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

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. 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.

Comparative Effects of the Marine Algal Toxins Azaspiracid-1, -2, and -3 on Jurkat T Lymphocyte Cells

“…This was suspected early on by the Botana group when studying secondary messenger systems (28,29,38,41) and also reported more recently by the Murray group when they observed a discrepancy between the effects of AZA1 and fragments of AZA1 on cytotoxicity and spontaneous calcium ion oscillations (31). We have documented previously the total retraction of pseudopodia in T lymphocyte cells caused by AZA1 in a time-dependent manner that was specific to AZA1 but not elicited by a wide variety of other marine phycotoxins (24).…”

“…At relatively high concentrations (i.e., 50-1000 nM), AZA2, AZA3, AZA4, and AZA5 have been shown to have various effects on intracellular pH, cytosolic calcium, and cAMP in lymphocytes (29,41,42 (31). This study provided the first side-by-side evidence for potency differences among these regulated toxins.…”

Marine toxins and the cytoskeleton: azaspiracids

“…AZA‐1 induced an increase in the intracellular free Ca 2+ concentration in human lymphocytes and mouse primary cerebellar granule neurons [14,32]. AZA‐2 modulated the intracellular Ca 2+ concentration by enhancing the intracellular Ca 2+ rise induced by pharmacological means in lymphocytes, and AZA‐3 induced a Ca 2+ increase by itself, similarly to AZA‐1 [33]. AZAs also induced an increase in intracellular cAMP levels in lymphocytes [32,33].…”

Marine Algal Toxin Azaspiracid Is an Open-State Blocker of hERG Potassium Channels

“…11, 12 In vitro studies have shown the AZAs to be highly cytotoxic causing a variety of cytoskeletal effects 13-18 and stimulation of cAMP production and cytosolic calcium release. 19, 20 In spinal cord neurons, AZA1 was shown to inhibit bioelectrical activity through a mechanism that was independent of voltage-gated sodium (Na + ) or calcium (Ca 2+ ) current. 21 Recent findings suggest the AZAs cause apoptosis, 13, 22, 23 although this toxin class may have multiple molecular targets 13, 14 and also induce necrotic pathways concurrently.…”