Stable expression and heterologous coupling of the kappa opioid receptor in cell lines of neural and nonneural origin.
Signal transduction cascades initiated by the neuronal kappa 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 kappa opioid receptor was stably expressed in the two cell lines by electroporation mediated transfer of a Cytomegalovirus-promoter driven construct, pCMV-kappa, harboring the kappa-opioid receptor cDNA. Positive clones (HN2 kappa 24 and CHO kappa 18) from both lines showed high expression of the kappa opioid receptor, as identified by [3H] U-69,593 binding to membranes prepared from HN2 kappa 24 and CHO kappa 18. Scatchard analysis revealed the presence of high affinity kappa opioid receptors in both engineered cell lines (KD=1.3 nM for HN2 kappa 24 and 2.1 nM for CHO kappa 18). Functional coupling to adenylate cyclase was displayed by 1 microM 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 kappa opioid receptor to phospholipases C (PL-C) and D (PL-D). U-69,593 (1 microM) treatment stimulated PL-C, but not PL-D, in HN2 kappa 24 cells, whereas PL-D, but not PL-C, was stimulated following such treatment of CHO kappa 18 cells. Our results using the model neuronal system, HN2 kappa 24, 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.
Banerjee
P
P
Department of Pediatrics, University of Chicago, Illinois, USA.
Chromy
B A
BA
Berry-Kravis
E
E
Hammond
D
D
Singh
J K
JK
Dawson
G
G
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D000906:Antibodies; D016466:CHO Cells / Q000378:metabolism / Q000648:ultrastructure; D002460:Cell Line; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis / Q000276:immunology; D006624:Hippocampus / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D007700:Kinetics; D051379:Mice; D016900:Neurofilament Proteins / Q000032:analysis / Q000276:immunology; D009457:Neuroglia / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure*; D009474:Neurons / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure*; D010739:Phospholipase D / Q000378:metabolism; D017473:Receptors, Opioid, kappa / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D013194:Staining and Labeling / Q000379:methods; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
D000906:Antibodies; D018076:DNA, Complementary; D005904:Glial Fibrillary Acidic Protein; D016900:Neurofilament Proteins; D017473:Receptors, Opioid, kappa; D010738:Type C Phospholipases; D010739:Phospholipase D; D000262:Adenylyl Cyclases