Basal Substance P Release Research Articles

Noradrenaline regulates substance P release from rat dorsal root ganglion neurons in vitro

To determine whether activation and/or inhibition of α-adrenoreceptors influences substance P (SP) release from dorsal root ganglion (DRG) primary sensory neurons in vitro. DRGs were dissected from 15-day embryonic Wistar rats. DRG neurons were dissociated and cultured for 2 d and then exposed to noradrenaline (NA) alone (1×10(-4) mol/L), or along with the α1-adrenoreceptor antagonist prazosin (1×10(-6) mol/L) or the α2-adrenoreceptor antagonist yohimbine (1×10(-5) mol/L) for 4 d. Then, RT-PCR was used to determine the levels of preprotachykinin (PPT) mRNA encoding for SP and Western blot to assess the protein levels of SP. Basal and capsaicin (CAP)-evoked SP release were measured by enzyme-linked immunosorbent assay (ELISA). CAP-evoked SP release was sensitized by NA and this effect was inhibited by pre-incubation with prazosin but not with yohimbine. The levels of PPT mRNA, SP peptide, and basal SP release did not change significantly in any of the experimental conditions. NA may significantly increase CAP-evoked SP release through activation of α-adrenoreceptors, which may contribute to noradrenergic pain modulation.

Effects of sangre de drago in an in vitro model of cutaneous neurogenic inflammation

Sangre de drago (SD) is a viscous bright red resin collected from Croton lechleri trees that grow in the South American jungle. This sap is used extensively in the native pharmacopoeia to treat skin disorders. Its effectiveness as an inhibitor of neurogenic inflammation has been recently demonstrated. To understand the underlying mechanisms of these effects, we examined the ability of SD to reduce substance P (SP) release in an in vitro model of cutaneous neurogenic inflammation (CNI). This model is based on an enzyme immunoassay of SP (an inducer of CNI) in a porcine co-culture of dorsal root ganglion neurons and keratinocytes. After incubation with different concentrations of SD, we noted an immediate and significant dose-dependent decrease in basal SP release, with average values of 32% at 1% SD (v/v) and 26% at 0.1% (v/v). On the other hand, pretreatment (72 or 1 h) of the co-culture with 1% SD (v/v) was sufficient to induce a 111% (72 h) or 65% (1 h) inhibition of capsaicin-induced SP release, while 0.1% SD (v/v) triggered a 109% (72 h) or 30% (1 h) inhibition. We conclude that sangre de drago is a potent inhibitor of CNI through direct inhibition of neuropeptide release by sensory afferent nerves.

Spinal N-methyl- d-aspartate receptors and nociception-evoked release of primary afferent substance P

Dorsal horn N-methyl- d-aspartate (NMDA) receptors contribute significantly to spinal nociceptive processing through an effect postsynaptic to non-primary glutamatergic axons, and perhaps presynaptic to the primary afferent terminals. The present study sought to examine the regulatory effects of NMDA receptors on primary afferent release of substance P (SP), as measured by neurokinin 1 receptor (NK1r) internalization in the spinal dorsal horn of rats. The effects of intrathecal NMDA alone or in combination with d-serine (a glycine site agonist) were initially examined on basal levels of NK1r internalization. NMDA alone or when co-administered with d-serine failed to induce NK1r internalization, whereas activation of spinal TRPV1 receptors by capsaicin resulted in a notable NK1r internalization. To determine whether NMDA receptor activation could potentiate NK1r internalization or pain behavior induced by a peripheral noxious stimulus, intrathecal NMDA was given prior to an intraplantar injection of formalin. NMDA did not alter the formalin-induced NK1r internalization nor did it enhance the formalin paw flinching behavior. To further characterize the effects of presynaptic NMDA receptors, the NMDA antagonists dl-2-amino-5-phosphonopentanoic acid (AP-5) and MK-801 were intrathecally administered to assess their regulatory effects on formalin-induced NK1r internalization and pain behavior. AP-5 had no effect on formalin-induced NK1r internalization, whereas MK-801 produced only a modest reduction. Both antagonists, however, reduced the formalin paw flinching behavior. In subsequent in vitro experiments, perfusion of NMDA in spinal cord slice preparations did not evoke basal release of SP or calcitonin gene-related peptide (CGRP). Likewise, perfusion of NMDA did not enhance capsaicin-evoked release of the two peptides. These results suggest that presynaptic NMDA receptors in the spinal cord play little if any role on the primary afferent release of SP.

Left vagal stimulation induces dynorphin release and suppresses substance P release from the rat thoracic spinal cord during cardiac ischemia.

Electrostimulatory forms of therapy can reduce angina that arises from activation of cardiac nociceptive afferent fibers during transient ischemia. This study sought to determine the effects of electrical stimulation of left thoracic vagal afferents (C(8)-T(1) level) on the release of putative nociceptive [substance P (SP)] and analgesic [dynorphin (Dyn)] peptides in the dorsal horn at the T(4) spinal level during coronary artery occlusion in urethane-anesthetized Sprague-Dawley rats. Release of Dyn and SP was measured by using antibody-coated microprobes. While Dyn and SP had a basal release, occlusion of the left anterior descending coronary artery only affected SP release, causing an increase from lamina I-VII. Left vagal stimulation increased Dyn release, inhibited basal SP release, and blunted the coronary artery occlusion-induced release of SP. Dyn release reflected activation of descending pathways in the thoracic spinal cord, because vagal afferent stimulation still increased the release of Dyn after bilateral dorsal rhizotomy of T(2)-T(5). These results indicate that electrostimulatory therapy, using vagal afferent excitation, may induce analgesia, in part, via inhibition of the release of SP in the spinal cord, possibly through a Dyn-mediated neuronal interaction.

Basal and activity-induced release of substance P from primary afferent fibres in NK 1 receptor knockout mice: evidence for negative feedback

The concept that NK 1 receptors are located pre-junctionally on substance P (SP)-containing nerves, acting as autoreceptors to inhibit SP release, has been suggested, but remains a controversial issue. To further investigate the existence of this receptor on central and peripheral terminals of primary afferent fibres, NK 1 receptor knockout mice and an NK 1 receptor antagonist were used in nerve-attached tissue preparations. These were the isolated dorsal horn of the spinal cord with dorsal roots attached, and the hairy skin of the hind paw with attached saphenous nerve. The results reveal that in the dorsal horn preparation, basal release of SP is significantly higher in NK 1 −/− mice than NK 1 +/+ mice ( P<0.05, n=7 mice/strain). However, a difference in SP release evoked in the dorsal horn by electrical stimulation of the dorsal roots or capsaicin application was not observed. In contrast, antidromic electrical stimulation of the saphenous nerve caused a substantially greater release of SP in the skin of NK 1 −/− mice than in NK 1 +/+ mice ( P<0.05, n=5 to 6 mice/strain). These results provide evidence for the existence of NK 1 autoreceptors on sensory nerves in skin, which may be relevant to the modulation of their peripheral pathophysiological effector functions.

Influence of painful chronic neuropathy on neurogenic inflammation

The effect of topical application of capsaicin cream on neurogenic inflammation was investigated in a neuropathic pain model in rat. The neuropathic state was induced by loose ligation of the sciatic nerve with a chromic gut suture. A marked thermal hyperalgesia was observed in response to heat stimulation applied to the operated side from 3 days through 2 weeks, followed by a gradual return to the control level 35 days after surgery. In sham-operated animals, topical application of capsaicin cream to both sides of the hind paw, the instep and sole, as well as antidromic stimulation of the sciatic nerve led to a significant increase in the amounts of Evans blue and substance P (SP) released into the perfusates. This stimulus-induced extravasation was significantly suppressed by pretreatment with RP67580, an NK 1 antagonist. On day 7 after ligation, capsaicin- and antidromic stimulation-induced extravasation were significantly reduced. At this time, both amount of SP released immediately after application of capsaicin and during antidromic stimulation were almost similar to that in sham-operated rats, whereas the basal amount of SP release significantly increased in ligated animals. In particular a major release of SP was detected immediately after the start of the perfusion compared with that in sham-operated rats. Plasma extravasation evoked by SP (10 −4 M) applied to the subcutaneous perfusate was significantly less in ligated than in sham-operated rats. These results suggest that nerve injury with chronic pain may produce increase in basal SP release into the peripheral tissues, and then such enhanced SP release cause reduction of SP-induced extravasation.

Release of substance P by low oxygen in the rabbit carotid body: evidence for the involvement of calcium channels

Carotid bodies from diverse species contain substance P (SP), an 11-residue peptide that belongs to the tachykinin peptide family. Previous studies indicated that SP is excitatory to the carotid body and is associated with sensory response to hypoxia. However, release of SP from the carotid body during hypoxia has not been documented. In the present study, we determined whether hypoxia releases SP from the carotid body and further characterized the mechanism(s) associated with SP release by low oxygen. The release of SP from superfused rabbit carotid body was determined by an enzyme immunoassay (EIA). SP-like immunoreactivity was localized to many glomus cells and nerve fibers and the concentration of SP in the rabbit carotid body was 1.5±0.1 ng/mg protein. For release studies, carotid bodies ( n=56) were superfused with a modified Tyrode medium containing Hepes buffer, pH 7.4, saturated with either room air (normoxia) or hypoxic gas mixtures. The basal release of SP during normoxia was 51.0±1.5 fmol/min per mg protein. Hypoxia increased SP release from the carotid body and the magnitude of release is dependent on the severity of hypoxic stimulus. Moderate hypoxia (pO 2, 79±4 mmHg) stimulated SP release by ∼50%, whereas SP release during severe hypoxia (pO 2, 11±6 mmHg) was 2-fold higher than the normoxic control. A similar pattern of SP release was also observed when superfusion medium containing CO 2–HCO 3 buffer, pH 7.4, was used for release studies. To examine the mechanism(s) associated with hypoxia-induced SP release from the carotid body, moderate level of hypoxia (12% O 2+N 2) was used. Omission of calcium in the superfusion medium markedly attenuated hypoxia-induced SP release (>95%), whereas the basal release of SP was unaffected. Cd 2+ (100 μM), a voltage-dependent Ca 2+-channel blocker, abolished hypoxia-induced SP release. About 85% of SP release by hypoxia was inhibited by ω-conotoxin GVIA (1 μM), an N-type Ca 2+ channel blocker, whereas nitrendipine (1.5 μM), an inhibitor of L-type Ca 2+ channel partially attenuated (∼65%) hypoxia-induced SP release. By contrast, ω-agatoxin TK (50 nM), a P/Q-type Ca 2+-channel inhibitor, had no significant effect ( P>0.05, n=6). These results suggest that SP is released from the rabbit carotid body by hypoxia that depends on the severity of the hypoxic stimulus. Further, SP release by hypoxia is a calcium-dependent process and is primarily mediated by N- and L-type Ca 2+ channels.

Effect of brain-derived neurotrophic factor on the release of substance P from rat spinal cord.

Brain-derived neurotrophic factor (BDNF) can produce hyperalgesia in the adult rat. Here we assessed whether changes in the spinal release of the nociceptive peptide substance P (SP) contributes to this effect. Antibody-coated microprobes revealed a significant basal release of SP in the dorsal horn in vivo that was increased following acute knee inflammation. Microinjection of BDNF into the grey matter (0.5 microl, 10(-5) M) altered SP release neither in rats with normal knees nor in rats with inflamed knee joints. In the lumbar dorsal horn slice preparation in vitro, superfusion with BDNF (100 ng/ml) could reduce SP release evoked by electrical dorsal root stimulation without modyfing SP basal outflow. It is unlikely, therefore, that enhanced spinal SP release mediates the hyperalgesic effect of BDNF.

NK 3 receptor blockade prevents hyperalgesia and the associated spinal cord substance P release in monoarthritic rats

Previous studies in vitro have shown that NK 3 receptors exist on primary afferent terminals in rat spinal cord and mediate potentiation of the depolarisation-evoked substance P (SP) release. In the present study we have investigated the role of the NK 3 receptor-mediated SP release system in a model of inflammatory pain. Monoarthritis was induced in rats by unilateral injection of complete Freund's adjuvant (CFA); withdrawal latencies to a thermal stimulus were subsequently measured at various times following CFA. The CFA-treated paw displayed hyperalgesia as early as 4 h after CFA injection and hyperalgesia was maintained until day 4 but had disappeared by day 21. The thermal hyperalgesia was associated with an increase in basal SP release from spinal cord synaptosomes. The possible involvement of endogenous neurokinin B acting at NK 3 receptors was tested by using SB 223412-A [(S)-(−)- N-(α-ethylbenzyl)-3-hydroxy-2-phenylquinoline-4-carboxamide hydrochloride], a novel, potent ( K i=30 nM) and selective ( K i>10 000 nM for NK 1 and NK 2 receptors), non-peptidic NK 3 receptor antagonist. In vitro SB 223412-A antagonised the potentiation of SP release produced by senktide in spinal cord synaptosomes. Administered systemically to monoarthritic rats (50 mg/kg, p.o., b.i.d., for 4 days), the NK 3 receptor antagonist SB 223412-A significantly reduced thermal hyperalgesia and normalised the basal release of SP from spinal cord synaptosomes. The data suggest that neurokinin B acting at NK 3 receptors that mediate SP release within the spinal cord play a role in inflammation. These NK 3 receptors may represent, therefore, appropriate targets in the therapy of inflammatory pain.

Activated eosinophils elicit substance P release from cultured dorsal root ganglion neurons.

This study was performed to test the hypothesis that activated eosinophils or their secretory products can directly stimulate sensory neurons to release their neuropeptides. Neurons derived from neonatal rat dorsal root ganglia (DRG), which synthesize and store sensory neuropeptides, were placed in primary cell culture and were exposed to eosinophils or their bioactive mediators. The resultant release of substance P (SP) was measured by enzyme-linked immunosorbent assay and was expressed as a percent (mean +/- SE) of total neuronal SP content. Eosinophils were isolated from human volunteers with a history of allergic rhinitis and/or mild asthma and were activated by incubation with cytochalasin B (5 micrograms/ml) and N-formyl-methionyl-leucyl-phenylalanine (FMLP, 1 microM). Activated eosinophils [6 x 10(6)/ml, suspended in Hanks' buffered salt solution (HBSS)] applied to cultured DRG neurons for 30 min increased basal SP release 2.4-fold compared with HBSS-exposed neurons (activated eosinophils 11.10 +/- 2.48% vs. HBSS 4.59 +/- 0.99%; P = 0.002), whereas neither nonactivated eosinophils nor cytochalasin B and FMLP in HBSS influenced SP release. Additional cultured DRG neurons were exposed to soluble products made by eosinophils. Compared with SP release under control conditions (2.37 +/- 0.34%), major basic protein (MBP) increased release in a concentration-related fashion (e.g., 3 microM MBP: 6.23 +/- 0.67%, P = 0.006 vs. control), whereas neither eosinophil cationic protein (3 microM), eosinophil-derived neurotoxin (3 microM), leukotriene D4 (500 nM), platelet-activating factor (100 nM), nor H2O2 (100 microM) affected SP release. These studies demonstrate that activated eosinophils can stimulate cultured DRG neurons directly and suggest that MBP may be the responsible mediator.

Subchronic haloperidol treatment decreases the in vivo release of tachykinins in rat substantia nigra

Microdialysis combined with sensitive radioimmunoassays was used to measure the in vivo release of substance P (SP) and neurokinin A (NKA) in the rat substantia nigra. The effect of acute and subchronic haloperidol treatment (0.5 mg/kg) on the basal and potassium-evoked release was studied. No significant effect was observed after a single injection. However, pretreatment with haloperidol for 10 days decreased the potassium-induced release of SP and NKA by 25 and 27%, respectively. The basal overflow of NKA was reduced by 29%, while no significant effect could be seen on the basal release of SP.

Rapid release of substance P and LH-RH from synaptosomes prepared from the medial basal hypothalamus and substantia nigra

We investigated Ca2+-dependent, depolarization-induced release of substance P (SP) and LH-RH from medial basal hypothalamic (MBH) and substantia nigra (SN) synaptosomes prepared from male rat brain. Depolarization of MBH synaptosomes evoked significant release of SP from 10.0 +/- 0.1 (5 mM K+) to 28.0 +/- 2.4 (75 mM K+) pg released/10 seconds. Fractional release was 1.0% and 2.7% respectively. In contrast, LH-RH was not released by depolarization of MBH synaptosomes: 11.6 +/- 0.9 (5 mM K+) to 11.0 +/- 0.7 (75 mM K+) pg released/10 seconds. Fractional release was 1.1 and 1.0% respectively. Depolarization-induced LH-RH release also did not occur in the presence of 10(-4) or 10(-6) M norepinephrine, 10(-7) M 12-O-tetradecanoylphorbol-13-acetate (TPA, PMA), 10(-5) M forskolin or in female rats. The inability of depolarizing concentrations of K+ to stimulate LH-RH release in physiological buffers remains an enigma. Significant depolarization-induced SP release was seen from MBH and SN synaptosomes at 20, 15, 10, 5 and only 1 second of release. Despite comparable basal release of SP from MBH and SN synaptosomes, the rate and magnitude of evoked release were much more pronounced in SN synaptosomes. The initial rate (0-1 second) of SP release was 4.5-fold greater from SN than from MBH synaptosomes [krel = 0.027(-1) (SN), krel = 0.006(-1) (MBH)]. The magnitude of SP release from SN synaptosomes was 2- to 3-fold greater at any given time interval compared with release from MBH synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Laminar localization of the sites of release of immunoreactive substance P in the dorsal horn with antibody-coated microelectrodes

The localization of the sites of release of immunoreactive substance P (SP) in the spinal cord after peripheral nerve stimulation has been examined in the anaesthetized cat. A new technique using antibodies bound to the outside of glass microelectrodes has allowed the identification of these sites with a spatial precision previously unobtainable. A basal release of SP was detected and this was not increased by electrical stimulation of large myelinated primary afferent fibres. Excitation of unmyelinated primary afferents resulted in the release of high concentrations of SP confined to the region of the substantia gelatinosa and lamina V–VI.