Impact of adenosine receptors on immunoglobulin production by human peripheral blood B lymphocytes.

Adenosine is an endogenous compound that regulates function of several immune cells including lymphocytes by activating adenosine receptors (ARs). Several reports indicate that stimulation of ARs on lymphocytes affects lymphocyte activation, proliferation and lymphocyte-mediated cytolysis. Unfortunately, most studies focused on T lymphocytes and little information exists on involvement of ARs in B cells regulation. In this study we elucidated the impact of ARs activation on immunoglobulin M (IgM) production by purified human peripheral blood B lymphocytes stimulated in vitro with Staphyloccocus aureus Covan I (SAC) plus IL-2. Performed experiments showed that endogenous adenosine that is released/produced by human B lymphocytes is able to induce cAMP accumulation in the cell through activation of A2A-AR however, this takes place only when other ARs are inhibited by selective antagonists. We observed that accumulated intracellular cAMP suppressed IgM production by B cells stimulated with SAC plus IL-2. Our experiments showed that human B cells cultured at 25 mM glucose produced significantly less IgM in response to stimulation with SAC comparing to cells maintained in media containing 5 mM glucose. However, the high glucose effect on IgM production by B cells stimulated with SAC depended on other factor/s than ARs.

Adenosine 5'-triphosphate is the predominant source of peripheral adenosine in human B lymphoblasts.

Adenosine 5'-triphosphate (ATP) and adenosine are the crucial endogenous signaling molecules in immunity and inflammation. In this study we identified the source of extracellular adenosine in human B lymphoblasts, and evaluate the ATP release and metabolism. We observed that the B cells continuously released substantial quantities of ATP (35 pmol/10(6) cells) when subjected to slow motion in the incubation medium. The adenosine level in the B cell incubation medium was very low, and increased (5-fold) upon inhibition of adenosine deaminase activity with 10 muM of 2-deoxycoformycin (DCF). Inclusion of an inhibitor of equilibrate nucleoside transport (nitrobenzylthioinosine) in the incubation medium in the presence of DCF resulted in the elevation of adenosine level by 9-fold. Inhibition of ecto-ATPase activity with 100 muM of ARL67156 was associated with a 2-fold increase of the extracellular ATP level and a 3-fold decrease of adenosine concentration in the cell culture media. Inclusion of alpha,alpha-methyleneadenosine 5'-diphosphate, a selective inhibitor of ecto-5'-nucleotidase in the incubation medium resulted in a significant decrease (7-fold) the adenosine concentration. In conclusion, our results indicate that ATP released from the B cell is the primary source of peripheral adenosine, and that the activities of ecto enzymes and the efficiency of Ado uptake through the nucleoside transporters determine the Ado level on the B cell surface.

Protein kinase C mediated high glucose effect on adenosine receptors expression in rat B lymphocytes.

Hyperglycemia-induced alterations of adenosine receptors (ARs) expression are implicated in the pathomechanism leading to impaired function of the lymphocytes in diabetes. However, the signaling pathways utilized by glucose to regulate ARs expression are unknown. This work was undertaken to investigate the impact of high glucose level on the ARs expression in rat B lymphocytes. The results presented in this report demonstrate that rat B lymphocytes express all four types of ARs at the mRNA and protein level. Exposing B cells to high glucose (25 mM) suppressed the expression of A(1)-AR, A(2B)-AR, and A(3)-AR, but had no effect on the expression of A(2)A-AR. A selective inhibitor of Ca(2+)-dependent protein kinase C (PKC) isoforms suppressed the high glucose effect on A(1)-AR expression. Inhibition of PKC-delta with rottlerin blocked the high glucose effect on A(1)-AR mRNA level. An inhibitor of Raf-1 kinase completely blocked the high glucose effect on A(2B)-AR expression. The suppression of A(1)-AR and A(2B)-AR mRNA expression induced by high glucose was blocked by an inhibitor (PD98059) of MAPK kinase (MEK). In conclusion, high glucose utilizes a signaling pathway involving some elements of the MAPK pathway and different PKC isoforms to suppress the expression of A(1)-AR, A(2B)-AR, and A(3)-AR in rat B lymphocytes.

Prevalence of unidirectional Na+-dependent adenosine transport and altered potential for adenosine generation in diabetic cardiac myocytes.

Adenosine is an important physiological regulator of the cardiovascular system. The goal of our study was to assess the expression level of nucleoside transporters (NT) in diabetic rat cardiomyocytes and to examine the activities of adenosine metabolizing enzymes. Isolated rat cardiomyocytes displayed the presence of detectable amounts of mRNA for ENT1, ENT2, CNT1, and CNT2. Overall adenosine (10 microM) transport in cardiomyocytes isolated from normal rat was 36 pmol/mg/min. The expression level of equilibrative transporters (ENT1, ENT2) decreased and of concentrative transporters (CNT1, CNT2) increased in myocytes isolated from diabetic rat. Consequently, overall adenosine transport decreased by 30%, whereas Na(+)-dependent adenosine uptake increased 2-fold, and equilibrative transport decreased by 60%. The activity ratio of AMP deaminase/5'-nucleotidase in cytosol of normal cardiomyocytes was 11 and increased to 15 in diabetic cells. The activity of ecto-5'-nucleotidase increased 2-fold in diabetic cells resulting in a rise of the activity ratio of ecto-5'-nucleotidase/adenosine deaminase from 28 to 56.These results indicate that in rat cardiomyocytes diabetes alters activities of adenosine metabolizing enzymes in such a way that conversion of AMP to IMP is favored in the cytosolic compartment, whereas the capability to produce adenosine extracellularly is increased. This is accompanied by an increased unidirectional Na(+)-dependent uptake of adenosine and significantly reduced bidirectional adenosine transport.