Development of diabetes is associated with altered expression of adenosine receptors (ARs). Some of these alterations might be attributed to changes in insulin concentration. This study was undertaken to investigate the possible insulin effect on ARs level, and to determine the signaling pathway utilized by insulin to regulate the expression of ARs in rat B lymphocytes. Western blot analysis of B lymphocytes protein extracts indicated that all four ARs were present at detectable levels in the cells cultured for 24 h without insulin (<or=10(-11) M), although the protein band of A(2A)-AR was barely visible. Inclusion of insulin (10(-8) M) in the culture medium resulted in an increase of A(1)-AR and A(2A)-AR protein levels and a significant decrease of A(2B)-AR protein, whereas the protein level of A(3)-AR remained unchanged. Alterations in the ARs protein content were accompanied by changes in the ARs mRNA levels. Increase of the insulin concentration from 10(-11) to 10(-8) M resulted in 50% decrease of A(2B)-AR mRNA level and two-, and threefold increase of A(1)-AR and A(2A)-AR mRNA levels, respectively. Pretreatment of B cells with cycloheximide completely blocked the insulin action on A(1)-AR and A(2A)-AR mRNA, but not on A(2B)-AR expression. Detailed pharmacological analysis demonstrated that insulin-induced A(1)-AR and A(2A)-AR mRNA expression through the Ras/Raf-1/MEK/ERK pathway. The insulin effect on A(2B)-AR expression was blocked by p38 MAP kinase inhibitor (SB 203580). Concluding, elevated insulin concentration differentially affects the expression of ARs in B lymphocytes in a fashion that might enhance the various immunomodulatory effects of adenosine.