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

P. Banerjee, B. A. Chromy, E. Berry-Kravis, D. Hammond, J. K. Singh, G. Dawson

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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 κ 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 κ opioid receptor, as identified by [3H] U-69,593 binding to membranes prepared from HN2κ24 and CHOκ18. Scatchard analysis revealed the presence of high affinity κ 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 E1-stimulated 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 CHOκ18 cells. Our results using the model neuronal system, HN2κ24, demonstrate cell-type specific, positive coupling of the kappa opioid receptor to the major Ca2+ mobilizing system, the PL-C cascade, which regulates neuronal firing.

Original languageEnglish (US)
Pages (from-to)1277-1284
Number of pages8
JournalLife Sciences
Volume58
Issue number15
DOIs
StatePublished - Mar 8 1996
Externally publishedYes

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Keywords

  • Kappa opioid receptor
  • PL-C
  • PL-D
  • Signal transduction
  • Transfection

ASJC Scopus subject areas

  • Pharmacology

Cite this

Banerjee, P., Chromy, B. A., Berry-Kravis, E., Hammond, D., Singh, J. K., & Dawson, G. (1996). Stable expression and heterologous coupling of the kappa opioid receptor in cell lines of neural and nonneural origin. Life Sciences, 58(15), 1277-1284. https://doi.org/10.1016/0024-3205(96)00089-6