inositol-1-4-5-trisphosphate and Pain

BACKGROUND Fractalkine is widely expressed throughout the brain and spinal cord, where it can exert effects on pain enhancement and hyperalgesia by activating microglia through CX3C chemokine receptor 1 (CX3CR1), which triggers the release of several pro-inflammatory cytokines in the spinal cord. Fractalkine has also been shown to increase cytosolic calcium ([Ca2+]i) in microglia. MATERIAL AND METHODS Based on the characteristics of CX3CR1, a G protein-coupled receptor, we explored the role of inositol 1,4,5-trisphosphate (IP3) signaling in fractalkine-induced inflammatory response in BV-2 cells in vitro. The effect and the underlying mechanism induced by fractalkine in the brain were observed using a mouse model with intracerebroventricular (i.c.v.) injection of exogenous fractalkine. RESULTS [Ca2+]i was significantly increased and IL-1β and TNF-α levels were higher in the fractalkine-treated cell groups than in the farctalkine+ 2-APB groups. We found that i.c.v. injection of fractalkine significantly increased p-p38MAPK, IL-1β, and TNF-α expression in the brain, while i.c.v. injection of a fractalkine-neutralizing antibody (anti-CX3CR1), trisphosphate receptor (IP3R) antagonist (2-APB), or p38MAPK inhibitor (SB203580) prior to fractalkine addition yielded an effective and reliable anti-allodynia effect, following the reduction of p-p38MAPK, IL-1β, and TNF-α expression. CONCLUSIONS Our results suggest that fractalkine leads to hyperalgesia, and the underlying mechanism may be associated with IP3/p38MAPK-mediated calcium signaling and its phlogogenic properties.

Topics: Animals; Calcium; Calcium Signaling; Cell Line; Chemokine CX3CL1; China; CX3C Chemokine Receptor 1; Hyperalgesia; Injections, Spinal; Inositol 1,4,5-Trisphosphate; Interleukin-1beta; Macrophage Activation; Mice; Microglia; Mitogen-Activated Protein Kinase 14; Pain; Receptors, Chemokine; Signal Transduction; Spinal Cord; Tumor Necrosis Factor-alpha

Bradykinin (Bk) is a potent inflammatory mediator that causes hyperalgesia. The action of Bk on the sensory system is well documented but its effects on motoneurons, the final pathway of the motor system, are unknown. By a combination of patch-clamp recordings and two-photon calcium imaging, we found that Bk strongly sensitizes spinal motoneurons. Sensitization was characterized by an increased ability to generate self-sustained spiking in response to excitatory inputs. Our pharmacological study described a dual ionic mechanism to sensitize motoneurons, including inhibition of a barium-sensitive resting K(+) conductance and activation of a nonselective cationic conductance primarily mediated by Na(+). Examination of the upstream signaling pathways provided evidence for postsynaptic activation of B2 receptors, G protein activation of phospholipase C, InsP3 synthesis, and calmodulin activation. This study questions the influence of motoneurons in the assessment of hyperalgesia since the withdrawal motor reflex is commonly used as a surrogate pain model.

Topics: Action Potentials; Animals; Animals, Newborn; Bradykinin; Calcium; Calmodulin; Female; Ganglia, Spinal; Gene Expression; Hyperalgesia; Inflammation; Inositol 1,4,5-Trisphosphate; Male; Molecular Imaging; Motor Neurons; Pain; Patch-Clamp Techniques; Potassium Channels; Rats; Rats, Wistar; Receptor, Bradykinin B2; Signal Transduction; Sodium Channels; Spinal Cord; Type C Phospholipases

Reactive oxygen species (ROS) such as superoxide are emerging as important signaling molecules in physiological plasticity but also in peripheral and spinal cord pain pathology. Underlying mechanisms and pain-related ROS signaling in the brain remain to be determined. Neuroplasticity in the amygdala plays a key role in emotional-affective pain responses and depends on group I metabotropic glutamate receptors (mGluRs) and protein kinases. Using patch-clamp, live-cell imaging, and behavioral assays, we tested the hypothesis that mitochondrial ROS links group I mGluRs to protein kinase activation to increase neuronal excitability and pain behavior. Agonists for mGluR1/5 (DHPG) or mGluR5 (CHPG) increased neuronal excitability of neurons in the laterocapsular division of the central nucleus of the amygdala (CeLC). DHPG effects were inhibited by an mGluR5 antagonist (MTEP), IP(3) receptor blocker (xestospongin C), or ROS scavengers (PBN, tempol), but not by an mGluR1 antagonist (LY367385) or NO synthase inhibitor (l-NAME). Tempol inhibited the effects of IP(3) but not those of a PKC activator, indicating that ROS activation was IP(3) mediated. Live-cell imaging in CeLC-containing brain slices directly showed DHPG-induced and synaptically evoked mitochondrial superoxide production. DHPG also increased pain-related vocalizations and spinal reflexes through a mechanism that required mGluR5, IP(3), and ROS. Combined application of inhibitors of ERK (U0126) and PKA (KT5720) was necessary to block completely the excitatory effects of a ROS donor (tBOOH). A PKC inhibitor (GF109203X) had no effect. Antagonists and inhibitors alone did not affect neuronal excitability. The results suggest an important role for the novel mGluR5- IP(3)-ROS-ERK/PKA signaling pathway in amygdala pain mechanisms.

Topics: Amygdala; Analysis of Variance; Animals; Antioxidants; Benzoates; Cyclic AMP-Dependent Protein Kinases; Cyclic N-Oxides; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Extracellular Signal-Regulated MAP Kinases; Glycine; Inositol 1,4,5-Trisphosphate; Macrocyclic Compounds; Mitochondria; Neurons; NG-Nitroarginine Methyl Ester; Oxazoles; Pain; Pain Measurement; Pain Perception; Patch-Clamp Techniques; Pyridines; Reactive Oxygen Species; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Signal Transduction; Spin Labels; Superoxides; Thiazoles; Vocalization, Animal

To clarify the molecular mechanism of substance P (SP) release from dorsal root ganglion (DRG) neurons, we investigated the involvement of several intracellular effectors in the regulation of SP release evoked by capsaicin, potassium or/and bradykinin. Bradykinin-evoked SP release from cultured adult rat DRG neurons was attenuated by either the mitogen-activated protein kinase kinase (MEK) inhibitor (U0126) or cycloheximide. As the long-term exposure of DRG neurons to bradykinin (3 h) resulted in extracellular signal-regulated kinase (ERK) phosphorylation at an early stage and thereafter induced cyclooxygenase-2 (COX-2) protein expression, which both contribute to the SP release triggered by bradykinin B2 receptor. The long-term exposure of DRG neurons to bradykinin enhanced the SP release by capsaicin, but attenuated that by potassium. Interestingly, the inositol 1,4,5-triphosphate (IP3)-induced calcium release blocker [2-aminoethyl diphenylborinate (2-APB)] not only inhibited the potassium-evoked SP release, but also completely abolished the enhancement of capsaicin-induced SP release by bradykinin from cultured DRG neurons. Together, these findings suggest that the molecular mechanisms of SP release by bradykinin involve the activation of MEK, and also require the de novo protein synthesis of COX-2 in DRG neurons. The IP3-dependent calcium release could be involved in the processes of the regulation by bradykinin of capsaicin-triggered SP release.

Topics: Animals; Boron Compounds; Bradykinin; Capsaicin; Cells, Cultured; Cyclooxygenase 2; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Ganglia, Spinal; Inositol 1,4,5-Trisphosphate; MAP Kinase Kinase 1; MAP Kinase Signaling System; Neurons; Pain; Potassium; Potassium Chloride; Protein Synthesis Inhibitors; Rats; Rats, Wistar; Receptor, Bradykinin B2; Signal Transduction; Substance P; Type C Phospholipases

The role of intracellular calcium in acute thermal nociception was investigated in the mouse hot-plate test. Intracerebroventricular (i.c.v.) administration of TMB-8, a blocker of Ca++ release from intracellular stores, produced hypernociception. By contrast, i.c.v. pretreatment with thapsigargin, a depletor of Ca++ intracellular stores, produced an increase of the mouse pain threshold. Furthermore, non-analgesic doses of thapsigargin prevented the hypernociception produced by TMB-8. In mice undergoing treatment with heparin, an InsP3-receptor antagonist, or ryanodine, a ryanodine receptor (RyR) antagonist, a dose-dependent reduction of the pain threshold was observed. Pretreatment with D-myo inositol, compound which produces InsP3, and 4-chloro-m-cresol, a RyR agonist, induced an antinociceptive effect. The heparin hypernociception was prevented by D-myo inositol, but not by L-myo inositol, used as negative control. In the same experimental conditions, the antinociception induced by D-myo inositol was prevented by a non-hyperalgesic dose of heparin. Similarly, the reduction of pain threshold produced by ryanodine was reversed by non-analgesic doses of 4-chloro-m-cresol, whereas the antinocicpetion induced by 4-chloro-m-cresol was prevented by non-hyperalgesic doses of ryanodine. The pharmacological treatments employed did not produce any behavioral impairment of mice as revealed by the rota-rod and hole-board tests. These results indicate that a variation of intracellular calcium contents at a supraspinal level is involved in the modulation of acute thermal nociception. In particular, the stimulation of both InsP3- and Ry-receptors appears to play an important role in the induction of antinociception in mice, whereas a blockade of these receptors is involved in an hypernociceptive response to acute thermal pain.

Topics: Acute Disease; Animals; Behavior, Animal; Calcium; Calcium Channel Blockers; Calcium-Transporting ATPases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gallic Acid; Hot Temperature; Hyperalgesia; Injections, Intraventricular; Inositol 1,4,5-Trisphosphate; Male; Mice; Pain; Pain Threshold; Postural Balance; Reaction Time; Ryanodine Receptor Calcium Release Channel; Thapsigargin

Kyotorphin is a dipeptidic neuropeptide (tyrosine-arginine) that has specific receptor coupled to G(i) and phospholipase C and elicits Met-enkephalin release. Here, we attempted to demonstrate the in vivo evidence for the presynaptic mechanism by analyzing its nociceptive responses after peripheral application. Kyotorphin elicited potent nociceptive flexor responses at extremely low doses between 0.1 and 100 fmol after the intraplantar injection into the hind-limb of mice. The site of action of kyotorphin-induced responses was identified to be on nociceptor endings, because the responses were markedly attenuated by intrathecal pretreatments with Galpha(i1) or Galpha(i2) antisense-oligodeoxynucleotides. Similar mechanisms were observed with histamine-induced nociceptive responses, except for the use of different antagonist and Galpha(q/11) antisense-oligodeoxynucleotide. Both responses were characterized to be mediated through inositol trisphosphate receptor-gated Ca(2+) influx, because they were blocked by xestospongin C, an allosteric antagonist for inositol trisphosphate receptor and EGTA, but not thapsigargin. Because the nociceptive responses by compound 48/80 through histamine-release from mast cells were completely abolished by thapsigargin, it is unlikely that the dose of thapsigargin is not sufficient to block both responses. All of these in vivo findings strongly support our previous view that kyotorphin elicits Ca(2+) influx through inositol trisphosphate receptor located at presynaptic plasma membranes.

Topics: Analgesics; Animals; Calcium; Calcium Channels; Endorphins; GTP-Binding Protein alpha Subunits, Gi-Go; Histamine; Injections, Spinal; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Male; Mice; Neurokinin-1 Receptor Antagonists; Pain; Pain Measurement; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Virulence Factors, Bordetella

Stimulation of delta-opioid receptors has been shown to activate phospholipase C via the activation of G-proteins in vitro. The present study was designed to determine, with the tail-flick method, whether the stimulatory effect of delta-opioid receptor agonists on phospholipase C and inositol lipid turnover participates in the mechanisms of the delta-opioid receptor-mediated antinociception in the mouse spinal cord. Intrathecal pretreatment with the phospholipase C inhibitors neomycin and U73122, which produced no changes in the basal tail-flick latencies when they were injected alone, significantly attenuated the antinociception induced by intrathecal administration of the selective delta-opioid receptor agonist [D-Ala(2)]deltorphin II in mice. The selective phosphatidylinositol-specific phospholipase C inhibitor ET-18-OCH(3) inhibited the antinociception induced by intrathecal administration of [D-Ala(2)]deltorphin II in a dose-dependent manner. In mice undergoing treatment with LiCl, which impairs phosphatidylinositol synthesis, the antinociception induced by intrathecal administration of [D-Ala(2)]deltorphin II was significantly reduced. Co-administration of D-myo-inositol-1,4,5-trisphosphate restored the [D-Ala(2)]deltorphin II-induced antinociception in LiCl-pretreated mice. On the other hand, intrathecal pretreatment with the selective protein kinase C inhibitor calphostin C, but not the protein kinase A inhibitor KT5720, resulted in a dose-dependent enhancement of the [D-Ala(2)]deltorphin II-induced antinociception. These results indicate a potential role for the phospholipase C-inositol-1,4, 5-trisphosphate pathway in the expression of delta-opioid receptor-mediated antinociception in the mouse spinal cord. Furthermore, activation of protein kinase C by the stimulation of delta-opioid receptors may constitute a significant pathway involved in negative modulation of spinal delta-opioid receptor-mediated antinociception.

Topics: Analgesics; Animals; Carbazoles; Cyclic AMP-Dependent Protein Kinases; Estrenes; Indoles; Inositol 1,4,5-Trisphosphate; Male; Mice; Mice, Inbred ICR; Naphthalenes; Oligopeptides; Pain; Pain Measurement; Phosphatidylinositols; Protein Kinase C; Pyrroles; Pyrrolidinones; Receptors, Opioid, delta; Signal Transduction; Spinal Cord; Type C Phospholipases

The C57BL/6J-bgJ/bgJ (beige-J) mutation imparts a blunted response to intracerebroventricular (i.c.v.) morphine in the tail-flick test, without altered micro-opioid receptor number or morphine affinity. We now report that co-administration of IP3 (36.1 nmol) restored morphine responsiveness of beige-J mice to essentially that of normal littermates (bg+/bg-; ED50 = 3.9 and 3.5 nmol, respectively). IP3 had no effect on morphine-induced antinociception in control animals. Neither myoinositol at 36.1 nmol nor IP6 at the highest testable dose (4.5 nmol) had a similar effect. Myo-inositol at 5.5 mumol restored beige-J responsiveness to that of littermates. These findings implicate some component of the phosphoinositide cycle in the antinociceptive defect of beige-J mice.

Topics: Analgesics; Animals; Dose-Response Relationship, Drug; Injections, Intraventricular; Inositol; Inositol 1,4,5-Trisphosphate; Male; Mice; Mice, Inbred C57BL; Morphine; Pain; Pain Measurement; Reaction Time; Second Messenger Systems