The antioxidant butylated hydroxytoluene stimulates platelet protein kinase C and inhibits subsequent protein phosphorylation induced by thrombin.
The phenolic antioxidant 2,6-bis(1,1-dimethyl ethyl)-4-methylphenol (BHT) evokes a transient phosphorylation of two platelet proteins of Mr 20,000 and 47,000 that are well-known substrates of protein kinase C (PKC) and, similarly to phorbol esters, a slight but persistent phosphorylation of a protein of Mr 26,000. These effects are observed both in the presence and in the absence of extracellular calcium, but are abolished in the presence of the protein kinase C inhibitor staurosporine. The phosphorylation of the 47 kDa protein takes place mostly at the serine and, to a lesser extent, at threonine residues. BHT induces an increased binding of tritiated phorbol dibutyrate to platelets indicating a PKC translocation from cytosol to plasma membrane. Addition of BHT (20 microM) a few min prior to thrombin causes inhibition of both agonist-evoked protein phosphorylation and increase in the Ca2+ concentration, the latter inhibition being counteracted by staurosporine. The inhibitory effect lasts for several minutes even after removal of BHT from the cellular suspending medium. Similar results are obtained with nordihydroguaiaretic acid, whereas 2- and 3-tert-butyl-4-methoxyphenol (BHA) produce only slight effects. BHT activates the protein kinase C purified from pig brain in a concentration-dependent manner (up to 200 microM), whereas it does not affect the activity of other purified protein kinases such as type 1 and 2 casein kinases, type II A, II B and III tyrosine protein kinases from rat spleen and the catalytic subunit of cyclic AMP-dependent protein kinase. It is concluded that, similarly to diacylglycerols and phorbol esters, these phenolic antioxidants activate the protein kinase C, which in turn desensitizes platelets towards subsequent phospholipase C activation.
Ruzzene
M
M
Department of Biological Chemistry, University of Padova, Italy.
Donella-Deana
A
A
Alexandre
A
A
Francesconi
M A
MA
Deana
R
R
D000818:Animals; D000975:Antioxidants; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D001921:Brain / Q000187:drug effects / Q000201:enzymology; D002083:Butylated Hydroxyanisole / Q000494:pharmacology; D002084:Butylated Hydroxytoluene / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D009637:Masoprocol / Q000494:pharmacology; D008970:Molecular Weight; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000097:blood*; D013552:Swine; D013917:Thrombin / Q000494:pharmacology*
D000975:Antioxidants; D002084:Butylated Hydroxytoluene; D002083:Butylated Hydroxyanisole; D009637:Masoprocol; D011493:Protein Kinase C; D013917:Thrombin