title: Rapid desensitization of the thyrotropin-releasing hormone receptor expressed in single human embryonal kidney 293 cells.
abstract: This study uses fluorescence microscopy combined with dynamic video imaging to examine the events associated with the rapid desensitization of the thyrotropin-releasing hormone receptor (TRH-R). In single non-pituitary human embryonic kidney 293 (HEK-293) cells, expressing either the rat or human TRH-Rs, TRH produced a rapid dose-dependent monophasic rise in [Ca2+]i. This Ca2+ transient was completely abolished by pretreatment of cells with the intracellular Ca2+ antagonists thapsigargin or cyclopiazonic acid, but not EGTA, the voltage-operated Ca2+ channel (VOCC) antagonist nifedipine or the second-messenger-operated Ca2+ channel antagonist SK&F 96365. These results suggest that TRH causes the mobilization of Ca2+ from thapsigargin/cyclopiazonic acid-sensitive intracellular Ca2+ stores but not the influx of extracellular Ca2+. HEK-293 cells also failed to respond to KCl or the slow Ca(2+)-channel activator BAY K 8644, suggesting that they lack L-type VOCCs. Rat and human TRH-Rs are highly conserved except at the C-terminus where the sequence differs. The C-terminus is believed to be important in receptor desensitization. Despite differences in this region, rat and human TRH-Rs expressed in HEK-293 cells underwent rapid (within 1 min) desensitization. This desensitization was dose-dependent and did not involve receptor loss. Similarly the bradykinin receptor endogenous to HEK-293 cells also displays a rapid desensitization. We conclude that in TRH-R-expressing non-pituitary HEK-293 cells, TRH mobilizes intracellular Ca2+ resulting in a monophasic Ca2+ transient. The rat and human TRH-Rs as well as the endogenous bradykinin receptor also displayed rapid receptor desensitization.
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D015870:Gene Expression; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000196:embryology / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D051381:Rats; D018002:Receptors, Bradykinin / Q000378:metabolism; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000235:genetics / Q000378:metabolism*; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology; D014162:Transfection; D014741:Video Recording
chemical_list: D002121:Calcium Channel Blockers; D005456:Fluorescent Dyes; D018002:Receptors, Bradykinin; D018025:Receptors, Thyrotropin-Releasing Hormone; C049925:fura-2-am; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D013973:Thyrotropin-Releasing Hormone; D019204:GTP-Binding Proteins; D002118:Calcium; D016257:Fura-2