Up-regulation of mu-, delta- and kappa-opioid receptors in concanavalin A-stimulated rat spleen lymphocytes
Abstrakt
Regulation of mu-, delta- and kappa-opioid receptor protein level in spleen lymphocytes when stimulated by mitogen is not known. To answer the question whether these cells do express opioid receptor (OR) proteins, primary, fresh rat spleen lymphocytes were prepared and stimulated for 48 h with mitogenic dose of Con A.
The unstimulated lymphocytes did not express mu- and delta-OR proteins in detectable amounts, however, stimulation with Con A resulted in appearance of clearly detectable immunoblot signals of both mu-OR and delta-OR. kappa-OR were detected already in primary cells and increased 2.4-fold in Con A-stimulated cells. These results were supported by data obtained by flow cytometry analysis indicating a dramatic increase in number of mu-, delta- and kappa-OR expressing cells after mitogen stimulation.
The newly synthesized mu-, delta- and kappa-OR in Con A-stimulated spleen lymphocytes were present in the cells interior and not functionally mature, at least in terms of their ability to enhance activity of trimeric G proteins determined by three different protocols of agonist-stimulated, high-affinity [S-35]GTP gamma S binding assay. The up-regulation of mu-, delta- and kappa-OR was associated with specific decrease of their cognate trimeric G proteins, G(i)1 alpha/G(i)2 alpha; the other G alpha and G beta subunits were unchanged.
The level of beta-arrestin-1/2 was also decreased in Con A-stimulated splenocytes. We conclude that up-regulation of OR expression level in spleen lymphocytes by Con A proceeds in conjunction with down-regulation of their intracellular signaling partners, G(i)1 alpha/G(i)2 alpha proteins and beta-arrestin-1/2.
These regulatory proteins are expressed in high amounts already in unstimulated cells and decreased by mitogen stimulation.