Modulation of peripheral inflammation by the substance P N-terminal metabolite substance P1–7

peptides 27 (2006) 1490–1497

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Modulation of peripheral inflammation by the substance P N-terminal metabolite substance P1–7 Daniel Wiktelius a,1, Zeinab Khalil b, Fred Nyberg a,* a

Department of Pharmaceutical Biosciences, Division of Biological Research on Drug Dependence, Uppsala University, P.O. Box 591, S-751 24 Uppsala, Sweden b National Ageing Research Institute, University of Melbourne, Poplar Road, P.O. Box 31, Parkville 3052, Melbourne, Australia

article info

abstract

Article history:

The N-terminal metabolite of the undecapeptide substance P (SP), substance P1–7 (SP1–7), is

Received 26 April 2005

known to modulate nociception in the central nervous system (CNS) and often has opposite

Received in revised form

effects from SP. This study investigated the ability of SP1–7 to modulate the vasodilatation

6 December 2005

response to SP in anaesthetized rats under different injury conditions using a blister model

Accepted 7 December 2005

of inflammation on the hind footpad. The results indicated that SP1–7 inhibited the vascular

Published on line 18 January 2006

response to SP in a dose-dependent manner. The putative antagonists naloxone and D-Pro27 D-Phe -SP1–7 (D-SP1–7)

reversed the effect of SP1–7. D-SP1–7 improved the responsiveness to SP

Keywords:

under chronic nerve injury, which suggests a role for endogenous SP1–7 in this model. SP1–7

Inflammation

did not inhibit the response to electrical stimulation of the sciatic nerve, which indicates

Nerve injury

that the heptapeptide interacts at a post-terminal binding site. The current results suggest

Opioid antagonist

that SP1–7 may have inhibitory properties in inflammation, analogous to its antinociceptive

Substance P (SP)

role in the central nervous system.

Substance P fragments SP1–7, SP5–11

# 2005 Elsevier Inc. All rights reserved.

and SP9–11 Rats Vasodilatation

1.

Introduction

The tachykinin substance P (SP) is composed of 11 amino acids and perhaps it represents one of the most extensively characterized neuropeptides in a number of different species. Together with its preferred receptor (NK-1) SP is widely distributed within the central nervous system (CNS), where it is attributed to a variety of functions [48], foremost as a transmitter and modulator of pain signals [10,43]. The main mechanism for inactivation of SP in the synapses appears to involve various steps of enzymatic degradation. Several

enzymes capable of converting or degrading SP have been described (for review, see Ref. [37]). Moreover, some of these peptidases are known to release SP fragments retaining biological activity [7,18,39]. We have earlier described an enzyme hydrolyzing SP at its Phe7–Phe8 bond generating the N-terminal fragment SP1–7 [39]. This heptapeptide has been demonstrated to share some, but oppose other, effects of the parent compound. SP induces aversive behavior when administered i.t. into mice [41,49]. C-terminal metabolites of SP elicit the same type of effects, most likely through the action on the NK-1 receptor,

* Corresponding author. Tel.: +46 18 4714166; fax: +46 18 501920. E-mail address: [email protected] (F. Nyberg). 1 Present address: Department of Chemistry, Medicinal Chemistry, Go¨teborg University, Kemiva¨gen 10, S-412 96 Go¨teborg, Sweden. 0196-9781/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2005.12.004

peptides 27 (2006) 1490–1497

whereas the N-terminal fragment SP1–7 has antagonistic effects on SP induced behavior, and also exhibits antinociceptive effects [15,31,36,41]. SP1–7 is known to modulate pain in a more complex way, showing both hyperalgesic and antinociceptive effects depending on assay, dose, and the time span of the experiment [14,16,33]. Furthermore, SP receptor agonists are known to augment the intensity of opioid withdrawal syndrome [23], while SP1–7 counteracts this effect [29,51,52,53]. No clear mechanism for how N-terminal SP fragments exert their effects has so far been laid out. SP1–7 has one population of binding sites in brain and two in the spinal cord of the mouse [5,20]. The N-terminus of SP is essential for complete homologous desensitization of the NK-1 receptor; however, N-terminal SP-fragments themselves do not desensitize, and have no affinity for, NK-1 receptors expressed by rat kidney cells [47]. The inhibitory effects of SP1–7 on SP induced behavior can be blocked by naloxone [41,43], and it was initially thought that SP1–7 acted on m-opioid receptors. It has been showed however, that no specific antagonist for m-, d- or k-receptors inhibits the effects of SP1–7 [35] and that naloxone cannot displace SP1–7 from its binding sites [5,20]. Therefore, a separate receptor for SP1–7 has been proposed. The observed modulatory effect of SP1–7 on morphine tolerance and withdrawal [29,53] suggests an inter-regulatory link between the opioid and SPergic systems [21]. Modulation of opioid receptor conformation by SP1–7 could be a possible explanation of the effects of the heptapeptide [6,30]. N-terminal fragments have also been shown to down-regulate the SP receptor NK-1 after intrathecal administration into mice [49] and to affect the expression of the gene transcript of this receptor in rat [46]. Inflammatory processes that take place in peripheral tissues involve a number of mediators that are delivered by the circulation and liberated from resident and immigrated cells at the site of inflammation. These algesic-inducing compounds include proinflammatory cytokines, chemokines, protons, nerve growth factor, and prostaglandins [40]. Less well known is that analgesic mediators, which counteract pain, are also produced in inflamed tissues. These include anti-inflammatory cytokines and opioid peptides. With regard to the role of SP in the inflammatory process, the peptide is suggested to function as an initiator and regulator of inflammation. In the axon reflex process, vasoactive peptides such as SP and calcitonin gene-related peptide (CGRP) are exuded from primary sensory nerve termini at the site of injury leading to an increase in local tissue blood flow, capillary permeability and inflammatory cell activities. This component of inflammation is referred to as ‘‘neurogenic’’. Antidromic electrical stimulation of sensory nerves gives rise to a cutaneous inflammatory response [32] involving both SP and CGRP [17]. The vascular response to SP is rapid in onset, briefly it involves stimulation of the NK-1 receptor of endothelial cell which triggers the phosphatidyl inositol phosphate–diacylglycerol pathway with subsequent release of nitric oxide and smooth muscle relaxation [21]. SP elicits a wheal and flare response through direct actions on blood vessels, but also indirectly by stimulation of mast cells and sensory nerves [1,9,13,25]. The ability to stimulate histamine release appears to reside within the N-terminus of SP [33]. Considering the known modulatory effect of SP1–7 on pain transmission involving SP, we designed this study to examine

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whether the N-terminal SP metabolite may also affect or modulate the inflammatory response. We also examined the ability of SP1–7 to affect the possible response of substance P fragments lacking the N-terminal, e.g. SP5–11 and SP9–11. An established blister model of inflammation was used to investigate the effect and possible involvement of SP1–7 in acute, late phase acute, recurrent and chronic injury conditions. Inflammation was induced at the base of a blister on the rat hind footpad by perfusion of SP or electrical stimulation of the sciatic nerve. A chronic constrictive nerve injury (CCI) model was used to study the inflammatory response under chronic injury conditions. Furthermore, the possible location of possible sites for the SP1–7 interaction was also undertaken in these studies.

2.

Methods

2.1.

Animals

Out bred young male Sprague–Dawley rats with an average weight of 250–350 g were used. Animals were housed in groups of four on a 12 h:12 h dark:light cycle and had access to food and water ad libitum. Surgical anesthesia was induced with pentobarbitone sodium (Nembutal 65 mg/kg i.p.) and maintained by supplementary injections (15 mg/kg). Absence of the eyelid reflex was used to monitor the level of consciousness. Body temperature was maintained at 37 8C. Animals were sacrificed by anesthetic overdose at the end of the experiment. All experimental animal techniques and surgical procedures have been approved by the Royal Melbourne Hospital Research Foundation Animal Ethics Committee and adhered to IASP guidelines.

2.2.

Peptides and chemicals

Substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetNH2), substance P5–11, substance SP9–11 and substance P1–7 were purchased from Auspep Pty. Ltd., Melbourne, Australia. DSubstance P1–7 (Arg-D-Pro-Lys-Pro-Gln-Gln-D-Phe) was prepared by Dr. Gunnar Lindeberg at the Department of Organic Chemistry, Uppsala University (Uppsala, Sweden), using standard solid-phase peptide synthesis techniques with Fmoc/tbutyl protection and was purified by RP-HPLC on a Vydac C18 column (10 mm, 2.2 cm  25 cm) with an acetonitrile–water gradient in the presence of 0.1% trifluoroacetic acid (TFA), Plasma desorption mass spectrometry (PDMS) of the purified material gave two major peaks (MW 900.1): 902.4 (M + H)+ and 924.9 (M + Na)+. Naloxone hydrochloride and sodium nitroprusside were obtained from SIGMA Chemical Co., St. Louis, USA. Heparin sodium was purchased from Fisons Pty. Ltd., Sydney, Australia. Nembutal (pentobarbitone sodium) was from Rhone Merieux Australia Pty. Ltd., Qld, Australia.

2.3.

General experimental procedure and set-up

A blister was induced on the glabrous hind footpad skin of the anaesthetized rat by application of a 40 kPa vacuum pressure via a metal cup pre-heated to 40 8C. This method of blister induction is known to separate the epidermis from the underlying skin layers with minimal damage to the dermis

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and nerve endings [19]. The surface epidermis of the established blister was carefully removed. The foot was secured in a perspex perfusion chamber with inlet and outlet ports. The chamber was sealed in place with silicon grease. A peristaltic pump (Microperspex S 2232, LKB, Stockholm, Sweden) calibrated to 4 ml/h was used to perfuse solutions over the blister base. A laser-Doppler probe (PF303, Perimed, Stockholm, Sweden) was positioned vertically above the blister base. Relative blood flow was measured over time by a laser-Doppler flowmeter (Periflux PF2B, Perimed, Bromma, Sweden) and recorded by a chart recorder (Unicorder U-2288, Pantos, Nippon Denshi Kagaku, Japan). The inflammatory vasodilator response was measured as the area under the response curve using a digital planimeter (Planix, Tamaya, Tokyo, Japan).

2.4.

Acute and late acute injury model

The inflammatory response was studied in newly established blisters; perfusion began no later than 20 min after blister induction was completed. This was to ensure that the endogenous inflammatory response triggered by blister formation itself was minimal. In studies of late phase acute injury, a blister was induced, and the animal was kept under light anesthesia (Nembutal, 15 mg/kg i.p. at 2 and 4 h postblister induction) for 5 h. This model allowed us to study the inflammatory response in presence of inflammatory cells and endogenous mediators. Surgical anesthesia (Nembutal, 65 mg/ kg i.p.) was re-induced before the experiment proceeded.

2.5.

perfusion of sodium nitroprusside (100 mM in Ringers solution). Sodium nitroprusside is a direct smooth muscle dilator and was used to correct for inter-individual differences in smooth muscle reactivity. Perfusion of peptides began after reestablishment of basal blood flux. The inflammatory vasodilator response to SP (control) under different injury conditions was assessed by perfusion of SP at 1 mM for 30 min. The dose response of SP5–11 and SP9–11 was assessed by perfusion of SP5– 11 at 0.1, 1 and 10 mM and SP9–11 at 10, 100 and 1 mM for 30 min each. The dose response effect of SP1–7 on SP induced vasodilatation (VD) was assessed by perfusion of SP1–7 at 1, 10 and 100 mM for 30 min prior to SP administration under acute injury conditions. The effect of SP1–7 on the inflammatory VD response to SP5–11 under acute injury was assessed by perfusion of SP1–7 (10 mM) for 10 min prior to and 30 min together with SP5–11 at 1 and 10 mM. The effect of SP1–7 on the inflammatory vasodilator response to SP under different injury conditions was assessed by perfusion of SP1–7 (10 mM) for 10 min prior to and together with the SP peptides. The putative antagonist D-SP1–7 was perfused at 10 mM for 10 min, followed by concomitant perfusion with SP1–7 (10 min) and/or SP for 30 min. Naloxone was dissolved in saline and administered (1 mg/kg) via a catheter inserted into the jugular vein 10 min before peptide perfusion was commenced. Rats treated with naloxone were also given 12.5 U heparin dissolved in 1 ml of saline throughout the experiment. SP was perfused for 30 min at 1 mM in all experiments. The vascular inflammatory response was measured as the area (in cm2) under the response curve to SP or its C-terminal fragments also examined in this study.

Recurrent injury model 2.8.

A blister was induced as mentioned above, and the animals were allowed to recover for 2 weeks. The inflammatory response was studied in a new blister established at the same site.

2.6.

Chronic injury model

A chronic constrictive nerve injury (CCI) model was used to study the inflammatory response under chronic injury conditions. CCI surgery was performed as described earlier [4]. The rats were left to recover for 2 weeks. The animals were observed to ensure that peripheral neuropathy was developed. The operated rats displayed signs of neuropathic pain. This behavior included reluctance to place weight on the foot of the operated side, and it was only used for support via the heel or the medial edge. This behavioral response to CCI has previously been observed in our lab [27]. It is also known that changes in the vascular response and pain behavior coincide after CCI-surgery [2,27]. The inflammatory response was studied in a blister induced on the rat footpad (an area innervated by the injured nerve) 2 weeks post the CCI induction.

2.7.

Peptide perfusion protocols

All peptides were dissolved in Ringers solution (9.0 g NaCl, 0.2 g NaHCO3, 0.42 g KCl, 0.48 g CaCl2 in 1000 ml of H2O). A stable baseline was established by perfusion of Ringers solution for at least 20 min. This was followed by 10 min

Electrical stimulation protocol

A blister was induced and a perfusion chamber was used as previously described. The sciatic nerve was exposed in the midthigh region of the anesthetized rat by blunt dissection and cut as proximally as possible. The skin flaps of the wound were raised to form a pool, which was filled with liquid paraffin preheated to 37 8C. The distal end of the cut nerve was placed over bipolar platinum electrodes and immersed into the oil. It was ensured that the electrodes or the nerve did not make contact with any other structure. A stable baseline was obtained by perfusion of Ringers solutions for at least 20 min. Sodium nitroprusside was perfused for 10 min to test for smooth muscle reactivity as mentioned above. Basal blood flow was reestablished by perfusion of Ringers solution. Electrical stimulation was commenced immediately thereafter to assess the control response. In the experimental group, SP1–7 was perfused 10 min prior to, and continuously during the stimulation and post-stimulation periods. The nerve was stimulated with a Grass S48 stimulator (Grass instruments, MA, USA) at 20 V, 5 Hz and 2 ms square waves for 1 min. These stimulation parameters are known to activate C-fibers [31]. The vascular inflammatory response was measured as the area (in cm2) under the response curve for 20 min after stimulation.

2.9.

Statistics

The vasodilatation response (in cm2) to SP or electrical stimulation of the sciatic nerve was corrected for inter-individual

peptides 27 (2006) 1490–1497

variability according to the following formula [{average height of group sodium nitroprusside response (cm)/height of individual sodium nitroprusside response (cm)}  individual vasodilator response (cm2)]. Since sodium nitroprusside is a direct smooth muscle vasodilator, this correction effectively uses the response to sodium nitroprusside to correct for inter-individual differences in smooth muscle reactivity that is independent of sensory nerve activation. The adjusted vasodilator responses were compared between treated groups and control using the independent samples Students two-tailed t-test or one way analysis of variance (ANOVA) followed by the Student–Newman–Keuls post hoc test. Results are expressed as mean vasodilator response of group  S.E.M. p < 0.05 was considered as significant.

3.

Results

3.1. Effect of SP1–7 on the vascular inflammatory response to SP and SP-fragments Perfusion of SP (1 mM) alone for 30 min over the blister base produced an immediate vasodilator response that underwent tachyphylaxis (Fig. 1). The measurements were done under acute injury conditions. The vascular inflammatory control response to SP (area under the response curve) was 50.4  4.3 cm2. Perfusion of SP1–7 for 30 min prior to SP inhibited the vasodilator response in a dose-dependent manner (Fig. 2). SP1–7 was perfused at 1, 10 and 100 mM and significantly reduced the vascular response to 34.1  3.2, 31.2  3.5 and 17.1  5.5 cm2 respectively, corresponding to reductions of 32%, 38% and 66%. Moreover, it had an equipotent inhibitory effect when perfused for 10 min at 10 mM prior to and together with SP, the vascular response was 28.9  4.8 cm2 (57% of control). The heptapeptide did not exhibit an intrinsic effect on basal blood flow at doses of 1 or 10 mM. At 100 mM however, SP1–7 gave a vasodilator response of 46.4  9.9 cm2 by itself that appeared to undergo slow tachyphylaxis.

Fig. 1 – Digitized scan of a typical record of a vasodilatation response measured by laser-Doppler flowmetry to perfusion of SP (1 mM) over the base of a blister on the rat hind footpad. It shows an immediate increase in local blood flow following the start of perfusion. This is followed by a rapid decline (tachyphylaxis) despite continuous perfusion of SP.

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Fig. 2 – The dose–response effect of SP1–7 (1, 10 and 100 mM) on the vasodilator response to SP (1 mM). The peptides were perfused sequentially for 30 min each over the base of a blister on the hind footpad of anaesthetized rats. Relative blood flow was measured as the area under the response curve obtained by laser-Doppler flowmetry. Columns represent mean response (cm2) W S.E.M. * denotes significant difference from control (by ANOVA, n = 6, 10 and 4 at the doses 1, 10 and 100 mM, respectively).

In a parallel experiment SP5–11 (1 mM) perfused for 30 min over the blister base produced an immediate vasodilator response that underwent tachyphylaxis in similarity to that for SP (Fig. 1). The measurements were done under acute injury conditions. The vascular inflammatory control response to SP5–11 (area under the response curve) was 30.2  3.4 cm2 (n = 4). To construct a dose–response curve for SP5–11, the peptide was perfused over the blister base at 0.1, 1 and 10 mM and this resulted in a vascular response in a dosedependent manner (28.2  2.4, 30.2  3.4 and 57.8  7.4 cm2, respectively (n = 4 in all groups)). Furthermore, SP1–7 perfused at 10 mM concentration significantly inhibited the response of SP5–11, both at the 1 and 10 mM concentration of the C-terminal SP fragment, under acute injury conditions. In the presence of 10 mM SP1–7 the vascular response to SP5–11 at 1 and 10 mM was 18.7  0.9 cm2 and 27.3  1.8 cm2, representing 62% and 47% of the control response respectively (n = 4 in both groups). SP9–11 was not able to induce any vascular response (data not shown), although high concentrations were used. The concentrations used were 10, 100 and 1000 mM. To further establish the inability of SP9–11 to stimulate an inflammatory response, the perfusates were scanned for protein content using the Bradford Protein Assay and the Commassie Blue Reagent as a standard procedure for detection of protein extravasation. SP9–11 did not cause any significant change in plasma extravasation (n = 4 in all experiments), which is in contrast to SP1–7 which is known to produce histamine dependent plasma extravasation in this model [25].

3.2. Reversal of SP1–7 induced inhibition of the vascular inflammatory response to SP by putative antagonists under acute injury conditions The heptapeptide antagonist D-SP1–7 (10 mM) partially reversed the effects of SP1–7 from 57% to 81% of the control response to SP (Fig. 3). The vascular response to SP in presence of both

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3.3. Effect of SP1–7 and D-SP1–7 on the vascular inflammatory response to SP under different injury conditions The response to SP (1 mM) under late phase acute and recurrent injury conditions was elevated compared to the response under acute conditions. The response under chronic injury conditions (CCI) was suppressed. SP1–7 (10 mM) significantly inhibited the inflammatory vascular response to SP under acute and recurrent injury conditions, but not during the late phase of acute injury or under chronic injury conditions. D-SP1–7 (10 mM) was tested for its intrinsic effect on SP induced vasodilatation. The vascular response to SP under acute, late phase acute and recurrent injury conditions was unaltered by the heptapeptide antagonist. The response to SP under chronic injury conditions was significantly improved by D-SP1–7. The results are summarized in Table 1.

Fig. 3 – Reversal of SP1–7 (10 mM) induced inhibition of the vasodilator response to SP (1 mM) by D-SP1–7 (10 mM) and naloxone (1 mg/kg i.v.). The peptides were perfused over the base of a blister on the hind footpad of anaesthetized rats. D-SP1–7 was perfused for 10 min alone, followed by 10 min perfusion together with SP1–7. Both peptides were then co-perfused with SP. Naloxone was administered 10 min before peptide perfusion was commenced. Relative blood flow was measured as the area under the response curve obtained by laser-Doppler flowmetry. Columns represent mean response (cm2) W S.E.M. * denotes significant difference from control (by t-test, n = 4 in each group).

3.4. Effect of SP1–7 on the vascular inflammatory response to electrical stimulation of the sciatic nerve Electrical stimulation of the sciatic nerve produced an immediate increase in blood flow. The elevated blood flow was maintained for the full 20 min post-stimulation period. The vascular response was 30.2  1.1 cm2 (n = 4). Perfusion of SP1–7 (10 mM) for 10 min prior to, and throughout the post-stimulation period had no significant effect on the vasodilator response to electrical stimulation. The response was 32.4  8.1 cm2 (n = 4).

peptides was 40.9  6.7 cm2. Likewise, naloxone (1 mg/kg i.v.) partially reversed the inhibitory effects of SP1–7, the vascular response to SP was restored to 79% of the control response (39.6  3.8 cm2). Naloxone has previously been shown not to alter the vasodilator response to SP on its own [42]. D-SP1–7 alone had no apparent effect on the vascular response to SP. The reversing effects of D-SP1–7 and naloxone were nonadditive. The vascular response to SP in presence of SP1–7 and both antagonists was 36.2  1.9 cm2, corresponding to 72% of the control response. Statistically, the overall analysis of variance of this data set was not significant; by t-tests, groups treated with antagonists did not differ significantly from control (SP alone) or from the group treated with SP1–7.

4.

Discussion

The pharmacology of SP is complicated by its cleavage into active metabolites. It is well established that these metabolites modulate pain transmission in the CNS, and that N-terminal fragments often have opposite effects from their parent molecule [14–16,18,31,36,41,43,44,53]. The involvement of the SPergic system and sensory nerves in both neurogenic inflammation and pain transmission, in addition to the known effects of SP fragments, makes it reasonable to speculate that SP metabolites may play a role in the modulation of inflammation. We designed this study to investigate whether

Table 1 – Effects of SP1–7 and D-SP1–7 on the vasodilator response to SP under different injury conditions Condition

Treatment SP control Response (cm2)

Acute Late phase acute Recurrent Chronic

50.4  4.3, n = 13 95.6  4.1, n = 4 86.4  11.2, n = 4 17.5  1.6, n = 4

SP + D-SP1–7

SP + SP1–7 Response (cm2) *

28.9  4.8 , n = 4 102  16, n = 4 43.1  9.2*, n = 4 16.6  1.8, n = 4

% of control 57 107 50 95

Response (cm2) 39.5  5, 4 n = 4 102  22, n = 4 105  10, n = 4 51.1  8.3*, n = 5

% of control 78 107 122 292

SP1–7 or D-SP1–7 were perfused for 10 min at 10 mM followed by concomitant perfusion with SP (1 mM) for 30 min over the base of a blister on the hind footpad of anaesthetized rats. Perfusion was commenced immediately after blister induction in the acute injury model, and 5 h after blister induction in the model of late phase acute injury. The recurrent injury model involved repeated blister induction on the same site after a recovery period of 2 weeks. The model for chronic injury was obtained by surgical chronic constrictive nerve injury. The vascular response was measured as the area under the response curve obtained by laser-Doppler flowmetry. The results are expressed as the vascular response in cm2  S.E.M. and percent of SP control for the respective injury condition. * denotes significant difference from control (by t-test, n of each group is given in the table).

peptides 27 (2006) 1490–1497

the N-terminal metabolite SP1–7 has functions as a modulator of peripheral neurogenic inflammation. This heptapeptide was chosen because it is the most extensively studied Nterminal fragment. It has also been detected in comparatively high levels in CNS areas related to pain and inflammation [50]. No study to date has focused on the role of SP1–7 in different inflammatory conditions in skin. We examined the effect of SP1–7 on SP induced vasodilatation. The fragment given in the dose range 1–100 mM inhibited the response to SP when perfused prior to or prior to and together with SP in a dose-dependent manner (Fig. 2). Two perfusion protocols were used in this study. The first protocol, where SP1–7 was perfused for 30 min prior to SP, was based on a protocol used by other investigators to test the effect of SP1–7 on morphine tolerance and withdrawal symptoms [29]. The second protocol, where SP1–7 was perfused 10 min before and concomitant with SP, is the protocol commonly used in our lab to examine modulating effects on inflammation in the periphery. The potency of SP1–7 was not different between the perfusion regimens. When SP1–7 was perfused at 100 mM, it induced an increase in blood flow on its own, besides inhibiting the vascular response to SP. The present results also indicated that the SP C-terminal fragment SP5–11, but not SP9–11, mimics the effect of SP in a dose-dependent manner with regard to the inflammatory VD response. Further, this effect was found to be inhibited by the N-terminal heptapeptide fragment, SP1–7. This observation was considered to indicate that the VD promoting effect seen for SP is related to the C-terminal part of its structure. SP5–11 has been shown to exhibit equal or even increased potency relative to SP in bioassay systems [25,45,47]. However, it has further been shown that it represents the shortest amino acid sequence in the SP structure that retains full ability to desensitize the NK-1 receptor in vivo [45]. The inability of SP9–11 to induce VD despite the high concentrations used in this study correlates with the finding that the tripeptide has no affinity for the NK-1 receptor [8]. Previous studies have concluded that the behavioral effects of SP1–7 can be blocked by naloxone [41,43]. This made us examine whether this is true also in a peripheral tissue under inflammatory stress. Naloxone (1 mg/kg i.v.) reversed the inhibitory effect of SP1–7 (10 mM) on SP induced vasodilatation, but not completely (Fig. 3). It has been speculated that SP1–7 modulates opioid receptor conformation to increase the binding affinity for opioid ligands [6,30]. Endogenous opioids are not activated in our model of acute injury [26], and the fact that naloxone was effective may argue against modulation of opioid binding by SP1–7. D-SP1–7 (10 mM) was also effective in reversing the effect of SP1–7 on SP induced vasodilatation. The potency of D-SP1–7 was similar to that of naloxone, and the reversal was incomplete (Fig. 3). The reversing effects of the two antagonists were non-additive, which suggests that they may act on a common site. The consistency of the data concerning the antagonists did not allow a full statistical confirmation of the effects; however, the observed tendency urged us to examine the effect of D-SP1–7 under different injury conditions (see below). In the present work our studies of the vascular response to electrical stimulation with or without perfusion of SP1–7 was also aimed as an attempt to locate the binding site for SP1–7.

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The binding sites could either be located on the nerve, or on the vasculature itself. Sites located on neurons could mediate inhibition of peptide release in response to injury, analogous to the pre-terminal effect of opioids. We used parameters known to stimulate C-fibers and cause release of sensory neuropeptides [32]. These peptides include SP itself, but the main transmitter released is CGRP [34]. SP1–7 was not effective in inhibiting the response to electrical stimulation, but inhibited the response to exogenously perfused SP. This might suggest that SP1–7 interacts with post-terminal sites located on endothelial cells, and that the inhibition of SP induced vasodilatation by SP1–7 may be confined to actions within the microvasculature supplying the injured area. The ability of a tissue to respond to an inflammatory challenge depends on the injury condition under which the stress is imposed. Our study includes four different injury conditions, namely acute, late phase acute, recurrent and chronic, each exhibiting different responses to inflammatory stimuli (Table 1). The effects of SP1–7 under acute injury conditions have already been discussed. The involvement of endogenous inflammatory mediators and inhibitory systems are minimal in this model [24]. The late phase acute injury model allowed us to study the effect of SP1–7 in a more matured inflammatory process, probably with involvement of local tissue factors. Experiments were done 5 h after blister induction, and it has been shown that activated inflammatory cells accumulate by the blister base at this stage [11,12]. SP1–7 had no significant effects on SP induced vasodilatation in this model (Table 1). We raised the possibility that endogenous SP1–7 may already be affecting the vasculature, and wanted to test this hypothesis by perfusing D-SP1–7 together with SP; however, no significant alteration in the response was found (Table 1). It is possible that other inflammatory mediators controlling blood flow may mask the activity of SP1–7 in this model. The responsiveness to SP under recurrent injury conditions was also elevated (Table 1). It has been suggested that hypersensitivity of NK-1 receptors contribute to the increased sensitivity [28]. SP1–7 was approximately equally effective in inhibiting the vascular response to SP under recurrent and acute conditions (50% and 42% inhibition respectively). SP1–7 and SP were perfused at the same concentrations in these models, and the fact that the heptapeptide had similar potency in both cases might suggest that the effect of SP1–7 is independent of the degree of activation of NK-1 receptors. The vascular response to SP under chronic injury conditions is suppressed (Table 1) by activated inhibitory systems such as the endothelins and endogenous opioids [3]. Chronic injury also involves constant release of SP and we speculated that this would ultimately lead to high levels of SP metabolites, and possibly SP1–7. Endogenously formed SP1–7 could further limit the suppressed vascular response to SP observed in chronic injury. To test this, SP1–7 and D-SP1–7 were perfused together with SP. D-SP1–7 significantly improved the response, while SP1–7 had no effect (Table 1). These findings indicate that endogenous SP1–7, or SP1–7-like agonists are present in this model. We suggest that the diminished vascular responsiveness to SP in chronic injury may be related to the formation of inhibiting metabolites. The improved responsiveness to SP in the presence of D-SP1–7 supports the hypothesis that SP1–7 modulates opioid receptor conformation; the antagonist would obliterate the

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SP1–7-induced increase in affinity of endogenous opioids for their receptor. Our results do not elicit any further indication regarding the underlying mechanism by which SP1–7 exerts its inhibitory effect on the vascular response to SP. The possibility that the effect of SP1–7 depends on alterations in intracellular transduction pathways cannot be excluded. The results of this study indicate that SP1–7 modulates the neurogenic inflammatory process in skin under different injury conditions. SP1–7 inhibits the vascular inflammatory response to its parent molecule, SP, in a dose-dependent manner via receptor sites that are sensitive to D-SP1–7 and functionally affected by naloxone. SP1–7 exhibited equal inhibitory potency in models of acute and recurrent injury, but had no effect during the late phase of acute injury. We also conclude that SP1–7 may be involved in suppression of the vascular response to SP under chronic injury conditions. That SP1–7 did not alter the vascular response to electrical stimulation of the sciatic nerve is suggestive of a post-terminal site of action. Different N-terminal metabolites such as SP1–4 and SP1–9 have been shown to have similar properties as SP1–7 in several studies [15,22,38]. The metabolism of SP has not been investigated in rat skin, but it is likely that the inactivation involves enzymatic degradation. Such metabolism would generate fragments that may include SP1–7, or other N-terminal fragments with similar characteristics. Based on the current study, we hypothesize that these metabolites (in particular SP1– 7) play an important role in modulating the activity of the parent molecule, SP, under different injury conditions. The current study also supports the notion, that the generation of Nterminal metabolites with opposite effects from SP may have inter-regulatory importance in the peripheral SPergic system, in a similar fashion to that reported in the CNS [18]. Further characterization of the SP1–7 receptor, and the development of non-peptide ligands for the binding site may reveal the mechanism of action and perhaps even allow exploitation of the SP1–7 anti-inflammatory effects in the treatment of painful inflammatory diseases. The design and development of SP1–7 mimetics may be opportune in the search of drugs that improve healing capacity in chronic and impaired inflammatory processes.

Acknowledgements This study was supported by the Swedish Medical Research Council (Grant 9459) and by Svenska Reumatikerfo¨rbundet. We thank Dr. Maryam Bassirat and Ms. Bereha Khodr for expert technical assistance and Dr. Gunnar Lindeberg for the synthesis of D-SP1–7.

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