Adenosine metabolism in phytohemagglutinin-stimulated human lymphocytes.

Abstract

A B S T R A C T The association of a human genetic deficiency of adenosine deaminase activity with combined immunodeficiency prompted a study of the effects of adenosine and of inhibition of adenosine deaminase activity on human lymphocyte transformation and a detailed study of adenosine metabolism throughout phytohemagglutinin-induced blastogenesis. The adenosine deaminase inhibitor, coformycin, at a concentration that inhibited adenosine deaminase activity more than 95%, or 50 uM adenosine, did not prevent blastogenesis by criteria of morphology or thymidine incorporation into acid-precipitable material. The combination of coformycin and adenosine, however, substantially reduced both the viable cell count and the incorporation of thymidine into DNA in phytohemagglutinin-stimulated lymphocytes.Incubation of lymphocytes with phytohemagglutinin for 72 h produced a 12-fold increase in the rate of deamination and a 6-fold increase in phosphorylation of adenosine by intact lymphocytes. There was no change in the apparent affinity for adenosine with either deamination or phosphorylation. The increased rates of metabolism, apparent as early as 3 h after addition of mitogen, may be due to increased entry of the nucleoside into stimulated lymphocytes. Increased adenosine metabolism was not due to changes in total enzyme activity; after 72 h in culture, the ratios of specific activities in extracts of stimulated to unstimulated lymphocytes were essentially unchanged for adenosine kinase, 0.92, and decreased for adenosine deaminase, 0.44. As much as 38% of the initial lymphocyte adenosine deaminase activity accumulated extracellularly after a 72-h culture with phytohemag- glutinin. In phytohemagglutinin-stimulated lymphocytes, the principal route of adenosine metabolism was phosphorylation at less than 5 FM adenosine, and deamination at concentrations greater than 5 WM. In unstimulated lymphocytes, deamination was the principal route of adenosine metabolism over the range of adenosine concentrations studied (0.5-250 MM). These studies demonstrate the dependence of both the unstimulated and stimulated lymphocyte on adenosine deaminase activity for the metabolism of adenosine and may account for the observed sensitivity of mitogen-stimulated lymphocytes to the toxic effects of exogenously supplied adenosine in the presence of the adenosine deaminase inhibitor coformycin.A single case of immunodeficiency disease has been reported in association with purine nucleoside phosphorylase deficiency. The catabolism of guanosine was also found to be enhanced in stimulated normal lymphocytes; phosphorolysis of guanosine to guanine by intact lymphocytes increased six fold after 72-h culture with phytohemagglutinin. The specific activity of purine nucleoside phosphorylase in extracts, with guanosine as substrate, was essentially the same in stimulated and unstimulated lymphocytes after 72 h of culture.

T lymphocyte ontogeny in adenosine deaminase-deficient severe combined immune deficiency after treatment with polyethylene glycol-modified adenosine deaminase.

“…Plasma levels of ADA activity, levels oftotal erythrocyte adenine ribo-and deoxyribonucleotides (dAXP) and erythrocyte levels of S-adenosylhomocysteine hydrolase (SAHase) activity were determined as previously de-scribed (6)(7)(8)(9) (normal, 8-84). Red blood cell ADA activity was absent (normal, 33-100 umol/h per g hemoglobin), diagnostic of ADA deficiency.…”

In vitro of adenosine on lymphocytes and erythrocytes from horses with combined immunodeficiency.

“…Although the exact biochemical mechanism is not known (12), elevated levels of adenosine and its metabolites are thought to be selective inhibitors for differentiation and function of lymphoid tissue. This hypothesis is based in part on the in vitro observations that lymphoid tissues normally contain high levels of ADA activity (13)(14)(15) and that adenosine inhibits mitogen-induced stimulation of normal human lymphocytes (14)(15)(16)(17)(18)(19)(20)(21), the growth of human and mouse lymphoblast cell lines (22), and the destruction oftumor cells by specifically sensitized mouse lymphocytes (23). Simultaneous addition ofADA inhibitors and adenosine potentiates these inhibitory effects (17-21, 23).…”

Conformational basis for the activation of adenylate cyclase by adenosine

“…8-Aminoadenosine and 8-bromoadenosine are unable to stimulate cAMP accumulation in guinea pig ventricular slice preparations (14). The inactivity of ribavirin derivatives with methyl, amino, or chloro substituents at the 5 position (50) has been attributed to the destabilization of the high anti conformation required for the inhibition of IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.2.1.14) by these purine analogs (48; unpublished data).…”

“…The enzymes adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) and adenosine kinase (ATP:adenosine-5'-phosphotransferase, EC 2.7.1.20) compete for adenosine. At higher concentrations of adenosine, adenosine deaminase dominates (11)(12)(13)(14) and protects the body from the effects of an excess of adenosine. One of these effects is the suppression of immune responses associated with the activation by adenosine of adenylate cyclase ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.11 which increases cAMP levels (15, 16).…”