cytosol - drug effects; Fluorescent Dyes - chemistry; Humans; Hydrogen-Ion Concentration; Intracellular Fluid - drug effects; Intracellular Fluid - metabolism; Lymphocytes - drug effects; Lymphocytes - metabolism; Marine Toxins - toxicity; Microscopy, Fluorescence; Spiro Compounds - toxicity; Time Factors
• ISSN: 0893-228X; 1520-5010
• DOI: 10.1021/tx0341862

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 ([Ca2+]i), and cytosolic pH levels (pHi) 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:  Ca2+ release from internal stores and Ca2+ influx from extracellular medium. AZ-2 sensitive Ca2+ stores seem to be different from the thapsigargin sensitive one. AZ-2-induced Ca2+ influx is mediated through Ni2+ and SKF96365 blockable channels, and it is additive with Tg-induced Ca2+ influx. Surprisingly, AZ-3 does not empty intracellular stores but also increases cytosolic calcium levels. This AZ-3-induced Ca2+ influx is mediated through Ni2+ blockable channels, and it is not additive with Tg-induced Ca2+ 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 Ca2+ increase and AZ-3-induced pHi rise. Thus, both analogues seem to involve an AC pathway, although its effects on [Ca2+]i and pHi are quite different.