Effects of methylxanthine derivatives on red cell phosphorylation

Methylxanthines interact with cyclic nucleotide-dependent phosphorylation of proteins: they are potent inhibitors of cyclic nucleotide phosphodiesterase; such that they increase cAMP level and indirectly stimulate the cAMP-dependent phosphorylation. Conversely, in the course of our studies on human red blood cell protein phosphorylation we observed that methylxanthines derivatives inhibited cAMP-independent phosphorylation by casein-kinases. Intact human red blood cells were incubated in presence or inorganic 32P with and without cAMP with various concentrations of caffeine or pentoxifylline and for various times. Haemolysis was then performed and ghosts were prepared according to Dodge et al's method. Overall phosphorylation of membrane proteins was measured and SDS-PAGE was performed with the aim to determine the phosphorylation level of separate polypeptides. We observed that cAMP-dependent phosphorylations were normal but that cAMP-independent phosphorylations were decreased proportionally to the methylxanthine derivative concentration; phosphorylation of spectrin band II and protein III were decreased. Same results were observed studying phosphorylation of ghost' proteins with gamma (32P)ATP. The mechanism of this inhibition was studied using pure casein-kinase from erythrocyte cytosol, with casein or spectrin as substrates and gamma (32P)-ATP as phosphoryl donor. The results clearly showed that caffeine and pentoxifylline were competitive inhibitors of ATP in the enzymatic phosphorylation. This would be expected since the methylxanthines are adenine analogues. If the spectrin phosphorylation is necessary to the equilibrium of cytoskeleton, methylxanthine derivatives could act on erythrocyte membrane upsetting this equilibrium.