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

Brain Research 935 (2002) 59–64 www.elsevier.com / locate / bres

Research report

Role of TRH receptors as possible mediators of analeptic actions of TRH-like peptides a, b,c b b,c,d Patricia M. Hinkle *, A. Eugene Pekary , Shayani Senanayaki , Albert Sattin a

Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Box 711, 601 Elmwood Ave., Rochester, NY 14642, USA b Psychiatry and Research Services, VA Greater Los Angeles Healthcare System, Los Angeles, CA 90073, USA c UCLA Department of Medicine, Los Angeles, CA, USA d Department of Psychiatry and Biobehavioral Sciences, Los Angeles, CA 90073, USA Accepted 22 November 2001

Abstract A large family of TRH-like peptides in the limbic region of rat brain including pGlu-Glu-Pro-NH 2 (EEP), pGlu-Val-Pro-NH 2 (Val 2 -TRH), Leu 2 -TRH, Phe 2 -TRH and Tyr 2 -TRH has recently been discovered. TRH (pGlu-His-Pro-NH 2 ) 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, Val 2 -TRH and Leu 2 -TRH were analeptics, like TRH, but Phe 2 -TRH and Tyr 2 -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 IC 50 values of TRH-like peptides for displacement of [ 3 H]TRH from TRHR2 were TRHD(Leu 2 -, Phe 2 -TRH),(Gln 2 -, Ser 2 -TRH)<(Val 2 -, Tyr 2 -, Arg 2 -, Thr 2 -, and Glu 2 -TRH). The IC 50 for Leu 2 -TRH was about 100 times that for TRH. When tested at the calculated IC 50 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.  2002 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Peptide receptor structure and function Keywords: TRH; EEP; TRH receptor; Calcium signaling

1. Introduction Thyrotropin-releasing hormone (pGlu-His-Pro-NH 2 ), EEP (pGlu-Glu-Pro-NH 2 ), and Leu 2 -TRH (pGlu-Leu-ProNH 2 ) have been reported to have antidepressant, analeptic, neuroprotective, euphoric, and antiamnesic effects [7– 9,11–13,15]. Peptides with the structure pGlu-X-ProNH 2 are referred to here as TRH-like peptides. These, and other TRH-like peptides including Val 2 -TRH, Phe 2 -TRH, and *Corresponding author. Tel.: 11-716-275-4933; fax: 11-716-4610397. E-mail address: patricia [email protected] (P.M. Hinkle). ]

Tyr 2 -TRH, occur in high concentration within rat brain regions associated with the regulation of mood [11–13]. 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

0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )02454-X

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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, Val 2 -TRH and Leu 2 -TRH are analeptics following intracisternal (i.c.) injection while Phe 2 -TRH and Tyr 2 -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.

2. Material and methods

previously described [18]. Cells were grown in DMEM containing 5% FBS at 37 8C in a humidified atmosphere containing 5% CO 2 and 95% air.

2.4. Affinity measurements Peptides were tested for affinity for either TRHR1 or TRHR2 in stably transfected HEK293 cells. Cells on 10-cm dishes were rinsed twice with 0.15 M NaCl and then scraped gently into 0.5-2 ml HBSS buffered with 15 mM Hepes to pH 7.4. Aliquots (100 ml) of suspended cells were incubated with 2 nM [ 3 H]TRH and different concentrations of peptides at room temperature for 45 min. The reaction mixtures were diluted with 2.5 ml ice-cold buffered HBSS and filtered through 2.5-cm Whatman GF / A glass fiber filters, which were rinsed three times with 2.5 ml cold HBSS, dried, and counted. Each peptide was tested in at least three different experiments. Maximal binding was between 1500 and 36 000 dpm.

2.1. Materials 2.5. Calcium measurement pGlu-Glu-Pro-NH 2 was purchased from Sigma (St Louis, MO). pGlu-Phe-Pro-NH 2 and pGlu-Gln-Pro-NH 2 were prepared by Peninsula Laboratories. All other TRHlike 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). [ 3 H]TRH was from Dupont / New England Nuclear Corporation (Boston, MA). A plasmid encoding TRHR2 in pcDNA3 was provided by Dr Marvin C. Gershengorn (NIH, Bethesda, MD).

2.2. Analeptic experiments Groups of 10 male Wistar rats, 125–250 g, from Harlan Laboratories (Indianapolis, IN) were anesthetized with 35 mg / kg pentobarbital. Twenty minutes after loss of the righting reflex, 10 ml of either sterile saline or TRH-like peptide (1 or 10 mg / 10 ml sterile saline) was injected i.c. [14]. The time from i.c. injection to recovery of the righting reflex, as measured three times within 10 s, was recorded.

2.3. Cell culture HEK293 cells stably expressing TRHR1 have been described previously [16]. The late passage cells used in these experiments expressed TRHR1 at approximately 20% the concentration measured in earlier studies. HEK293 cells stably expressing TRHR2 were prepared by transfecting HEK293 cells with a plasmid encoding TRHR2 in pcDNA3 using lipofectamine (Gibco, Grand Island, NY) and selecting with 0.5 mg / ml G418 as

Measurements of intracellular calcium were performed at 37 8C as previously described [10]. Cells grown on 25-mm glass coverslips were loaded at room temperature with 4 mM Fura2-AM, 0.2% bovine serum albumin and 20 mg / ml cyclosporin A for 30 min in buffered HBSS. The coverslip was then rinsed and placed in a Sykes-Moore chamber from Bellco (Vineland, NJ) covered with 1 ml Ca 21 - and Mg 21 -free HBSS. Imaging was performed on a Nikon inverted microscope with a DAGE CCD72 camera and Geniisys intensifier system (Michigan City, IN). Cells were alternately excited at 340 and 380 nm and emission was measured at 490 nm; 340 / 380 ratios were obtained every 1.2 s and analyzed using Image-1 software from Universal Imaging Corporation (Media, PA). After a stable baseline was obtained, peptide was added to the incubation chamber and responses were followed for at least 5 min. Peak responses from between 33 and 120 individual cells were averaged, and responses to TRH were measured with each set of cells as a standard. Most of the peptides caused a small increase in intracellular Ca 21 when added to control HEK293 cells bathed in Ca 21 -containing medium, but with the exception of the Leu 2 analog, none of them evoked an appreciable Ca 21 response in Ca 21 -free HBSS, which was used for all experiments reported here.

2.6. Statistical analysis Statistical comparisons were made with the aid of Statview (Abacus Concepts, Berkeley, CA), a statistical software package for the Macintosh computer. All multigroup comparisons were carried out by one-way ANOVA using post-hoc Scheffe contrasts with the control group.

P.M. Hinkle et al. / Brain Research 935 (2002) 59 – 64

3. Results Peptides were tested for their analeptic effect, defined as reduction in pentobarbital-induced sleep time. Injection (i.c.) of 1 mg 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 mg TRH, EEP, Leu 2 -TRH or Val 2 -TRH had an analeptic effect. Interestingly, the injection of TRH, Leu 2 -TRH and Val 2 -TRH was frequently accompanied by ‘wet dog’ shaking while the animal was still anesthetized. On the other hand, EEP never produced shaking but was an analeptic. Tyr 2 -TRH and Phe 2 -TRH did not reduce pentobarbital-induced sleep time and did not produce ‘wet dog’ shakes. To assess the affinity of peptides substituted at His 2 of TRH for the known TRH receptors, we used HEK293 cells stably expressing either TRHR1 or TRHR2. The density of receptors was calculated to be 0.30 pmol / mg protein for cells expressing TRHR1 and 0.13 pmol / mg protein for cells expressing TRHR2. Peptides were tested for the ability to compete with [ 3 H]TRH for binding to cells expressing either TRHR1 or TRHR2. There was essentially no binding of [ 3 H]TRH to HEK293 cells not transfected with any receptor. Typical competition displacement curves from a single experiment are shown in Fig. 1, and average IC 50 values in Table 2. Most analogs bound TRHR2 with slightly higher affinity than TRHR1, but none of the TRH-like peptides had high affinity for either receptor. The peptides containing Leu and Phe in the 2-position were the most potent, with affinities between 0.1 and 1% that of TRH. The Ser and Gln peptides also had measurable affinities, whereas other peptides, including Thr 2 -TRH, did not. Because [ 3 H]TRH was used at a very low concentration, the IC 50 values are close to the Ki values based on the equation Ki 5 IC 50 /(1 1 [L*] /Kd(L* ) ) where [L*] and Kd( L*) are the concentration of radioligand L* and the Kd for L*, respectively. TRH acts via both TRHR1 and TRHR2 to stimulate phospholipase C and increase inositol (1,4,5)trisphosphate (IP3 ), which acts on IP3 receptors in the endoplasmic 21 reticulum to release Ca from intracellular stores [3,6].

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To determine whether the peptides were agonists or antagonists, we tested them for their ability to evoke a Ca 21 transient in control HEK293 cells or HEK293 cells expressing TRHR1 or TRHR2. Cells were loaded with the intracellularly trapped Ca 21 indicator fura2 and then challenged in Ca 21 -free buffers with peptides at concentrations equal to the calculated IC 50 values, where these were measurable, or at 50 mg / ml. Averaged responses from multiple individual cell traces are shown in Fig. 2. In non-transfected control HEK293 cells, TRH caused no response at concentrations up to 1 mg / ml, and TRH-like peptides caused little or no Ca 21 response except for the Leu peptide, which consistently produced a small Ca 21 transient. In cells expressing TRHR1 or TRHR2, all of the peptides caused an increase in intracellular Ca 21 . The average amplitudes are shown in Fig. 3. The Ca 21 transients were greater in cells expressing TRHR1 than TRHR2, probably reflecting the higher receptor density in the TRHR1 cell line. The peak heights were comparable for all of the peptides when these were tested at mid-range concentrations. In addition, we tested the Leu-substituted TRH peptide for its ability to block the Ca 21 response to low concentrations of TRH mediated by both receptors, and we found no effect, indicating that it does not have antagonistic activity.

4. Discussion We have shown that EEP, Val 2 -TRH and Leu 2 -TRH are analeptics, like TRH, but Phe 2 -TRH and Tyr 2 -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, His 2 -substituted peptides had not previously been known to exert this activity. The receptor mediating the analeptic effects of TRH and related peptides has not been identified, and TRHR2 was a particularly attractive candidate based on its distribution within the CNS. It is evident from comparing the results in Tables 1 and

Table 1 Analeptic effects of TRH and TRH-like peptides Exp. [1

Sleep time (min) n P-value b

Exp. [3 a

Exp. [2

Saline

TRH

EEP

Saline

Leu 2 -TRH

Phe 2 -TRH

Saline

Tyr 2 -TRH

Val 2 -TRH

76.5619.7

48.6617.2

52.8613.3

74.2618.5

56.5630.5

70.8627.5

100.0%64.7

92.2%643.1

72.7%619.2

19

10 ,0.0001

21 ,0.0001

21

20 ,0.05

23 N.S.

11

14 N.S.

14 ,0.05

Shown are the effects of intracisternal injection of 10 mg of TRH or TRH-like peptide in 10 ml sterile saline on pentobarbital-induced sleep time compared to a saline-injected control group. Results are expressed as mean6S.D. a A fixed dose of pentobarbital was used for this experiment. The mean sleep times decreased 53% over the course of this experiment, due, in part, to the 26% increase in the mean body weights. This necessitated normalization of sleep results by the corresponding mean control sleep time measured on a given day. b One-way ANOVA using post-hoc Scheffe contrasts with the control group.

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Fig. 1. Affinity of His-substituted TRH peptides for TRH receptors. HEK293 cells stably expressing TRHR1 or TRHR2 were incubated with 1 nM [ 3 H]TRH and different concentrations of peptide, as shown. Data are expressed as the percent of Bmax , the amount of [ 3 H]TRH bound in the presence of no competitor, and results shown give the mean of duplicate determinations, which typically differed by less than 5%.

Table 2 Affinity of peptides for TRHR1 and TRHR2 pGlu-X a -ProNH 2

TRHR1

TRHR2

Leu Phe Val Tyr Ser Thr Gln Glu (EEP) Arg His (TRH)

1.8560.60 1.5860.68 .100 .100 21.763.3 .100 9.8061.40 .100 .100 0.008560.0016

0.6660.24 1.2560.44 .100 .100 12.362.9 .100 7.5760.47 .100 .100 0.008860.0024

Shown are IC 50 values (mg / ml), the concentrations of peptides causing 50% inhibition of binding, for various peptides. Values given are the mean and range or S.E.M. of 2–6 determinations. a X refers to the substituents in the first column of the table.

2 that TRH-like peptides that are analeptics do not bind effectively to either TRHR1 or TRHR2. TRH and Leu 2 TRH produced an equivalent antisedative response following injection of the same i.c. dose. Although the Leu 2 -TRH peptide bound to TRH receptors with the highest affinity of the peptides tested, its affinity was still less than 1% that of TRH. Furthermore, both EEP and Val 2 -TRH displayed significant analeptic activity but had immeasurably low affinity for TRHR1 and TRHR2. Based on these observations, it is unlikely that the analeptic activity of TRH-like peptides occurs via the known TRH receptors. Each of the TRH-like peptides that occur abundantly in the CNS and peripheral tissues may have its own, yet to be identified, receptor, or these peptides may act via a common but still unidentified signal pathway. EEP, also known as the fertilization promoting peptide, has been

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Fig. 2. Typical calcium responses to His 2 -substituted TRH peptides. Shown are Ca 21 responses to TRH and several peptides in HEK293 cells or HEK293 cells stably expressing TRHR1 or TRHR2. Peptides were tested at concentrations equal to the IC 50 values obtained in experiments like those shown in Fig. 1. Traces are averages from 20–40 individual cells. Note that the scale for responses of HEK293 / TRHR1 cells is larger than the scales for control HEK293 cells and HEK293 / TRHR2 cells, which are the same.

shown to have sperm capacitating activity via the TCP-11 receptor and the adenylyl cyclase / cAMP pathway [1]. TRH increases intracellular calcium and activates protein kinase C but does not normally increase cAMP [3,6]. The

molecular mechanisms underlying the analeptic actions of TRH have not been identified. The binding data reveal that TRHR1 and TRHR2 have stringent requirements for the residue in the 2-position of

Fig. 3. Calcium responses of cells expressing TRHR1 and TRHR2. Average increases in intracellular Ca 21 , expressed as 340 / 380 fluorescence ratio, were calculated from traces such as those shown in Fig. 2; values give the mean and S.E.M. for 33–120 cells from multiple experiments. Peptides were tested at concentrations equal to the IC 50 or, where the IC 50 was too high to measure, at 50 mg / ml.

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the peptide. In a model developed by Gershengorn et al. [4], the imidazole ring of the His in TRH is predicted to sit in a stack with Tyr282 in the 6th transmembrane domain of TRHR1. The current finding of low affinity for Hissubstituted residues is consistent with the importance of van der Waals interactions between the imidazole of the ligand and the receptor. The results are also consistent with previous investigations showing that substitutions on the His residue severely reduce receptor affinity [17]. The TRH-like peptides tested all elicited calcium transients at concentrations sufficient to occupy half of the receptors. These responses were mediated by TRHR1 and TRHR2, because they were not observed in parental HEK293 cells. Because TRH causes a maximal Ca 21 response at doses well below its Kd [10], it is not surprising that even those TRH-related peptides with poor affinity were able to evoke a response. The TRH-like peptides tested here either display very little activity or act as weak agonists via TRHR1 and TRHR2 in a cell culture model; we found no evidence that any of them can act as an antagonist. In summary, EEP, Val 2 -TRH and Leu 2 -TRH, like TRH, are analeptics. These novel TRH-like peptides, which exert important effects in the CNS and peripheral tissues, do not exert their antisedative activities through the two known TRH receptors, TRHR1 and TRHR2. The data suggest that novel receptors mediate some of the neuronal actions of TRH-like peptides.

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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