Stable expression and heterologous coupling of the kappa opioid receptor in cell lines of neural and nonneural origin

Abstract

Signal transduction cascades initiated by the neuronal κ opioid receptor were studied following transfection of a neuronal (hippocampal) line, HN2, and the non-neural CHOs. Retinoic-acid mediated differentiation resulted in intense staining of the HN2 cells with a neurofilament protein antibody SMI 33 but not with an antibody to GFAP, thus establishing neuronal characteristics of the HN2 cell line. The k opioid receptor was stably expressed in the two cell lines by electroporation mediated transfer of a Cytomegalovirus-promoter driven construct, pCMV-κ, harboring the κ-opioid receptor cDNA. Positive clones (HN2κ24 and CHOκ18) from both lines showed high expression of the k opioid receptor, as identified by [ 3H] U-69,593 binding to membranes prepared from HN2κ24 and CHOκ 18. Scatchard analysis revealed the presence of high affinity k opioid receptors in both engineered cell lines (K D = 1.3 nM for HN2κ24 and 2.1 nM for CHOκ18). Functional coupling to adenylate cyclase was displayed by 1 μM U-69,593 mediated inhibition (55–63%) of prostaglandin eistimulated intracellular cAMP levels. A major difference between the two clones was observed in functional coupling of the expressed κ opioid receptor to phospholipases C (PL-C) and D (PL-D). U-69,593 (1 μM) treatment stimulated PL-C, but not PL-D, in HN2κ24 cells, whereas PL-D, but not PL-C, was stimulated following such treatment of CHOκ 18 cells. Our results using the model neuronal system, HN2κ24, demonstrate cell-type specific, positive coupling of the kappa opioid receptor to the major Ca 2+ mobilizing system, the PL-C cascade, which regulates neuronal firing.