Characterization of serine/threonine protein phosphatases in RINm5F insulinoma cells

1993 ◽  
Vol 13 (6) ◽  
pp. 349-358 ◽  
Author(s):  
Åke Sjöhom ◽  
Richard E. Honkanen ◽  
Per-Olof Berggren
Keyword(s):  
Okadaic Acid ◽  
Potent Inhibitor ◽  
Protein Phosphatases ◽  
Marine Sponges ◽  
Rinm5f Cell ◽  
Ic50 Values ◽  
Marine Dinoflagellates ◽  
Insulinoma Cells

This study investigates the occurrence and regulation of serine/threonine protein phosphatases (PPases) in insulin-secreting RINm5F insulinoma cells. PPases types 1 and 2A were identified in crude RINm5F cell homogenates by both enzymatic assay and Western blot analysis. We then characterized and compared the inhibitory actions of several compounds isolated from cyanobacteria, marine dinoflagellates and marine sponges, (viz. okadaic acid, microcystin-LR, calyculin-A and nodularin) cation-independent PPase activities in RINm5F cell homogenates. It was found that okadaic acid was the least potent inhibitor (IC50 ≈ 10−9M, IC100 ≈ 10−6M), while the other compounds exhibited IC50 values of ≈ 5·10−10 M and IC100 ≈ 5·10−9 M. The findings indicate that the inhibitory substances employed in this study may be used pharmacologically to investigate the role of serine/threonine PPases in RINm5F cell insulin secretion, a process that is likely to be regulated to a major extent by protein phosphorylation.

Okadaic acid-induced actin assembly in neutrophils: Role of protein phosphatases

1993 ◽  
Vol 155 (3) ◽  
pp. 505-519 ◽  
Author(s):  
Gregory P. Downey ◽  
Akira Takai ◽  
Ricardo Zamel ◽  
Sergio Grinstein ◽  
Chi Kin Chan
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatases ◽  
Actin Assembly

Role of kinases and phosphatases in the regulation of fluid secretion and Cl-/HCO3- exchange in cholangiocytes

1997 ◽  
Vol 273 (2) ◽  
pp. G303-G313 ◽  
Author(s):  
D. Alvaro ◽  
A. Mennone ◽  
J. L. Boyer
Keyword(s):  
Bile Duct ◽  
Protein Kinase ◽  
Okadaic Acid ◽  
Protein Phosphatases ◽  
Specific Inhibitor ◽  
Fluid Secretion ◽  
Kinase C ◽  
Bile Duct Epithelium ◽  
Exchange Activity

The role of protein kinase A (PKA), protein kinase C (PKC), and protein phosphatases in the process of secretin stimulation of fluid and bicarbonate secretion from biliary epithelium was examined using a novel isolated bile duct unit (IBDU) model from rat liver. Sp-adenosine 3',5'-cyclic monophosphothiolate (Sp-cAMPS), 100 microM, a PKA-specific agonist, significantly increased secretion during a 30-min perfusion (+61%, P < 0.01). In contrast, preincubation and perfusion of Rp-cAMPS, 100 microM, a specific PKA inhibitor, reduced the ability of secretin to stimulate both fluid secretion (111 vs. 25%; P < 0.01) and Cl-/HCO3- exchanger activity (80 vs. 28%). Neither the PKC agonist phorbol 12-myristate 13-acetate, 10 microM, nor the PKC antagonist staurosporine showed any effect on either basal or secretin-stimulated fluid secretion or Cl-/HCO3- exchange activity in IBDU. Okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, also had no effect on basal fluid secretion or on the basal activity of the Cl-/HCO3- exchanger. However, okadaic acid resulted in persistence of secretion after removal of secretin, in contrast to the reduction in secretion observed in controls. These findings indicate that PKA but not PKC is involved in the signal transduction of secretin-stimulated fluid secretion and Cl-/HCO3- exchange activity in rat bile duct epithelium, a process inactivated by dephosphorylation by protein phosphatases 1 and/or 2A.

scholarly journals Inhibition of myeloperoxidase by benzoic acid hydrazides

1995 ◽  
Vol 308 (2) ◽  
pp. 559-563 ◽  
Author(s):  
A J Kettle ◽  
C A Gedye ◽  
M B Hampton ◽  
C C Winterbourn
Keyword(s):  
Benzoic Acid ◽  
Potent Inhibitor ◽  
Acid Hydrazide ◽  
Bacterial Killing ◽  
Ic50 Value ◽  
Ic50 Values ◽  
Substituent Constants ◽  
O2 Production ◽  
Benzoic Acid Hydrazide

Myeloperoxidase is the most abundant protein in neutrophils and catalyses the conversion of H2O2 and chloride into HOCl. To help clarify the role of this enzyme in bacterial killing and inflammation, a specific and potent inhibitor needs to be identified. We have studied a series of benzoic acid hydrazides and found that in general they inhibit the peroxidation activity of myeloperoxidase with an IC50 value of less than 10 microM. The IC50 values of derivatives with substituents containing oxygen or nitrogen were related to their Hammett substituent constants. This indicates that myeloperoxidase oxidizes the hydrazide group of these compounds, and the degree to which they inhibit the enzyme is dependent on the ease of their oxidation. Unsubstituted benzoic acid hydrazide and its 4-chloro derivative were poor inhibitors of peroxidation. Thus it is likely that hydrogen-bonding of the enzyme to substituents containing oxygen or nitrogen increases the binding affinity of the hydrazides and enhances their oxidation by myeloperoxidase. 4-Aminobenzoic acid hydrazide (ABAH) was the most potent inhibitor of peroxidation. It irreversibly inhibited HOCl production by the purified enzyme, having an IC50 value of 0.3 microM. With neutrophils stimulated with opsonized zymosan or phorbol myristate acetate, ABAH inhibited HOCl production by up to 90% and the IC50 values were 16 microM and 2.2 microM respectively. In the presence of superoxide dismutase, these values decreased to 6.4 microM and 0.6 microM respectively. ABAH had no effect on superoxide radical (O2-.) production and degranulation by neutrophils, nor did it inhibit catalase or glutathione peroxidase. Thus ABAH is an effective and selective inhibitor that should be useful for determining the contribution of myeloperoxidase to oxidant-mediated reactions of neutrophils.

scholarly journals Characterization of microcystin-LR, a potent inhibitor of type 1 and type 2A protein phosphatases.

1990 ◽  
Vol 265 (32) ◽  
pp. 19401-19404 ◽  
Author(s):  
R E Honkanen ◽  
J Zwiller ◽  
R E Moore ◽  
S L Daily ◽  
B S Khatra ◽  
...  
Keyword(s):  
Potent Inhibitor ◽  
Protein Phosphatases

scholarly journals Effects of intrahippocampal administration of the phosphatase inhibitor okadaic acid: Dual effects on memory formation

2010 ◽  
Vol 4 (1) ◽  
pp. 23-27 ◽  
Author(s):  
Monica R.M. Vianna ◽  
Adriana Coitinho ◽  
Luciana Izquierdo ◽  
Ivan Izquierdo
Keyword(s):  
Okadaic Acid ◽  
Memory Consolidation ◽  
Potent Inhibitor ◽  
Inhibitory Avoidance ◽  
Memory Formation ◽  
Long Term Memory ◽  
Term Memory ◽  
Step Down

Abstract Protein phosphorylation mediated by serine-threonine kinases in the hippocampus is crucial to the synaptic modifications believed to underlie memory formation. The role of phosphatases has been the focus of comparatively little study. Objectives: Here we evaluate the contribution of the serine-threonine protein phosphatases 1 and 2A (PP1, PP2A) on memory consolidation. Methods: We used immediate post-training bilateral hippocampal infusions of okadaic acid (OA, 0.01 and 10 pmol/side), a potent inhibitor of PP1 and PP2A, and measured short- [3 h] and long-term memory [24 h] (STM, LTM) of step-down inhibitory avoidance. Results: At the lower dose, OA inhibited both STM and LTM whereas at the higher dose it instead enhanced LTM. Pre-test infusion of these two doses of OA had no effect on retrieval. Conclusions: These two doses of OA are known to selectively inhibit PP1 and PP2A respectively. These findings point to the importance of these enzymes in memory formation and also suggest a deleterious influence of endogenous hippocampal PP2A on LTM formation.

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