Analysis of delta-opioid receptor activities stably expressed in CHO cell lines: function of receptor density?
The question of whether short- and long-term opioid agonist activities could be affected by receptor density is being addressed with Chinese hamster ovary (CHO) cell lines stably expressing delta-opioid receptors. CHO cells expressing different levels of delta-opioid binding sites were isolated and characterized. The opioid binding sites in these cell lines have stereoselective high affinity for 3H-diprenorphine (0.18 to 0.91 nM), with agonist binding sensitive to both Na+ and GTP gamma S, and are of the delta-2-opioid receptor subtype. This is conclude from observations that the delta-2-opioid receptor-selective agonist naltriben (NTB) has higher affinity than the delta-1-opioid receptor-selective agonist 7-benzylidenenaltrexone (BNTX). It also could be demonstrated that delta-opioid agonists inhibited forskolin-stimulated intracellular 3H-cAMP production and that agonist inhibition could be blocked by pretreating cells with pertussis toxin. Again, NTB is more potent than BNTX or naloxone in reversing DPDPE inhibition of intracellular 3H-cAMP production. When the ability of [D-Ala2,D-Leu5]enkephalin (DADLE) to regulate adenylyl cyclase activity was examined in three separate CHO cell lines: CHODORX1-15, CHODORX1-8 and CHODORX1-4, which express 1.42 +/- 0.08, 0.74 +/- 0.07 and 0.27 +/- 0.04 pmol/mg-protein of receptor, respectively, maximal inhibitory levels in clones X1-15 and X1-8 were similar, whereas maximal inhibitory level for clone X1-4 was 66% of the other two clones. However, when maximal inhibitory levels of other opioid receptor-selective agonists were examined, different levels of inhibition were observed among these three CHO clones.(ABSTRACT TRUNCATED AT 250 WORDS)
Law
P Y
PY
Department of Pharmacology, University of Minnesota Medical School, Minneapolis.
McGinn
T M
TM
Wick
M J
MJ
Erikson
L J
LJ
Evans
C
C
Loh
H H
HH
D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000032:analysis; D020881:Enkephalin, D-Penicillamine (2,5)-; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives / Q000378:metabolism; D016308:Enkephalin, Leucine-2-Alanine / Q000494:pharmacology; D004745:Enkephalins / Q000378:metabolism; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D017465:Receptors, Opioid, delta / Q000032:analysis* / Q000235:genetics; D014162:Transfection
D000067956:Adenylyl Cyclase Inhibitors; D004745:Enkephalins; D012333:RNA, Messenger; D017465:Receptors, Opioid, delta; D004743:Enkephalin, Leucine; D016308:Enkephalin, Leucine-2-Alanine; C034318:enkephalin, Ser(2), Leu(5), Thr(6)-; D020881:Enkephalin, D-Penicillamine (2,5)-; D000242:Cyclic AMP