kn-93 and Pain

Topics: Analgesia; Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Electroacupuncture; Freund's Adjuvant; Inflammation; Injections, Intraventricular; Male; Mice; Mice, Inbred C57BL; Pain; Pain Management; Sulfonamides

Pain is one of the most challenging and stressful conditions to patients with sickle cell disease (SCD) and their clinicians. Patients with SCD start experiencing pain as early as 3 months old and continue having it throughout their lives. Although many aspects of the disease are well understood, little progress has been made in understanding and treating pain in SCD. This study aimed to investigate the functional involvement of Ca/calmodulin-dependent protein kinase II (CaMKIIα) in the persistent and refractory pain associated with SCD. We found that nonevoked ongoing pain as well as evoked hypersensitivity to mechanical and thermal stimuli were present in Berkeley sickle cell transgenic mice (BERK mice), but not nonsickle control littermates. Prominent activation of CaMKIIα was observed in the dorsal root ganglia and spinal cord dorsal horn region of BERK mice. Intrathecal administration of KN93, a selective inhibitor of CaMKII, significantly attenuated mechanical allodynia and heat hyperalgesia in BERK mice. Meanwhile, spinal inhibition of CaMKII elicited conditioned place preference in the BERK mice, indicating the contribution of CaMKII in the ongoing spontaneous pain of SCD. We further targeted CaMKIIα by siRNA knockdown. Both evoked pain and ongoing spontaneous pain were effectively attenuated in BERK mice. These findings elucidated, for the first time, an essential role of CaMKIIα as a cellular mechanism in the development and maintenance of spontaneous and evoked pain in SCD, which can potentially offer new targets for pharmacological intervention of pain in SCD.

Topics: Anemia, Sickle Cell; Anesthetics, Local; Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Conditioning, Operant; Disease Models, Animal; Enzyme Inhibitors; Formaldehyde; Ganglia, Spinal; Gene Expression Regulation; Hemoglobins; Hyperalgesia; Inflammation; Lidocaine; Mice; Mice, Inbred C57BL; Mice, Transgenic; Pain; Pain Threshold; Physical Stimulation; Spinal Cord; Sulfonamides

The N-methyl-d-aspartate receptor (NMDAR) containing subunit 2B (NR2B) is critical for the regulation of nociception in bone cancer pain, although the precise molecular mechanisms remain unclear. KIF17, a kinesin motor, plays a key role in the dendritic transport of NR2B. The up-regulation of NR2B and KIF17 transcription results from an increase in phosphorylated cAMP-response element-binding protein (CREB), which is activated by calcium/calmodulin-dependent protein kinase II (CaMKII). In this study, we hypothesized that CaMKII-mediated KIF17/NR2B trafficking may contribute to bone cancer pain. Osteosarcoma cells were implanted into the intramedullary space of the right femurs of C3H/HeJ mice to induce progressive bone cancer-related pain behaviors. The expression of spinal t-CaMKII, p-CaMKII, NR2B and KIF17 after inoculation was also evaluated. These results showed that inoculation of osteosarcoma cells induced progressive bone cancer pain and resulted in a significant up-regulation of p-CaMKII, NR2B and KIF17 expression after inoculation. Intrathecal administration of KN93, a CaMKII inhibitor, down-regulated these three proteins and attenuated bone cancer pain in a dose- and time-dependent manner. These findings indicated that CaMKII-mediated KIF17/NR2B trafficking may contribute to bone cancer pain, and inhibition of CaMKII may be a useful alternative or adjunct therapy for relieving cancer pain.

Topics: Animals; Benzylamines; Bone Neoplasms; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Line, Tumor; Injections, Spinal; Kinesins; Mice; Mice, Inbred C3H; Pain; Protein Kinase Inhibitors; Protein Transport; Receptors, N-Methyl-D-Aspartate; Sulfonamides

Calcium/calmodulin-dependent protein kinase II (CaMKII) is considered an important enzyme contributing to the pathogenesis of persistent pain. The aim of this study was to test whether intrathecal injection of CaMKII inhibitors may reduce pain-related behavior in diabetic rats. Male Sprague-Dawley rats were used. Diabetes was induced with intraperitoneal injection of 55mg/kg streptozotocin. Two weeks after diabetes induction, CaMKII inhibitor myristoil-AIP or KN-93 was injected intrathecally. Behavioral testing with mechanical and thermal stimuli was performed before induction of diabetes, the day preceding the injection, as well as 2h and 24h after the intrathecal injection. The expression of total CaMKII and its alpha isoform in dorsal horn was quantified using immunohistochemistry. Intrathecal injection of mAIP and KN-93 resulted in significant decrease in expression of total CaMKII and CaMKII alpha isoform activity. Also, mAIP and KN93 injection significantly increased sensitivity to a mechanical stimulus 24h after i.t. injection. Intrathecal inhibition of CaMKII reduced the expression of total CaMKII and its CaMKII alpha isoform activity in diabetic dorsal horn, which was accompanied with an increase in pain-related behavior. Further studies about the intrathecal inhibition of CaMKII should elucidate its role in nociceptive processes of diabetic neuropathy.

Topics: Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Diabetes Mellitus, Experimental; Diabetic Neuropathies; Injections, Spinal; Male; Pain; Peptides; Physical Stimulation; Posterior Horn Cells; Rats; Rats, Sprague-Dawley; Sulfonamides; Temperature

It has been proposed, but not directly tested, that persistent inflammatory nociception enhances excitatory glutamatergic inputs to neurons in the rostral ventromedial medulla (RVM), altering the activity and function of these neurons. This study used whole cell patch-clamp methods to record evoked excitatory postsynaptic currents (eEPSCs) in spinally projecting RVM neurons from rats injected with saline or complete Freund's adjuvant (CFA) 3-4 days earlier and to examine the role of substance P (SP) in modulating excitatory synaptic transmission. Input-output relationships demonstrated that CFA treatment facilitated fast excitatory glutamatergic inputs to type 1 and type 2 nonserotonergic spinally projecting RVM neurons, but not to type 3 neurons. The facilitation in type 1 and 2 neurons was dependent on neurokinin-1 (NK1) and N-methyl-d-aspartate (NMDA) receptors and prevented by the PKC inhibitor GF109203X. In a subset of neurons from naïve rats, SP mimicked the effects of CFA and increased the potency and efficacy of glutamatergic synaptic transmission. The facilitation was prevented by 10 microM GF109203X, but not by 10 microM KN93, a CaMKII inhibitor. SP (0.3-3 microM) by itself produced concentration-dependent inward currents in most nonserotonergic, but not serotonergic neurons. The present study is the first demonstration, at the cellular level, that persistent inflammatory nociception leads to a sustained facilitation of fast excitatory glutamatergic inputs to RVM neurons by an NK1 and NMDA receptor-dependent mechanism that involves PKC. Further, it demonstrates that the facilitation is restricted to specific populations of RVM neurons that by inference may be pain facilitatory neurons.

Topics: Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Glutamic Acid; In Vitro Techniques; Indoles; Inflammation; Male; Maleimides; Medulla Oblongata; Neural Pathways; Neurons; Neurotransmitter Agents; Pain; Protein Kinase C; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Receptors, Neurokinin-1; Serotonin; Substance P; Sulfonamides; Synaptic Transmission

In the present study, we characterized differential expressions of phosphorylated Ca(2+)/calmodulin-dependent protein kinase IIalpha (pCaMKIIalpha) and phosphorylated extracellular signal-regulated protein (pERK) in the mouse hippocampus induced by various nociceptive stimuli. In an immunoblot study, s.c. injection of formalin and intrathecal (i.t.) injections of glutamate, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1 beta) significantly increased pCaMKIIalpha expression in the hippocampus, but i.p. injections of acetic acid did not. pERK1/2 expression was also increased by i.t. injection of glutamate, TNF-alpha, and IL-1beta but not by s.c. injections of formalin or i.p. injections of acetic acid. In an immunohistochemical study, we found that increased pCaMKIIalpha and pERK expressions were mainly located at CA3 or the dentate gyrus of the hippocampus. In a behavioral study, we assessed the effects of PD98059 (a MEK 1/2 inhibitor) and KN-93 (a CaMKII inhibitor) following i.c.v. administration on the nociceptive behaviors induced by i.t. injections of glutamate, pro-inflammatory cytokines (TNF-alpha or IL-1beta), and i.p. injections of acetic acid. PD98059 as well as KN-93 significantly attenuated the nociceptive behavior induced by glutamate, pro-inflammatory cytokines, and acetic acid. Our results suggest that (1) pERKalpha and pCaMK-II located in the hippocampus are important regulators during the nociceptive processes induced by s.c. formalin, i.t. glutamate, i.t. pro-inflammatory cytokines, and i.p. acetic acid injection, respectively, and (2) the alteration of pERK and pCaMKIIalpha in nociceptive processing induced by formalin, glutamate, pro-inflammatory cytokines and acetic acid was modulated in a different manner.

Topics: Acetic Acid; Analysis of Variance; Animals; Behavior, Animal; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Formaldehyde; Gene Expression Regulation, Enzymologic; Glutamic Acid; Hippocampus; Interleukin-1beta; Male; Mice; Mice, Inbred ICR; Pain; Pain Measurement; Phosphorylation; Protein Kinase Inhibitors; Sulfonamides; Time Factors; Tumor Necrosis Factor-alpha

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is a major protein kinase that is capable of regulating the activities of many ion channels and receptors. In the present study, the role of CaMKII in the complete Freund's adjuvant (CFA)-induced inflammatory pain was investigated. Intraplantarly injected CFA was found to induce spinal activity of CaMKII (phosphorylated CaMKII), which was blocked by KN93 [[2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine)], a CaMKII inhibitor. Pretreatment with KN93 (i.t.) dose-dependently prevented the development of CFA-induced thermal hyperalgesia and mechanical allodynia. Acute treatment with KN93 (i.t.) also dose-dependently reversed CFA-induced thermal hyperalgesia and mechanical allodynia. The action of KN93 started in 30 min and lasted for at least 2 to 4 h. KN92 (45 nmol i.t.) [2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine], an inactive analog of KN93, showed no effect on CFA-induced CaMKII activation, allodynia, or hyperalgesia. Furthermore, our previous studies identified trifluoperazine, a clinically used antipsychotic drug, to be a potent CaMKII inhibitor. Inhibition of CaMKII activity by trifluoperazine was confirmed in the study. In addition, trifluoperazine (i.p.) dose-dependently reversed CFA-induced mechanical allodynia and thermal hyperalgesia. The drug was also effectively when given orally. In conclusion, our findings support a critical role of CaMKII in inflammatory pain. Blocking CaMKII or CaMKII-mediated signaling may offer a novel therapeutic target for the treatment of chronic pain.

Topics: Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Dose-Response Relationship, Drug; Freund's Adjuvant; Hyperalgesia; Inflammation; Mice; Mice, Inbred ICR; Pain; Remission Induction; Spine; Sulfonamides

Previous studies have suggested that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) can modulate opioid tolerance and dependence via its action on learning and memory. In this study, we examined whether CaMKII could directly regulate opioid tolerance and dependence. CaMKII activity was increased after the treatment with morphine (100 mg/kg s.c. or 75 mg s.c. of morphine/pellet/mouse); the effect exhibited a temporal correction with the development of opioid tolerance and dependence. In mice treated with morphine (100 mg/kg s.c.), morphine tolerance and dependence developed in 2 to 6 h. An acute supraspinal administration of KN93 [2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine)], a CaMKII inhibitor, was able to dose-dependently reverse the already-established antinociceptive tolerance to morphine (p < 0.001 for 15-30 nmol; not significant for 5 nmol). KN92 [2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine] (30 nmol i.c.v.), a kinase-inactive analog of KN93, did not affect opioid tolerance. Neither KN92 nor KN93 affected basal nociception or acute morphine antinociception (1-10 nmol i.c.v.). Likewise, dependence on morphine was abolished by the acute administration of KN93, but not KN92, in a dose-dependent manner. Pretreatment of mice with KN93 also prevented the development of morphine tolerance and dependence. The effect of acute CaMKII inhibition was not limited to the particular experimental model, because KN93 also acutely reversed the established opioid tolerance and dependence in mice treated with morphine (75 mg/pellet/mouse s.c.) for 6 days. Taken together, these data strongly support the hypothesis that CaMKII can act as a key and direct factor in promoting opioid tolerance and dependence. Identifying such a direct mechanism may be useful for designing pharmacological treatments for these conditions.

Topics: Analgesics, Opioid; Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinases; Drug Tolerance; Male; Mice; Mice, Inbred ICR; Morphine; Morphine Dependence; Pain; Protein Kinase Inhibitors; Sulfonamides

This study aimed to explore the modulatory effect of substance P (SP) on the current response mediated by N-methyl-D-aspartate (NMDA) receptor in rat primary sensory neurons and its time course using whole-cell patch clamp technique. The majority of neurons (179/213, 84.0%) examined were sensitive to NMDA (0.1-1000 microM) with an inward current, and a proportion of the NMDA-sensitive neurons also responded to SP (78/98, 80.0%) with an inward current. Pretreatment with SP potentiated the NMDA-activated current (INMDA) in a non-competitive manner, which is shown in that SP shifted the concentration-response curve for NMDA upwards compared with the control; the maximal value of INMDA increased fourfold, while the EC50 values for both curves were very close (28 vs. 30 microM). Furthermore, this potentiating effect was time-dependent: the amplitude of INMDA reached its maximum 20 min after SP preapplication, and thereafter maintained a steady level of about 2-3 times its control for 2 or even 3 h. This sustained potentiation by SP of INMDA could be blocked by extracellular application of WIN51708, a selective non-peptide antagonist of NK-1 receptor; and abolished by intracellular application of either BAPTA, or H-7, or KN-93. Though NMDA applied alone also induced a short-term (less than 20 min) self-potentiation of INMDA, it could be abolished by intracellular dialysis of BAPTA or KN-93 completely. As is known, the cell body of dorsal root ganglion (DRG) neurons is generally used as an accessible model for studying the characteristics of the membrane of primary afferent terminals in the dorsal horn of spinal cord. Therefore, these results may offer a clue to the explanation of the symptoms of chronic pain.

Topics: Animals; Benzylamines; Calcium Signaling; Cell Membrane; Cells, Cultured; Chelating Agents; Chronic Disease; Dose-Response Relationship, Drug; Drug Synergism; Egtazic Acid; Enzyme Inhibitors; Ganglia, Spinal; Membrane Potentials; Models, Biological; N-Methylaspartate; Neurokinin-1 Receptor Antagonists; Neurons, Afferent; Pain; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Reaction Time; Receptors, N-Methyl-D-Aspartate; Receptors, Neurokinin-1; Substance P; Sulfonamides

It has been reported that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) can modulate opioid tolerance via its action on learning and memory. In this study, we examine if CaMKII can directly affect opioid tolerance. We found that spinal CaMKII activity was increased in rats tolerant to morphine. In these rats, acute spinal administration of 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) (KN93), a CaMKII inhibitor, was able to reverse the already-established antinociceptive tolerance. These results suggest that CaMKII may directly promote opioid tolerance.

Topics: Analgesics, Opioid; Animals; Benzylamines; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Drug Tolerance; Enzyme Inhibitors; Injections, Spinal; Male; Morphine; Nociceptors; Pain; Pain Measurement; Rats; Rats, Sprague-Dawley; Spinal Cord; Sulfonamides