The testing of T lymphocytes isolated from the blood of almost 100 human donors has not established that cocaine in the 1 muM range suppresses PHA- induced proliferation. Drug suppression is related to an inhibition of the mobilization of cytosolic free calcium. In addition, we have established that catecholamines and cocaine interact antagonistically in modulating PHA-induced proliferation. Furthermore, we have demonstrated that cocaine enhances proliferation in response to anti-CD3 antibodies rather than suppressing proliferation as with PHA. In the studies proposed, we will examine in detail several signal transduction molecular pathways involved in lymphocyte activation in order to obtain a clearer understanding of the molecular mechanisms involved in these cocaine effects. Molecular studies will examine the influence of cocaine and other local anesthetics on cytosolic calcium mobilization, and enzyme and Western blotting studies will examine the activity of phospholipase C, protein kinase C, tyrosine protein kinase. Also, drug effects on the upregulation of IL-2 gene expression will be determined by Northern blotting. Co-cultivation studies with various monokines will also be instituted to determine whether these accessory factors can attenuate the drug effects on T? lymphocyte proliferation. In addition to studying the effects of cocaine on PHA- induced and anti-CD3 antibody-induced lymphocyte proliferation, we will also examine drug effects on lymphocyte activation through other ligand mediated pathways (i.e., CD2, LFA 1 and CD4). These studies will define the diversity of receptor activation pathways susceptible to modulation by cocaine. Regarding drug antagonizing effects with cocaine and catecholamines, we will examine cellular metabolism of cAMP and cGMP along with the activity of phosphodiesterase in order to test the hypothesis that the drugs may be modulating lymphocyte proliferation by altering the levels of these cyclic nucleotides. The demonstration that cocaine can directly affect T lymphocyte functions suggests that this drug may also directly affect the function of other immune cells. Therefore, we will examine the effects of the drug on other immune cells, including human B lymphocyte proliferation to a polyclonal activator, an cytokine (IL-6/TNF/IL1) production by NK cells and monocytes. the proposed studies will answer important questions concerning cocaine effects on lymphocyte membrane function, signal transduction, gene expression, and cyclic nucleotide metabolism, all of which represent various stages in lymphocyte activation. In addition, we will investigate the cellular diversity of cocaine effects by testing the potency of this drug in modulating the function of immune cells other than T lymphocytes.
