Production of antibodies and development of a radioimmunoassay for okadaic acid

, , , , and . Production of antibodies and development of a radioimmunoassay for okadaic acid. Toxicon 26, 1123–1128, 1988.—An okadaic acid immunogen, prepared by conjugation of okadaic acid to bovine albumin with carbodiimide, was used to immunize two rabbits. The rabbits responded by producing antibodies that neutralized okadaic acid's stimulation of arachidonic acid metabolism and this neutralization increased during the course of immunization. The immune sera bound ³H-okadaic acid and this binding also increased with repeated immunization. After absorption of the rabbit IgG with a goat anti-rabbit IgG, binding was reduced greater than 99%. The binding of okadaic acid to the antibodies in one antiserum was inhibited by as little as 0.2 pmoles of unlabelled okadaic acid. The apparent association constant for binding with this antiserum was 4.17 × 10⁹ M⁻¹ (35°C). Maitotoxin, teleocidin, 12-O-tetradecanoylphorbol-13-acetate, aplysiatoxin, palytoxin and breve-toxin B when tested at 29, 228, 168, 169, 3.7 and 112 pmole levels, respectively, did not inhibit binding. The serologic and biological activities of okadaic acid after incubation for 60 min in 0.01 N HCl at 35°C or at 100°C at pH 7.2 were unaffected.     

Okadaic acid: the archetypal serine/threonine protein phosphatase inhibitor

As the first recognized member of the "okadaic acid class" of phosphatase inhibitors, the marine natural product okadaic acid is perhaps the most well-known member of a diverse array of secondary metabolites that have emerged as valuable probes for studying the roles of various cellular protein serine/threonine phosphatases. This review provides a historical perspective on the role that okadaic acid has played in stimulating a broad spectrum of modern scientific research as a result of the natural product's ability to bind to and inhibit important classes of protein serine / threonine phosphatases. The relationships between the structure and biological activities of okadaic acid are briefly reviewed, as well as the structural information regarding the particular cellular receptors protein phosphatases 1 (PP1) and 2A. Laboratory syntheses of okadaic acid and its analogs are thoroughly reviewed. Finally, an interpretation of the critical contacts observed between okadaic acid and PP1 by X-ray crystallography is provided, and specific molecular recognition hypotheses that are testable via the synthesis and assay of non-natural analogs of okadaic acid are suggested.     

Protective effect of carvedilol on oxidative stress induced by okadaic acid in N1E-115 cells.

The effect of carvedilol on oxidative and cell damage induced by okadaic acid in N1E-115 cells were studied. The effects of okadaic acid were evaluated as changes in: the quantity of lipid peroxidation products, protein carbonyl groups, reduced glutathione content (GSH), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase and total lactate dehydrogenase (cell LDH). Additionally, a dose of carvedilol (10(-5)M) was added 2h before incubation with okadaic acid (50 nM) and was present until the end of the experiment (2h later added okadaic acid). Our results reveal that okadaic acid induces oxidative stress and an increase of cell LDH in N1E-115 cells, whereas carvedilol prevented the changes prompted by okadaic acid. In conclusion, the data show the protective effect of carvedilol, as well as its ability to modify cell response to okadaic acid, involving like cytoprotective mechanism its antioxidative properties.     

Stimulation of hepatocyte growth factor production in human fibroblasts by the protein phosphatase inhibitor okadaic acid

In this study, we examined whether the production of hepatocyte growth factor (HGF) in fibroblasts is regulated by protein phosphatase(s). Inhibitors of the enzymes okadaic acid and calyculin A were used for this purpose. Both inhibitors markedly stimulated HGF production in human skin fibroblasts in a dose-dependent manner. The effects of okadaic acid and calyculin A were maximal at 25-37.5 and 1.25 nM, respectively. Highly active HGF production in MRC-5 human embryonic lung fibroblasts was also promoted by both inhibitors. The effect of okadaic acid was accompanied by an up-regulation of HGF gene expression. The stimulating effect of okadaic acid on HGF production was synergistic with that of phorbol 12-myristate 13-acetate (PMA) and epidermal growth factor (EGF), whereas it was additive to the effect of cholera toxin. The protein kinase C (PKC) inhibitor GF 109203X inhibited the effect of PMA, but not of okadaic acid and EGF. The effect of okadaic acid as well as EGF was not inhibited, but rather enhanced in human skin fibroblasts pretreated for 24 hr with a high dose of PMA to deplete PKC, as compared with its effect in untreated cells. PD 98059, an inhibitor of mitogen-activated protein (MAP) kinase kinase, suppressed the effects of okadaic acid and EGF, but not those of cholera toxin and 8-bromo-adenosine 3',5'-cyclic monophosphate (cAMP). These results suggest that HGF production in human skin fibroblasts is down-regulated by protein phosphatase(s) and that HGF production stimulated by okadaic acid is, at least in part, dependent on the activation of the MAP kinase cascade.     

Okadaic acid is taken-up into the cells mediated by human hepatocytes transporter OATP1B3

Okadaic acid is known as a diarrheal shellfish poison. It is thought that there is no specific target organ for okadaic acid after it has been absorbed into the body. However, the details of its pharmacokinetics are still unknown.In this study, we demonstrated that okadaic acid was more toxic to the hepatocyte-specific uptake transporter OATP1B1- or OATP1B3-expressing cells than control vector-transfected cells. In addition, PP2A activity, which is a target molecule of okadaic acid, was more potently inhibited by okadaic acid in OATP1B1- or OATP1B3-expressing cells compared with control vector-transfected cells. The cytotoxicity of okadaic acid in OATP1B1- or OATP1B3-expressing cells was attenuated by known substrates of OATP1B1- and OATP1B3, but not in control vector-transfected cells. Furthermore, after uptake inhibition study using OATP1B3-expressing cells, Dixon plot showed that okadaic acid inhibited the uptake of hepatotoxin microcystin-LR, which is a substrate for OATP1B1 and OATP1B3, in a competitive manner. These results strongly suggested that okadaic acid is a substrate for OATP1B3 and probably for OATP1B1, and could be involved in unknown caused liver failure and liver cancer. Since okadaic acid possesses cytotoxicity and cell proliferative activity by virtue of its known phosphatase inhibition activity.     

冈田软海绵酸研究进展

本文概念了冈田软海绵酸的来源、分布、化学结构及其生物学特性。结合我国的研究现状,对其发展前景进行了展望,并提出了冈田软海绵酸的重点研究方向。     

Effect of okadaic acid on glucose regulation

Okadaic acid is the main toxin responsible for the natural phenomena known as diarrheic shellfish poisoning (DSP). This toxin is a tumor promoter C38 polyether fatty acid that contains acidic and hydrophobic moieties and is cyclic. Okadaic acid is a potent inhibitor of important classes of protein serine/threonine phosphatases such as protein phosphatase 1 and 2A. The toxin binds in a hydrophobic groove adjacent to the active site of the protein phosphatases and interacts with basic residues within the active site. Therefore okadaic acid causes increases in phosphorylation of proteins that affect a diverse array of cellular processes. For instance, this toxin modulates metabolic parameters in intact cells. In this sense it stimulates lipolysis, and inhibits fatty acid synthesis in adipocytes however increases glucose output and gluconeogenesis in hepatocytes. Additionally, okadaic acid reaches cytotoxic concentrations in the intestinal tissues in accordance with the diarrhea. Recent studies suggested that toxic effects of okadaic acid might be related to modification of nutrients, ionic and water absorption across the small intestine presumably by altering the transporter system. The subject of this review is limited to the effect of okadaic acid on glucose regulation and its cellular as well as clinical implications.     

Apoptotic events induced by the phosphatase inhibitor okadaic acid in normal human lung fibroblasts.

We have studied different biochemical indicators of apoptosis in okadaic acid-treated normal human lung fibroblasts (NHLF). Apoptosis was identified by fluorimetric microplate measurements of DNA content, caspase-3 activation and changes in mitochondrial and plasma membrane after 1-48-h treatments with 1-1000 nM okadaic acid. Cells exposed to okadaic acid showed activation of caspase-3, decreased DNA content (<50% of controls at >100 nM okadaic acid after 12 h of incubation) and translocation of phosphatidylserine to the outer leaflet of the plasma membrane, as indicated by the increase in Merocyanine 540 fluorescence after 4 h of incubation with more than 250 nM okadaic acid. Decreased mitochondrial membrane potential (53-98% of controls) was observed with MitoTracker Red CMXRos in all cases, which indicated an active role of mitochondria during the early phase of apoptosis. However, reactive oxygen species were significantly reduced in okadaic acid-treated fibroblasts (50-70% of controls at 1000 nM after 3 h of incubation), which indicates that ROS cannot be considered as a hallmark of apoptosis in okadaic acid-treated cells. These results provide evidence of apoptotic events induced by okadaic acid in NHLF, which can be detected by means of sensitive and reliable fluorimetric microplate assays.     

Effect of okadaic acid on histamine release from rat peritoneal mast cells activated by anti-IgE

Abstract— The effect of okadaic acid, a potent inhibitor of protein phosphatase 1 and 2A, on histamine release from mast cells has been investigated. Okadaic acid strongly and dose-dependently inhibited histamine release from mast cells induced by anti-IgE. The IC50 value of okadaic acid on histamine release induced by anti-IgE was 3·2 Nm. However, okadaic acid failed to inhibit histamine release induced by A23187 and compound 48/80. Moreover, okadaic acid showed no effect on the initial rise in intracellular Ca²⁺, Ca²⁺-mobilization from intracellular Ca²⁺-stores and the generation of inositol trisphosphate. These results suggest a possible involvement of protein phosphatase 2A in the histamine release from mast cells induced by anti-IgE.     

Isolation and identification of a cis-C8-diol-ester of okadaic acid from Dinophysis acuta in New Zealand.

A cis-isomer of a C(8)-diol ester of okadaic acid (1) was isolated during large-scale purification of pectenotoxins (PTXs) from extracts of Dinophysis acuta collected from the west coast of South Island, New Zealand. The compound was identified by NMR spectroscopic and liquid chromatography-mass spectrometry (LC-MS) studies, and is the first reported cis-isomer of an okadaic acid C(8)-diol-ester identified in Dinophysis. The more abundant trans-C(8)-diol ester of okadaic acid (2) isolated from the same Dinophysis extract was rapidly hydrolyzed to okadaic acid in vitro by the supernatant from green-lipped mussel hepatopancreas.     

Insulin alters the sensitivity of glycogen synthesis to inhibition by okadaic acid, a protein phosphatase inhibitor.

We investigated the interactions of insulin and okadaic acid, an inhibitor of protein phosphatases type-2A and type-1, on glycogen synthesis in rat, guinea pig and rabbit hepatocytes. Insulin stimulated glycogen synthesis in rat and guinea pig but not in rabbit hepatocytes. In rat and guinea pig hepatocytes, the stimulation of glycogen synthesis by insulin was inhibited by low concentrations of okadaic acid (2.5-5 nM), which did not inhibit glycogen synthesis in the absence of insulin. In rabbit hepatocytes, insulin increased the sensitivity of glycogen synthesis to inhibition by low concentrations of okadaic acid even though it did not stimulate glycogen synthesis, and in the presence of insulin and okadaic acid (5 nM) glycogen synthesis was significantly lower than in the presence of okadaic acid alone. An increase in extracellular pH from 7.4 to 7.8 in a bicarbonate-free buffer, decreased the concentration of okadaic acid causing half-maximal inhibition of glycogen synthesis. It is suggested that an increase in cytosolic pH may be one mechanism by which insulin alters the sensitivity of glycogen synthesis to phosphatase inhibition.     

Preparation of monoclonal antibodies against okadaic acid prepared from the sponge Halichondria okadai

, , , and . Preparation of monoclonal antibodies against okadaic acid prepared from the sponge Halichondria okadai. Toxicon 27, 1323–1330, 1989.—Three murine monoclonal antibodies, OA-1, OA-2 and OA-3, against okadaic acid were prepared from hybridoma clones obtained by fusion of mouse 653 myeloma cells with mouse immune spleen cells sensitized to okadaic acid-ovalbumin conjugate. Each antibody reacted with dinophysistoxin-1 ( = 35-methylokadaic acid) as well as okadaic acid, but did not react with the other diarrhetic shellfish poisons or related compounds, such as 7-O-palmitoyl-okadaic acid (analogue of dinophysistoxin-3), pectenotoxin-1 and yessotoxin. A competitive inhibition enzyme-linked immunosorbent assay which employed OA-3 antibody was performed and showed a sensitivity of about 10ppb (10ng/ml) for okadaic acid. This simple and time-saving ELISA assay system may be useful for the specific detection of diarrhetic shellfish poisons.     

Liquid chromatography-linked protein phosphatase bioassay; a highly sensitive marine bioscreen for okadaic acid and related diarrhetic shellfish toxins.

Okadaic acid and dinophysistoxin-1 were resolved by liquid chromatography, then identified and quantitated by specific inhibition of both protein phosphatase-1 and -2A (PP1/PP2A) catalytic subunits in a 32P-phosphorylase a phosphatase radioassay. Based on the IC50 for PP2A inhibition (0.2 nM), the procedure has a detection sensitivity of less than 10 pg okadaic acid. Confirmative identification by PP1 inhibition (IC50 = 19 nM) requires 500 pg okadaic acid. Analyses of methanolic extracts from control, "okadaic acid spiked" and suspected diarrhetic mussels showed the bioscreen to be accurate, reproducible and identified okadaic acid/dinophysistoxin-1 in Canadian shellfish for the first time. In addition, a protein phosphatase inhibitor distinct from okadaic acid/dinophysistoxin-1 was identified in diarrhetic mussels with a potency equivalent to 900 ng okadaic acid/g digestive tract. Protein phosphatase inhibition probably underlies the biological activity of okadaic acid as a diarrhetic shellfish toxin and tumour promoter (Cohen, P., Holmes, C. F. B. and Tsukitani, Y. (1990), TIBS 15, 98-102). The liquid chromatography-linked protein phosphatase bioscreen should therefore facilitate identification of novel toxins comprising diarrhetic profiles in infested shellfish.     

Inhibitory effect of a toxin okadaic acid, isolated from the black sponge on smooth muscle and platelets.

1. Effects of okadaic acid, a toxin isolated from marine sponges, on smooth muscle contraction and platelet activation were examined. 2. Contractions in rabbit aorta induced by high concentrations of K+ and noradrenaline were inhibited by 0.1-1 microM okadaic acid in a concentration-dependent manner. Spontaneous rhythmic contractions as well as high K+-induced contraction in guinea-pig taenia caeci were also inhibited by 1 microM okadaic acid. 3. High K+-induced contraction in rabbit aorta was accompanied by increased Ca2+ influx measured with 45Ca2+ and increased cytosolic Ca2+ [( Ca2+]cyt) measured with fura-2-Ca2+ fluorescence. Okadaic acid inhibited the contraction without inhibiting Ca2+ influx and produced only a small decrease in [Ca2+]cyt. 4. In a saponin-skinned taenia, Ca2+-induced contraction was not inhibited but rather potentiated by okadaic acid. 5. Okadaic acid, 1 microM, inhibited aggregation, ATP release and increased in [Ca2+]cyt induced by thrombin in washed rabbit platelets. Okadaic acid itself did not change the platelet activities. 6. Okadaic acid did not change the cyclic AMP content of rabbit aorta although the inhibitory effects of okadaic acid were similar to those of cyclic AMP. 7. Although the mechanism of the inhibitory effect of okadaic acid was not clarified in the present experiments, it is suggested that okadaic acid acts by inhibiting protein phosphatases resulting in an indirect activation of cyclic AMP-dependent protein phosphorylation.     

Contrasting effects of calyculin A and okadaic acid on the respiratory burst of human neutrophils

The involvement of serine/threonine protein-phosphatases in the production of superoxide (respiratory burst) by human neutrophils was investigated using calyculin A, a potent inhibitor of both protein phosphatases type 1 and 2A, and okadaic acid, which preferentially inhibits protein phosphatase type 2A. Treatment of neutrophils with calyculin A (25–75 nM) or okadaic acid (1–4 μM) had no stimulatory effect but potently enhanced total superoxide production induced by an optimal fMLP (N-formyl-methionyl-leucoyl-phenylalanine) concentration (0.1 μM). The maximal increase platacuaed with 50–75 nM calyculin A and 2–4 μM okadaic acid, reaching approximately 120 and 200% of control values, respectively. Unlike calyculin A, okadaic acid also primed the initial rate of superoxide production, suggesting that protein phosphatases may down-regulate both initiation and termination of respiratory burst. Optimal stimulation of the respiratory burst by PMA (160 nM) was inhibited by calyculin A and okadaic acid, with an IC₅₀ of 60 nM and 2 μM, respectively, although both drugs caused protein hyperphosphorylation. The inhibition was partially prevented by a nonstimulatory concentration of A23187, indicating a role of calcium in the inhibitory effects of the drugs. Unlike the optimal respiratory burst, suoptimal respiratory burst induced by PMA (1–7 nM) was enhanced by calyculin A and okadaic acid. Unprimed and primed respiratory burst were depressed by a selective antagonist of protein kinase C (GF 109203X), indicating positive regulation of these responses by protein kinase C. Thus, the use of calyculin A and okadaic acid distinguishes two regulatory processes of superoxide production. The respiratory burst induced by low PMA concentrations of fMLP was up-regulated by both calyculin A and okadaic acid, in keeping with a down-regulatory role of protein phosphatases in these responses. By contrast, intense protein kinase C activation by PMA triggered a respiratory burst which was depressed by both drugs, pointing to positive regulation of the respiratory burst by protein phosphatases.     

Inhibitory and contractile effects of okadaic acid on rat uterine muscle.

The effects of okadaic acid and its interactions with various agents known to increase, by different mechanisms, the intracellular levels of cyclic AMP and/or cyclic GMP were investigated in isolated strips of rat myometrium. Okadaic acid showed inhibitory effects at concentrations between 10(-7) M and 3 x 10(-6) M. At higher concentrations, a biphasic, contractile and then relaxant response was observed. The results obtained suggest that, in rat uterine smooth muscle, the inhibitory effects of okadaic acid are not entirely mediated by the activation of cyclic AMP- and/or cyclic GMP-dependent pathways. The data also point to the existence of a clear interaction between okadaic acid and methylxanthines, although further studies are needed to clarify the mechanisms involved in this interaction.     

Identification of okadaic acid production in the marine dinoflagellate Prorocentrum rhathymum from Florida Bay

Extracts of fifty-seven newly isolated strains of dinoflagellates and raphidophytes were screened for protein phosphatase (PP2A) inhibition. Five strains, identified by rDNA sequence analysis as Prorocentrum rhathymum, tested positive and the presence of okadaic acid was confirmed in one strain by HPLC-MS/MS and by HPLC with fluorescence detection and HPLC-MS of the okadaic acid ADAM derivative. Quantitation of the ADAM derivative indicated that the concentration of okadaic acid in the culture medium is 0.153 μg/L.     

Protein phosphatase-protein kinase interplay modulates alpha 1b-adrenoceptor phosphorylation: effects of okadaic acid

In the present work we studied the effect of protein phosphatase inhibitors on the phosphorylation state and function of alpha(1b)-adrenoceptors. Okadaic acid increased receptor phosphorylation in a time- and concentration-dependent fashion (maximum at 30 min, EC(50) of 30 nM). Other inhibitors of protein phosphatases (calyculin A, tautomycin and cypermethrin) mimicked this effect. Staurosporine and Ro 31-8220, inhibitors of protein kinase C, blocked the effect of okadaic acid on receptor phosphorylation. Neither genistein nor wortmannin altered the effect of okadaic acid. The intense adrenoceptor phosphorylation induced by okadaic acid altered the adrenoceptor-G protein coupling, as evidenced by a small decreased noradrenaline-stimulated [(35)S]GTPgammaS binding. Okadaic acid did not alter the noradrenaline-stimulated increases in intracellular calcium or the production of inositol trisphosphate. Our data indicate that inhibition of protein phosphatases increases the phosphorylation state of alpha(1b)-adrenoceptors; this effect seems to involve protein kinase C. In spite of inducing an intense receptor phosphorylation, okadaic acid alters alpha(1b)-adrenergic actions to a much lesser extent than the direct activation of protein kinase C by phorbol myristate acetate.     

Structurally different members of the okadaic acid class selectively inhibit protein serine/threonine but not tyrosine phosphatase activity.

The relative potencies of four main types of okadaic acid class compounds as inhibitors of the catalytic subunits of protein serine/threonine phosphatases 1 and 2A and the protein tyrosine phosphatase 1 were determined. These four types of compounds are okadaic acid, calyculin A, microcystin-LR, and tautomycin, which are isolated from different natural sources, a black sponge Halichondria okadai, a marine sponge Discodermia calyx, a blue-green alga Microcystis aeruginosa, and Streptomyces spirover ticillatus, respectively. While okadaic acid was a more effective inhibitor of protein phosphatase 2A (IC50, 0.07 nM) than protein phosphatase 1 (IC50, 3.4 nM), other compounds of the okadaic acid class were equally effective against the two protein serine/threonine phosphatases. The order of potency was microcystin greater than calyculin A greater than tautomycin, and the IC50S ranged from 0.1 to 0.7 nM. None of the okadaic acid class compounds inhibited protein tyrosine phosphatase 1 activity at concentrations up to 0.01 mM. These results indicate that the compounds of the okadaic acid class are selective inhibitors of protein serine/threonine but not tyrosine phosphatases.     

Preparation of monoclonal antibodies against okadaic acid prepared from the sponge Halichondria okadai

T. Usagawa, M. Nishimura, Y. Itoh, T. Uda and T. Yasumoto. Preparation of monoclonal antibodies against okadaic acid prepared from the sponge Halichondria okadai. Toxicon27, 1323–1330, 1989.—Three murine monoclonal antibodies, OA-1, OA-2 and OA-3, against okadaic acid were prepared from hybridoma clones obtained by fusion of mouse 653 myeloma cells with mouse immune spleen cells sensitized to okadaic acid-ovalbumin conjugate. Each antibody reacted with dinophysistoxin-1 ( = 35-methylokadaic acid) as well as okadaic acid, but did not react with the other diarrhetic shellfish poisons or related compounds, such as 7-O-palmitoyl-okadaic acid (analogue of dinophysistoxin-3), pectenotoxin-1 and yessotoxin. A competitive inhibition enzyme-linked immunosorbent assay which employed OA-3 antibody was performed and showed a sensitivity of about 10ppb (10ng/ml) for okadaic acid. This simple and time-saving ELISA assay system may be useful for the specific detection of diarrhetic shellfish poisons.