Beta-Endorphin is an endogenous opioid peptide, secreted from the pituitary gland in response to stressors and synthesized by subpopulations of splenic mononuclear cells. Although its peripheral targets are not well defined, the existence of receptors selective for Beta-endorphin on cells involved in host defense provides a likely site for the action of Beta-endorphin. Indeed, several groups have reported that mitogen-induced proliferation of mixed lymphoid cells is affected by Beta-endorphin in a naloxone-resistant manner. Elucidating mechanisms whereby a naloxone-resistant receptor and/or opiate receptor (OR) mediate these effects on the cell cycle of T lymphocytes will require a relatively homogenous population of normal cells. We plan to compare CD4+ vs. CD8+ T-cells (more than 99% pure by FAC sorting) from different mouse strains, in order to characterize (i) the pharmacology and (ii) the effects of opioids on signal transduction pathways mediating cell proliferation in response to cross-linking the T-cell antigen receptor (TCR)/CD3 complex with anti-CD3-epsilon. The effects of Beta-endorphin on anti-CD3-induced proliferation will be measured with respect to: cell doubling time, distribution of cells by phase of the cell cycle, 3H- thymidine incorporation and IL-2 secretion. The concentration-dependent effects of Beta-endorphin will be compared to other OR-specific agonists such as DAGO (mu), DPDP-enkephalin and D-ala2,Glu4-deltorphin (delta1vs.delta2) and U50,488 (kappa); specific OR antagonists will also be evaluated. The hypothesis underlying studies of the effects of opiates on early signal transduction pathways mediating T cell proliferation is that opiates stimulate the synthesis of cAMP, which is well established as a direct, anti-proliferative signal in highly purified normal CD4+ and CD8+ T cells. Experiments will measure the effects of opiates on 3H-cAMP formation, and on the inositol 1,4,5 triphosphate (IP3) and Ras-MAPK (mitogen activated protein kinases) pathways. Both of these pathways mediating TCR-induced proliferation are inhibited by cAMP. Thus, effects of opiates vs forskolin on the TCR- dependent stimulation of phospholipase cgamma1 (PLCgamma1) and consequent formation of IP3 will be compared. Since tyrosine phosphorylation activates PLC-gamma1, measuring the extent of its phosphorylation will provide an activity index. Similarly, effects of opiates vs. forskolin on anti-CD3-induced Ras-MAPK activation can be measured. The amount of activated, hyperphosphorylated MAPK can be quantified, since activation of MAPK retards its mobility on SDS-polyacrylamide gel electrophoresis. Also, bioactivity of the MAPK, ERK2, can be assayed by quantifying the phosphorylation of added substrate, myelin basic protein. Activation of high affinity IL-2 receptor complexes by IL-2 is required for progression from G1 into S phase (DNA duplication); these later events are also involved in TCR-mediated proliferation. Since cAMP is known to inhibit expression of IL-2 receptor subunits, the effects of opiates vs. forskolin on expression IL-2 alpha and Beta receptor subunits and mRNAs will be determined. Together, these investigators will clarify mechanisms underlying opiate modulation of TCR-induced proliferation by identifying effects on both early and later signaling pathways.
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