Cryptic peptide derived from the rat neuropeptide FF precursor affects G-proteins linked to opioid receptors in the rat brain

peptides 29 (2008) 1988–1993

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Cryptic peptide derived from the rat neuropeptide FF precursor affects G-proteins linked to opioid receptors in the rat brain Piotr Suder a,*, Dominika Nawrat b, Adam Bielawski b, Agnieszka Zelek-Molik b, Hana Raoof a, Tomasz Dylag a, Jolanta Kotlinska c, Irena Nalepa b, Jerzy Silberring a a Department of Neurobiochemistry, Faculty of Chemistry and Regional Laboratory, Jagiellonian University, Ingardena 3 Street, 30-060 Krakow, Poland b Department of Brain Biochemistry, Institute of Pharmacology, Polish Academy of Sciences, Smetna 12 Street, 31-343 Krakow, Poland c Department of Pharmacology and Pharmacodynamics, Medical University, Staszica 4 Street, 20-081 Lublin, Poland

article info

abstract

Article history:

Recently, we reported the discovery of a novel amino acid sequence derived from the NPFF

Received 28 April 2008

precursor NAWGPWSKEQLSPQA, which blocked the expression of conditioned place pre-

Received in revised form

ference induced by morphine and reversed the antinociceptive activity of morphine (5 mg/

1 July 2008

kg, s.c.) in the tail-immersion test in rats. Here, we name it as NPNA (Neuropeptide NA from

Accepted 15 July 2008

its flanking amino acid residues). The synthetic peptide influenced the expression of mRNA

Published on line 24 July 2008

coding for Ga(i1), (i2), and (i3) subunits. The results provide further evidence that yet another bioactive sequence might be present within the NPFF precursor. # 2008 Elsevier Inc. All rights reserved.

Keywords: G-proteins Opioids GPCR NPFF mRNA Metabolism

1.

Introduction

Recently, we published data showing the existence of a novel bioactive sequence within the NPFF precursor [4]. The selected peptide NAWGPWSKEQLSPQA spans residues 85–99, and is flanked by the sequences of well-known NPFF and NPSF. The sequence of fragment 85–99 differs between species, therefore we tested the rat sequence in rats only. In contrast to the two other peptides already described in the literature, NPSF and NPFF, this peptide is not amidated at its C-terminus due to the lack of a glycine residue necessary for amidation. Therefore, we anticipate that NPFF precursor (85–99) exerts anti-opioid

actions by a mechanism different from that of NPFF, which has been widely described in the literature [7,16,18]. Here, we denote this sequence as NPNA (Neuropeptide NA due to its flanking amino acids). According to the classification recently proposed [1], the peptide described here can be categorized as type 3 cryptein, i.e. a novel bioactive peptide fragment that was generated in vitro and might not necessarily be naturally occurring. NPNA (residues 85–99 in the NPFF precursor) was shown to be bioactive in several pharmacological tests. Synthetic NPNA (10 and 20 nmol, i.c.v.) blocked the expression of conditioned place preference induced by morphine (5 mg/kg, s.c.) [4]. A

* Corresponding author. Tel.: +48 12 663 56 03; fax: +48 12 634 05 15. E-mail address: [email protected] (P. Suder). 0196-9781/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2008.07.012

peptides 29 (2008) 1988–1993

functionally similar effect was observed after i.c.v. injection of stable FF receptor agonist [9]. Neuropeptide FF receptor antagonist (dansyl-PQRamide) significantly reversed this effect [6]. NPNA alone (10 and 20 nmol, i.c.v.) had no influence on the baseline latency of a nociceptive reaction, but reversed the antinociceptive activity of morphine (5 mg/kg, s.c.) in the tail-immersion test in rats. These findings and comparison to activities of other peptides from NPFF precursor support the thesis that NPNA belongs to bioactive peptides derived from the NPFF precursor. The results triggered us to search for its further possible biological role in the CNS. Here, we investigated the influence of NPNA on the expression of Ga(i) proteins (subunits i1, i2, and i3) linked to opioid receptors.

2.

Materials and methods

2.1.

Animals

All experiments were performed in agreement with Polish and European ethical regulations and were approved by the Local Ethics Committee. Male Wistar rats (150–220 g) were purchased from a local distributor (HZL, Warszawa, Poland) and housed in groups of five per cage, with standard food and free access to water. Animals were maintained in a 12:12 h light:dark cycle (light on at 08:00 h) in an air-conditioned room. After 1 week of adaptation and handling, the rats were divided into groups (8 animals/group) and prepared for the test.

2.2.

Drugs and chemicals

Rat neuropeptide NA was synthesized by the standard Fmoc solid-phase method with the use of a semi-automatic peptide synthesizer (Advanced ChemTech Model 90) and purified by a HPLC system (Shimadzu, Japan) using a semi-preparative reversed-phase C18 column (Phenomenex, Kromasil 10 mm, 10 mm  250 mm). The identity and purity of the peptide were confirmed by electrospray (Esquire 3000, Bruker Daltonics, Leipzig, Germany) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Reflex IV, Bruker Daltonics, Leipzig, Germany). Its purity was greater than 98%. The peptide was dissolved in physiological saline (0.9% NaCl) and injected intracerebroventricularly (i.c.v.) in a volume of 5 ml.

2.3.

Injection procedure

After 1 week of adaptation and handling, rats were prepared for i.c.v. injections, 2 days before the experiment. Surgery was done under pentobarbital (50 mg/kg, i.p., Vetbutal, Biowet, Pulawy, Poland) anesthesia. The coordinates for i.c.v. injections were measured from the bregma, according to the atlas of the rat brain [13], and were as follows: 1.5 mm lateral, 1.0 mm caudal, 3.5 mm ventral from the surface of the skull into the right lateral ventricle. On the day of experiment, the rats received a single injection (free-hand) of either saline (controls), or 10 nmol NPNA in a volume of 5 ml. The liquid was injected within 20 s. Rats were divided into three groups: (1) control group, N = 8; (2) peptide injected group, and (3) NPNA

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injected groups (both N = 8). Control (1) and peptide injected (2) groups were decapitated 90 min after injection. The last group of rats (3) was decapitated 240 min after the injection. Immediately after decapitation, the skulls were opened, and brains were removed and placed on ice-cold plastic plates. From every brain, the hippocampus, striatum and frontal cortex were dissected, and immediately frozen on dry ice. Structures were collected and stored for further analyses at 86 8C.

2.4.

Total RNA isolation and cDNA synthesis

Tissues were homogenized using a glass-Teflon homogenizer (Glas-Col, Terra Haute, IN, USA) and total tissue RNA was isolated using 1 ml of Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The quantity of RNA was determined spectrophotometrically at 260 nm and 260 nm/280 nm (Pharmacia Ultraspec 2000 UV/Vis, Pharmacia Biotech, UK) and its quality was confirmed by electrophoresis on agarose (Amresco, Solon, OH, USA) gel. Some amount of RNA from each sample was converted into cDNA using T3 Thermocycler (Biometra, Germany). The reaction of reverse transcription (RT) was carried out in a total volume of 20 ml. Briefly, 2 mg of total RNA was incubated for 5 min at 65 8C with 2 U RNAse inhibitor (Fermentas, Vilnius, Lithuania) and 6 ml RNase-free water and the samples were chilled on ice. Then, 5x buffer for AMV reverse transcriptase (Finnzymes OY, Finland), 2 mM of custom-synthesized oligo(dT)15 primers (TiB MolBiol, Poland), 1 mM dNTP mix (Fermentas, Vilnius, Lithuania), 10 U AMV reverse transcriptase (Finnzymes OY, Finland) made up to a final volume of 10 ml (RNase-free water), were added before incubation steps of 90 min at 42 8C and 10 min at 70 8C.

2.5. Real-time quantitative reverse transcriptase-PCR (qRT-PCR) assay for mRNAs of Ga(i1), Ga(i2), Ga(i3) and HPRT Real-time qRT-PCR was performed in the Chromo4TM system for real-time PCR detection (MJ Research, Waltham, MA, USA) using double-stranded DNA-intercalating dye, SYBR Green I. Amplification was carried out in 20 ml reaction mixture containing 10 ml of FastStart SYBR Green Master (Rox) mix (Roche Diagnostics, Germany), 2 ml of cDNA template (1:4 diluted), 3 mM forward and reverse specific primers, and 7.4 ml RNase-free water. All reactions were run in duplicates, and samples containing no template were included as negative controls. Specific primers were selected from the sequence of rat mRNA for Ga(i1) (NM 013145); Ga(i2) (NM 031035); Ga(i3) (NM 013106); and HPRT (NM 012583) by using OLIGO 5.0 Primer Analysis Software (MBI, West Cascade, CO, USA). Detailed data about specific forward- and reverse primers, as well as the length of amplified G-proteins and control protein regions, can be seen in Table 1. The PCR program was as follows: one hold at 95 8C for 15 min, followed by 40 cycles of 94 8C for 30 s, 60 8C (Ga(i1) and HPRT) or 58 8C (Ga(i2)) or 56 8C (Ga(i3)) for 30 s, and 72 8C for 1 min. The PCR products specificity was verified by melting curve analysis at a range of 50–95 8C, and the presence of a single band of appropriate molecular size detected by agarose (Amresco, Solon, OH, USA) gel electrophoresis. The standard curve was generated for

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5 -GTC AAC GGG GGA CAT AAA AGT-30 50 - CAA GGG CAT ATC CAA CAA CA-30 254 bp region of HPRT

each target gene by serial dilutions of pulled cDNA from samples of the control group (corresponding to a range of 1 mg/ml to 15 ng/ml) of total RNA used for RT. The cycle threshold C(t) line for standards and experimental samples were set using the Opticon Monitor 3.1 Software (MJ Research, Waltham, MA, USA) to calculate the relative abundance of each target gene. There was no difference in the HPRT (hypoxanthine–guanine phosphoribosyl transferase) mRNA expression between the treated groups.

2.6.

Statistical analysis

Ga(i3) (NM 013106)

5 -GAT GTG GGT GGT CAG CGA TCT-3 50 -TTG GTG TCT TTG CGT TTA TTC A-30 353 bp region of Ga(i2)

0 0

Ga(i2) (NM 031035)

5 -TGT GGC CCT GAG TGA CTA TGA-3 50 -GCG CAA GTG AAG TGG GTG TAA-30 306 bp region of Ga(i1)

0 0

Ga(i1) (NM 013145)

Forward primer Reverse primer Amplification

0

mRNA for

Table 1 – Sequence of specific forward- and reverse primers, and the length of amplified G-proteins and control gene regions

0

5 -AGA TGT TCT TCG GAC GAG AGT-3 50 -TCA AAC TGG CAC TGG ATG TAA G-30 408 bp region of Ga(i3)

0

HPRT (NM 012583)

peptides 29 (2008) 1988–1993

Data were analyzed by one-way analysis of variances (ANOVA) followed by a Fisher’s LSD (Least Significant Difference) post hoc comparison (Statistica 7.0 software). Difference was considered to be significant if P < 0.05. The data represent the means  S.E.M. of 6–8 rats per group.

3.

Results

Expression of mRNA of genes coding for three subtypes of Ga(i) subunit: Ga(i1), Ga(i2), and Ga(i3) were investigated in the hippocampus, striatum, and frontal cortex of rats. The mRNA levels were assessed at two time-points: 90 min and 240 min after the NPNA injection. Every group contained 8 animals but, as a result of experimental procedures, imperfections in i.c.v. injections and structures isolation, we were forced to remove some animals or some structures from the groups. This caused changes in the quantity of material received. The final quantity of every investigated structure is given within the descriptions of the figures. In the hippocampus, one-way ANOVA indicated significant differences between groups for all three subunits: F(2, 20) = 3.598, P < 0.05, for Ga(i1); F(2, 21) = 15.275, P < 0.001, for Ga(i2) and F(2, 20) = 5.875, P < 0.01, for Ga(i3). Post hoc test showed that injection of the peptide induced a significant decrease of Ga(i1) mRNA expression by 18% (P < 0.05), the Ga(i2) and Ga(i3) by 21% (P < 0.001 and P < 0.01) compared to the saline controls. The effect was observed at 240 min while no change was present in a shorter time, i.e., at 90 min after the peptide injection (Figs. 1A–C). In the striatum and the frontal cortex, the changes were well-marked in the case of Ga(i2) mRNA expression. One-way ANOVA indicated significant differences between the groups: F(2, 21) = 4.167, P < 0.05 and F(2, 21) = 3.984, P < 0.05 (for striatum and frontal cortex, respectively). Post hoc comparison showed that 90 min after the peptide injection, the expression of Ga(i2) mRNA was decreased in both brain structures by 20% and 18%, respectively, while the effect was not observed after 240 min (Fig. 2B and Fig. 3B). A similar pattern of changes was noticed for Ga(i3) protein expression (P < 0.05, 90 min vs. control) [F(2, 21) = 3.048, P = 0.071 and F(2, 21) = 3.277, P = 0.059, in striatum and frontal cortex, respectively] (Fig. 2C and Fig. 3C). In the striatum Ga(i1) mRNA expression was not affected by peptide in either of two treatment groups, while in the frontal cortex the decrease (by 21%, P < 0.05 vs. saline) [F(2, 21) = 2.540, P = 0.105] was transient and not observed after 240 min (Fig. 2A and Fig. 3A).

peptides 29 (2008) 1988–1993

Fig. 1 – Effect of single NPNA injection on the expression of Ga(i) mRNA subunits in hippocampus of rats measured at 90 and 240 min after the peptide injection. (A) Ga(i1); (B) Ga(i2); (C) Ga(i3). Data are expressed as a percent of saline control, and the bars represent the mean values W S.E.M. of 6–8 rats per group: white bars—saline; light grey— 90 min, and dark grey bars—240 min after NPNA. *P < 0.05; **P < 0.01; ***P < 0.001 vs. saline control.

4.

Discussion

G-proteins play a crucial role in linking 7-transmembrane Gprotein coupled receptors (GPCRs) binding to changes in specific intracellular signaling pathways, whose signaling ultimately affects the regulation of genomic changes. All of the

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Fig. 2 – Effect of single NPNA injection on the expression of Ga(i) mRNA subunits in striatum of rats measured at 90 and 240 min after the peptide injection. (A) Ga(i1); (B) Ga(i2); (C) Ga(i3). *P < 0.05 vs. saline control. For other descriptions, see Fig. 1.

several known classes of opioid receptors belong to the GPCR superfamily (for review see [22]). G-proteins consist of three subunits: a, b, and g. The a subunit dissociation from the abg complex is an important process for activation of specific signaling cascades. A dimer bg facilitates interactions between receptor kinases and GPCRs, particularly opioid receptors.

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peptides 29 (2008) 1988–1993

Fig. 3 – Effect of single NPNA injection on the expression of Ga(i) mRNA subunits in frontal cortex of rats measured at 90 and 240 min after the peptide injection. (A) Ga(i1); (B) Ga(i2); (C) Ga(i3). *P < 0.05 vs. saline control. For other descriptions, see Fig. 1.

This, in turn, promotes phosphorylation of the receptor and activates the pathway linked to arrestin [15]. All the principal opioid receptors (m, d, and k), are associated with Ga(o) and Ga(i) proteins. There are three Ga(i) subunits, i1, i2, and i3, which are coded by separate genes and have been shown to mediate receptor-dependent inhibition of various types of adenylyl cyclases [20]. Although all three subtypes of

opioid receptors appear to preferentially activate Ga(i2), there is an evidence that mOR show some preference for Ga(i3) [3]. Treatment with morphine induces changes in the expression of many genes coding for various cellular proteins, including G-proteins [12], and others, e.g., c-Fos [21]. It is interesting that morphine action on its receptors is distinct from other opioid ligands. Although morphine induces strong tolerance, it does not trigger significant internalization of the mOR [14]. A novel cryptic peptide NPNA derived from the NPFF precursor and recently described by us [4] possesses biological activity in several pharmacological tests, but its mechanism of action and binding site remains unknown. The C-terminal end of the NPNA sequence cannot be amidated, in contrast to other peptides derived from this precursor (NPFF, NPAF, NPSF), where a glycine residue from the C-terminal side of the peptide’s sequence is a donor of –NH2 group for amidation. Therefore, a similarity in binding is still obscure. Recently, Gouarderes et al. [5], described functional differences between NPFF1 and NPFF2 receptors expressed in CHO cells, and those receptors are coupled differently to Gproteins. This group also demonstrated that Ga(i3) and Ga(s) are the main transducers of NPFF1 receptors while NPFF2 are probably coupled with Ga(i2), Ga(i3), Ga(o) and Ga(s) proteins. It should be noted here that the G(i) proteins investigated in our study (as in other studies as well) are not exclusively linked to opioid receptors. NPNA in our work showed differences in its action on various G-protein subunits, and it seems that this effect is also tissue- or even CNS structurespecific. NPFF modulates the opioid system by a mechanism exerting anti-opioid activity [17]. It is likely that by increasing the mobility of mOR, NPFF could weaken their association with G-protein and channel subunits, resulting in a reduced inhibition of the calcium channel by the opioid agonist, i.e. an anti-opioid effect [11]. Also, NPFF modulates the opioid system by promoting NPF–MOP receptors heteromerization, resulting in a reduction of opioid response. Such an observation is strengthened by recent data from Simonin et al. [19], who showed that by the use of a selective NPFF receptor antagonist it was possible to prove that NPFF receptors are, in fact, a part of the anti-opioid system. The main and the most important finding of the current study was that i.c.v. injection of NPNA modulates the activity of genes coding for three alpha subunits of G(i) protein in three brain regions (hippocampus, frontal cortex, and striatum). Although an upstream mechanism leading to this effect is presently unclear, at least three possible actions may be considered. They are based on an analogy to known properties of NPFF and its interaction with various neurotransmitter systems: (1) interaction at the level of membrane receptors (i.e., upstream to the signaling cascade—heteromerization of NPFF with opioid receptors) [17], (2) interaction at a level downstream from the receptor—an influence of NPNA on the system of other neurotransmitters that share the same G(i) proteins, e.g., serotonin or glutamate. NPFF was shown to modulate the level of neurotramsmitters that are linked to GPCRs [2,8].

peptides 29 (2008) 1988–1993

However, an explanation of the observed NPNA effects according to the above-mentioned mechanisms seems to be premature and speculative in respect to the current data, so we postulate the following mechanism as the most probable explanation:

[6]

[7]

(3) Data coming from the SH-SY5Y neuroblastoma cell line model indicate that NPFF receptors can be coupled to G(i) proteins, and in this way they induce cAMP inhibition [10]. Thus, assuming that NPNA receptor(s) represent the NPFFlike (i.e., G(i)-coupled) receptor, we propose that the NPNAinduced decrease in mRNA expression of Ga(i1, i2, i3) occurs as a consequence of NPNA receptor stimulation. In such a case, the stimulation of an inhibitory G(i) protein (resulted from NPNA receptor activation) is followed by inhibition of adenylyl cyclase/cyclic AMP/protein kinase; a pathway, which in turn, modulates transcriptional activity of various genes, including those coding for Ga(i) subunits.

[8]

[9]

[10]

[11]

Further speculations on similarities between NPFF and NPNA effect on the second messenger cascade, as well as a detailed mechanism of NPNA action would be premature. More detailed studies are needed, including identification of its endogenous sequence and receptor specificity. Nevertheless, our results confirm the presence of another bioactive sequence within NPFF precursor molecule. Moreover, regardless of mechanism(s) leading to the observed phenomenon, our data provide the first evidence that NPNA is able to modulate the mRNA expression of Ga(i1, i2, i3) in several regions of rat brain.

Acknowledgements This work was supported by the International Centre for Genetic Engineering and Biotechnology (ICGEB) grant no. CRP/ POL05-02, Jagiellonian University CRBW/2008 funds and by the statutory funds of the Institute of Pharmacology, Polish Academy of Sciences.

[12]

[13] [14]

[15]

[16]

[17]

references

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