kt-5720 and Pain

Pain is a complex experience involving sensory and affective components. Although the neuronal mechanisms underlying the sensory component of pain have been extensively studied, those underlying its affective component have yet to be elucidated. Recently, we reported that corticotrophin-releasing factor (CRF)-induced depolarization in type II neurons within the dorsolateral bed nucleus of the stria terminalis (dlBNST) is critical for pain-induced aversive responses in rats. However, the intracellular signaling underlying the excitatory effects of CRF and the contribution of such signaling to the induction of pain-induced aversion remain unclear. In the present study, we addressed these issues by conducting whole-cell patch-clamp recordings in rat brain slices and by undertaking behavioral pharmacological analyses. Intracellular perfusion of protein kinase A (PKA) inhibitor Rp-cyclic adenosine monophosphorothioate (Rp-cAMPS) or KT5720 suppressed the excitatory effects of CRF in type II dlBNST neurons, and bath application of Rp-cAMPS also suppressed it. In addition, bath application of forskolin, an adenylate cyclase (AC) activator, mimicked the effects of CRF, and pretreatment with forskolin diminished the excitatory effects of CRF. Furthermore, a conditioned place aversion (CPA) test showed that co-administration of Rp-cAMPS with CRF into the dlBNST suppressed CRF-induced CPA. Intra-dlBNST injection of Rp-cAMPS also suppressed pain-induced CPA. These results suggest that CRF increases excitability of type II dlBNST neurons through activation of the AC-cAMP-PKA pathway, thereby causing pain-induced aversive responses. The present findings shed light on the neuronal mechanisms underlying the negative affective component of pain and may provide therapeutic targets for treating intractable pain accompanied by psychological factors.

Topics: Adenylyl Cyclases; Animals; Carbazoles; Colforsin; Conditioning, Classical; Corticotropin-Releasing Hormone; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Male; Pain; Pyrroles; Rats; Rats, Sprague-Dawley; Septal Nuclei; Signal Transduction

Proinflammatory agents trypsin and mast cell tryptase cleave and activate protease-activated receptor-2 (PAR-2), which is expressed on sensory nerves and causes neurogenic inflammation. P2X3 is a subtype of the ionotropic receptors for adenosine 5'-triphosphate (ATP), and is mainly localized on nociceptors. Here, we show that a functional interaction of the PAR-2 and P2X3 in primary sensory neurons could contribute to inflammatory pain. PAR-2 activation increased the P2X3 currents evoked by α, β, methylene ATP in dorsal root ganglia (DRG) neurons. Application of inhibitors of either protein kinase C (PKC) or protein kinase A (PKA) suppressed this potentiation. Consistent with this, a PKC or PKA activator mimicked the PAR-2-mediated potentiation of P2X3 currents. In the in vitro phosphorylation experiments, application of a PAR-2 agonist failed to establish phosphorylation of the P2X3 either on the serine or the threonine site. In contrast, application of a PAR-2 agonist induced trafficking of the P2X3 from the cytoplasm to the plasma membrane. These findings indicate that PAR-2 agonists may potentiate the P2X3, and the mechanism of this potentiation is likely to be a result of translocation, but not phosphorylation. The functional interaction between P2X3 and PAR-2 was also confirmed by detection of the α, β, methylene-ATP-evoked extracellular signal-regulated kinases (ERK) activation, a marker of neuronal signal transduction in DRG neurons, and pain behavior. These results demonstrate a functional interaction of the protease signal with the ATP signal, and a novel mechanism through which protease released in response to tissue inflammation might trigger the sensation to pain through P2X3 activation.

Topics: Adenosine Triphosphate; Animals; Carbazoles; Colforsin; Cyclic AMP-Dependent Protein Kinases; Ganglia, Spinal; Indoles; Inflammation; Male; Maleimides; MAP Kinase Signaling System; Membrane Potentials; Neurons; Pain; Phosphorylation; Protein Kinase C; Protein Transport; Purinergic P2X Receptor Agonists; Purinergic P2X Receptor Antagonists; Pyrroles; Rats, Sprague-Dawley; Receptor, PAR-2; Receptors, Purinergic P2X3; Tetradecanoylphorbol Acetate

The laterocapsular division of the central nucleus of the amygdala (CeLC) has emerged as an important site of pain-related plasticity and pain modulation. Glutamate and neuropeptide receptors in the CeLC contribute to synaptic and behavioral changes in the arthritis pain model, but the intracellular signaling pathways remain to be determined. This study addressed the role of PKA, PKC, and ERK in the CeLC. Adult male Sprague-Dawley rats were used in all experiments. Whole-cell patch-clamp recordings of CeLC neurons were made in brain slices from normal rats and from rats with a kaolin/carrageenan-induced monoarthritis in the knee (6 h postinduction). Membrane-permeable inhibitors of PKA (KT5720, 1 microM; cAMPS-Rp, 10 microM) and ERK (U0126, 1 microM) activation inhibited synaptic plasticity in slices from arthritic rats but had no effect on normal transmission in control slices. A PKC inhibitor (GF109203x, 1 microM) and an inactive structural analogue of U0126 (U0124, 1 microM) had no effect. The NMDA receptor-mediated synaptic component was inhibited by KT5720 or U0126; their combined application had additive effects. U0126 did not inhibit synaptic facilitation by forskolin-induced PKA-activation. Administration of KT5720 (100 microM, concentration in microdialysis probe) or U0126 (100 microM) into the CeLC, but not striatum (placement control), inhibited audible and ultrasonic vocalizations and spinal reflexes of arthritic rats but had no effect in normal animals. GF109203x (100 microM) and U0124 (100 microM) did not affect pain behavior. The data suggest that in the amygdala PKA and ERK, but not PKC, contribute to pain-related synaptic facilitation and behavior by increasing NMDA receptor function through independent signaling pathways.

Topics: Amygdala; Animals; Arthritis; Behavior; Butadienes; Carbazoles; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Disease Models, Animal; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Indoles; Male; Maleimides; Neuronal Plasticity; Neurons; Nitriles; Pain; Protein Kinase C; Protein Kinase Inhibitors; Pyrroles; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Synaptic Transmission; Thionucleotides

Male Swiss Webster mice exhibited antinociception, hypothermia and Straub tail 3h following a 75mg morphine pellet implantation. These signs disappeared by 72h, and the morphine-pelleted mice were indistinguishable from placebo-pelleted ones, although brain morphine concentrations ranged from 200 to 400ng/gm. We previously demonstrated that chemical inhibitors of protein kinase C (PKC) and A (PKA) are able to reverse morphine tolerance in acutely morphine-challenged mice. However, it was not known whether the reversal of tolerance was due to the interaction of kinase inhibitors with the morphine released from the pellet, the acutely injected morphine to challenge tolerant mice, or both. The present study aimed at determining the interaction between the PKC and PKA inhibitors and the morphine released "solely" from the pellet to reinstate the morphine-induced behavioral and physiological effects, 72h after implantation of morphine pellets. Placebo or 75mg morphine pellets were surgically implanted, and testing was conducted 72h later. Our results showed that the intracerebroventricular (i.c.v.) administration of the PKC inhibitors, bisindolylmaleimide I and Gö-6976 as well as the PKA inhibitors, 4-cyano-3-methylisoquinoline and KT-5720, restored the morphine-induced behaviors of antinociception, Straub tail and hypothermia in morphine-pelleted mice to the same extent observed 3h following the pellet implantation. The tail withdrawal and the hot plate reaction time expressed as percent maximum possible effect (%MPE) was increased to 80-100 and 41-90%, respectively, in PKC and PKA inhibitor-treated morphine tolerant mice compared to 2-10% in non-treated mice. Similarly, a significant hypothermia (1.3-4.0 degrees C decrease in body temperature) was detected in PKC and PKA inhibitor-treated morphine tolerant mice compared to an euthermic state in non-treated morphine tolerant mice. Finally, the Straub tail score was increased to 1.1-1.6 in PKC and PKA inhibitor-treated tolerant mice, whereas it was totally absent in non-treated animals. It is noticeably that the kinase inhibitors used in the study had no effect in placebo-pelleted mice. Our results provide the first evidence on the ability of PKC and PKA inhibitors to reinstate the behavioral and physiological effects of morphine in non-challenged morphine-tolerant animals.

Topics: Analgesics, Opioid; Animals; Behavior, Animal; Body Temperature; Carbazoles; Cyclic AMP-Dependent Protein Kinases; Drug Implants; Drug Tolerance; Enzyme Inhibitors; Hot Temperature; Hypothermia; Indoles; Injections, Intraventricular; Isoquinolines; Male; Maleimides; Mice; Morphine; Pain; Protein Kinase C; Pyrroles; Reaction Time

We have previously reported that intracerebroventricular (i.c.v.) injection of either a PKC or PKA inhibitor completely reversed the expression of 5- to 8-fold morphine antinociceptive tolerance. We developed a model of 45-fold morphine tolerance that included a 75-mg morphine pellet and twice daily morphine injections. PKC inhibitor doses of bisindolylmaleimide I and Gö-7874 that completely reversed 8-fold tolerance only partly reversed the 45-fold level of antinociceptive tolerance. A component of tolerance was resistant to PKC inhibition, since even higher inhibitor doses failed to further reverse the high level of morphine tolerance. In addition, the 45-fold tolerance was only partly reversed by the PKA inhibitor KT-5720 at a dose previously cited by others to reverse 5-fold tolerance. Another PKA inhibitor 4-cyano-3-methylisoquinoline only partly reversed the morphine tolerance as well. In other experiments PKC and PKA inhibitors were co-administered together to determine their effectiveness for completely reversing the 45-fold level of morphine tolerance. Co-administering either bisindolylmaleimide I with KT-5720, or Gö-7874 with KT-5720, completely reversed the high level of tolerance. The high level of morphine tolerance was also completely reversed by co-administering Gö-7874 with 4-cyano-3-methylisoquinoline. Thus, high levels of morphine tolerance may reflect increases in protein phosphorylation by the terminal kinases of both the adenylyl cyclase and phosphatidylinositol cascades in brain and spinal cord areas critical to the expression of antinociception.

Topics: Analgesics; Animals; Brain; Carbazoles; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Drug Interactions; Drug Therapy, Combination; Drug Tolerance; Enzyme Inhibitors; Indoles; Injections, Intraventricular; Isoquinolines; Male; Maleimides; Mice; Morphine; Morphine Dependence; Pain; Protein Kinase C; Protein Serine-Threonine Kinases; Pyrroles

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

We examined the tail-flick response to various heat intensities in diabetic and non-diabetic mice. Heat intensities were set to one of five values by adjusting the source voltage of a 50-W projection bulb to 25, 35, 50, 65 and 80 V. These heat intensities produced surface skin heating rates of 0.1, 0.4, 0.9, 3.0 and 7.3 degrees C/s, respectively. Tail-flick latencies at source voltages of 35 and 50 V in diabetic mice were significantly shorter than those in non-diabetic mice. However, there were no significant differences in tail-flick latencies at 25, 65 and 80 V. In non-diabetic mice, tail-flick latencies were not affected by intrathecal (i.t.) pretreatment with capsaicin 24 h before testing. Tail-flick latencies at 35 and 50 V in diabetic mice were increased by pretreatment with capsaicin. Moreover, although tail-flick latencies in non-diabetic mice were not affected by i.t. pretreatment with calphostin C, a selective protein kinase C inhibitor, those at 35 and 50 V in diabetic mice were increased. However, i.t. pretreatment with (8R, 9S, 11S)-(-)-9-hydroxy-9-n-hexyloxy-carbonyl-8-methyl-2, 3, 9, 10-tetrahydro-8, 11-epoxy-1H, 8H, 11H-2, 7b, 11a-triazadibenzo [a, g]cycloocta[cde]-trinden-1-one (KT5720), a selective protein kinase A inhibitor, did not affect tail-flick latencies in either diabetic or non-diabetic mice. In non-diabetic mice, i.t. pretreatment with phorbol 12,13-dibutyrate (PDB), a protein kinase C activator, decreased tail-flick latencies at 35 and 50 V. Tail-flick latencies in diabetic mice were not affected by i.t. pretreatment with PDB 60 min before testing. Furthermore, the attenuation of tail-flick latencies induced by i.t. pretreatment with PDB in non-diabetic mice was reversed by i.t. pretreatment with capsaicin 24 h before testing. These results indicate that diabetic mice exhibit thermal allodynia and hyperalgesia. Furthermore, this thermal allodynia and hyperalgesia in diabetic mice may be due to the enhanced release of substance P followed by activation of protein kinase C in the spinal cord.

Topics: Analysis of Variance; Animals; Capsaicin; Carbazoles; Carcinogens; Cyclic AMP-Dependent Protein Kinases; Diabetes Mellitus; Enzyme Inhibitors; Hot Temperature; Hyperalgesia; Indoles; Male; Mice; Mice, Inbred ICR; Naphthalenes; Pain; Pain Measurement; Phorbol 12,13-Dibutyrate; Protein Kinase C; Pyrroles; Spinal Cord