Effects of SKF-96365, a TRPC inhibitor, on melittin-induced inward current and intracellular Ca2+ rise in primary sensory cells.

OBJECTIVE: Melittin (MEL) is a major component of bee venom and can produce both persistent spontaneous nociception and pain hypersensitivity when injected subcutaneously in the periphery. The present study aimed to examine the roles of transient receptor potential canonical (TRPC) channels in mediation of MEL-induced activation of primary nociceptive cells. METHODS: Whole-cell patch-clamp and laser scanning confocal calcium detection were used to evaluate the effects of SKF-96365, a TRPC inhibitor, applied on the acutely isolated dorsal root ganglion (DRG) cells of rat, on MEL-induced increase in intracellular calcium concentration ([Ca(2+)](i)) and inward current. RESULTS: Under voltage-clamp mode, 43.9% (40/91) DRG cells were evoked to give rise to the inward current by 2 µmol/L MEL, which could be significantly suppressed by 3 doses of SKF-96365 (1, 5 and 10 µmol/L) in a dose-dependent manner. Of the other 210 cells, 67.6% responded to MEL with an intracellular Ca(2+) rise, as revealed by confocal calcium imaging. Of these MEL-sensitive cells, 46.5% (66/142) were suppressed by the highest dose of SKF-96365. CONCLUSION: MEL-induced activation of small to medium-sized DRG cells can be suppressed by SKF-96365, suggesting the involvement of TRPC channels in the mediation of MEL-induced activation of primary nociceptive cells.

Differential roles of ERK, JNK and p38 MAPK in pain-related spatial and temporal enhancement of synaptic responses in the hippocampal formation of rats: multi-electrode array recordings.

It is known that chronic pain affects various higher brain functions including perception, emotion, cognition, and memory. However, few studies have been performed to examine pain-induced synaptic plastic changes in the hippocampal formation (HF), an important region subserving affective-motivational component of pain. Our previous study has revealed a strong impact of peripheral persistent nociception on synaptic connection, transmission and function in the HF of rats, in both temporal and spatial domains, by using a newly developed MED64 multichannel recording system. However, the underlying signaling mechanisms for this pain-related spatial and temporal plasticity are still less understood. As an initial investigation, the present study attempted to examine potential different roles of the mitogen-activated protein kinase (MAPK) members in mediating this plastic phenomenon. By virtue of the three well-known MAPK inhibitors targeting extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK), respectively, in combination with the well-established MED64 multisite recording system, we found that pharmacological inhibition of the ERK- and JNK-mediated signaling pathway, at the plateau phase of the long-term potentiation (LTP), significantly decreased pain-enhanced LTP maintenance whereas similar blockade of p38 MAPK pathway dramatically further increased the potentiation. Regarding the spatial magnification of pain, ERK and p38 MAPK seemed to play opposing roles, with the former positively involved and the latter negatively involved, without any detectable effect of the JNK signaling pathway. Together, these results suggest differential roles of the specific members of the MAPK family in mediating pain-associated spatial and temporal plasticity in the HF, which are in good agreement with previous observations. In addition, a possible mechanistic separation between spatial and temporal magnification of pain is also indicated in this study.

Mediating roles of the vanilloid receptor TRPV1 in activation of rat primary afferent nociceptive neurons by formaldehyde.

The formalin test is a commonly used animal model of acute and tonic pain. However, the molecular targets of formaldehyde (FA, the main ingredient of the formalin solution) on primary nociceptor cells remain controversial. In this report, the effects of FA on electrophysiologically-identified primary nociceptor cells were evaluated in vitro and the roles of the vanilloid receptor TRPV1 in FA-produced activation of primary nociceptors were also examined at both cellular and behavioral levels. Of 92 acutely dissociated dorsal root ganglion (DRG) cells recorded by current patch-clamp technique, 34% were discharged by FA application with the mean onset latencies of the first action potential (AP) being (367.34+/-32.96) s. All the FA-sensitive cells were identified as nociceptor cells by their distinguishable features of AP including longer duration, existence of a hump (a shoulder or inflection) on the repolarizing phase, and longer after-hyperpolarization of APs. Co-application of capsazepine (CPZ), a competitive antagonist of TRPV1 receptors, could block FA-evoked firing with partial inhibition on the membrane depolarization of all cells tested. Of another 160 cells examined by confocal calcium imaging, 32% were shown to respond to FA with an intracellular Ca(2+) rise. Of 51 FA-sensitive cells, 67% were suppressed by CPZ, suggesting partial involvement of TRPV1 in mediation of the FA-evoked intracellular Ca(2+) rise. Under voltage-clamp mode, 41% of DRG cells were evoked to give rise to inward current with the remaining 59% being unchanged. In separate experiments on the other 56 FA-sensitive cells, concentration-dependent increase in the FA-evoked current amplitude was demonstrated. In comparison with controls, the FA-evoked inward current could be significantly suppressed by CPZ that was further enhanced by HC-030031, a TRPA1 selective antagonist. Finally, local effects of CPZ were confirmed in the formalin test and it was shown that the formalin-induced paw flinches were strongly suppressed by CPZ in phase 1 but with phase 2 being significantly suppressed only during 25-55 min. It is therefore concluded that FA can directly activate a subpopulation of primary nociceptor cells and the FA-induced AP discharges are likely to contribute mainly to phase 1, but not phase 2 of the formalin-induced nociception. The activation of primary nociceptor cells by FA is likely to be mediated, at least in part, through TRPV1 and/or TRPA1 receptors.

Imbalance between excitatory and inhibitory amino acids at spinal level is associated with maintenance of persistent pain-related behaviors.

Although the postsynaptic events responsible for development of pathological pain have been intensively studied, the relative contribution of presynaptic neurotransmitters to the whole process remains less elucidated. In the present investigation, we sought to measure temporal changes in spinal release of both excitatory amino acids (EAAs, glutamate and aspartate) and inhibitory amino acids (IAAs, glycine, ?-aminobutyric acid and taurine) in response to peripheral inflammatory pain state. The results showed that following peripheral chemical insult induced by subcutaneous bee venom (BV) injection, there was an initial, parallel increase in spinal release of both EAAs and IAAs, however, the balance between them was gradually disrupted when pain persisted longer, with EAAs remaining at higher level but IAAs at a level below the baseline. Moreover, the EAAs-IAAs imbalance at the spinal level was dependent upon the ongoing activity from the peripheral injury site. Intrathecal blockade of ionotropic (NMDA and non-NMDA) and metabotropic (mGluRI, II, III) glutamate receptors, respectively, resulted in a differential inhibition of BV-induced different types of pain (persistent nociception vs. hyperalgesia, or thermal vs. mechanical hyperalgesia), implicating that spinal antagonism of any specific glutamate receptor subtype fails to block all types of pain-related behaviors. This result provides a new line of evidence emphasizing an importance of restoration of EAAs-IAAs balance at the spinal level to prevent persistence or chronicity of pain.

Differential activation of p38 and extracellular signal-regulated kinase in spinal cord in a model of bee venom-induced inflammation and hyperalgesia.

BACKGROUND: Honeybee's sting on human skin can induce ongoing pain, hyperalgesia and inflammation. Injection of bee venom (BV) into the intraplantar surface of the rat hindpaw induces an early onset of spontaneous pain followed by a lasting thermal and mechanical hypersensitivity in the affected paw. The underlying mechanisms of BV-induced thermal and mechanical hypersensitivity are, however, poorly understood. In the present study, we investigated the role of mitogen-activated protein kinase (MAPK) in the generation of BV-induced pain hypersensitivity. RESULTS: We found that BV injection resulted in a quick activation of p38, predominantly in the L4/L5 spinal dorsal horn ipsilateral to the inflammation from 1 hr to 7 d post-injection. Phosphorylated p38 (p-p38) was expressed in both neurons and microglia, but not in astrocytes. Intrathecal administration of the p38 inhibitor, SB203580, prevented BV-induced thermal hypersensitivity from 1 hr to 3 d, but had no effect on mechanical hypersensitivity. Activated ERK1/2 was observed exclusively in neurons in the L4/L5 dorsal horn from 2 min to 1 d, peaking at 2 min after BV injection. Intrathecal administration of the MEK inhibitor, U0126, prevented both mechanical and thermal hypersensitivity from 1 hr to 2 d. p-ERK1/2 and p-p38 were expressed in neurons in distinct regions of the L4/L5 dorsal horn; p-ERK1/2 was mainly in lamina I, while p-p38 was mainly in lamina II of the dorsal horn. CONCLUSION: The results indicate that differential activation of p38 and ERK1/2 in the dorsal horn may contribute to the generation and development of BV-induced pain hypersensitivity by different mechanisms.

Phosphorylation of c-Jun N-terminal kinase isoforms and their different roles in spinal cord dorsal horn and primary somatosensory cortex.

The present study was undertaken to investigate whether isoforms of c-Jun N-terminal kinase (JNK 46 kDa and 54 kDa), one component of the mitogen-activated protein kinase (MAPK) family, might show region-related differential activation patterns in both naïve and pain-experiencing rats. In naïve rats, no significant difference was observed in total expression level of the two JNK isoforms between spinal cord and primary somatosensory cortex (S1 area). However, phosphorylated JNK 46 kDa was normally expressed in the S1 area, but not in the spinal cord, while neither of the two structures contained phosphorylated JNK 54 kDa. Subcutaneous bee venom (BV)-induced persistent pain stimulation resulted in a significant increase in the phosphorylation of both JNK isoforms in each area for a long period (lasting at least 48 h). Nevertheless, JNK 46 kDa exhibited a much higher activation than JNK 54 kDa in the spinal cord, whereas the same noxious stimulation elicited evident activation of JNK 54 kDa in the S1 area, leaving JNK 46 kDa less affected. Intraplantar injection of sterile saline solution, causing acute and transient pain, produced almost the same changes in activation profile of the two JNK isoforms as found in the BV-treated rats. These results implicate that individual members of the JNK family may be associated with specific regions of nociceptive processing. Also, the two JNK isoforms are supposed to function differently according to their locations within the rat central nervous system.

Region- or state-related differences in expression and activation of extracellular signal-regulated kinases (ERKs) in naïve and pain-experiencing rats.

BACKGROUND: Extracellular signal-regulated kinase (ERK), one member of the mitogen-activated protein kinase (MAPK) family, has been suggested to regulate a diverse array of cellular functions, including cell growth, differentiation, survival, as well as neuronal plasticity. Recent evidence indicates a role for ERKs in nociceptive processing in both dorsal root ganglion and spinal cord. However, little literature has been reported to examine the differential distribution and activation of ERK isoforms, ERK1 and ERK2, at different levels of pain-related pathways under both normal and pain states. In the present study, quantitative blot immunolabeling technique was used to determine the spatial and temporal expression of ERK1 and ERK2, as well as their activated forms, in the spinal cord, primary somatosensory cortex (SI area of cortex), and hippocampus under normal, transient pain and persistent pain states. RESULTS: In naïve rats, we detected regional differences in total expression of ERK1 and ERK2 across different areas. In the spinal cord, ERK1 was expressed more abundantly than ERK2, while in the SI area of cortex and hippocampus, there was a larger amount of ERK2 than ERK1. Moreover, phosphorylated ERK2 (pERK2), not phosphorylated ERK1 (pERK1), was normally expressed with a high level in the SI area and hippocampus, but both pERK1 and pERK2 were barely detectable in normal spinal cord. Intraplantar saline or bee venom injection, mimicking transient or persistent pain respectively, can equally initiate an intense and long-lasting activation of ERKs in all three areas examined. However, isoform-dependent differences existed among these areas, that is, pERK2 exhibited stronger response than pERK1 in the spinal cord, whereas ERK1 was more remarkably activated than ERK2 in the S1 area and hippocampus. CONCLUSION: Taken these results together, we conclude that: (1) under normal state, while ERK immunoreactivity is broadly distributed in the rat central nervous system in general, the relative abundance of ERK1 and ERK2 differs greatly among specific regions; (2) under pain state, either ERK1 or ERK2 can be effectively phosphorylated with a long-term duration by both transient and persistent pain, but their response patterns differ from each other across distinct regions; (3) The long-lasting ERKs activation induced by bee venom injection is highly correlated with our previous behavioral, electrophysiological, morphological and pharmacological observations, lending further support to the functional importance of ERKs-mediated signaling pathways in the processing of negative consequences of pain associated with sensory, emotional and cognitive dimensions.

Nociceptive stimulus modality-related difference in pharmacokinetic-pharmacodynamic modeling of morphine in the rat.

Pharmacokinetics (PK)-pharmacodynamics (PD) modeling, the mathematical description of the relationship between PK and PD, can estimate and predict relevant parameters associated with onset, magnitude and time courses of dose-concentration-effect of a drug. In this report, we introduce a new nonsteady-state and time-dependent PK-PD modeling of a single dose of morphine in which time courses of concentration of unconjugated and estimated conjugated morphine in compartments of either plasma or biophase (cerebrospinal fluid, CSF) and multiple anti-nociceptive effects across thermal and mechanical stimulus modalities in rats were studied. The results showed that: (1) both intragastric and intraperitoneal administration of a single dose of morphine resulted in a differential anti-nociceptive effect in both magnitude and time course of the drug between thermal and mechanical painful stimuli (anti-mechanical pain effect was 2-3 fold stronger than anti-thermal pain effect, P < 0.01); (2) the PK data showed that the area under concentration-time curves of conjugated morphine was 4.5 and 2.0 fold bigger than unconjugated morphine in either plasma and biophase compartments, suggesting that the PK processes of unconjugated morphine are different from that of conjugated morphine; (3) the PD data also showed a change in PD characteristics of unconjugated and conjugated morphine across systemic and biophasic compartments for anti-mechanical pain effect, while there was no change at all for anti-thermal pain effect; (4) the difference in analgesia of a single dose of morphine across thermal and mechanical stimulus modalities was well reflected by the difference in the nonsteady-state and time-dependent PK-PD modeling, namely, the clockwise hysteresis loop model well represents the relationship of the time course between unconjugated/conjugated morphine concentration (both plasma and biophase) and anti-thermal pain effect, while the counter-clockwise hysteresis loop model well represents that between conjugated morphine concentration (mainly in biophase) and anti-mechanical pain effect. Taken together, the multiple PD-PK modeling is more useful in estimation and prediction of onset, magnitude and time courses of concentration-multiple pharmacological effects of morphine than simple PK or PD models, and establishment of various multiple PD-PK modeling might also be more useful in optimizing clinical use of existing drugs as well as new drugs for analgesia or treatment of other diseases.

[Effect of repetitive hypoxia on MMP-2/9 expression and activation in murine brain].

AIM: To explore the changes of MMP-2/9 protein expression and excitation in brain of repetitive hypoxic mice. METHODS: The biochemistry techniques of SDS-PAGE, Western bolt and Gel Goc Image Analysis System were applied to determine the level of MMP-2 and MMP-9 expression and activation in cortex and hippocampus of mice. The animals were randomly divided into 5 groups: the normal control group (H0), acute hypoxic (H1, hypoxic exposure once), repetitive hypoxic groups (H2-H4, repetitive hypoxia for 2-4 runs respectively). RESULTS: (1) The MMP- 2 expression level was increased first then decreased in hippocampus and the significant decrease was found in H4 group (P < 0.05, n=6), but no significant changes among the 5 groups in cortex. In addition, no activated form of 66 kD MMP-2 had been detected both in hippocampus and cortex. (2) Along with the development of brain hypoxic preconditioning, the level MMP-9 protein expression also increased first then decreased gradually in hippocampus, and the significant changes were found both in H1 and H4 groups (P < 0.05, n=7 for each group). The same trace of changes was also found in the activation of MMP-9 (include 82 and 78 kD forms) in hippocampus, and the significance both in H1 and H4 (P < 0.05, n=7 for each group) were detected. However, there was not any significant change in the level of MMP-9 protein expression or activation to be found in cortex. CONCLUSION: These results suggested that MMP-2 and MMP-9 might play certain role in the development of cerebral hypoxic preconditioning, the different changes of MMP-2/9 protein expression and activation both in cortex and hippocampus might be involved in their selective vulnerability to hypoxia.

Hypoxic preconditioning: a novel intrinsic cytoprotective strategy.

A concept of tissue-cell adaptation to hypoxia (hypoxic preconditioning) is raised and its corresponding animal model is introduced. A significantly strengthened tolerance to hypoxia and a protective effect of the brain extracts from the preconditioned animals are presented. Changes in animals' behavior, neuromorphology, neurophysiology, neurochemistry and molecular neurobiology during preconditioning are described. Energy saving, hypometabolism, and cerebral protection in particular are thought to be involved in the development of hypoxic tolerance and tissue-cell protection. The essence and significance of the hypoxic tissue-cell adaptation or preconditioning are discussed in terms of biological evolution and practical implication.

[Hypoxic preconditioning increases cPKCgamma membrane translocation in murine brain].

Cerebral hypoxic preconditioning (CHP), which was induced by repetitive sub-lethal hypoxic insult, is an endogenous protection of neuron against subsequent severe hypoxic injury. Although a number of possible induction pathways have been investigated, such as neuroactive cytokines, activation of glutamate receptors, the ATP-sensitive potassium channel, nitric oxide and oxidative stress, the exact mechanism underlying CHP-induced protection remains unclear. It is interesting that all the above-mentioned mechanisms are involved in the activation of protein kinases C (PKC). Recently we reported that the level of PKCs membrane translocation was significantly increased in the brain of hypoxic preconditioned mice. In order to explore the role of conventional protein kinases C (cPKC) in the development of cerebral hypoxic preconditioning, biochemical techniques of SDS-PAGE and Western bolt were applied to observe the effects of repetitive hypoxic exposure (H1-H4) on the level of cPKCalpha and gamma membrane translocation in the cortex and hippocampus of mice. Experiments were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals. The hypoxic preconditioned mice model was adapted with minor modification from our previous report. In brief, healthy adult BALB/C mice weighing 18-20 g of either sex were randomly divided into 5 groups: control group (H0), hypoxic control group (H1, hypoxic exposure once ), hypoxic preconditioned group (H2-H4, repetitive hypoxic exposure for 2-4 times respectively). The first sign of gasping breath was taken as the end of each hypoxic exposure, and then the mice were kept in normal control condition for a 30-min interval to recover before the following hypoxic insult. We found that the level of cPKCgamma membrane translocation was increased significantly (*P<0.05, n=6) with the increase of the hypoxic exposure times in both hippocampus (H0: 100% vs H1 approximately H4: 119.2%+/-7.0% *, 139.3% +/-7.4%*, 134.2% +/-8.95%*, 184.0% +/-10.8%*) and cortex (H0: 100% vs H1-H4: 129.7% +/-13.8%, 143.3% +/-13.9%*, 204.0% +/-12.1%*, 229.5% +/-14.6%*) of mice. But there were no significant changes in cPKCalpha membrane translocation in cortex and hippocampi of hypoxic preconditioned mice. These results suggest that cPKCgamma plays an important role in the development of cerebral hypoxic preconditioning. The changes in some other forms of novel and atypical PKCs are still under investigation.

[Measurement of nitrate and nitrite concentrations in biological fluids using high performance liquid chromatography].

AIM AND METHODS: New method to analyse nitrate and nitrite concentrations in saliva, serum and urine was developed using high performance liquid chromatography. RESULTS: The whole isolation process was completed in less than 7 minutes, the determination linearity of nitrate and nitrite were 0.7 ng-100 ng and 5 ng-100 ng, respectively. MINIMUM: Detectable limits of nitrate and nitrite were 0.3 ng and 2 ng, respectively. Nitrate recovery ratio was 99%-102% and nitrite recovery ratio was 99%-104%. The RSD of nitrate and nitrite was 0.8% and 1.7%, respectively. CONCLUSION: In comparison with other methods available, the present method seems to be simpler, more sensitive and specific.