Triggering of various cells by mitogens leads to a complex biochemical cascade culminating in their activation and, in some instances, to pathophysiology. Free radicals are known to mediate certain signal transduction events initiated by various mitogens in lymphocytes. We have identified a new class of lymphocyte mitogens and termed them ferromitogens. These are iron-containing compounds whose mitogenic potentials are related to their oxidative properties. Further, we found that various thiol-modifying agents such as mercury and phenylarsine oxide also activate human peripheral blood mononuclear cells (PBMC). Hence, we examined whether nitric oxide (NO), a biologically relevant oxidant, also induces biochemical events characteristic of an activated phenotype in resting human PBMC. We discovered that NO induces TNF-alpha secretion, NF-kappaB nuclear transcription factor translocation and activation of p56/lck protein tyrosine kinase and membrane protein tyrosine phosphatase activities, all indicators of cell activation. These activation events were cGMP-independent. Thus NO may have immune-stimulatory properties which are unrelated to its direct cytotoxic nature and independent of its ability to generate cGMP. Recently, we reported that one ferromitogen, hemin, acts as a potent inducer of cAMP in resting human PBMC. The kinetics of cAMP generation were similar to those found in hormonally- stimulated cells, suggesting a guanine-nucleotide binding protein (G protein)-mediated event. Thus, G proteins may be a target of NO; we now have new preliminary data to support this postulate. Accordingly, we propose to test the hypothesis that NO activates G proteins in human lymphocytes and this mediates some of the cell-stimulatory properties of NO. First, we will determine at the molecular level how NO activates G proteins using pure recombinant p21/ras as a model. Next, we will identify which G proteins in human T cells are targets of NO. This will include using 32/P-NAD/pertussis toxin labeling and [32/P]azidoanilido GTP crosslinking techniques. Next, we will identify the role that NO- activated G proteins play in generating an activated phenotype. Also, we will determine whether NO participates in inflammation by providing an accessory signal; for this, we will assess the ability of NO generated by activated macrophages to activate G proteins and stimulate T cells. Finally, we will extend these findings to an in vivo mouse system to determine whether NO-donating drugs such as the organic nitrates have hitherto undefined cell-stimulatory effects when given in vivo. The proposed experiments are designed to 1) characterize the NO-G protein signaling pathway and 2) define the role of this pathway in mediating NO- induced lymphocyte activation. The elucidation of such a pathway will continue to provide new insight into the mechanisms of lymphocyte activation and inflammatory conditions, and thus improve our understanding in several important areas of pathophysiology.
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