Contribution of response kinetics to the response pattern: Studies of responses to thyrotropin‐releasing hormone in Xenopus oocytes

Abstract

In Xenopus oocytes injected with total rat pituitary GH3 cell RNA, thyrotropin-releasing hormone (TRH) causes the activation of the inositol lipid transduction pathway and the induction of chloride conductance via calcium-activated channels (Oron et al., 1987, Mol. Endocrinol., 1:918-925). This response exhibits characteristic prolonged latency (Oron et al., 1988, Proc. Natl. Acad. Sci. U.S.A., 85:3820-3824; Lipinsky et al., 1993, Pflugers Arch., 425:140-149). We examined the role of agonist diffusion in the extracellular medium in the generation of latency and the determination of response amplitude. An increase in the viscosity of the medium markedly prolonged the latency and decreased the amplitude of the response. Moreover, an increase in the viscosity of the medium in the immediate vicinity of the oocyte had a major effect on both the latency and the amplitude of the response, which appeared to be a result of desensitization rather than restricted diffusion of chloride to the medium. Extrapolation to [TRH] infinity yielded a diffusion-dependent latency value of 0 and a diffusion-independent latency value of 4 seconds. In low viscosity medium, at all TRH concentrations, diffusion contributed less than 2% to the latency of the response. This implied that events distal to ligand binding are responsible for a major part of latency. Analysis of the dependence of latency and amplitude of the response on [TRH] yielded Hill coefficients markedly smaller than unity, suggesting postreceptor negative modulation of the response. Preincubation of cells with a specific inhibitor of protein kinase C, chelerythrine, increased the Hill coefficients to unity and changed the shape of the Hill plot of response amplitudes. Our results suggest that at low agonist concentrations, even in a low viscosity medium, the prolonged latency allows negative effects on both latency and amplitude by a simultaneous activation of a protein kinase C.

Inverse agonist abolishes desensitization of a constitutively active mutant of thyrotropin‐releasing hormone receptor: role of cellular calcium and protein kinase C

“…The involvement of [Ca 2+ ] i in desensitization suggested that one of the calcium‐sensitive steps in the signalling pathway might be mediating this effect. Since our previous results suggested that rapid desensitization of the response to TRH in Xenopus oocytes was partially mediated by PKC ( Lipinsky et al ., 1995a , 1995b ), we tested the role of PKC in desensitization of cells expressing C335Stop TRH‐Rs. Two different approaches were employed: down‐regulation of PKC by chronic exposure to PMA ( Glikes et al ., 1994 ), and inhibition by chelerythrine, a specific inhibitor of PKC ( McFerran & Guild, 1994 ; McFerran et al ., 1995 ).…”

“…Our previous observations in oocytes led us to postulate that the very rapid desensitization caused by limited occupancy of TRH‐R is mediated, at least in part, by PKC ( Lipinsky et al ., 1995a , 1995b ). In cells expressing WT TRH‐Rs, acute activation of PKC appears to dramatically inhibit TRH responses: in AtT20 cells by 75–96% ( Perlman & Gershengorn, 1991 ), and in Xenopus oocytes by more than 90% ( Lipinsky et al ., 1995b ).…”

Alkaline pH facilitates the exchange of guanine nucleotides: A possible mechanism for modulation of the kinetics of responses mediated by guanine nucleotide-binding proteins

“…It is unlikely that changes in extracellular pH can significantly improve the binding of TRH to its receptor and, thus, shorten the latency of the response, without affecting its amplitude. Moreover, our previous results indicate that the binding process does not contribute to the latency of the response (Lipinsky et al, 1995a). Also, Perlman et al (1992) have proposed that the binding of TRH to its receptor does not involve ionic interactions and is mostly pH-insensitive between pH 6.6 and 9.5.…”

“…Furthermore, the response latency provides a temporal window for integration of external signals that affect the amplitude of the response (Lipinsky et al, 1995a) and its desensitization (Lipinsky et al, 199513). The binding of guanine nucleotides to G-proteins may be affected by …”