title: Azaspiracids modulate intracellular pH levels in human lymphocytes.
abstract: The azaspiracids (AZAs) are a group of marine toxins implicated in several intoxications whose mechanism of action is unknown. These phycotoxins include the five compounds shown in : AZA-1 (1), AZA-2 (2), AZA-3 (3), AZA-4 (4), and AZA-5 (5). The aim of this work was to study the effects of the five naturally occurring azaspiracids (AZA-1 to -5, Fig. 1) and four synthetic analogues (6-9, Fig. 2) on intracellular pH, and the influence of Ca2+ upon this effect. The AZAs (1-5) were found to modulate cytosolic Ca2+ levels in human lymphocytes, while some of them, but not all, had effects on the intracellular pH. AZA-1 (1) and AZA-2 (2) did not modify intracellular pH in a Ca2+-containing or a Ca2+-free medium. AZA-3 (3) increased intracellular pH by 0.16 units in the presence of extracellular Ca2+, an effect that was blocked when a 1 mM solution of Ni2+ was added. In a Ca2+-free medium, the increase in pH induced by AZA-3 (3) was reduced to 0.08 pH units. AZA-4 (4) inhibited the basal pH increase even in the presence of a 1 mM solution of Ni2+. In a Ca2+-free medium, the inhibition caused by AZA-4 (4) was small, but when Ca2+ was added back to the medium, the pH basal increase was again significantly inhibited. The alkalinization was also inhibited when AZA-4 (4) was added simultaneously, 10 min before or 10 min after thapsigargin (Tg), and also when the Ca2+-influx induced by Tg was inhibited by Ni2+. AZA-5 (5), on the other hand, did not modulate the intracellular pH profile in either a Ca2+-containing or a Ca2+-free medium. Finally, we investigated four synthetic analogues (6-9, Fig. 2) whose structures were based on the four originally proposed structures of azaspiracid-1, with an opened E-ring. Compound 6 induced a small cytosolic Ca2+ increase, but did not modify intracellular pH in saline solution. In a Ca2+-free medium, compound 6 blocked the pH fall when Ca2+ was added back to the medium. Compound 7 also did not modify intracellular pH in saline solutions, however it significantly blocked basal pH increases in a Ca2+-free medium. Compound 8 did not alter intracellular pH, however compound 9 induced a small acidification when Ca2+ was present in the extracellular medium. These results point to a structure-activity relationship in AZAs pH effect that affects the modulation and the coupling of intracellular pH and Ca2+.
authors.affiliation: Departamento de Farmacología, Facultad de Veterinaria, USC, Campus Universitario s/n, 27002 Lugo, Spain.
authors.forename: Amparo
authors.initials: A
authors.lastname: Alfonso
authors.forename: Mercedes R
authors.initials: MR
authors.lastname: Vieytes
authors.forename: Katsuya
authors.initials: K
authors.lastname: Ofuji
authors.forename: Masayuki
authors.initials: M
authors.lastname: Satake
authors.forename: K C
authors.initials: KC
authors.lastname: Nicolaou
authors.forename: Michael O
authors.initials: MO
authors.lastname: Frederick
authors.forename: L M
authors.initials: LM
authors.lastname: Botana
mesh_terms: D002118:Calcium / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008214:Lymphocytes / Q000187:drug effects* / Q000378:metabolism*; D008387:Marine Toxins / Q000737:chemistry / Q000494:pharmacology*; D015394:Molecular Structure; D009532:Nickel / Q000494:pharmacology; D013141:Spiro Compounds / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D019284:Thapsigargin / Q000494:pharmacology; D000095702:Polyether Toxins
chemical_list: D002118:Calcium; D008387:Marine Toxins; D009532:Nickel; D013141:Spiro Compounds; D019284:Thapsigargin; C406592:azaspiracid; D000095702:Polyether Toxins