Bulgarian Chemical Communications, Volume 41, Number 2 (pp. 116-121) 2009

Received July 16, 2008

The aim of the present study was to investigate the involvement of endogenous nitric oxide (NO) in the effects of neuropeptide kyotorphin (Kyo) and its synthetic analogue D-kyotorphin (D-Kyo) on immobilization and cold stress-induced analgesia (SIA). In scientific literature, Kyo is considered as neuromodulator. D-Kyo is more stable enzimatically. Some studies demonstrate that Kyo is a possible substrate for neuronal and inducible NO synthase, also the NO system is stress-limiting and plays an important role in initiation and maintenance of pain.

Kyo and D-kyo were synthesized by the Group of Antimetabolites at the Institute of Molecular Biology, Bulgarian Academy of Sciences. The proposed synthetic route was based on well-established liquid-phase methods of peptide synthesis – active esters, using tert-butyloxycarbonyl (Boc) group for protection of the Nα-amino group according to the protocol previously described in the literature. Male Wistar rats were used in acute immobilization and cold stress models. The evaluation of nociceptive effects was carried out using the paw pressure (PP) and hot plate (HP) tests. Kyo and D-Kyo (both in dose 5 mg/kg), L-NAME (10 mg/kg), and L-Arginine (1 mg/kg) were dissolved in saline and were injected intraperitoneally.

The result showed that endogenous NO is differently involved in the effects of peptides, which may be due to different implication of opioid and non-opioid components of SIA. Probably, L- or D-form of the peptide is also important, as well as the type of the stressor and its neurochemical signature.

Key words: kyotorphin, D-kyotorphin, nitric oxide, stress-induced analgesia.

Introduction

Kyotorphin (Kyo) is a dipeptide (L-Tyr-L-Arg) (Fig. 1A) synthesized in specific brain regions [1]. The highest levels were found in the lower brain stem and dorsal spinal cord, areas closely associated with the pain regulatory system [2, 3]. D-Kyo-torphin (D-Kyo or L-Tyr-D-Arg) (Fig. 1B) is a synthetic analogue of Kyo. Both peptides bind to a specific Kyo-receptor and induced Met-enkephalin release at rates of approximately 4 times basal release [4]. Literature data showed that Kyo-receptor is identified in the membrane-preparations of the brain, which suggests that it plays a physio-logical significance in the neurotransmission as a neurotransmitter/neuromodulator [5]. However, D-Kyo shows enhanced analgesic activity, i.e. 5.6-fold higher than that observed with Kyo. Takagi and co-workers [1] suggested that this effect is a result of protease resistance conferred by the substitution of L-arginine (L-Arg) with a D-arginine residue [6].

It is also known that acute and chronic stress induces biochemical changes affecting both pain threshold and behaviour [7]. Stressors such as immobilization and cold exposure can cause stress-induced analgesia (SIA) [8]. It can be resolved into an opioid, when it is antagonized by naloxone, but also a non-opioid component [9].

Fig. 1. Structural formula of: A. kyotorphin

Rat experiments have demonstrated that the nitric oxide (NO) system fulfils the main criteria of a stress-limiting system [10]. Earlier it was reported that the mechanism of NO-induced antinociception involved opioid components and was also dependent on brain NO [11]. NO is an unique neurotransmitter was synthesized by the enzyme nitric oxide synthase (NOS) and plays an important role in initiation and maintenance of pain [12]. Also, it is known that Kyo, as well as L-Arg, are possible substrates for neuronal and inducible NOS [13, 14]. The aim of the present study was to investigate the involvement of endogenous NO in the effects of kyotorphin and D-kyotorphin on immobilization and cold stress-induced analgesia.

Experimental

Chemistry. All reagents and solvents were of reagent grade and used without further purification. Amino acid derivative Boc-Tyr-OH was prepared according to the general procedure of Pozdnev [15] and Boc-Tyr-OSu – according to [16].

Active ester procedure (AE). Nα-protected tyrosine succinimide ester (1 mmol) was dissolved in l ml dimethylformamide (DMF), and 1.2 mmol arginine (L- or D- form) in 1 ml DMF was added to it. The pH was adjusted to 8–9 with 5% NaHCO₃, and the mixture was stirred at room temperature overnight. After reduced pressure evaporation, the residue was partitioned between 0.5 M NaHSO₄ and n-BuOH. The acidic aqueous layer was extracted twice with n-BuOH (2 × 30 ml) and the combined organic extracts were washed with 10% NaHCO₃ and brine. The organic phase was dried over Na₂SO₄ and evaporated in vaccuo to a smaller volume;

Trifluoroacetic acid (TFA) deprotection. The fully protected peptides were treated with TFA (1/10 molar ratio) in the presence of anisole for 30 min at room temperature to remove the Boc-pro-tecting groups. The crude peptides were purified by suitable crystallization. The pure TFA salt obtained in this way was converted to the target product by treatment with ion-exchange resin Amberlite IR-45, dissolved in water and lyophilized.

Analyses. The purity of the peptides and struc-tural identity was established by TLC, analytical HPLC and electrospray mass spectometry. TLC was carried out on silicagel 6OF254 pre-coated (Merck) aluminum plates, with the use of the following solvent systems: A = n-butanol:acetic acid:water (4:1:5); B = chloroform:methanol:water (80:30:5). Visualization was done with either UV, or nin-hydrin. For the analytical gradient RP-HPLC, a Merck-Hitachi liquid chromatograph, was used. A water/0.1% TFA buffer, pH 2.25, and a linear acetonitrile gradient were used for elution on a
100-5 Nucleosil Ci8 column. To check the purity of compounds, a 5–100% acetonitrile gradient in 30 min was performed. Optical rotation was measured with a Perkin-Elmer Model 141 polarimeter.

Animals. Male Wistar rats (180–200 g) were used. The animals were housed in groups of 6 per cage and kept under a normal 12 h light/dark cycle and 22 ± 2°C temperature. Rats had free access to food and water.

E. B. Dzambazova et al.: Involvement of endogenous nitric oxide in the effects of kyotorphin

The experiments were approved by the Animal Care and Use Committee of the Medical University, Sofia.

Results and Discussion

The proposed synthetic route was based on well-established liquid-phase methods of peptide syn-thesis – active esters, using tert-butyloxycarbonyl (Boc) group for protection of the Nα-amino group (Fig. 2) according to the protocol previously described in literature [18]. The strategy of the minimal side-chain protection was adopted. Thus, the phenolic group of tyrosine and δ-guanidino group of arginine were unprotected.

Fig. 2. Scheme for the synthesis of kyothorphin and

The Kyo and D-Kyo were synthesized by con-densation of the preliminary prepared Boc-Tyr-OSu with L-Arg or D-Arg, respectively. The reaction carried out for 16–24 hours at room temperature resulted in high yields.

After isolation and purification, the peptides were identified and characterized by optical rotation,

TLC, analytical HPLC, mass-spectra and elemental analysis.

E. B. Dzambazova et al.: Involvement of endogenous nitric oxide in the effects of kyotorphin

The investigations started 15 min after intra-peritoneal injection of each of the peptides. Our results showed that in PP test Kyo (p < 0.01) and D- Kyo (p < 0.01) (both at a dose of 5 mg/kg, i.p.) administered immediately after stress procedure significantly inhibited 1 hour immobilization stress-induced analgesia (ISIA) at the beginning of the experiment. On the 30^th min only Kyo had kept this effect, while D-Kyo significantly potentated pain threshold (p < 0.05) (Fig. 3).

L-NAME (10 mg/kg, i.p.) and L-Arg (1 mg/kg, i.p.) were injected 20 min before investigated peptides and their interactions have been studied after each stress model. Co-administration of Kyo and D-Kyo with L-NAME or L-Arg significantly decreased analgesia induced by IS and CS and measured by PP and HP tests (Figs. 3, 4, 5 and 6).

In PP test L-NAME or L-Arg significantly decreased inhibiting effect of Kyo on ISIA. Their effect on D-Kyo was the same only on the 15th min, while on the 30^th min they significantly decreased its increasing pain threshold (Fig. 3).

Injected immediately after 1 hour CS, Kyo (p < 0.01) significantly inhibited cold stress-induced analgesia (CSIA) during the whole investigated period, while D-Kyo injected after 1 hour CS procedure decreased significantly the analgesic effect of CS on the 45^th min (p < 0.01) from the beginning of the experiment (Fig. 4).

Fig. 3. Effects of Kyo and D-Kyo (both in 5 mg/kg, i.p.) and their combination with L-NAME (10 mg/kg, i.p.) and

L-Arg (1 mg/kg, i.p.) on nociception measured with paw pressure test after 1 hour immobilisation stress (IS) in male Wistar rats (n = 5). Mean values ± S.E.M. are presented. *p < 0.05, **p < 0.01 vs. control; ⁺p < 0.05, ⁺⁺p < 0.01 vs. IS.

Fig. 4. Effects of Kyo and D-Kyo (both in 5 mg/kg, i.p.) and their combination with L-NAME (10 mg/kg, i.p.) and

L-Arg (1 mg/kg, i.p.) on nociception measured with paw pressure test after 1 hour cold stress (CS) in male Wistar rats (n = 5). Mean values ± S.E.M. are presented. *p < 0.05, **p < 0.01 vs. control; ⁺p < 0.05, ⁺⁺p < 0.01 vs. CS.

Fig. 5. Effects of Kyo and D-Kyo (both in 5 mg/kg, i.p.) and their combination with L-NAME (10 mg/kg, i.p.) and

L-Arg (1 mg/kg, i.p.) on nociception measured with hot plate test after 1 hour immobilisation stress (IS) in male Wistar rats (n = 5). Mean values ± S.E.M. are presented. *p < 0.05, **p < 0.01 vs. control; ⁺p < 0.05, ⁺⁺p < 0.01 vs. IS.

Fig. 6. Effects of Kyo and D-Kyo (both in 5 mg/kg, i.p.) and their combination with L-NAME (10 mg/kg, i.p.) and

L-Arg (1 mg/kg, i.p.) on nociception measured with hot plate test after 1 hour cold stress (CS) in male Wistar rats
(n = 5). Mean values ± S.E.M. are presented. *p < 0.05, **p < 0.01 vs. control; ⁺p < 0.05, ⁺⁺p < 0.01 vs. CS.