Abstract
To address the role of protein phosphatases in regulating hormonal responses in mammalian cells, we investigated the effects of okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A, on epinephrine and prostaglandin E1 stimulation of cAMP accumulation and adenylyl cyclase in S49 WT and kin- lymphoma cells. Depending on the dose and time of okadaic acid pretreatment of both cell lines, there were two distinguishable effects on cAMP accumulation, an augmentation and an inhibition. The augmentation occurred rapidly (t1/2 < 1 min), was maximal with 3 microM okadaic acid, and was observed with concentrations of okadaic acid as low as 0.3 microM. Prolonged (t1/2 of 5-15 min) pretreatment of cells with okadaic acid caused an inhibition of epinephrine-stimulated cAMP accumulation, which was characterized by a 2-3-fold increase in the EC50 for the response to epinephrine. The EC50 for the okadaic acid-mediated inhibition was similar to that for the augmentation. In assays of adenylyl cyclase in membrane fractions prepared from okadaic acid-pretreated cells the inhibitory, but not the stimulatory, effects of okadaic acid pretreatment were observed. The data demonstrate that protein phosphatases play an important role in regulating adenylyl cyclase and suggest that cAMP-dependent protein kinase is not involved in either of its actions.
Highlights
To address the role of protein phosphatases in regu- regulation of a number of cellular processes: for example, the lating hormonal responses in mammalian cells, we in- Na+/H+ exchangeirn thymic lymphocytes [10];glucose transvestigated the effects of okadaic acid, a potent inhibitor port,cAMPphosphodiesterase [12], and myelin basic of protein phosphatases 1 and 2A, on epinephrine and protein kinase activity [13] in adipose tissue; and phosphatiprostaglandin El stimulation ofcAMP accumulation dylinositol metabolismand Ca2+transients in human platelets and adenylyl cyclase in S49 wild type (WT) and kin- lymphoma cells
Studies of the mechanisms involved in the regulation of hormonal stimulation of adenylyl cyclase have relied for the most part on experimentswhere variousprotein kinaseswere activated or inactivated, both in the intact cell and in vitro
For example the role of cAPK in thedesensitization of hormonal stimulation of adenylyl cyclase in intact cells was determined through the use of the kin- mutant of the S49 lymphoma cell line which lacked cAPK activity, and through the use of forskolin and cAMPanalogs to activate the kinase in W T cellsin the absence of receptor level activation of adenylyl cyclase [15, 16]
Summary
Materials-Okadaic acid was purchased from Moana BioProducts, demonstrated thata 30-min pretreatment with 2 FM okadaic Inc., and was dissolved in N,N-dimethyl formamide (DMF), dimethylacid caused an inhibition of the initial rate of cAMP accusulfoxide, ethanol, or by suspension directly in the cell incubation mulation in response to 3 nM epinephrine. Aswe havedescribed pM okadaic acid (data not shown) resulted in effects similar previously [15, 16], thecAPK-mediated desensitization of t o those seen with 3 p~ okadaic acid In seven such experi- hormonal stimulation of adenylyl cyclase does not occur in ments, we have found that pretreatment with0.3 p~ okadaic the intact kin- cells; there is no rapid rise and fall in cAMP acid for 30 min caused only an augmentation of epinephrine levels in response to stimulation with relatively low concenstimulation. These results demonstrate that the okadaic acid-induced tion of cAMP in response to 100 nM epinephrine as shown in augmentation develops rapidly, whereas the inhibitory effect Fig. 4. Cells were stimulated for 2 min (WT) or 2-3 min (kin-) with either 20 p M forskolin or 2 g M PGE,, and cAMP levels were measured
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