calyculin-a and microcystin

Okadaic acid (OA) and microcystin (MC) as well as several other microbial toxins like nodularin and calyculinA are known as tumor promoters as well as inducers of apoptotic cell death. Their intracellular targets are the major serine/threonine protein phosphatases. This review summarizes mechanisms believed to be responsible for the death induction and tumor promotion with focus on the interdependent production of reactive oxygen species (ROS) and activation of Ca(2+)/calmodulin kinase II (CaM-KII). New data are presented using inhibitors of specific ROS producing enzymes to curb nodularin/MC-induced liver cell (hepatocyte) death. They indicate that enzymes of the arachidonic acid pathway, notably phospholipase A2, 5-lipoxygenase, and cyclooxygenases, may be required for nodularin/MC-induced (and presumably OA-induced) cell death, suggesting new ways to overcome at least some aspects of OA and MC toxicity.

Topics: Animals; Apoptosis; Carcinogens; Cell Death; Enzyme Inhibitors; Humans; Marine Toxins; Microcystins; Okadaic Acid; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Reactive Oxygen Species

Topics: Animals; Antifungal Agents; Marine Toxins; Microcystins; Neoplasms, Experimental; Okadaic Acid; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Protein Phosphatase 1; Pyrans; Spiro Compounds

Topics: Alkenes; Animals; Antifungal Agents; Cantharidin; Marine Toxins; Microcystins; Okadaic Acid; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Polyenes; Pyrans; Pyrones; Spiro Compounds

Protein phosphatase 5 (Ppp5) is a serine/threonine protein phosphatase comprising a regulatory tetratricopeptide repeat (TPR) domain N-terminal to its phosphatase domain. Ppp5 functions in signalling pathways that control cellular responses to stress, glucocorticoids and DNA damage. Its phosphatase activity is suppressed by an autoinhibited conformation maintained by the TPR domain and a C-terminal subdomain. By interacting with the TPR domain, heat shock protein 90 (Hsp90) and fatty acids including arachidonic acid stimulate phosphatase activity. Here, we describe the structure of the autoinhibited state of Ppp5, revealing mechanisms of TPR-mediated phosphatase inhibition and Hsp90- and arachidonic acid-induced stimulation of phosphatase activity. The TPR domain engages with the catalytic channel of the phosphatase domain, restricting access to the catalytic site. This autoinhibited conformation of Ppp5 is stabilised by the C-terminal alphaJ helix that contacts a region of the Hsp90-binding groove on the TPR domain. Hsp90 activates Ppp5 by disrupting TPR-phosphatase domain interactions, permitting substrate access to the constitutively active phosphatase domain, whereas arachidonic acid prompts an alternate conformation of the TPR domain, destabilising the TPR-phosphatase domain interface.

Topics: Acyl Coenzyme A; Arachidonic Acid; Catalytic Domain; Crystallography, X-Ray; Enzyme Activation; Enzyme Inhibitors; HSP90 Heat-Shock Proteins; Humans; Marine Toxins; Microcystins; Models, Molecular; Nuclear Proteins; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Protein Conformation; Repetitive Sequences, Nucleic Acid; Signal Transduction

A method is described to measure threonine phosphorylation of the Na-K-2Cl cotransporter in ferret erythrocytes using readily available antibodies. We show that most, if not all, cotransporter in these cells is NKCC1, and this was immunoprecipitated with T4. Cotransport rate, measured as 86Rb influx, correlates well with threonine phosphorylation of T4-immunoprecipitated protein. The cotransporter effects large fluxes and is significantly phosphorylated in cells under control conditions. Transport and phosphorylation increase 2.5- to 3-fold when cells are treated with calyculin A or Na+ arsenite. Both fall to 60% control when cell [Mg2+] is reduced below micromolar or when cells are treated with the kinase inhibitors, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine or staurosporine. Importantly, these latter interventions do not abolish either phosphorylation or transport suggesting that a phosphorylated form of the cotransporter is responsible for residual fluxes. Our experiments suggest protein phosphatase 1 (PrP-1) is extremely active in these cells and dephosphorylates key regulatory threonine residues on the cotransporter. Examination of the effects of kinase inhibition after cells have been treated with high concentrations of calyculin indicates that residual PrP-1 activity is capable of rapidly dephosphorylating the cotransporter. Experiments on cotransporter precipitation with microcystin sepharose suggest that PrP-1 binds to a phosphorylated form of the cotransporter.

Topics: Animals; Erythrocytes; Ferrets; Genistein; Magnesium; Marine Toxins; Microcystins; Molecular Weight; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphorylation; Phosphothreonine; Protein Phosphatase 1; Pyrazoles; Pyrimidines; Sodium Potassium Chloride Symporter Inhibitors; Sodium-Potassium-Chloride Symporters; Staurosporine; Threonine

Topics: Animals; Base Sequence; Binding Sites; Conserved Sequence; DNA Primers; Gene Expression Regulation, Enzymologic; Isoenzymes; Mammals; Marine Toxins; Microcystins; Nuclear Proteins; Okadaic Acid; Oligonucleotides, Antisense; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Recombinant Proteins; RNA, Antisense; RNA, Messenger; Transfection

We studied the cellular phosphatase inhibitors okadaic acid (OKA), calyculin A, and microcystin on the epithelial sodium channel (ENaC) in A6 renal cells. OKA increased the amiloride-sensitive current after approximately 30 min with maximal stimulation at 1-2 h. Fluctuation analysis of cell-attached patches containing a large number of ENaC yielded power spectra with corner frequencies in untreated cells almost two times as large as in cells pretreated for 30 min with OKA, implying an increase in single channel open probability (P(o)) that doubled after OKA. Single channel analysis showed that, in cells pretreated with OKA, P(o) and mean open time approximately doubled. Two other phosphatase inhibitors, calyculin A and microcystin, had similar effects on P(o) and mean open time. An analog of OKA, okadaone, that does not inhibit phosphatases had no effect. Pretreatment with 10 nM OKA, which blocks protein phosphatase 2A (PP2A) but not PP1 in mammalian cells, had no effect even though both phosphatases are present in A6 cells. Several proteins were differentially phosphorylated after OKA, but ENaC subunit phosphorylation did not increase. We conclude that, in A6 cells, there is an OKA-sensitive phosphatase that suppresses ENaC activity by altering the phosphorylation of a regulatory molecule associated with the channel.

Topics: Animals; Cell Line; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Epithelial Sodium Channels; Ion Channel Gating; Isomerism; Kidney; Marine Toxins; Microcystins; Okadaic Acid; Oxazoles; Patch-Clamp Techniques; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase C; Protein Phosphatase 2; Sodium Channels

Phosphorylation of serine, threonine, and tyrosine controls fundamental mammalian cell events and is achieved by kinases which, in turn, are in dynamic relationship with phosphatases. Few selective inhibitors of protein tyrosine and dual specificity phosphatases are readily available. Based on SAR studies of naturally occurring phosphatase inhibitors and following up on previously published research, we have designed a new pharmacophore model V and synthesized a new library of functional analogues of V. All synthetic steps were carried out and optimized employing combinatorial chemistry methods on Wang resin. All compounds were tested in vitro for their ability to inhibit recombinant human protein tyrosine (PTP1B) and dual-specificity (Cdc25B(2) and VHR) phosphatases. Three of the approximately 70 compounds in our library inhibited Cdc25B(2) by 50% at 375-490 microM. No compounds inhibited PTP1B, and only one blocked VHR. Cell-culture studies revealed no toxicity to human breast cancer cells with two of the phosphatase inhibitors.

Topics: Databases as Topic; Drug Design; Enzyme Inhibitors; Ethers, Cyclic; Humans; Indicators and Reagents; Kinetics; Marine Toxins; Microcystins; Models, Molecular; Okadaic Acid; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Structure-Activity Relationship

Microcystin-induced ser/thr protein phosphatase (PP) inhibition and toxicity were examined in the little skate (Raja erinacea), an evolutionarily primitive marine vertebrate. As in mammals, PP inhibition and toxicity were exclusively hepatocellular, but were much more persistent in the skate. A dose of 63 microg/kg given iv to adult male skates resulted in the near complete inhibition of hepatic PP activity at 24 h. PP activity was still 95% inhibited 7 days after dosing in skates given 125 microg/kg microcystin. Mortality occurred at doses of 500 microg/kg or more. Hepatic lesions were only seen in animals with fully inhibited PP activity in liver. The histological changes seen at 125 microg/kg were mild periportal inflammatory changes increasing in severity together with hepatocyte necrosis at higher doses of microcystin. Microcystin persisted and could be detected in plasma up to 7 days after dosing. This finding shows that, in the skate, as in mammals, the liver is the only organ capable of uptake of microcystin, since there was no significant inhibition of PP activity in the rectal gland and small decreases in PP activity of the kidney that were not time or dose dependent. In vitro microcystin caused dose-dependent inhibition of PP activity in isolated skate hepatocytes, while it was without effect in cultured rectal glands. Uptake of microcystin and the accompanying inhibition of PP activity in skate hepatocytes was prevented by the addition of a series of organic dyes and bile acids. The spectrum of inhibitors of microcystin uptake in skate is similar to that seen in the rat, indicating common features of the carrier(s) in these diverse species.

Topics: Animals; Cell Adhesion; Cell Size; Cells, Cultured; Cholic Acids; Coloring Agents; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Hemorrhage; Kidney; Liver; Male; Marine Toxins; Microcystins; Necrosis; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Salt Gland; Sharks; Skates, Fish

The current research examined the regulation of synaptic strength by protein phosphorylation during aging. Bath application of the protein phosphatase 1 and 2A (PP1 and PP2A) inhibitor calyculin A (1 microM) enhanced CA3-CA1 synaptic strength in hippocampal slices from aged male (20-24 mo) but not from young adult male (4-6 mo) Fischer 344 rats. Similarly, injection of the PP1 and PP2A inhibitor microcystin-L,R (5 microM) into CA1 cells caused an increase in the intracellular synaptic response only in slices from aged rats. In contrast, bath application of the serine/threonine kinase inhibitor H-7 (10 microM) induced a decrease in synaptic strength only in slices from the young adult group. These results demonstrate that phosphorylation dependent regulation of intrinsic synaptic efficacy changes during aging.

Topics: Aging; Animals; Brain Chemistry; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Hippocampus; Male; Marine Toxins; Microcystins; Organ Culture Techniques; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Protein Kinases; Protein Phosphatase 1; Rats; Rats, Inbred F344; Synapses

The hepatotoxic cyclic heptapeptide microcystins and cyclic pentapeptide nodularins are powerful liver tumor promoters and potent inhibitors of the catalytic subunits of protein phosphatase-1 and -2A (PP-1c and PP-2Ac). In marked contrast to microcystins, which interact covalently with PP-1 and PP-2A, the nodularins do not bind covalently to PP-1 and PP-2A and may additionally possess unique carcinogenic properties. The conformation of microcystin-LR has been determined in solution and bound to PP-1c. We show here that the free NMR solution structures of two distinct microcystin structural congeners (microcystin-LR and -LL) are remarkably similar to the bound crystal structure of microcystin-LR. We have exploited this finding by using Metropolis Monte Carlo modeling to dock the solution structures of microcystin-LL and the marine toxin motuporin (nodularin-V) onto the crystal structure of PP-1c. Both of these toxins occupy a position similar to that of microcystin-LR when bound to PP-1c. However, although there are relatively minor differences in the structural orientation of microcystin-LL compared with microcystin-LR, there is a striking difference in the position of the N-methyldehydrobutyrine residue in motuporin relative to the comparable N-methyldehydroalanine residue in microcystin-LR. We propose that this difference in orientation provides a molecular explanation for why nodularins are incapable of forming a covalent linkage with PP-1c. Furthermore, the predicted position of N-methyldehydrobutyrine in motuporin is at the surface of the PP-1c-toxin complex, which may thus facilitate chemical interaction with a further macromolecule(s) possibly relating to its carcinogenic properties. PP-1c and PP-2Ac are also targets for other marine toxins such as okadaic acid and calyculin A. It was therefore of interest to use Metropolis Monte Carlo modeling to dock the known free crystal structures of okadaic acid and calyculin A to the crystal structure of PP-1c. These experiments predict that both okadaic acid and calyculin A are strikingly similar to microcystins and motuporin in their tertiary structure and relative PP-1c binding position.

Topics: Animals; Marine Toxins; Microcystins; Models, Molecular; Monte Carlo Method; Okadaic Acid; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Protein Phosphatase 1; Toxins, Biological

The microcystins (Mcyst) are cyclic peptide hepatotoxins produced by cyanobacteria. They are chemically very stable and represent a public health threat when they occur in water bodies used for human consumption. Mice injected ip with Mcyst (LD50 50-100 micrograms/kg) accumulate Mcyst in the liver and die within 2-4 hr with massive intrahepatic hemorrhage. Pretreatment of mice with cyclosporin A (CP), rifamycin (Rif), trypan blue (TB), and trypan red (TR) protected the animals from a lethal dose of the toxin. The studies reported here using the freshly isolated rat hepatocyte model were undertaken in order (1) to evaluate the contribution of Mcyst transport in hepatocytes to the mechanism of chemoprotection for Mcyst in vivo toxicity by CP, Rif, TB, and TR and (2) to better characterize the hepatic Mcyst transporter in this model and determine its relationship to other bile acid/organic anion transporters that have already been fully described. Incubations with 125I-Mcyst were used to measure Mcyst uptake and accumulation in hepatocytes. It has been shown that at the cellular level Mcyst binds to and inhibits protein phosphatases 1 and 2A (PP) at nanomolar concentrations. Lethal doses of Mcyst in mice resulted in rapid profound inhibition of hepatic PP activity. PP activity was also inhibited in hepatocytes incubated with 100-500 nM Mcyst. PP inhibition in these studies was used as a marker of metabolic effects of the toxin. The chemoprotectants CP (5 microM), Rif (50 microM), TR (20 microM), and TB (20 microM) decreased accumulation of Mcyst (320 nM, a toxic concentration) after 30 min incubation to 37, 26, 30, and 66%, respectively, of that of cells treated with Mcyst only. Inhibition of PP activity in these cells was decreased. Inhibition of PP activity in hepatocytes was also decreased by known inhibitors of Mcyst transport: 50 microM of the bile acids cholate and taurocholate (TC) and 50 microM sulfobromophthalein. For all compounds tested the amount of Mcyst accumulated in the hepatocytes correlated qualitatively with the extent of PP inhibition. From these results it can be concluded that inhibition of Mcyst uptake by hepatocytes is the most likely mechanism of chemoprotection for Mcyst in vivo toxicity for TR, TB, CP, and Rif. Uptake of Mcyst was unaffected by changes in the ionic composition of the uptake buffer but was significantly decreased when PP activity of hepatocytes was inhibited by preincubation with Mcyst.(ABSTRACT TRUNCATED AT 400 WOR

Topics: Animals; Biological Transport; Cyclosporine; Enzyme Inhibitors; Liver; Male; Marine Toxins; Mice; Microcystins; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Rats; Rats, Sprague-Dawley; Taurocholic Acid

Microcystin (Mcyst) and calyculin A (CalA) in vitro inhibit protein phosphatases (PP)1 and 2A activity (IC50 0.1-2.0 nM). This study was aimed at determining the contribution of PP inhibition to Mcyst hepatotoxicity by comparing the effect of these two chemically different inhibitors in perfused rat livers. Both compounds (60 micrograms Mcyst and 6 micrograms CalA/150 ml perfusate) caused cessation of bile flow and inhibition of PP activity after 20 min of perfusion to 8% and 37% of control activity for Mcyst and CalA treatments, respectively. Histopathological findings included loss of cord sinusoidal pattern and of normal liver architecture. There also was hepatocyte swelling, pyknotic changes and necrosis. Mcyst caused a modest increase in perfusion pressure of 1.2 cm of water, whereas CalA caused a 3-fold increase. The most likely explanation for this hemodynamic effect is direct action of CalA on the vascular endothelium and/or sinusoidal and perisinusoidal cells. This possibility was explored with hepatocytes and sinusoidal endothelial cells. PP activity of both cell types was inhibited by 10 to 100 nM CalA followed later by cell lysis, whereas Mcyst (500 nM-2 microM) had no effect on sinusoidal endothelial cells, but inhibited PP activity and caused later lysis in hepatocytes (Mcyst 20-160 nM). Mcyst hepatotoxicity is therefore a direct consequence of PP inhibition in hepatocytes, the loss of sinusoidal integrity following from the primary toxic insult to the hepatocyte. Inhibition of PP activity of the cells of the presinusoidal vasculature and/or nonparenchymal cells results in hepatic hypertension.

Topics: Animals; Cells, Cultured; Endothelium, Vascular; Liver; Male; Marine Toxins; Microcystins; Oxazoles; Peptides, Cyclic; Perfusion; Phosphoprotein Phosphatases; Rats; Rats, Sprague-Dawley

Calcium- and lipid-dependent protein kinase (PKC) activity in the ovary of the pseudopregnant rat is masked by an endogenous inhibitor of PKC. These studies were undertaken to examine the mechanism of action of the endogenous inhibitor of PKC in the rat ovary. The addition of the phosphatase inhibitors calyculin-A (0.09 nM), microcystin-LR (6.4 nM), and okadaic acid (10 nM) resulted in the loss of PKC inhibitory activity and an increase in basal PKC activity in rat ovarian cytosol. In phosphatase assays, significant dephosphorylation of histone-III-S or myelin basic protein that had been phosphorylated by PKC occurred within 4 min after the addition of ovarian cytosol from the pseudopregnant rat. This dephosphorylation was prevented from the pseudopregnant rat. This dephosphorylation was prevented by the addition of calyculin-A (0.73 nM) and was removed by fractionation of ovarian cytosol on diethylaminoethyl cellulose. No inhibition of PKC activity was observed when the PKC-specific peptides AcMBP-(4-14) and [Ser25]PKC-(19-31) were used as the substrate for phosphorylation. In addition, rat ovarian cytosol did not exhibit phosphatase activity when the peptide AcMBP-(4-14) was used as the substrate. Addition of ovarian cytosol resulted in dephosphorylation of phosphorylase-alpha phosphorylated by phosphorylase kinase, but not dephosphorylation of histone-II-A or histone-VIII-S phosphorylated by PKA. The data suggest that the endogenous inhibitor of PKC in the rat ovary is a protein phosphatase.

Topics: Animals; Brain; Cytosol; Ethers, Cyclic; Female; Histones; Marine Toxins; Microcystins; Myelin Basic Protein; Okadaic Acid; Ovary; Oxazoles; Peptides, Cyclic; Phosphoprotein Phosphatases; Protein Kinase C; Pseudopregnancy; Rats; Rats, Sprague-Dawley