cyclic-gmp and Peripheral-Nervous-System-Diseases

A large body of evidence indicates that nitric oxide (NO) and cGMP contribute to central sensitization of pain pathways during inflammatory pain. Here, we investigated the distribution of cyclic nucleotide-gated (CNG) channels in the spinal cord, and identified the CNG channel subunit CNGA3 as a putative cGMP target in nociceptive processing. In situ hybridization revealed that CNGA3 is localized to inhibitory neurons of the dorsal horn of the spinal cord, whereas its distribution in dorsal root ganglia is restricted to non-neuronal cells. CNGA3 expression is upregulated in the superficial dorsal horn of the mouse spinal cord and in dorsal root ganglia following hindpaw inflammation evoked by zymosan. Mice lacking CNGA3 (CNGA3(-/-) mice) exhibited an increased nociceptive behavior in models of inflammatory pain, whereas their behavior in models of acute or neuropathic pain was normal. Moreover, CNGA3(-/-) mice developed an exaggerated pain hypersensitivity induced by intrathecal administration of cGMP analogs or NO donors. Our results provide evidence that CNGA3 contributes in an inhibitory manner to the central sensitization of pain pathways during inflammatory pain as a target of NO/cGMP signaling.

Topics: Analysis of Variance; Animals; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Disease Models, Animal; Enzyme Inhibitors; Ganglia, Spinal; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Inflammation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microdissection; Naphthalenes; Natriuretic Peptides; Nitric Oxide; Pain; Pain Measurement; Pain Perception; Peripheral Nervous System Diseases; Physical Stimulation; RNA, Messenger; Signal Transduction; Spinal Cord; Stathmin; Statistics, Nonparametric; Thionucleotides; Triazenes; Vesicular Inhibitory Amino Acid Transport Proteins

The cGMP/cGMP-dependent protein kinase I (cGKI) signaling pathway plays an important role in spinal nociceptive processing. However, downstream targets of cGKI in this context have not been identified to date. Using a yeast two-hybrid screen, we isolated cysteine-rich protein 2 (CRP2) as a novel cGKI interactor in the spinal cord. CRP2 is expressed in laminas I and II of the mouse spinal cord and is colocalized with cGKI, calcitonin gene-related peptide, and isolectin B4. Moreover, the majority of CRP2 mRNA-positive dorsal root ganglion (DRG) neurons express cGKI and peripherin. CRP2 is phosphorylated in a cGMP-dependent manner, and its expression increases in the spinal cord and in DRGs after noxious stimulation of a hindpaw. To elucidate the functional role of CRP2 in nociception, we analyzed mice with a targeted deletion of CRP2. CRP2-deficient (CRP2-/-) mice demonstrate normal behavioral responses to acute nociception and after axonal injury of the sciatic nerve, but increased nociceptive behavior in models of inflammatory hyperalgesia compared with wild-type mice. Intrathecal administration of cGMP analogs increases the nociceptive behavior in wild-type but not in CRP2-/- mice, indicating that the presence of CRP2 is important for cGMP-mediated nociception. These data suggest that CRP2 is a new downstream effector of cGKI-mediated spinal nociceptive processing and point to an inhibitory role of CRP2 in the generation of inflammatory pain.

Topics: Animals; Chronic Disease; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type I; Cyclic GMP-Dependent Protein Kinases; Ganglia, Spinal; Inflammation Mediators; LIM Domain Proteins; Mice; Mice, Knockout; Muscle Proteins; Nuclear Proteins; Pain; Peripheral Nervous System Diseases; Rats; Signal Transduction; Spinal Cord

The possible involvement of the nitric oxide (NO)-cyclic GMP (cGMP)-protein kinase G (PKG) pathway on bovine lactoferrin (BLF)-induced spinal antihyperalgesic activity was elucidated in sciatic nerve injured rats. Intrathecal BLF reduced thermal hyperalgesia in a dose-dependent manner. Pretreatment with NG-L-nitro-arginine methyl ester (L-NAME, non-specific inhibitor of NO synthase), 7-nitroindazole (7-NI, neuronal NO synthase inhibitor), 1H-[1,2,4]-oxadiazolo [4,3-a] quinoxalin-1-one (ODQ, guanylyl-cyclase inhibitor), (9S, 10R, 12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2, 9-dimethyl-1-oxo-9, 12-epoxy-1H-diindolo-[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT-5823, specific PKG inhibitor) or glybenclamide (ATP-sensitive K+ channel blocker), but not NG-D-nitro-arginine methyl ester (D-NAME, an inactive enantiomer of l-NAME), d-Phe-Cys-Tyr-d-Trp-Orn-Thr-NH2 (CTOP, selective mu-opioid receptor antagonist) or naloxone (nonselective opioid receptor antagonist) prevented BLF-induced antihyperalgesia. Data suggest that BLF-induced spinal antihyperalgesia could be due to activation of the NO-cGMP-PKG-K+ channel pathway and it is not mediated by mu-opioid receptor in a model of neuropathic pain.

Topics: Analgesics; Animals; Cattle; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hyperalgesia; Injections, Spinal; Lactoferrin; Male; Narcotic Antagonists; Neurons, Afferent; Nitric Oxide; Peripheral Nervous System Diseases; Potassium Channel Blockers; Potassium Channels; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Signal Transduction

Injury or inflammation affecting sensory neurons in dorsal root ganglia (DRG) causes hyperexcitability of DRG neurons that can lead to spontaneous firing and neuropathic pain. Recent results indicate that after chronic compression of DRG (CCD treatment), both hyperexcitability of neurons in intact DRG and behaviorally expressed hyperalgesia are maintained by concurrent activity in cAMP-protein kinase A (PKA) and cGMP-protein kinase G (PKG) signaling pathways. We report here that when tested under identical conditions, dissociation produces a pattern of hyperexcitability in small DRG neurons similar to that produced by CCD treatment, manifest as decreased action potential (AP) current threshold, increased AP duration, increased repetitive firing to depolarizing pulses, increased spontaneous firing and resting depolarization. A novel feature of this hyperexcitability is its early expression-as soon as testing can be conducted after dissociation (approximately 2 h). Both forms of injury increase the electrophysiological responsiveness of the neurons to activation of cAMP-PKA and cGMP-PKG pathways as indicated by enhancement of hyperexcitability by agonists of these pathways in dissociated or CCD-treated neurons but not in control neurons. Although inflammatory signals are known to activate cAMP-PKA pathways, dissociation-induced hyperexcitability is unlikely to be triggered by signals released from inflammatory cells recruited to the DRG because of insufficient time for recruitment during the dissociation procedure. Inhibition by specific antagonists indicates that continuing activation of cAMP-PKA and cGMP-PKG pathways is required to maintain hyperexcitability after dissociation. The reduction of hyperexcitability by blockers of adenylyl cyclase and soluble guanylyl cyclase after dissociation suggests a continuing release of autocrine and/or paracrine factors from dissociated neurons and/or satellite cells, which activate both cyclases and help to maintain acute, injury-induced hyperexcitability of DRG neurons.

Topics: Action Potentials; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Artifacts; Cells, Cultured; Cyclic AMP; Cyclic GMP; Dissection; Enzyme Inhibitors; Ganglia, Spinal; Guanylate Cyclase; Hyperalgesia; Male; Neurons, Afferent; Nociceptors; Peripheral Nervous System Diseases; Rats; Rats, Sprague-Dawley; Signal Transduction; Stress, Mechanical; Stress, Physiological; Up-Regulation

Topics: Animals; Artifacts; Cyclic AMP; Cyclic GMP; Ganglia, Spinal; Humans; Models, Biological; Neurons, Afferent; Nociceptors; Pain; Peripheral Nervous System Diseases; Signal Transduction; Stress, Physiological

Axotomised dorsal root ganglia (DRG) neurons show an increased expression of neuronal nitric oxide synthase (nNOS) compared with neurons from the intact ganglia. Increased nNOS expression resulted in synthesis of nitric oxide (NO) and the subsequent activation of cGMP in satellite glia cells surrounding the DRG neuron soma. In dissociated DRG we have demonstrated that the increase in nNOS expression is regulated by nerve growth factor and that the subsequent inhibition of NO production or cGMP synthesis precipitates apoptosis of neurons expressing nNOS and some non-nNOS neurons. Hence, NO or the NO-cGMP cascade appears to have a neuroprotective action in trophic factor-deprived DRG neurons. In the present study, using immunocytochemistry, we have investigated some of the factors associated with apoptosis that are activated when nNOS activity is blocked with NOS inhibitor in DRG neurons in vitro. Marked elevation of bax was observed within a few hours of NOS inhibition in nNOS containing neurons, whereas pretreatment of cultures with l-arginine completely abolished this effect in almost all nNOS neurons and 8-bromo-cGMP in some neurons. The apoptosis precipitated by NOS inhibition was also partially prevented by a number of caspase inhibitors; of those a caspase-9 blocker was the most effective. These observations further support the neuroprotective role of NO/NO-cGMP in stressed DRG neurons in an autocrine fashion that involves the suppression of bax, caspase-3 and -9 activation.

Topics: Animals; Animals, Newborn; Apoptosis; Arginine; Autocrine Communication; bcl-2-Associated X Protein; Caspase Inhibitors; Caspases; Cells, Cultured; Cyclic GMP; Enzyme Inhibitors; Female; Ganglia, Spinal; Immunohistochemistry; Male; Nerve Degeneration; Neurons, Afferent; Nitric Oxide; Nitric Oxide Synthase; Peripheral Nervous System Diseases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Signal Transduction; Stress, Physiological