Use of radioreceptor assay and cell superfusion system for in vitro screening of analogs of growth hormone-releasing hormone

Abstract

In the search for more active agonists and antagonists of human growth hormone-releasing hormone (hGH-RH), various analogs are being synthesized. In order to follow the binding affinity of these analogs, we have developed a sensitive in vitro radioreceptor assay for GH-RH based on binding of labeled [His1,Nle27]hGH-RH(1-32)NH2 to rat anterior pituitary membrane homogenates by adapting and modifying earlier methods. Scatchard analysis of saturation binding data demonstrated the presence of a single class of specific binding sites for GH-RH in membranes of rat anterior pituitaries with a Bmax of 33.3 +/- 5.2 fmol/mg protein and an apparent Kd of 0.19 +/- 0.02 nM. In displacement analyses, we compared the binding affinity of [His1,Nle27]hGH-RH(1-32)NH2 with its iodinated derivative. No significant differences were detected in IC50 concentrations ranging from 0.97 to 3.4 nM between labeled and nonlabeled hGH-RH analogs. These findings demonstrate the validity of the radioreceptor assay. To evaluate the biological activities of hGH-RH derivatives, we applied a sensitive, dispersed rat pituitary cell superfusion system. This dynamic in vitro system eliminates the drawbacks of the static pituitary cell culture. No differences were observed in biological activities of the iodinated and noniodinated hGH-RH analogs. GH-releasing activity obtained from the superfusion assay correlated well with GH-RH receptor binding affinity for all nonlabeled and labeled hGH-RH analogs examined. These two methods are fast, simple, and relatively inexpensive, and provide quantitative data on receptor affinities, biological activities, and hence structure-affinity and structure-activity relationships. Joint use of these two in vitro systems appears to be suitable for screening newly synthesized GH-RH analogs.