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(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.
Departamentos de Farmacología and Departamento de Fisiología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain.
Yolanda
Y
Román
Amparo
A
Alfonso
Mercedes R
MR
Vieytes
Katsuya
K
Ofuji
Masayuki
M
Satake
Takeshi
T
Yasumoto
Luis M
LM
Botana
D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism; D002413:Cations, Divalent; D002478:Cells, Cultured; D003600:Cytosol / Q000737:chemistry / Q000187:drug effects; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism*; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D008387:Marine Toxins / Q000633:toxicity*; D008856:Microscopy, Fluorescence; D013141:Spiro Compounds / Q000633:toxicity*; D013997:Time Factors; D000095702:Polyether Toxins
D002118:Calcium; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D002413:Cations, Divalent; D005456:Fluorescent Dyes; D008387:Marine Toxins; D013141:Spiro Compounds; C406592:azaspiracid; D000095702:Polyether Toxins