Real-time analysis of the activities of GnRH and GnRH analogs in alpha T3-1 cells by the cytosensor microphysiometer

Gonadotropin-releasing hormone (GnRH), acting via the GnRH receptor, elicit ed rapid extracellular acidification responses in mouse gonadotrope-derived alpha T3-1 cells as measured by the Cytosensor microphysiometer, which ind irectly monitors cellular metabolic rates. GnRH increased the extracellular acidification rate of the cells in a dose-dependent manner (EC50 = 1.81 +/ - 0.24 nM). The GnRH-stimulated acidification rate could be attenuated by p rotein kinase C (PKC) down-regulation, extracellular Ca2+ depletion, and th e voltage-sensitive Ca2+ channel (VSCC) blocker nifedipine, indicating that the acidification response is activated by both Ca2+ and PKC-mediated path ways. Upon continuous exposure to 100 nM GnRH or periodic stimulation by 10 nM GnRH at 40 min intervals, homologous desensitization was more pronounce d in the absence of extracellular Ca2+, suggesting that desensitization of GnRH activity may be mediated via depletion of intracellular Ca2+ stores. W e have also compared the potency of eight GnRH analogs on alpha T3-1 cells. No acidification response was detected for GnRH free acid, consistent with the idea that the C-terminal amide is a critical structural determinant fo r GnRH activity. Replacement of Gly-NH2 at the C-terminus by N-ethylamide d ramatically reduced the EC50 value, suggesting that substitution of the Gly -NH2 moiety by N-ethylamide increases the potency of Cn RH analogs. Substit ution of Gly at position 6 by D-Trp significantly reduced the EC50 value, w hereas D-Lys at the same position slightly increased the EC50 value, implyi ng that either an aromatic amino acid or a non-basic amino acid at position 6 may be essential for potent GnRH agonists. In summary, our results demon strate that the Cytosensor microphysiometer can be used to evaluate the act ions of GnRH and GnRH analogs in alpha T3-1 cells in a real-time and noninv asive manner. This silicon-based microphysiometric system should provide ne w information on the structure-function studies of GnRH and is an invaluabl e tool for the screening of new GnRH agonists and antagonists in the future . (C) 2001 Wiley-Liss, Inc.