Environmental enrichment reduces mechanical hypersensitivity in neuropathic mice,but fails to abolish the phenotype of CCK2 receptor deficient mice

Genetic invalidation of CCK₂ receptors abolishes chronic constriction injury (CCI) induced mechanical hypersensitivity in mice. However, housing in environmentally enriched conditions significantly alters the phenotype of CCK₂ receptor deficient mice in all major behavioral domains. Furthermore, environmental enrichment itself has been reported to have protective effects in several rodent models of neurological diseases (brain and spinal trauma, ischemic stroke, Alzheimer's disease, etc.). In the present study we reproduced the earlier finding that mice, lacking CCK₂ receptors (−/−) are resistant to CCI-induced hypersensitivity. On the other hand, environmental enrichment substantially reduced CCI-induced mechanical hypersensitivity in wild-type (+/+) mice. Nevertheless, the phenotypic differences between wild-type (+/+) and mutant (−/−) mice in mechanical sensitivity before and after CCI-surgery were not eliminated by alternative housing conditions. These observations suggest that environmental enrichment has beneficial effects in neuropathic conditions and reinforce the causal link between CCK₂ receptors, mechanical sensitivity and the development of CCI-induced hypersensitivity.     

Impaired P2X and P2Y receptor-mediated signaling in HPRT-deficient B103 neuroblastoma cells

Defect of hypoxanthine phosphoribosyl transferase (HPRT) causes Lesch-Nyhan disease (LND), but the link between HPRT deficiency and the self-injurious behavior of LND is unknown. In a previous study (Pinto et al., J. Neurochem. 72 (2005) 1579-1586) we reported on a decrease in nucleotidase activity in membranes of several HPRT⁻ cell lines and fibroblasts from LND patients. Since nucleotidases are involved in ATP-induced signal transduction, in the present study, we tested the hypothesis that P2X and P2Y receptor-mediated signal transduction is impaired in HPRT deficiency. As model we studied rat B103 neuroblastoma cells. Compared to control cells, in HPRT⁻ cells, NTP and NDP-induced Ca²⁺ influx across the membrane and Ca²⁺ mobilization from intracellular stores were impaired. Both P2X and P2Y receptors were involved in the responses. Quantitative real-time PCR revealed reduced expression of receptors P2X₃, P2X₅, P2Y₂, P2Y₄, P2Y₁₂, P2Y₁₃ and P2Y₁₄ in HPRT deficiency. Collectively, HPRT deficiency is associated with abnormal purinergic signaling, encompassing P2X and P2Y receptors and nucleotidases.     

Anatibant,a selective non-peptide bradykinin B2 receptor antagonist,reduces intracranial hypertension and histopathological damage after experimental traumatic brain injury

Bradykinin, the main metabolite of the kallikrein-kinin system and one of the first mediators released during inflammation, is well known to increase the permeability of the blood brain barrier (BBB) by activation of kinin B₂ receptors and hence promote brain edema formation following traumatic brain injury (TBI). Anatibant^® (LF 16-0687), a selective non-peptide bradykinin B₂ receptor antagonist, reduces brain edema after experimental TBI, however, so far no data are available if Anatibant^® reduces also the sequels of brain edema formation, i.e. morphological brain damage. Therefore, we investigated the effect of Anatibant (3.0 mg/kg b.w.) on intracranial pressure (ICP) and contusion volume after experimental TBI. Male C57/Bl6 mice (25–28 g) were subjected to Controlled Cortical Impact trauma (CCI). Anatibant^® was administrated as a subcutaneous bolus 15 min and 8 h after TBI. ICP was measured 3, 6, and 10 h after injury and contusion volume was quantified 24 h after trauma. Our data demonstrate a significant reduction of ICP (16.6 ± 1.67 mmHg vs. 24.40 ± 3.58 mmHg; n = 6; p = 0.002) and of contusion volume 24 h after trauma (28.28 ± 5.18 mm³ vs. 35.0 ± 3.32 mm³n = 7; p = 0.003) in treated mice. Therefore we conclude, that inhibition of bradykinin B₂ receptors seems to be a promising treatment option, and might therefore be investigated in clinical trails for the treatment of TBI.     

Up-regulation of P2X7 receptor-immunoreactivity by in vitro ischemia on the plasma membrane of cultured rat cortical neurons

Mixed neuronal/astrocytic cortical cell cultures of the rat were incubated for 2 or 12 h under normoxic or ischemic conditions. Subsequent flow cytometric analysis with an anti-P2X₇ receptor antibody directed against an extracellular epitope indicated the up-regulation of these receptors at the plasma membrane by 12 h of ischemia. Labelling of MAP-2 immunopositive neurons by an anti-P2X₇ antibody directed against a C-terminal epitope, documented the selectivity of the ischemia-induced increase in receptor-density for the neuronal population. By contrast, staining of GFAP immunopositive astrocytes by the same anti-P2X₇ antibody excluded any effect of ischemia on the astrocytic density of P2X₇ receptors. The ischemic up-regulation of neuronal P2X₇ receptors is in perfect agreement with the previously reported facilitation of transmitter release from the GABAergic non-pyramidal cell type in such cultures [K. Wirkner, A. Köfalvi, W. Fischer, A. Günther, H. Franke, H. Gröger-Arndt, W. Nörenberg, E. Madarasz, E.S. Vizi, D. Schneider, B. Sperlagh, P. Illes, Supersensitivity of P2X₇ receptors in cerebrocortical cell cultures after in vitro ischemia, J. Neurochem. 95 (2005) 1421–1437].     

Involvement of enkephalins in the inhibition of osteosarcoma-induced thermal hyperalgesia evoked by the blockade of peripheral P2X3 receptors

Although previous studies describe the up-regulation of purinergic P2X₃ receptors expressed at peripheral nociceptive fibers in experimental painful neoplastic processes, the analgesic efficacy of P2X₃ receptor antagonists has not been tested in these settings. We study here the effect of the P2X₃ receptor antagonist, A-317491, on thermal hyperalgesia produced by the intratibial inoculation of NCTC 2472 fibrosarcoma cells to C3H/HeJ mice. The peritumoral administration of A-317491 (10–100 μg) dose-dependently attenuated osteosarcoma-induced thermal hyperalgesia without modifying thermal latencies measured in the contralateral paws. This antihyperalgesic effect was inhibited by the coadministration of naloxone-methiodide (0.1–1 μg) or the systemic injection of the selective μ-opioid receptor antagonist cyprodime (1 mg/kg), demonstrating the involvement of peripheral μ-opioid receptors. Furthermore, the antihyperalgesic effect induced by A-317491, was antagonised by the coadministration of an anti-enkephalin antibody supporting the participation of endogenous enkephalins. Consistent with this result, the antihyperalgesic effect induced by A-317491 was dramatically enhanced by the administration of an enkephalin-degrading inhibitor, Debio 0827, as demonstrated by isobolographic analysis. This synergism opens the theoretical possibility that the combination of both types of drugs could be useful to counteract some nociceptive symptoms derived from tumor development.     

1,8-桉叶素对背根神经节内P2X3受体介导神经病理痛的作用

目的:探讨1,8-桉叶素对大鼠背根神经节(DRG)神经元P2X3受体介导神经病理痛的作用。方法:建立大鼠坐骨神经慢性压迫性损伤模型(CCI)。SD大鼠随机分为假手术(Sham)组,坐骨神经慢性压迫性损伤(模型组,CCI)组、低剂量1,8-桉叶素治疗组、高剂量1,8-桉叶素治疗组、二甲亚砜对照组。检测大鼠术后7、14 d机械缩足反射(MWT)及热缩足反射潜伏期(TWL),观察大鼠行为学变化。免疫组织化学和原位杂交观察神经病理痛大鼠第4~5腰(L_(4-5))DRG神经元P2X3受体表达变化。结果:术后第7和14天,模型组大鼠MWT和TWL明显低于假手术组,低、高剂量治疗组较模型组明显升高,二甲亚砜组与模型组比较无差别;L_(4-5)DRG内P2X3受体表达模型组明显高于假手术组,低、高剂量治疗组较模型组均明显降低,二甲亚砜组与模型组比较无明显区别。结论:1,8-桉叶素抑制CCI大鼠L_(4-5)DRG神经元P2X3受体过表达,从而缓解神经病理性疼痛症状。     

Calcium responses mediated by type 2 IP3-receptors are required for osmotic volume regulation of retinal glial cells in mice

Prevention of osmotic swelling of retinal glial (Müller) cells is required to avoid detrimental decreases in the extracellular space volume during intense neuronal activity. Here, we show that glial cells in slices of the wildtype mouse retina maintain the volume of their somata constant up to ∼4 min of perfusion with a hypoosmolar solution. However, calcium chelation with BAPTA/AM induced a rapid swelling of glial cell bodies. In glial cells of retinas from inositol-1,4,5-trisphosphate-receptor type 2-deficient (IP₃R2^−/−) mice, hypotonic conditions caused swelling of the cell bodies without delay. Exogenous ATP (acting at P2Y₁ receptors) prevented the swelling of glial cells in retinal slices from wildtype but not from IP₃R2^−/− mice. Müller cells from IP₃R2^−/− mice displayed a strongly reduced amplitude of the ATP-evoked calcium responses as compared to cells from wildtype mice. It is concluded that endogenous calcium signaling mediated by IP₃R2 is required for the osmotic volume regulation of retinal glial cells.     

Inflammatory mediators potentiate high affinity GABAA currents in rat dorsal root ganglion neurons

Following acute tissue injury action potentials may be initiated in afferent processes terminating in the dorsal horn of the spinal cord that are propagated back out to the periphery, a process referred to as a dorsal root reflex (DRR). The DRR is dependent on the activation of GABA_A receptors. The prevailing hypothesis is that DRR is due to a depolarizing shift in the chloride equilibrium potential (E_Cl) following an injury-induced activation of the Na⁺–K⁺–Cl⁻-cotransporter. Because inflammatory mediators (IM), such as prostaglandin E₂ are also released in the spinal cord following tissue injury, as well as evidence that E_Cl is already depolarized in primary afferents, an alternative hypothesis is that an IM-induced increase in GABA_A receptor mediated current (I_GABA) could underlie the injury-induced increase in DRR. To test this hypothesis, we explored the impact of IM (prostaglandin E₂ (1 μM), bradykinin (10 μM), and histamine (1 μM)) on I_GABA in dissociated rat dorsal root ganglion (DRG) neurons with standard whole cell patch clamp techniques. IM potentiated I_GABA in a subpopulation of medium to large diameter capsaicin insensitive DRG neurons. This effect was dependent on the concentration of GABA, manifest only at low concentrations (<10 μM). THIP evoked current were also potentiated by IM and GABA (1 μM) induced tonic currents enhanced by IM were resistant to gabazine (20 μM). The present data are consistent with the hypothesis that an acute increase in I_GABA contributes to the emergence of injury-induced DRR.     

Comparison of three models of neuropathic pain in mice using a new method to assess cold allodynia: The double plate technique

The recent identification of receptors sensitive to cold stimuli increased the significance of using mice to study cold allodynia, one of the important features of neuropathic pain. However, commonly used techniques (simple cold plate and acetone technique) may be inappropriate to study cold allodynia in mice because of problems of interpretation. We have developed a new method for assessing aversion to a cold non-noxious stimulus. It consists of calculating the time that mice spend on a non-noxious cold plate during their explorative behavior versus a thermoneutral one. We used three different models of neuropathic pain: chronic constriction injury of the sciatic nerve (CCI), partial sciatic nerve ligation (PSL) and chronic constriction of the saphenous nerve (CCS) with their respective sham groups and naive animals to assess the double plate in comparison to the acetone drop technique. All operated mice displayed cold allodynia with both methods. The response to acetone and the time spent on the cold plate were correlated (r = −0.93) and we also showed that the CCI mice were more sensitive to cold. Pharmacological validation of this technique showed that CCI induced cold allodynia was alleviated by gabapentin. In conclusion, the double plate technique provides a new, relevant method for assessing cold allodynia in mice. The advantages and drawbacks with the other techniques are discussed.     

Negative crosstalk between M1 and M2 muscarinic autoreceptors involves endogenous adenosine activating A1 receptors at the rat motor endplate

At the rat motor nerve terminals, activation of muscarinic M₁ receptors negatively modulates the activity of inhibitory muscarinic M₂ receptors. The present work was designed to investigate if the negative crosstalk between muscarinic M₁ and M₂ autoreceptors involved endogenous adenosine tonically activating A₁ receptors on phrenic motor nerve terminals. The experiments were performed on rat phrenic nerve-hemidiaphragm preparations loaded with [³H]-choline (2.5 μCi/ml). Selective activation of muscarinic M₁ and adenosine A₁ receptors with 4-(N-[3-clorophenyl]-carbamoyloxy)-2-butyryltrimethylammonium (McN-A-343, 3 μM) and R-N⁶-phenylisopropyladenosine (R-PIA, 100 nM), respectively, significantly attenuated inhibition of evoked [³H]-ACh release induced by muscarinic M₂ receptor activation with oxotremorine (10 μM). Attenuation of the inhibitory effect of oxotremorine (10 μM) by R-PIA (100 nM) was detected even in the presence of pirenzepine (1 nM) blocking M₁ autoreceptors, suggesting that suppression of M₂-inhibiton by A₁ receptor activation is independent on muscarinic M₁ receptor activity. Conversely, the negative crosstalk between M₁ and M₂ autoreceptors seems to involve endogenous adenosine tonically activating A₁ receptors. This was suggested, since attenuation of the inhibitory effect of oxotremorine (10 μM) by McN-A-343 (3 μM) was suppressed by the A₁ receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (2.5 nM), and by reducing extracellular adenosine with adenosine deaminase (0.5 U/mL) or with the adenosine transport blocker, S-(p-nitrobenzyl)-6-thioinosine (NBTI, 10 μM). The results suggest that the negative crosstalk between muscarinic M₁ and M₂ autoreceptors involves endogenous adenosine outflow via NBTI-sensitive (es) nucleoside transport system channelling to the activation of presynaptic inhibitory A₁ receptors at the rat motor endplate.     

Time-course of neuropathic pain in mice deficient in neuronal or inducible nitric oxide synthase

Nitric oxide which is synthesised by nitric oxide synthase (NOS) is involved in processes related to regeneration after nerve injury and neuropathic pain. Here we investigated functional aspects of the nociceptive system. For that purpose, the chronic constriction injury (CCI) model induced by loose ligation of the sciatic nerve was employed in C57Bl/6J wild-type (WT), nNOS and iNOS knock-out (−/−) mice. Their thermal and mechanical pain thresholds were then measured over a period of six weeks. In addition, ³H-DAMGO, ³H-CP 55.940, and ³H-l-glutamate binding, and neuronal (NeuN-immunostained) and astroglial (GFAP-immunostained) cell composition were studied. There were no significant differences in cell composition between the three strains used. Significant differences between CCI and sham-operated animals were found in nNOS−/− after day 6, in WT mice after day 10, and in iNOS−/− after day 17 post surgery. The mechanical pain threshold was normalised after day 45 post surgery in WT mice only. There were no changes in DAMGO and glutamate binding. However, we found significant differences in CP 55.940 binding in the spinal cord. It was concluded that NOS–cannabinoid interaction contributes to differences in nociceptive behaviour.     

Effect of high trait anxiety on mechanical hypersensitivity in male rats

Recently, we have shown that neuropathic pain is associated with anxiety-like behaviour in rats with sciatic nerve lesion. An enhanced pain perception has also been described in patients with anxiety disorders. However, there is only limited knowledge about the relationship between anxiety and pain in animals. To investigate whether trait anxiety influences nociception, we measured mechanical hypersensitivity following chronic constriction injury (CCI) in rats selectively bred for high (HAB) or low (LAB) anxiety-like behaviour. Pain sensitivity was assessed before surgery and at day 7, 14, 21, 36 and 57 after CCI by determination of withdrawal thresholds. Additionally, we examined pain-induced anxiety-like behaviour using the elevated plus-maze (EPM). HAB and LAB rats exhibited similar levels of mechanical hypersensitivity 7 days post-injury. However, at day 14 and 21 after surgery, mechanical-induced pain thresholds were significantly decreased in HAB rats (p < 0.05) in comparison to LAB rats. From day 21 onward HAB rats displayed a faster return of paw withdrawal threshold to baseline level when compared to LAB rats (p < 0.01). In the EPM anxiety-like behaviour was observed following CCI injury in HAB and LAB rats on top of low and high trait anxiety reflected in a reduced number of entries in open arms. These findings indicate that trait anxiety increases mechanical hypersensitivity in CCI rats during the chronic phase of pain, thereby suggesting that affective processes modulate even simple pain-related behaviour. Furthermore, we demonstrated that neuropathic pain in the CCI model increases anxiety-like behaviour even in LAB rats.     

Modulation of histamine H3 receptor function by monovalent ions

Monovalent ions differently affect ligand binding to G protein-coupled receptors (GPCRs) by as yet poorly defined mechanisms. In particular, NaCl often decreases the affinity of agonists but increases it for antagonists. We examined the effect of various monovalent ions on human histamine H₃ receptor (hH₃R), co-expressed with mammalian G proteins (Gα_i1, Gα_i2, Gα_i3 or Gα_o1, and β₁γ₂ dimers, respectively) in Sf9 insect cell membranes, with respect to agonist binding and G protein activation. NaCl (100 mM) had no effect on affinity of the agonist [³H]N^α-methylhistamine ([³H]NAMH). In steady-state GTPase assays, the endogenous agonist histamine had a lower potency and the inverse agonist thioperamide had a higher potency, when NaCl (100 mM) was present. Monovalent ions reduced H₃R-regulated signalling in the order of efficacy Li⁺ ∼ Na⁺ ∼ K⁺ < Cl⁻ < Br⁻ < I⁻. NaCl had a stronger effect on basal hH3R-signalling when Gα_i3 was co-expressed. Asp80^2.50, a putative interaction site for Na⁺, was mutated to Asn80^2.50 (D2.50N-hH₃R). Strikingly, the mutation was unable to activate Gα_i3 at all. The effects can be explained by a model, where (i) monovalent ions as well as a charge-neutralizing mutation of Asp80^2.50 generally reduce the interaction of hH₃R with G proteins, (ii) monovalent anions increase the affinity of G proteins for GDP and thus, indirectly affect their interaction with hH₃R and, (iii) Asp80^2.50 is a key residue for hH₃R/Gα_i3-protein activation. The latter result suggests that hH₃R/G protein-coupling interfaces may differ even between closely related subunits.     

Augmented peripheral chemoreflex in patients with heart failure and inspiratory muscle weakness

We hypothesized that heart failure patients with inspiratory muscle weakness (IMW) present greater peripheral chemoreflex responsiveness and augmented exercise ventilatory oscillation compared to patients with preserved inspiratory muscle strength. We studied 19 heart failure patients: 9 with IMW (maximal inspiratory pressure [PImax] < 70% of predicted) and 10 with preserved inspiratory muscle strength. Inspiratory muscle strength was measured via pressure transducer. Peripheral chemoreflex was evaluated by the single-breath CO₂ test. Exercise ventilatory oscillation was determined as the ratio between amplitude and mean of each oscillation during incremental exercise. Patients with IMW had greater peripheral chemoreflex response (0.11 ± 0.03 l min⁻¹ Torr⁻¹) than those with preserved inspiratory muscle strength (0.07 ± 0.03 l min⁻¹ Torr⁻¹, p = 0.02). Moreover, there was a significant and inverse correlation between PImax and peripheral chemoreflex response (r = −0.57, p = 0.01). Likewise, there was a significant and inverse correlation between PImax and ventilatory oscillations (r = −0.46, p = 0.04). Our findings indicate that IMW is linked to increased peripheral chemoreflex and augmented exercise ventilatory oscillation in patients with chronic heart failure.     

Unilateral intra-perilymphatic infusion of substance P enhances ipsilateral vestibulo-ocular reflex gains in the sinusoidal rotation test

Previous studies have reported localization of substance P (SP) within the inner ear and that SP exists abundantly within vestibular endorgans. While SP's functional role in the inner ear remains unclear, SP can act as a neuromodulator in the CNS and directly influences neuronal excitability. We hypothesized that SP might influence neuronal excitability within the vestibular periphery. The present study used the sinusoidal rotation test to investigate the influence of SP after its local application in the guinea pig unilateral inner ear. A tiny hole was made adjacent to the round window in the right ears of Hartley white guinea pigs that had normal tympanic membranes and Preyer reflexes. An osmotic pump infused SP (10⁻⁴ M, 10⁻³ M, and 10⁻² M), neurokinin-1 (NK-1) receptor antagonist (10⁻³ M) alone, or SP (10⁻³ M) + NK-1 receptor antagonist (10⁻³ M) through this hole, with rotation tests performed before, and 12 h and 24 h after the treatment. Results were used to calculate the vestibulo-ocular reflex (VOR) gains. After administration of 10⁻³ M and 10⁻² M SP, significant increases in the VOR gains were noted at 12 h after treatment, with these gains disappearing by 24 h after treatment. This increase was not observed when there was simultaneous NK-1 receptor antagonist administration. There were also no changes in the VOR gains noted after administration of 10⁻⁴ M SP or the NK-1 receptor antagonist alone. These results indicate the possibility that SP may act on vestibular endorgans as an excitatory factor via the NK-1 receptors.     

Aspirin-triggered lipoxin induces CB1-dependent catalepsy in mice

Evidence are that inhibition of cyclooxygenase 2 (COX-2) enhances endocannabinoid signaling, indicating a crosstalk between these two eicosanoid pathways. Aspirin, a non-selective COX inhibitor, acetylates COX-2 with generation of a lipoxygenase (LOX) substrate, whose end product is the 15-epi-lipoxin A₄ (15-epi-LXA₄), an aspirin-triggered lipoxin. Our objective was to investigate whether 15-epi-LXA₄ would potentiate in vivo effects of the endocannabinoid anandamide (AEA). Catalepsy was selected as a behavioral parameter and tested 5 min after AEA injection in all experiments. AEA induced dose-dependent (200 pmol/2 μl, i.c.v.) catalepsy. A sub-dose of AEA (10 pmol/2 μl, i.c.v.) was potentiated by aspirin (300 mg/kg, p.o.) via a 5-LOX-dependent step. The cataleptic effect induced by the interaction between sub-doses of 15-epi-LXA₄ (0.01 pmol/2 μl, i.c.v.) and AEA (10 pmol/2 μl, i.c.v.) was prevented by the cannabinoid CB₁ receptors antagonist SR141716A (1 mg/kg, i.p.), but not by the antagonist of lipoxin ALX receptors Boc-2 (10 μg/kg, i.p.). While previous studies have shown that COX inhibition itself may enhance endocannabinoid effects, here we add another piece of evidence revealing that a LOX-derivative produced in consequence of COX-2 acetylation participates in this process.     

Comparison of sympathetic sprouting in sensory ganglia in three animal models of neuropathic pain

Sympathetic postganglionic fibers sprout in the dorsal root ganglion (DRG) after peripheral nerve injury. Therefore, one possible contributing factor of sympathetic dependency of neuropathic pain is the extent of sympathetic sprouting in the DRG after peripheral nerve injury. The present study compared the extent of sympathetic sprouting in the DRG as well as in the injured peripheral nerve in three rat neuropathic pain models: (1) the chronic constriction injury model (CCI); (2) the partial sciatic nerve ligation injury model (PSI); and (3) the segmental spinal nerve ligation injury model (SSI). All three methods of peripheral nerve injury produced behavioral signs of ongoing and evoked pain with some differences in the magnitude of each pain component. The density of sympathetic fibers in the DRG was significantly higher at all examined postoperative times than controls in the SSI model, while it was somewhat higher than controls only at the last examined postoperative time (20 weeks) in the CCI and PSI models. Therefore, data suggest that, although sympathetic changes in the DRG may contribute to neuropathic pain syndromes in the SSI model, other mechanisms seem to be more important in the CCI and PSI models at early times following peripheral nerve injury.     

Modulation of P2X receptors in dorsal root ganglion neurons of streptozotocin-induced diabetic neuropathy

Painful diabetic neuropathy causes hyperalgesia and does not respond to commonly used analgesics such as non-steroidal anti-inflammatory drugs or opioids at doses below those producing disruptive side effects. In the present study, we examined the effect of P2X receptor antagonists, which are known to modulate the pain pathway, on mechanical hyperalgesia in streptozotocin (STZ)-induced diabetic mice. The paw withdrawal frequency measured by von Frey filaments, began to significantly increase 5 days after STZ injection and was maintained for more than 14 days. Intrathecal administration of P2X receptor antagonists (PPADS and TNP-ATP) inhibited the mechanical allodynia in diabetic mice. The levels of P2X₂ and P2X₃ receptors mRNA were significantly increased in diabetic mice at 14 days after the intravenous injection of STZ. These results suggest that the upregulation of P2X₂, P2X₃ and/or P2X_2/3 receptor in DRG neurons is associated with mechanical allodynia in STZ-induced diabetic mice.     

Involvement of prostaglandin F2α receptor in ATP-induced mechanical allodynia

Nociceptive primary afferents have the capacity to induce a state of increased excitability in the dorsal horn neurons of the spinal cord. It is well accepted that capsaicin-sensitive C-fibers transduce noxious stimulation and acute pain and that capsaicin-insensitive Aβ-fibers are responsible for touch and innocuous sensation. It has been reported that the intrathecal (i.t.) administration of prostaglandin F_2α (PGF_2α) and ATP induces mechanical allodynia via the capsaicin-insensitive primary afferent pathway. In the present study, we investigated the interaction of purinoceptor P2X and the PGF_2α receptor (FP) in the induction of allodynia by use of mice lacking FP (FP^−/−). Both PGF_2α and the P2X receptor agonist αβ-methylene ATP administered i.t. strongly induced allodynia for 50 min by tactile stimuli to the flank of mice. The allodynia induced by αβ-methylene ATP, but not that by PGF_2α, was suppressed by simultaneous i.t. administration of P2X receptor antagonists pyridoxalphosphate-6-azophenyl-2,4-disulphonic acid and A-317491. In contrast, the allodynia induced by αβ-methylene ATP as well as that by PGF_2α was not observed in FP^−/− mice. Immunostaining of β-galactosidase, a reporter knocked into the endogenous FP locus in FP^−/− mice, showed that the FP receptor was co-localized with P2X₂ and P2X₃ receptors in neurons of the spinal cord. αβ-Methylene ATP evoked a transient or sustained [Ca²⁺]_i increase in most of the PGF_2α-responsive cells in the deeper layer of the spinal cord, and the αβ-methylene ATP-evoked increase was blocked by the FP receptor antagonist AL-8810 in two-thirds of the cells. Neither PGF_2α nor αβ-methylene ATP induced the activation of spinal microglia. The present study demonstrates that the αβ-methylene ATP-evoked allodynia is mediated by the FP receptor, possibly via the functional coupling between the activation of P2X_2/3 receptors on the central terminal of capsaicin-insensitive fibers and FP receptors on spinal neurons.     

Altered Ca homeostasis in the skeletal muscle of DJ − 1 null mice

Loss-of-function mutations in DJ − 1 are associated with early-onset of Parkinson's disease. Although DJ − 1 is ubiquitously expressed, the functional pathways affected by it remain unresolved. Here we demonstrate an involvement of DJ − 1 in the regulation of Ca²⁺ homeostasis in mouse skeletal muscle. Using enzymatically dissociated flexor digitorum brevis muscle fibers from wild-type (wt) and DJ − 1 null mice, we examined the effects of DJ − 1 protein on resting, cytoplasmic [Ca²⁺] ([Ca²⁺]_i) and depolarization-evoked Ca²⁺ release in the mouse skeletal muscle. The loss of DJ − 1 resulted in a more than two-fold increase in resting [Ca²⁺]_i. While there was no alteration in the resting membrane potential, there was a significant decrease in depolarization-evoked Ca²⁺ release from the sarcoplasmic reticulum in the DJ − 1 null muscle cells. Consistent with the role of DJ − 1 in oxidative stress regulation and mitochondrial functional maintenance, treatments of DJ − 1 null muscle cells with resveratrol, a mitochondrial activator, or glutathione, a potent antioxidant, reversed the effects of the loss of DJ − 1 on Ca²⁺ homeostasis. These results provide evidence of DJ − 1's association with Ca²⁺ regulatory pathways in mouse skeletal muscle, and suggest the potential benefit of resveratrol to functionally compensate for the loss of DJ − 1.