Desensitization of thyrotropin-releasing hormone receptor-mediated responses involves multiple steps

Desensitization and recovery of the inositol 1,4,5-trisphosphate (IP3) and intracellular free calcium concentration ([Ca2+](i)) responses to thyrotropin-releasing hormone (TRH) were measured in HEK298 cells stably expressing the G protein coupled TRH receptor, TRH caused a large, rapid, and transient increase in IP3 and a biphasic increase in [Ca2+](i). Desensitization of the TRH response was measured by exposing cells to TRH, washing, and then incubating the cells in hormone-free medium before reintroducing TRH and measuring IP3, [Ca2+](i), and intracellular Ca2+ pool size, When cells were incubated with 1 mu M TRH for 10 s or 10 min and reexposed to TRH, there was almost no IP3, or [Ca2+](i) increase, The IP3, response recovered first, followed by the [Ca2+](i) response, The ionomycin-releasable intracellular Ca2+ pool was almost completely depleted by TRH, and pool refilling was slow, Thrombin, endothelin, and carbachol, when combined, stimulated large increases in IP3 and [Ca2+](i), but did not block the IP3 or [Ca2+](i) responses to TRH measured 10 min later. In contrast, cells exposed to TRH first responded to combined agonists with a nearly normal increase in IP3, but no rise in [Ca2+](i), Thus, the IP3 response to TRH displays homologous desensitization, whereas the [Ca2+](i) response displays heterologous desensitization because depletion of intracellular Ca2+ pools prevents responses to other hormones.