Fluorescein-labeled naloxone binding to mu opioid receptors on live Chinese hamster ovary cells using confocal fluorescent microscopy

A general method of confocal laser scanning microscopy was used to demonstr ate specific binding of fluorescein-labeled naloxone (FNAL, 10-50 nM) to st ably transfected mu opioid receptors on live Chinese hamster ovary cells. N onspecific binding was visually indistinguishable from autofluorescence in cells with intact cell membranes. Fluorescent labeling of cell perimeters, not present in control nontransfected cells, reversed in transfected cells upon washout of FNAL or following the addition of either unlabeled naloxone (25 mu M) or the mu specific antagonist CTOP (1 mu M). The addition of the delta and kappa specific agonists DPDPE (1 mu M) and U50 488 (1 mu M), res pectively, failed to reverse the labeling. Further evidence of specific bin ding was obtained from kinetic experiments, where it was observed that only transfected cells showed a time-dependent exponential change in fluorescen ce that permitted estimation of association and dissociation binding rate c onstants of (5.8 +/- 0.5, mean +/- S.E.M.) x 10(5) M-1 s(-1) and (3.3 +/- 0 .6) x 10(-3) s(-1), respectively and a kinetically derived dissociation con stant of 5.7 +/- 1.4 nM. These estimates were comparable to those obtained under similar conditions in radioligand binding experiments using [H-3]-nal oxone. (C) 2000 Elsevier Science B.V. All rights reserved.