Maho Tsubota

Bladder pain relief by HMGB1 neutralization and soluble thrombomodulin in mice with cyclophosphamide-induced cystitis.

High mobility group box 1 (HMGB1), one of damage-associated molecular patterns (DAMPs), plays roles in not only inflammation but also processing of somatic pain. Given that no evidence for roles of HMGB1 in visceral pain signaling is available, we asked if HMGB1 participates in bladder pain accompanying cystitis caused by cyclophosphamide in mice, using the anti-HMGB1 neutralizing antibody and recombinant human soluble thrombomodulin (rhsTM) that sequesters HMGB1 and promotes its degradation by thrombin. Cyclophosphamide, administered i.p., caused bladder pain-like nociceptive behavior and referred hyperalgesia accompanying cystitis symptoms including increased bladder weight, an indicator of edema, in mice. The cyclophosphamide-induced bladder pain and referred hyperalgesia, but not increased bladder weight, were prevented by i.p. preadministration of the anti-HMGB1 neutralizing antibody or rhsTM. HMGB1, given i.p., facilitated the bladder pain and referred hyperalgesia caused by a subeffective dose of cyclophosphamide, an effect blocked by rhsTM. In the cyclophosphamide-treated mice, HMGB1 levels greatly decreased in the bladder tissue, particularly in the urothelial cells, but did not change in the plasma. Low molecular weight heparin, known to inhibit the receptor for advanced glycation end products (RAGE), but not lipopolysaccharide from Rhodobacter sphaeroides, an inhibitor of toll-like receptor 4 (TLR4), blocked the cyclophosphamide-induced bladder pain and referred hyperalgesia. Thus, our data indicate involvement of HMGB1 in the cyclophosphamide-induced bladder pain signaling, but not cystitis itself, and suggest that targeting HMGB1 with rhsTM or blocking RAGE might serve as a novel therapeutic strategy for the management of bladder pain.

Roles of Cav3.2 and TRPA1 channels targeted by hydrogen sulfide in pancreatic nociceptive processing in mice with or without acute pancreatitis

Hydrogen sulfide (H2S), formed by multiple enzymes, including cystathionine‐γ‐lyase (CSE), targets Cav3.2 T‐type Ca2+ channels (T channels) and transient receptor potential ankyrin‐1 (TRPA1), facilitating somatic pain. Pancreatitis‐related pain also appears to involve activation of T channels by H2S formed by the upregulated CSE. Therefore, this study investigates the roles of the Cav3.2 isoform and/or TRPA1 in pancreatic nociception in the absence and presence of pancreatitis. In anesthetized mice, AP18, a TRPA1 inhibitor, abolished the Fos expression in the spinal dorsal horn caused by injection of a TRPA1 agonist into the pancreatic duct. As did mibefradil, a T‐channel inhibitor, in our previous report, AP18 prevented the Fos expression following ductal NaHS, an H2S donor. In the mice with cerulein‐induced acute pancreatitis, the referred hyperalgesia was suppressed by NNC 55‐0396 (NNC), a selective T‐channel inhibitor; zinc chloride; or ascorbic acid, known to inhibit Cav3.2 selectively among three T‐channel isoforms; and knockdown of Cav3.2. In contrast, AP18 and knockdown of TRPA1 had no significant effect on the cerulein‐induced referred hyperalgesia, although they significantly potentiated the antihyperalgesic effect of NNC at a subeffective dose. TRPA1 but not Cav3.2 in the dorsal root ganglia was downregulated at a protein level in mice with cerulein‐induced pancreatitis. The data indicate that TRPA1 and Cav3.2 mediate the exogenous H2S‐induced pancreatic nociception in naïve mice and suggest that, in the mice with pancreatitis, Cav3.2 targeted by H2S primarily participates in the pancreatic pain, whereas TRPA1 is downregulated and plays a secondary role in pancreatic nociceptive signaling.

Recombinant human soluble thrombomodulin prevents peripheral HMGB1‐dependent hyperalgesia in rats

High‐mobility group box 1 (HMGB1), a nuclear protein, is actively or passively released during inflammation. Recombinant human soluble thrombomodulin (rhsTM), a medicine for treatment of disseminated intravascular coagulation (DIC), sequesters HMGB1 and promotes its degradation. Given evidence for involvement of HMGB1 in pain signalling, we determined if peripheral HMGB1 causes hyperalgesia, and then asked if rhsTM modulates the HMGB1‐dependent hyperalgesia.

Peripheral HMGB1-induced hyperalgesia in mice: Redox state-dependent distinct roles of RAGE and TLR4

Nuclear HMGB1 that contains 3 cysteine residues is acetylated and secreted to the extracellular space, promoting inflammation via multiple molecules such as RAGE and TLR4. We thus evaluated and characterized the redox state-dependent effects of peripheral HMGB1 on nociception. Intraplantar (i.pl.) administration of bovine thymus-derived HMGB1 (bt-HMGB1), all-thiol HMGB1 (at-HMGB1) or disulfide HMGB1 (ds-HMGB1) caused long-lasting mechanical hyperalgesia in mice. The hyperalgesia following i.pl. bt-HMGB1 or at-HMGB1 was attenuated by RAGE inhibitors, while the ds-HMGB1-induced hyperalgesia was abolished by a TLR4 antagonist. Thus, nociceptive processing by peripheral HMGB1 is considered dependent on its redox states.

Therapeutic potential of RQ-00311651, a novel T-type Ca2+ channel blocker, in distinct rodent models for neuropathic and visceral pain.

Abstract T-type Ca2+ channels (T channels), particularly Cav3.2 among the 3 isoforms, play a role in neuropathic and visceral pain. We thus characterized the effects of RQ-00311651 (RQ), a novel T-channel blocker, in HEK293 cells transfected with human Cav3.1 or Cav3.2 by electrophysiological and fluorescent Ca2+ signaling assays, and also evaluated the antiallodynic/antihyperalgesic activity of RQ in somatic, visceral, and neuropathic pain models in rodents. RQ-00311651 strongly suppressed T currents when tested at holding potentials of −65 ∼ −60 mV, but not −80 mV, in the Cav3.1- or Cav3.2-expressing cells. RQ-00311651 also inhibited high K+-induced Ca2+ signaling in those cells. In mice, RQ, administered intraperitoneally (i.p.) at 5 to 20 mg/kg or orally at 20 to 40 mg/kg, significantly suppressed the somatic hyperalgesia and visceral pain-like nociceptive behavior/referred hyperalgesia caused by intraplantar and intracolonic administration of NaHS or Na2S, H2S donors, respectively, which involve the enhanced activity of Cav3.2 channels. RQ-00311651, given i.p. at 5 to 20 mg/kg, exhibited antiallodynic or antihyperalgesic activity in rats with spinal nerve injury–induced neuropathy or in rats and mice with paclitaxel-induced neuropathy. Oral and i.p. RQ at 10 to 20 mg/kg also suppressed the visceral nociceptive behavior and/or referred hyperalgesia accompanying cerulein-induced acute pancreatitis and cyclophosphamide-induced cystitis in mice. The analgesic and antihyperalgesic/antiallodynic doses of oral and i.p. RQ did not significantly affect the locomotor activity and motor coordination. Together, RQ is considered a state-dependent blocker of Cav3.1/Cav3.2 T channels and may serve as an orally available analgesic for treatment of neuropathic and inflammatory pain including distinct visceral pain with minimum central side effects.

Involvement of high mobility group box 1 in the development and maintenance of chemotherapy-induced peripheral neuropathy in rats

Given that high mobility group box 1 (HMGB1), a nuclear protein, once released to the extracellular space, promotes nociception, we asked if inactivation of HMGB1 prevents or reverses chemotherapy-induced painful neuropathy in rats and also examined possible involvement of Toll-like receptor 4 (TLR4) and the receptor for advanced glycation endproduct (RAGE), known as targets for HMGB1. Painful neuropathy was produced by repeated i.p. administration of paclitaxel or vincristine in rats. Nociceptive threshold was determined by the paw pressure method and/or von Frey test in the hindpaw. Tissue protein levels were determined by immunoblotting. Repeated i.p. administration of the anti-HMGB1-neutralizing antibody or recombinant human soluble thrombomodulin (rhsTM), known to inactivate HMGB1, prevented the development of hyperalgesia and/or allodynia induced by paclitaxel or vincristine in rats. A single i.p. or intraplantar (i.pl.) administration of the antibody or rhsTM reversed the chemotherapy-induced neuropathy. A single i.pl. administration of a TLR4 antagonist or low molecular weight heparin, known to inhibit RAGE, attenuated the hyperalgesia caused by i.pl. HMGB1 and also the chemotherapy-induced painful neuropathy. Paclitaxel or vincristine treatment significantly decreased protein levels of HMGB1 in the dorsal root ganglia, but not sciatic nerves. HMGB1 thus participates in both development and maintenance of chemotherapy-induced painful neuropathy, in part through RAGE and TLR4. HMGB1 inactivation is considered useful to prevent and treat the chemotherapy-induced painful neuropathy.

Repeated Cold Stress Enhances the Acute Restraint Stress-Induced Hyperthermia in Mice

The rodents exposed to repeated cold stress according to a specific schedule, known as specific alternation of rhythm in temperature (SART), exhibit autonomic imbalance, and is now used as an experimental model of fibromyalgia. To explore the susceptibility of SART-stressed animals to novel acute stress, we tested whether exposure of mice to SART stress for 1 week alters the extent of acute restraint stress-induced hyperthermia. Mice were subjected to 7-d SART stress sessions; i.e., the mice were alternately exposed to 24 and 4°C at 1-h intervals during the daytime (09:00-16:00) and kept at 4°C overnight (16:00-09:00). SART-stressed and unstressed mice were exposed to acute restraint stress for 20-60 min, during which rectal temperature was monitored. Serum corticosterone levels were measured before and after 60-min exposure to restraint stress. SART stress itself did not alter the body temperature or serum corticosterone levels in mice. Acute restraint stress increased the body temperature and serum corticosterone levels, both responses being greater in SART-stressed mice than unstressed mice. The enhanced hyperthermic responses to acute restraint stress in SART-stressed mice were significantly attenuated by SR59230A, a β3 adrenoceptor antagonist, but unaffected by diazepam, an anxiolytic, mifepristone, a glucocorticoid receptor antagonist, or indomethacin, a cyclooxygenase inhibitor. These results suggest that SART stress enhances the susceptibility of mice to acute restraint stress, characterized by increased hyperthermia and corticosterone secretion, and that the increased hyperthermic responses to acute stress might involve accelerated activation of sympathetic β3 adrenoceptors, known to regulate non-shivering thermogenesis in the brown adipose tissue.

Tacrolimus Triggers Transient Receptor Potential Vanilloid-1-Dependent Relapse of Pancreatitis-Related Pain in Mice

Transient receptor potential vanilloid-1 (TRPV1) expressed in nociceptors is directly phosphorylated and activated by protein kinase C, and involved in the signaling of pancreatic pain. On the other hand, Cav3.2 T-type Ca2+ channels expressed in nociceptors are functionally upregulated by phosphorylation with protein kinase A and also play a role in pancreatitis-related pain. Calcineurin, a phosphatase, negatively regulates various channel functions including TRPV1, and calcineurin inhibitor-induced pain syndrome by tacrolimus, a calcineurin inhibitor, used as an immunosuppressant, has been a clinical problem. We thus examined the effect of tacrolimus on pancreatitis-related pain in mice. Repeated treatment with cerulein caused referred hyperalgesia accompanying acute pancreatitis, which was unaffected by tacrolimus. Pancreatitis-related symptoms disappeared in 24 h, whereas the referred hyperalgesia recurred following the administration of tacrolimus, which was abolished by the blockers of TRPV1 but not T-type Ca2+ channels. Thus, tacrolimus appears to cause the TRPV1-dependent relapse of pancreatitis-related pain, suggesting the involvement of calcineurin in the termination of pancreatic pain.

Blockade of T-type calcium channels by 6-prenylnaringenin, a hop component, alleviates neuropathic and visceral pain in mice

ABSTRACT Since Cav3.2T‐type Ca2+ channels (T‐channels) expressed in the primary afferents and CNS contribute to intractable pain, we explored T‐channel‐blocking components in distinct herbal extracts using a whole‐cell patch‐clamp technique in HEK293cells stably expressing Cav3.2 or Cav3.1, and purified and identified sophoraflavanone G (SG) as an active compound from SOPHORAE RADIX (SR). Interestingly, hop‐derived SG analogues, (2S)‐6‐prenylnaringenin (6‐PNG) and (2S)‐8‐PNG, but not naringenin, also blocked T‐channels; IC50 (&mgr;M) of SG, (2S)‐6‐PNG and (2S)‐8‐PNG was 0.68–0.75 for Cav3.2 and 0.99–1.41 for Cav3.1. (2S)‐6‐PNG and (2S)‐8‐PNG, but not SG, exhibited reversible inhibition. The racemic (2R/S)‐6‐PNG as well as (2S)‐6‐PNG potently blocked Cav3.2, but exhibited minor effect on high‐voltage‐activated Ca2+ channels and voltage‐gated Na+ channels in differentiated NG108–15cells. In mice, the mechanical allodynia following intraplantar (i.pl.) administration of an H2S donor was abolished by oral or i.p. SR extract and by i.pl. SG, (2S)‐6‐PNG or (2S)‐8‐PNG, but not naringenin. Intraperitoneal (2R/S)‐6‐PNG strongly suppressed visceral pain and spinal ERK phosphorylation following intracolonic administration of an H2S donor in mice. (2R/S)‐6‐PNG, administered i.pl. or i.p., suppressed the neuropathic allodynia induced by partial sciatic nerve ligation or oxaliplatin, an anti‐cancer agent, in mice. (2R/S)‐6‐PNG had little or no effect on open‐field behavior, motor performance or cardiovascular function in mice, and on the contractility of isolated rat aorta. (2R/S)‐6‐PNG, but not SG, was detectable in the brain after their i.p. administration in mice. Our data suggest that 6‐PNG, a hop component, blocks T‐channels, and alleviates neuropathic and visceral pain with little side effects. HIGHLIGHTSCav3.2T‐type Ca2+ channels contribute to intractable pain.We identified sophoraflavanone G from SOPHORAE RADIX as a T‐channel blocker.The analogues, 6‐ and 8‐prenylnaringenin, derived from hops, also blocked T‐channels.6‐Prenylnaringenin had the most preferable characteristics as a T‐channel blocker.6‐Prenylnaringenin alleviated neuropathic and visceral pain with little side effects.

Repeated Cold Stress Reduces Cyclophosphamide-Induced Cystitis/Bladder Pain and Macrophage Activity in Mice

We examined the effect of repeated cold (RC) stress on cyclophosphamide (CPA)-induced cystitis/bladder pain in mice, in relation to macrophage activity. CPA, given i.p. at 400 mg/kg, caused bladder pain symptoms accompanying cystitis in both unstressed and RC-stressed mice, which were prevented by the macrophage inhibitor minocycline. A low dose, that is, 200 mg/kg, of CPA still produced bladder pain symptoms in unstressed but not RC-stressed mice. Lipopolysaccharide-induced cytokine production in peritoneal macrophages from RC-stressed mice was less than that from unstressed mice. Thus, RC stress appears to reduce CPA-induced bladder pain in mice, which may be associated with the decreased macrophage activity.