h-89 and Pain

Recent studies have shown the 5-HT

Topics: Adenine; Animals; Behavior, Animal; Gene Expression Regulation; Genes, fos; Indoles; Isoquinolines; Male; Methylamines; Nociception; Pain; Pain Measurement; Piperazines; Prefrontal Cortex; Protein Kinase Inhibitors; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Serotonin; Serotonin Receptor Agonists; Spinal Cord; Sulfonamides

It has been shown that spinal PKA/CREB signaling pathway is involved in neuropathic and inflammatory pain, but its effects on bone cancer pain have not previously been investigated. The aim of this study was to examine the potential role of the spinal PKA/CREB signaling pathway in the development of bone cancer pain.. A bone cancer pain model was made by inoculation of Walker 256 cells into the intramedullary space of rat tibia. Western blot analysis examined the expression of PKAca (PKA catalytic subunit) and phospho-CREB (p-CREB) protein levels. The authors further investigated effects of intrathecal treatment with H-89 (a PKA inhibitor, 8 nmol) or forskolin (a PKA agonist, 10 nmol) on nociceptive behavior and the expression of PKAca and p-CREB.. On days 6, 9, and 15 after inoculation, the expression of PKAca and p-CREB protein levels were higher in the bone cancer pain rats compared to the sham rats. On day 9, intrathecal administration of H-89 significantly attenuated bone cancer-induced mechanical allodynia as well as upregulation of PKAca and p-CREB protein levels. These effects were completely abolished by intrathecal pretreatment with the PKA agonist forskolin.. The results suggest that the spinal PKA/CREB signaling pathway may participate in the development of bone cancer pain. The findings of this study may provide an evidence for developing novel analgesics to block bone cancer pain.

Topics: Animals; Bone Neoplasms; Colforsin; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Female; Isoquinolines; Pain; Pain Measurement; Posterior Horn Cells; Rats; Rats, Sprague-Dawley; Signal Transduction; Sulfonamides; Up-Regulation

Bone cancer pain is difficult to treat and has a strong impact on the quality of life of patients. Few therapies have emerged because the molecular mechanisms underlying bone cancer pain are poorly understood. Recently, T-cell death-associated gene 8 (TDAG8) has been shown to participate in complete Freund's adjuvant-induced chronic inflammatory pain. In this study, we aimed to examine whether TDAG8 and its downstream protein kinase A (PKA) pathway are involved in bone cancer pain. A bone cancer pain model was made by inoculation of Walker 256 cells into the intramedullary space of rat tibia. Spinal TDAG8 expression was increased after inoculation with tumor cells. Intrathecal TDAG8 siRNA attenuated bone cancer pain behaviors during the initiation and maintenance phases; there were also concomitant decreases in TDAG8 mRNA and protein levels in spinal cord. Moreover, we found spinal PKA and phosphorylated cAMP response element-binding (pCREB) protein levels were up-regulated in the rat model of bone cancer pain. Knockdown of TDAG8 resulted in reduced bone cancer pain-induced spinal PKA and pCREB protein expression in two procedures. Furthermore, intrathecal H-89 (a PKA inhibitor) significantly attenuated bone cancer pain behaviors in rats. Our results suggest a causal relationship between TDAG8 expression and the initiation and maintenance of bone cancer pain. Activation of spinal TDAG8 contributes to bone cancer pain through the PKA signaling pathway in rats. These findings may lead to novel strategies for the treatment of bone cancer pain.

Topics: Animals; Bone Neoplasms; Carcinoma 256, Walker; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Female; Isoquinolines; Neoplasm Transplantation; Pain; Posterior Horn Cells; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled; RNA, Small Interfering; Sulfonamides; Up-Regulation

We have recently shown that gabapentin generates protein kinase A (PKA)-dependent presynaptic inhibition of GABAergic synaptic transmission in locus coeruleus (LC) neurons only under neuropathic states. To verify behaviorally this in vitro electrophysiological finding, the PKA inhibitor H-89 was injected intracerebroventricularly (i.c.v.) before supraspinal application of gabapentin in mice developing thermal and mechanical hypersensitivity after peripheral nerve injury. H-89 dose-dependently attenuated the analgesic effects of i.c.v.-injected gabapentin, suggesting that PKA-dependent removal of GABAergic inhibition of LC neurons is the most plausible synaptic mechanism underlying the supraspinally mediated analgesic effects of gabapentin involving activation of the descending noradrenergic pain-inhibitory system.

Topics: Amines; Analgesics; Animals; Cyclic AMP-Dependent Protein Kinases; Cyclohexanecarboxylic Acids; Dose-Response Relationship, Drug; Gabapentin; gamma-Aminobutyric Acid; Injections, Intraventricular; Isoquinolines; Locus Coeruleus; Male; Mice; Pain; Pain Measurement; Protein Kinase Inhibitors; Sulfonamides

The roles of central protein kinases A and C (PKA and PKC) in various pain states have intensively been investigated during the past decade. The aim of the present study was to investigate the peripheral involvement of PKA and PKC in persistent nociceptive response, evoked pain behaviors, and inflammation induced by subcutaneous (s.c.) injection of bee venom (BV, 0.2mg/50 microl) in rats. The effects of intraplantar injection of H-89 (a PKA inhibitor, 5-100 microg/50 microl) and chelerythrine chloride (a PKC inhibitor, 5-100 microg/50 microl) on BV-elicited persistent nociception (nociceptive flinching reflex), mechanical hyperalgesia, and inflammation were systematically investigated. Pre-treatment with H-89 dose-dependently inhibited only BV-induced mechanical hyperalgesia, but not the persistent nociception and inflammation. In contrast, pre-treatment with chelerythrine chloride dose-dependently inhibited BV-induced sustained nociception and inflammation, but not the mechanical hyperalgesia. Topical pre-treatment of the sciatic nerve with 1% capsaicin significantly blocked the inhibitory effects of the PKC inhibitor on BV-induced inflammation, but not the persistent flinching response. These results indicate that peripheral PKA and PKC involvements in BV-induced pain behaviors differ, and capsaicin-sensitive afferents appear to participate in the pro-inflammatory role of PKC in the BV pain model. Findings from the present study also suggest that targeting specific peripheral protein kinases might prove effective in the treatment of persistent pain and inflammation.

Topics: Analysis of Variance; Animals; Bee Venoms; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Functional Laterality; Hyperalgesia; Inflammation; Injections, Subcutaneous; Isoquinolines; Male; Pain; Pain Measurement; Plethysmography; Protein Kinase C; Random Allocation; Rats; Rats, Sprague-Dawley; Reaction Time; Sulfonamides

Visceral noxious stimulation induces central neuronal plasticity changes and suggests that the c-AMP-dependent protein kinase (PKA) signal transduction cascade contributes to long-term changes in nociceptive processing at the spinal cord level. Our previous studies reported the clinical neurosurgical interruption of post synaptic dorsal column neuron (PSDC) pathway by performing midline myelotomy effectively alleviating the intractable visceral pain in patients with severe pain. However, the intracellular cascade in PSDC neurons mediated by PKA nociceptive neurotransmission was not known. In this study, by using multiple experimental approaches, we investigated the role of PKA in nociceptive signaling in the spinal cord and PSDC neurons in a visceral pain model in rats with the intracolonic injection of mustard oil. We found that mustard oil injection elicited visceral pain that significantly changed exploratory behavior activity in rats in terms of decreased numbers of entries, traveled distance, active and rearing time, rearing activity and increased resting time when compared to that of rats receiving mineral oil injection. However, the intrathecal infusion of PKA inhibitor, H89 partially reversed the visceral pain-induced effects. Results from Western blot studies showed that mustard oil injection significantly induced the expression of PKA protein in the lumbosacral spinal cord. Immunofluorescent staining in pre-labeled PSDC neurons showed that mustard oil injection greatly induces the neuronal profile numbers. We also found that the intrathecal infusion of a PKA inhibitor, H89 significantly blocked the visceral pain-induced phosphorylation of c-AMP-responsive element binding (CREB) protein in spinal cord in rats. The results of our study suggest that the PKA signal transduction cascade may contribute to visceral nociceptive changes in spinal PSDC pathways.

Topics: Animals; Behavior, Animal; Blotting, Western; Catheters, Indwelling; Cyclic AMP-Dependent Protein Kinases; Disease Models, Animal; Injections, Spinal; Isoquinolines; Male; Mustard Plant; Neurons; Pain; Plant Oils; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Signal Transduction; Spinal Cord; Sulfonamides

Glutamatergic inputs arising from the parabrachial nucleus to neurons in the lateral sector of the central amygdala were studied in vitro. Tetanic stimulation of these inputs led to LTP that did not require activation of NMDA receptors or a rise of postsynaptic calcium. LTP was accompanied by a reduction in the paired-pulse ratio, indicating that LTP results from an increase in transmitter release probability. Activation of adenylyl cyclase with forskolin potentiated these inputs with a similar reduction in paired-pulse facilitation and occluded LTP induction. LTP was inhibited by the protein kinase A blocker H89. Low-frequency stimulation led to LTD that required activation of postsynaptic NMDA receptors and a rise in postsynaptic calcium. There was no change in paired-pulse facilitation with LTD. LTD was blocked by protein phosphatase blockers calyculin and okadaic acid. We conclude that parabrachial inputs to the lateral sector of the central amygdala show presynaptic LTP that requires activation of a presynaptic protein kinase A via a calcium-dependent adenylyl cyclase while LTD at the same synapses is postsynaptic and requires a rise in postsynaptic calcium and activation of protein phosphatase.

Topics: Adenylyl Cyclases; Amygdala; Animals; Calcium Signaling; Colforsin; Cyclic AMP-Dependent Protein Kinases; Electric Stimulation; Enzyme Activation; Enzyme Activators; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Glutamic Acid; In Vitro Techniques; Isoquinolines; Long-Term Potentiation; Long-Term Synaptic Depression; Neuronal Plasticity; Neurons, Afferent; Nociceptors; Okadaic Acid; Pain; Phosphoprotein Phosphatases; Presynaptic Terminals; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Sulfonamides; Synaptic Transmission

Chronic opioid-induced drug dependence and withdrawal syndrome after opioid cessation remain a severe obstacle in clinical treatment of chronic pain and opioid drug addiction. One of the key symptoms during opioid withdrawal is a state of sensitized pain. The most significant molecular adaptation induced by chronic opioids in the brain is upregulation of the cAMP-signaling pathway. Although the cAMP system is known to have multiple effects on central neuron functions, how its upregulation mediates behavioral opioid dependence and withdrawal-induced pain in vivo remains unclear. In this study, we demonstrate that withdrawal from chronic morphine significantly upregulates the mRNA level of adenylyl cyclase (AC) VI and VIII isoforms and immunoreactivity of ACV/VI in the nucleus raphe magnus (NRM), a brainstem site critically involved in opioid modulation of pain. In cellular studies of NRM neurons containing mu-opioid receptors, we show that morphine withdrawal significantly increases glutamate synaptic transmission via a presynaptic mechanism mediated by an upregulated cAMP pathway. Morphine withdrawal also enhances the hyperpolarization-activated current in these neurons by increased intracellular cAMP. Both of the withdrawal-induced cAMP actions increase the excitability of these mu-receptor-containing neurons, which are thought to facilitate spinal pain transmission. Furthermore, in morphine-dependent rats in vivo, blocking the cAMP pathway significantly reduces withdrawal-induced pain sensitization. These results illustrate neurobiological mechanisms for the cAMP-mediated withdrawal pain and provide potential therapeutic targets for the treatment of opioid dependence and withdrawal-related problems.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; 8-Bromo Cyclic Adenosine Monophosphate; Adenylyl Cyclases; Animals; Animals, Newborn; Behavior, Animal; Blotting, Western; Colforsin; Cyclic AMP; Dose-Response Relationship, Radiation; Drug Administration Schedule; Drug Interactions; Electric Stimulation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Gene Expression Regulation; Imines; In Vitro Techniques; Isoquinolines; Male; Membrane Potentials; Microinjections; Morphine; Naloxone; Narcotic Antagonists; Narcotics; Opioid-Related Disorders; Oxidoreductases; Pain; Pain Measurement; Patch-Clamp Techniques; Pyrimidines; Raphe Nuclei; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulfonamides