| This study was designed to investigate the involvement of delta opioid receptor (DOR)-mediated signaling in cell proliferation and cell death. A murine fibroblast cell line, NIH3T3, stably transfected with the murine DOR1 cDNA, was selected as a model system. Plating quiescent cells of this cell line into serum free medium, caused them to die via an apoptotic process. The addition of basic fibroblast growth factor (bFGF) rescued cells from death and elicited a proliferative response. Treatment of cells with a DOR agonist, [D-Pen2,5]-enkephalin (DPDPE) alone, caused a small, but significant, increase in cell viability. When added along with bFGF, DPDPE enhanced bFGF-induced proliferation, but did not enhance its anti-apoptotic effect. This increase was antagonist- and pertussis toxin-reversible and dependent on the concentration of DPDPE. The enhancement of bFGF's effect by DPDPE, was exerted entirely through the PI3 kinase pathway and was independent of ERK activation.; During the induction of quiescence, the level of DOR expressed by the stably transfected cell line, decreased. This was confirmed by radioligand binding assays. A green fluorescent protein tagged DOR1 was also used to confirm this effect using FACS. This agonist-independent loss of receptor did not require the cells to be contact inhibited and was entirely dependent on the gradual serum depletion that occurred during quiescence induction. This loss was partially attenuated by the expression of a dominant negative (K44A) dynamin, indicating that the DOR internalizes, at least partially, by a dynamin dependent pathway, in this agonist-independent model.; These studies are among the first to dissect the molecular mechanisms of opioid signaling in cell proliferation. This report links the DOR to the activation of the PI3 kinase pathway, and demonstrates its possible physiological relevance. This study also indicates a model for agonist independent DOR internalization. It brings up the issue that clinically relevant opioid agonists should be evaluated for their proliferation-inducing effects in vivo. |
