Neurokinin-1 Receptor Activation Research Articles

Fos expression induced by activation of NMDA and neurokinin-1 receptors in the trigeminal subnucleus caudalis in vitro: Role of protein kinases

Activity-induced neuronal plasticity is partly facilitated by the expression of the immediate-early gene c-fos and the resulting transcription factor Fos. Expression of Fos is associated with nociceptive afferent activation, but a detailed stimulation-transcription pathway for Fos expression has not yet been determined in the trigeminal system. This study utilized a novel in vitro model to determine whether Fos expression can be induced in trigeminal subnucleus caudalis by NMDA or neurokinin-1 receptor activation, and whether inhibition of intracellular kinases has any effect on Fos expression induced by activation of these receptors. Brainstems of male Wistar rats were excised and maintained in artificial cerebrospinal fluid at 37 °C. NMDA or the specific neurokinin-1 receptor agonist [Sar 9,Met(O 2) 11]-SP was applied. These agonists were subsequently tested in the presence of the protein kinase A inhibitor Rp-cAMP or protein kinase C inhibitor chelerythrine chloride. In all experiments the sodium channel blocker tetrodotoxin was used to prevent indirect neuronal activation. Brainstems were processed immunocytochemically for Fos expression, and positive cells were counted in the trigeminal subnucleus caudalis. NMDA and [Sar 9,Met(O 2) 11]-SP significantly increased Fos expression, but these increases could be prevented by chelerythrine chloride. Rp-cAMP had no effect on Fos induced by NMDA but caused a significant reduction in Fos induced by [Sar 9,Met(O 2) 11]-SP. These data demonstrate that in trigeminal subnucleus caudalis activation of either NK1 or NMDA receptors alone induces Fos expression; protein kinases A and C are involved in NK1R-induced Fos while protein kinase A is not required for NMDA receptor-induced Fos.

Transgenic Mice Lacking Serotonin Neurons Have Severe Apnea and High Mortality during Development

Central serotonin (5-HT) neurons modulate many vital brain functions, including respiratory control. Whether breathing depends critically on 5-HT neurons, or whether their influence is excitatory or inhibitory, remains controversial. Here we show that neonatal Lmx1b(flox/flox;ePet-Cre/+) mice (also called Lmx1b(f/f/p) mice), which selectively lack serotonin neurons, display frequent and severe apnea lasting as long as 55 s. This was associated with a marked decrease in ventilation to less than one-half of normal. These respiratory abnormalities were most severe during the postnatal period, markedly improving by the time the pups were 2-4 weeks old. Despite the severe breathing dysfunction, many of these mice survived, but there was a high perinatal mortality, and those that survived had a decrease in growth rate until the age at which the respiratory defects resolved. Consistent with these in vivo observations, respiratory output was markedly reduced in isolated brainstem-spinal cord preparations from neonatal Lmx1b(f/f/p) mice and completely blocked in perfused brain preparations from neonatal rats treated with selective antagonists of 5-HT(2A) and neurokinin 1 (NK-1) receptors. The ventilatory deficits in neonatal Lmx1b(f/f/p) mice were reversed in vitro and in vivo with agonists of 5-HT(2A) and/or NK-1 receptors. These results demonstrate that ventilatory output in the neonatal period is critically dependent on serotonin neurons, which provide excitatory drive to the respiratory network via 5-HT(2A) and NK-1 receptor activation. These findings provide insight into the mechanisms of sudden infant death syndrome, which has been associated with abnormalities of 5-HT neurons and of cardiorespiratory control.

Paclitaxel and vinorelbine, evoked the release of substance P from cultured rat dorsal root ganglion cells through different PKC isoform-sensitive ion channels

Many patients suffer from serious adverse effects including respiratory distress and pulmonary edema during and after chemotherapy with paclitaxel or vinorelbine. These effects appear to be due to the activation of neurokinin-1 receptors. The present study investigated the influences of paclitaxel and vinorelbine on the substance P (sP) release from cultured dorsal root ganglion (DRG) cells using a radioimmunoassay. Both paclitaxel and vinorelbine evoked sP release in a dose- and time-dependent manner within 60 min at a concentration range of 0.1–10 μM. The sP release levels induced by the two drugs were attenuated by pretreatment with the protein kinase Cs (PKCs) inhibitors (bisindolylmaleimide I and Gö6976). Moreover, the paclitaxel- or vinorelbine-induced sP release was diminished in the absence of extracellular Ca 2+ or the presence of LaCl 3 (an extracellular Ca 2+ influx blocker). A Ca 2+ imaging assay further indicated that both paclitaxel and vinorelbine gradually increased the intracellular Ca 2+ concentration, and these increases lasted for at least 15 min and were suppressed by Gö6976. Paclitaxel caused the membrane translocation of only PKCβ within 10 min after stimulation, whereas vinorelbine induced the translocation of both PKCα and β. The paclitaxel- and vinorelbine-induced sP release levels were separately inhibited by ruthenium red (a transient receptor potential (TRP) channel blocker) and gabapentin (an inhibitor of voltage-gated Ca 2+ channels (VGCCs)). These findings suggest that paclitaxel and vinorelbine evoke the sP release from cultured DRG cells by the extracellular Ca 2+ influx through TRP channels activated by PKCβ and VGCCs activated by both PKCα and β, respectively.

Neurokinin-1 receptor activation in globus pallidus

The undecapeptide substance P has been demonstrated to modulate neuronal activity in a number of brain regions by acting on neurokinin-1 receptors. Anatomical studies revealed a moderate level of neurokinin-1 receptor in rat globus pallidus. To determine the electrophysiological effects of neurokinin-1 receptor activation in globus pallidus, whole-cell patch-clamp recordings were performed in the present study. Under current-clamp recordings, neurokinin-1 receptor agonist, [Sar9, Met(O2)11] substance P (SM-SP) at 1 μM, depolarized globus pallidus neurons and increased their firing rate. Consistently, SM-SP induced an inward current under voltage-clamp recording. The depolarization evoked by SM-SP persisted in the presence of tetrodotoxin, glutamate and GABA receptor antagonists, indicating its direct postsynaptic effects. The neurokinin-1 receptor antagonist, SR140333B, could block SM-SP-induced depolarization. Further experiments showed that suppression of potassium conductance was the predominant ionic mechanism of SM-SP-induced depolarization. To determine if neurokinin-1 receptor activation exerts any effects on GABAergic and glutamatergic neurotransmission, the action of SM-SP on synaptic currents was studied. SM-SP significantly increased the frequency of spontaneous inhibitory postsynaptic currents, but only induced a transient increase in the frequency of miniature inhibitory postsynaptic currents. No change was observed in both spontaneous and miniature excitatory postsynaptic currents. Based on the direct excitatory effects of SM-SP on pallidal neurons, we hypothesize that neurokinin-1 receptor activation in globus pallidus may be involved in the beneficial effect of substance P in Parkinson's disease.

PKC∊-Dependent Potentiation of TTX-Resistant NaV1.8 Current by Neurokinin-1 Receptor Activation in Rat Dorsal Root Ganglion Neurons

BackgroundSubstance P (SP), which mainly exists in a subtype of small-diameter dorsal root ganglion (DRG) neurons, is an important signal molecule in pain processing in the spinal cord. Our previous results have proved the expression of SP receptor neurokinin-1 (NK-1) on DRG neurons and its interaction with transient receptor potential vanilloid 1 (TRPV1) receptor.ResultsIn this study we investigated the effect of NK-1 receptor agonist on Nav1.8, a tetrodotoxin (TTX)-resistant sodium channel, in rat small-diameter DRG neurons employing whole-cell patch clamp recordings. NK-1 agonist [Sar9, Met(O2)11]-substance P (Sar-SP) significantly enhanced the Nav1.8 currents in a subgroup of small-diameter DRG neurons under both the normal and inflammatory situation, and the enhancement was blocked by NK-1 antagonist Win51708 and protein kinase C (PKC) inhibitor bisindolylmaleimide (BIM), but not the protein kinase A (PKA) inhibitor H89. In particular, the inhibitor of PKCε, a PKC isoform, completely blocked this effect. Under current clamp model, Sar-SP reduced the amount of current required to evoke action potentials and increased the firing rate in a subgroup of DRG neurons.ConclusionThese data suggest that activation of NK-1 receptor potentiates Nav1.8 sodium current via PKCε-dependent signaling pathway, probably participating in the generation of inflammatory hyperalgesia.

Phosphorylation of TRPV1 by neurokinin-1 receptor agonist exaggerates the capsaicin-mediated substance P release from cultured rat dorsal root ganglion neurons

The present study was conducted to determine whether the activation of neurokinin-1 receptor (NK-1R) by its agonist (GR73632) enhances the capsaicin-evoked substance P (SP) release using a radioimmunoassay. A pre-exposure to GR73632 enhanced the capsaicin-evoked SP release in a time- and dose-dependent manner. The augmentation of capsaicin-evoked SP release by GR73632 was completely inhibited by pharmacological blockade of NK-1R or transient receptor potential vanilloid receptor subtype 1 (TRPV1), and was partially attenuated by the inhibition of either protein kinase C (PKC), cyclooxygenase (COX) or phospholipase C (PLC), p38 or p42/44 mitogen-activated protein (MAP) kinase, but not protein kinase A. This augmentation of SP release was further increased by inhibition of c-Jun NH2-terminal kinase. A short-term (10min) exposure to GR73632 resulted in an increase in the TRPV1 phosphorylation. The increase in the TRPV1 phosphorylated forms induced by a 60-min exposure to GR73632 was completely abolished by the inhibition of either PKC, COX or PLC, p38 or p42/44 MAP kinases. Immunocytochemistry study demonstrated that the NK-1R and TRPV1 were mainly co-expressed in the small-sized neurons. These findings suggest that the activation of NK-1R by its agonist, by sensitizing the TRPV1 through the PKC phosphorylation of TRPV1, may play a role in the enhancement of the capsaicin-evoked SP release from cultured rat DRG neurons.

Modulation of basal and stress‐induced amygdaloid substance P release by the potent and selective NK1 receptor antagonist L‐822429

It has been shown that anxiety and stress responses are modulated by substance P (SP) released within the amygdala. However, there is an important gap in our knowledge concerning the mechanisms regulating extracellular SP in this brain region. To study a possible self-regulating role of SP, we used a selective neurokinin-1 (NK1) receptor antagonist to investigate whether blockade of NK1 receptors results in altered basal and/or stress-evoked SP release in the medial amygdala (MeA), a critical brain area for a functional involvement of SP transmission in enhanced anxiety responses induced by stressor exposure. In vitro binding and functional receptor assays revealed that L-822429 represents a potent and selective rat NK1 receptor antagonist. Intra-amygdaloid administration of L-822429 via inverse microdialysis enhanced basal, but attenuated swim stress-induced SP release, while the low-affinity enantiomer of L-822429 had no effect. Using light and electron microscopy, synaptic contacts between SP-containing fibres and dendrites expressing NK1 receptors was demonstrated in the medial amygdala. Our findings suggest self-regulatory capacity of SP-mediated neurotransmission that differs in the effect on basal and stress-induced release of SP. Under basal conditions endogenous SP can serve as a signal that tonically inhibits its own release via a NK1 receptor-mediated negative feedback action, while under stress conditions SP release is further facilitated by activation of NK1 receptors, likely leading to high local levels of SP and activation of receptors to which SP binds with lower affinity.

Differences in the length of the carboxyl terminus mediate functional properties of neurokinin-1 receptor

The neurokinin-1 receptor (NK1R) has two naturally occurring forms that differ in the length of the carboxyl terminus: a full-length receptor consisting of 407 aa and a truncated receptor consisting of 311 aa. We examined whether there are differential signaling properties attributable to the carboxyl terminus of this receptor by using stably transfected human embryonic kidney (HEK293) cell lines that express either full-length or truncated NK1R. Substance P (SP) specifically triggered intracellular calcium increase in HEK293 cells expressing full-length NK1R but had no effect in the cells expressing the truncated NK1R. In addition, in cells expressing full-length NK1R, SP activated NF-kappaB and IL-8 mRNA expression, but in cells expressing the truncated NK1R, SP did not activate NF-kappaB, and it decreased IL-8 mRNA expression. In cells expressing full-length NK1R, SP stimulated phosphorylation of PKCdelta but inhibited phosphorylation of PKCdelta in cells expressing truncated NK1R. There are also differences in the timing of SP-induced ERK activation in cells expressing the two different forms of the receptor. Full-length NK1R activation of ERK was rapid (peak within 1-2 min), whereas truncated NK1R-mediated activation was slower (peak at 20-30 min). Thus, the carboxyl terminus of NK1R is the structural basis for differences in the functional properties of the full-length and truncated NK1R. These differences may provide important information toward the design of new NK1R receptor antagonists.

Characterization of a conformationally sensitive TOAC spin-labeled substance P

To probe the binding of a peptide agonist to a G-protein coupled receptor in native membranes, the spin-labeled amino acid analogue 4-amino-4-carboxy-2,2,6,6-tetramethylpiperidino-1-oxyl (TOAC) was substituted at either position 4 or 9 within the substance P peptide (RPKPQQFFGLM-NH2), a potent agonist of the neurokinin-1 receptor. The affinity of the 4-TOAC analog is comparable to the native peptide while the affinity of the 9-TOAC derivative is ∼250-fold lower. Both peptides activate receptor signaling, though the potency of the 9-TOAC peptide is substantially lower. The utility of these modified ligands for reporting conformational dynamics during the neurokinin-1 receptor activation was explored using EPR spectroscopy, which can determine the real-time dynamics of the TOAC nitroxides in solution. While the binding of both the 4-TOAC substance P and 9-TOAC substance P peptides to isolated cell membranes containing the neurokinin-1 receptor is detected, a bound signal for the 9-TOAC peptide is only obtained under conditions that maintain the receptor in its high-affinity binding state. In contrast, 4-TOAC substance P binding is observed by solution EPR under both low- and high-affinity receptor states, with evidence of a more strongly immobilized peptide in the presence of GDP. In addition, to better understand the conformational consequences of TOAC substitution into substance P as it relates to receptor binding and activation, atomistic models for both the 4- and 9-TOAC versions of the peptide were constructed, and the molecular dynamics calculated via simulated annealing to explore the influence of the TOAC substitutions on backbone structure.

NK-1 receptors in the rostral ventromedial medulla contribute to hyperalgesia produced by intraplantar injection of capsaicin

The rostral ventromedial medulla (RVM) is an area of the brainstem involved in the descending modulation of nociception at the level of the spinal cord. Although the RVM is involved in the inhibition or facilitation of nociception, the underlying mechanisms are not understood. Here we examined the role of the neuropeptide substance P and neurokinin-1 (NK-1) receptors located in the RVM on withdrawal responses evoked by mechanical and heat stimuli applied to the rat hindpaw under normal conditions and during hyperalgesia produced by capsaicin. The mechanical withdrawal threshold was obtained using von Frey monofilaments applied to the plantar surface of the hindpaw. Sensitivity to heat was determined by measuring the latency to withdrawal from radiant heat applied to the plantar surface. Mechanical and heat hyperalgesia were defined as a decrease in withdrawal response threshold or latency, respectively. Rats were prepared with a chronic cannula and either vehicle or the NK-1 receptor antagonists, L-733,060 or RP-67580, was injected into the RVM. Paw withdrawal responses were obtained before and after RVM injection, and then at 5, 30, and 60 min after an intraplantar injection of capsaicin (10 μg). Injection of the NK-1 antagonists at doses of 0.5 pmol or higher did not alter withdrawal responses to mechanical or heat stimuli under normal conditions but reduced the duration of nocifensive behavior and the mechanical and heat hyperalgesia produced by capsaicin. These findings suggest that the activation of NK-1 receptors in the RVM contributes to the hyperalgesia produced by capsaicin.

Activation of Neurokinin-1 Receptors during Ozone Inhalation Contributes to Epithelial Injury and Repair

We investigated the importance of neurokinin (NK)-1 receptors in epithelial injury and repair and neutrophil function. Conscious Wistar rats were exposed to 1 ppm ozone or filtered air for 8 hours, followed by an 8-hour postexposure period. Before exposure, we administered either the NK-1 receptor antagonist, SR140333, or saline as a control. Ethidium homodimer was instilled into lungs as a marker of necrotic airway epithelial cells. After fixation, whole mounts of airway dissected lung lobes were immunostained for 5-bromo-2'-deoxyuridine, a marker of epithelial proliferation. Both ethidium homodimer and 5-bromo-2'-deoxyuridine-positive epithelial cells were quantified in specific airway generations. Rats treated with the NK-1 receptor antagonist had significantly reduced epithelial injury and epithelial proliferation compared with control rats. Sections of terminal bronchioles showed no significant difference in the number of neutrophils in airways between groups. In addition, staining ozone-exposed lung sections for active caspase 3 showed no apoptotic cells, but ethidium-positive cells colocalized with the orphan nuclear receptor, Nur77, a marker of nonapoptotic, programmed cell death mediated by the NK-1 receptor. An immortalized human airway epithelial cell line, human bronchial epithelial-1, showed no significant difference in the number of oxidant stress-positive cells during exposure to hydrogen peroxide and a range of SR140333 doses, demonstrating no antioxidant effect of the receptor antagonist. We conclude that activation of the NK-1 receptor during acute ozone inhalation contributes to epithelial injury and subsequent epithelial proliferation, a critical component of repair, but does not influence neutrophil emigration into airways.

Neurokinin‐1 receptor expressing neurons in the nucleus of the solitary tract are required for full expression of post‐exercise hypotension in spontaneously hypertensive rats

A single bout of exercise decreases blood pressure (post‐exercise hypotension; PEH) in both hypertensive humans and spontaneously hypertensive rats (SHR). Our recent studies suggest that in SHR, PEH may be dependent in part on activation of neurokinin‐1 receptors (NK1‐R) in the nucleus of the solitary tract (NTS). The current experiments were performed to test the hypothesis that selective elimination of NK1‐R expressing neurons in the NTS would attenuate PEH in SHR. The NK1‐R targeted neurotoxin, stable substance‐P saporin (SSP‐SAP) was microinjected into the NTS to ablate NK1‐R neurons in adult male SHR implanted with telemetry transmitters to measure blood pressure (BP) and heart rate. SSP‐SAP induced a transient (5 day) increase in BP after ablation relative to control (blank saporin; p<0.01) rats. Blood pressure variability, assessed as the standard deviation of daily systolic BP, increased by the second week after NK1‐R neuron ablation (p<0.05). Two weeks after NK1‐R neuron ablation, rats were subjected to a single bout of dynamic exercise or sham exercise. SSP‐SAP significantly blunted PEH (p<0.005, 2‐way RM ANOVA). For example, by 120 min post‐exercise blood pressure was decreased by −15.2 ± 2 mmHg in control (n=4) versus 0.5 ± 2.7 mmHg in SSP‐SAP (n=6) treated rats. We conclude that ablation of NTS NK1‐R expressing neurons increases blood pressure variability and attenuates PEH in SHR. (Support: HL‐67183)

Neurokinin-1 Receptors Modulate the Excitability of Expiratory Neurons in the Ventral Respiratory Group

We studied the role of neurokinin-1 receptors (NK1-R) on the excitability of expiratory (E) neurons (tonic discharge, E(TONIC); augmenting, E(AUG); decrementing, E(DEC)) throughout the ventral respiratory group, including Bötzinger Complex (BötC) using extracellular single-unit recording combined with pressurized picoejection in decerebrate, arterially perfused juvenile rats. Responses evoked by picoejection of the NK1-R agonist, [Sar9-Met(O2)11]-substance P (SSP) were determined before and after the selective NK1-R antagonist, CP99,994. SSP excited 20 of 35 expiratory neurons by increasing the number of action potentials per burst (+33.7 +/- 6.5% of control), burst duration (+20.6 +/- 7.9% of control), and peak firing frequency (+16.2 +/- 4.8% of control; means +/- SE). Pretreatment with CP99,994 completely blocked SSP-evoked excitation in a subset of neurons tested, supporting the notion that SSP excitation was mediated through NK1-R activation. Because we had previously shown that E(AUG) neurons were crucial to locomotor-respiratory coupling (LRC), we reasoned that blockade of NK1-R would alter LRC by preventing somatic-evoked excitation of E(AUG) neurons. Blockade of NK1-Rs by CP99,994 in the BötC severely disrupted LRC and prevented somatic-evoked excitation of E(AUG) neurons. These findings demonstrate that LRC is dependent on endogenous SP release acting via NK1-Rs on E(AUG) neurons of the BötC. Taken together with our earlier finding that inspiratory off-switching by the Hering-Breuer Reflex requires endogenous activation of NK1-Rs through activation of NK1-Rs on E(DEC) neurons, we suggest that endogenous release of substance P in the BötC provides a reflex pathway-dependent mechanism to selectively modulate respiratory rhythm.

Dorsal horn NK1-expressing neurons control windup of downstream trigeminal nociceptive neurons

Windup is a progressive, frequency-dependent increase in the excitability of trigeminal and spinal dorsal horn wide dynamic range (WDR) nociceptive neurons to repetitive stimulation of primary afferent nociceptive C-fibers. Superficial dorsal horn neurokinin 1 receptor (NK1R)-expressing neurons were recently shown to regulate sensitization of WDR nociceptive neurons through activation of a defined spino-bulbo-spinal loop. However, the windup of WDR nociceptive neurons was not regulated through this loop. In the present study, we sought to identify the alternative circuit activated by dorsal horn NK1Rs that mediates WDR neuron windup. As a model we used the rat spinal trigeminal nucleus, in which the subnucleus oralis (Sp5O) contains a pool of WDR neurons that receive their nociceptive C-input indirectly via interneurons located in the medullary dorsal horn (MDH). First, we found that intravenous injection of NK1R antagonists (SR140333 and RP67580) produced a reversible inhibition of Sp5O WDR neuron windup. Second, we anatomically identified in the MDH lamina III a subpopulation of NK1R-expressing local interneurons that relay nociceptive information from the MDH to downstream Sp5O neurons. Third, using microinjections of NK1R antagonists during in vivo electrophysiological recordings from Sp5O WDR neurons, we showed that WDR neuron windup depends on activation of NK1Rs located in the MDH laminae I–III. We conclude that, in contrast to central sensitization that is controlled by a spino-bulbo-spinal loop, Sp5O WDR neuron windup is regulated through a local circuit activated by MDH lamina III NK1Rs.

Substance P excites globus pallidus neurons in vivo

Substance P is a member of the neurokinin family. Previous studies have reported the existence of substance P and its high-affinity receptor, neurokinin-1 receptor, in globus pallidus. Employing in vivo extracellular recording combined with behavioural tests, the effects of substance P in globus pallidus of rats were studied. Micropressure ejection of the selective neurokinin-1 receptor agonist [Sar9,Met(O2)11] substance P increased the spontaneous firing rate of pallidal neurons in a concentration-dependent manner, with increases of 27.3% at 0.01, 33.4% at 0.03, 45.5% at 0.1, 38.4% at 0.3 and 36.4% at 1.0 mm. The selective neurokinin-1 receptor antagonist SR140333B prevented the excitatory effects induced by [Sar9,Met(O2)11] substance P. In behaving rats, we observed the postural effects of neurokinin-1 receptor activation in the globus pallidus. Consistent with electrophysiological results, unilateral microinjection of [Sar9,Met(O2)11] substance P (0.1 mm) led to a SR140333B-sensitive contralateral deflection in the presence of systemic haloperidol administration. Combining electrophysiological and behavioural findings, we concluded that substance P produces excitatory effects on globus pallidus neurons via neurokinin-1 receptors.

Effect of CP-96,345 on the expression of adhesion molecules in acute pancreatitis in mice

We investigated the effect of a specific neurokinin-1 receptor (NK1R) antagonist, CP-96,345, on the regulation of the expression of adhesion molecules ICAM-1, VCAM-1, E-selectin, and P-selectin as well as leukocyte recruitment during acute pancreatitis (AP). AP was induced in male Balb/C mice by 10 consecutive hourly intraperitoneal injections of caerulein. In the treatment groups, CP-96,345 was administered at 2.5 mg/kg ip either 30 min before or 1 h after the first caerulein injection. Animals were killed, and the lungs and pancreas were isolated for RNA extraction and RT-PCR or for immunohistochemical staining. mRNA expression of the four adhesion molecules was upregulated in the pancreas during AP. Treatment with CP-96,345 effectively reduced the mRNA expression of P-selectin and E-selectin but not ICAM-1 and VCAM-1. In the lung, ICAM-1, E-selectin, and P-selectin mRNA expression increased during AP. Antagonist treatment suppressed this elevation. Similar expression patterns were seen in the immunohistochemical stainings. Intravital microscopy of the pancreatic microcirculation revealed the effect of CP-96,345 on leukocyte recruitment. The present study provides important information on the relationship between NK1R activation and the regulation of adhesion molecules. Also, this study points to the differential regulation of inflammation in the pancreas and lung with AP.

Activation of the Neurokinin-1 Receptor by Substance P Triggers the Release of Substance P from Cultured Adult Rat Dorsal Root Ganglion Neurons

BackgroundAlthough substance P (SP) is an important primary afferent modulator in nociceptive processes, it is unclear whether SP regulates its own release from primary sensory neurons.ResultsUsing a highly sensitive radioimmunoassay for SP, we have demonstrated that the activation of neurokinin-1 receptor by SP or GR73632 (a potent neurokinin-1 receptor agonist) triggered an increase of SP release from cultured adult rat dorsal root ganglion (DRG) neurons depending on the dose and exposure time within 60 min, and thereafter, the SP release level gradually decreased over 360 min. Accompanying the SP release, a significant reduction in the percentage of neurons expressing neurokinin-1 receptor on their membranes during exposure to SP (200 pg/dish) occurred time dependently (56 ± 5% and 32 ± 2% at 180 and 360 min, respectively). The GR73632-evoked (10 nM, 60 min) SP release was attenuated by several inhibitors for mitogen-activated protein kinase kinase, p38 mitogen-activated protein (MAP) kinase and cyclooxygenase-2 (COX-2), protein kinase C (PKC), respectively. In contrast, a c-Jun NH2-terminal kinase inhibitor increased the GR73632-evoked SP release.ConclusionThese results indicate that the neurokinin-1 receptor activation by its agonists regulates the SP release process involving the activation of MAP kinases, PKCs and COX-2 from cultured DRG neurons.

Activation of Spinal Cholecystokinin and Neurokinin-1 Receptors Is Associated With the Attenuation of Intrathecal Morphine Analgesia Following Electroacupuncture Stimulation in Rats

We previously demonstrated that electroacupuncture (EA) stimulation both produced antinociception and attenuated intrathecal (i.t.) morphine analgesia, suggesting that EA is capable of inducing two opposing systems, that is, opioid and anti-opioid mechanisms. This study examined the involvement of cholecystokinin (CCK) in the anti-opioid effects following EA in the spinal cord. EA was applied to commonly used acupoints for antinociception, ST-36 located 5-mm lateral to the anterior tubercle of the tibia, and analgesia was assessed by the hind-paw pressure test in male Sprague-Dawley rats. I.t. administration of CCK (0.01 – 10 µg) attenuated i.t. morphine analgesia (10 µg) dose-dependently. The attenuation of morphine analgesia following EA was reversed by i.t. proglumide, a CCK-receptor antagonist (0.01 µg). CCK-like immunoreactivity was increased in lamina I and II in the dorsal horn, and expression of spinal CCK mRNA increased after EA. Moreover, i.t. pretreatment with the neurokinin-1 (NK1)-receptor antagonist L-703,606 (18 µg) reversed both EA- and CCK-induced attenuation of morphine analgesia. These results suggest that CCK-mediated neural systems in the spinal cord may be involved in the attenuation of morphine analgesia following EA and that substance P-induced activation of NK1 receptors may be responsible for the downstream neuronal transmission of the CCK-mediated neuronal system.

NK-1 Receptors Modulate the Excitability of on Cells in the Rostral Ventromedial Medulla

The role of neurokinin-1 (NK-1) receptors in the rostral ventromedial medulla (RVM) was studied using extracellular single-unit recording combined with microiontophoresis. In rats, on- and off-type neurons were identified using noxious heat or mechanical stimuli applied to the tail. Responses evoked by iontophoretic application of N-methyl-d-aspartate (NMDA) were determined before and after intraplantar injection of capsaicin or iontophoretic application of substance P. In off cells, capsaicin produced an extended pause in ongoing activity but did not alter the subsequent spontaneous discharge rate or NMDA-evoked responses. In contrast, spontaneous discharge rates of on cells increased after capsaicin, and their responses to NMDA increased >100% above control values. The increased responses to NMDA after capsaicin were attenuated by iontophoretic application of the selective NK-1 receptor antagonist L-733,060. Similarly to capsaicin, iontophoretic application of the selective NK-1 receptor agonist, [Sar(9),Met(O(2))(11)]-substance P (SM-SP), increased the spontaneous discharge rate and NMDA-evoked responses of on cells by >100% of control values. These effects were antagonized by L-733,060. Immunohistochemical studies showed that a subset of neurons in the RVM labeled NK-1 receptors and that nearly all of these neurons were immunoreactive for the NMDAR1 subunit of the NMDA receptor. These results demonstrate that activation of NK-1 receptors in the RVM enhances responses of on cells evoked by NMDA. It is suggested that activation of NK-1 receptors in the RVM and the ensuing sensitization of on cells may contribute to the development of central sensitization and hyperalgesia after tissue injury and inflammation.

Involvement of Substance P and Neurogenic Inflammation in Arsenic-Induced Early Vascular Dysfunction

Vascular-related diseases, including Blackfoot Disease and atherosclerosis, are prominent clinical findings among populations residing in arseniasis areas. While oxidative stress provided a general but nonspecific mechanistic base for arsenic-induced endothelial cell damage in vitro, more specific mechanism is needed to explain the highly targeted vascular lesions induced by arsenic in vivo. Based on our previous studies, we hypothesized that arsenic exerted its action on blood vessels via the neurogenic inflammation process involving release of a neuropeptide (substance P) and activation of endothelial Neurokinin 1 (NK-1) receptor in vivo. Indeed, our present study demonstrated a significantly higher substance P levels in arsenic-treated tissues when compared to saline-treated controls indicating a rapid release of substance P under the influence of arsenic. Furthermore, the arsenic-induced vascular leakage could be significantly reduced when the neurogenic inflammation process was interrupted (via either disruption on the release of substance P, interference on the action of substance P, or blockage of endothelial NK-1 receptor) showing that the neurogenic inflammation process was indeed involved. Histamine release was not found to play a significant role in arsenic-induced vascular permeability change. Our present study affirmed a de novo concept that a pathophysiological mechanism involving the neurogenic release of substance P and activation of endothelial NK-1 receptor underlies the arsenic-induced vascular injury and dysfunction in vivo. This pathophysiological process constituted a two-tiered biological interaction between the nervous system and vascular system and therefore was not readily unveiled by traditional in vitro studies in the past. Our present finding unveiled an important de novo concept on arsenic vascular toxicity in vivo.