Release Of Substance P Research Articles

Substance P spinal signaling induces glial activation and nociceptive sensitization after fracture

Tibia fracture in rodents induces substance P (SP)-dependent keratinocyte activation and inflammatory changes in the hindlimb, similar to those seen in complex regional pain syndrome (CRPS). In animal pain models spinal glial cell activation results in nociceptive sensitization. This study tested the hypothesis that limb fracture triggers afferent C-fiber SP release in the dorsal horn, resulting in chronic glial activation and central sensitization. At 4weeks after tibia fracture and casting in rats, the cast was removed and hind paw allodynia, unweighting, warmth, and edema were measured, then the antinociceptive effects of microglia (minocycline) or astrocyte (L-2-aminoadipic acid (LAA)) inhibitors or an SP receptor antagonist (LY303870) were tested. Immunohistochemistry and PCR were used to evaluate microglial and astrocyte activation in the dorsal horn. Similar experiments were performed in intact rats after brief sciatic nerve electric stimulation at C-fiber intensity. Microglia and astrocytes were chronically activated at 4weeks after fracture and contributed to the maintenance of hind paw allodynia and unweighting. Furthermore, LY303870 treatment initiated at 4weeks after fracture partially reversed both spinal glial activation and nociceptive sensitization. Similarly, persistent spinal microglial activation and hind paw nociceptive sensitization were observed at 48h after sciatic nerve C-fiber stimulation and this effect was inhibited by treatment with minocycline, LAA, or LY303870. These data support the hypothesis that C-fiber afferent SP signaling chronically supports spinal neuroglial activation after limb fracture and that glial activation contributes to the maintenance of central nociceptive sensitization in CRPS. Treatments inhibiting glial activation and spinal inflammation may be therapeutic for CRPS.

Abstract P230: Glucagon-like Peptide-1-induced Increases in Afferent Renal Nerve Activity and Renal Sodium Excretion: Role of Trpv1

Glucagon-like peptide 1 (GLP-1), an incretin hormone that has clinically been used to treat type 2 diabetic patients, may cause diuresis and natriuresis. However, the mechanism of GLP-1 effects on the kidney is largely unknown. We test the hypothesis that GLP-1 increases afferent renal nerve activity (ARNA) and renal sodium excretion by activation of the transient receptor potential vanilloid 1 (TRPV1) channels expressed in afferent renal nerves innervating the kidney. Exendin-4 (Ex4, 3*10-7M), a GLP-1 receptor agonist perfused into the left renal pelvis, increased ipsilateral ARNA in wild type (WT) mice when compared to TRPV1-null mutant (TRPV1–/–) mice (ARNA % integrated activity, WT:160±22 vs TRPV1-/-: 109±5, p<0.05). Ex4-induced increases in ARNA in WT mice were abolished by capsazepine, a selective antagonist of TRPV1, or by RP67580, a selective antagonist of the neurokinin 1 (NK1) receptor, but not by calcitonin-gene related peptide (CGRP)8-37, a selective antagonist of the CGRP receptor pre-perfused into the renal pelvis. Ex4 increased substance P (SP) and CGRP release from the renal pelvis isolated from WT but not TRPV1-/- mice. Ex4-induced increases in SP and CGRP in WT mice were prevented by Ex9-39, a GLP-1 receptor antagonist, 2,5-dideoxyadenosine, an adenylate cyclase inhibitor (ACI), and brefeldin A (BFA), an EPAC inhibitor, or attenuated by bisindolylmaleimide I (BIM) , a PKC inhibitor, and H89, a PKA inhibitor. Wortmannin (Wort), a PI3K inhibitor, had no effect on Ex4-induced SP or CGRP release. Acute Ex4 treatment (3ug/kg, i.p.) increased renal sodium excretion in both strains with greater degree of increases in WT compared to TRPV1-/- mice (increased % rate, WT: 160±36 vs. TRPV1-/-: 82±7, p<0.05). Thus, our data show that Ex4 increases ARNA, renal SP and CGRP release, and urinary sodium excretion in WT mice, and these functions of Ex4 are impaired in TRPV1-/- mice. These data indicate that Ex4-induced enhancement of ARNA and natriuresis attributes to, at least in part, activation of TRPV1-positive afferent nerves possibly via stimulating the GLP-1R/cAMP-PKA/PKC-TRPV1/SP pathway.

Inflammation-induced abnormalities in the subcellular localization and trafficking of the neurokinin 1 receptor in the enteric nervous system.

Activated G protein-coupled receptors traffic to endosomes and are sorted to recycling or degradative pathways. Endosomes are also a site of receptor signaling of sustained and pathophysiologically important processes, including inflammation. However, the mechanisms of endosomal sorting of receptors and the impact of disease on trafficking have not been fully defined. We examined the effects of inflammation on the subcellular distribution and trafficking of the substance P (SP) neurokinin 1 receptor (NK1R) in enteric neurons. We studied NK1R trafficking in enteric neurons of the mouse colon using immunofluorescence and confocal microscopy. The impact of inflammation was studied in IL10(-/-)-piroxicam and trinitrobenzenesulfonic acid colitis models. NK1R was localized to the plasma membrane of myenteric and submucosal neurons of the uninflamed colon. SP evoked NK1R endocytosis and recycling. Deletion of β-arrestin2, which associates with the activated NK1R, accelerated recycling. Inhibition of endothelin-converting enzyme-1 (ECE-1), which degrades endosomal SP, prevented recycling. Inflammation was associated with NK1R endocytosis in myenteric but not submucosal neurons. Whereas the NK1R in uninflamed neurons recycled within 60 min, NK1R recycling in inflamed neurons was delayed for >120 min, suggesting defective recycling machinery. Inflammation was associated with β-arrestin2 upregulation and ECE-1 downregulation, which may contribute to the defective NK1R recycling. We conclude that inflammation evokes redistribution of NK1R from the plasma membrane to endosomes of myenteric neurons through enhanced SP release and defective NK1R recycling. Defective recycling may be secondary to upregulation of β-arrestin2 and downregulation of ECE-1. Internalized NK1R may generate sustained proinflammatory signals that disrupt normal neuronal functions.

H2O2 generated by NADPH oxidase 4 contributes to transient receptor potential vanilloid 1 channel-mediated mechanosensation in the rat kidney.

The presence of NADPH oxidase (Nox) in the kidney, especially Nox4, results in H2O2 production, which regulates Na(+) excretion and urine formation. Redox-sensitive transient receptor potential vanilloid 1 channels (TRPV1s) are distributed in mechanosensory fibers of the renal pelvis and monitor changes in intrapelvic pressure (IPP) during urine formation. The present study tested whether H2O2 derived from Nox4 affects TRPV1 function in renal sensory responses. Perfusion of H2O2 into the renal pelvis dose dependently increased afferent renal nerve activity and substance P (SP) release. These responses were attenuated by cotreatment with catalase or TRPV1 blockers. In single unit recordings, H2O2 activated afferent renal nerve activity in response to rising IPP but not high salt. Western blots revealed that Nox2 (gp91(phox)) and Nox4 are both present in the rat kidney, but Nox4 is abundant in the renal pelvis and originates from dorsal root ganglia. This distribution was associated with expression of the Nox4 regulators p22(phox) and polymerase δ-interacting protein 2. Coimmunoprecipitation experiments showed that IPP increases polymerase δ-interacting protein 2 association with Nox4 or p22(phox) in the renal pelvis. Interestingly, immunofluorescence labeling demonstrated that Nox4 colocalizes with TRPV1 in sensory fibers of the renal pelvis, indicating that H2O2 generated from Nox4 may affect TRPV1 activity. Stepwise increases in IPP and saline loading resulted in H2O2 and SP release, sensory activation, diuresis, and natriuresis. These effects, however, were remarkably attenuated by Nox inhibition. Overall, these results suggest that Nox4-positive fibers liberate H2O2 after mechanostimulation, thereby contributing to a renal sensory nerve-mediated diuretic/natriuretic response.

A role for neuropeptides in innate immune inflammation of the nose

Background The airway epithelium constitutes the first line of defense in the protection against invading pathogens. It acts as a barrier, but it is also is a major source of early released inflammatory mediators, which help shape the inflammatory response. Neuropeptides, such as substance P (SP), have long been considered to be early contributors to the inflammatory response, causing pain hypersensitivity and vasodilation, as well as activation and infiltration of various immune cells. Toll-like receptor 7 (TLR7) is found on the epithelial cells and is known to be activated by viruses. The present study has investigated the relationship between TLR7 activation/expression and SP release/ stimulation.

Inflammation in acute CNS injury: a focus on the role of substance P

Recently, a number of reports have shown that neurogenic inflammation may play a role in the secondary injury response following acute injury to the CNS, including traumatic brain injury (TBI) and stroke. In particular substance P (SP) release appears to be critically involved. Specifically, the expression of the neuropeptide SP is increased in acute CNS injury, with the magnitude of SP release being related to both the frequency and magnitude of the insult. SP release is associated with an increase in blood-brain barrier permeability and the development of vasogenic oedema as well as neuronal injury and worse functional outcome. Moreover, inhibiting the actions of SP through use of a NK1 receptor antagonist is highly beneficial in both focal and diffuse models of TBI, as well as in ischaemic stroke, with a therapeutic window of up to 12 h. We propose that NK1 receptor antagonists represent a novel therapeutic option for treatment of neurogenic inflammation following acute CNS injury.

Ozone Exposure Initiates a Sequential Signaling Cascade in Airways Involving Interleukin-1Beta Release, Nerve Growth Factor Secretion, and Substance P Upregulation

Previous studies demonstrated that interleukin-1β (IL-1β) and nerve growth factor (NGF) increase synthesis of substance P (SP) in airway neurons both after ozone (O3) exposure and by direct application. It was postulated that NGF mediates O3-induced IL-1β effects on SP. The current study specifically focused on the influence of O3 on IL-1β, NGF, and SP levels in mice bronchoalveolar lavage fluid (BALF) and whether these mediators may be linked in an inflammatory-neuronal cascade in vivo. The findings showed that in vivo O3 exposure induced an increase of all three proteins in mouse BALF and that O3-induced elevations in both NGF and SP are mediated by the inflammatory cytokine IL-1β. Further, inhibition of NGF reduced O3 induced increases of SP in both the lung BALF and lung tissue, demonstrating NGF serves as a mediator of IL-1β effects on SP. These data indicate that IL-1β is an early mediator of O3-induced rise in NGF and subsequent SP release in mice in vivo.

RETRACTED: Antinociceptive effects of endomorphin-2: Suppression of substance P release in the inflammatory pain model rat

Endomorphin-2 (EM2) and Substance P (SP) exert suppressive and facilitative influences upon nociception, respectively. Although EM2 and SP were often co-expressed in single neurons in dorsal root ganglion (DRG), it is still unknown if and how the nociception-suppressive influences of EM2 might be exerted upon nociception-facilitative effects of SP in the DRG neurons. We examined these issues in the inflammatory pain model rats produced by subcutaneous injection of the complete Freund's adjuvant into the hind paw. The paw withdrawal threshold for mechanical allodynia was measured. Changes of EM2 and SP release were estimated by measuring intrathecal levels of EM2 and SP through in vivo microdialysis analysis of cerebrospinal fluid. The mechanical allodynia was dose-dependently attenuated by intrathecal injection of EM2 or a neurokinin-1 receptor antagonist, and facilitated by intrathecal injection of SP or a mu-opioid receptor (MOR) antagonist. Importantly, intrathecal level of SP was found to be lowered by intrathecal injection of EM2. Morphologically, colocalization of EM2-, MOR- and SP-immunoreactivity in single DRG neurons was observed by immunofluorescent histochemistry, and co-expression of EM2 and SP in large, dense-cored presynaptic vesicles in primary afferents, as well as localization of MOR on pre- and postsynaptic membrane in spinal dorsal horn, was also confirmed electron miscroscopically. Thus, the results indicated that analgesic influences of EM2 upon inflammatory pain might be exerted through suppression of SP release, supporting the assumptions that binding of EM2 to presynaptic MOR might induce such effects.

A dual delivery of substance P and bone morphogenetic protein-2 for mesenchymal stem cell recruitment and bone regeneration.

Implantation of ex vivo expanded and osteogenically differentiated mesenchymal stem cells (MSCs) for bone regeneration has drawbacks for clinical applications, such as poor survival of implanted cells and increased treatment expenses. As a new approach for bone regeneration that can circumvent these limitations, we propose dual delivery of substance P (SP) and bone morphogenetic protein-2 (BMP-2) to facilitate endogenous stem cell recruitment to bone defects by SP and subsequent in situ osteogenic differentiation of those cells by BMP-2. A heparin-conjugated fibrin (HCF) gel enabled dual delivery with fast release of SP and slow release of BMP-2, which would be ideal for prompt recruitment of endogenous stem cells in the first stage and time-consuming osteogenic differentiation of the recruited stem cells in the second stage. The HCF gels with SP and/or BMP-2 were implanted into mouse calvarial defects for 8 weeks. Local delivery of SP to the calvarial defects using HCF gel was more effective in recruiting MSCs to the calvarial defects than intraperitoneal or intravenous administration of SP. Many of the cells recruited by SP underwent osteogenic differentiation through local delivery of BMP-2. The efficacy of in vivo bone regeneration was significantly higher in the SP/BMP-2 dual delivery group. The dual delivery of SP and BMP-2 using the HCF gel therefore has potential as an effective bone regeneration strategy.

Abstract 18521: Sensory Neuropathy Jeopardizes Nociceptive-mediated Liberation and Homing of Stem Cells in Mice and Patients With Diabetes

Introduction: Diabetic sensory neuropathy (SN) causes either unbearable pain or lack of ischemic pain perception. It remains unknown if SN affects bone marrow (BM) with consequence for stem cell (SCs) liberation upon tissue injury. We reported that ischemia induces the liberation of Lineage - Sca-1 + c-kit + (LSK)SCs co-expressing the SP receptor NK1 (LSK-NK1) via a neuronal circuitry projecting to the BM and a humoral mechanism consisting of the generation of a chemoattractant gradient of Substance P (SP) between the circulation and BM. We hypothesize that SN might contribute to faulty SC liberation in diabetes. Results: Nociceptive fibers coexpressing SP and NGF are reduced in BM of type 1 (T1D, streptozotocin-induced) and type 2 diabetic (T2D, Leptin-receptor knockout) mice compared with controls. BM SN was prevented by systemic AAV-mediated NGF gene therapy. Upon induction of limb ischemia, a gradient of SP is created between skeletal muscle>circulation>BM in non-diabetic mice, which associates with LSK-NK1 cell liberation and homing to the ischemic limb. These phenomena are abrogated in T1D mice. Furthermore, in vitro SP-induced migration of BM LSK-NK1 cells is reduced in T1D mice ( P <0.05 vs. ND mice). For clinical translation, we studied the association of SN and SC liberation in T2D with or without vascular complications. Sensory fibers were found reduced in BM of T2D patients. In addition, critical limb ischemia fails to create an SP gradient and induce CD34+NK1+ SC release. Pain is an acknowledged side effect of BM stimulation by granulokines. We found that BM stimulation with G-CSF fails to induce CD34+NK1+ SC liberation in T2D patients, with this phenomenon being directly correlated to pain score and increase in SP levels. Conclusions: We are the first to show that diabetes is associated with NGF-sensitive BM SN in animal models and humans. Two nociceptive mechanisms fail following ischemia or granulokine stimulation: the ability of nerve terminals to coordinate SP release between periphery and BM and responsiveness of NK1 SCs to SP-induced chemoattraction. These findings open new avenues to rescue diabetic SC mobilopathy and thereby encourage vascular repair.

The selective PAC1 receptor agonist maxadilan inhibits neurogenic vasodilation and edema formation in the mouse skin

We have earlier shown that PACAP-38 decreases neurogenic inflammation. However, there were no data on its receptorial mechanism and the involvement of its PAC1 and VPAC1/2 receptors (PAC1R, VPAC1/2R) in this inhibitory effect.Neurogenic inflammation in the mouse ear was induced by topical application of the Transient Receptor Potential Ankyrin 1 (TRPA1) receptor activator mustard oil (MO). Consequent neurogenic edema, vasodilation and plasma leakage were assessed by measuring ear thickness with engineer's micrometer, detecting tissue perfusion by laser Doppler scanning and Evans blue or indocyanine green extravasation by intravital videomicroscopy or fluorescence imaging, respectively. Myeloperoxidase activity, an indicator of neutrophil infiltration, was measured from the ear homogenates with spectrophotometry. The selective PAC1R agonist maxadilan, the VPAC1/2R agonist vasoactive intestinal polypeptide (VIP) or the vehicle were administered i.p. 15 min before MO. Substance P (SP) concentration of the ear was assessed by radioimmunoassay.Maxadilan significantly diminished MO-induced neurogenic edema, increase of vascular permeability and vasodilation. These inhibitory effects of maxadilan may be partially due to the decreased substance P (SP) levels. In contrast, inhibitory effect of VIP on ear swelling was moderate, without any effect on MO-induced plasma leakage or SP release, however, activation of VPAC1/2R inhibited the increased microcirculation caused by the early arteriolar vasodilation. Neither the PAC1R, nor the VPAC1/2R agonist influenced the MO-evoked increase in tissue myeloperoxidase activity.These results clearly show that PAC1R activation inhibits acute neurogenic arterial vasodilation and plasma protein leakage from the venules, while VPAC1/2R stimulation is only involved in the attenuation of vasodilation.

Involvement of substance P in the development of cisplatin‐induced acute and delayed pica in rats

Although substance P (SP) and neurokinin NK1 receptors have been reported to be involved in cisplatin-induced acute and delayed emesis, their precise roles remain unclear. Pica, the consumption of non-nutrient materials such as kaolin in rats, can be used as a model of nausea in humans. We investigated the time-dependent changes in cisplatin-induced pica and the involvement of SP and NK1 receptors in this behaviour. Rats were administered cisplatin with or without a daily injection of a 5-HT3 receptor antagonist (granisetron) or an NK1 receptor antagonist (aprepitant), and kaolin intake was then monitored for 5 days. The effects of granisetron on the cisplatin-induced expression of preprotachykinin-A (PPT-A) mRNA, which encodes mainly for SP, and on SP release in the medulla, measured by in vivo brain microdialysis, were also investigated. Cisplatin induced pica within 8 h of its administration that continued for 5 days. Granisetron inhibited the acute phase (day 1), but not the delayed phase (days 2-5), of pica, whereas aprepitant abolished both phases. Within 24 h of the injection of cisplatin, PPT-A mRNA expression and SP release in the medulla were significantly increased; these findings lasted during the observation period and were inhibited by granisetron for up to 24 h. The profiles of cisplatin-induced pica in rats are similar to clinical findings for cisplatin-induced emesis in humans, and we showed that SP production in the medulla and activation of NK1 receptors are involved in this cisplatin-induced pica.

Signaling via neurokinin 1 and 2 receptors exerts opposing effects on mast cell pro-inflammatory and Th2-biasing functions. (HYP3P.347)

Abstract Following activation via FcεRI, peripheral tissue mast cells (MCs) initiate and sustain atopic diseases, by secreting proinflammatory and Th2 biasing cytokines. MC express neuropeptide receptors and are exposed to neuroinflammatory mediators. However, the role of neuropeptides in FcεRI-activated MCs remains mainly unknown. Two of the most potent pro-inflammatory neuropeptides, substance-P (SP) and hemokinin-1 (HK-1), bind with high affinity to the neurokinin 1 receptor (NK1R) and with lower affinity to the NK2R. We addressed in vitro and in vivo the role of the NK1R and NK2R in MC responses initiated via FcεRI. In vitro, FcεRI activation of bone marrow derived (BM)MCs from wild type (WT) mice increased NK1R and NK2R expression and HK-1 release, but not release of SP. Utilizing NK1R-/- BMMCs, we determined that the NK1R was necessary for Akt-NFκB signaling and for the secretion of the pro-inflammatory TNF-α, IL-6 and Th2 biasing IL-4 and IL-13. Conversely, inhibition of NK2R signaling in WT BMMCs with GR159897 decreased the release of proinflammatory and Th2 cytokines. In vivo, NK1R signaling was necessary for amplifying IgE-Ag mediated cutaneous and systemic anaphylaxis and chronic asthma. We conclude that NK1R and the NK2R display opposing effects on the inflammatory function of FcεRI-activated MCs relevant for the control of MC derived atopic disorders.

5-Aminosalicylic Acid Inhibits Acute Clostridium difficile Toxin A-Induced Colitis in Rats.

We tested the hypothesis that 5-aminosalicylic acid (5-ASA) inhibits toxin A-induced generation of colonic leukotriene B4 (LTB4) and toxin A colitis in rats. Isolated colonic segments in anesthetized rats were treated intraluminally with toxin A for 3 hours with or without 30 minutes of pretreatment with either 5-ASA or sulfapyridine and then colonic tissue levels of LTB4 were measured and inflammation was assessed. Separately, sulfasalazine was administered to rats in their drinking water for 5 days, isolated colonic segments were then prepared, toxin A was administered, and inflammation was assessed as before. Pretreatment with 5-ASA inhibited toxin A-induced increased tissue LTB4 concentration in the colon. Sulfasalazine and 5-ASA but not sulfapyridine significantly inhibited toxin A colitis. However, pretreatment with 5-ASA did not protect against direct TRPV1-mediated colitis caused by capsaicin. Toxin A stimulated the release of substance P (SP), and this effect was also inhibited by sulfasalazine and 5-ASA but not by sulfapyridine. Thus, toxin A stimulates colonic LTB4 resulting in activation of TRPV1, release of SP, and colitis. Inhibition of 5-LO by 5-ASA disrupts this pathway and supports the concept that LTB4 activation of TRPV1 plays a role in toxin A colitis.

Botulinum toxin B in the sensory afferent: Transmitter release, spinal activation, and pain behavior

We addressed the hypothesis that intraplantar botulinum toxin B (rimabotulinumtoxin B: BoNT-B) has an early local effect upon peripheral afferent terminal releasing function and, over time, will be transported to the central terminals of the primary afferent. Once in the terminals it will cleave synaptic protein, block spinal afferent transmitter release, and thereby prevent spinal nociceptive excitation and behavior. In mice, C57Bl/6 males, intraplantar BoNT-B (1 U) given unilaterally into the hind paw had no effect upon survival or motor function, but ipsilaterally decreased: 1) intraplantar formalin-evoked flinching; 2) intraplantar capsaicin-evoked plasma extravasation in the hind paw measured by Evans blue in the paw; 3) intraplantar formalin-evoked dorsal horn substance P (SP) release (neurokinin 1 [NK1] receptor internalization); 4) intraplantar formalin-evoked dorsal horn neuronal activation (c-fos); 5) ipsilateral dorsal root ganglion (DRG) vesicle-associated membrane protein (VAMP); 6) ipsilateral SP release otherwise evoked bilaterally by intrathecal capsaicin; 7) ipsilateral activation of c-fos otherwise evoked bilaterally by intrathecal SP. These results indicate that BoNT-B, after unilateral intraplantar delivery, is taken up by the peripheral terminal, is locally active (blocking plasma extravasation), is transported to the ipsilateral DRG to cleave VAMP, and is acting presynaptically to block release from the spinal peptidergic terminal. The observations following intrathecal SP offer evidence for a possible transsynaptic effect of intraplantar BoNT. These results provide robust evidence that peripheral BoNT-B can alter peripheral and central terminal release from a nociceptor and attenuate downstream nociceptive processing via a presynaptic effect, with further evidence suggesting a possible postsynaptic effect.

Effect of Intranasal Azelastine on Substance P Release in Perennial Nonallergic Rhinitis Patients

Rhinitis symptoms can be produced or augmented by neural mechanisms. Azelastine, a pharmacologic agent with potent H1-receptor blocking activity can inhibit the release of various mediators implicated in the pathogenesis of nasal hyperresponsiveness. The therapeutic benefits of topical intranasal azelastine on symptoms of perennial nonallergic rhinitis (NAR) are in part because of an impact on neural mechanisms. Assessment of changes in the concentration of substance P (SP) in nasal lavage fluid before and after saline hypertonic challenge may be a means of assessing the effect of intranasal azelastine on neuropeptide release and severity of rhinitis symptoms. Twenty-three patients with perennial NAR (negative skin-prick tests with inhalant allergens and concentration of total IgE in the normal range) were studied. Thirteen of 23 patients were treated with intranasal azelastine 0.15% spray at a dosage of 2 sprays (137 micrograms/spray) twice daily for 10 days. The control group consisted of 10 untreated patients with rhinitis. Nasal provocation using 4.5% saline solution was after 15 minutes by lavage before and after 10 days of treatment with intranasal azelastine. The concentration of SP in nasal lavage fluid was determined by enzyme immunoassay methods. Nasal lavage fluid baseline concentrations of SP were similar in both groups. After azelastine treatment, significantly greater concentrations of SP were seen in nasal lavage fluid 15 minutes after hypertonic saline challenge in the untreated patients (56.8 ± 13.8 pg/mL) in comparison with azelastine-treated patients (44.5 ± 16.5 pg/mL; p < 0.05). Total vasomotor rhinitis symptoms scores were substantially reduced in the azelastine-treated subjects compared with the control group. Azelastine intranasal spray reduces SP release into nasal lavage fluid of NAR patients immediately after hypertonic nasal saline challenge. Reduction of neuropeptide release may be an important aspect of the clinical efficacy of topical azelastine in perennial NAR patients.

Inflammation enhances Y1 receptor signaling, neuropeptide Y-mediated inhibition of hyperalgesia, and substance P release from primary afferent neurons

Neuropeptide Y (NPY) is present in the superficial laminae of the dorsal horn and inhibits spinal nociceptive processing, but the mechanisms underlying its anti-hyperalgesic actions are unclear. We hypothesized that NPY acts at neuropeptide Y1 receptors in the dorsal horn to decrease nociception by inhibiting substance P (SP) release, and that these effects are enhanced by inflammation. To evaluate SP release, we used microdialysis and neurokinin 1 receptor (NK1R) internalization in rat. NPY decreased capsaicin-evoked SP-like immunoreactivity in the microdialysate of the dorsal horn. NPY also decreased non-noxious stimulus (paw brush)-evoked NK1R internalization (as well as mechanical hyperalgesia and mechanical and cold allodynia) after intraplantar injection of carrageenan. Similarly, in rat spinal cord slices with dorsal root attached, [Leu31, Pro34]-NPY inhibited dorsal root stimulus-evoked NK1R internalization. In rat dorsal root ganglion neurons, Y1 receptors colocalized extensively with calcitonin gene-related peptide (CGRP). In dorsal horn neurons, Y1 receptors were extensively expressed and this may have masked the detection of terminal co-localization with CGRP or SP. To determine whether the pain inhibitory actions of Y1 receptors are enhanced by inflammation, we administered [Leu31, Pro34]-NPY after intraplantar injection of complete Freund’s adjuvant (CFA) in rat. We found that [Leu31, Pro34]-NPY reduced paw clamp-induced NK1R internalization in CFA rats but not uninjured controls. To determine the contribution of increased Y1 receptor-G protein coupling, we measured [35S]GTPγS binding simulated by [Leu31, Pro34]-NPY in mouse dorsal horn. CFA inflammation increased the affinity of Y1 receptor G-protein coupling. We conclude that Y1 receptors contribute to the anti-hyperalgesic effects of NPY by mediating the inhibition of SP release, and that Y1 receptor signaling in the dorsal horn is enhanced during inflammatory nociception.

Diacetyl Increases Sensory Innervation and Substance P Production in Rat Trachea

Inhalation of diacetyl, a butter flavoring, causes airway responses potentially mediated by sensory nerves. This study examines diacetyl-induced changes in sensory nerves of tracheal epithelium. Rats (n = 6/group) inhaled 0-, 25-, 249-, or 346-ppm diacetyl for 6 hr. Tracheas and vagal ganglia were removed 1-day postexposure and labeled for substance P (SP) or protein gene product 9.5 (PGP9.5). Vagal ganglia neurons projecting to airway epithelium were identified by axonal transport of fluorescent microspheres intratracheally instilled 14 days before diacetyl inhalation. End points were SP and PGP9.5 nerve fiber density (NFD) in tracheal epithelium and SP-positive neurons projecting to the trachea. PGP9.5-immunoreactive NFD decreased in foci with denuded epithelium, suggesting loss of airway sensory innervation. However, in the intact epithelium adjacent to denuded foci, SP-immunoreactive NFD increased from 0.01 ± 0.002 in controls to 0.05 ± 0.01 after exposure to 346-ppm diacetyl. In vagal ganglia, SP-positive airway neurons increased from 3.3 ± 3.0% in controls to 25.5 ± 6.6% after inhaling 346-ppm diacetyl. Thus, diacetyl inhalation increases SP levels in sensory nerves of airway epithelium. Because SP release in airways promotes inflammation and activation of sensory nerves mediates reflexes, neural changes may contribute to flavorings-related lung disease pathogenesis.

Intrathecal P/Q- and R-type calcium channel blockade of spinal substance P release and c-Fos expression

Intrathecal (IT) studies have shown that several voltage sensitive calcium channels (VSCCs), such as the L-, N- and T-type may play roles in nociception and that of these only the N-type regulates primary afferent substance P (SP) release. However, the actions of other VSCCs at the spinal level are not well known. We investigated the roles of spinal P/Q- and R-type VSCCs, by IT administration of R-type (SNX-482) and P/Q-type (ω-agatoxin IVA) VSCC blockers on intraplantar formalin-evoked flinching, SP release from primary afferents and c-Fos expression in spinal dorsal horn. Intraplantar injection of formalin (2.5%, 50 μL) produced an intense, characteristic biphasic paw flinching response. In rats with IT catheters, IT SNX-482 (0.5 μg) reduced formalin-evoked paw flinching in both phase 1 and 2 compared with vehicle. Intraplantar formalin caused robust neurokinin 1 receptor (NK1r) internalization (indicating SP release) and c-Fos expression in the ipsilateral dorsal horn, which were blocked by IT SNX-482. IT ω-agatoxin IVA (0.03, 0.125 and 0.5 μg) did not reduce formalin-evoked paw flinching or c-Fos expression at any doses, with higher doses resulting in motor dysfunction. Thus, we demonstrated that blockade of spinal R-type, but not P/Q type VSCCs attenuated formalin-induced pain behavior, NK1r internalization and c-Fos expression in the superficial dorsal horn. This study supports a role for Cav2.3 in presynaptic neurotransmitter release from peptidergic nociceptive afferents and pain behaviors.

1016 PARTIAL BLADDER OUTLET OBSTRUCTION MODULATES THE RELEASE OF SENSORY NEUROPEPTIDES SUBSTANCE P AND CALCITONIN GENE RELATED PEPTIDE FROM PENILE AFFERENT FIBERS

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research (II)1 Apr 20131016 PARTIAL BLADDER OUTLET OBSTRUCTION MODULATES THE RELEASE OF SENSORY NEUROPEPTIDES SUBSTANCE P AND CALCITONIN GENE RELATED PEPTIDE FROM PENILE AFFERENT FIBERS Qi Lei, Xiao-Qing Pan, Shaohua Chang, Ariana Smith, Allen Seftel, and Anna Malykhina Qi LeiQi Lei Glenolden, PA More articles by this author , Xiao-Qing PanXiao-Qing Pan Glenolden, PA More articles by this author , Shaohua ChangShaohua Chang Camden, NJ More articles by this author , Ariana SmithAriana Smith Philadelphia, PA More articles by this author , Allen SeftelAllen Seftel Camden, NJ More articles by this author , and Anna MalykhinaAnna Malykhina Glenolden, PA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.601AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Erectile dysfunction (ED) and benign prostatic hyperplasia (BPH) are common problems in aging males worldwide. Despite the epidemiological evidence of the link between BPH and ED, the mechanisms underlying this co-morbidity are still unclear. The objective of this work was to evaluate the effects of partial bladder outlet obstruction (PBOO) on function of sensory terminals innervating the corpus cavernosum (CC), and release of sensory neuropeptides Substance P (SP) and calcitonin gene related peptide (CGRP) in the CC using an animal model of PBOO. METHODS Experiments were conducted on adult male Sprague Dawley rats divided into two groups: sham operated and PBOO surgery. PBOO was surgically induced by a partial ligation of the bladder neck during laparotomy. Concentration of the sensory neuropeptides SP and CGRP was measured by ELISA. Contractility and relaxation of the CCSM was studied in vitro and expression of nitric oxide synthase (NOS) was evaluated by Western blotting. RESULTS The results of neuropeptide analysis established that the release of SP and CGRP in the pelvic viscera is tissue dependent. Concentration of SP in the CC was higher (2.01±0.23 ng/ml, n=10) in comparison to the urinary bladder (0.3±0.05 ng/ml, n=8, p≤0.001) under normal physiological conditions. However, basal concentration of CGRP was significantly elevated in the urinary bladder reaching 120.6±37.2 pg/ml in comparison to 14.5±0.9 pg/ml in the CC (p≤0.01). PBOO caused impairment in SP release from sensory terminals as evidenced by a 5-fold down-regulation of SP in the CC (n=10, p≤0.001) without significant changes in the obstructed bladder. Additionally, concentration of CGRP was down-regulated in the urinary bladder by PBOO without parallel changes in the CC, however, the decrease did not reach the level of statistical significance. The contractile responses of CC muscle strips to KCl and phenylephrine were decreased after PBOO, followed by a reduced relaxation response to nitroprusside. A significant decrease in nNOS expression was also detected in the CC of obstructed animals. CONCLUSIONS Our results provide evidence that PBOO leads to the impairment of bladder neck afferent innervation followed by a decrease in CC smooth muscle relaxation, down-regulation of nNOS expression and reduced release of sensory neuropeptides in the CC smooth muscle. These results suggest that nerve damage in PBOO may contribute to LUTS-ED comorbidity and trigger secondary changes in the contraction/relaxation mechanisms of CCSM. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e416-e417 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Qi Lei Glenolden, PA More articles by this author Xiao-Qing Pan Glenolden, PA More articles by this author Shaohua Chang Camden, NJ More articles by this author Ariana Smith Philadelphia, PA More articles by this author Allen Seftel Camden, NJ More articles by this author Anna Malykhina Glenolden, PA More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...