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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.

SUBMITTER: Anderson L

PROVIDER: S-EPMC1136012 | biostudies-other | 1995 Oct

REPOSITORIES: biostudies-other

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Project description:Thyrotropin releasing hormone (TRH: Glp-His-Pro-NH2) is a peptide mainly produced by brain neurons. In mammals, hypophysiotropic TRH neurons of the paraventricular nucleus of the hypothalamus integrate metabolic information and drive the secretion of thyrotropin from the anterior pituitary, and thus the activity of the thyroid axis. Other hypothalamic or extrahypothalamic TRH neurons have less understood functions although pharmacological studies have shown that TRH has multiple central effects, such as promoting arousal, anorexia and anxiolysis, as well as controlling gastric, cardiac and respiratory autonomic functions. Two G-protein-coupled TRH receptors (TRH-R1 and TRH-R2) transduce TRH effects in some mammals although humans lack TRH-R2. TRH effects are of short duration, in part because the peptide is hydrolyzed in blood and extracellular space by a M1 family metallopeptidase, the TRH-degrading ectoenzyme (TRH-DE), also called pyroglutamyl peptidase II. TRH-DE is enriched in various brain regions but is also expressed in peripheral tissues including the anterior pituitary and the liver, which secretes a soluble form into blood. Among the M1 metallopeptidases, TRH-DE is the only member with a very narrow specificity; its best characterized biological substrate is TRH, making it a target for the specific manipulation of TRH activity. Two other substrates of TRH-DE, Glp-Phe-Pro-NH2 and Glp-Tyr-Pro-NH2, are also present in many tissues. Analogs of TRH resistant to hydrolysis by TRH-DE have prolonged central efficiency. Structure-activity studies allowed the identification of residues critical for activity and specificity. Research with specific inhibitors has confirmed that TRH-DE controls TRH actions. TRH-DE expression by β2-tanycytes of the median eminence of the hypothalamus allows the control of TRH flux into the hypothalamus-pituitary portal vessels and may regulate serum thyrotropin secretion. In this review we describe the critical evidences that suggest that modification of TRH-DE activity in tanycytes, and/or in other brain regions, may generate beneficial consequences in some central and metabolic disorders and identify potential drawbacks and missing information needed to test these hypotheses.

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Rapid desensitization of the thyrotropin-releasing hormone receptor expressed in single human embryonal kidney 293 cells.

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