LATENCY IN THE INOSITOL LIPID TRANSDUCTION PATHWAY - THE ROLE OF CELLULAR EVENTS IN RESPONSES TO THYROTROPIN-RELEASING-HORMONE IN XENOPUS-OOCYTES

To dissect the cellular events responsible for the prolonged latency o f the response to thyrotropin-releasing hormone (TRH) in Xenopus oocyt es we interfered with different steps of the signal transduction pathw ay. Preincubation of oocytes with cis-vaccenic acid (a membrane-fluidi zing agent) shortened the latency, suggesting a contribution of membra nal processes. TRH-induced depletion of cellular calcium stores prolon ged latency (up to threefold), which returned to control levels upon r epletion of the stores. Injection of D-2,3-diphosphoglycerate (PGA), w hich inhibits inositol (1,4,5)-trisphosphate (InsP(3)) dephosphorylati on, alone evoked a small, prolonged depolarizing current and significa ntly shortened the latency of the response to TRH. Injection of guanos ine 5'-0-(2-thiodiphosphate) (GDP S-beta), which inactivates guanine n ucleotide-binding regulatory proteins, decreased the amplitude of the response and increased latency. Injection of guanosine 5-0-(3-thiotrip hosphate) (GTP S-gamma) immediately before the challenge with TRH did not shorten the latency of the response. Decreasing the effective rece ptor density with chlorodiazepoxide, an antagonist of the TRH receptor , resulted in an extension of latency, whereas the expression of a lar ge number of TRH receptors by injection of RNA transcribed from cloned receptor DNA (20-100ng/oocyte) shortened the latency to below 2 s. Ou r results suggest that the latency of the response to TRH reflects the activation of a late step in the signal transduction sequence, most l ikely the release of calcium by InsP(3). We propose that this process is kinetically controlled by an early rate-limiting event, involving t he activation of a guanine nucleotide-binding protein by the TRH recep tor.