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Up-regulation of μ-, δ- and κ-opioid receptors in concanavalin A-stimulated rat spleen lymphocytes.

Journal of neuroimmunology (2018-06-30)
Kristina Cechova, Martina Hlouskova, Eliska Javorkova, Lenka Roubalova, Hana Ujcikova, Vladimir Holan, Petr Svoboda
ABSTRACT

Regulation of μ-, δ- and κ-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 μ- and δ-OR proteins in detectable amounts, however, stimulation with Con A resulted in appearance of clearly detectable immunoblot signals of both μ-OR and δ-OR. κ-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 μ-, δ- and κ-OR expressing cells after mitogen stimulation. The newly synthesized μ-, δ- and κ-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 [35S]GTPγS binding assay. The up-regulation of μ-, δ- and κ-OR was associated with specific decrease of their cognate trimeric G proteins, Gi1α/Gi2α; the other Gα and Gβ subunits were unchanged. The level of β-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, Gi1α/Gi2α proteins and β-arrestin-1/2. These regulatory proteins are expressed in high amounts already in unstimulated cells and decreased by mitogen stimulation.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
RPMI-1640 Medium, With L-glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Concanavalin A from Canavalia ensiformis (Jack bean), biotin conjugate, Type IV, lyophilized powder
Sigma-Aldrich
[D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt, ≥97% (HPLC)
Sigma-Aldrich
U-69593, solid
Sigma-Aldrich
[D-Ala2, D-Leu5]-Enkephalin acetate salt, ≥95% (HPLC)
Sigma-Aldrich
Adenosine 3′,5′-cyclic monophosphate tris salt, ≥97% (HPLC), powder