Elsevier

Brain Research

Volume 935, Issues 1–2, 10 May 2002, Pages 59-64
Brain Research

Research report
Role of TRH receptors as possible mediators of analeptic actions of TRH-like peptides

https://doi.org/10.1016/S0006-8993(02)02454-XGet rights and content

Abstract

A large family of TRH-like peptides in the limbic region of rat brain including pGlu-Glu-Pro-NH2 (EEP), pGlu-Val-Pro-NH2 (Val2-TRH), Leu2-TRH, Phe2-TRH and Tyr2-TRH has recently been discovered. TRH (pGlu-His-Pro-NH2) has antidepressant, neuroprotective, analeptic, anticonvulsant, antiamnesic and euphoric properties, and other TRH-like peptides such as EEP exert several of these effects. A new TRH receptor (TRHR2) has been reported which is highly expressed in regions of rat brain that regulate attention and learning, arousal, sleep and processing of sensory information. The TRHR1 predominates in limbic structures involved in regulation of mood and in pituitary. This study examined the possibility that some of the newly discovered TRH-like peptides bind with high affinity to TRHR2, and that this receptor acts as the transducer for some of the CNS effects of this new class of neuropeptides. EEP, Val2-TRH and Leu2-TRH were analeptics, like TRH, but Phe2-TRH and Tyr2-TRH were not. The affinity and efficacy of TRH-like peptides for TRHR1 and TRHR2 were measured in HEK293 cells stably expressing these receptors. The IC50 values of TRH-like peptides for displacement of [3H]TRH from TRHR2 were TRH⋘(Leu2-, Phe2-TRH)<(Gln2-, Ser2-TRH)≪(Val2-, Tyr2-, Arg2-, Thr2-, and Glu2-TRH). The IC50 for Leu2-TRH was about 100 times that for TRH. When tested at the calculated IC50 values, TRH-like peptides stimulated calcium responses in cells expressing TRHR1 and TRHR2, indicating that the peptides act as weak agonists at both receptors. These results indicate that TRHR1 and TRHR2 do not mediate the behavioral effects of TRH-like peptides.

Introduction

Thyrotropin-releasing hormone (pGlu-His-Pro-NH2), EEP (pGlu-Glu-Pro-NH2), and Leu2-TRH (pGlu-Leu-Pro-NH2) have been reported to have antidepressant, analeptic, neuroprotective, euphoric, and antiamnesic effects [7], [8], [9], [11], [12], [13], [15]. Peptides with the structure pGlu-X-ProNH2 are referred to here as TRH-like peptides. These, and other TRH-like peptides including Val2-TRH, Phe2-TRH, and Tyr2-TRH, occur in high concentration within rat brain regions associated with the regulation of mood [11], [12], [13]. A recently described TRH receptor 2 (TRHR2) is highly expressed in rat brain regions involved in the regulation of attention, learning, arousal, sleep, central motor control and processing of sensory information [5]. In limbic regions such as the perirhinal cortex, entorhinal cortex and shell of the accumbens, areas associated with mood regulation, TRHR1 predominates [5]. We have inquired whether TRHR2 has physiologically significant affinity for TRH-like peptides with known CNS effects and whether these interactions contribute to the analeptic effect of TRH-like peptides. We began our survey of possible mediators of TRH-like peptide action using transformed cells expressing the known TRH receptors. We measured the affinity and signal transduction capacity of TRH-like peptides in cell lines stably transfected with either TRHR1 or TRHR2, allowing us to identify effects mediated by the two receptors selectively. The binding specificity of TRHR2 has not yet been characterized as extensively as it has for TRHR1 [2]. We also tested the ability of TRH-like peptides to stimulate a calcium response, allowing us to determine whether the peptides behave as agonists. We show for the first time that EEP, Val2-TRH and Leu2-TRH are analeptics following intracisternal (i.c.) injection while Phe2-TRH and Tyr2-TRH are not. The present results suggest that these novel TRH-like peptides with important action in the CNS and peripheral tissues do not function via TRHR1 or TRHR2.

Section snippets

Materials

pGlu-Glu-Pro-NH2 was purchased from Sigma (St Louis, MO). pGlu-Phe-Pro-NH2 and pGlu-Gln-Pro-NH2 were prepared by Peninsula Laboratories. All other TRH-like peptides were custom synthesized by Phoenix Pharmaceuticals (Belmont, CA). Cell culture media, sera, and HBSS were from Gibco-BRL (Grand Island, NY). Fura2AM was from Molecular Probes (Eugene, OR), and TRH from Calbiochem (La Jolla, CA). [3H]TRH was from Dupont/New England Nuclear Corporation (Boston, MA). A plasmid encoding TRHR2 in pcDNA3

Results

Peptides were tested for their analeptic effect, defined as reduction in pentobarbital-induced sleep time. Injection (i.c.) of 1 μg of TRH or TRH-like peptides did not result in a significant analeptic effect. The results in Table 1 show that i.c. injections of 10 μg TRH, EEP, Leu2-TRH or Val2-TRH had an analeptic effect. Interestingly, the injection of TRH, Leu2-TRH and Val2-TRH was frequently accompanied by ‘wet dog’ shaking while the animal was still anesthetized. On the other hand, EEP

Discussion

We have shown that EEP, Val2-TRH and Leu2-TRH are analeptics, like TRH, but Phe2-TRH and Tyr2-TRH are not. These results indicate that the imidazole ring is not necessary for the analeptic activity of this novel family of peptides, and a hydrophobic residue in the 2-position is not sufficient. Although the analeptic effect of TRH is well established, His2-substituted peptides had not previously been known to exert this activity. The receptor mediating the analeptic effects of TRH and related

Acknowledgements

This work was supported in part by NIH grant DK19974 (PMH) and the Research Service of the US Department of Veterans Affairs (AEP and AS). The authors are grateful to John A. Puskas for excellent technical assistance.

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