Neuropeptide--receptor interactions studied with the aid of fluorescence-activated cell sorting and video...

Studies of interactions between neurotransmitters and their receptors would be greatly facilitated by a method for obtaining cell fractions enriched with cells that contain a high density of receptors specific for the neutrotransmitter. Here we report the use of a fluorescence-activated cell sorter (FACS) to prepare fractions of pituitary cells that contain a high density of specific receptors for the decapeptide, luteinizing hormone-releasing hormone (LHRH). The fluorescence probe, an agonist of LHRH coupled to rhodamine isothiocyanate [(rhod-D-Lys6)-LHRH], was 75% as potent as LHRH in terms of releasing LH from dispersed pituitary cells. Cells, dispersed mechanically from the anterior pituitary glands of either immature rats or ovariectomized rats treated with oestrogen and progesterone, were exposed to (rhod-D-Lys6)-LHRH at 4 degrees C for 1 h and passed through a FACS. The pituitary cells sorted into two major peaks, one of which contained cells that fluoresced brightly and showed a significant LH response to LHRH, and a second which contained cells that did not fluoresce and showed no LH response to LHRH. Using photon counting techniques, the population of fluorescent cells was found to bind LHRH in a saturable and specific manner. In the immature rats the percentage of the large cell population that bound (rhod-D-Lys6)-LHRH was about 80%, approximately the same proportion of the cell population that is made up of gonadotrophs.