Inhibitors of ser/thr phosphatases 1 and 2A induce apoptosis in pituitary GH3 cells

Abstract

Two structurally different inhibitors of ser/thr phosphatases 1 and 2A, okadaic acid and calyculin A, time- and concentration-dependently stimulated and inhibited cell-specific function (hormone gene expression) in pituitary GH3 cells. The negative effect was associated with the appearance of apoptotic cell death. Nanomolar concentrations of both agents produced the characteristic morphological alterations and a DNA fragmentation ladder. Calyculin A treatment resulted in comparable changes with 10fold lower concentrations than okadaic acid. Observations with derivatives of okadaic acid with no or lower phosphatase inhibitory potency supported the conclusion that apoptosis induction is related to inhibition of ser/thr phosphatases, presumably types 1 and 2A. Membrane damage as measured by lactate dehydrogenase liberation into medium was significantly lower in apoptotic vs. necrotic cells. DNA fragmentation could be reduced by the addition of zinc but not by removal of extracellular calcium with EGTA. Apoptotic changes were reduced by the concomitant activation of protein kinase A by a membrane permeable cAMP analogue. Incubation of cells for 4 months in successively increased concentrations of okadaic acid resulted in a population that proliferated at the initially lethal concentration of 30 nM.

Effect of Therapeutic Doses of Ionising Radiation on the Somatomammotroph Pituitary Cell Line, GH3

“…In another report, p38 MAP kinase is activated in GH3 cells during bromocryptine-induced apoptosis [26]. The apoptosis of GH3 cells was also observed following treatment with a dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium ion [27], and with inhibitors of serine/threonine phosphatases 1 and 2A [28,29].…”

Mode of action of membrane-disruptive lytic compounds from the marine dinoflagellate Alexandrium tamarense

“…microcystins bind and inhibit catalytic subunits of the wellconserved intracellular serine/threonine protein phosphatase (PP) 1 and 2A (Holmes and Boland, 1993;Goldberg et al, 1995), and consequently cause a series of chain reactions, and finally apoptosis of target cells. By a similar mechanism, OA and its analogs also induce apoptosis of target cells (Tergau et al, 1997;Rossini, 2000;Klumpp and Krieglstein, 2002). Here it is important to note that the allelochemical lytic activity of Alexandrium is unrelated to phosphatase inhibition activity, at least as mediated by these toxins (they are absent).…”

A Fast and Inexpensive Procedure for Drying Polyacrylamide Gels

“…Follow- 1 This work was supported by National Institutes of Health Grant GM-53638. 2 To whom correspondence should be addressed at Department of Pharmacology, Vanderbilt University School of Medicine, 23rd Avenue at Pierce, Nashville, TN 37232-6602. Fax: 615-322-4707.…”

“…Steric analysis of hydroxyeicosatetraenoates (HETEs) 3 and other fatty acid derivatives plays a vital part in the characterization of lipoxygenase, cyclooxygenase, and P450 reactions, and there is also often a need for preparative resolution of oxylipin and eicosanoid enantiomers. Assignment of the absolute configuration can be accomplished using spectroscopic techniques such as NMR (1) and circular dichroism (2). However, chiral column chromatography represents one of the most straightforward methods, and it can be performed with basic HPLC equipment.…”